US20030199467A1

US20030199467A1 - Antisense modulation of human Rho family gene expression

Info

Publication number: US20030199467A1
Application number: US10/178,325
Authority: US
Inventors: M. Roberts; Lex Cowsert
Original assignee: Individual
Current assignee: Individual
Priority date: 1998-09-18
Filing date: 2002-06-21
Publication date: 2003-10-23
Also published as: AU6647700A; US6410323B1; WO2001015739A1

Abstract

This invention provides compositions and methods for modulating expression of members of the human Rho gene family, which encode low molecular weight GTPases that act as molecular switches in signal transduction. In preferred embodiments, Rho family members include RhoA, RhoB, RhoC, RhoG, Rac1 and cdc42. This invention is also directed to methods for inhibiting hyperproliferation of cells; these methods can be used diagnostically or therapeutically. Furthermore, this invention is directed to treatment of conditions associated with expression of the human Rho family members, particularly in hyperproliferative disorders.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. Ser. No. 09/387,341 filed Aug. 31, 1999 which is a continuation-in-part application of U.S. Ser. No. 09/156,424 filed Sep. 18, 1998, now issued as U.S. Pat. No. 5,945,290, and U.S. Ser. No. 09/156,979 filed Sep. 18, 1998, now issued as U.S. Pat. No. 5,962,672, and U.S. Ser. No. 09/156,807 filed Sep. 18, 1998, now issued as U.S. Pat. No. 6,030,786, and U.S. Ser. No. 09/161,015, filed on Sep. 25, 1998, now issued as U.S. Pat. No. 5,965,370.[0001]

FIELD OF THE INVENTION

This invention relates to compositions and methods for modulating expression of members of the human Rho gene family, which encode low molecular weight GTPases that act as molecular switches in signal transduction. This invention is also directed to methods for inhibiting hyperproliferation of cells; these methods can be used diagnostically or therapeutically. Furthermore, this invention is directed to treatment of conditions associated with expression of the human Rho family member genes.

BACKGROUND OF THE INVENTION

The Rho family of genes are a sub-family of low molecular weight GTPases and are related to each other based on sequence homology and function (Vojtek, A. B., and Cooper, J. A., Cell 1995, 82, 527-529). Other sub-families include Ras, Rab, Arf, and Ran. As GTPases, these proteins bind and hydrolyze GTP. In an active state, they bind to GTP and transduce signals of other proteins in signal transduction pathways. In their inactive state, they are bound to GDP. Members of the Rho family are typically involved in regulation of the actin cytoskeleton. Members of the Rho family include RhoA, RhoB, RhoC, RhoD, RhoE, RhoG, Rac1, Rac2, Rac3 and Cdc42.

Each class appears to have a unique function in actin reorganization. Rho has been shown to be essential for the formation of stress fibers and focal adhesions (Ridley, A. J. and Hall, A., Cell 1992, 70, 389-399). Focal adhesions are an area of the cell where integrin receptors cluster and extracellular matrix proteins such as fibronectin and collagen are bound. Stress fibers attach at these focal adhesions within a cell. Rac has been shown to be essential for the formation of membrane ruffles, which results from the formation of large vesicles within the cell (Ridley, A. J., et al., Cell 1992, 70, 401-410). Cdc42 (also known as cdc42Hs and G25K) regulates the formation of filopodia, short bundles of actin filaments that protrude from a cell (Nobes, C. D. and Hall, A., Cell 1995, 81, 53-62). Such activities on cell morphology may play an important role in cell motility, cytokinesis, and endocytosis.

Additional functions for the Rho family have begun to be elucidated. Rac and Rho have been found to promote cadherin-based cell-cell adhesion (Takaishi, K., et al., J. Cell Biol. 1997, 139, 1047-1059). Rac1 and Cdc42 play a critical role in the c-jun amino-terminal kinase (JNK)/stress-activated protein kinase (SAPK) signaling pathway, thereby, potentially having an important role in gene transcription (Coso, O. A. et al., Cell 1995, 81, 1137-1146). RhoA, Rac1 and Cdc42 also regulate transcription through JNK-independent pathways by binding to either serum response factor (SRF; Hill, C. S., et al., Cell 1995, 81, 1159-1170) or NF-κB (Perona, R., et al., Genes and Develop. 1997, 11, 463-475).

Members of the Rac subfamily have also been found to regulate oxygen radical production. Both Rac1 (Sundaresan, M., et al., Biochem. J. 1996, 318, 379-382) and Rac2 (Knaus, U. G., et al., Science 1991, 254, 1512-1515) are involved in this process.

Members of the Rho family are thought to be involved in various disease processes, including cancer. Rho, Rac and Cdc42 all play a role in Ras transformation. Rac was found to essential for transformation by Ras, but not RafCAAX, a modified Raf kinase with a localization signal from K-ras (Qiu, R.-G., et al., Nature 1995 374, 457-459). Rho is not essential for Ras transformation, but acts cooperatively in transformation by Ras and RafCAAX (Qiu, R.-G., et al., Proc. Natl. Acad. Sci. USA 1995, 92, 11781-11785). Cdc42 was also found to be essential for Ras transformation, but its role is distinct from that of Rac (Qiu, R.-G., et al., Mol. Cell Biol. 1997, 17, 3449-3458). In addition to transformation, members of the Rho family may also play a role in invasion and metastasis. Michiels, F. et al. (Nature 1995, 375, 338-340) demonstrated that T-lymphoma cells that constitutively expressed Rac1 became invasive. Yoshioka, K. et al. (J. Biol. Chem. 1998, 273, 5146-5154) found that cells stably transfected with RhoA were also invasive. The RhoB gene has been classified as an immediate-early gene, which means that its transcription is rapidly activated upon exposure to certain growth factors or mitogens. The factors shown to activate RhoB transcription include epidermal growth factor (EGF), platelet-derived growth factor (PDGF), genotoxic stress from UV light, alkylating xenobiotics and the retroviral oncogene v-fps. Each of these stimuli triggers DNA synthesis in cultures of high cell density (Engel et al., J. Biol. Chem., 1998, 273, 9921-9926). The response of RhoB to these factors implies a role for RhoB in wound repair and tissue regeneration upon growth factor stimulation and tumorigenesis upon mitogen stimulation.

The involvement of Rho family proteins in ras-mediated transformation and tumor cell invasion suggests that they could be novel targets for cancer treatment (Ridley, A. J., Int. J. Biochem. Cell Biol. 1997, 29, 1225-1229). In particular, overexpression of the RhoC gene has been associated with pancreatic cancer. Suwa, H. et al. (Br. J. Cancer, 1998, 77, 147-152) looked for a role of RhoA, RhoB and RhoC genes in ductal adenocarcinoma of the pancreas. They found that expression levels of RhoC were higher in tumors than in normal tissue and that metastatic tumors expressed RhoC at higher levels than primary tumors. Rho C expression is also elevated in a megakaryocytic leukemia cell line, CMK. Takada et al., Exp. Hematol., 1996, 24, 524-530. Manifestations of altered RhoB regulation also appear in disease states, including the development of cancer. Cellular transformation and acquisition of the metastatic phenotype are the two main changes normal cells undergo during the progression to cancer. Expression of constitutively activated forms of RhoB have been shown to cause tumorigenic transformation of NIH 3T3 and Rat1 rodent fibroblasts (Khosravi-Far et al., Adv. Cancer Res., 1998, 72, 57-107). RhoB has also been shown to be overexpressed in human breast cancer tissues (Zalcman et al., Oncogene, 1995, 10, 1935-1945). RhoA is also believed to be involved in the development of cancer. Cellular transformation and acquisition of the metastatic phenotype are the two main changes normal cells undergo during the progression to cancer. Recent studies demonstrate that RhoA-regulated pathways can induce both changes in cells. Injecting cells transformed with rhoA genes directly into the bloodstream of mice produced metastasis, or tumor growth, in distant organs (del Peso et al., Oncogene, 1997, 15, 3047-3057).

It has also been suggested that inhibition of Rac genes may be useful for preventing reoxygenation injury as it occurs when ischemic cells undergo reperfusion (Kim, K.-S., et al., J. Clin. Invest. 1998, 101, 1821-1826). With reoxygenation, reactive oxygen species are presented to the cell, greatly augmenting cell death. Kim, K.-S., et al. showed that adenoviral-mediated transfer of a dominant negative Rac1 could inhibit the formation of reactive oxygen species and protect cells against hypoxia/reoxygenation-induced cell death. They suggest that inhibition of rac1 would be useful, clinically, in treatment in cases where there is the possibility of reperfusion injury.

Manifestations of altered RhoA regulation also appear in both injury and disease states. It has been proposed that acute central nervous system trauma may contribute to the development of Alzheimer's disease. Findings that show a high concentration of thrombin, a serine-protease in the blood clotting cascade, localized to the plaques of Alzheimer's disease brains support this claim. An excess of thrombin has been shown to stimulate Rho A activity with a concomitant increase in apoptosis (programmed cell death) (Donovan et al., J. Neurosci., 1997, 17, 5316-5326). These studies also imply a role for RhoA in wound repair and clotting disorders.

Although members of the Rho family have been implicated in various disease processes including cancer and reoxygenation injury, no effective therapy specifically targeting these proteins is available. Antisense oligonucleotides have been used to study the role of some Rho family members in various physiological processes. Dorseuil, O., et al. ( J. Biol. Chem. 1992, 267, 20540-20542) used an 16-mer antisense oligonucleotide targeted to the start site of both Rac1 and Rac2 and demonstrated a dose-dependent reduction in superoxide production in whole cells. Brenner, B., et al. (Biochem. Biophys. Res. Commun. 1997, 231, 802-807) used a similar oligonucleotide (a 15-mer targeted to the start site) and showed that inhibition of Rac2 protein expression prevented L-selectin-induced actin polymerization. An 45-mer antisense oligonucleotide targeted to the 3′-UTR has also been used as a probe for rac1 (Didsbury, J., et al., J. Biol. Chem. 1989, 264, 16378-16382).

Thus, there remains an unmet need for compositions and methods targeting expression of Rho family members, and disease processes associated there-with.

SUMMARY OF THE INVENTION

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

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

The present invention also comprises methods for diagnosing and treating cancer and preventing reoxygenation injury. These methods are believed to be useful, for example, in diagnosing Rho family member-associated disease progression. These methods employ the oligonucleotides of the invention. These methods are believed to be useful both therapeutically, including prophylactically, and as clinical research and diagnostic tools.

DETAILED DESCRIPTION OF THE INVENTION

Members of the Rho family of GTPases are essential for transformation by Ras and play a role in tumor cell invasion. In addition, the Rac subfamily is a regulator of oxygen radical formation. As such, they represent attractive targets for antineoplastic therapy and preventative agents for radical deoxygenation. In particular, modulation of the expression of RhoC may be useful for the treatment of pancreatic carcinomas and modulation of Rac1 may be useful for preventing ischemia/reperfusion injury.

Antisense oligonucleotides targeting members of the Rho family represent a novel therapeutic approach.

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

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

In accordance with this invention, persons of ordinary skill in the art will understand that messenger RNA includes not only the information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the 5′-untranslated region, the 3′-untranslated region, the 5′ cap region and intron/exon junction ribonucleotides. Thus, oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the informational ribonucleotides. The oligonucleotide may therefore be specifically hybridizable with a transcription initiation site region, a translation initiation codon region, a 5′ cap region, an intron/exon junction, coding sequences, a translation termination codon region or sequences in the 5′- or 3′-untranslated region. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon.” A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding a Rho family member, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region,” “AUG region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. This region is a preferred target region. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon. This region is a preferred target region. The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other preferred target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene. The 5′ cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5′ cap region may also be a preferred target region.

Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a pre-mRNA transcript to yield one or more mature mRNA. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., exon-exon or intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. Targeting particular exons in alternatively spliced mRNAs may also be preferred. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.

Once the target site or sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired modulation.

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

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

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

Hybridization of antisense oligonucleotides with mRNA interferes with one or more of the normal functions of mRNA. The functions of mRNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA. Binding of specific protein(s) to the RNA may also be interfered with by antisense oligonucleotide hybridization to the RNA.

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

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

The present invention is also suitable for diagnosing abnormal proliferative states in tissue or other samples from patients suspected of having a hyperproliferative disease such as cancer. The ability of the oligonucleotides of the present invention to inhibit cell proliferation may be employed to diagnose such states. A number of assays may be formulated employing the present invention, which assays will commonly comprise contacting a tissue sample with an oligonucleotide of the invention under conditions selected to permit detection and, usually, quantitation of such inhibition. In the context of this invention, to “contact” tissues or cells with an oligonucleotide or oligonucleotides means to add the oligonucleotide(s), usually in a liquid carrier, to a cell suspension or tissue sample, either in vitro or ex vivo, or to administer the oligonucleotide(s) to cells or tissues within an animal. Similarly, the present invention can be used to distinguish a Rho family member-associated tumor from tumors having other etiologies, or those associated with one rho family member from another, in order that an efficacious treatment regimen can be designed.

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

In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent intersugar (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced binding to target and increased stability in the presence of nucleases.

The antisense compounds in accordance with this invention preferably comprise from about 5 to about 50 nucleobases. Particularly preferred are antisense oligonucleotides comprising from about 8 to about 30 nucleobases (i.e. from about 8 to about 30 linked nucleosides). As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, 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 U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. No. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361 and 5,625,050.

Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH ₂component parts.

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

In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNA compounds can be found in Nielsen et al. ( Science, 1991, 254, 1497-1500).

Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH ₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl, O-alkyl-O-alkyl, O—, S—, or N-alkenyl, or O—, S—or N-alkynyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C ₁to C₁₀alkyl or C₂to C₁₀alkenyl and alkynyl. Particularly preferred are O[(CH₂)_nO]_mCH₃, O(CH₂)_nOCH₃, O(CH₂)₂ON(CH₃)₂, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH₂)_nON[(CH₂)_n(CH₃)]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁to C₁₀lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′—O—CH₂CH₂OCH₃, also known as 2′—O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in U.S. patent application Ser. No. 09/016,520, filed on Jan. 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′—O—CH ₃), 2′-aminopropoxy (2′—OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′—F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugars structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920.

Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C or m5c), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering 1990, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, those disclosed by Englisch et al. (Angewandte Chemie, International Edition 1991, 30, 613-722), and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications 1993, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications 1993, CRC Press, Boca Raton, pages 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941.

Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett. 1994, 4, 1053-1059), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci. 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let. 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J. 1991, 10, 1111-1118; Kabanov et al., FEBS Lett. 1990, 259, 327-330; Svinarchuk et al., Biochimie 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett. 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res. 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett. 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al. , J. Pharmacol. Exp. Ther. 1996, 277, 923-937).

Representative U.S. patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

The present invention also includes oligonucleotides which are chimeric oligonucleotides. “Chimeric” oligonucleotides or “chimeras,” in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of antisense inhibition of gene expression. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. This RNAse H-mediated cleavage of the RNA target is distinct from the use of ribozymes to cleave nucleic acids. Ribozymes are not comprehended by the present invention.

Examples of chimeric oligonucleotides include but are not limited to “gapmers,” in which three distinct regions are present, normally with a central region flanked by two regions which are chemically equivalent to each other but distinct from the gap. A preferred example of a gapmer is an oligonucleotide in which a central portion (the “gap”) of the oligonucleotide serves as a substrate for RNase H and is preferably composed of 2′-deoxynucleotides, while the flanking portions (the 5′ and 3′ “wings”) are modified to have greater affinity for the target RNA molecule but are unable to support nuclease activity (e.g., 2′-fluoro- or 2′-O-methoxyethyl-substituted). Other chimeras include “wingmers,” also known in the art as “hemimers,” that is, oligonucleotides with two distinct regions. In a preferred example of a wingmer, the 5′ portion of the oligonucleotide serves as a substrate for RNase H and is preferably composed of 2′-deoxynucleotides, whereas the 3′ portion is modified in such a fashion so as to have greater affinity for the target RNA molecule but is unable to support nuclease activity (e.g., 2′-fluoro- or 2′-O-methoxyethyl-substituted), or vice-versa. In one embodiment, the oligonucleotides of the present invention contain a 2′-O-methoxyethyl (2′—O—CH ₂CH₂OCH₃) modification on the sugar moiety of at least one nucleotide. This modification has been shown to increase both affinity of the oligonucleotide for its target and nuclease resistance of the oligonucleotide. According to the invention, one, a plurality, or all of the nucleotide subunits of the oligonucleotides of the invention may bear a 2′-O-methoxyethyl (—O—CH₂CH₂OCH₃) modification. Oligonucleotides comprising a plurality of nucleotide subunits having a 2′-O-methoxyethyl modification can have such a modification on any of the nucleotide subunits within the oligonucleotide, and may be chimeric oligonucleotides. Aside from or in addition to 2′-O-methoxyethyl modifications, oligonucleotides containing other modifications which enhance antisense efficacy, potency or target affinity are also preferred. Chimeric oligonucleotides comprising one or more such modifications are presently preferred.

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

The antisense compounds of the present invention include bioequivalent compounds, including pharmaceutically acceptable salts and prodrugs. This is intended to encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of the nucleic acids of the invention and prodrugs of such nucleic acids. APharmaceutically acceptable salts@ are physiologically and pharmaceutically acceptable salts of the nucleic acids of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma. Sci. 1977, 66, 1-19).

For oligonucleotides, examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

The oligonucleotides of the invention may additionally or alternatively be prepared to be delivered in a Aprodrug@ form. The term Aprodrug@ indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993.

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

Pharmaceutical compositions comprising the oligonucleotides of the present invention may include penetration enhancers in order to enhance the alimentary delivery of the oligonucleotides. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., fatty acids, bile salts, chelating agents, surfactants and non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, 8, 91-192; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1-33). One or more penetration enhancers from one or more of these broad categories may be included.

Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, recinleate, monoolein (a.k.a. 1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, mono- and di-glycerides and physiologically acceptable salts thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1; El-Hariri et al., J. Pharm. Pharmacol. 1992 44, 651-654).

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

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

Chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)[Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1-33; Buur et al., J. Control Rel. 1990, 14, 43-51). Chelating agents have the added advantage of also serving as DNase inhibitors.

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

Non-surfactants include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol. 1987, 39, 621-626).

As used herein, “carrier compound” refers to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor.

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 mind so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinyl-pyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrates (e.g., starch, sodium starch glycolate, etc.); or wetting agents (e.g., sodium lauryl sulphate, etc.). Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are described in U.S. Pat. No. 4,704,295; 4,556,552; 4,309,406; and 4,309,404.

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional compatible pharmaceutically-active materials such as, e.g., antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the 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 oligonucleotides of the invention are introduced into a patient, colloidal dispersion systems may be used as delivery vehicles to enhance the in vivo stability of the oligonucleotides and/or to target the oligonucleotides to a particular organ, tissue or cell type. Colloidal dispersion systems include, but are not limited to, macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes and lipid:oligonucleotide complexes of uncharacterized structure. A preferred colloidal dispersion system is a plurality of liposomes. Liposomes are microscopic spheres having an aqueous core surrounded by one or more outer layers made up of lipids arranged in a bilayer configuration (see, generally, Chonn et al., Current Op. Biotech. 1995, 6, 698-708).

The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, epidermal, and transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration, e.g., by inhalation or insufflation, or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.

Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

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

Compositions for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. In some cases it may be more effective to treat a patient with an oligonucleotide of the invention in conjunction with other traditional therapeutic modalities in order to increase the efficacy of a treatment regimen. In the context of the invention, the term “treatment regimen” is meant to encompass therapeutic, palliative and prophylactic modalities. For example, a patient may be treated with conventional chemotherapeutic agents, particularly those used for tumor and cancer treatment. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. Preferred are chemotherapeutic agents which are direct or indirect inhibitors of a Rho family member. These include MTX, Tomudex and fluorinated pyrimidines such as 5-FU and 5-FUdR. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide).

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 ₅₀s found to be effective in vitro and in in vivo animal models. In general, dosage is from 0.01 μg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every 20 years.

Thus, in the context of this invention, by “therapeutically effective amount” is meant the amount of the compound which is required to have a therapeutic effect on the treated individual. This amount, which will be apparent to the skilled artisan, will depend upon the age and weight of the individual, the type of disease to be treated, perhaps even the gender of the individual, and other factors which are routinely taken into consideration when designing a drug treatment. A therapeutic effect is assessed in the individual by measuring the effect of the compound on the disease state in the animal. For example, if the disease to be treated is cancer, therapeutic effects are assessed by measuring the rate of growth or the size of the tumor, or by measuring the production of compounds such as cytokines, production of which is an indication of the progress or regression of the tumor.

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

EXAMPLES

Example 1

Synthesis of Oligonucleotides

Unmodified oligodeoxynucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine. β-cyanoethyldiisopropyl-phosphoramidites are purchased from Applied Biosystems (Foster City, Calif.). For phosphorothioate oligonucleotides, the standard oxidation bottle was replaced by a 0.2 M solution of [0071] ³H-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′-methoxyβ-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham, Mass.) 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. [0072]
2′-fluoro oligonucleotides were synthesized as described in Kawasaki et al. ([0073] J. Med. Chem. 1993, 36, 831-841). Briefly, the protected nucleoside N⁶-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′-α-fluoro atom is introduced by a S_N2-displacement of a 2′-β-O-trifyl group. Thus N⁶-benzoyl-9-β-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N⁶-benzoyl groups was accomplished using standard methodologies and standard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.
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. [0074]
Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by the modification of a known 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. [0075]
2′-deoxy-2′-fluorocytidine was synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N[0076] 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, P. ([0077] Helv. Chim. Acta 1995, 78, 486-506). For ease of synthesis, the last nucleotide was a deoxynucleotide. 2′—O—CH₂CH₂OCH_3— cytosines may be 5-methyl cytosines.
Synthesis of 5-Methyl Cytosine Monomers [0078]
2,2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]: [0079]
5-Methyluridine (ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released in a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure. The resulting syrup was poured into diethylether (2.5 L), with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL). The solution was poured into fresh ether (2.5 L) to yield a stiff gum. The ether was decanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for 24 h) to give a solid which was crushed to a light tan powder (57 g, 85% crude yield). The material was used as is for further reactions. [0080]
2′-O-Methoxyethyl-5-methyluridine: [0081]
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[0082] ₃CN (600 mL) and evaporated. A silica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂(250 mL) and adsorbed onto silica (150 g) prior to loading onto the column. The product was eluted with the packing solvent to give 160 g (63%) of product.
2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine: [0083]
2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporated with pyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L). A first aliquot of dimethoxy-trityl 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[0084] ₃CN (200 mL). The residue was dissolved in CHCl₃(1.5 L) and extracted with 2×500 mL of saturated NaHCO₃and 2×500 mL of saturated NaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated. 275 g of residue was obtained. The residue was purified on a 3.5 kg silica gel column, packed and eluted with EtOAc/-Hexane/Acetone (5:5:1) containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 g of product. Approximately 20 g additional was obtained from the impure fractions to give a total yield of 183 g (57%).
3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine: [0085]
2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room temperature for 24 hours. The reaction was monitored by tlc by first quenching the tlc sample with the addition of MeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL) was added and the mixture evaporated at 35° C. The residue was dissolved in CHCl[0086] ₃(800 mL) and extracted with 2×200 mL of saturated sodium bicarbonate and 2×200 mL of saturated NaCl. The water layers were back extracted with 200 mL of CHCl₃. The combined organics were dried with sodium sulfate and evaporated to give 122 g of residue (approx. 90% product). The residue was purified on a 3.5 kg silica gel column and eluted using EtOAc/Hexane(4:1). Pure product fractions were evaporated to yield 96 g (84%).
3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine: [0087]
A first solution was prepared by dissolving 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in CH[0088] ₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L), cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solution maintained at 0-10° C., and the resulting mixture stirred for an additional 2 hours. The first solution was added dropwise, over a 45 minute period, to the later solution. The resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The filtrate was washed with 1×300 mL of NaHCO₃and 2×300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound.
2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine: [0089]
A solution of 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH[0090] ₄OH (30 mL) was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2×200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. MeOH (400 mL) saturated with NH₃gas was added and the vessel heated to 100° C. for 2 hours (tlc showed complete conversion). The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL). The organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound.
N[0091] ⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine:
2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, tlc showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MEOH (200 mL). The residue was dissolved in CHCl[0092] ₃(700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄and evaporated to give a residue (96 g). The residue was chromatographed on a 1.5 kg silica column using EtOAc/Hexane (1:1) containing 0.5% Et₃NH as the eluting solvent. The pure product fractions were evaporated to give 90 g (90%) of the title compound.
N[0093] ⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite:
N[0094] ⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) was dissolved in CH₂Cl₂(1 L) Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere. The resulting mixture was stirred for 20 hours at room temperature (tlc showed the reaction to be 95% complete). The reaction mixture was extracted with saturated NaHCO₃(1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes were back-extracted with CH₂Cl₂(300 mL), and the extracts were combined, dried over MgSO₄and concentrated. The residue obtained was chromatographed on a 1.5 kg silica column using EtOAc\Hexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g (87%) of the title compound.
5-methyl-2′-deoxycytidine (5-me-C) containing oligonucleotides were synthesized according to published methods (Sanghvi et al., [0095] Nucl. Acids Res. 1993, 21, 3197-3203) using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.).
Oligonucleotides having methylene (methylimino) (MMI) backbones are synthesized according to U.S. Pat. No. 5,378,825, which is coassigned to the assignee of the present invention and is incorporated herein in its entirety. For ease of synthesis, various nucleoside dimers containing MMI linkages were synthesized and incorporated into oligonucleotides. Other nitrogen-containing backbones are synthesized according to WO 92/20823 which is also coassigned to the assignee of the present invention and incorporated herein in its entirety. [0096]
Oligonucleotides having amide backbones are synthesized according to De Mesmaeker et al. ([0097] Acc. Chem. Res. 1995, 28, 366-374). The amide moiety is readily accessible by simple and well-known synthetic methods and is compatible with the conditions required for solid phase synthesis of oligonucleotides.
Oligonucleotides with morpholino backbones are synthesized according to U.S. Pat. No. 5,034,506 (Summerton and Weller). [0098]
Peptide-nucleic acid (PNA) oligomers are synthesized according to P. E. Nielsen et al. ([0099] Science 1991, 254, 1497-1500).
After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55° C. for 18 hours, the oligonucleotides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on denaturing gels and judged to be at least 85% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in synthesis were periodically checked by [0100] ³¹P nuclear magnetic resonance spectroscopy, and for some studies oligonucleotides were purified by HPLC, as described by Chiang et al. (J. Biol. Chem. 1991, 266, 18162). Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 2

Human RhoA Oligonucleotide Sequences

Antisense oligonucleotides were designed to target human RhoA. Target sequence data are from the RhoA cDNA sequence published by Yeramian, P., et al. ([0101] Nucleic Acids Res. 1987, 15, 1869); Genbank accession number X05026, provided herein as SEQ ID NO: 1. Oligonucleotides were synthesized primarily with phosphorothioate linkages. Oligonucleotide sequences are shown in Table 1.
A549 cells, human lung carcinoma cells (obtained from American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (DMEM) low glucose, 10% fetal calf serum, and penicillin (50 units/ml)/streptomycin (50 mg/ml). Cells were passaged at 90-95% confluency. All culture reagents were obtained from Life Technologies (GIBCO BRL, Rockville, Md). [0102]
A549 cells were plated at a starting cell number of approximately 2×10[0103] ⁵cells per well. After twenty-four hours, at 80-90% confluency, the cells were washed twice with Opti-Mem (GIBCO BRL) and the oligonucleotide formulated in LIPOFECTIN (GIBCO BRL), a 1:1 (w/w) liposome formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA), and dioleoyl phosphotidylethanolamine (DOPE) in membrane filtered water, at a constant ratio of 2.5 mg/ml LIPOFECTIN to 100 nM oligonucleotide, in Opti-Mem. For an initial screen, the oligonucleotide concentration was 300 nM. Treatment was for four hours. After treatment, the media was removed and the cells were further incubated in DMEM containing 10% FCS, and penicillin/streptomycin for 24 or 48 hours.
mRNA was isolated using the MICRO-FASTTRACK kit (Invitrogen, Carlsbad, Calif.), separated on a 1% agarose gel, transferred to Hybond-N+ membrane (Amersham, Arlington Heights, Ill.), a positively charged nylon membrane, and probed. A RhoA probe was generated using asymmetric PCR, in the presence of a[[0104] ³²P]-dCTP (Amersham), with the following primers:

Forward: 5′-TGCAAGCACAGCCCTTATG-3′ SEQ ID NO. 2

Reverse: 5′-TGTCAAAAGGACCCTGGTG-3′ SEQ ID NO. 3

A glyceraldehyde 3-phosphate dehydrogenase (G3PDH) probe was purchased from Clontech (Palo Alto, Calif.), Catalog Number 9805-1. The probe was labeled by random primer using the Large Fragment of DNA polymerase (Klenow fragment) (GIBCO BRL) as described in Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, 1989. mRNA was quantitated by a PhosphoImager (Molecular Dynamics, Sunnyvale, Calif.).

TABLE 1


Nucleotide Sequences of RhoA Oligonucleotides

			TARGET GENE
		SEQ	NUCLEOTIDE	GENE
ISIS	NUCLEOTIDE SEQUENCE	ID	CO-	TARGET
NO.	(5′ -> 3′)	NO:	ORDINATES¹	REGION

16191	AGTCGCAAACTCGGAGAC	4	0085-0102	5′-UTR

16192	TTGCTCAGGCAACGAATC	5	0142-0159	AUG

16193	CTGAACACTATCACCAAGCATG	6	0214-0235	Coding

16194	CTCATCATTCCGAAGATCC	7	0515-0533	Coding

16195	CCAATCCTGTTTGCCATATCTC	8	0592-0613	Coding

16196	CCATCTTTGGTCTTTGCTGAAC	9	0634-0655	Coding

16197	CCAGAGCAGCTCTCGTAGCCA	10	0676-0696	Coding

16198	TCACAAGACAAGGCAACCAG	11	0721-0740	Stop

16199	AGGCCAGTAATCATACACTA	12	0799-0818	3′-UTR

16200	GTTGGCTTCTAAATACTGCT	13	0871-0890	3′-UTR

16201	GGCTGTTAGAGCAGTGTCAA	14	0937-0956	3′-UTR

16202	AGCGCCTGGTGTGTCAGGTG	15	0971-0990	3′-UTR

16203	TAGTTACAGCCTAATTGACA	16	1051-1073	3′-UTR

16913	GGCACCTGTTGGGTGAGCTG	17	16202 control

16914	ACACTCTTGCTTACCGTACCTT	18	16195 control

16915	TCCCGTAAGTGCGGTATCAA	19	16201 control

Results are shown in Table 2. Oligonucleotides 16193 (SEQ. ID NO. 6), 16195 (SEQ ID NO. 8), 16196 (SEQ ID NO. 9), 16197 (SEQ ID NO. 10), 16198 (SEQ ID NO. 11), 16199 (SEQ ID NO. 12), 16200 (SEQ ID NO. 13), 16201 (SEQ ID NO. 14), and 16202 (SEQ ID NO. 15) gave better than 50% inhibition of RhoA expression. Oligonucleotides 16195 (SEQ ID NO. 8), 16197 (SEQ ID NO. 10), 16199 (SEQ ID NO. 12), 16201 (SEQ ID NO. 14), and 16202 (SEQ ID NO. 15) gave better than 75% inhibition of RhoA expression.

TABLE 2


Activities of Phosphorothioate Oligonucleotides Targeted to
Human RhoA

	SEQ	GENE
ISIS	ID	TARGET	% mRNA	% mRNA
No:	NO:	REGION	EXPRESSION	INHIBITION

LIPOFECTIN	—	—	100.0%	0.0%
only
16191	4	5′-UTR	66.4%	33.6%
16192	5	AUCG	68.0%	32.0%
16193	6	Coding	31.9%	68.1%
16194	7	Coding	79.9%	20.1%
16195	8	Coding	3.9%	96.1%
16196	9	Coding	31.4%	68.6%
16197	10	Coding	19.2%	81.8%
16198	11	Stop	46.4%	53.6%
16199	12	3′-UTR	22.9%	77.1%
16200	13	3′-UTR	36.9%	63.1%
16201	14	3′-UTR	22.0%	78.0%
16202	15	3′-UTR	14.4%	85.6%
16203	16	3′-UTR	88.0%	12.0%

Example 3

Dose Response and Specificity of Antisense Oligonucleotide Effects on Human RhoA mRNA Levels in A549 Cells

Three of the most active oligonucleotides from the initial screen were chosen for dose response assays. These include oligonucleotides 16195 (SEQ ID NO. 8), 16201 (SEQ ID NO. 14), and 16202 (SEQ ID NO. 15). A549 cells were grown, treated and processed as described in Example 2. LIPOFECTIN was added at a ratio of 2.5 mg/ml per 100 nM of oligonucleotide. The control included LIPOFECTIN at a concentration of 7.5 mg/ml. Results are shown in Table 3. Each oligonucleotide showed a dose response effect with maximal inhibition greater than 90%. [0107]

The specificity of these oligonucleotides was investigated using scrambled controls, i.e. oligonucleotides with the same base composition and a scrambled sequence. Oligonucleotide 16915 (SEQ ID NO. 19) is a scrambled control for 16201 (SEQ ID NO. 14) and oligonucleotide 16913 (SEQ ID NO. 17) is a scrambled control for 16202 (SEQ ID NO. 15). Both antisense oligonucleotides showed a dose dependent effect on mRNA expression, while scrambled controls showed much less inhibition which was only seen at higher does.

TABLE 3


Dose Response of A549 Cells to RhoA
Antisense Oligonucleotides (ASOs)

	SEQ ID	ASO Gene		% mRNA	% mRNA
ISIS #	NO:	Target	Dose	Expression	Inhibition

control	—	LIPOFECTIN	—	100.0%	0.0%
		only
16195	8	Coding	75 nM	72.7%	27.3%
16195	8	″	150 nM	35.0%	65.0%
16195	8	″	300 nM	20.3%	79.7%
16201	14	3′-UTR	75 nM	79.1%	20.9%
16201	14	″	150 nM	35.7%	64.3%
16201	14	″	300 nM	9.5%	90.5%
16202	15	3′-UTR	75 nM	68.7%	31.3%
16202	15	″	150 nM	28.8%	71.2%
16202	15	″	300 nM	6.1%	93.7%

TABLE 4


Specificity of RhoA Antisense Oligonucleotides (ASOs) in
A549 Cells

	SEQ ID	ASO Gene		% mRNA	% mRNA
ISIS #	NO:	Target	Dose	Expression	Inhibition

control	—	LIPOFECTIN	—	100%	0%
		only
16201	14	3′-UTR	75 nM	64.4%	35.6%
16201	14	″	150 nM	35.3%	64.7%
16201	14	″	300 nM	5.7%	94.3%
16915	19	control	75 nM	89.9%	10.1%
16915	19	″	150 nM	98.3%	1.7%
16915	19	″	300 nM	84.8%	15.2%
16202	15	3′-UTR	75 nM	39.9%	60.1%
16202	15	″	150 nM	20.2%	79.8%
16202	15	″	300 nM	10.8%	89.2%
16913	17	control	75 nM	97.6%	2.4%
16913	17	″	150 nM	89.8%	10.2%
16913	17	″	300 nM	55.6%	44.4%

Example 4

Design and Testing of Chimeric (Deoxy Gapped) 2′-O-methoxyethyl RhoA Antisense Oligonucleotides on RhoA Levels in A549 Cells

Oligonucleotides having SEQ ID NO: 14 were synthesized as a uniformly phosphorothioate or mixed phosphorothioate/phosphodiester chimeric oligonucleotides having variable regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides. All 2′-MOE cytosines were 5-methyl-cytosines. Additionally, some oligonucleotides were synthesized with deoxycytosines as 5-methyl-cytosines. Additional oligonucleotides were synthesized, with similar chemistries, as scrambled controls.

TABLE 5


Nucleotide Sequences of 16201 Analogues

		SEQ	TARGET GENE	GENE
ISIS	NUCLEOTIDE SEQUENCE	ID	NUCLEOTIDE	TARGET
NO.	(5′ -> 3′)¹	NO:	CO-ORDINATES²	REGION

17130	GsGcCsTsGsTsTsAsGsAsGsCsAsGsTs GsTsCsAsA	14	0937-0956	3′-UTR

17131	GsGsCsTsGsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA	14	0937-0956	3′-UTR

17132	GsGsCsTsGsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA	14	0937-0956	3′-UTR

17133	GsGsCsTsGsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA	14	0937-0956	3′-UTR

17134	GsGsCsTsGsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA	14	0937-0956	3′-UTR

17818	GoGoCsTsGsTsTsAsGsAsGsCsAoGoToGoToCoAoA	14	0937-0956	all 5-meC

17819	ToGoCsGsCsTsAsAsGsTsGsCsGoGoToAoToCoAoA	19	16201 control	all 5-meC

18550	TsGsCsGsGsTsAsAsCsTsGsCsGsGsTsAsTsCsAsA	19	16201 control

20459	GsGsCsTsCsTsTsAsGsAsGsCsAsGsTsGsTsCsAsA	14	0937-0956	all 5-meC

21919	GsTsCsGsTsTsAsCsTsCsGsGsAsAsAsTsGsGsAsGsGsC	20	16201 control

21920	AsGsCsTsTsGsTsTsGsAsAsCsGsAsGsTsGsTsCsGsA	21	16201 control

21921	TsGsCsAsGsTsTsCsGsCsAsGsAsGsTsCsTsGsAsA	22	16201 control

Dose response experiments were performed using chimeric oligonucleotides as discussed in Example 3. Results are shown in Table 6. The introduction of 2′-MOE nucleotides into the sequence improved the maximum inhibition from 60%, with a phosphorothioate oligodeoxynucleotide, to greater than 75%. The exception was the fully modified 2-MOE oligonucleotide which was less effective than the oligodeoxynucleotide.

TABLE 6


Dose Response of A549 Cells to RhoA
Antisense Gapmer Oliqonucleotides (ASOs)

	SEQ ID	ASO Gene		% mRNA	% mRNA
ISIS #	NO:	Target	Dose	Expression	Inhibition

control	—	LIPOFECTIN	—	100%	0%
		only
16201	14	3′-UTR	75 nM	119.5%	—
16201	14	″	150 nM	54.5%	45.5%
16201	14	″	300 nM	39.5%	60.5%
17130	14	3′-UTR	75 nM	56.2%	43.8%
17130	14	″	150 nM	31.5%	68.5%
17130	14	″	300 nM	14.1%	85.9%
17131	14	3′-UTR	75 nM	55.5%	44.5%
17131	14	″	150 nM	35.4%	64.6%
17131	14	″	300 nM	24.7%	75.3%
17132	14	3′-UTR	75 nM	71.3%	28.7%
17132	14	″	150 nM	31.3%	68.7%
17132	14	″	300 nM	13.1%	86.9%
17133	14	3′-UTR	75 nM	41.7%	58.3%
17133	14	″	150 nM	33.8%	66.2%
17133	14	″	300 nM	14.4%	85.6%
17134	14	3′-UTR	75 nM	76.6%	23.4%
17134	14	″	150 nM	35.9%	64.1%
17134	14	″	300 nM	68.5%	31.5%

Example 5

Time Course of Antisense Oligonucleotide Effects on Human RhoA Protein Levels in A549 Cells

Oligonucleotide 17131 was tested by treating for varying times and measuring the effect of the oligo on RhoA protein levels. A549 cells were grown and treated with oligonucleotide (300 nM) as described in Example 2. Cells were harvested at 24, 48 and 72 hours after treatment. RhoA protein levels were measured by Western blotting. After oligonucleotide treatment, cells were washed twice in phosphate-buffered saline (PBS) and lysed in 25 mM Tris-HCl pH 7.5, 1% Triton X-100, 0.2% SDS, 0.5% sodium deoxycholate, 450 mM NaCl, and 10 mg/ml aprotinin and leupeptin. After 15 minutes on ice, the samples were centrifuged at maximum speed in a microfuge. Protein concentration was determined by Bradford reagent (Bio-Rad Laboratories, Hercules, Calif.). Fifty mg of protein was separated by SDS-PAGE (15%). Following electrophoresis, proteins were transferred to IMMOBILON-P membranes (Millipore, Bedford, Mass.) The membrane was blocked in 5% fish gelatin (Sigma Chemicals, St. Louis, Mo.) and RhoA specific antibodies were used to visualize the proteins. After incubation with the appropriate secondary antibody, proteins were visualized using either LUMIGLO Reagent (New England Biolabs, Beverly, Mass.) or ECL PLUS (Amersham Pharmacia Biotech, Piscataway, N.J.) . Inhibition of RhoA protein was observable after 24 hours. After 48 hours, RhoA protein concentration was reduced by 80% using 17131 (SEQ ID NO. 14). Minimal inhibition was seen with 17163 (SEQ ID NO. 190), an oligonucleotide targeted to Rac1. Results are shown in Table 7.

TABLE 7


Time course of RhoA Antisense Oligonucleotides (ASOs) in
A549 Cells

			Time
	SEQ ID	ASO Gene	after	% protein	% protein
ISIS #	NO:	Target	treatment	Expression	Inhibition

control	—	LIPOFECTIN	—	100%	0%
		only
17131	14	3′-UTR	24 hr	46.2%	53.8%
17131	14	″	48 hr	16.0%	84.0%
17131	14	″	72 hr	12.4%	87.6%
17163	190	Rac1 control	24 hr	104.1%	—
17163	190	″	48 hr	82.3%	17.7%
17163	190	″	72 hr	95.2%	4.8%

Example 6

Dose Response of Antisense Oligonucleotide Effects on Human RhoA Protein Levels in A549 Cells

Oligonucleotide 17131 was tested for a dose response by using varying concentrations of oligonucleotide and measuring the effect of the oligonucleotide on RhoA protein levels. A549 cells were grown and treated with oligonucleotide (concentrations indicated in Table 8) as described in Example 2. Western blotting was performed to measure protein levels as described in Example 5. A dose response effect is seen with 17131 (SEQ ID NO. 14), whereas the scrambled control 18550 (SEQ ID NO. 19) had no effect on RhoA protein levels.

TABLE 8


Dose response of RhoA antisense oligonucleotide on protein
levels in A549 cells

	SEQ ID	ASO Gene		% protein	% protein
ISIS #	NO:	Target	Dose	Expression	Inhibition

control	—	LIPOFECTIN	—	100%	0%
		only
17131	14	3′-UTR	75 nM	51%	49%
17131	14	″	150 nM	23%	77%
17131	14	″	300 nM	20%	80%
18550	19	control	75 nM	101%	—
18550	19	″	150 nM	101%	—
18550	19	″	300 nM	104%	—

Example 7

Inhibition of JNK Activation by RhoA Antisense Oligonucleotides in A549 Cells Stimulated with H₂O₂

Oligonucleotide 17131 (SEQ ID NO. 14) was tested for its ability to inhibit JNK activation by stimulation with H[0114] ₂O₂or Il-1b. A549 cells were grown as described in Example 2. Cells were treated with 150 nM of oligonucleotide for four hours. After treatment, the media was replaced with DMEM, 0.1% FCS, and the cells were left in culture for 48 hours prior to stimulation. Stimulation was with either 30 ng/ml IL-1b or 1 mM H₂O₂for 30 minutes. After stimulation, the cells were washed twice in PBS, and lysed in 25 mM Hepes pH 7.7, 0.3 M NaCl, 1.5 mM MgCl₂, 0.1% Triton X-100, 20 mM b-glycerophosphate, 0.1 mM sodium orthovanadate (Na₃VO₄), 0.5 mM PMSF, and 10 mg/ml of aprotinin and leupeptin. After 20 minutes on ice, the lysates were centrifuged at maximum speed in a microfuge for 20 minutes. The protein concentration in the supernatant was determined using Bradford reagent (Bio-Rad Laboratories, Hercules, Calif.). To 150 mg of protein, 25 ml of c-Jun fusion beads (New England Biolabs, Beverly, Mass.) were added and incubated at 4° C. on a rotating wheel overnight. The samples were then washed four times in 20 mM Hepes pH 7.7, 50 mM NaCl, 0.1 mM EDTA, 2.5 mM MgCl₂, and 0.05% Triton X-100 (HIBI buffer). The kinase reaction was run for 20 minutes at 30° C. in 20 mM Hepes pH 7.7, 20 mM MgCl₂, 20 mM b-glycerophosphate, 20 mM p-nitrophenyl phosphate, 0.1 mM Na₃VO₄, 2 mM DTT, 20 mM ATP, and 5 mCi of g[³²P]-ATP. The reaction was stopped with 500 ml of ice cold HIBI buffer. The beads were pelleted, resuspended in PAGE loading buffer, boiled for 5 minutes, and the products separated on a 12% SDS gel (Novex, La Jolla, Calif.). Bands were quantitated using a PhosphorImager.
Results are shown in Table 9. Oligonucleotide 17131 (SEQ ID NO. 14) showed moderate but specific inhibition of H[0115] ₂O₂-induced JNK activation.

TABLE 9

Inhibition of JNK activation by RhoA antisense

oligonucleotides

SEQ

ID ASO Gene % inhibition % inhibition

ISIS # NO: Target Dose Il-1b induced H₂O₂induced

control 13 LIPOFECTIN — 0% 0%

only

17131 14 3′-UTR 150 nM — 37.6%

18550 19 control 150 nM 2.2% 5.8%

Example 8

Synthesis of Additional RhoA Sequences

Additional oligonucleotides were synthesized in 96 well plate format 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-cyanoethyl-di-isopropyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per published methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites. [0116]
Oligonucleotides were cleaved from support and deprotected with concentrated NH[0117] ₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.
A series of oligonucleotides were designed to target different regions of the human RhoA RNA, using published sequences (GenBank accession number X05026, incorporated herein as SEQ ID NO: 1). The oligonucleotides are shown in Table 10. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X05026), to which the oligonucleotide binds. [0118]

All compounds in Table 10 are oligodeoxynucleotides with phosphorothioate backbones (internucleoside linkages) throughout. All compounds in Table 11 are chimeric oligonucleotides (“gapmers”) 18 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 four-nucleotide “wings.” The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. Cytidine residues in the 2′-MOE wings are 5-methylcytidines.

TABLE 10


Nucleotide Sequences of Human RhoA
Phosphorothioate Oligodeoxynucleotides

		SEQ	TARGET GENE	GENE
ISIS	NUCLEOTIDE SEQUENCE	ID	NUCLEOTIDE	TARGET
NO.	(5′ -> 3′)	NO:	CO-ORDINATES¹	REGION

25544	AGAGAACCGACGGAGCAC	23	0030-0047	5′-UTR

25545	GTCGACTAATGAGAGAAC	24	0041-0058	5′-UTR

25546	GACCGTCCACTAATCAGA	25	0045-0062	5′-UTR

25547	AGCTGAAGACCAGACCGT	26	0057-0074	5′-UTR

25548	AGTCGCAAACTCGGAGAC	4	0085-0102	5′-UTR

25549	AATCCGAGTCCAGCCTCT	27	0128-0145	5′-UTR

25550	AACGAATCCGAGTCCACC	28	0132-0149	5′-UTR

25551	TCAGGCAACGAATCCCAG	29	0138-0155	5′-UTR

25552	CACCAACAATCACCAGTT	30	0178-0195	Coding

25553	AAGACTATGAGCAAGCAT	31	0215-0232	Coding

25554	ATACACCTCTGGGAACTG	32	0243-0260	Coding

25555	ACATAGTTCTCAAACACT	33	0269-0286	Coding

25556	ACTCTACCTGCTTTCCAT	34	0304-0321	Coding

25557	CACAAACCCAACTCTACC	35	0314-0331	Coding

25558	AACATCGCTATCTGGGTA	36	0378-0395	Coding

25559	TTCTGGGATGTTTTCTAA	37	0432-0449	Coding

25560	GGACAGAAATGCTTGACT	38	0464-0481	Coding

25561	GTGCTCATCATTCCGAAG	39	0519-0536	Coding

25562	CTTCTCTGCTCATCATTC	40	0524-0541	Coding

25563	TAGCTCCCGCCTTGTGTG	41	0534-0551	Coding

25564	CCAATCCTGTTTGCCATA	42	0596-0613	Coding

25565	GTCTTTCCTGAACACTCC	43	0629-0646	Coding

25566	AAAACCTCTCTCACTCCA	44	0653-0670	Coding

25567	AAGACAAGGCAACCAGAT	45	0719-0736	Coding

25568	TTTCACAAGACAAGGCAA	46	0725-0742	Stop

25569	GCAAGGTTTCACAAGACA	47	0731-0748	Stop

25570	ATTAACCGCATAAGGGCT	48	0758-0775	3′-UTR

25571	TAATAAACAGCACTTCAA	49	0777-0794	3′-UTR

25572	CCAGTAATCATACACTAA	50	0798-0815	3′-UTR

25573	ATGACTTCTGATTTGTAA	51	0847-0864	3′-UTR

25574	TAGCAAGATGACTTCTGA	52	0854-0871	3′-UTR

25575	CTGGTAGOAAGATGACTT	53	0858-0875	3′-UTR

25576	CTAAATACTGGTAGCAAG	54	0865-0882	3′-UTR

25577	TTGGCTTCTAAATACTGG	55	0872-0889	3′-UTR

25578	TCATAGTTGGCTTCTAAA	56	0878-0895	3′-UTR

25579	AATAATCATAGTTGGCTT	57	0883-0900	3′-UTR

25580	TCAAAAGGACCCTGGTGG	58	0923-0940	3′-UTR

25581	GTGCAGAGGAGGGCTGTT	59	0950-0967	3′-UTR

25582	CCAACTGTTTCTCTTTCT	60	1026-1043	3′-UTR

25583	AAGTAGTTACAGCCTAAT	61	1056-1073	3′-UTR

Example 9

Total RNA Isolation

Total mRNA was isolated using an RNEASY 96 kit and buffers purchased from Qiagen Inc. (Valencia Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 100 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 μL of 70% ethanol was then added to each well and the contents mixed by pippeting 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. [0120]
Poly(A)+ mRNA may be isolated according to Miura et al., Clin. Chem., 42, 1758 (1996). 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 ml cold PBS. 60 ml lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 ml of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 ml 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 ml 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° hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate. [0121]
Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions. [0122]

Example 10

Real-Time Quantitative PCR Analysis of RhoA mRNA Levels

Quantitation of RhoA mRNA levels was determined by real-time quantitative PCR using the ABI PRISM 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR, in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., JOE or FAM, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular (six-second) intervals by laser optics built into the ABI PRISM 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples. [0123]

PCR reagents were obtained from PE-Applied Biosystems, Foster City, Calif.. RT-PCR reactions were carried out by adding 25 μL PCR cocktail (1×TAQMAN buffer A, 5.5 mM MgCl ₂, 300 μM each of dATP, dCTP and dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD, and 12.5 Units MuLV reverse transcriptase) to 96 well plates containing 25 μL poly(A) mRNA solution. The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the AMPLITAQ GOLD, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension). RhoA probes and primers were designed to hybridize to the human RhoA sequence, using published sequence information (GenBank accession number X05026, incorporated herein as SEQ ID NO: 1).


For RhoA the PCR primers were:
forward primer:
GGCTGGACTCGGATTCGTT	(SEQ ID NO: 62)

reverse primer:
CCATCACCAACAATCACCAGTT	(SEQ ID NO: 63)

and the PCR probe was:
FAM-CCTGAGCAATGGCTGCCATCCG-TAMRA

(SEQ ID NO: 64) where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye. [0125]

For GAPDH the PCR primers were:

forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 65)

reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 66)

and the PCR probe was:

5′ JOE-CAAGCTTCCCGTTCTCAGCC- TAMPA 3′ (SEQ ID NO: 67)
ID NO: 67) where JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye. [0126]

Example 11

Antisense Inhibition of RhoA Expression-Phosphorothioate Oligodeoxynucleotides

In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human RhoA RNA, using published sequences (GenBank accession number X05026, incorporated herein as SEQ ID NO: 1). The oligonucleotides are shown in Table 10. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X05026), to which the oligonucleotide binds. All compounds in Table 10 are oligodeoxynucleotides with phosphorothioate backbones (internucleoside linkages) throughout. The compounds were analyzed for effect on RhoA mRNA levels by quantitative real-time PCR as described in other examples herein. Data are shown in Table 11 and are averages from three experiments. If present, “N.D.” indicates “no data”.

TABLE 11


Inhibition of RhoA mRNA levels by phosphorothioate
oligodeoxynucleotides

SEQ

		TARGET		% Inhi-	ID
ISIS#	REGION	SITE	SEQUENCE	bition	NO.

25544	5′ UTR	30	AGACAACCGACGGAGGAC	47	23

25545	5′ UTR	41	GTGGACTAATGAGAGAAC	0	24

25546	5′ UTR	45	GACCGTGGACTAATGAGA	40	25

25547	5′ UTR	57	AGCTGAAGACCAGACCGT	76	26

25548	5′ UTR	85	AGTCGCAAACTCGGAGAC	36	4

25549	5′ UTR	128	AATCCGAGTCCAGCCTCT	67	27

25550	5′ UTR	132	AACGAATCCGAGTCCAGC	34	28

25551	5′ UTR	138	TCAGGCAACGAATCCGAG	59	29

25552	CODING	178	CACCAACAATCACCAGTT	47	30

25553	CODING	215	AAGACTATGAGCAAGCAT	36	31

25554	CODING	243	ATACACCTCTGGGAACTG	74	32

25555	CODING	269	ACATAGTTCTCAAACACT	31	33

25556	CODING	304	ACTCTACCTGCTTTCCAT	64	34

25557	CODING	314	CACAAAGCCAACTCTACC	25	35

25558	CODING	378	AACATCGGTATCTGGGTA	35	36

25559	CODING	432	TTCTGGGATGTTTTCTAA	21	37

25560	CODING	464	GGACAGAAATGCTTGACT	64	38

25561	CODING	519	GTGCTCATCATTCCGAAG	71	39

25562	CODING	524	CTTGTGTGCTCATCATTC	38	40

25563	CODING	534	TAGCTCCCGCCTTGTGTG	78	41

25564	CODING	596	CCAATCCTGTTTGCCATA	82	42

25565	CODING	629	GTCTTTGCTGAACACTCC	56	43

25566	CODING	653	AAAACCTCTCTCACTCCA	68	44

25567	CODING	719	AAGACAAGGCAACCAGAT	55	45

25568	STOP	725	TTTCACAAGACAAGGCAA	0	46

25569	STOP	731	GCAAGGTTTCACAAGACA	37	47

25570	3′ UTR	758	ATTAACCGCATAAGGGCT	77	48

25571	3′ UTR	777	TAATAAACAGCACTTCAA	19	49

25572	3′ UTR	798	CCAGTAATCATACACTAA	26	50

25573	3′ UTR	847	ATGACTTCTGATTTGTAA	27	51

25574	3′ UTR	854	TAGCAAGATGACTTCTGA	62	52

25575	3′ UTR	858	CTGGTAGCAAGATGACTT	59	53

25576	3′ UTR	865	CTAAATACTGGTAGCAAG	29	54

25577	3′ UTR	872	TTGGCTTCTAAATACTGG	57	55

25578	3′ UTR	878	TCATAGTTGGCTTCTAAA	60	56

25579	3′ UTR	883	AATAATCATAGTTGGCTT	33	57

25580	3′ UTR	923	TCAAAAGGACCCTGGTGG	25	58

25581	3′ UTR	950	GTGCAGAGGAGGGCTGTT	68	59

25582	3′ UTR	1026	CCAACTGTTTCTCTTTCT	52	60

25583	3′ UTR	1056	AAGTAGTTACAGCCTAAT	26	61

As shown in Table 11, SEQ ID NOs 23, 26, 27, 29, 30, 32, 34, 38, 39, 41, 42, 43, 44, 45, 48, 52, 53, 56, 57, 59 and 60 demonstrated at least 45% inhibition of RhoA expression in this assay and are therefore preferred. [0128]

Example 12

Antisense Inhibition of RhoA Expression-Phosphorothioate 2′-MOE Gapmer Oligonucleotides

In accordance with the present invention, a second series of oligonucleotides targeted to human RhoA were synthesized. The oligonucleotide sequences are shown in Table 12. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X05026), to which the oligonucleotide binds. [0129]

All compounds in Table 12 are chimeric oligonucleotides (“gapmers”) 18 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 four-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. Cytidine residues in the 2′-MOE wings are 5-methylcytidines.

TABLE 12


Nucleotide Sequences of Human RhoA Gapmer
Oligonucleotides

25584	AGAGAACCGACGGAGGAC	23	0030-0047	5′-UTR

25585	GTGGACTAATGAGAGAAC	24	0041-0058	5′-UTR

25586	GACCGTGGACTAATGAGA	25	0045-0062	5′-UTR

25587	AGCTGAAGACCAGACCGT	26	0057-0074	5′-UTR

25588	AGTCGCAAACTCGGAGAC	4	0085-0102	5′-UTR

25589	AATCCGAGTCCAGCCTCT	27	0128-0145	5′-UTR

25590	AACGAATCCGAGTCCAGC	28	0132-0149	5′-UTR

25591	TCAGGCAACGAATCCGAG	29	0138-0155	5′-UTR

25592	CACCAACAATCACCAGTT	30	0178-0195	Coding

25593	AAGACTATGAGCAAGCAT	31	0215-0232	Coding

25594	ATACACCTCTGGGAACTG	32	0243-0260	Coding

25595	ACATAGTTCTCAAACACT	33	0269-0286	Coding

25596	ACTCTACCTGCTTTCCAT	34	0304-0321	Coding

25597	CACAAAGCCAACTCTACC	35	0314-0331	Coding

25598	AACATCGGTATCTGGGTA	36	0378-0395	Coding

25599	TTCTGGGATGTTTTCTAA	37	0432-0449	Coding

25600	GGACAGAAATGCTTGACT	38	0464-0481	Coding

25601	GTGCTCATCATTCCGAAG	39	0519-0536	Coding

25602	CTTGTGTGCTCATCATTC	40	0524-0541	Coding

25603	TAGCTCCCGCCTTGTGTG	41	0534-0551	Coding

25604	CCAATCCTGTTTGCCATA	42	0596-0613	Coding

25605	GTCTTTGCTGAACACTCC	43	0629-0646	Coding

25606	AAAACCTCTCTCACTCCA	44	0653-0670	Coding

25607	AAGACAACCCAACCAGAT	45	0719-0736	Coding

25608	TTTCACAAGACAAGGCAA	46	0725-0742	Stop

25609	GCAACCTTTCACAAGACA	47	0731-0748	Stop

25610	ATTAACCCCATAACGGCT	48	0758-0775	3′-UTR

25611	TAATAAACAGCACTTCAA	49	0777-0794	3′-UTR

25612	CCAGTAATCATACACTAA	50	0798-0815	3′-UTR

25613	ATGACTTCTGATTTGTAA	51	0847-0864	3′-UTR

25614	TAGCAAGATGACTTCTGA	52	0854-0871	3′-UTR

25615	CTGGTAGCAAGATGACTT	53	0858-0875	3′-UTR

25616	CTAAATACTGGTAGCAAG	54	0865-0882	3′-UTR

25617	TTGGCTTCTAAATACTGG	55	0872-0889	3′-UTR

25618	TCATAGTTCGCTTCTAAA	56	0878-0895	3′-UTR

25619	AATAATCATAGTTGGCTT	57	0883-0900	3′-UTR

25620	TCAAAAGGACCCTCGTGG	58	0923-0940	3′-UTR

25621	GTGCAGAGGAGGCCTGTT	59	0950-0967	3′-UTR

25622	CCAACTCTTTCTCTTTCT	60	1026-1043	3′-UTR

25623	AAGTAGTTACAGCCTAAT	61	1056-1073	3′-UTR

The oligonucleotides shown in Table 12 were tested by real-time quantitative PCR as described in other examples herein and data are shown in Table 13 (averaged from three experiments). If present, “N.D.” indicates “no data”.

TABLE 13


Inhibition of RhoA mRNA levels by chimeric
phosphorothioate oligonucleotides having 2′-MOE
wings and a deoxy gap

					SEQ
		TARGET		% Inhi-	ID
ISIS#	REGION	SITE	SEQUENCE	bition	NO.

25584	5′ UTR	30	AGAGAACCGACGGAGGAC	44	23

25585	5′ UTR	41	GTGGACTAATGAGAGAAC	35	24

25586	5′ UTR	45	GACCGTGGACTAATGAGA	53	25

25587	5′ UTR	57	AGCTGAAGACCAGACCGT	62	26

25588	5′ UTR	85	AGTCGCAAACTCGGAGAC	54	4

25589	5′ UTR	128	AATCCGAGTCCAGCCTCT	38	27

25590	5′ UTR	132	AACGAATCCGAGTCCAGC	47	28

25591	5′ UTR	138	TCAGGCAACGAATCCGAG	31	29

25592	CODING	178	CACCAACAATCACCAGTT	0	30

25593	CODING	215	AAGACTATGAGCAAGCAT	43	31

25594	CODING	243	ATACACCTCTGGGAACTG	23	32

25595	CODING	269	ACATAGTTCTCAAACACT	16	33

25596	CODING	304	ACTCTACCTGCTTTCCAT	0	34

25597	CODING	314	CACAAAGCCAACTCTACC	0	35

25598	CODING	378	AACATCGGTATCTGGGTA	65	36

25599	CODING	432	TTCTGGGATGTTTTCTAA	53	37

25600	CODING	464	GGACAGAAATGCTTGACT	50	38

25601	CODING	519	GTGCTCATCATTCCGAAG	45	39

25602	CODING	524	CTTGTGTGCTCATCATTC	26	40

25603	CODING	534	TAGCTCCCGCCTTGTGTG	59	41

25604	CODING	596	CCAATCCTGTTTGCCATA	40	42

25605	CODING	629	GTCTTTGCTGAACACTCC	47	43

25606	CODING	653	AAAACCTCTCTCACTCCA	30	44

25607	CODING	719	AAGACAAGGCAACCAGAT	0	45

25608	STOP	725	TTTCACAAGACAAGGCAA	7	46

25609	STOP	731	GCAAGGTTTCACAAGACA	53	47

25610	3′ UTR	758	ATTAACCGCATAAGGGCT	56	48

25611	3′ UTR	777	TAATAAACAGCACTTCAA	7	49

25612	3′ UTR	798	CCAGTAATCATACACTAA	41	50

25613	3′ UTR	847	ATGACTTCTGATTTGTAA	53	51

25614	3′ UTR	854	TAGCAAGATGACTTCTGA	59	52

25615	3′ UTR	858	CTGGTAGCAAGATGACTT	67	53

25616	3′ UTR	865	CTAAATACTGGTAGCAAG	65	54

25617	3′ UTR	872	TTGGCTTCTAAATACTGG	74	55

25618	3′ UTR	878	TCATAGTTGGCTTCTAAA	52	56

25619	3′ UTR	883	AATAATCATACTTGGCTT	49	57

25620	3′ UTR	923	TCAAAAGGACCCTGGTGG	58	58

25621	3′ UTR	950	GTGCAGAGGAGGGCTGTT	60	59

25622	3′ UTR	1026	CCAACTGTTTCTCTTTCT	62	60

25623	3′ UTR	1056	AAGTAGTTACACCCTAAT	44	61

As shown in Table 13, SEQ ID NOs 23, 24, 25, 26, 4, 27, 28, 31, 36, 37, 38, 39, 41, 42, 43, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 and 61 demonstrated at least 35% inhibition of RhoA expression in this experiment and are therefore preferred. [0132]

Example 13

Synthesis of RhoB Antisense Oligonucleotide Sequences

Oligonucleotide sequences were synthesized as described in previous examples. Antisense oligonucleotides were designed to target human RhoB. Target sequence data are from the RhoB cDNA sequence published by Chardin, P., et al. ( Nucleic Acids Res. 1988, 16, 2717); Genbank accession number X06820, provided herein as SEQ ID NO: 68.

TABLE 14


Nucleotide Sequences of Human RhoB
Phosphorothioate Oligodeoxynucleotides

25384	CCACCACCACCTTCTTCC	69	0014-0031	Coding

25385	CCCTCCCCCACCACCACC	70	0024-0041	Coding

25386	GCACGTCTTGCCACACGC	71	0043-0060	Coding

25387	ACTGAACACGATCAGCAG	72	0061-0078	Coding

25388	TTACTGAACACGATCAGC	73	0063-0080	Coding

25389	CCTTACTGAACACGATCA	74	0065-0082	Coding

25390	GTCCTTACTGAACACGAT	75	0067-0084	Coding

25391	CTCGTCCTTACTGAACAC	76	0070-0087	Coding

25392	AACTCGTCCTTACTGAAC	77	0072-0089	Coding

25393	CATAGTTCTCGAAGACGG	78	0110-0127	Coding

25394	TCGCCCACATAGTTCTCG	79	0117-0134	Coding

25395	CCGTCCACCTCAATGTCG	80	0132-0149	Coding

25396	AAGCACATGAGAATGACG	81	0234-0251	Coding

25397	GAGTCCGGGCTGTCCACC	82	0255-0272	Coding

25398	ATGTTCTCCAGCGAGTCC	83	0267-0284	Coding

25399	GGGATGTTCTCCAGCGAG	84	0270-0287	Coding

25400	GACATGCTCGTCGCTGCG	85	0364-0381	Coding

25401	CCGACATGCTCGTCGCTG	86	0366-0383	Coding

25402	TGTGCGGACATGCTCGTC	87	0370-0387	Coding

25403	CTCTGTGCGGACATGCTC	88	0373-0390	Coding

25404	CCAGCTCTGTGCGGACAT	89	0377-0394	Coding

25405	CGCCCCACCTCTGTGCGG	90	0381-0398	Coding

25406	TGCGGGCCAGCTCTGTGC	91	0383-0400	Coding

25407	GTTCCTGCTTCATGCGGG	92	0395-0412	Coding

25408	ACGGGTTCCTGCTTCATG	93	0399-0416	Coding

25409	GTAGTCGTAGGCTTGGAT	94	0451-0468	Coding

25410	CGACGTAGTCGTAGCCTT	95	0455-0472	Coding

25411	GTCTTCCCACACCACTCG	96	0471-0488	Coding

25412	ACCTCGCGCACGCCTTCC	97	0492-0509	Coding

25413	AGACCTCGCGCACGCCTT	98	0494-0511	Coding

25414	CGAAGACCTCCCGCACGC	99	0497-0514	Coding

25415	CTCGAAGACCTCGCGCAC	100	0499-0516	Coding

25416	GCCGTCTCGAAGACCTCG	101	0504-0521	Coding

25417	CCTGGCCGTCTCGAACAC	102	0508-0525	Coding

25418	GTTCTGGGAGCCGTAGCG	103	0544-0561	Coding

25419	GCCGTTCTGGGAGCCGTA	104	0547-0564	Coding

25420	GATGCAGCCGTTCTGGGA	105	0553-0570	Coding

25421	GTTGATGCAGCCGTTCTG	106	0556-0573	Coding

25422	CAGCAGTTGATCCAGCCG	107	0561-0578	Coding

25423	AGCACCTTGCAGCAGTTG	108	0570-0587	Coding

Example 14

Antisense Inhibition of RhoB Expression-Phosphorothioate Oligodeoxynucleotides

In accordance with the present invention, the oligonucleotides shown in Table 14 were analyzed for effect on RhoB mRNA levels by quantitative real-time PCR as described in examples herein. Data are averages from three experiments. If present, “N.D.” indicates “no data”.

TABLE 15


Inhibition of RhoB mRNA levels by phosphorothioate
oligodeoxynucleotides

					SEQ
		TARGET		% Inhibi-	ID
ISIS#	REGION	SITE	SEQUENCE	tion	NO.

25384	Coding	14	CCACCACCAGCTTCTTGC	0	69

25385	CODING	24	CCGTCGCCCACCACCACC	0	70

25386	CODING	43	GCACGTCTTGCCACACGC	0	71

25387	CODING	61	ACTGAACACGATCAGCAG	0	72

25388	CODING	63	TTACTGAACACGATCAGC	0	73

25389	CODING	65	CCTTACTGAACACGATCA	0	74

25390	CODING	67	GTCCTTACTGAACACGAT	5	75

25391	CODING	70	CTCGTCCTTACTGAACAC	1	76

25392	CODING	72	AACTCGTCCTTACTGAAC	30	77

25393	CODING	110	CATAGTTCTCGAAGACGG	0	78

25394	CODING	117	TCGGCCACATAGTTCTCG	13	79

25395	CODING	132	CCGTCCACCTCAATGTCG	0	80

25396	CODING	234	AAGCACATGAGAATGACG	0	81

25397	CODING	255	GAGTCCGGGCTGTCCACC	0	82

25398	CODING	267	ATGTTCTCCAGCGAGTCC	0	83

25399	CODING	270	GGGATGTTCTCCAGCGAG	33	84

25400	CODING	364	GACATGCTCGTCGCTGCG	0	85

25401	CODING	366	CGGACATGCTCGTCGCTG	0	86

25402	CODING	370	TGTGCGGACATGCTCGTC	0	87

25403	CODING	373	CTCTGTGCGGACATGCTC	39	88

25404	CODING	377	CCAGCTCTGTGCGGACAT	21	89

25405	CODING	381	CGGGCCAGCTCTGTGCGG	38	90

25406	CODING	383	TGCGGGCCAGCTCTGTGC	31	91

25407	CODING	395	GTTCCTGCTTCATGCGGG	27	92

25408	CODING	399	ACGGGTTCCTGCTTCATG	0	93

25409	CODING	451	GTAGTCGTAGGCTTGGAT	29	94

25410	CODING	455	CGAGGTAGTCGTAGGCTT	39	95

25411	CODING	471	GTCTTGGCAGAGCACTCG	20	96

25412	CODING	492	ACCTCGCGCACGCCTTCC	0	97

25413	CODING	494	AGACCTCGCGCACGCCTT	16	98

25414	CODING	497	CGAAGACCTCGCGCACGC	0	99

25415	CODING	499	CTCGAAGACCTCGCGCAC	0	100

25416	CODING	504	GCCGTCTCGAAGACCTCG	0	101

25417	CODING	508	CGTGGCCGTCTCGAAGAC	0	102

25418	CODING	544	GTTCTGGGAGCCGTAGCG	36	103

25419	CODING	547	GCCGTTCTGGGAGCCGTA	0	104

25420	CODING	553	GATGCAGCCGTTCTGGGA	0	105

25421	CODING	556	GTTGATGCAGCCGTTCTG	7	106

25422	CODING	561	CAGCAGTTGATGCAGCCG	31	107

25423	CODING	570	AGCACCTTGCAGCAGTTG	0	108

As shown in Table 15, SEQ ID Nos 77, 84, 88, 90, 91, 92, 94, 95, 103 and 107 demonstrated at least 25% inhibition of RhoB expression in this assay and are therefore preferred. [0135]

Example 15

Antisense Inhibition of RhoB Expression-Phosphorothioate 2′-MOE Gapmer Oligonucleotides

In accordance with the present invention, a second series of oligonucleotides targeted to human RhoB were synthesized. The oligonucleotide sequences are shown in Table 16. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X06820), to which the oligonucleotide binds. [0136]

All compounds in Table 16 are chimeric oligonucleotides (“gapmers”) 18 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 four-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. Cytidine residues in the 2′-MOE wings are 5-methylcytidines.

TABLE 16


Nucleotide Sequences of Human RhoB Gapmer
Oligonucleotides

	NUCLEOTIDE		TARGET GENE	GENE
ISIS	SEQUENCE	SEQ ID	NUCLEOTIDE	TARGET
NO.	(5′→3′)	NO:	CO-ORDINATES¹	REGION

25424	CCACCACCAGCTTCTTGC	69	0014-0031	Coding

25425	CCGTCGCCCACCACCACC	70	0024-0041	Coding

25426	GCACGTCTTGCCACACGC	71	0043-0060	Coding

25427	ACTGAACACGATCAGCAG	72	0061-0078	Coding

25428	TTACTGAACACGATCAGC	73	0063-0080	Coding

25429	CCTTACTGAACACGATCA	74	0065-0082	Coding

25430	GTCCTTACTGAACACGAT	75	0067-0084	Coding

25431	CTCGTCCTTACTGAACAC	76	0070-0087	Coding

25432	AACTCGTCCTTACTGAAC	77	0072-0089	Coding

25433	CATAGTTCTCGAAGACGG	78	0110-0127	Coding

25434	TCGGCCACATAGTTCTCG	79	0117-0134	Coding

25435	CCGTCCACCTCAATGTCG	80	0132-0149	Coding

25436	AAGCACATGAGAATGACG	81	0234-0251	Coding

25437	GAGTCCGGGCTGTCCACC	82	0255-0272	Coding

25438	ATGTTCTCCAGCGAGTCC	83	0267-0284	Coding

25439	GGGATGTTCTCCAGCGAG	84	0270-0287	Coding

25440	GACATGCTCGTCGCTGCG	85	0364-0381	Coding

25441	CGGACATGCTCGTCGCTG	86	0366-0383	Coding

25442	TGTGCGGACATGCTCGTC	87	0370-0387	Coding

25443	CTCTGTGCGGACATGCTC	88	0373-0390	Coding

25444	CCAGCTCTGTGCGGACAT	89	0377-0394	Coding

25445	CGGGCCAGCTCTGTGCGG	90	0381-0393	Coding

25446	TGCGGGCCAGCTCTGTGC	91	0383-0400	Coding

25447	GTTCCTGCTTCATGCGGG	92	0395-0412	Coding

25448	ACGGGTTCCTCCTTCATG	93	0399-0416	Coding

25449	GTAGTCGTAGGCTTGGAT	94	0451-0468	Coding

25450	CGAGGTAGTCGTAGGCTT	95	0455-0472	Coding

25451	GTCTTGGCAGAGCACTCG	96	0471-0488	Coding

25452	ACCTCGCGCACGCCTTCC	97	0492-0509	Coding

25453	AGACCTCGCGCACGCCTT	98	0494-0511	Coding

25454	CGAAGACCTCGCGCACGC	99	0497-0514	Coding

25455	CTCGAAGACCTCGCGCAC	100	0499-0516	Coding

25456	GCCGTCTCGAAGACCTCG	101	0504-0521	Coding

25457	CGTGGCCGTCTCGAAGAC	102	0508-0525	Coding

25458	GTTCTGGGAGCCGTAGCG	103	0544-0561	Coding

25459	GCCGTTCTGGGAGCCGTA	104	0547-0564	Coding

25460	GATGCACCCGTTCTGGGA	105	0553-0570	Coding

25461	GTTGATGCAGCCGTTCTG	106	0556-0573	Coding

25462	CAGCAGTTGATGCAGCCG	107	0561-0578	Coding

25463	AGCACCTTCCACCAGTTG	108	0570-0587	Coding

Data for the compounds in Table 16 were obtained by real-time quantitative PCR as described in other examples herein and are averaged from three experiments. Results are shown in Table 17. If present, “N.D.” indicates “no data”.

TABLE 17


Inhibition of RhoB mRNA levels by chimeric
phosphorothioate oligonucleotides having
2′-MOE wings and a deoxy gap

				%
		TARGET		Inhi-	SEQ ID
ISIS#	REGION	SITE	SEQUENCE	bition	NO.

25424	Coding	14	CCACCACCAGCTTCTTGC	29	69

25425	CODING	24	CCGTCGCCCACCACCACC	23	70

25426	CODING	43	GCACGTCTTGCCACACGC	46	71

25427	CODING	61	ACTGAACACGATCAGCAG	37	72

25428	CODING	63	TTACTGAACACGATCAGC	47	73

25429	CODING	65	CCTTACTGAACACGATCA	7	74

25430	CODING	67	GTCCTTACTGAACACGAT	46	75

25431	CODING	70	CTCGTCCTTACTGAACAC	52	76

25432	CODING	72	AACTCGTCCTTACTGAAC	35	77

25433	CODING	110	CATAGTTCTCGAAGACGG	29	78

25434	CODING	117	TCGGCCACATAGTTCTCG	65	79

25435	CODING	132	CCGTCCACCTCAATGTCG	40	80

25436	CODING	234	AAGCACATGAGAATGACG	44	81

25437	CODING	255	GAGTCCGGGCTGTCCACC	36	82

25438	CODING	267	ATGTTCTCCAGCGAGTCC	28	83

25439	CODING	270	GGGATGTTCTCCAGCGAG	54	84

25440	CODING	364	GACATGCTCGTCGCTGCG	49	85

25441	CODING	366	CGGACATGCTCGTCGCTG	46	86

25442	CODING	370	TGTGCGGACATGCTCGTC	65	87

25443	CODING	373	CTCTGTGCGGACATGCTC	39	88

25444	CODING	377	CCAGCTCTGTGCGGACAT	19	89

25445	CODING	381	CGGGCCAGCTCTGTGCGG	21	90

25446	CODING	383	TGCGGGCCAGCTCTGTGC	9	91

25447	CODING	395	GTTCCTGCTTCATGCGGG	16	92

25448	CODING	399	ACGGGTTCCTGCTTCATG	7	93

25449	CODING	451	GTAGTCGTAGGCTTGGAT	38	94

25450	CODING	455	CGAGGTAGTCGTAGGCTT	0	95

25451	CODING	471	GTCTTGGCAGAGCACTCG	42	96

25452	CODING	492	ACCTCGCGCACGCCTTCC	9	97

25453	CODING	494	AGACCTCGCGCACGCCTT	7	98

25454	CODING	497	CGAAGACCTCGCGCACGC	12	99

25455	CODING	499	CTCGAAGACCTCGCGCAC	23	100

25456	CODING	504	GCCGTCTCGAAGACCTCG	34	101

25457	CODING	508	CGTGGCCGTCTCGAAGAC	27	102

25458	CODING	544	GTTCTGGGAGCCGTAGCG	58	103

25459	CODING	547	GCCGTTCTGGGAGCCGTA	63	104

25460	CODING	553	GATGCAGCCGTTCTGGGA	17	105

25461	CODING	556	GTTGATGCAGCCGTTCTG	21	106

25462	CODING	561	CAGCAGTTGATGCAGCCG	50	107

25463	CODING	570	AGCACCTTGCAGCAGTTG	55	108

As shown in Table 17, SEQ ID Nos 71, 62, 63, 75, 76, 77, 79, 80, 81, 82, 84, 85, 86, 87, 88, 94, 96, 101, 103, 104, 107 and 108 demonstrated at least 30% inhibition of RhoB expression in this experiment and are therefore preferred. [0139]

Example 16

Synthesis of RhoC Antisense Oligonucleotide Sequences

Oligonucleotide sequences were synthesized as described in previous examples. Antisense oligonucleotides were designed to target human RhoC. Target sequence data are from the RhoC cDNA sequence determined by Fagan, K. P., et al.; Genbank accession number L25081, provided herein as SEQ ID NO: 109.

TABLE 18


Nucleotide Sequences of Human RhoC
Phosphorothioate Oligonucleotides

25304	GAGCTGAGATGAAGTCAA	110	0004-0021	5′-UTR

25305	GCTGAAGTTCCCAGGCTG	111	0044-0061	5′-UTR

25306	CCGGCTGAAGTTCCCAGG	112	0047-0064	5′-UTR

25307	GGCACCATCCCCAACGAT	113	0104-0121	Coding

25308	AGGCACCATCCCCAACGA	114	0105-0122	Coding

25309	TCCCACAGGCACCATCCC	115	0111-0128	Coding

25310	AGGTCTTCCCACAGGCAC	116	0117-0134	Coding

25311	ATGAGGAGGCACCTCTTC	117	0127-0144	Coding

25312	TTGCTGAAGACGATGAGG	118	0139-0156	Coding

25313	TCAAAGACAGTAGGGACG	119	0173-0195	Coding

25314	TTCTCAAAGACAGTAGGG	120	0181-0193	Coding

25315	ACTTCTCAAAGACAGTAG	121	0183-0200	Coding

25316	TGTTTTCCAGGCTGTCAG	122	0342-0359	Coding

25317	TCGTCTTGCCTCAGGTCC	123	0433-0450	Coding

25318	GTGTGCTCGTCTTGCCTC	124	0439-0456	Coding

25319	CTCCTGGTGTGCTCGTCT	125	0445-0462	Coding

25320	CAGACCCAACGGGCTCCT	126	0483-0500	Coding

25321	TTCCTCAGACCGAACGGG	127	0488-0505	Coding

25322	ACTCAAGGTAGCCAAAGG	128	0534-0551	Coding

25323	CTCCCGCACTCCCTCCTT	129	0566-0583	Coding

25324	CTCAAACACCTCCCGCAC	130	0575-0592	Coding

25325	GGCCATCTCAAACACCTC	131	0581-0598	Coding

25326	CTTGTTCTTGCGGACCTG	132	0614-0631	Coding

25327	CCCCTCCGACGCTTGTTC	133	0625-0642	Coding

25328	GTATGGAGCCCTCAGGAG	134	0737-0754	3′-UTR

25329	GAGCCTTCAGTATGGAGC	135	0746-0763	3′-UTR

25330	GAAAATGGAGCCTTCAGT	136	0753-0770	3′-UTR

25331	GGAACTGAAAATGGAGCC	137	0759-0776	3′-UTR

25332	GGAGGGAACTGAAAATGG	138	0763-0780	3′-UTR

25333	GCAGGAGGGAACTGAAAA	139	0766-0783	3′-UTR

25334	AGGGCAGGGCATAGGCGT	140	0851-0868	3′-UTR

25335	GGAAGGGCAGGGCATAGG	141	0854-0871	3′-UTR

25336	CATGAGGAAGGGCAGGGC	142	0859-0876	3′-UTR

25337	TAAAGTGCTGGTGTGTGA	143	0920-0937	3′-UTR

25338	CCTGTGAGCCAGAAGTGT	144	0939-0956	3′-UTR

25339	TTCCTGTGAGCCAGAAGT	145	0941-0958	3′-UTR

25340	CACTTTCCTGTGAGCCAG	146	0945-0962	3′-UTR

25341	AGACACTTTCCTGTGAGC	147	0948-0965	3′-UTR

25342	ACTCTGGGTCCCTACTGC	148	0966-0983	3′-UTR

25343	TGCAGAAACAACTCCAGG	149	0992-1009	3′-UTR

The compounds shown in Table 18 were analyzed for effect on RhoC mRNA levels by quantitative real-time PCR as described in examples herein. Data are shown in Table 19 and are averages from three experiments. If present, “N.D.” indicates “no data”.

TABLE 19


Inhibition of RhoC mRNA levels by phosphorothioate
oligodeoxynucleotides

				%
		TARGET		Inhi-	SEQ ID
ISIS#	REGION	SITE	SEQUENCE	bition	NO.

25304	5′ UTR	4	GAGCTGAGATGAAGTCAA	29	110

25305	5′ UTR	44	GCTGAAGTTCCCAGGCTG	25	111

25306	5′ UTR	47	CCGGCTGAAGTTCCCAGG	42	112

25307	CODING	104	GGCACCATCCCCAACGAT	81	113

25308	CODING	105	AGGCACCATCCCCAACGA	81	114

25309	CODING	111	TCCCACAGGCACCATCCC	70	115

25310	CODING	117	AGGTCTTCCCACAGGCAC	40	116

25311	CODING	127	ATGAGGAGGCAGGTCTTC	41	117

25312	CODING	139	TTGCTGAAGACGATGAGG	23	118

25313	CODING	178	TCAAAGACAGTAGGGACG	0	119

25314	CODING	181	TTCTCAAAGACAGTAGGG	2	120

25315	CODING	183	AGTTCTCAAAGACAGTAG	38	121

25316	CODING	342	TGTTTTCCAGGCTGTCAG	59	122

25317	CODING	433	TCGTCTTGCCTCAGGTCC	79	123

25318	CODING	439	GTGTGCTCGTCTTGCCTC	67	124

25319	CODING	445	CTCCTGGTGTGCTCGTCT	67	125

25320	CODING	483	CAGACCGAACGGGCTCCT	65	126

25321	CODING	488	TTCCTCAGACCGAACGGG	57	127

25322	CODING	534	ACTCAAGGTAGCCAAAGG	33	128

25323	CODING	566	CTCCCGCACTCCCTCCTT	91	129

25324	CODING	575	CTCAAACACCTCCCGCAC	34	130

25325	CODING	581	GGCCATCTCAAACACCTC	64	131

25326	CODING	614	CTTGTTCTTGCGGACCTG	72	132

25327	CODING	625	CCCCTCCGACGCTTGTTC	66	133

25328	3′ UTR	737	GTATGGAGCCCTCAGGAG	60	134

25329	3′ UTR	746	GAGCCTTCAGTATGGAGC	63	135

25330	3′ UTR	753	GAAAATGGAGCCTTCAGT	24	136

25331	3′ UTR	759	GGAACTGAAAATGGAGCC	2	137

25332	3′ UTR	763	GGAGGGAACTGAAAATGG	13	138

25333	3′ UTR	766	GCAGGAGGGAACTGAAAA	27	139

25334	3′ UTR	851	AGGGCAGGGCATAGGCGT	31	140

25335	3′ UTR	854	GGAAGGGCAGGGCATAGG	21	141

25336	3′ UTR	859	CATGAGGAAGGGCAGGGC	0	142

25337	3′ UTR	920	TAAAGTGCTGGTGTGTGA	39	143

25338	3′ UTR	939	CCTGTGAGCCAGAAGTGT	69	144

25339	3′ UTR	941	TTCCTGTGAGCCAGAAGT	69	145

25340	3′ UTR	945	CACTTTCCTGTGAGCCAG	82	146

25341	3′ UTR	948	AGACACTTTCCTGTGAGC	69	147

25342	3′ UTR	966	ACTCTGGGTCCCTACTGC	20	148

25343	3′ UTR	992	TGCAGAAACAACTCCAGG	0	149

As shown in Table 19, SEQ ID NOs 113, 114, 115, 122, 123, 124, 125, 126, 127, 129, 131, 132, 133, 134, 135, 144, 145, 146 and 147 demonstrated at least 50% inhibition of RhoC expression in this assay and are therefore preferred. [0142]

Example 17

Antisense Inhibition of RhoC Expression-Phosphorothioate 2′-MOE Gapmer Oligonucleotides

In accordance with the present invention, a second series of oligonucleotides targeted to human RhoC were synthesized. The oligonucleotide sequences are shown in Table 20. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. L25081), to which the oligonucleotide binds. [0143]

All compounds in Table 20 are chimeric oligonucleotides (“gapmers”) 18 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 four-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. Cytidine residues in the 2′-MOE wings are 5-methylcytidines.

TABLE 20


Nucleotide Sequences of Human RhoC Gapmer
Oligonucleotides

25344	GAGCTGAGATGAAGTCAA	110	0004-0021	5′-UTR
25345	GCTGAAGTTCCCAGGCTG	111	0044-0061	5′-UTR
25346	CCGGCTGAAGTTCCCAGG	112	0047-0064	5′-UTR
25347	GGCACCATCCCCAACGAT	113	0104-0121	Coding
25348	AGGCACCATCCCCAACGA	114	0105-0122	Coding
25349	TCCCACAGGCACCATCCC	115	0111-0128	Coding
25350	AGGTCTTCCCACAGGCAC	116	0117-0134	Coding
25351	ATGAGGAGGCAGGTCTTC	117	0127-0144	Coding
25352	TTGCTGAAGACGATGAGG	118	0139-0156	Coding
25353	TCAAAGACAGTAGGGACG	119	0178-0195	Coding
25354	TTCTCAAAGACAGTAGGG	120	0181-0198	Coding
25355	AGTTCTCAAAGACAGTAG	121	0183-0200	Coding
25356	TGTTTTCCAGGCTGTCAG	122	0342-0359	Coding
25357	TCGTCTTGCCTCAGGTCC	123	0433-0450	Coding
25358	GTGTCCTCGTCTTGCCTC	124	0439-0456	Coding
25359	CTCCTGGTGTGCTCGTCT	125	0445-0462	Coding
25360	CAGACCGAACGGCCTCCT	126	0483-0500	Coding
25361	TTCCTCAGACCGAACGGG	127	0488-0505	Coding
25362	ACTCAAGGTAGCCAAAGG	128	0534-0551	Coding
25363	CTCCCGCACTCCCTCCTT	129	0566-0583	Coding
25364	CTCAAACACCTCCCGCAC	130	0575-0592	Coding
25365	GGCCATCTCAAACACCTC	131	0581-0598	Coding
25366	CTTGTTCTTGCGGACCTG	132	0614-0631	Coding
25367	CCCCTCCGACGCTTGTTC	133	0625-0642	Coding
25368	GTATGGAGCCCTCAGGAG	134	0737-0754	3′-UTR
25369	GAGCCTTCAGTATGGAGC	135	0746-0763	3′-UTR
25370	GAAAATGGAGCCTTCAGT	136	0753-0770	3′-UTR
25371	GGAACTGAAAATGGAGCC	137	0759-0776	3′-UTR
25372	GGAGGGAACTGAAAATGG	138	0763-0780	3′-UTR
25373	GCAGGAGGGAACTGAAAA	139	0766-0783	3′-UTR
25374	AGGGCAGGGCATAGGCGT	140	0851-0868	3′-UTR
25375	GGAAGGGCAGGGCATAGG	141	0854-0871	3′-UTR
25376	CATGAGGAAGGGCAGGGC	142	0859-0876	3′-UTR
25377	TAAAGTGCTGGTGTGTGA	143	0920-0937	3′-UTR
25378	CCTGTGAGCCAGAAGTGT	144	0939-0956	3′-UTR
25379	TTCCTGTGAGCCAGAAGT	145	0941-0958	3′-UTR
25380	CACTTTCCTGTGAGCCAG	146	0945-0962	3′-UTR
25381	AGACACTTTCCTGTGAGC	147	0948-0965	3′-UTR
25382	ACTCTGGGTCCCTACTGC	148	0966-0983	3′-UTR
25383	TGCAGAAACAACTCCAGG	149	0992-1009	3′-UTR

RhoC inhibition data for these compounds were obtained by real-time quantitative PCR as described in other examples herein and are averaged from three experiments. Data are shown in Table 21. If present, “N.D.” indicates “no data”.

TABLE 21


Inhibition of RhoC mRNA levels by chimeric
phosphorothioate oligonucleotides having
2′-MOE wings and a deoxy gap

				%
		TARGET		Inhi-	SEQ ID
ISIS#	REGION	SITE	SEQUENCE	bition	NO.

25344	5′ UTR	4	GAGCTGAGATGAAGTCAA	0	110

25345	5′ UTR	44	GCTGAAGTTCCCAGGCTG	35	111

25346	5′ UTR	47	CCGGCTGAAGTTCCCAGG	53	112

25347	Coding	104	GGCACCATCCCCAACGAT	50	113

25348	Coding	105	AGGCACCATCCCCAACGA	56	114

25349	Coding	111	TCCCACAGGCACCATCCC	4	115

25350	Coding	117	AGGTCTTCCCACAGGCAC	11	116

25351	Coding	127	ATGAGGAGGCAGGTCTTC	6	117

25352	Coding	139	TTGCTGAAGACGATGAGG	15	118

25353	Coding	178	TCAAAGACAGTAGGGACG	32	119

25354	Coding	181	TTCTCAAAGACAGTAGGG	7	120

25355	Coding	183	AGTTCTCAAAGACAGTAG	39	121

25356	Coding	342	TGTTTTCCAGGCTGTCAG	59	122

25357	Coding	433	TCGTCTTGCCTCAGGTCC	48	123

25358	Coding	439	GTGTGCTCGTCTTGCCTC	36	124

25359	Coding	445	CTCCTCGTGTGCTCGTCT	61	125

25360	Coding	483	CAGACCGAACGGGCTCCT	50	126

25361	Coding	488	TTCCTCAGACCGAACGGG	14	127

25362	Coding	534	ACTCAAGGTAGCCAAAGG	32	128

25363	Coding	566	CTCCCGCACTCCCTCCTT	21	129

25364	Coding	575	CTCAAACACCTCCCGCAC	9	130

25365	Coding	581	GGCCATCTCAAACACCTC	66	131

25366	Coding	614	CTTGTTCTTGCGGACCTG	61	132

25367	Coding	625	CCCCTCCGACGCTTGTTC	0	133

25368	3′ UTR	737	GTATGGAGCCCTCAGGAG	28	134

25369	3′ UTR	746	GAGCCTTCAGTATGGAGC	32	135

25370	3′ UTR	753	GAAAATGGAGCCTTCAGT	0	136

25371	3′ UTR	759	GGAACTGAAAATGGAGCC	40	137

25372	3′ UTR	763	GGAGGGAACTGAAAATGG	45	133

25373	3′ UTR	766	GCAGGAGGGAACTGAAAA	35	139

25374	3′ UTR	351	AGGGCAGGGCATAGGCGT	5	140

25375	3′ UTR	854	GGAAGGGCAGGGCATAGG	0	141

25376	3′ UTR	859	CATGAGGAAGGGCAGGGC	0	142

25377	3′ UTR	920	TAAAGTGCTGGTGTGTGA	20	143

25378	3′ UTR	939	CCTGTGAGCCAGAAGTGT	67	144

25379	3′ UTR	941	TTCCTGTGAGCCAGAAGT	61	145

25380	3′ UTR	945	CACTTTCCTGTGAGCCAG	80	146

25381	3′ UTR	943	AGACACTTTCCTGTGAGC	0	147

25382	3′ UTR	966	ACTCTGGGTCCCTACTGC	0	148

25383	3′ UTR	992	TGCAGAAACAACTCCAGG	0	149

As shown in Table 21, SEQ ID NOs 111, 112, 113, 114, 119, 121, 122, 123, 124, 125, 126, 128, 131, 132, 134, 135, 137, 138, 139, 144, 145 and 146 demonstrated at least 25% inhibition of RhoC expression in this experiment and are therefore preferred. [0146]

Example 18

Synthesis of RhoG Antisense Oligonucleotide Sequences

Oligonucleotide sequences designed to target human RhoG were synthesized as described in previous examples and are shown in Table 22. Target sequence data are from the RhoG cDNA sequence published by Vincent, S., et al. ( Mol. Cell. Biol. 1992, 12, 3138-3148); Genbank accession number X61587, provided herein as SEQ ID NO: 150.

TABLE 22


Nucleotide Sequences of Human RhoG
Phosphorothioate Oligodeoxynucleotide

25464	GACCTGGTGCCCCTCCCG	151	0048-0065	5′-UTR

25465	TCTTCTGGACCCCTCTGG	152	0073-0090	5′-UTR

25466	GGCAGTGCCTCCTCTCTC	153	0089-0106	5′-UTR

25467	GTGCAGTTGCTGTAGTGA	154	0107-0124	5′-UTR

25468	GCATCGTGGGTGCAGTTG	155	0116-0133	AUG

25469	CCACCACGCACTTGATGC	156	0137-0154	Coding

25470	TTGTGTAGCAGATGAGCA	157	0185-0202	Coding

25471	AAAGCGTTAGTTGTGTAG	158	0195-0212	Coding

25472	GCGCGCTGTAATTGTCGA	159	0239-0256	Coding

25473	GGTTCACTGTGCGCCCGT	160	0269-0286	Coding

25474	GTCCCACAGGTTCAGGTT	161	0283-0300	Coding

25475	TGTACGGAGGCGGTCATA	162	0319-0336	Coding

25476	ACGTTGGTCTGAGGGTAG	163	0342-0359	Coding

25477	CAATGGAGAAACAGATGA	164	0365-0382	Coding

25478	CATAGGACGGCGGACTGG	165	0383-0400	Coding

25479	CGCACGTTCTCATAGGAC	166	0393-0410	Coding

25480	ACCTCTGGATGCCACTTG	167	0414-0431	Coding

25481	AGGGCAGTGGTGGCACAC	168	0430-0447	Coding

25482	CAGCAGGATGGCCACATC	169	0448-0465	Coding

25483	GGGTGTCAGGCTGGGCTC	170	0488-0505	Coding

25484	CCCTGCTGCGGTGTGATG	171	0537-0554	Coding

25485	CGCGAGTGCCTGGCCCTG	172	0550-0567	Coding

25486	GTAGCGCACAGCGTGGAT	173	0574-0591	Coding

25487	CATTCGAGGTAGCGCACA	174	0582-0599	Coding

25488	ACACCATCCTGTTGCAGG	175	0606-0623	Coding

25489	GAACACTTCCTTGACACC	176	0619-0636	Coding

25490	ACAGCCTCGGCGAACACT	177	0630-0647	Coding

25491	AAGAGGATGCAGGACCGC	178	0684-0701	Coding

25492	GCAGCCTCCAAGCCAAGT	179	0713-0730	3′-UTR

25493	AAAAGGCATTCAGGGAAC	180	0818-0835	3′-UTR

25494	GGGTCCAACCTTGGCTTG	181	0936-0953	3′-UTR

25495	GTCAGTAGCGGAAAATGG	182	0984-1001	3′-UTR

25496	AGCTGGATGAACTGGTCA	183	0998-1015	3′-UTR

25497	AACTGTGTGGAAAGCTGG	184	1010-1027	3′-UTR

25498	ACCACAATAGGCAGCAAC	185	1028-1045	3′-UTR

25499	GAGGGCAGAGGTTAGAGA	186	1074-1091	3′-UTR

25500	CAATTCCAAGAGCAGCGA	187	1090-1107	3′-UTR

25501	TGGAGAAGGGAGAGAGCA	188	1119-1136	3′-UTR

25502	ACATTCACCTTCTCAGGA	189	1154-1171	3′-UTR

25503	GTCAGCAAATGCGTAAGG	190	1199-1216	3′-UTR

The compounds in Table 22 were analyzed for effect on RhoG mRNA levels by quantitative real-time PCR as described in other examples herein. Data, shown in Table 23, are averages from three experiments. If present, “N.D.” indicates “no data”.

TABLE 23


Inhibition of RhoG mRNA levels by phosphorothioate
oligodeoxynucleotides

				%
		TARGET		Inhi-	SEQ ID
ISIS#	REGION	SITE	SEQUENCE	bition	NO.

25464	5′ UTR	48	GACCTGGTGCCCCTCCCG	35	151

25465	5′ UTR	73	TCTTCTGGACCCCTCTGG	36	152

25466	5′ UTR	89	GGCAGTGCCTCCTCTCTC	35	153

25467	5′ UTR	107	GTGCAGTTGCTGTAGTGA	10	154

25468	5′ UTR	116	GCATCGTGGGTGCAGTTG	47	155

25469	CODING	137	CCACCACGCACTTGATGC	14	156

25470	CODING	185	TTGTGTAGCAGATGAGCA	35	157

25471	CODING	195	AAAGCGTTAGTTGTGTAG	0	158

25472	CODING	239	GCGCGCTGTAATTGTCGA	36	159

25473	CODING	269	GGTTCACTGTGCGCCCGT	16	160

25474	CODING	283	GTCCCACAGGTTCAGGTT	31	161

25475	CODING	319	TGTACGGAGGCGGTCATA	37	162

25476	CODING	342	ACGTTGGTCTGAGGGTAG	38	163

25477	CODING	365	CAATGGAGAAACAGATGA	0	164

25478	CODING	383	CATAGGACGGCGGACTGG	17	165

25479	CODING	393	CGCACGTTCTCATAGGAC	24	166

25480	CODING	414	ACCTCTGGATGCCACTTG	35	167

25481	CODING	430	AGGGCAGTGGTGGCACAC	15	168

25482	CODING	448	CAGCAGGATGGGCACATC	20	169

25483	CODING	488	GGGTGTCAGGCTGGGCTC	15	170

25484	CODING	537	CCCTGCTGCGGTGTGATG	44	171

25464	5′ UTR	48	GACCTGGTGCCCCTCCCG	35	151

25465	5′ UTR	73	TCTTCTGGACCCCTCTGG	36	152

25466	5′ UTR	89	GGCAGTGCCTCCTCTCTC	35	153

25485	CODING	550	CGCGAGTGCCTGGCCCTG	9	172

25486	CODING	574	GTAGCGCACAGCGTGGAT	35	173

25487	CODING	582	CATTCGAGGTAGCGCACA	39	174

25488	CODING	606	ACACCATCCTGTTGCAGG	23	175

25489	CODING	619	GAACACTTCCTTGACACC	31	176

25490	CODING	630	ACAGCCTCGGCGAACACT	6	177

25491	CODING	684	AAGAGGATGCAGGACCGC	18	178

25492	3′ UTR	713	GCAGCCTCCAAGCCAAGT	42	179

25493	3′ UTR	818	AAAAGGCATTCAGGGAAC	0	180

25494	3′ UTR	936	GGGTCCAACCTTGGCTTG	58	181

25495	3′ UTR	984	GTCAGTAGCGGAAAATGG	0	182

25496	3′ UTR	998	AGCTGGATGAACTGGTCA	23	183

25497	3′ UTR	1010	AACTGTGTGGAAAGCTGG	8	184

25498	3′ UTR	1028	ACCACAATAGGCAGCAAC	31	185

25499	3′ UTR	1074	GAGGGCAGAGGTTAGAGA	21	186

25500	3′ UTR	1090	CAATTCCAAGAGCAGCGA	18	187

25501	3′ UTR	1119	TGGAGAAGGGAGAGAGCA	32	188

25502	3′ UTR	1154	ACATTCACCTTCTCAGGA	20	189

25503	3′ UTR	1199	GTCAGCAAATGCGTAAGG	24	190

As shown in Table 23, SEQ ID NOs 151, 152, 153, 155, 157, 159, 161, 162, 163, 167, 171, 173, 174, 176, 179, 181, 185 and 188 demonstrated at least 25% inhibition of RhoG expression in this assay and are therefore preferred. [0149]

Example 19

Antisense Inhibition of RhoG Expression-Phosphorothioate 2′-MOE Gapmer Oligonucleotides

In accordance with the present invention, a second series of oligonucleotides targeted to human RhoG were synthesized. The oligonucleotide sequences are shown in Table 24. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X61587), to which the oligonucleotide binds. [0150]

All compounds in Table 24 are chimeric oligonucleotides (“gapmers”) 18 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 four-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. Cytidine residues in the 2′-MOE wings are 5-methylcytidines.

TABLE 24


Nucleotide Sequences of Human RhoG Gapmer
Oligonucleotides

25504	GACCTGGTGCCCCTCCCG	151	0048-0065	5′-UTR

25505	TCTTCTGGACCCCTCTGG	152	0073-0090	5′-UTR

25506	GGCAGTGCCTCCTCTCTC	153	0089-0106	5′-UTR

25507	GTGCAGTTGCTCTAGTGA	154	0107-0124	5′-UTR

25508	GCATCGTGGGTGCAGTTG	155	0116-0133	AUG

25509	CCACCACGCACTTGATGC	156	0137-0154	Coding

25510	TTGTGTAGCACATGAGCA	157	0185-0202	Coding

25511	AAAGCGTTAGTTGTGTAG	158	0195-0212	Coding

25512	GCGCGCTGTAATTGTCGA	159	0239-0256	Coding

25513	GGTTCACTGTGCGCCCGT	160	0269-0286	Coding

25514	GTCCCACAGGTTCAGGTT	161	0283-0300	Coding

25515	TGTACGGAGGCGGTCATA	162	0319-0336	Coding

25516	ACGTTGGTCTGAGGGTAG	163	0342-0359	Coding

25517	CAATGGAGAAACAGATGA	164	0365-0382	Coding

25518	CATAGGACGGCGGACTGG	165	0383-0400	Coding

25519	CGCACGTTCTCATAGGAC	166	0393-0410	Coding

25520	ACCTCTGGATGCCACTTG	167	0414-0431	Coding

25521	AGGGCAGTGGTGGCACAC	168	0430-0447	Coding

25522	CAGCAGGATGCGCACATC	169	0448-0465	Coding

25523	GGGTGTCAGGCTGGGCTC	170	0488-0505	Coding

25524	CCCTGCTGCGGTGTGATG	171	0537-0554	Coding

25525	CGCGAGTGCCTGGCCCTG	172	0550-0567	Coding

25526	GTAGCGCACAGCGTGGAT	173	0574-0591	Coding

25527	CATTCGAGGTAGCGCACA	174	0582-0599	Coding

25528	ACACCATCCTGTTGCAGG	175	0606-0623	Coding

25529	GAACACTTCCTTGACACC	176	0619-0636	Coding

25530	ACAGCCTCGGCGAACACT	177	0630-0647	Coding

25531	AAGAGGATGCAGGACCGC	178	0684-0701	Coding

25532	GCAGCCTCCAAGCCAAGT	179	0713-0730	3′-UTR

25533	AAAAGGCATTCAGGGAAC	180	0818-0835	3′-UTR

25534	GGGTCCAACCTTGGCTTG	181	0936-0953	3′-UTR

25535	GTCAGTAGCGGAAAATGG	182	0984-1001	3′-UTR

25536	AGCTGGATGAACTGGTCA	183	0998-1015	3′-UTR

25537	AACTGTGTGGAAAGCTGG	184	1010-1027	3′-UTR

25538	ACCACAATAGGCAGCAAC	185	1028-1045	3′-UTR

25539	GAGGGCAGAGGTTAGAGA	186	1074-1091	3′-UTR

25540	CAATTCCAAGAGCAGCGA	187	1090-1107	3′-UTR

25541	TGGAGAAGGGAGAGAGCA	188	1119-1136	3′-UTR

25542	ACATTCACCTTCTCAGGA	189	1154-1171	3′-UTR

25543	GTCAGCAAATGCGTAAGG	190	1199-1216	3′-UTR

RhoG inhibition data for compounds in Table 24 were obtained by real-time quantitative PCR as described in other examples herein and are averaged from three experiments. Data are shown in Table 25. If present, “N.D.” indicates “no data”.

TABLE 25


Inhibition of RhoG mBNA levels by chimeric
phosphorothioate oligonucleotides having 2′-MOE
wings and a deoxy gap

		TAR-			SEQ
		GET		%	ID
ISIS#	REGION	SITE	SEQUENCE	Inhibition	NO.

25504	5′UTR	48	GACCTGGTGCCCCTCCCG	0	151

25505	5′UTR	73	TCTTCTGGACCCCTCTGG	32	152

25506	5′UTR	89	GGCAGTGCCTCCTCTCTC	28	153

25507	5′UTR	107	GTGCAGTTGCTGTAGTGA	0	154

25508	5′UTR	116	GCATCGTGGGTGCAGTTG	12	155

25509	Coding	137	CCACCACGCACTTGATGC	0	156

25510	Coding	185	TTGTGTAGCAGATGAGCA	0	157

25511	Coding	195	AAAGCGTTAGTTGTGTAG	33	158

25512	Coding	239	GCGCGCTGTAATTGTCGA	0	159

25513	Coding	269	GGTTCACTGTCCGCCCGT	82	160

25514	Coding	283	GTCCCACAGGTTCAGGTT	0	161

25515	Coding	319	TGTACGCACGCCGTCATA	13	162

25516	Coding	342	ACGTTGGTCTCAGGGTAG	53	163

25517	Coding	365	CAATGGAGAAACAGATGA	0	164

25518	Coding	383	CATAGGACGGCGGACTGG	55	165

25519	Coding	393	CGCACGTTCTCATAGGAC	9	166

25520	Coding	414	ACCTCTCGATGCCACTTG	56	167

25521	Coding	430	AGGGCAGTGGTGGCACAC	0	168

25522	Coding	448	CAGCAGGATGGGCACATC	0	169

25523	Coding	488	GGGTCTCAGGCTGGGCTC	27	170

25524	Coding	537	CCCTGCTGCGGTGTGATG	55	171

25525	Coding	550	CGCGAGTGCCTGGCCCTG	41	172

25526	Coding	574	GTAGCGCACAGCGTGGAT	41	173

25527	Coding	582	CATTCGAGGTAGCGCACA	0	174

25528	Coding	606	ACACCATCCTCTTGCAGG	37	175

25529	Coding	619	GAACACTTCCTTCACACC	23	176

25530	Coding	630	ACAGCCTCGGCGAACACT	59	177

25531	Coding	684	AAGAGGATGCAGGACCGC	39	178

25532	3′UTR	713	GCAGCCTCCAAGCCAAGT	13	179

25533	3′UTR	818	AAAAGGCATTCAGGGAAC	43	180

25534	3′UTR	936	GGGTCCAACCTTGGCTTG	78	181

25535	3′UTR	984	GTCAGTAGCGGAAAATGG	54	182

25536	3′UTR	998	AGCTGGATGAACTGGTCA	54	183

25537	3′UTR	1010	AACTCTGTGCAAAGCTGG	59	184

25538	3′UTR	1028	ACCACAATAGGCAGCAAC	43	185

25539	3′UTR	1074	GAGGGCAGAGGTTAGAGA	0	186

25540	3′UTR	1090	CAATTCCAAGAGCAGCGA	26	187

25541	3′UTR	1119	TGGAGAACGGAGAGAGCA	0	188

25542	3′UTR	1154	ACATTCACCTTCTCAGGA	26	189

25543	3′UTR	1199	GTCACCAAATGCGTAAGG	73	190

As shown in Table 25, SEQ ID NOs 152, 158, 160, 163, 165, 167, 171, 172, 173, 175, 177, 178, 180, 181, 182, 183, 184, 185 and 190 demonstrated at least 30% inhibition of RhoG expression in this experiment and are therefore preferred. [0153]

Example 20

Human Rac1 Oligonucleotide Sequences

Antisense oligonucleotides were designed to target human Rac1. Target sequence data are from the Rac1 cDNA sequence published by Didsbury, J., et al. ([0154] J. Biol. Chem. 1989, 264, 16378-16382); Genbank accession number M29870, provided herein as SEQ ID NO: 191. Oligonucleotides were synthesized primarily with phosphorothioate linkages. Oligonucleotide sequences are shown in Table 26.
Cells were cultured, treated with oligonucleotides, and mRNA was isolated and quantitated as described in Example 2. A 45-mer antisense oligonucleotide to Rac1 (5′-ATAGAATGTGAGTCTGAACTCTTACATTTAGAACAAACAAAACCT-3′ SEQ ID NO. 192) was used as a probe as described in Didsbury, J., et al. ([0155] J. Biol. Chem. 1989, 264, 16378-16382).
An initial screen of Rac1 specific antisense oligonucleotides was performed using a oligonucleotide concentration of 300 nM. [0156]

Results are shown in Table 27. Oligonucleotides 16052 (SEQ ID NO. 195), 16056 (SEQ ID NO. 199), 16058 (SEQ ID NO. 201), 16062 (SEQ ID NO. 204) and 16063 (SEQ ID NO. 205) gave better than 50% inhibition of Rac1 mRNA levels. Oligonucleotides 16052 (SEQ ID NO. 195), 16058 (SEQ ID NO. 201) and 16062 (SEQ ID NO. 204) gave better than 75% inhibition.

TABLE 26


Nucleotide Sequences of Rac-1 Phosphorothioate Oligonucleotides

			TARGET GENE	GENE
	NUCLEOTIDE SEQUENCE		NUCLEOTIDE	TARGET
ISIS NO.	(5′ ->3′)	SEQ ID NO:	CO-ORDINATES¹	REGION

16050	CAAATGATGCAGGACTCACA	193	0252-0271	Coding

16051	CACCACCACACACTTGATC	194	0009-0027	Coding

16052	ATAAGCCCAGATTCACCG	195	0149-0166	Coding

16053	TCTTTGCGGATAGGATAGG	196	0207-0225	Coding

16054	GCTTCTTCTCCTTCAGTTTCTC	197	0379-0400	Coding

16055	CAGCACCAATCTCCTTAGC	198	0436-0454	Coding

16056	CTCTTCCTCTTCTTCACCC	199	0542-0560	Coding

16057	CCTAAGATCAAGTTTAGTTC	200	0341-0360	Coding

16058	CGCACCTCAGGATACCACTT	201	0286-0305	Coding

16059	ATCTACCATAACATTGGCAG	202	0122-0141	Coding

16060	TAATTGTCAAAGACAGTAGG	203	0100-0119	Coding

16062	GAGCGCCGAGCACTCCAGGT	204	0461-0480	Coding

16063	CTCAAACACTGTCTTGAGGC	205	0491-0510	Coding

16143	ATAGAATGTGAGTCTCAACT	206	unknown³	3′-UTR

16144	CTTACATTTAGAACAAACAAAACCT	207	unknown³	3′-UTR

16849	CCCAGCTAAGAATTCCGCTC	208	16058 control

16850	TAAACGCCGAATCTACGC	209	16052 control

TABLE 27


Activities of Phosphorothioate Oligonucleotides Targeted to
Human Rac1

	SEQ	GENE
ISIS	ID	TARGET	% mRNA	% mRNA
No:	NO:	REGION	EXPRESSION	INHIBITION

LIPOFECTIN	—	—	100.0%	0.0%
only
16051	194	Coding	77.1%	22.9%
16052	195	Coding	3.7%	96.3%
16053	196	Coding	68.4%	31.6%
16054	197	Coding	67.6%	32.4%
16055	198	Coding	70.8%	29.2%
16056	199	Coding	48.0%	52.0%
16057	200	Coding	97.3%	2.7%
16058	201	Coding	22.2%	77.8%
16059	202	Coding	57.7%	42.3%
16060	203	Coding	91.6%	8.4%
16062	204	Coding	21.7%	78.3%
16063	205	Coding	32.4%	67.6%
16143	206	3′-UTR	56.1%	43.9%
16144	207	3′-UTR	72.9%	27.1%

Example 21

Dose Response and Specificity of Antisense Oligonucleotide Effects on Human Rac1 mRNA Levels in A549 Cells

Oligonucleotides 16050 (SEQ ID NO. 193), 16052 (SEQ ID NO. 195)16058 (SEQ ID NO. 201), 16062 (SEQ ID NO. 204) and 16143 (SEQ ID NO. 206) were chosen for dose response studies. Oligonucleotide 16057 (SEQ ID NO. 200) was chosen as a negative control because it was inactive in the initial screen. Results are shown in Table 28. Oligonucleotides 16050, 16052, 16058 and 16062 inhibited Rac1 mRNA expression in a dose dependent manner with maximum expression of 65% to 82%. [0159]

The specificity of oligonucleotides 16052 and 16058 was tested using scrambled controls. Results are shown in Table 29. Both sequences inhibited Rac1 mRNA expression in a dose dependent manner and were significantly better than their scrambled controls.

TABLE 28


Dose Response of A549 Cells to Rac1
Antisense Oligonucleotides (ASOs)

	SEQ ID	ASO Gene		% mRNA	% mRNA
ISIS #	NO:	Target	Dose	Expression	Inhibition

control	—	LIPOFECTIN	—	100%	0%
		only
16050	193	coding	75 nM	71.1%	28.9%
16050	193	″	150 nM	53.6%	46.4%
16050	193	″	300 nM	33.6%	66.4%
16052	195	coding	75 nM	68.2%	31.8%
16052	195	″	150 nM	40.5%	59.5%
16052	195	″	300 nM	28.3%	71.7%
16057	200	coding	75 nM	81.7%	18.3%
16057	200	″	150 nM	80.2%	19.8%
16057	200	″	300 nM	85.8%	14.2%
16058	201	coding	75 nM	60.1%	39.9%
16058	201	″	150 nM	42.9%	57.1%
16058	201	″	300 nM	17.7%	82.3%
16062	204	coding	75 nM	50.5%	49.5%
16062	204	″	150 nM	40.2%	59.8%
16062	204	″	300 nM	25.2%	74.8%
16143	206	3′-UTR	75 nM	294.8%	—
16143	206	″	150 nM	100.8%	—
16143	206	″	300 nM	88.6%	11.4%

TABLE 29


Specificity of Rac1 Antisense Oligonucleotides (ASOs) in
A549 Cells

	SEQ ID	ASO Gene		% mRNA	% mRNA
ISIS #	NO:	Target	Dose	Expression	Inhibition

control	—	LIPOFECTIN	—	100%	0%
		only
16052	195	coding	75 nM	86.6%	13.4%
16052	195	″	150 nM	52.8%	47.2%
16052	195	″	300 nM	18.5%	81.5%
16850	209	control	75 nM	88.9%	11.1%
16850	209	″	150 nM	97.2%	2.8%
16850	209	″	300 nM	107.4%	—
16058	201	coding	75 nM	82.7%	17.3%
16058	201	″	150 nM	36.8%	63.2%
16058	201	″	300 nM	21.1%	78.9%
16849	208	control	75 nN	90.7%	9.3%
16849	208	″	150 nM	70.2%	29.8%
16849	208	″	300 nM	68.2%	31.8%

Example 22

Design and Testing of Chimeric (Deoxy Gapped) 2′-O-Methoxyethyl Rac1 Antisense Oligonucleotides on Rac1 mRNA Levels in A549 Cells

Oligonucleotides targeted to Rac1 were synthesized as a uniformly phosphorothioate or mixed phosphorothioate/phosphodiester chimeric oligonucleotides having variable regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides. All 2′-MOE cytosines were 5-methyl-cytosines. Additionally, some oligonucleotides were synthesized with deoxycytosines as 5-methyl-cytosines. Additional oligonucleotides were synthesized, with similar chemistries, as scrambled controls. Oligonucleotide sequences and chemistries are shown in Tables 30 and 31. A dose response experiment was performed using a number of these oligonucleotides as described in Example 3. [0162]

Results are shown in Table 32. All of the chimeric oligonucleotides tested showed a dose dependent effect and showed inhibition of Rac mRNA levels comparable to that of the phosphorothioate oligodeoxynucleotide.

TABLE 30


Nucleotide Sequences of Rac1 Gapmer
Oligonucleotides

		SEQ	TARGET GENE	GENE
ISIS	NUCLEOTIDE SEQUENCE	ID	NUCLEOTIDE	TARGET
NO.	(5′ -> 3′)	NO:	CO-ORDINATES¹	REGION

16899	ATAAGCCCAGATTCACCG	195	0149-0166	Coding
16900	CAAATGATGCAGGACTCACA	193	0252-0271	Coding
16901	CGCACCTCAGGATACCACTT	201	0286-0305	Coding
17161	ATAAGCCCAGATTCACCG	195	0149-0166	Coding
17162	ATAAGCCCAGATTCACCG	195	0149-0166	Coding
17163	ATAAGCCCAGATTCACCG	195	0149-0166	Coding
17164	ATAAGCCCAGATTCACCG	195	0149-0166	Coding

18540	ATAAGCCCTGATTCACCG	210	16899 mismatch
18541	ATACGCCCTGATTCACCG	211	16899 mismatch
18542	ATACGCCCTGATTAACCG	212	16899 mismatch
18549	TAAACGCCGAATCTACGC	213	16899 control

TABLE 31


Nucleotide Sequences of Rac1 Mixed Backbone Oligonucleotides

		SEQ	TARGET GENE	GENE
ISIS	NUCLEOTIDE SEQUENCE	ID	NUCLEOTIDE	TARGET
NO.	(5′ ->3′)	NO:	CO-ORDINATES¹	REGION

17814

ToAoAoAoCoGoCoCoGsAsAsTsCsTsAsCsGsC

213

16899 control

17815	AoToAoAoGoCoCoCoAsGsAsTsTsCsAsCsCsC	195	0149-0166	Coding
17816	CoAoAoAoToGsAsTsGsCsAsGsGsAsCsToCoAoCoA	193	0252-0271	Coding
17817	AoAoAoCoToGsCsTsGsAsAsGsTsAsCsGoCoAoCoA	214	17816 control

24686	ToAoAoAoCoGoCoCoGoAoAoToCoToAoCoGoC	213	16899 control
24687	TsAsAsAsCsGsCsCsGsAsAsTsCsTsAsCsGsC	213	16899 control

TABLE 32


Dose Response of A549 Cells to Rac1
Antisense Gapmer Oligonucleotides (ASOs)

	SEQ ID	ASO Gene		% mRNA	% mRNA
ISIS #	NO:	Target	Dose	Expression	Inhibition

control	—	LIPOFECTIN	—	100	0.0%
		only
16899	195	coding	75 nM	79.9%	20.1%
″	″	″	150 nM	40.8%	59.2%
″	″	″	300 nM	21.8%	78.2%
17161	195	coding	75 nM	31.3%	68.7%
″	″	″	150 nM	16.9%	83.1%
″	″	″	300 nM	12.3%	87.7%
17162	195	coding	75 nM	89.2%	10.8%
″	″	″	150 nM	63.0%	37.0%
″	″	″	300 nM	18.4%	81.6%
17163	195	coding	75 nM	93.4%	6.6%
″	″	″	150 nM	67.3%	32.7%
″	″	″	300 nM	34.4%	65.6%
17164	195	coding	75 nM	94.7%	5.3%
″	″	″	150 nM	65.9%	34.1%
″	″	″	300 nM	36.2%	63.8%

Example 23

Human cdc42 Chimeric (Deoxy Gapped) 2′-O-methoxyethyl Oligonucleotide Sequences

Antisense oligonucleotides were designed to target human cdc42. Target sequence data are from the cdc42 cDNA sequence published by Shinjo, K. et al. ([0166] Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 9853-9857); Genbank accession number M57298, provided herein as SEQ ID NO: 215. Oligonucleotides were synthesized as uniformly phosphorothioate chimeric oligonucleotides having a centered deoxy gap of eight nucleotides flanked by 2′-O-methoxyethyl (2′-MOE) regions. All 2′-MOE cytosines were 5-methyl-cytosines. Oligonucleotide sequences are shown in Table 33.
A549 cells were cultured and treated with oligonucleotide as described in Example 2. Quantitation of cdc42 mRNA levels was determined by real-time PCR (RT-PCR) as described in previous examples. [0167]

For cdc42 the PCR primers were:


For cdc42 the PCR primers were:
Forward:
5′-TTCAGCAATGCACACAATTAAGTGT-3′	SEQ ID NO. 216

Reverse:
5′-TGTTGTGTAGGATATCAGGAGACATGT-3′	SEQ ID NO. 217

and the PCR probe was:
FAM-TTGTGGGCGATGGTGCTGTTGGTA-TAMRA

(SEQ ID NO. 218) where FAM or JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye. [0169]

For GAPDH the PCR primers were:


For GAPDH the PCR primers were:
Forward primer:
5′-GAAGGTGAAGGTCGGAGTC-3′	SEQ ID NO. 65

Reverse primer:
5′-GAAGATGGTGATGGGATTTC-3′	SEQ ID NO. 66

and the PCR probe was:
5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′

(SEQ ID NO. 67) where FAM or JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye. [0171]

Results are shown in Table 34. All oligonucleotides tested gave greater than 40% inhibition of cdc42 mRNA expression.

TABLE 33


Nucleotide Sequences of cdc42 Oligonucleotides

		SEQ	TARGET GENE	GENE
ISIS	NUCLEOTIDE SEQUENCE	ID	NUCLEOTIDE	TARGET
NO.	(5′ ->3′)	NO: CO-ORDINATES¹	REGION

17203	TAATTGTCTGCATTGCTGAA	219	0063-0082	AUG

17209	TTACCAACAGCACGATCGCC	220	0097-0116	Coding

17210	CCACCAATCATAACTGTGAC	221	0193-0212	Coding

17211	GTGGATAACTCAGCGGTCGT	222	0270-0289	Coding

17212	GAAGATGGAGAGACCACTGA	223	0316-0335	Coding

17213	GTGAGTTATCTCAGGCACCC	224	0359-0378	Coding

17214	GCTTCTGTTTGTTCTTGGCA	225	0456-0475	Coding

17215	TGACAGCCTTCAGGTCACGG	226	0507-0526	Coding

17216	CACCTGCGGCTCTTCTTCGG	227	0613-0632	Coding

17217	TTGTCTCACACGAGTGCATG	228	0774-0793	3′-UTR

17218	TTCTGACAATACAATTACTC	229	0844-0863	3′-UTR

17219	TTACAGAGTCATCCACAAGC	230	0961-0980	3′-UTR

20457	CGATAGTCTCCACGTGAGGC	231	17215 control

21668	CGATAGTCTCCACGTGAGGC	231	17215 control

21917	GTAACGCTCCTATGGCCAGG	232	17215 control

21918	AGACTGACTGCTCGTCGCGA	233	17215 control

TABLE 34


Activities of Phosphorothioate Oligonucleotides Targeted to
Human Cdc42

	SEQ	GENE
ISIS	ID	TARGET	% mRNA	5 mRNA
No:	NO:	REGION	EXPRESSION	INHIBITION

LIPOFECTIN	—	—	100%	0%
only
17208	219	AUG	40.6%	59.4%
17209	220	Coding	43.4%	56.6%
17210	221	Coding	55.4%	44.6%
17211	222	Coding	25.5%	74.5%
17212	223	Coding	31.1%	68.9%
17213	224	Coding	14.0%	86.0%
17214	225	Coding	27.4%	72.6%
17215	226	Coding	16.9%	83.1%
17216	227	Coding	26.0%	74.0%
17217	228	3′-UTR	28.4%	71.6%
17218	229	3′-UTR	17.2%	82.8%
17219	230	3′-UTR	20.2%	79.8%

Example 24

Dose Response of Antisense Oligonucleotide Effects on Human cdc42 mRNA Levels in A549 Cells

Oligonucleotides 17213 (SEQ ID NO. 224), 17215 (SEQ ID NO. 226), 17218 (SEQ ID NO. 229), and 17219 (SEQ ID NO. 230) were chosen for dose response studies. Results are shown in Table 35.

TABLE 35


Dose Response of A549 Cells to Cdc42
Antisense Oligonucleotides (ASOs)

	SEQ ID	ASO Gene		% mRNA	% mRNA
ISIS #	NO:	Target	Dose	Expression	Inhibition

control	—	LIPOFECTIN	—	100%	0%
		only
17213	224	coding	75 nM	158%	—
17213	″	″	300 nM	16%	84%
17215	226	coding	75 nM	90%	10%
17215	″	″	300 nM	21%	79%
17218	229	3′-UTR	75 nM	53%	47%
17218	″	″	300 nM	38%	62%
17219	230	3′-UTR	75 nM	102%	—
17219	″	″	300 nM	41%	59%

Example 25

Additional cdc42 Chimeric Oligonucleotides

Oligonucleotides having SEQ ID NO: 226 were synthesized as mixed phosphorothioate/phosphodiester chimeric oligonucleotides having variable wing regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and a central stretch of nine deoxynucleotides. All 2′-MOE cytosines were 5-methyl-cytosines. Oligonucleotide sequences and chemistries are shown in Table 36.

TABLE 36


Nucleotide Sequence of 17215 Analog

		SEQ	TARGET GENE	GENE
ISIS	NUCLEOTIDE SEQUENCE	ID	NUCLEOTIDE	TARGET
NO.	(5′ ->3′)	NO:	CO-ORDINATES¹	REGION

22276	ToGoAoCoAoGsCsCsTsTsCsAsGsGsTsCoAoCoGoG	226	0507-0526	Coding

22277	CoGoAoToAoGsTsCsTsCsCsAsCsGsTsGoAoGoGoC	231	22276 control

[0176]
1 233 1 1074 DNA Homo sapiens 1 gaattcgggc taccctcgcc ccgcccgcgg tcctccgtcg gttctctcat tagtccacgg 60 tctggtcttc agctacccgc cttcgtctcc gagtttgcga ctcgcgggac cggcgtcccc 120 ggcgcgaaga ggctggactc ggattcgttg cctgagcaat ggctgccatc cggaagaaac 180 tggtgattgt tggtgatgga gcctgtggaa agacatgctt gctcatagtc ttcagcaagg 240 accagttccc agaggtgtat gtgcccacag tgtttgagaa ctatgtggca gatatcgagg 300 tggatggaaa gcaggtagag ttggctttgt gggacacagc tgggcaggaa gattatgatc 360 gcctgaggcc cctctcctac ccagataccg atgttatact gatgtgtttt tccatcgaca 420 gccctgatag tttagaaaac atcccagaaa agtggacccc agaagtcaag catttctgtc 480 ccaacgtgcc catcatcctg gttgggaata agaaggatct tcggaatgat gagcacacaa 540 ggcgggagct agccaagatg aagcaggagc cggtgaaacc tgaagaaggc agagatatgg 600 caaacaggat tggcgctttt gggtacatgg agtgttcagc aaagaccaaa gatggagtga 660 gagaggtttt tgaaatggct acgagagctg ctctgcaagc tagacgtggg aagaaaaaat 720 ctggttgcct tgtcttgtga aaccttgctg caagcacagc ccttatgcgg ttaattttga 780 agtgctgttt attaatctta gtgtatgatt actggccttt ttcatttatc tataatttac 840 ctaagattac aaatcagaag tcatcttgct accagtattt agaagccaac tatgattatt 900 aacgatgtcc aacccgtctg gcccaccagg gtccttttga cactgctcta acagccctcc 960 tctgcactcc cacctgacac accaggcgct aattcaagga atttcttaac ttcttgcttc 1020 tttctagaaa gagaaacagt tggtaacttt tgtcaattag gctgtaacta cttt 1074 2 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 2 tgcaagcaca gcccttatg 19 3 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 3 tgtcaaaagg accctggtg 19 4 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 4 agtcgcaaac tcggagac 18 5 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 5 ttgctcaggc aacgaatc 18 6 22 DNA Artificial Sequence Description of Artificial SequenceSynthetic 6 ctgaagacta tgagcaagca tg 22 7 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 7 ctcatcattc cgaagatcc 19 8 22 DNA Artificial Sequence Description of Artificial SequenceSynthetic 8 ccaatcctgt ttgccatatc tc 22 9 22 DNA Artificial Sequence Description of Artificial SequenceSynthetic 9 ccatctttgg tctttgctga ac 22 10 21 DNA Artificial Sequence Description of Artificial SequenceSynthetic 10 gcagagcagc tctcgtagcc a 21 11 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 11 tcacaagaca aggcaaccag 20 12 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 12 aggccagtaa tcatacacta 20 13 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 13 gttggcttct aaatactggt 20 14 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 14 ggctgttaga gcagtgtcaa 20 15 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 15 agcgcctggt gtgtcaggtg 20 16 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 16 tagttacagc ctaattgaca 20 17 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 17 ggcacctgtt gggtgagctg 20 18 22 DNA Artificial Sequence Description of Artificial SequenceSynthetic 18 acactcttgc ttaccgtacc tt 22 19 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 19 tgcggtaagt gcggtatcaa 20 20 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 20 gtcgttagtc gaaatgagg 19 21 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 21 agcttgtgaa cgagtgtcga 20 22 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 22 tgcagttggc agagtctgaa 20 23 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 23 agagaaccga cggaggac 18 24 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 24 gtggactaat gagagaac 18 25 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 25 gaccgtggac taatgaga 18 26 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 26 agctgaagac cagaccgt 18 27 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 27 aatccgagtc cagcctct 18 28 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 28 aacgaatccg agtccagc 18 29 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 29 tcaggcaacg aatccgag 18 30 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 30 caccaacaat caccagtt 18 31 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 31 aagactatga gcaagcat 18 32 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 32 atacacctct gggaactg 18 33 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 33 acatagttct caaacact 18 34 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 34 actctacctg ctttccat 18 35 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 35 cacaaagcca actctacc 18 36 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 36 aacatcggta tctgggta 18 37 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 37 ttctgggatg ttttctaa 18 38 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 38 ggacagaaat gcttgact 18 39 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 39 gtgctcatca ttccgaag 18 40 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 40 cttgtgtgct catcattc 18 41 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 41 tagctcccgc cttgtgtg 18 42 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 42 ccaatcctgt ttgccata 18 43 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 43 gtctttgctg aacactcc 18 44 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 44 aaaacctctc tcactcca 18 45 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 45 aagacaaggc aaccagat 18 46 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 46 tttcacaaga caaggcaa 18 47 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 47 gcaaggtttc acaagaca 18 48 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 48 attaaccgca taagggct 18 49 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 49 taataaacag cacttcaa 18 50 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 50 ccagtaatca tacactaa 18 51 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 51 atgacttctg atttgtaa 18 52 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 52 tagcaagatg acttctga 18 53 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 53 ctggtagcaa gatgactt 18 54 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 54 ctaaatactg gtagcaag 18 55 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 55 ttggcttcta aatactgg 18 56 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 56 tcatagttgg cttctaaa 18 57 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 57 aataatcata gttggctt 18 58 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 58 tcaaaaggac cctggtgg 18 59 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 59 gtgcagagga gggctgtt 18 60 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 60 ccaactgttt ctctttct 18 61 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 61 aagtagttac agcctaat 18 62 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 62 ggctggactc ggattcgtt 19 63 22 DNA Artificial Sequence Description of Artificial SequenceSynthetic 63 ccatcaccaa caatcaccag tt 22 64 22 DNA Artificial Sequence Description of Artificial SequenceSynthetic 64 cctgagcaat ggctgccatc cg 22 65 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 65 gaaggtgaag gtcggagtc 19 66 20 DNA Homo sapiens Description of Artificial SequenceSynthetic 66 gaagatggtg atgggatttc 20 67 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 67 caagcttccc gttctcagcc 20 68 591 DNA Homo sapiens Description of Artificial SequenceSynthetic 68 atggcggcca tccgcaagaa gctggtggtg gtgggcgacg gcgcgtgtgg caagacgtgc 60 ctgctgatcg tgttcagtaa ggacgagttc cccgaggtgt acgtgcccac cgtcttcgag 120 aactatgtgg ccgacattga ggtggacggc aagcaggtgg agctggcgct gtgggacacg 180 gcgggccagg aggactacga ccgcctgcgg ccgctctcct acccggacac cgacgtcatt 240 ctcatgtgct tctcggtgga cagcccggac tcgctggaga acatccccga gaagtgggtc 300 cccgaggtga agcacttctg tcccaatgtg cccatcatcc tggtggccaa caaaaaagac 360 ctgcgcagcg acgagcatgt ccgcacagag ctggcccgca tgaagcagga acccgtgcgc 420 acggatgacg gccgcgccat ggccgtgcgc atccaagcct acgactacct cgagtgctct 480 gccaagacca aggaaggcgt gcgcgaggtc ttcgagacgg ccacgcgcgc cgcgctgcag 540 aagcgctacg gctcccagaa cggctgcatc aactgctgca aggtgctatg a 591 69 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 69 ccaccaccag cttcttgc 18 70 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 70 ccgtcgccca ccaccacc 18 71 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 71 gcacgtcttg ccacacgc 18 72 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 72 actgaacacg atcagcag 18 73 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 73 ttactgaaca cgatcagc 18 74 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 74 ccttactgaa cacgatca 18 75 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 75 gtccttactg aacacgat 18 76 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 76 ctcgtcctta ctgaacac 18 77 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 77 aactcgtcct tactgaac 18 78 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 78 catagttctc gaagacgg 18 79 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 79 tcggccacat agttctcg 18 80 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 80 ccgtccacct caatgtcg 18 81 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 81 aagcacatga gaatgacg 18 82 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 82 gagtccgggc tgtccacc 18 83 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 83 atgttctcca gcgagtcc 18 84 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 84 gggatgttct ccagcgag 18 85 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 85 gacatgctcg tcgctgcg 18 86 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 86 cggacatgct cgtcgctg 18 87 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 87 tgtgcggaca tgctcgtc 18 88 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 88 ctctgtgcgg acatgctc 18 89 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 89 ccagctctgt gcggacat 18 90 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 90 cgggccagct ctgtgcgg 18 91 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 91 tgcgggccag ctctgtgc 18 92 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 92 gttcctgctt catgcggg 18 93 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 93 acgggttcct gcttcatg 18 94 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 94 gtagtcgtag gcttggat 18 95 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 95 cgaggtagtc gtaggctt 18 96 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 96 gtcttggcag agcactcg 18 97 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 97 acctcgcgca cgccttcc 18 98 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 98 agacctcgcg cacgcctt 18 99 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 99 cgaagacctc gcgcacgc 18 100 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 100 ctcgaagacc tcgcgcac 18 101 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 101 gccgtctcga agacctcg 18 102 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 102 cgtggccgtc tcgaagac 18 103 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 103 gttctgggag ccgtagcg 18 104 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 104 gccgttctgg gagccgta 18 105 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 105 gatgcagccg ttctggga 18 106 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 106 gttgatgcag ccgttctg 18 107 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 107 cagcagttga tgcagccg 18 108 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 108 agcaccttgc agcagttg 18 109 1058 DNA Homo sapiens 109 gccttgactt catctcagct ccagagcccg ccctctcttc ctgcagcctg ggaacttcag 60 ccggctggag cccaccatgg ctgcaatccg aaagaagctg gtgatcgttg gggatggtgc 120 ctgtgggaag acctgcctcc tcatcgtctt cagcaaggat cagtttccgg aggtctacgt 180 ccctactgtc tttgagaact atattgcgga cattgaggtg gacggcaagc aggtggagct 240 ggctctgtgg gacgcggaca ttgaggtgga cggcaagcag gtggagctgg ctctgtggga 300 cgacactgat gtcatcctca tgtgcttctc catcgacagc cctgacagcc tggaaaacat 360 tcctgagaag tggaccccag aggtgaagca cttctgcccc aacgtgccca tcatcctggt 420 ggggaataag aaggacctga ggcaagacga gcacaccagg agagagctgg ccaagatgaa 480 gcaggagccc gttcggtctg aggaaggccg ggacatggcg aaccggatca gtgcctttgg 540 ctaccttgag tgctcagcca agaccaagga gggagtgcgg gaggtgtttg agatggccac 600 tcgggctggc ctccaggtcc gcaagaacaa gcgtcggagg ggctgtccca ttctctgaga 660 tccccccaaa gggccctttt cctacatgcc ccctcccttc acaggggtac agaaattatc 720 cccctacaac cccagcctcc tgagggctcc atactgaagg ctccattttc agttccctcc 780 tgcccaggac tgcattgttt tctagccccg aggtgtggca cgggccctcc ctcccagcgc 840 tctgggagcc acgcctatgc cctgcccttc ctcatgggcc cctggggatc ttgccccttt 900 gaccttcccc aaaggatggt cacacaccag cactttatac acttctggct cacaggaaag 960 tgtctgcagt agggacccag agtcccaggc ccctggagtt gtttctgcag gggccttgtc 1020 tctcactgca tttggtcagg ggggcatgaa taaaggct 1058 110 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 110 gagctgagat gaagtcaa 18 111 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 111 gctgaagttc ccaggctg 18 112 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 112 ccggctgaag ttcccagg 18 113 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 113 ggcaccatcc ccaacgat 18 114 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 114 aggcaccatc cccaacga 18 115 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 115 tcccacaggc accatccc 18 116 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 116 aggtcttccc acaggcac 18 117 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 117 atgaggaggc aggtcttc 18 118 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 118 ttgctgaaga cgatgagg 18 119 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 119 tcaaagacag tagggacg 18 120 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 120 ttctcaaaga cagtaggg 18 121 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 121 agttctcaaa gacagtag 18 122 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 122 tgttttccag gctgtcag 18 123 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 123 tcgtcttgcc tcaggtcc 18 124 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 124 gtgtgctcgt cttgcctc 18 125 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 125 ctcctggtgt gctcgtct 18 126 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 126 cagaccgaac gggctcct 18 127 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 127 ttcctcagac cgaacggg 18 128 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 128 actcaaggta gccaaagg 18 129 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 129 ctcccgcact ccctcctt 18 130 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 130 ctcaaacacc tcccgcac 18 131 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 131 ggccatctca aacacctc 18 132 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 132 cttgttcttg cggacctg 18 133 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 133 cccctccgac gcttgttc 18 134 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 134 gtatggagcc ctcaggag 18 135 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 135 gagccttcag tatggagc 18 136 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 136 gaaaatggag ccttcagt 18 137 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 137 ggaactgaaa atggagcc 18 138 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 138 ggagggaact gaaaatgg 18 139 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 139 gcaggaggga actgaaaa 18 140 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 140 agggcagggc ataggcgt 18 141 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 141 ggaagggcag ggcatagg 18 142 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 142 catgaggaag ggcagggc 18 143 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 143 taaagtgctg gtgtgtga 18 144 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 144 cctgtgagcc agaagtgt 18 145 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 145 ttcctgtgag ccagaagt 18 146 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 146 cactttcctg tgagccag 18 147 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 147 agacactttc ctgtgagc 18 148 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 148 actctgggtc cctactgc 18 149 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 149 tgcagaaaca actccagg 18 150 1284 DNA Homo sapiens 150 gcttctcgag cccggagccg ctgccgccgc ccccagctcc cccgcctcgg gaggggcacc 60 aggtcactgc agccagaggg gtccagaaga gagaggaggc actgcctcac tacagcaact 120 gcacccacga tgcagagcat caagtgcgtg gtggtgggtg atggggctgt gggcaagacg 180 tgcctgctca tctgctacac aactaacgct ttccccaaag agtacatccc caccgtgttc 240 gacaattaca gcgcgcagag cgcagttgac gggcgcacag tgaacctgaa cctgtgggac 300 actgcgggcc aggaggagta tgaccgcctc cgtacactct cctaccctca gaccaacgtt 360 ttcgtcatct gtttctccat tgccagtccg ccgtcctatg agaacgtgcg gcacaagtgg 420 catccagagg tgtgccacca ctgccctgat gtgcccatcc tgctggtggg caccaagaag 480 gacctgagag cccagcctga caccctacgg cgcctcaagg agcagagcca ggcgcccatc 540 acaccgcagc agggccaggc actcgcgaaa cagatccacg ctgtgcgcta cctcgaatgc 600 tcagccctgc aacaggatgg tgtcaaggaa gtgttcgccg aggctgtccg ggctgtgctc 660 aaccccacgc cgatcaagcg tgggcggtcc tgcatcctct tgtgaccctg gcacttggct 720 tggaggctgc ccctgccctc cccccaccag ttgtgccttg gtgccttgtc cgcctcagct 780 gtgccttaag gactaattct ggcacccctt tccaggggtt ccctgaatgc ctttttctct 840 gagtgccttt ttctccttaa ggaggcctgc agagaaaggg gctttgggct ctgcccctct 900 ggcttgggaa cactgggtat tctcatgagc tcatccaagc caaggttgga cccctcccca 960 agaggccaac ccagtgcccc ctcccatttt ccgctactga ccagttcatc cagctttcca 1020 cacagttgtt gctgcctatt gtggtgccgc ctcaggttag gggctctcag ccatctctaa 1080 cctctgccct cgctgctctt ggaattgcgc ccccaagatg ctctctccct tctccaatga 1140 gggagccaca gaatcctgag aaggtgaatg taccctaacc tgctcctctg tgcctaggcc 1200 ttacgcattt gctgactgac tcagccccca tgcttctggg gacctttcct acccccatca 1260 gcatcaataa aacctcctgt ctcc 1284 151 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 151 gacctggtgc ccctcccg 18 152 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 152 tcttctggac ccctctgg 18 153 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 153 ggcagtgcct cctctctc 18 154 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 154 gtgcagttgc tgtagtga 18 155 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 155 gcatcgtggg tgcagttg 18 156 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 156 ccaccacgca cttgatgc 18 157 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 157 ttgtgtagca gatgagca 18 158 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 158 aaagcgttag ttgtgtag 18 159 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 159 gcgcgctgta attgtcga 18 160 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 160 ggttcactgt gcgcccgt 18 161 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 161 gtcccacagg ttcaggtt 18 162 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 162 tgtacggagg cggtcata 18 163 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 163 acgttggtct gagggtag 18 164 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 164 caatggagaa acagatga 18 165 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 165 cataggacgg cggactgg 18 166 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 166 cgcacgttct cataggac 18 167 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 167 acctctggat gccacttg 18 168 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 168 agggcagtgg tggcacac 18 169 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 169 cagcaggatg ggcacatc 18 170 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 170 gggtgtcagg ctgggctc 18 171 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 171 ccctgctgcg gtgtgatg 18 172 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 172 cgcgagtgcc tggccctg 18 173 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 173 gtagcgcaca gcgtggat 18 174 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 174 cattcgaggt agcgcaca 18 175 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 175 acaccatcct gttgcagg 18 176 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 176 gaacacttcc ttgacacc 18 177 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 177 acagcctcgg cgaacact 18 178 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 178 aagaggatgc aggaccgc 18 179 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 179 gcagcctcca agccaagt 18 180 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 180 aaaaggcatt cagggaac 18 181 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 181 gggtccaacc ttggcttg 18 182 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 182 gtcagtagcg gaaaatgg 18 183 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 183 agctggatga actggtca 18 184 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 184 aactgtgtgg aaagctgg 18 185 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 185 accacaatag gcagcaac 18 186 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 186 gagggcagag gttagaga 18 187 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 187 caattccaag agcagcga 18 188 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 188 tggagaaggg agagagca 18 189 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 189 acattcacct tctcagga 18 190 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 190 gtcagcaaat gcgtaagg 18 191 579 DNA Artificial Sequence Description of Artificial SequenceSynthetic 191 atgcaggcca tcaagtgtgt ggtggtggga gacggagctg taggtaaaac ttgcctactg 60 atcagttaca caaccaatgc atttcctgga gaatatatcc ctactgtctt tgacaattat 120 tctgccaatg ttatggtaga tggaaaaccg gtgaatctgg gcttatggga tacagctgga 180 caagaagatt atgacagatt acgcccccta tcctatccgc aaacagatgt gttcttaatt 240 tgcttttccc ttgtgagtcc tgcatcattt gaaaatgtcc gtgcaaagtg gtatcctgag 300 gtgcggcacc actgtcccaa cactcccatc atcctagtgg gaactaaact tgatcttagg 360 gatgataaag acacgatcga gaaactgaag gagaagaagc tgactcccat cacctatccg 420 cagggtctag ccatggctaa ggagattggt gctgtaaaat acctggagtg ctcggcgctc 480 acacagcgag gcctcaagac agtgtttgac gaagcgatcc gagcagtcct ctgcccgcct 540 cccgtgaaga agaggaagag aaaatgcctg ctgttgtaa 579 192 45 DNA Artificial Sequence Description of Artificial SequenceSynthetic 192 atagaatgtg agtctgaact cttacattta gaacaaacaa aacct 45 193 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 193 caaatgatgc aggactcaca 20 194 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 194 caccaccaca cacttgatg 19 195 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 195 ataagcccag attcaccg 18 196 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 196 tgtttgcgga taggatagg 19 197 22 DNA Artificial Sequence Description of Artificial SequenceSynthetic 197 gcttcttctc cttcagtttc tc 22 198 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 198 cagcaccaat ctccttagc 19 199 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic 199 ctcttcctct tcttcacgg 19 200 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 200 cctaagatca agtttagttc 20 201 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 201 cgcacctcag gataccactt 20 202 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 202 atctaccata acattggcag 20 203 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 203 taattgtcaa agacagtagg 20 204 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 204 gagcgccgag cactccaggt 20 205 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 205 gtcaaacact gtcttgaggc 20 206 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 206 atagaatgtg agtctgaact 20 207 25 DNA Artificial Sequence Description of Artificial SequenceSynthetic 207 cttacattta gaacaaacaa aacct 25 208 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 208 cccagctaag aattccgctc 20 209 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 209 taaacgccga atctacgc 18 210 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 210 ataagccctg attcaccg 18 211 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 211 atacgccctg attcaccg 18 212 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 212 atacgccctg attaaccg 18 213 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic 213 taaacgccga atctacgc 18 214 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 214 aaactgctga agtacgcaca 20 215 1175 DNA Artificial Sequence Description of Artificial SequenceSynthetic 215 ccccggtgga gaagctgagg tcatcatcag atttgaaata tttaaagtgg atacaaaatt 60 atttcagcaa tgcagacaat taagtgtgtt gttgtgggcg atggtgctgt tggtaaaaca 120 tgtctcctga tatcctacac aacaaacaaa tttccatcgg aatatgtacc gactgttttt 180 gacaactatg cagtcacagt tatgattggt ggagaaccat atactcttgg actttttgat 240 actgcagggc aagaggatta tgacagatta cgaccgctga gttatccaca aacagatgta 300 tttctagtct gtttttcagt ggtctctcca tcttcatttg aaaacgtgaa agaaaagtgg 360 gtgcctgaga taactcacca ctgtccaaag actcctttct tgcttgttgg gactcaaatt 420 gatctcagag atgacccctc tactattgag aaacttgcca agaacaaaca gaagcctatc 480 actccagaga ctgctgaaaa gctggcccgt gacctgaagg ctgtcaagta tgtggagtgt 540 tctgcactta cacagaaagg cctaaagaat gtatttgacg aagcaatatt ggctgccctg 600 gagcctccag aaccgaagaa gagccgcagg tgtgtgctgc tatgaacatc tctccagagc 660 cctttctgca cagctggtgt cggcatcata ctaaaagcaa tgtttaaatc aaactaaaga 720 ttaaaaatta aaattcgttt ttgcaataat gacaaatgcc ctgcacctac ccacatgcac 780 tcgtgtgaga caaggcccat aggtatggcc ccccccttcc ccctcccagt actagttaat 840 tttgagtaat tgtattgtca gaaaagtgat tagtactatt tttttttgtt gtttcaaaaa 900 aaaaattttt gtgtgtctgt tttttttttt tttttttttt gttgtttaaa aggaaggcat 960 gcttgtggat gactctgtaa cagactaatt ggaattgttg aagctgctcc ctggttccac 1020 tctggagagt aatctgggac atcttagtgt tttgttttgt ttttttccct cctctttttt 1080 ttggggggga gtgtgtgggg ggtttgtttt ttagtcttgt ttttttaatt cattaaccag 1140 tggttaagcc cttaagggag gaggacggat tgatt 1175 216 25 DNA Artificial Sequence Description of Artificial SequenceSynthetic 216 ttcagcaatg cagacaatta agtgt 25 217 27 DNA Artificial Sequence Description of Artificial SequenceSynthetic 217 tgttgtgtag gatatcagga gacatgt 27 218 24 DNA Artificial Sequence Description of Artificial SequenceSynthetic 218 ttgtgggcga tggtgctgtt ggta 24 219 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 219 taattgtctg cattgctgaa 20 220 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 220 ttaccaacag caccatcgcc 20 221 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 221 ccaccaatca taactgtgac 20 222 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 222 gtggataact cagcggtcgt 20 223 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 223 gaagatggag agaccactga 20 224 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 224 gtgagttatc tcaggcaccc 20 225 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 225 gcttctgttt gttcttggca 20 226 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 226 tgacagcctt caggtcacgg 20 227 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 227 cacctgcggc tcttcttcgg 20 228 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 228 ttgtctcaca cgagtgcatg 20 229 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 229 ttctgacaat acaattactc 20 230 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 230 ttacagagtc atccacaagc 20 231 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 231 cgatagtctc cacgtgaggc 20 232 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 232 gtaacgctcc tatggccagg 20 233 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 233 agactgactg ctcgtcgcga 20

Claims

What is claimed is:

1. An antisense compound targeted to a nucleic acid molecule encoding a member of the human Rho family of small GTP binding proteins, wherein said antisense compound inhibits the expression of said member of the human Rho family.

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

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

4. The antisense compound of claim 3 wherein the modified internucleoside linkage is a phosphorothioate linkage.

5. The antisense compound of claim 2 wherein the oligonucleotide comprises at least one modified sugar moiety.

6. The antisense compound of claim 5 wherein the modified sugar moiety is a 2′-O-methoxyethyl sugar moiety.

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

8. The antisense compound of claim 7 wherein the modified nucleobase is a 5-methylcytosine.

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

10. The antisense compound of claim 1 which is from 8 to 30 nucleobases in length.

11. The antisense compound of claim 1 wherein said member of the human Rho family of small GTP binding proteins is RhoA, RhoB, RhoC, or RhoG.

12. The antisense compound of claim 1 wherein said member of the human Rho family of small GTP binding protein is rac1.

13. The antisense compound of claim 12 wherein the oligonucleotide comprises SEQ ID NO: 193, 195, 199, 201, 204 or 205.

14. The antisense compound of claim 1 wherein said member of the human Rho family of small GTP binding protein is cdc42.

15. The antisense compound of claim 14 wherein the oligonucleotide comprises SEQ ID NO: 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229 or 230.

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

17. The pharmaceutical composition of claim 16 further comprising a colloidal dispersion system.

18. The pharmaceutical composition of claim 16 wherein the antisense compound is an antisense oligonucleotide.

19. A method of inhibiting the expression of a member of the human Rho family of small GTP binding proteins in human cells or tissues comprising contacting said cells or tissues with the antisense compound of claim 1 so that expression of said human Rho family member is inhibited.

20. A method of treating a human having a disease or condition associated with a member of the human Rho family of small GTP binding proteins comprising administering to said animal a therapeutically or prophylactically effective amount of the antisense compound of claim 1 so that expression of said human Rho family member is inhibited.

21. The method of claim 20 wherein the disease or condition is a hyperproliferative condition.

22. The method of claim 21 wherein the hyperproliferative condition is cancer.

23. The method of claim 20 wherein the disease or condition is abnormal wound healing or clotting.

24. The method of claim 20 wherein the disease or condition is ischemia/reperfusion or reoxygenation injury.

25. A compound which inhibits JNK activation by a non-cytokine activator but does not inhibit JNK activation by a cytokine.

26. The compound of claim 25 wherein the non-cytokine activator is a stress signal.

27. The compound of claim 26 wherein the stress signal is hydrogen peroxide or ultraviolet radiation.

28. The compound of claim 25 wherein the cytokine is IL-1β.

29. The compound of claim 25 which is an inhibitor of rhoA.

30. The compound of claim 29 which is an antisense inhibitor of rhoA.

31. A method of inhibiting JNK activation by a non-cytokine activator without inhibiting JNK activation by a cytokine comprising contacting JNK or cells or tissues containing JNK with an inhibitor of rhoA.

32. The method of claim 31 wherein the non-cytokine activator is a stress signal.

33. The method of claim 32 wherein the stress signal is hydrogen peroxide or ultraviolet radiation.

34. The method of claim 31 wherein the cytokine is IL-1β.

35. The method of claim 31 wherein the Inhibitor of rhoA is an antisense oligonucleotide inhibitor of rhoA.