WO2007064853A2 - Locked nucleic acid oligonucleotides - Google Patents

Abstract

The present invention provides oligonucleotides and compositions thereof for the treatment and prevention of cancer, wherein said oligonucleotides comprise locked nucleic acid nucleotides.

Description

LOCKED NUCLEIC ACID OLIGONUCLEOTIDES

[001] This application claims the benefit of the U.S. Provisional application No. 60/741,228, filed on December 1, 2005 and the U.S. Provisional application No. 60/778,488, filed on March 2, 2006, both of which are herein incorporated by reference.

FIELD OF THE INVENTION

[002] The present invention relates to methods and compositions for cancer therapy. In particular, the present invention provides oligonucleotides for inhibition of oncogene expression.

BACKGROUND OF THE INVENTION

[003] Oncogenes have become the central concept in understanding cancer biology and may provide valuable targets for therapeutic drugs. In many types of human tumors, including lymphomas and leukemias, oncogenes are overexpressed, and may be associated with tumorigenicity (Tsujimoto et al., Science 228:1440-1443 [1985]). For instance, high levels of expression of the human bcl-2 gene have been found in all lymphomas with a t(14; 18) chromosomal translocations including most follicular B cell lymphomas and many large cell non-Hodgkin's lymphomas. High levels of bcl-2 gene expression have also been found in certain leukemias that do not have a t(14; 18) chromosomal translation, including most cases of chronic lymphocytic leukemia acute, many lymphocytic leukemias of the pre-B cell type, neuroblastomas, nasophryngeal carcinomas, and many adenocarcinomas of the prostate, breast and colon. (Reed et al., Cancer Res. 51 :6529 [1991]; Yunis et al., New England J. Med. 320:1047; Campos et al., Blood 81:3091-3096 [1993]; McDonnell et al., Cancer Res. 52:6940- 6944 [1992); Lu et al., Int. J Cancer 53:29-35 [1993]; Bonner et al., Lab Invest. 68:43A [1993]. Other oncogenes include TGF-α, c-ki-ras, c-Ha-ras, Her-2, and c-myc. [004] Gene expression, including oncogene expression, can be inhibited by molecules that interfere with promoter function. Accordingly, the expression of oncogenes may be inhibited by single stranded oligonucleotides. Nucleic acid therapeutics, however, often lack therapeutic efficacy due to instability in body fluids or inefficient uptake into cells. Thus, there is a need for stable and effective oligonucleotides for the treatment of cancer. SUMMARY OF THE INVENTION

[005] The present invention relates to methods and compositions for treating cancer. In particular, the present invention provides oligomeric compounds and methods for modulating the expression of certain genes such as, for example, the Bcl-2 gene, in humans. In one aspect, the invention relates to oligomeric compounds of about 10 to about 60 nucleotides in length j which comprise a target binding domain which incorporates at least two locked nucleic acid (LNA) nucleotides or LNA analogue (LNA*) nucleotides. In some embodiments, the oligonucleotide is SEQ. ID. No. 1251. In other embodiments, the oligonucleotide is selected form SEQ ID NOs 2-281, 283-461, 463-935, 937-1080, 1082-1248, 1250-1254 and 1270-1477 and the complements thereof. In another aspect, the oligonucleotides is selected from SEQ ID NOs 2-22, 283-301, 463-503, 937-958, 1082-1109, 1250-1254 and 1270-1477 and the complements thereof.

[006] The present invention further relates to pharmaceutical compositions comprising such oligomeric compounds, uses thereof and methods of treatment and diagnosis utilizing such oligomeric compounds.

[007] In one aspect, the invention includes an oligonucleotide compound that hybridizes to SEQ. ID. NO:1249 or 1254, wherein the oligonucleotide comprises from about 10 to about 60 nucleotides in which one or more nucleotides are LNA or LNA* nucleotides. Embodiments of this aspect include one or more of the following features. The oligonucleotide is comprised of 15 to 40 nucleotides wherein one or more nucleotides are LNA or LNA* nucleotides. The oligonucleotide is comprised of 20 to 30 nucleotides wherein one or more nucleotides are LNA or LNA* nucleotides. The oligonucleotide includes 10 to 15 nucleotides wherein the number of LNA or LNA* nucleotides is from 1 to 15. The oligonucleotide includes 15 to 40 nucleotides wherein the number of LNA or LNA* nucleotides is from 1 to 16. The oligonucleotide includes 20 to 30 nucleotides wherein the number of LNA or LNA* nucleotides is from 1 to 12. The oligonucleotide, has up to 10 LNA or LNA* sequential nucleotides. An oligonucleotide wherein the LNA and LNA* nucleotides are separated by one or more DNA or RNA nucleotides. The oligonucleotide includes from 1 to 3 sequential LNA and LNA* nucleotides. The oligonucleotide includes 15 to 40 nucleotides wherein the number of LNA or LNA* nucleotides is 4. The oligonucleotide includes 15 to 40 nucleotides wherein the oligomer includes 2 pair of sequential LNA or LNA* nucleotides. The oligonucleotide includes 15 to 40 nucleotides wherein one pair is at the 3' terminus. The oligonucleotide includes 15 to 40 nucleotides wherein one pair is at the 5' terminus. The oligonucleotide ϊlfS^Qffl'ή^Φ©⁸ wherein one pair is at the 3' terminus and the other pair is at the ' terminus. The oligonucleotide includes at least one LNA of Formula 1

i wherein

B constitutes a nucleobase;

Z* is selected from an internucleoside linkage and a terminal group;

Z is selected from a bond to the internucleoside linkage of a preceding nucleotide/nucleoside and a terminal group, provided that only one of Z and Z* can be a terminal group.

The oligonucleotide includes at least one LNA* of Formula 2

2 wherein

B constitutes a nucleobase;

Z* is selected from an internucleoside linkage and a terminal group;

Z is selected from a bond to the internucleoside linkage of a preceding nucleotide/nucleoside and a terminal group, provided that only one of Z and Z* can be a terminal group;

X and Y are independently selected from -O-, -S-, -N(H)-, -N(R)-, -CH₂- or -C(H)=, -CH₂-

O-, -CH₂-S-, -CH₂-N(H)-, -CH₂-N(R)-, -CH₂-CH₂- or -CH₂-C(H)=, -CH=CH- ; provided that X and Y are not both O.

R is selected from hydrogen and C_1-4-alkyl.

[008] In an embodiment, the oligonucleotide hybridizes to nucleotides 500-2026, 500-1525, 800-1225, 900-1125, 950-1075 or 970-1045 of SEQ ID NO:1249. In yet another embodiment, fφeigliiμjpfle.Θtϊ.φfpaiibj^gplected from SEQ ID NOs: 1250-1253, and 1267-1477, and in still another embodiment, the oligonucleotide is SEQ ID NO: 1251.

[009] In another aspect, the invention includes an oligonucleotide compound that hybridizes to SEQ. ID. NO:936, wherein the oligonucleotide comprises from about 10 to about 60 nucleotides in which one or more nucleotides are LNA or LNA* nucleotides. Embodiments of this aspect include one or more of the following features. The oligonucleotide is comprised of an oligonucleotide that hybridizes with nucleotides 1-1000 of SEQ ID NO:936 and the oligonucleotide can comprise SEQ ID NO:940 or SEQ ID NO:943. [010] In an additional embodiment, the oligonucleotide further comprises another oligonucleotide. The second oligonucleotide can be selected from SEQ ID NOs: 1250-1253, 1267-1477, 2-281, 283-461, 463-935, 937-1080, and 1082-1248.

[011] In yet another aspect, the present inventions provides a pharmaceutical composition comprising an oligonucleotide compound that hybridizes to SEQ. ID. NO:1249, 1254 or 936, wherein the oligonucleotide comprises from about 10 to about 60 nucleotides in which one or more nucleotides are LNA or LNA* nucleotides.

[012] In still further aspects, the present invention provides a method, comprising administering a pharmaceutical composition comprising an oligonucleotide compound that hybridizes to SEQ. ID. NO: 1249, 1254 or 936, wherein the oligonucleotide comprises from about 10 to about 60 nucleotides in which one or more nucleotides are LNA or LNA* nucleotides to a cell or an animal. The administration of the composition can result in a reduction of tumor burden in an animal having a tumor, decrease in the growth rate of a tumor, or other results. In some embodiments, the animal is a non-human mammal. In other embodiments, the animal is a human. In some embodiments, the pharmaceutical composition is introduced to the host animal one or more times per day. In other embodiments, the pharmaceutical composition is introduced to the host animal continuously. In some embodiments, the method further comprises the step of administering a chemotherapeutic agent (e.g., docetaxel, Taxol, or Vincristine) to the subject. In one embodiment, the chemotherapeutic agent is not formulated with the pharmaceutical composition. In another embodiment, the chemotherapeutic agent is formulated with the pharmaceutical composition comprising the oligonucleotide.

BRIEF DESCRIPTION OF THE DRAWING

[013] Figure 1 shows inhibition of PC-3 cell proliferation by LNA-PNT-100 (SEQ ID NO: 1251 and Thio-PNT-100. DESCRIPTION OF SEQUENCE LISTING

[014] SEQ ID NO: 1 c-erb-2 (her-2) upstream region

[015] SEQ ID NOs:2-281 c-erb-2 (her-2) oligonucleotides

[016] SEQ ID NO:282 c-ki-ras upstream region

[017] SEQ ID NOs:283-461 c-ki-ras oligonucleotides

[018] SEQ ID NO:462 c-Ha-ras upstream region

[019] SEQ ID NOs:463-935 c-Ha-ras oligonucleotides

[020] SEQ ID NO:936 c-myc upstream region

[021] SEQ ID NOs:937-1080 c-myc oligonucleotides

[022] SEQ ID NO: 1081 TGF-α upstream region

[023] SEQ ID NOs:1082-1248 TGF-α oligonucleotides

[024] SEQ ID NO: 1249 bcl-2 upstream region

[025] SEQ ID NO: 1250 PNT- 100 oligonucleotide methylated

[026] SEQ ID NO: 1251 PNT- 100 oligonucleotide not methylated

[027] SEQ ID NO: 1252 bcl-2 oligonucleotide methylated

[028] SEQ ID NO : 1253 bcl-2 oligonucleotide not methylated

[029] SEQ ID NO: 1254 bcl-2 secondary promoter sequence

[030] SEQ ID NOs: 1255-1266 bcl-2 sequences

[031] SEQ ID NOs: 1250-1254 bcl-2 oligonucleotides and 1267-1477

[032] SEQ ID NOs: 1448-1461 bcl-2 control oligonucleotides

DEFINITIONS

[033] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

[034] As used herein, the term "LNA" (Locked Nucleic Acid) (or "LNA oligonucleotide") refers to an oligonucleotide containing one or more bicyclic, tricyclic or polycyclic nucleoside analogues also referred to as LNA nucleotides and LNA analogue nucleotides.

[035] As used herein, the term "under conditions such that expression of said gene is inhibited" refers to conditions in which an oligonucleotide of the present invention hybridizes to a gene (e.g., a regulatory region of the gene) and inhibits transcription of the gene by at least

10%, at least 25%, at least 50%, or at least 90% relative to the level of transcription in the absence of the oligonucleotide. The present invention is not limited to the inhibition of

Exemplary genes include, but are not limited to, c-ki-Ras, c- Ha-ras, c-myc, her-2, TGF-α, and bcl-2.

[036] As used herein, the term "under conditions such that growth of said cell is reduced" refers to conditions where an oligonucleotide of the present invention, when administered to a cell (e.g., a cancer) reduces the rate of growth of the cell by at least 10%, at least 25%, even at least 50%, and at least 90% relative to the rate of growth of the cell in the absence of the oligonucleotide.

[037] As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[038] As used herein, the term "non-human animals" refers to all non-human animals including, but are not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc. and non-vertebrate animals such as drosophila and nematode.

[039] As used herein, the term "nucleic acid molecule" refers to any nucleic acid containing molecule, including but not limited to, DNA or RNA. The term encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxyl-methyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminornethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2- thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine. [040] The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full-length or fragjfiep£m yetafiϊgdyfilhiiterm also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. [041] As used herein, the term "heterologous gene" refers to a gene that is not in its natural environment. For example, a heterologous gene includes a gene from one species introduced into another species. A heterologous gene also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to non-native regulatory sequences, etc). Heterologous genes are distinguished from endogenous genes in that the heterologous gene sequences are typically joined to DNA sequences that are not found naturally associated with the gene sequences in the chromosome or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).

[042] As used herein, the "regulatory region" of a gene is any part of a gene that regulates the expression of a gene, including, without limitation, transcriptional and translational regulation. The regions include without limitation the 5' and 3' regions of genes, binding sites for regulatory factors, including without limitation transcription factor binding sites. The regions also include regions that are as long as 20,000 or more base pairs upstream or downstream of translational start sites, so long as the region is involved in any way in the regulation of the expression of the gene. The region may be as short as 20 base pairs or as long as thousands of base pairs. '

[043] As used herein, the term "gene expression" refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of the gene (i.e., via the enzymatic action of an RNA polymerase), and for protein encoding genes, into protein through "translation" of mRNA. Gene expression can be fregfj^tf <|y||naϊi|,®.gøs.|]||he process. "Up-regulation" or "activation" refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while "down- regulation" or "repression" refers to regulation that decrease production. Molecules (e.g., transcription factors) that are involved in up-regulation or down-regulation are often called "activators" and "repressors," respectively.

[044] In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3¹ end of the sequences that are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct the termination of transcription, post-transcriptional cleavage and polyadenylation. [045] The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene. In contrast, the term "modified" or "mutant" refers to a gene or gene product that displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild- type gene or gene product. It is noted that naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics (including altered nucleic acid sequences) when compared to the wild-type gene or gene product. [046] As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.

[047] As used herein, the terms "an oligonucleotide having a nucleotide sequence encoding a gene" and "polynucleotide having a nucleotide sequence encoding a gene," means a nucleic acid sequence comprising the coding region of a gene or in other words the nucleic acid sequence that encodes a gene product. The coding region may be present in a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide or polynucleotide may be single-stranded (i.e., the sense strand) or double-stranded. Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Alternatively, the coding region i utilised ip |he exprέSss|dή;-f©ctors of the present invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.

[048] As used herein, the term "oligonucleotide," refers to a short length of single-stranded polynucleotide chain. Oligonucleotides are typically less than 200 residues long (e.g., between 8 and 100), however, as used herein, the term is also intended to encompass longer polynucleotide chains (e.g., as large as 5000 residues). Oligonucleotides are often referred to by their length. For example a 24 residue oligonucleotide is referred to as a "24-mer". Oligonucleotides can form secondary and tertiary structures by self-hybridizing or by hybridizing to other polynucleotides. Such structures can include, but are not limited to, duplexes, hairpins, cruciforms, bends, and triplexes.

[049] In some embodiments, oligonucleotides are "antigenes." As used herein, the term "antigene" refers to an oligonucleotide that hybridizes to a regulatory region of a gene. In some embodiments, the hybridization of the antigene to a regulatory region inhibits expression of the gene.

[050] As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence "A-G-T," is complementary to the sequence "T-C-A." Complementarity maybe "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids.

[051] As used herein, the term "completely complementary," for example when used in reference to an oligonucleotide of the present invention refers to an oligonucleotide where all of the nucleotides are complementary to a target sequence (e.g., a gene). [052] As used herein, the term "partially complementary," for example when used in reference to an oligonucleotide of the present invention, refers to an oligonucleotide where at least one nucleotide is not complementary to the target sequence. Some partially complementary oligonucleotides are those that can still hybridize to the target sequence under physiological conditions. The term "partially complementary" refers to oligonucleotides that have regions of one or more non-complementary nucleotides both internal to the

o^lfilf irøii^έ'^iSI/fitMiend. Oligonucleotides with mismatches at the ends may still hybridize to the target sequence.

[053] The term "homology" refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence is a nucleic acid molecule that at least partially inhibits a completely complementary nucleic acid molecule from hybridizing to a target nucleic acid is "substantially homologous." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous nucleic acid molecule to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target that is substantially non-complementary (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non- complementary target.

[054] When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term "substantially homologous" refers to any probe that can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.

[055] A gene may produce multiple RNA species that are generated by differential splicing of the primary RNA transcript. cDNAs that are splice variants of the same gene will contain regions of sequence identity or complete homology (representing the presence of the same exon or portion of the same exon on both cDNAs) and regions of complete non-identity (for example, representing the presence of exon "A" on cDNA 1 wherein cDNA 2 contains exon "B" instead). Because the two cDNAs contain regions of sequence identity they will both hybridize to a probe derived from the entire gene or portions of the gene containing sequences found on both cDNAs; the two splice variants are therefore substantially homologous to such a probe and to each other.

[056] When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" refers to any probe that can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.

"hybridization" is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the T_m of the formed hybrid, and the G:C ratio within the nucleic acids. A single molecule that contains pairing of complementary nucleic acids within its structure is said to be "self-hybridized." [058] As used herein, the term "T_m" is used in reference to the "melting temperature." The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the T_m of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of the T_m value may be calculated by the equation: T_m = 81.5 + 0.41(% G + C), when a nucleic acid is in aqueous solution at 1 M NaCl (See e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization [1985]). Other references include more sophisticated computations that take structural as well as sequence characteristics into account for the calculation of T_m.

[059] As used herein the term "stringency" is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. Under "low stringency conditions" a nucleic acid sequence of interest will hybridize to its exact complement, sequences with single base mismatches, closely related sequences (e.g., sequences with 90% or greater homology), and sequences having only partial homology (e.g., sequences with 50-90% homology). Under "medium stringency conditions," a nucleic acid sequence of interest will hybridize only to its exact complement, sequences with single base mismatches, and closely related sequences (e.g., 90% or greater homology). Under "high stringency conditions," a nucleic acid sequence of interest will hybridize only to its exact complement, and (depending on conditions such as temperature) sequences with single base mismatches. In other words, under conditions of high stringency the temperature can be raised so as to exclude hybridization to sequences with single base mismatches.

[060] "High stringency conditions" when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH₂PO₄ H₂O and 1.85 g/1 EDTA, pH adjusted to 7.4 with

NaOH), 0.5% SDS, 5X Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA fpltpMMM^Wg^$~$®teύon comprising 0.1X SSPE, 1.0%-SDS at 42⁰C when a probe of about 500 nucleotides in length is employed.

[061] "Medium stringency conditions" when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH₂PC^ H₂O and 1.85 g/1 EDTA, pH adjusted to 7.4 with

NaOH), 0.5% SDS, 5X Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising l.OX SSPE, 1.0% SDS at 42⁰C when a probe of about 500 nucleotides in length is employed.

[062] "Low stringency conditions" comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH₂Pθ4

H₂O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reagent

[5OX Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE, 0.1% SDS at 42°C when a probe of about 500 nucleotides in length is employed.

[063] The present invention is not limited to the hybridization of probes of about 500 nucleotides in length. The present invention contemplates the use of probes between approximately 8 nucleotides up to several thousand (e.g., at least 5000) nucleotides in length. It will be recognized that stringency conditions maybe altered for probes of other sizes (See e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization [1985] and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY [1989]).

[064] It is known in the art that numerous equivalent conditions may be employed to comprise low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions. In addition, the art knows conditions that promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.) (see definition above for "stringency").

[065] As used herein, the term "physiological conditions" refers to specific stringency conditions that approximate or are conditions inside an animal (e.g., a human). Exemplary

use in vitro include, but are not limited to, 37°C, 95% air, 5% CO₂, commercial medium for culture of mammalian cells (e.g., DMEM media available from Gibco, MD), 5-10% serum (e.g., calf serum or horse serum), additional buffers, and optionally hormone (e.g., insulin and epideπnal growth factor).

[066] The term "isolated" when used in relation to a nucleic acid, as in "an isolated oligonucleotide" or "isolated polynucleotide" refers to a nucleic acid sequence that is identified and separated from at least one component or contaminant with which it is ordinarily associated in its natural source. Isolated nucleic acid is such present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids as nucleic acids such as DNA and RNA found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. However, isolated nucleic acid encoding a given protein includes, by way of example, such nucleic acid in cells ordinarily expressing the given protein where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid, oligonucleotide, or polynucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid, oligonucleotide or polynucleotide is to be utilized to express a protein, the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide or polynucleotide may be single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide or polynucleotide may be double-stranded).

[067] As used herein, the term "purified" or "to purify" refers to the removal of components (e.g., contaminants) from a sample. For example, antibodies are purified by removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulin that does not bind to the target molecule. The removal of non- immunoglobulin proteins and/or the removal of immunoglobulins that do not bind to the target molecule results in an increase in the percent of target-reactive immunoglobulins in the sample. In another example, recombinant polypeptides are expressed in bacterial host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample.

[068] The term "epitope" as used herein refers to that portion of an antigen that makes contact with a particular antibody. fϊP^ξlSrø-i&^ftcS^iSδfrfiiagment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as "antigenic determinants". An antigenic determinant may, compete with the intact antigen (i.e., the "immunogen" used to elicit the immune response) for binding to an antibody. [070] The term "Western blot" refers to the analysis of protein(s) (or polypeptides) immobilized onto a support such as nitrocellulose or a membrane. The proteins are run on acrylamide gels to separate the proteins, followed by transfer of the protein from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized proteins are then exposed to antibodies with reactivity against an antigen of interest. The binding of the antibodies may be detected by various methods, including the use of radiolabeled antibodies. [071] As used herein, the term "cell culture" refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro.

[072] As used, the term "eukaryote" refers to organisms distinguishable from "prokaryotes." It is intended that the term encompass all organisms with cells that exhibit the usual characteristics of eukaryotes, such as the presence of a true nucleus bounded by a nuclear membrane, within which lie the chromosomes, the presence of membrane-bound organelles, and other characteristics commonly observed in eukaryotic organisms. Thus, the term includes, but is not limited to such organisms as fungi, protozoa, and animals (e.g., humans). [073] As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell culture. The term "in vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.

[074] The terms "test compound" and "candidate compound" refer to any chemical entity, pharmaceutical, drug, and the like that is a candidate for use to treat or prevent a disease, illness, sickness, or disorder of bodily function (e.g., cancer). Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present invention. [075] As used herein, the term "known chemotherapeutic agents" refers to compounds known to be useful in the treatment of disease (e.g., cancer). Exemplary chemotherapeutic agents affective against cancer include, but are not limited to, daunorubicin, dactinomycin, 'dόllig^ifffl^jbleiillyclinl-llitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin and diethylstilbestrol (DES).

[076] As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Environmental samples include environmental material such as surface matter, soil, water, crystals and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present invention. [077] For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001.

[078] As used herein the term "aliphatic' encompasses the terms alkyl, alkenyl, alkynyl, each of which being optionally substituted as set forth below.

[079] As used herein, an "alkyl" group refers to a saturated aliphatic hydrocarbon group containing 1-8 (e.g., 1-6 or 1-4) carbon atoms. An alkyl group can be straight or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-heptyl or 2-ethylhexyl. An alkyl group can be substituted (i.e., optionally substituted) with one or more substituents such as halo, cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, alkoxy, aroyl, heteroaroyl, cycloaliphaticcarbonyl, (heterocycloaliphatic)carbonyl, nitro, cyano, amino, amido, acyl, sulfonyl, sulfϊnyl, sulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, carboxy, carbamoyl, cycloaliphaticoxy, heterocycloaliphaticoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroarylalkoxy, or hydroxy. Without limitation, some examples of substituted alkyls include carboxyalkyl (such as HOOC-alkyl, alkoxycarbonylalkyl and alkylcarbonyloxyalkyl), cyanoalkyl, hydroxyalkyl, alkoxyalkyl, acylalkyl, hydroxyalkyl, aralkyl, (alkoxyaryl)alkyl, (sulfonylamino)alkyl (such as (alkylsulfonylamino)alkyl), aminoalkyl, amidoalkyl, (cycloaliρhatic)alkyl, cyanoalkyl, or haloalkyl. [

group refers to an aliphatic carbon group that contains 2-8 (e.g., 2-6 or 2-4) carbon atoms and at least one double bond. Like an alkyl group, an alkenyl group can be straight or branched. Examples of an alkenyl group include, but are not limited to, allyl, isoprenyl, 2-butenyl and 2-hexenyl. An alkenyl group can be optionally substituted with one or more substituents such as halo, cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, alkoxy, aroyl, heteroaroyl, (cycloaliphatic)carbonyl, (heterocycloaliphatic)carbonyl, nitro, cyano, amino, amido, acyl, sulfonyl, sulfinyl, sulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, carboxy, carbamoyl, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy, aryloxy, heteroaryloxy, aralkyloxy, (heteroaryl)alkoxy, or hydroxy.

[081] As used herein, an "alkynyl" group refers to an aliphatic carbon group that contains 2-8 (e.g., 2-6 or 2-4) carbon atoms and has at least one triple bond. An alkynyl group can be straight or branched. Examples of an alkynyl group include, but are not limited to, propargyl and butynyl. An alkynyl group can be optionally substituted with one or more substituents such as halo, cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, alkoxy, aroyl, heteroaroyl, (cycloaliphatic)carbonyl, (heterocycloaliphatic)carbonyl, nitro, cyano, amino, amido, acyl, sulfonyl, sulfinyl, sulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, carboxy, carbamoyl, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy, aryloxy, heteroaryloxy, aralkyloxy, (heteroaryl)alkoxy, or hydroxy.

[082] As used herein, an "amido" encompasses both "aminocarbonyl" and "carbonylamino". These terms when used alone or in connection with another group refers to an amido group such as N(R^X)₂-C(O> or R^YC(O)-N(R^X)₂- when used terminally and -C(O)-N(R^X)- or -N(R^X)- C(O)- when used internally, wherein R^x and R^γ are defined below. Examples of amido groups include alkylamido (such as alkylcarbonylamino and alkylcarbonylamino), (heterocycloaliphatic) amido, (heteroaralkyl) amido, (heteroaryl) amido, (heterocycloalkyl)alkylamido, arylamido, aralkylamido, (cycloalkyl)alkylamido, and cycloalkylamido.

[083] As used herein, an "amino" group refers to -NR^XR^Y wherein each of R^x and R^γ is independently hydrogen, alkyl, cycloaliphatic, (cycloaliphatic)aliphatic, aryl, araliphatic, heterocycloaliphatic, (heterocycloaliphatic)aliphatic, heteroaryl, carboxy, sulfanyl, sulfinyl, sulfonyl, (aliphatic)carbonyl, (cycloaliphatic)carbonyl, ((cycloaliphatic)aliphatic)carbonyl, arylcarbonyl, (araliphatic)carbonyl, (heterocycloaliphatic)carbonyl,

((heterocycloaliphatic)aliphatic)carbonyl, (heteroaryl)carbonyl, or (heteroaraliphatic)carbonyl, each of which being defined herein and being optionally substituted. Examples of amino groups include alkylamino, dialkylamino, and arylamino.

is not the terminal group (e.g., alkylcarbonylamino), it is represented by -NR^X-. R^x has the same meaning as defined above.

[085] As used herein, an "aryl" group used alone or as part of a larger moiety as in "aralkyl", "aralkoxy", or "aryloxyalkyl" refers to monocyclic (e.g., phenyl); bicyclic (e.g., indenyl, naphthalenyl, tetrahydronaphthyl, tetrahydroindenyl); and tricyclic (e.g., fluorenyl tetrahydrofluorenyl, or tetrahydroanthracenyl, anthracenyl). The bicyclic and tricyclic groups include benzofused 2-3 membered carbocyclic rings. For example, a benzofused group includes phenyl fused with two or more C_4-8 carbocyclic moieties. An aryl is optionally substituted with one or more substituents including aliphatic [e.g., alkyl, alkenyl, or alkynyl]; cycloaliphatic; (cycloaliphatic)aliphatic; heterocycloaliphatic; (heterocycloaliphatic)aliphatic; aryl; heteroaryl; alkoxy; (cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy; heteroaryloxy; (araliphatic)oxy; (heteroaraliphatic)oxy; aroyl; heteroaroyl; amino; oxo (on a non-aromatic carbocyclic ring of a benzofused bicyclic or tricyclic aryl); nitro; carboxy; amido; acyl [ e.g., aliphaticcarbonyl; (cycloaliphatic)carbonyl;

((cycloaliphatic)aliphatic)carbonyl; (araliphatic)carbonyl; (heterocycloaliphatic)carbonyl; ((heterocycloaliphatic) aliphatic)carbonyl; and (heteroaraliphatic)carbonyl]; sulfonyl [e.g., aliphaticsulfonyl and aminosulfonyl]; sulfinyl [e.g., aliphaticsulfmyl]; sulfanyl [e.g., aliphaticsulfanyl]; nitro; cyano; halo; hydroxyl; mercapto; sulfoxy; urea; thiourea; sulfamoyl; sulfamide; and carbamoyl. Alternatively, an aryl can be unsubstituted. [086] Non-limiting examples of substituted aryls include haloaryl [e.g., mono-, di ( such as /?,7w-dihaloaryl), and (trihalo)aryl]; (carboxy)aryl [e.g., (alkoxycarbonyl)aryl, ((arylalkyl)carbonyloxy)aryl, and (alkoxycarbonyl)aryl]; (amido)aryl [e.g., (aminocarbonyl)aryl, (((alkylamino)alkyl)aminocarbonyl)aryl, (alkylcarbonyl)aminoaryl, (arylaminocarbonyl)aryl, and (((heteroaryl)amino)carbonyl)aryl]; aminoaryl [e.g., ((alkylsulfonyl)amino)aryl and ((dialkyl)amino)aryl]; (cyanoalkyl)aryl; (alkoxy)aryl; (sulfamoyl)aryl [e.g., (aminosulfonyl)aryl]; (alkylsulfonyl)aryl; (cyano)aryl; (hydroxyalkyl)aryl; ((alkoxy)alkyl)aryl; (hydroxyl)aryl, ((carboxy)alkyl)aryl; (((dialkyl)amino)alkyl)aryl; (nitroalkyl)aryl; (((alkylsulfonyl)amino)alkyl)aryl; ((heterocycloaliphatic)carbonyl)aryl; ((alkylsulfonyl)alkyl)aryl; (cyanoalkyl)aryl; (hydroxyalkyl)aryl; (alkylcarbonyl)aryl; alkylaryl; (trihaloalkyl)aryl; p-amino-m- alkoxycarbonylaryl; ^-amino-rø-cyanoaryl; j9-halo-7w-aminoaryl; and (rø-(heterocycloaliphatic)- ø-(alkyl))aryl.

t Q⁸Ht *MMfe^Mftt#'l3raliphatic" such as an "aralkyl" group refers to an aliphatic group (e.g., a Ci_-4 alkyl group) that is substituted with an aryl group. "Aliphatic," "alkyl," and "aryl" are defined herein. An example of an araliphatic such as an aralkyl group is benzyl. [088] As used herein, a "bicyclic ring system" includes 8-12 (e.g., 9, 10, or 11) membered structures that form two rings, wherein the two rings have at least one atom in common (e.g., 2 atoms in common). Bicyclic ring systems include bicycloaliphatics (e.g., bicycloalkyl or bicycloalkenyl), bicycloheteroaliphatics, bicyclic aryls, and bicyclic heteroaryls. [089] As used herein, a "cycloaliphatic" group encompasses a "cycloalkyl" group and a "cycloalkenyl" group, each of which being optionally substituted as set forth below. [090] As used herein, a "cycloalkyl" group refers to a saturated carbocyclic mono- or bicyclic (fused or bridged) ring of 3-10 (e.g., 5-10) carbon atoms. Examples of cycloalkyl groups include cyclopropyl, eyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, norbornyl, cubyl, octahydro-indenyl, decahydro-naphthyl, bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl, bicyclo[3.3.2.]decyl, bicyclo[2.2.2]octyl, adamantyl, azacycloalkyl, or ((aminocarbonyl)cycloalkyl)cycloalkyl. A "cycloalkenyl" group, as used herein, refers to a non-aromatic carbocyclic ring of 3-10 (e.g., 4-8) carbon atoms having one or more double bonds. Examples of cycloalkenyl groups include cyclopentenyl, 1,4-cyclohexa-di-enyl, cycloheptenyl, cyclooctenyl, hexahydro-indenyl, octahydro-naphthyl, cyclohexenyl, cyclopentenyl, bicyclo[2.2.2]octenyl, andbicyclo[3.3.1]nonenyl.

[091] A cycloalkyl or cycloalkenyl group can be optionally substituted with one or more substituents such as aliphatic [e.g., alkyl, alkenyl, or alkynyl], cycloaliphatic, (cycloaliphatic) aliphatic, heterocycloaliphatic, (heterocycloaliphatic) aliphatic, aryl, heteroaryl, alkoxy, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy, aryloxy, heteroaryloxy, (araliphatic)oxy, (heteroaraliphatic)oxy, aroyl, heteroaroyl, amino, amido [e.g., (aliphatic)carbonylamino, (cycloaliphatic)carbonylamino, ((cycloaliphatic)aliphatic)carbonylamino, (aryl)carbonylamino, (araliphatic)carbonylamino, (heterocycloaliphatic)carbonylamino, ((heterocycloaliphatic)aliphatic)carbonylamino, (heteroaryl)carbonylamino, and (heteroaraliphatic)carbonylamino], nitro, carboxy [e.g., HOOC-, alkoxycarbonyl, and alkylcarbonyloxy], acyl [e.g., (cycloaliphatic)carbonyl, ((cycloaliphatic) aliphatic)carbonyl, (araliphatic)carbonyl, (heterocycloaliphatic)carbonyl,

((heterocycloaliphatic)aliphatic)carbonyl, and (heteroaraliphatic)carbonyl], nitro, cyano, halo, hydroxy, mercapto, sulfonyl [e.g., alkylsulfonyl and arylsulfonyl], sulfinyl [e.g., alkylsulfmyl], sulfanyl [e.g., alkylsulfanyl], sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, or carbamoyl.

moiety" includes cycloaliphatic, heterocycloaliphatic, aryl, or heteroaryl, each of which has been defined previously.

[093] As used herein, the term "heterocycloaliphatic" encompasses a heterocycloalkyl group and a heterocycloalkenyl group, each of which being optionally substituted as set forth below. [094] As used herein, a "heterocycloalkyl" group refers to a 3-10 membered mono- or bicylic (fused or bridged) (e.g., 5- to 10-membered mono- or bicyclic) saturated ring structure, in which one or more of the ring atoms is a heteroatom (e.g., N, O, S, or combinations thereof). Examples of a heterocycloalkyl group include piperidyl, piperazyl, tetrahydropyranyl, tetrahydrofuryl, 1,4-dioxolanyl, 1,4-dithianyl, 1,3-dioxolanyl, oxazolidyl, isoxazolidyl, morpholinyl, thiomorpholyl, octahydro-benzofuryl, octahydro-chromenyl, octahydro- thiochromenyl, octahydro-indolyl, octahydro-pyrindinyl, decahydro-quinolinyl, octahydro- benzo[£]thiopheneyl, 2-oxa-bicyclo[2.2.2]octyl, l-aza-bicyclo[2.2.2]octyl, 3-aza- bicyclo[3.2.1]octyl, and 2,6-dioxa-tricy_#clo[3.3.1.0³'⁷]nonyl. A monocyclic heterocycloalkyl group can be fused with a phenyl moiety such as tetrahydroisoquinoline. A "heterocycloalkenyl" group, as used herein, refers to a mono- or bicylic (e.g., 5- to 10- membered mono- or bicyclic) non-aromatic ring structure having one or more double bonds, and wherein one or more of the ring atoms is a heteroatom (e.g., N, O, or S). Monocyclic and bicycloheteroaliphatics are numbered according to standard chemical nomenclature. [095] A heterocycloalkyl or heterocycloalkenyl group can be optionally substituted with one or more substituents such as aliphatic [e.g., alkyl, alkenyl, or alkynyl], cycloaliphatic, (cycloaliphatic) aliphatic, heterocycloaliphatic, (heterocycloaliphatic) aliphatic, aryl, heteroaryl, alkoxy, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy, aryloxy, heteroaryloxy, (araliphatic)oxy, (heteroaraliphatic)oxy, aroyl, heteroaroyl, amino, amido [e.g., (aliphatic)carbonylamino, (cycloaliphatic)carbonylamino, ((cycloaliphatic) aliphatic)carbonylamino, (aryl)carbonylamino, (araliphatic)carbonylamino, (heterocycloaliphatic)carbonylarnino, ((heterocycloaliphatic) aliphatic)carbonylamino, (heteroaryl)carbonylamino, and (heteroaraliphatic)carbonylarnino], nitro, carboxy [e.g., HOOC-, alkoxycarbonyl, and alkylcarbonyloxy], acyl [e.g., (cycloaliphatic)carbonyl, ((cycloaliphatic) aliphatic)carbonyl, (araliphatic)carbonyl, (heterocycloaliphatic)carbonyl, ((heterocycloaliphatic)aliphatic)carbonyl, and (heteroaraliphatic)carbonylj, nitro, cyano, halo, hydroxy, mercapto, sulfonyl [e.g., alkylsulfonyl and arylsulfonyl], sulfinyl [e.g., alkylsulfmyl], sulfanyl [e.g., alkylsulfanyl], sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, or carbamoyl. [096] A "heteroaryl" group, as used herein, refers to a monocyclic, bicyclic, or tricyclic ring structure having 4 to 15 ring atoms wherein one or more of the ring atoms is a heteroatom

thereof) and wherein one ore more rings of the bicyclic or tricyclic ring structure is aromatic. A heteroaryl group includes a benzofused ring system having 2 to 3 rings. For example, a benzofused group includes benzo fused with one or two 4 to 8 membered heterocycloaliphatic moieties (e.g., indolizyl, indolyl, isoindolyl, 3H-indolyl, indolinyl, benzo[έ]furyl, benzo[έ]thiophenyl, quinolinyl, or isoquinolinyl). Some examples of heteroaryl are azetidinyl, pyridyl, lH-indazolyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, tetrazolyl, benzofuryl, isoquinolinyl, benzthiazolyl, xanthene, thioxanthene, phenothiazine, dihydroindole, benzo[l,3]dioxole, benzo [b] furyl, benzo[b]thiophenyl, indazolyl, benzimidazolyl, benzthiazolyl, puryl, cinnolyl, quinolyl, quinazolyl,cinnolyl, phthalazyl, quinazolyl, quinoxalyl, isoquinolyl, 4H-quinolizyl, benzo-l,2,5-thiadiazolyl, or 1,8- naphthyridyl.

[097] Without limitation, monocyclic heteroaryls include furyl, thiophenyl, 2H-pyrrolyl, pyrrolyl, oxazolyl, thazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, 1,3,4-thiadiazolyl, 2H-pyranyl, 4-H-pranyl, pyridyl, pyridazyl, pyrimidyl, pyrazolyl, pyrazyl, or 1,3,5-triazyl. Monocyclic heteroaryls are numbered according to standard chemical nomenclature. [098] Without limitation, bicyclic heteroaryls include indolizyl, indolyl, isoindolyl, 3H- indolyl, indolinyl, benzo[Z>]furyl, benzo[έ]thiophenyl, quinolinyl, isoquinolinyl, indazolyl, benzimidazyl, benzthiazolyl, purinyl, 4H-quinolizyl, quinolyl, isoquinolyl, cinnolyl, phthalazyl, quinazolyl, quinoxalyl, 1,8-naphthyridyl, or pteridyl. Bicyclic heteroaryls are numbered according to standard chemical nomenclature.

[099] A heteroaryl is optionally substituted with one or more substituents such as aliphatic [e.g., alkyl, alkenyl, or alkynyl]; cycloaliphatic; (cycloaliphatic)aliphatic; heterocycloaliphatic; (heterocycloaliphatic)aliphatic; aryl; heteroaryl; alkoxy; (cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy; heteroaryloxy; (araliphatic)oxy; (heteroaraliphatic)oxy; aroyl; heteroaroyl; amino; oxo (on a non-aromatic carbocyclic or heterocyclic ring of a bicyclic or tricyclic heteroaryl); nitro; carboxy; amido; acyl [ e.g., aliphaticcarbonyl; (cycloaliphatic)carbonyl; ((cycloaliphatic)aliphatic)carbonyl; (araliphatic)carbonyl; (heterocycloaliphatic)carbonyl; ((heterocycloaliphatic) aliphatic)carbonyl; and (heteroaraliphatic)carbonyl]; sulfonyl [e.g., aliphaticsulfonyl and aminosulfonyl]; sulfinyl [e.g., aliphaticsulfinyl]; sulfanyl [e.g., aliphaticsulfanyl]; nitro; cyano; halo; hydroxyl; mercapto; sulfoxy; urea; thiourea; sulfamoyl; sulfamide; or carbamoyl. Alternatively, a heteroaryl can be unsubstituted.

[0100] Non-limiting examples of substituted heteroaryls include (halo)heteroaryl [e.g., mono- and di-(halo)heteroaryl]; (carboxy)heteroaryl [e.g., (alkoxycarbonyl)heteroaryl];

Cya|fte^|ryl<t|||i|iMeoaryl [e.g., ((alkylsulfonyl)amino)heteroaryl and((dialkyl)amino)heteroaryl]; (amido)heteroaryl [e.g., aminocarbonylheteroaryl, ((alkylcarbonyl)amino)heteroaryl, ((((alkyl)amino)alkyl)aminocarbonyl)heteroaryl, (((heteroaryl)amino)carbonyl)heteroaryl, ((heterocycloaliphatic)carbonyl)heteroaryl, and ((alkylcarbonyl)amino)heteroaryl] ; (cyanoalkyl)heteroaryl; (alkoxy)heteroaryl; (sulfamoyl)heteroaryl [e.g., (aminosulfonyl)heteroaryl]; (sulfonyl)heteroaryl [e.g., (alkylsulfonyl)heteroaryl] ; (hydroxyalkyl)heteroaryl; (alkoxyalkyl)heteroaryl; (hydroxyl)heteroaryl; ((carboxy)alkyl)heteroaryl; [((dialkyl)amino)alkyl]heteroaryl; (heterocycloaliphatic)heteroaryl; (cycloaliphatic)heteroaryl; (nitroalkyl)heteroaryl; (((alkylsulfonyl)amino)alkyl)heteroaryl; ((alkylsulfonyl)alkyl)heteroaryl; (cyanoalkyl)heteroaryl; (acyl)heteroaryl [e.g., (alkylcarbonyl)heteroaryl]; (alkyl)heteroaryl, and (haloalkyl)heteroaryl [e.g., trihaloalkylheteroaryl].

[0101] A "heteroaraliphatic (such as a heteroaralkyl group) as used herein, refers to an aliphatic group (e.g., a C_1-4 alkyl group) that is substituted with a heteroaryl group. "Aliphatic," "alkyl," and "heteroaryl" have been defined above. [0102] As used herein, an "acyl" group refers to a formyl group or R^X-C(O)- (such as -alkyl-C(O)-, also referred to as "alkylcarbonyl") where R^x and "alkyl" have been defined previously. Acetyl and pivaloyl are examples of acyl groups.

[0103] As used herein, an "alkoxy" group refers to an alkyl-O- group where "alkyl" has been defined previously.

[0104] As used herein, a "carbamoyl" group refers to a group having the structure -O-CO- NR^XR^Y or -NR^X-CO-O-R^Z wherein R^x and R^γ have been defined above and R^z can be aliphatic, aryl, araliphatic, heterocycloaliphatic, heteroaryl, or heteroaraliphatic. [0105] As used herein, a "carboxy" group refers to -COOH, -COOR^X, -OC(O)H,

-OC(O)R when used as a terminal group or -OC(O)- or -C(O)O-; when used as an internal group.

[0106] As used herein, a "haloaliphatic" group refers to an aliphatic group substituted with 1-3 halogen. For instance, the term haloalkyl includes the group -CF₃.

[0107] As used herein, a "mercapto" group refers to -SH.

[0108] As used herein, a "sulfo" group refers to -SO₃H or -SO₃R^X when used terminally or

-S(O)3- when used internally.

[0109] As used herein, a "sulfamide" group refers to the structure -NR^X-S(O)₂-NR^YR^Z when used terminally and -NR^X-S(O)₂-NR^Y- when used internally, wherein R^x, R^γ, and R^z have been defined above.

ltQlIQB&i #ed%rjfnja^lfamoyr group refers to the structure -S(O)₂-NR R or -NR^X - S(O)₂-R² when used terminally or -S(O)₂-NR^X- or -NR^X -S(O)₂- when used internally, wherein R^x, R^γ, and R^z are defined above.

[0111] As used herein a "sulfanyl" group refers to -S-R^x when used terminally and -S- when used internally, wherein R^x has been defined above. Examples of sulfanyls include alkylsulfanyl.

[0112] As used herein a "sulfinyl" group refers to -S(O)-R when used terminally and -S(O)- when used internally, wherein R^x has been defined above.

[0113] As used herein, a "sulfonyl" group refers to-S(O)₂-R^x when used terminally and - S(O)₂- when used internally, wherein R^x has been defined above. [0114] As used herein, a "sulfoxy" group refers to -O-SO-R^X or -SO-O-R^X, when used terminally and -O-S(O)- or -S(O)-O- when used internally, where R^x has been defined above. [0115] As used herein, a "halogen" or "halo" group refers to fluorine, chlorine, bromine or iodine.

[0116] As used herein, an "alkoxycarbonyl," which is encompassed by the term carboxy, used alone or in connection with another group refers to a group such as alkyl-O-C(O)-. [0117] As used herein, an "alkoxyalkyl" refers to an alkyl group such as alkyl-O-alkyl-, wherein alkyl has been defined above. [0118] As used herein, a "carbonyl" refers to -C(O)-. [0119] As used herein, an "oxo" refers to =0.

[0120] As used herein, an "aminoalkyl" refers to the structure (R^x)₂N-alkyl-. [0121] As used herein, a "cyanoalkyl" refers to the structure (NC)-alkyl-. [0122] As used herein, a "urea" group refers to the structure -NR^X-CO-NR^YR^Z and a "thiourea" group refers to the structure -NR^X-CS-NR^YR^Z when used terminally and -NR^X-CO- NR^Y- or -NR^X-CS-NR^Y- when used internally, wherein R^x, R^γ and R^z have been defined above.

[0123] As used herein, a "guanidino" group refers to the structure -N=C(N (R^x R^Y))N(R^XR^Y) wherein R and R have been defined above.

[0124] As used herein, the term "amidino" group refers to the structure -C=(NR^X)N(R^XR^Y) wherein R^x and R^γhave been defined above. [0125] The terms "terminally" and "internally" refer to the location of a group within a substituent. A group is terminal when the group is present at the end of the substituent not further bonded to the rest of the chemical structure. Carboxyalkyl, i.e., R^xO(O)C-alkyl is an example of a carboxy group used terminally. A group is internal when the group is present in

at the end of the substituent bound to the to the rest of the chemical structure. Alkylcarboxy (e.g., alkyl-C(O)O- or alkyl-OC(O)-) and alkylcarboxyaryl (e.g., alkyl-C(O)O-aryl- or alkyl-O(CO)-aryl-) are examples of carboxy groups used internally. [0126] The phrase "optionally substituted" is used interchangeably with the phrase "substituted or unsubstituted." As described herein, compounds of the invention can optionally be substituted with one or more substituents, such as are illustrated generally above, or as exemplified by particular classes, subclasses, and species of the invention. As described herein, the variables contained herein encompass specific groups, such as alkyl and aryl. Unless otherwise noted, each of the specific groups for the variables contained herein can be optionally substituted with one or more substituents described herein. Each substituent of a specific group is further optionally substituted with one to three of halo, cyano, oxoalkoxy, hydroxyl, amino, nitro, aryl, haloalkyl, and alkyl. For instance, an alkyl group can be substituted with alkylsulfanyl and the alkylsulfanyl can be optionally substituted with one to three of halo, cyano, oxoalkoxy, hydroxyl, amino, nitro, aryl, haloalkyl, and alkyl. As an additional example, the cycloalkyl portion of a (cycloalkyl)carbonylamino can be optionally substituted with one to three of halo, cyano, alkoxy, hydroxyl, nitro, haloalkyl, and alkyl. When two alkoxy groups are bound to the same atom or adjacent atoms, the two alkxoy groups can form a ring together with the atom(s) to which they are bound.

[0127] In general, the term "substituted," whether preceded by the term "optionally" or not, refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. Specific substituents are described above in the definitions and below in the description of compounds and examples thereof. Unless otherwise indicated, an optionally substituted group can have a substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituent can be either the same or different at every position. A ring substituent, such as a heterocycloalkyl, can be bound to another ring, such as a cycloalkyl, to form a spiro-bicyclic ring system, e.g., both rings share one common atom. As one of ordinary skill in the art will recognize, combinations of substituents envisioned by this invention are those combinations that result in the formation of stable or chemically feasible compounds.

[0128] The phrase "stable or chemically feasible," as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and preferably their recovery, purification, and use for one or more of the purposes disclosed herein.

Qϊlf JJ^Igjllls/όilelgrøl-llδed, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools or probes in biological assays.

[0130] As used herein, an effective amount is defined as the amount required to confer a therapeutic effect on the treated patient, and is typically determined based on age, surface area, weight and condition of the patient. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich et al., Cancer Chemother. Rep., 50: 219 (1966). Body surface area can be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, New York, 537 (1970).

DETAILED DESCRIPTION OF THE INVENTION

[0131] The present invention relates to methods and compositions for the treatment and prevention of cancer. In particular, the present invention provides methods and compositions comprising oligonucleotides for the treatment and prevention of cancer. I. Oncogene Targets

[0132] In some embodiments, the present invention provides antigene inhibitors of oncogenes. The present invention is not limited to the inhibition of a particular oncogene. Indeed, the present invention encompasses antigene inhibitors to any number of oncogenes including, but not limited to, those disclosed herein.

A. Ras

[0133] One gene that has captured the attention of many scientists is the human proto- oncogene, c-Ha-ras. This gene acts as a central dispatcher, relaying chemical signals into cells and controlling cell division. Ras gene alteration may cause the gene to stay in the "on"

believed to underlie up to 30% of cancer, including colon cancer, lung cancer, bladder and mammary carcinoma (Bos, Cancer Res. 49:4682-4689 [1989]). The ras oncogene has therefore become a target for therapeutic drugs.

[0134] There are several reports showing that oligonucleotides complementary to various sites of ras mRNA can inhibit synthesis of ras protein (p21), which decreases the cell proliferation rate in cell culture (U.S. Pat. No. 5,576,208; U.S. Pat. No. 5,582,986; Daska et al, Oncogene Res. 5:267-275 [1990]; Brown et al, Oncogene Res. 4:243-252 [1989]; Saison-Behmoaras et al, EMBO J. 10:1111-1116 [1991)]. Oligonucleotides complementary to the 5¹ flanking region of the c-Ha-ras RNA transcript have shown to inhibit tumor growth in nude mice for up to 14 days (Gray et al, Cancer Res. 53:577-580 [1993]). It was recently reported that an antisense oligonucleotide directed to a point mutation (G>C) in codon 12 of the c-Ha-ras mRNA inhibited cell proliferation as well as tumor growth in nude mice when it was injected subcutaneously (U.S. Pat. No. 5,576,208; U.S. Pat. No. 5,582,986; Schwab et al, Proc. Natl. Acad. Sci. USA 91 : 10460-10464 [1994]; each of which is herein incorporated by reference). Researchers have also reported that antisense drugs shrank ovarian tumors in small clinical trials (Roush et al, Science 276:1192-1194 [1997]).

B. Her-2

[0135] The HER-2 (also known as neu oncogene or erbB-2) oncogene encodes a receptor-like tyrosine kinase (RTK) that has been extensively investigated because of its role in several human carcinomas (Hynes and Stern, Biochim. et Biophy. Acta 1198:165-184 [1994]; Dougall et al, Oncogene 9:2109-2123 [1994]) and in mammalian development (Lee et al, Nature 378:394-398 [1995]). The sequence of the HER-2 protein was determined from a cDNA that was cloned by homology to the epidermal growth factor receptor (EGFR) mRNA from placenta (Coussens et al, Science 230:1132-1139 [1985]) and from a gastric carcinoma cell line (Yamamoto et al, Nature 319:230-234 [1986]). The HER-2 mRNA was shown to be about 4.5 kb (Coussens et al, Science 230:1132-1139 [1985]; Yamamoto et al, Nature 319:230-234 [1986]) and encodes a transmembrane glycoprotein of 185 IcDa in normal and malignant human tissues (pl85HER-2) (Hynes and Steen, Biochim. et Biophys. Acta 1198:165-184 [1994]; Dougall et al, Oncogene 9:2109-2123 [1994]). Overexpression of HER-2 causes phenotypic transformation of cultured cells (DiFiore et al, Science 237:178-182 [1987]; Hudziak et al, Proc. Natl. Acad. Sci. USA 84:7159-7163 [1987]) and has been associated with aggressive clinical progression of breast and ovarian cancer (Slamon et al, Science 235:177-182 [1987]; Slamon et al, Science 244:707-712 [1989]) f[$l§β$HMrTϊΦWW¥&XΑθst frequently altered genes in cancer. It encodes a transmembrane receptor (also known as pi 85) with tyrosine kinase activity and is a member of the epidermal growth factor (EGF) family, and thus is related to the epidermal growth factor receptor (EGFR or HER-I). Aberrant HER-2 gene expression is present in a wide variety of cancers and is most common in breast, ovarian and gastric cancers. HER-2 is overexpressed in 25-30% of all human breast and ovarian cancers. Levels of HER-2 overexpression correlate well with clinical stage of breast cancer, prognosis and metastatic potential. Overexpression of HER-2 is associated with lower survival rates, increased relapse rates and increased metastatic potential. Tan et al, (Cancer Res., 57:1199 [1997]) have shown that overexpression of the HER-2 gene increases the metastatic potential of breast cancer cells without increasing their transformation ability.

[0137] Aberrant expression of HER-2 includes both increased expression of normal HER-2 and expression of mutant HER-2. Activation of the HER-2 proto-oncogene can occur by any of three mechanisms-point mutation, gene amplification and overexpression. Gene amplification is the most common mechanism. Unlike the other EGF family members for whom ligand activation is necessary for promoting transformation, overexpression of HER-2 alone is sufficient for transformation (Cohen, et al, J. Biol. Chem., 271 :30897 [1996]). [0138] Several therapeutic approaches have been used to reduce levels of the HER-2 gene product. The adenovirus type 5 gene product ElA has been studied as a potential therapeutic using a breast cancer model in nude mice. This gene product can repress HER-2/neu overexpression by repressing HER-2/neu promoter activity, and suppress the tumorigenic potential of HER-2/neu-overexpressing ovarian cancer cells. In mice bearing HER-2/neu- overexpressing breast cancer xenografts, ElA delivered either by adenovirus or liposome significantly inhibited tumor growth and prolonged mouse survival compared with the controls (Chang et al, Oncogene 14:561 [1997]).

[0139] Clinical trials have been conducted to evaluate a bispecific antibody that targets the extracellular domains of both the HER-2/neu protein product and Fc gamma RIII (CD 16), the Fc gamma receptor expressed by human natural killer cells, neutrophils, and differentiated mononuclear phagocytes (Weiner et al, J. Hematotherapy, 4:471 [1995]). [0140] Overexpression of HER-2 has also been found to be associated with increased resistance to chemotherapy. Thus, patients with elevated levels of HER-2 respond poorly to many drugs. Methods used to inhibit HER-2 expression have been combined with commonly used chemotherapeutic agents (Ueno etal, Oncogone 15:953 [1997]). Combining the adenovirus type 5 gene product, ElA, with taxol showed a synergistic effect in human breast ψ$n$_gz$φ^$h^gipeμi$^Qncogene, 12:571 [1996]) demonstrated that emodin, a tyrosine- specific inhibitor, sensitized non-small cell lung cancer (NSCLC) cells to a variety of chemotherapeutic drugs, including cisplatin, doxorubicin and etoposide. A HER-2 antibody was found to increase the efficacy of tamoxifen in human breast cancer cells (Witters et ah, Breast Cancer Res. and Treatment, 42:1 [1997]).

[0141] Oligonucleotides have also been used to study the function of HER-2. A triplex- forming oligonucleotide targeted to the HER-2 promoter, 42 to 69 nucleotides upstream of the mRNA transcription start site was found to inhibit HER-2 expression in vitro (Ebbinghaus et ah, J. Clin. Invest., 92:2433 [1993]). Porumb et a (Cancer Res., 56:515 [1996]) also used a triplex-forming oligonucleotide targeted to the same HER-2 promoter region. Decreases in HER-2 mRNA and protein levels were seen in cultured cells. Juhl et a (J. Biol. Chem., 272:29482 [1997]) used anti-HER-2 ribozymes targeted to a central region of the HER-2 RNA just downstream of the transmembrane region of the protein to demonstrate a reduction in HER-2 mRNA and protein levels in human ovarian cancer cells. A reduction in tumor growth in nude mice was also seen.

[0142] An antigene approach has been used as a potential therapeutic for HER-2 overexpressing cancers. Pegues et a (Cancer Lett, 117:73 [1997]) cloned a 1.5 kb fragment of HER-2 in an antisense orientation into an expression vector; transfecting of this construct into ovarian cancer cells resulted in a reduction of anchorage-independent growth. Casalini et ah (Lit. J. Cancer 72:631 [1997]) used several human HER-2 antisense vector constructs, containing HER-2 fragments from 151 bp to 415 bp in length, to demonstrate reduction in HER-2 protein levels and anchorage-independent growth in lung adenocarcinoma cells. Colomer et a (Br. J. Cancer, 70:819 [1994]) showed that phosphodiester antisense oligonucleotides targeted at or immediately downstream of, the translation initiation codon inhibited proliferation of human breast cancer cells by up to 60%. Wiechen et a (Int. J. Cancer 63:604 [1995]) demonstrated that an 18-nucleotide phosphorothioate oligonucleotide targeted to the coding region, 33 nucleotides downstream of the translation initiation codon, of HER-2 reduced anchorage-independent growth of ovarian cancer cells. Bertram et ah (Biochem. Biophys. Res. Commun., 200:661 [1994]) used antisense phosphorothioate oligonucleotides targeted to the translation initiation region and a sequence at the 3' part of the translated region of the mRNA which has high homology to a tyrosine kinase consensus sequence, and demonstrated a 75% reduction in HER-2 protein levels in human breast cancer cells. Liu et ah, (Antisense and Nucleic Acid Drug Develop., 6:9 [1996]) used antisense phosphorothioate oligonucleotides targeted to the 5' cap site and coding region. The most ejMSrgeted to the 5' cap site, reduced HER-2 protein expression by 90%. Cell proliferation was also reduced by a comparable amount. Vaughn et al. (Nuc. Acids. Res., 24:4558 [1996]) used phosphorothioate, phosphorodithioate and chimeric antisense oligonucleotides targeted at or adjacent to (either side) the translation initiation region of HER- 2. An alternating dithioate/diester oligonucleotide targeted to the translation initiation region worked slightly better than an all phosphorothioate oligonucleotide. Brysch et al (Cancer Gene Ther., 1 : 99 [1994]) used chemically modified antisense oligonucleotides targeted to the translation initiation codon of HER-2 to reduce protein levels and cause growth arrest of human breast cancer cell line.

C. C-Myc

[0143] The c-myc gene product is encoded by an immediate early response gene, the expression of which can be induced by various mitogens. C-myc expression is involved in the signal transduction pathways leading to cell division. Studies have demonstrated that proliferating cells have higher levels of c-myc mRNA and c-myc protein than do quiescent cells. Antibodies directed against the human c-myc protein are known to inhibit DNA synthesis in nuclei isolated from human cells. Conversely, constitutive expression of c-myc produced by gene transfer inhibits induced differentiation of several cell lines. Constitutive expression of c-myc predisposes transgenic mice to the development of tumors. [0144] Some studies have suggested that the c-myc gene product may play a proliferative role in SMCs. Balloon de-endothelialization and injury of rat aortas is known to increase c-myc mRNA expression of vascular SMC prior to their subsequent proliferation and migration. Also, SMCs in culture proliferate when exposed to several mitogens, including PDGF, FGF, EGF, IGF-I and to serum. Each of these mitogens has been found to be capable of increasing the expression in other cell lines of either c-myc protein, c-myc mRNA, or both. Additionally, blood serum has been found to increase c-myc mRNA levels in SMCs. [0145] Harel-Bellan et al. (J. Immun. 140; 2431-2435 (1988)) demonstrated that antisense oligonucleotides complementary to c-myc mRNA effectively inhibited the translation thereof in human T cells. These T cells were prevented from entering the S phase of cell division, c- myc proto-oncogene sequences are described in Marcu et al, Ann. Rev. Biochem., 61:809-860 [1992]; Watt et al, Nature, 303:725-728 [1983)]; Battey et al, Cell, 34:779-787 (1983); and Epstein et al, NTIS publication PB93- 100576

D. Bcl2

[0146] In many types of human tumors, including lymphomas and leukemias, the human bcl-2 gene is overexpressed, and may be associated with tumorigenicity (Tsujimoto et al, Science

f ^WSrøflϊiϊfS]^ ^ifSf|h levels of expression of the human bcl-2 gene have been found in all lymphomas with t (14; 18) chromosomal translocations including most follicular B cell lymphomas and many large cell non-Hodgkin's lymphomas. High levels of expression of the bcl-2 gene have also been found in certain leukemias that do not have a t(14; 18) chromosomal translation, including most cases of chronic lymphocytic leukemia acute, many lymphocytic leukemias of the pre-B cell type, neuroblastomas, nasophryngeal carcinomas, and many adenocarcinomas of the prostate, breast, and colon. (Reed et at, Cancer Res. 51 :6529 [1991]; Yunis et at, New England J. Med. 320:1047; Campos et at, Blood 81 :3091-3096 [1993]; McDonnell et at, Cancer Res. 52:6940-6944 [1992); Lu et at, Int. J Cancer 53:29-35 [1993]; Bonner et at, Lab Invest. 68:43A [1993]).

E. TGF-α

[0147] Transforming Growth Factor Alpha (TGF-α) is a polypeptide of 50 amino acids. It was first isolated from a retrovirus-transformed mouse cell line and subsequently was identified in human tumor cells, in early rat embryo cells and in cell cultures from the human pituitary gland. TGF-α is closely related to Epidermal Growth Factor (EGF), both structurally and functionally, and both bind to the same receptor, i.e., Epidermal Growth Factor Receptor (EGFR).

[0148] The sequence and three dimensional structure of both EGF and TGF-α have been determined (Campbell et at, Prog. Growth Factor Res. 1:13 [1989]). TGF-α is a 50 amino acid polypeptide having about 40% homology of residues with EGF. Both peptides are characterized by three well defined loops (denoted A, B and C) and have three intramolecular disulphide bonds.

[0149] Several growth factors, including TGF-α and EGF, are believed to exert their biological effects via interaction with the Epidermal Growth Factor Receptor (EGF Receptor). The EGF Receptor is a Type 1 receptor tyrosine kinase. The EGF Receptor and its ligands are of interest for their roles in normal physiological processes as well as in hyperproliferative and neoplastic diseases.

[0150] The in vivo precursor of TGF-α is a 160 amino acid residue membrane-bound protein (pro-TGF-.alpha.) that is cleaved to yield a soluble compound (Massague, J. Biol. Chem., 265:21393-21396 [1990]). This cleavage removes an extracellular portion comprised of 50 amino acids with a molecular weight of 6 Kd and is considered to be an important regulatory event (Pandiella et at, Proc. Natl. Acad. Sci. USA, 88:1726-1730 [1990]) that can be stimulated by phorbol esters acting via protein kinase C (Pandiella et at, J. Biol. Chem., 266:5769-5773 [1991]). f['Ql'5JJ^g|_jgr?d«l|i±iiaSi^jrC!|tatic tumor lines contain elevated levels of TGF-α mRNA and proliferate in response to TGF-α (Wilding et al, The Prostate, 15:1-12 [1989]). TGF-α appears to have both autocrine and paracrine function, stimulating physiologic activities such as cell division and angiogenesis. When induced in transgenic mice, TGF-α produced epithelial hyperplasia and focal dysplastic changes that resembled carcinoma in situ (Sandgren et al, Cell, 61:1121-1135 [1990]).

F. c-ki-RAS

[0152] The c-Ki-RAS (KRAS) oncogene is expressed ubiquitously. KRAS, with a length of more than 30 kb, is much larger than HRAS or NRAS. Although the 3 ras genes, HRAS, KRAS, and NRAS, have different genetic structures, all code for proteins of 189 amino acid residues, genetically designated p21. These genes acquire malignant properties by single point mutations that affect the incorporation of the 12th or 61st amino acid residue of their respective p21. KRAS is involved in malignancy much more often than is HRAS. In a study of 96 human tumors or tumor cell lines in the NIH 3T3 transforming system, (Pulciani et al., Nature 300: 539 (1982) found a mutated HRAS locus only in T24 bladder cancer cells, whereas transforming KRAS genes were identified in 8 different carcinomas and sarcomas. [0153] In a serous cystadenocarcinoma of the ovary, Feig et al. (Science 223: 698 (1984)) showed the presence of an activated KRAS oncogene not activated in normal cells of the same patient. The transforming gene product displayed an electrophoretic mobility in SDS- polyacrylamide gels that differed from the mobility of KRAS transforming proteins in other tumors. Thus, a previously undescribed mutation was responsible for activation of KRAS in this ovarian carcinoma. To study the role of oncogenes in lung cancer, Rodenhuis et al. {New Eng. J. Med. 317: 929 (1987)) used an assay based on oligonucleotide hybridization following an in vitro amplification step. Genomic DNA was examined from 39 tumor specimens obtained at thoracotomy. The KRAS gene was found to be activated by point mutations in codon 12 in 5 of 10 adenocarcinomas. Two of these tumors were less than 2 cm in size and had not metastasized. No HRAS, KRAS, or NRAS mutations were observed in 15 squamous cell carcinomas, 10 large cell carcinomas, 1 carcinoid, 2 metastatic adenocarcinomas from primary tumors outside the lung, and 1 small cell carcinoma. An approximately 20-fold amplification of the unmutated KRAS gene was observed in a tumor that proved to be a solitary lung metastasis of a rectal carcinoma. Yanez et al. {Oncogene 1:315 (1987)) found mutations in codon 12 of the KRAS gene in 4 of 16 colon cancers, 2 of 27 lung cancers, and 1 of 8 breast cancers; no mutations were found at position 61. Of the 6 possible amino acid replacements in codon 12, all but one was represented in the 7 mutations identified. _*Mmm&®&& Targets

[0154] The present invention is not limited to the oncogenes described above. The methods of the present invention are suitable for use with any oncogene with a known upstream region. Exemplary oncogenes included, but are not limited to, BCR/ ABL, ABL1/BCR, ABL, BCLl, CD24, CDK4, EGFR/ERBB-1, HSTFl, INTl /WNTl, INT2, MDM2, MET, MYB, MYC, MYCN, MYCLl, RAFl, NRAS, REL, AKT2, APC, BCL2-ALPHA, BCL2-BETA, BCL3, BCR₃ BRCAl, BRCA2, CBL, CCNDl, CDKNlA, CDKNlC, CDKN2A, CDKN2B, CRK, CRK-II, CSF1R/FMS, DBL, DDOST, DCC, DPC4/SMAD4, E-CAD, E2F1/RBAP, ELKl, ELK3, EPH, EPHAl, E2F1, EPHA3, ERG, ETSl, ETS2, FER, FGR, FLI1/ERGB2, FOS, FPS/FES, FRAl, FRA2, FYN, HCK, HEK, HER3/ERBB-2, ERBB-3, HER4/ERBB-4, HST2, INK4A, INK4B, JUN, JUNB, JUND, KIP2, KIT, KRAS2A, KRAS2B, LCK, LYN, MAS, MAX, MCC, MLHl, MOS, MSH2, MYBA, MYBB, NFl, NF2, P53, PDGFB, PIMl, PTC, RBl, RET, ROSl, SKI, SRCl, TALI, TGFBR2, THRAl, THRB, TIAMl, TRK, VAV, VHL, WAFl, WNT2, WTl, YESl, ALK/NPM1, AMIl, AXL, FMS, GIP, GLI, GSP, HOXIl, HST, IL3, INT2, KS3, K-SAM, LBC, LMO-I, LMO-2, L-MYC, LYLl, LYT-IO, MDM-2, MLHl, MLL, MLM, N-MYC, OST, PAX-5, PMS-I, PMS-2, PRAD-I, RAF, RHOM-I, RHOM-2, SIS, TAL2, TANl, TIAMl, TSC2, TRK, TSCl, STKI l, PTCH, MENl, MEN2, P57/KIP2, PTEN, HPCl, ATM, XPA/XPG, BCL6, DEK, AKAP13, CDHl, BLM, EWSR1/FLI1, FES, FGF3, FGF4, FGF6, FANCA, FLI1/ERGB2, FOSLl, FOSL2, GLI, HRASl, HRX/MLLT1, HRX/MLLT2, KRAS2, MADH4, MASl, MCF2, MLLT1/MLL, MLLT2/HRX, MTG8/RUNX1, MYCLKl, MYHl 1/CBFB, NFKB2, NOTCHl, NPM1/ALK, NRG/REL, NTRKl, PBX1/TCF3, PML/RARA, PRCAl, RUNXl, RUNXl /CBF A2T1, SET, TCF3/PBX1, TGFBl, TLXl, P53, WNTl, WNT2, WTl, αv-β3, PKCα, TNFα, Clusterin, Surviving, TGFβ, c-fos, c-SRC, and INT-I.

H. Non-Oncogene Targets

[0155] The present invention is not limited to the targeting of oncogenes. The methods and compositions of the present invention are useful for targeting any gene of which it is desirable to down regulate its expression. For example, in some embodiments, the genes to be targeted include, but are not limited to, an immunoglobulin or antibody gene, a clotting factor gene, a protease, a pituitary hormone, a protease inhibitor, a growth factor, a somatomedian, a gonadotrophin, a chemotactin, a chemokine, a plasma protein, a plasma protease inhibitor, an interleukin, an interferon, a cytokine, a transcription factor, or a pathogen target (e.g., a viral gene, a bacterial gene, a microbial gene, a fungal gene). ]f'01'5®Sl,aiϋplέSfeEst>fe£itf!θ' genes include, but are not limited to, ADAMTS4, ADAMTS5, APOAl, APOE, APP, B2M, COX2, CRP, DDX25, DMCl, FKBP8, GHl, GHR, IAPP, IFNAl, IFNG, ILl, 1110, IL12, IL13, IL2, IL4, IL7, IL8, IPW, MAPK14, Meil, MMP13, MYD88, NDN, PACE4, PRNP, PSENl, PSEN2, RAD51, RAD51C, SAP, SNRPN, TLR4, TLR9, TTR, UBE3A, VLA-4, and PTP-IB, c-RAF, m-TOR, LDL, VLDL, ApoB-100, HDL, VEGF, rhPDGF-BB, NADs, ICAM-I, MUCl, 2-dG, CTL, PSGL-I, E2F, NF-kB, HIF, and GCPRs.

[0157] In other embodiments and gene from a pathogen is targeted. Exemplary pathogens include, but are not limited to, Human Immunodeficiency virus, Hepatitis B virus, hepatitis C virus, hepatitis A virus, respiratory syncytial virus, pathogens involved in severe acute respiratory syndrome, west nile virus, and food borne pathogens (e.g., E. colϊ).

II. Oligonucleotides

[0158] In some embodiments, the present invention provides antigene oligonucleotides for inhibiting the expression of oncogenes. Exemplary design and production strategies for antigenes are described below. The description below is not intended to limit the scope of antigene compounds suitable for use in the present invention and that other antigenes are within the scope of the present invention.

[0159] The oligonucleotides are made up of sequences of nucleotides. The bases on the nucleotides determine sequence of the oligonucleotides. The individual nucleotides consist of a sugar or sugar mimetic, a base bound to the sugar and a linking group that can connect the nucleotide to the next nucleotide in the sequence. The sugar can be in a "locked" conformation as described below.

A. Oligonucleotide Sequences

1. Regulatory Regions of the Oncogenes

[0160] The bcl-2 gene has two promoters designated Pl and P2. Pl from which most bcl-2 mRNA is transcribed is located approximately 1 A kb upstream of the translation initiation site and P2 is 1.3 kb downstream of Pl. (See Seto, M. et al. EMBOJ. 7, 123-131 (1988).) Pl is GC-rich, lacks a TATA box, has many transcription start sites and includes seven consensus binding sites for the SPl transcription factor. P2 includes a CCAAT box and a TATA box and has two different transcription initiation sites. There are multiple NF-ϋTB recognition sites and an SV40 enhancer-like octamer motif within P2. (See Heckman, C.A., et al. Oncogene 21, 3898-3908 (2002).) (See SEQ ID NO: 1254). Most human follicular lymphomas contain t(14;18) chromosomal translocations that result from 3' '-bcl-2 gene region breakpoints. (See

Tsujimoto, Y. et al. Proc. Natl. Acad. Sci. U. S. A 84, 1329-1331 (1987).) These translocations "fpKcM©Ilik|*eisISilMMl& control of the immunoglobulin heavy chain (IgH) locus enhancer resulting in upregulation of BCL2 expression. Alternatively, there are 5' -bcl-2 breakpoint regions that result from fusions with either the IgH locus or two different immunoglobulin light chain (IgL) loci that are found in some DLCL lymphoma patient isolates. (See Yonetani, N. et al. Jpn. J. Cancer Res. 92, 933-940 (2001).) These 5' -bcl-2 breakpoints have been mapped in separate heterogeneous patient isolates to a region spanning 378 to 2312 bp upstream of the translation initiation site. (See SEQ ID NOs: 1255-1266.) Regions around the breakpoints may be sequences that can be used for bcl-2 oligonucleotide design.

[0161] The upstream regions of TGF-α, c-ki-ras, c-myc, c-erb-2 (Her-2), and c-Ha-ras can also be investigated to find regions to which oligonucleotides could bind based on preferred design criteria.

2. Oligonucleotide Design

[0162] The oligonucleotides can include any oligomer that hybridizes to the upstream regions of the c-ki-ras, c-Ha-ras, c-myc, her-2, TGF-a, or bcl-2 gene. For the purposes of this invention, those upstream regions are defined as SEQ ID NO:1 (for her-2, or c-erb-2), SEQ ID NO:282 (for c-ki-ras), SEQ ID NO:462 (for c-Ha-ras), SEQ ID NO:936 (for c-myc), SEQ ID NO:1081 (for TGF-a) and SEQ ID NOs:1249 and 1254 (for bcl-2).

[0163] In some embodiments, oligonucleotides are designed based on preferred design criteria. Such oligonucleotides can then be tested for efficacy using the methods disclosed herein. For example, in some embodiments, the oligonucleotides are methylated on at least one, two or all of the CpG islands. In other embodiments, the oligonucleotides contain no methylation. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, it is contemplated that oligonucleotides in some embodiments are those that have at least a 50% GC content and at least two GC dinucleotides. Also, in some embodiments, the oligonucleotides do not self hybridize. In further embodiments, oligonucleotides are designed with at least 1 A or T to minimize self hybridization. In yet further embodiments, commercially available computer programs are used to survey oligonucleotides for the ability to self hybridize. In still other embodiments, oligonucleotides are at least 10, or 15 nucleotides and no more than 100 nucleotides in length. In further embodiments, oligonucleotides are 18-26 nucleotides in length. In additional embodiments, oligonucleotides comprise the universal protein binding sequences CGCCC and CGCG or the complements thereof.

[0164] In some embodiments, oligonucleotides hybridize to a promoter region of a gene upstream from the TATA box of the promoter. In further embodiments, oligonucleotides are ifions of a regulatory region of an oncogene known to be bound by proteins (e.g., transcription factors). In some embodiments, oligonucleotide compounds are not completely homologous to other regions of the human genome. The homology of the oligonucleotide compounds of the present invention to other regions of the genome can be determined using available search tools (e.g., BLAST, available at the Internet site of NCBI). [0165] The present invention is not limited to the oligonucleotides described herein. Other suitable oligonucleotides may be identified (e.g., using the criteria described above or other criteria). Candidate oligonucleotides may be tested for efficacy using any suitable method. For example, candidate oligonucleotides can be evaluated for their ability to prevent cell proliferation at a variety of concentrations. In some embodiments, oligonucleotides inhibit gene expression or cell proliferation at a low concentration (e.g., less that 20 μM, or 10 μM in in vitro assays.).

3. Oligonucleotide Zones

[0166] In some embodiments, regions within the promoter region of an oncogene are further defined as regions for hybridization of oligonucleotides. In some embodiments, these regions are referred to as "hot zones."

[0167] In some embodiments, hot zones are defined based on oligonucleotide compounds that are demonstrated to be effective (see above section on oligonucleotides) and those that are contemplated to be effective based on the criteria for oligonucleotides described above. In some embodiments, hot zones encompass 10 bp upstream and downstream of each compound included in each hot zone and have at least one CG or more within an increment of 40 bp further upstream or downstream of each compound, hi further embodiments, hot zones encompass a maximum of 100 bp upstream and downstream of each oligonucleotide compound included in the hot zone, hi additional embodiments, hot zones are defined at beginning regions of each promoter. These hot zones are defined either based on effective sequence(s) or contemplated sequences and have a preferred maximum length of 200 bp. Based on the above described criteria, exemplary hot zones were designed. These hot zones are shown in Table 1.

4. Description

[0168] In one aspect, the oligonucleotides can be any oligomer that hybridizes under physiological conditions to the following sequences: SEQ ID NO:1, SEQ ID NO:282, SEQ ID NO:462, SEQ ID NO:936, SEQ ID NO:1081, SEQ ID NOs:1249 and/or 1254. In another aspect, the oligonucleotides can be any oligomer that hybridizes under physiological conditions to exemplary hot zones in SEQ ID NO:1, SEQ ID NO:282, SEQ ID NO:462, SEQ ID NO:936, SEQ ID NO: 1081 and SEQ ID NO: 1249. Examples of oligomers include, without limitation, those oligomers listed in SEQ ID NOs 2-281, 283-461, 463-935, 937-1080, 1082-1248, 1250- 1253 and 1267-1477 and the complements thereof. In another aspect, the oligonucleotides are SEQ ID NOs 2-22, 283-301, 463-503, 937-958, 1082-1109, 1250-1254 and 1270-1477 and the complements thereof. In an embodiment of these aspects, the oligonucleotides are from 15-35 base pairs in length.

[0169] For the bcl-2 gene, the oligomer can be any oligomer that hybridizes to SEQ ID NOs: 1249 or 1254. In another aspect, the oligomer can be any oligomer that hybridizes to nucleotides 500-2026, nucleotides 500-1525, nucleotides 800-1225, nucleotides 900-1125, nucleotides 950-1075 or nucleotides 970-1045 of SEQ ID NO: 1249 or the complement thereof. [0170] In one embodiment, the oligomer can be SEQ ID NO:1250, 1251, 1252, 1253, 1267- 1477 or the complement thereof. In another embodiment, the oligomer can be SEQ ID NOs 1250, 1251, 1267, 1268, 1276, 1277, 1285, 1286 or the complement thereof. In yet another embodiment, the oligomer can be SEQ ID NOs 1250, 1251, 1289-1358 or the complement thereof. In still another embodiment the oligomer can be SEQ ID NO:1250 or 1251. [0171] In a further embodiment of these aspects, the oligomer has the sequence of the positive strand of the bcl-2 sequence, and thus, binds to the negative strand of the sequence. [0172] In other aspects, the oligomers can include mixtures of bcl-2 oligonucleotides. For instance, the oligomer can include multiple oligonucleotides each of which hybridizes to different parts of SEQ ID NOs:1249 and 1254. Oligomers can hybridize to overlapping regions on those sequences or the oligomers may hybridize to non-overlapping regions. In ptm:mn&M$&øi9MbMϋrs can be SEQ ID NOs:1250, 1251, 1252, 1253, 1267-1477 or the complement thereof, wherein the mixture of bcl-2 oligomers comprises oligomers of at least 2 different sequences.

[0173] In other embodiments, the oligomer can include a mixture of oligomers, each of which hybridizes to a regulatory region of different genes. For instance, the oligomer can include a first oligomer that hybridizes to SEQ ID NO: 1249 or 1254 and second oligomer that hybridizes to a regulatory region of a second gene. In some embodiments, the oligomer includes an oligomer of SEQ ID NOs 1250-1254 and 1267-1477 or the complements thereof, and an oligomer that hybridizes to SEQ ID NO:1, SEQ ID NO:282, SEQ ID NO:462, SEQ ID NO:936, or SEQ ID NO: 1081 or the complement thereof. In other embodiments, the oligomer includes SEQ ID NO 1250 or 1251 or the complement thereof and an oligomer that hybridizes to SEQ ID NO:1, SEQ ID NO:282, SEQ ID NO:462, SEQ ID NO:936, or SEQ ID NO: 1081 or the complement thereof. In yet other embodiments, the oligomer includes SEQ ID NO: 1250 or 1251 or the complement thereof and any of SEQ ID NOs 2-281, 283-461, 463-935, 937-1080 and 1082-1248, or the complement thereof.

[0174] The oligonucleotides may also include oligonucleotides in which one or more of the cytosine bases are 5-methylcytosine. In some embodiments, the oligonucleotides are between 15 and 35 base pairs in length.

B. Oligonucleotide Structure

[0175] The oligonucleotides comprise nucleotide units that include one or more LNA or LNA analogue nucleotides. The LNAs and LNA*s comprise a sugar moiety to which is linked a nucleobase and an internucleoside linkage.

1. LNAs

[0176] The oligomeric compound comprises a compound in which the target binding domain comprises at least two LNA nucleotides or LNA analogue nucleotides. [0177] When used herein, the expression "target binding domain" refers to a domain of an oligomeric compound (or even the oligomeric compound as such) which binds to specified target sequence.

[0178] As used herein, the term "oligomeric compounds" refers to LNA oligonucleotides, i.e. ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) modified by substitution of one or more (or all) nucleotides therein with LNA nucleotides or LNA nucleotides, in particular at least two LNA nucleotides with the possible further substitution of nucleotides with LNA analogue nucleotides and nucleotide derivatives/analogues. .b||e^tf-|41I||)^"M(lϊeotide" includes oligonucleotides composed of naturally occurring nucleobases, sugars and internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly or with specific improved functions.

[0180] The oligomeric compounds to be used in the context of the present invention are 10-60 nucleotides in length, e.g. 15-40, or 20-30 nucleotides in length.

[0181] The term "nucleotides in length" refers to the length in terms of number of nucleotides upon hybridization to a linear complementary nucleic acid molecule, i.e. the total number of nucleotides of the complementary nucleic acid in the region to which the oligomeric compound is hybridized. Thus, the length of the oligomeric compound includes any intermediate nucleotides where a nucleobase is absent.

[0182] In one main embodiment, the oligomeric compounds (LNA oligonucleotides) of the invention comprise at least two LNA nucleotides.

[0183] In a further embodiment, the oligomeric compounds (LNA oligonucleotides) of the invention comprise at least two LNA analogue nucleotides, and possibly one or more LNA nucleotides.

[0184] The term "LNA" (Locked Nucleic Acid) (or "LNA oligonucleotide") refers to an oligonucleotide containing one or more bicyclic, tricyclic or polycyclic nucleoside analogues also referred to as LNA nucleotides and LNA nucleotide analogues.

[0185] LNA oligonucleotides, LNA nucleotides and LNA analogue nucleotides are generally described in International Publication No. WO 99/14226 and subsequent applications; International Publication Nos. WO 00/56746, WO 00/56748, WO 00/66604, WO 01/25248, WO 02/28875, WO 02/094250, WO 03/006475; U.S. Patent Nos. 6,043,060, 6268490, 6770748, 6639051, and U.S. Publication Nos. 2002/0125241, 2003/0105309, 2003/0125241, 2002/0147332, 2004/0244840 and 2005/0203042, all of which are incorporated herein by reference. LNA oligonucleotides and LNA analogue oligonucleotides are commercially available from, for example, Proligo LLC, 6200 Lookout Road, Boulder, CO 80301 USA. [0186] In the context of the present application and claims, the inventors differentiate between "LNA nucleotides" and "LNA* analogue nucleotides". An "LNA nucleotide" is a nucleotide of formula 1

wherein:

B constitutes a nucleobase;

Z* is selected from an internucleoside linkage and a terminal group; and Z is selected from a bond to the internucleoside linkage of a preceding nucleotide/nucleoside and a terminal group, provided that only one of Z and Z* can be a terminal group.

[0187] Nucleobases comprise naturally occurring nucleobases as well as non-naturally occurring nucleobases. Illustrative examples of such nucleobases include without limitation adenine, cytosine, 5-methylcytosine, isocytosine, pseudoisocytosine, guanine, thymine, uracil, 5-bromouracil, 5-propynyluracil, 5-propynylcytosine, 5-propyny-6-fluoroluracil, 5- methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 8-azaguanine, 8-azaadenine, 7-propyne-7- deazaadenine, 7-propyne-7-deazaguanine, 2-chloro-6-aminopurine, 4-acetylcytosine, 5- hydroxymethylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, 5- (carboxyhydroxyl-methyl) uracil, 5-fluorouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5- carboxymethylaminomethyluracil, dihydrouracil, N6-isopentenyladenine, 1-methyladenine, 1- methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, N6-methyladenine, 7-methylguanine and other alkyl derivatives of adenine and guanine, 2-propyl adenine and other alkyl derivatives of adenine and guanine, 2-aminoadenine, 5-methylaminomethyluracil, 5-methoxyarninomethyl-2- tliiouracil, beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2- methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 2-thiothymine, 5-halouracil, 5- halocytosine, 6-azo uracil, cytosine and thymine, 5-methyl-2-thiouracil, 2-thiouracil, 4- thiouracil, 5-methyluracil, 8-halo, 8-amino, 8-thiol, 8-hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl uracil and cytosine, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, queosine, xanthine, hypoxanthine, 2-thiocytosine, 2,6- diaminopurine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. [0188] The internucleoside linkage Z* means an internucleoside linkage to a succeeding nucleotide/nucleoside. Non-limiting examples of internucleoside linkages include -0-P(O)₂- O-, -0-P(O₅S)-O-, -0-P(S)₂-O-, -S-P(O)₂-O-, -S-P(O₅S)-O-, -S-P(S)₂-O-, -0-P(O)₂-S-, -O- P(O₅S)-S-, -S-P(O)₂-S-, -O-PO(R^H)-O-, O-PO(OCH₃)-O-, -0-P0(NR^H)-0-, -O- tPO^de^GH₂S ^jft)Φ^^i;©tPO(BH₃)-O-, -O-PO(NHR^H)-O-, -O-P(O)₂-NR^H, -NR^H-P(O)₂-O-₃ - NR^H-CO-O-₅ -NR^H-CO-NR^H-, -O-CO-O-, -O-CO-NR^H-, -NR^H-CO-CH₂-, -O-CH₂-CO-NR^H-, - O-CH₂CH₂NR^H, -CO-NR^H-CH₂-, -CH₂NR^H-CO-, -0-CH₂-CH₂-S-, -S-CH₂-CH₂-O-, -S-CH₂- CH₂-S-, -CH₂-SO₂-CH₂-, -CH₂-CO-NR^H-, -O-CH₂-CH₂-NR^H-CO-, -CH₂-NCH₃-O-CH₂-, where R^H is selected from hydrogen and C_1-4-alkyl and C₁ to C₄ substituted halo, -0-P(O)₂-R, where R is selected from alkaryl or araalkyl, and combinations thereof. [0189] In the present context, the term "C_1-4-alkyl" is intended to mean a linear or branched saturated hydrocarbon chain wherein the chain has from one to four carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. [0190] The internucleoside linkage Z* can also include cycloalkyl groups, mixed heteroatom and alkyl or cycloalkyl groups, heterocyclic groups, siloxane groups, methyleneimino, methylenehydrazino and amide groups.

[0191] When the LNA nucleotide is the 5'-terminal nucleotide of the oligomeric compound, Z* is a terminal group; and if the LNA nucleotide is the 3 '-terminal nucleotide of the oligomeric compound, Z is a terminal group. Such terminal groups are typically selected from hydrogen, azido, halogen, cyano, nitro, hydroxy, Prot-O-, Act-O-, mercapto, Prot-S-, Act-S-, C_1-6- alkylthio, amino, Prot-N(R^H)-, Act-N(R^H)-, mono- or di(C_1-6-alkyl)amino, optionally substituted Ci_-6-alkoxy, optionally substituted C_1-6-alkyl, optionally substituted C_2-6-alkenyl, optionally substituted C_2-6-alkenyloxy, optionally substituted C_2-6-alkynyl, optionally substituted C_2-6-alkynyloxy, monophosphate, monothiophosphate, diphosphate, dithiophosphate triphosphate, trithiophosphate, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulphono, hydroxymethyl, PrOt-O-CH₂-, Act-O-CH₂-, aminomethyl, Prot-N(R^H)-CH₂-, Act- N(R^H)-CH₂-, carboxymethyl, and sulphonomethyl, where Prot is a protection group for -OH, - SH, and -NH(R^H), respectively, Act is an activation group for -OH, -SH, and -NH(R^H), respectively, and R^H is selected from hydrogen and C_1-6-alkyl.

[0192] Protection groups (Prot) of hydroxy (and sulphur) substituents comprise substituted trityl, such as 4,4'-dimethoxytrityloxy (DMT), 4-monomethoxytrityloxy (MMT), and trityloxy, optionally substituted 9-(9-phenyl)xanthenyloxy (pixyl), optionally substituted methoxytetra- hydropyranyloxy (mthp), silyloxy such as trimethylsilyloxy (TMS), triisopropylsilyloxy (TIPS), tert-butyldimethylsilyloxy (TBDMS), triethylsilyloxy, and phenyldimethylsilyloxy, tert-butylethers, acetals (including two hydroxy groups), acyloxy such as acetyl or halogen substituted acetyls, e.g. chloroacetyloxy or fluoroacetyloxy, isobutyryloxy, pivaloyloxy, l%ή|#f|c||^;|nd%|ιl|M|u!t|4^enzoyls, methoxymethyloxy (MOM), and benzyl ethers or substituted benzyl ethers such as 2,6-dichlorobenzyloxy (2,6-Cl₂BzI). [0193] Illustrative examples of protecting groups of amino and amido groups are without limitation fluorenylmethoxy-carbonylamino (Fmoc), tert-butyloxycarbonylam-ino (BOC), trifluoroacetylamino, allyloxycarbonylamino (alloc, AOC), Z benzyloxycarbonylamino (Cbz), substituted benzyloxycarbonylaminos such as 2-chloro benzyloxycarbonylamino (2-ClZ), monomethoxytritylamino (MMT), dimethoxytritylamino (DMT), phthaloylamino, and 9-(9- phenyl)xanthenylamin-o-(pixyl).

[0194] The group "Act" designates an activation group for -OH, -SH, and -NH(R^H), respectively, for coupling to further nucleotides, solid phases, proteins, etc. In the embodiment above, Act designates an activation group. Such activation groups are, e.g., selected from optionally substituted 0-phosphoramidite, optionally substituted 0-phosphotriester, optionally substituted O-phosphodiester, optionally substituted H-phosphonate, and optionally substituted O-phosphonate. In the present context, the term "phosphoramidite" means a group of the formula -P(OR^x)-N(R^y)₂, wherein R^x designates an optionally substituted alkyl group, e.g. methyl, 2-cyanoethyl, or benzyl, and each of R^y designate optionally substituted alkyl groups, e.g. ethyl or isopropyl, or the group -N(R^y)₂ forms a morpholino group (-N(CH₂CH₂)₂O). R^x in some embodiments designates 2-cyanoethyl and the two R^y are preferably identical and designate isopropyl. Thus, for example, a relevant phosphoramidite is N,N-diisopropyl-O-(2- cyanoethyl)phosphoramidite.

[0195] In addition to the LNA [2'-O,4'-C-methylene-β-D-ribofuranosyl] monomers depicted by formula 1 (a [2,2,1] bicycle nucleoside), an LNA nucleotide can also include "locked nucleic acids" with other furanose or other sugars and/or with a different monomer formulation, including 2'-0,3' linked and 3 '-0,4' linked, V -0,3' linked, l'-0,4' linked, V- 0,5' linked, 2'-O, 5'linked, 1 '-0,2' linked bicyclonucleosides and others. One example is the LNA [3'-0-4'-C-methylene-β-ribofuranosyl] monomer described in U.S. Patent No. 6.043,060, which is herein incorporated by reference. The 3-4 linkage as well as the 2-3, 1-3, 1-4, 2-5, 3-5 and 1-2 linkages can also be obtained with arabinose, L-ribose and xylose, resulting, for example, in LNA [3'-O-4'-C-methylene (or ethyl ene)-β (or α)-arabino-, xylo- or L-ribo-furanosyl] monomers. Examples are illustrated in Scheme 1 below. [0196] The term "LNA analogue nucleotide" refers to bicyclic, tricyclic or polycyclic nucleotide analogues as those generally described in International Publication No. WO 99/14226 and subsequent applications; International Publication Nos. WO 00/56746, WO 00/56748, WO 00/66604, WO 01/25248, WO 02/28875, WO 2002/094250, WO 03/006475;

f&SUfttø^≠ϊ6ffl°8β&&626&490, 6770748 and U.S. Publication Nos. 2002/0125241, 2003/0105309, 2003/0125241, 2002/0147332, 2004/0244840 and 2005/0203042, excluding, however, the already described "LNA nucleotides". [0197] Examples of LNA analogue nucleotides are exemplified in Formula 2:

2 [0198] wherein:

X and Y are independently selected from -O-, -S-, -N(H)-, -N(R)-, -CH₂- or -C(H)=, CH₂-O-, -CH₂-S-, -CH₂-N(H)-, -CH₂-N(R)-, -CH₂-CH₂- or -CH₂-C(H)=, -CH=CH- ; [0199] R is selected from hydrogen and C_1-4-alkyl; and [0200] B, Z* and Z are as previously described; [0201] provided that X and Y are not both O. [0202] The asymmetric groups may be found in either orientation.

[0203] In some embodiments, X is oxygen and Y is selected from -O-, -S-, -N(H)- and -N(R)-. [0204] The oligomeric compounds of the invention may further carry Z and Z* groups as those defined for the LNA nucleotides.

[0205] In Formula 2, the four chiral centers are shown in a fixed configuration. However, the invention contemplates compounds of the general Formula 2 in which the chiral centers are found in different configurations. Thus, each chiral center in Formula 2 can exist in either R or S configuration. The definition of R (rectus) and S (sinister) are described in the IUPAC 1974 Recommendations, Section E, Fundamental Stereochemistry: The rules can be found in Pure Appl. Chem. 45, 13-30, (1976) and in "Nomenclature of organic Chemistry" Pergamon, N. Y., 1979.

[0206] Particular examples of "LNA analogue nucleotides" are illustrated in by formulae I, II, III, IV, V, and VI:

[0207] One example is the "thio-LNA" nucleotide, i.e. an LNA analogue nucleotide in which at least one of X in Formulae I, III, IV or VI is selected from -S- or -CH₂-S-. Such thio-LNA can be in both beta-D-configuration (I and FV) and alpha-L-configuration (III and VI), respectively.

[0208] Another example is the "amino-LNA" nucleotide, i.e. an LNA analogue nucleotide in which at least one of X in Formulae I, III, IV or VI is selected from -N(H)-, -N(R)-, -CH₂-

N(H)-, -CH₂-N(R)-, where R is selected from hydrogen and C_1-4-alkyl. Such amino-LNA can be in both the beta-D-configuration (I and IV) and alpha-L-confϊguration (III and VI), respectively.

[0209] A further example is the "ena-LNA" nucleotide, i.e. an LNA analogue nucleotide in which at least one of X in Formulae II or V is -CH₂-O-.

[0210] In yet another embodiment, the oligomeric compound comprises an "alpha-L-LNA"

(i.e. "beta.-L-LNA") nucleotide, i.e. an LNA nucleotide as shown in Formulae III and VI.

[0211] In addition to the LNA [2'-7,4'-C-methylene-β-D-ribofuranosyl] monomers depicted by formula 2 (a [2,2,1] bicyclo nucleoside), an LNA or LNA* nucleotide can also include

_t luu^cHjrJiijmjtPic a^ijytjs ywiiuijuiucr furanose or other 5 or 6-membered rings and/or with a different monomer formulation, including 2'-7,3' linked and 3'-7,4' linked, V-Y,3 linked, 1'- 7,4' linked, 3 '-7,5' linked, T-Y, 5'linked, l'-7,2' linked bicyclonucleosides and others. All the above mentioned LNAs can be obtained with different chiral centers, resulting, for example, in LNA [3'-7-4'-C-methylene (or ethylene)-β (or α)-arabino-, xylo- or L-ribo- furanosyl] monomers. Examples are illustrated in Scheme 1.

Scheme 1

[0212] Wherein X, Y, Z, Z* and B are defined as for Formula 2. [0213] In some embodiments, the oligomeric compounds comprise LNA and/or LNA* nucleotides in combination with nucleotides that are not LNA nucleotides. Such nucleotides include, for example, deoxyribonucleotides (DNA nucleotides), ribonucleotides (RNA nucleotides), nucleotide derivatives, nucleotide analogues (other than LNA), and Peptide Nucleic Acid (PNA) units.

[0214] Nucleotide analogues and nucleotide derivatives are described in e.g. Freier & Altmann (Nucl. Acid Res., 1997, 25, 4429-4443), Kurreck (Eur. J. Biochem, 2003, 270, 1628-44) and Uhlmann (Curr. Opinion in Drug & Development (2000, 3(2): 293-213), which are incorporated herein by reference. The nucleotide analogues and derivatives include nucleotides with modified backbones or non-natural nucleoside linkages described above, nucleotides containing base modifications described above, and nucleotides containing substituted sugar moieties or sugar mimetics.

[0215] Oligonucleotides can also have sugars other than ribose and deoxy ribose, including arabinofuranose (described in International Publication number WO 99 '/ '67378, which is herein incorporated by reference), xyloarabinofuranose (described in U.S. Patent Nos 6,316,612 and 6,489465, which are herein incorporated by reference), α-threofuranose (Schoning, et al. (2000) Science, 290, 1347-51, which is herein incorporated by reference) and L-ribofuranose. Sugar mimetics can replace the sugar in the nucleotides. They include cyclohexene (Wang et al.(2000) J. Am. Chem. Soc. 122, 8595-8602; Vebeure et al. Nucl. Acids Res. (2001) 29, 4941- |4$4?ϋf WUMk'∞e' %^UWMόδkporated by reference), a tricyclo group (Steffens, et al. J. Am. Chem. Soc. (1997) 119, 11548-11549, which is herein incorporated by reference), a cyclobutyl group, ahexitol group (Maurinsh, et al. (1997) J. Org. Chem, 62, 2861-71; J. Am. Chem. Soc. (1998) 120, 5381-94, which are herein incorporated by reference), an altritol group (Allart, et al., Tetrahedron (1999) 6527-46, which is herein incorporated by reference), a pyrrolidine group (Scharer, et al., J. Am. Chem. Soc, 117, 6623-24, which is herein incorporated by reference), carbocyclic groups obtained by replacing the oxygen of the furnaose ring with a methylene group (Froehler and Ricca, J. Am. Chem. Soc. 114, 8230-32, which is herein incorporated by reference) or with an S to obtain 4'-thiofuranose (Hancock, et al., Nucl. Acids Res. 21, 3485-91, which is herein incorporated by reference), and/or morpholino group (Heasman, (2002) Dev. Biol., 243, 209-214, which is herein incorporated by reference) in place of the pentofuranosyl sugar. Morpholino oligonucleotides are commercially available from Gene Tools, LLC (Corvallis Oregon, USA).

[0216] The nucleotide derivatives can include nucleotides containing one of the following at the 2' sugar position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl maybe substituted or unsubstituted Ci to Cio ^alky* ^{or C}2 to Cio alkenyl and alkynyl, O[(CH2)_nO]_mCH3, O(CH2)_nOCH3, O(CH₂)nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH2)_nON[(CH₂)_nCH₃)]₂, where n and m are from 1 to about 10, Ci to Cio lower 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, 2'-methoxyethoxy (2'-0-CH₂CH₂OCH₃, also known as 2'-O-

(2-methoxyethyl) or 2'-MOE) (Martin et αl, HeIv. Chim. Acta 78:486 [1995]) i.e., an alkoxyalkoxy group, 2'-dimethylaminooxyethoxy {i.e., an O(CH₂)₂ON(CH₃)₂ group), also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O- dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH₂-O-CH₂-N(CH₂)₂, 2'-methoxy (T-

O-CH₃), 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. Scheme 2 Illustrates selected examples hereof.

2'-Fluoro

2'-Fluoro arabino nucleic acid XyIo nucleic acid Tricyclo nucleic acid Peptide nucleic acid

nucleic acid

Scheme 2

[0217] Z and Z* are as described for formula 2.

[0218] In some embodiments of the invention, the oligonucleotide compounds hybridize to regulatory regions of oncogenes and comprise from 10 to 60 nucleotides wherein one or more nucleotides are LNA or LNA* nucleotides.

[0219] In other embodiments wherein the oligonucleotide is comprised of 15 to 40 nucleotides wherein one or more nucleotides are LNA or LNA* nucleotides.

[0220] In other embodiments wherein the oligonucleotide is comprised of 20 to 30 nucleotides wherein one or more nucleotides are LNA or LNA* nucleotides.

[0221] In some embodiments the nucleotide is comprised of 10 to 15 nucleotides wherein the number of LNA or LNA* nucleotides is from 1 to 15. '[0232¹Jr₁HL₁HPmIe %Mh^~σ3irllπCs the nucleotide is comprised of 15 to 40 nucleotides wherein the number of LNA or LNA* nucleotides is from 1 to 16.

[0223] In some embodiments the nucleotide is comprised of 20 to 30 nucleotides wherein the number of LNA or LNA* nucleotides is from 1 to 12.

[0224] It will be recognized that the LNA and LNA* nucleotides may be located within the nucleotide in a plurality of ways. For example, up to 10 or more LNA and LNA* nucleotides may be sequential. The LNA and LNA* nucleotides may be separated by one or more DNA or

RNA nucleotides. In some embodiments, from 1 to 3 LNA and LNA* nucleotides are sequential.

[0225] In some embodiments the nucleotide is comprised of 15 to 40 nucleotides wherein the number of LNA or LNA* nucleotides is 4. For example, the oligomer includes 2 pair of LNA or LNA* nucleotides. In one embodiment, one pair is at the 3' terminus. In another embodiment, one pair is at the 5' terminus. In another embodiment one pair is at the 3' terminus and the other pair is at the 5' terminus.

2. Other Structures

[0226] While oligonucleotides are one form of compound, the present invention comprehends other oligomeric oligonucleotide compounds, including but not limited to oligonucleotide mimetics such as are described below. The oligonucleotide compounds in accordance with this invention preferably comprise from about 18 to about 30 nucleotides (i.e., from about 18 to about 30 linked bases), although both longer and shorter sequences may find use with the present invention.

[0227] Specific examples of compounds useful with the present 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, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0228] Modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5^f linkages, 2'-5' linked W f&ijaaistglϊ^ϊfltesef-lMttiδS^ϊiaving 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.

[0229] 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 CH2 component parts.

[0230] In other 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 nucleotides are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science 254:1497 (1991) and Neilsen, Methods in Enzymology, 313, 156-164 (1999). PNA compounds can be obtained commercially, for example, from Applied Biosystems (Foster City, CA, USA).

[0231] In some embodiments, oligonucleotides of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular -CH2, -NH-O-CH2-, -CE^-NtCE^-O-CH^- [known as a methylene (methylimino) or MMI backbone], -CH₂-O-N(CH₃)-CH₂-, -CH₂-N(CH3)-N(CH3)-CH₂-, and -0-N(CH₃)- CH₂-CH₂- [wherein the native phosphodiester backbone is represented as -0-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. hi some embodiments, oligonucleotides have a morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506. a 5SJJU:ϊ€ISfiέ(IM!g<|i.%feβ)tides may also contain one or more substituted sugar moieties. In some embodiments, oligonucleotides comprise one of the following at the 2' position: OH; F; O-, S-, orN-alkyl; O-, S-, orN-alkenyl; O-, S- orN-alkynyl; or O-alkyl-0-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cj to Cj Q alkyl or C₂ to CJQ alkenyl and alkynyl. Other embodiments include O[(CH2)_nO]_mCH3, O(CH2)_nOCH3, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH₂)_nON[(CH₂)_nCH₃)]₂, where n and m are from 1 to about 10. Yet other oligonucleotides comprise one of the following at the 2' position: C\ to C\ Q lower 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. One modification includes 2'-methoxyethoxy (2'-0-CH₂CH₂OCH₃, also known as

2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al, HeIv. Chim. Acta 78:486 [1995]) i.e., an alkoxyalkoxy group. A further modification includes 2'-dimethylaminooxyethoxy (i.e., an O(CH₂)₂ON(CH₃)₂ group), also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy

(also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-0-CH₂-O-

CH₂-N(CH₂)₂.

[0233] Other modifications include 2'-methoxy (2'-0-CH₃), 2'-aminopropoxy(2'-

OCH₂CH₂CH₂NH₂) and 2'-fiuoro (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.

[0234] Oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine laμdlllldiϊMSr^-WaSφlMaouracil), 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. 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 0.6-1.2⁰C, and are sometimes combined with 2'-0-methoxyethyl sugar modifications.

[0235] In some embodiments, the present invention provides oligonucleotide therapeutics that are methylated at specific sites. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, it is contemplated that one mechanism for the regulation of gene activity is methylation of cytosine residues in DNA. 5-methylcytosine (5-MeC) is the only naturally occurring modified base detected in DNA (Ehrlick et al, Science 212:1350-1357 (1981)). Although not all genes are regulated by methylation, hypomethylation at specific sites or in specific regions in a number of genes is correlated with active transcription (Doerfler, Annu. Rev. Biochem. 52:93-124 [1984]; Christman, Curr. Top. Microbiol. Immunol. 108:49-78 [1988]; Cedar, Cell 34:5503-5513 [1988]). DNA methylation in vitro can prevent efficient transcription of genes in a cell-free system or transient expression of transfected genes. Methylation of C residues in some specific cis-regulatory regions can also block or enhance binding of transcriptional factors or repressors (Doerfler, supra; Christman, supra; Cedar, Cell 34:5503-5513 (1988); Tate et al, Curr. Opin. Genet. Dev. 3:225-231 [1993]; Christman et al, Virus Strategies, eds. Doerfler, W. & Bohm, P. (VCH, Weinheim, N. Y.) pp. 319-333 [1993]). [0236] Disruption of normal patterns of DNA methylation has been linked to the development of cancer (Christman etal, Proc. Natl. Acad. Sci. USA 92:7347-7351 [1995]). The 5-MeC content of DNA from tumors and tumor derived cell lines is generally lower than normal tissues (Jones et al, Adv. Cancer Res 40:1-30 [1983]). Hypomethylation of specific oncogenes such as c-myc, c-Ki-ras and c-Ha-ras has been detected in a variety of human and animal tumors (Nambu et al, Jpn. J. Cancer (Gann) 78:696-704 [1987]; Feinberg et al, Biochem. Biophys. Res. Commun. 111:47-54 [1983]; Cheah etal, JNCI73: 1057- 1063 [1984]; Bhave et al, Carcinogenesis (Lond) 9:343-348 [1988]. In one of the best studied examples of human fumlf 'pydgmssfoijf,-S hasMbfen shown that hypomethylation of DNA is an early event in development of colon cancer (Goetz et al, Science 228:187-290 [1985]). Interference with methylation in vivo can lead to tumor formation. Feeding of methyl ation inhibitors such as L- methionine or 5-azacytodine or severe deficiency of 5-adenosine methionine through feeding of a diet depleted of lipotropes has been reported to induce formation of liver tumors in rats (Wainfan et al, Cancer Res. 52:2071s-2077s [1992]). Studies show that extreme lipotrope deficient diets can cause loss of methyl groups at specific sites in genes such as c-myc, ras and c-fos (Dizik et al, Carcinogenesis 12:1307-1312 [1991]). Hypomethylation occurs despite the presence of elevated levels of DNA MTase activity (Wainfan et al, Cancer Res. 49:4094-4097 [1989]). Genes required for sustained active proliferation become inactive as methylated during differentiation and tissue specific genes become hypomethylated and are active. Hypomethylation can then shift the balance between the two states, hi some embodiments, the present invention thus takes advantage of this naturally occurring phenomena, to provide compositions and methods for site specific methylation of specific gene promoters, thereby preventing transcription and hence translation of certain genes, hi other embodiments, the present invention provides methods and compositions for upregulating the expression of a gene of interest (e.g., a tumor suppressor gene) by altering the gene's methylation patterns. [0237] The present invention is not limited to the use of methylated oligonucleotides. Indeed, the use of non-methylated oligonucleotides for the inhibition of gene expression is specifically contemplated by the present invention. Experiments conducted during the course of development of the present invention (See e.g., Example 8) demonstrated that an unmethylated oligonucleotide targeted toward Bcl-2 inhibited the growth of lymphoma cells to a level that was comparable to that of a methylated oligonucleotide.

[0238] Another modification of the oligonucleotides of the present invention involves chemically linking to the oligonucleotide one or more moieties or conjugates that 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, cholic acid, a thioether, {e.g., hexyl-S-tritylthiol), a thiocholesterol, an aliphatic chain, {e.g., dodecandiol or undecyl residues), a phospholipid, {e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O- hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or a polyethylene glycol chain or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl- oxycholesterol moiety. I lCT23S]!ri3Mskillfe_!3Sj&%fei'rt!jevant art knows well how to generate oligonucleotides containing the above-described modifications. The present invention is not limited to the oligonucleotides described above. Any suitable modification or substitution may be utilized. [0240] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes pharmaceutical compositions and formulations that include the antisense compounds of the present invention as described below.

C. Preparation and Formulation of Oligonucleotides

[0241] Any of the known methods of oligonucleotide synthesis can be used to prepare the modified oligonucleotides of the present invention. In some embodiments utilizing methylated oligonucleotides the nucleotide, dC is replaced by 5-methyl-dC where appropriate, as taught by the present invention. The modified or unmodified oligonucleotides of the present invention are most conveniently prepared by using any of the commercially available automated nucleic acid synthesizers. They can also be obtained from commercial sources that synthesize custom oligonucleotides pursuant to customer specifications.

III. USES

A. Research

[0242] The present invention is not limited to therapeutic applications. For example, in some embodiments, the present invention provides compositions and methods for the use of oligonucleotides as a research tool.

B. Kits

[0243] In some embodiments, the present invention provides kits comprising oligonucleotides specific for inhibition of a gene of interest, and optionally cell lines (e.g., cancer cells lines) known to express the gene. Such kits find use, for example, in the identification of metabolic pathways or the involvement of genes in disease (e.g., cancer), as well as in diagnostic applications. In some embodiments, the kits further comprise buffer and other necessary reagents, as well as instructions for using the kits.

C. Target validation

[0244] In some embodiments, the present invention provides methods and compositions for use in the validation of gene targets (e.g., genes suspected of being involved in disease). For example, in some embodiments, the expression of genes identified in broad screening applications (e.g., gene expression arrays) as being involved in disease is downregulated using the methods and compositions of the present invention. The methods and compositions of the

for use in vitro and in vivo (e.g., in a non-human animal) for the purpose of target validation, hi other embodiments, the compounds of the present invention find use in transplantation research (e.g., HLA inhibition).

D. Drug Screening

[0245] In other embodiments, the methods and compositions of the present invention are used in drug screening applications. For example, in some embodiments, oligonucleotides of the present invention are administered to a cell (e.g., in culture or in a non-human animal) in order to inhibit the expression of a gene of interest. In some embodiments, the inhibition of the gene of interest mimics a physiological or disease condition. In other embodiments, an oncogene is inhibited. Test compounds (e.g., small molecule drugs or oligonucleotide mimetics) are then administered to the test cell and the effect of the test compounds is assayed. [0246] The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone, which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckennann et al, J. Med. Chem. 37: 2678-85 [1994]); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are sometimes used for use with peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).

[0247] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt etal, Proc. Natl. Acad. Sci. U.S.A. 90:6909 [1993]; Erb et al, Proc. Nat. Acad. Sci. USA 91:11422 [1994]; Zuckermann et al, J. Med. Chem. 37:2678 [1994]; Cho et al, Science 261:1303 [1993]; Carrell et al, Angew. Chem. Int. Ed. Engl. 33.2059 [1994]; Carell et al, Angew. Chem. Int. Ed. Engl. 33:2061 [1994]; and Gallop et al, J. Med. Chem. 37:1233 [1994].

[0248] Libraries of compounds may be presented in solution {e.g., Houghten, Biotechniques 13:412-421 [1992]), or on beads (Lam, Nature 354:82-84 [1991]), chips (Fodor, Nature 364:555-556 [1993]), bacteria or spores (U.S. Patent No. 5,223,409; herein incorporated by reference), plasmids (Cull et al, Proc. Nat. Acad. Sci. USA 89:18651869 [1992]) or on phage (Scott and Smith, Science 249:386-390 [1990]; Devlin Science 249:404-406 [1990]; Cwirla et al, Proc. Natl. Acad. Sci. 87:6378-6382 [1990]; Felici, J. MoI. Biol. 222:301 [1991]). [0249] In some embodiments, the oligonucleotide compounds of the present invention are formulated as pharmaceutical compositions for delivery to a subject as a pharmaceutical. The novel antigen compounds of the present invention find use in the treatment of a variety of disease states and conditions in which it is desirable to inhibit the expression of a gene or the growth of a cell. In some embodiments, the compounds are used to treat disease states resulting from uncontrolled cell growth, for example including, but not limited to, cancer. The present invention is not limited to the treatment of a particular cancer. The oligonucleotide compounds of the present invention are suitable for the treatment of a variety of cancers including, but not limited to, breast, colon, lung, stomach, pancreatic, bladder, leukemia and lymphoma. The below discussion provides exemplary, non-limiting examples of formulations and dosages.

A. Pharmaceutical Compositions

[0250] The present invention further provides pharmaceutical compositions (e.g., comprising the oligonucleotide compounds described above). The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, intraocular, epidermal and transdermal), oral, via a medical device or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

[0251] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0252] Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. [0253] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. if^S^ϊllfflagόifiSlalMΦIi'ositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

[0255] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0256] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. [0257] In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. [0258] Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (WO 97/30731), also enhance the cellular uptake of oligonucleotides.

[0259] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the fcΛjMSCMIs rfttSrpHI&Envention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation. [0260] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In one embodiment, oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. In other embodiments, surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Prefered bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro- fusidate, sodium glycodihydrofusidate. Prefered fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1- monocaprate, l-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also prefered are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly prefered combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9- lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. In one embodiment, complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE- hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,

olfU^wlgφ/WQl fiplffC^tUactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG).

[0261] Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more oligonucleotide compounds and (b) one or more other chemotherapeutic agents that function by a non-oligonucleotide mechanism. Examples of such chemotherapeutic agents include, but are not limited to, anticancer drugs such as daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin and diethylstilbestrol (DES). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Other non-oligonucleotide chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

B. Delivery

[0262] The oligonucleotide compounds of the present invention may be delivered using any suitable method. In some embodiments, naked DNA is administered. In other embodiments, lipofection is utilized for the delivery of nucleic acids to a subject. In still further embodiments, oligonucleotides are modified with phosphothiolates for delivery (See e.g., U.S. Patent 6,169,177, herein incorporated by reference).

[0263] In some embodiments, nucleic acids for delivery are compacted to aid in their uptake (See e.g., U.S. Patents 6,008,366, 6,383,811 herein incorporated by reference). In some embodiments, compacted nucleic acids are targeted to a particular cell type (e.g., cancer cell) via a target cell binding moiety (See e.g., U.S. Patents 5,844,107, 6,077,835, each of which is herein incorporated by reference).

[0264] In some embodiments, oligonucleotides are conjugated to other compounds to aid in their delivery. For example, in some embodiments, nucleic acids are conjugated to polyethylene glycol to aid in delivery (See e.g., U.S. Patents 6,177,274, 6,287,591, 6,447,752, 6,447,753, and 6,440,743, each of which is herein incorporated by reference). In yet other embodiments, oligonucleotides are conjugated to protected graft copolymers, which are chargeable" drug nano-carriers (Pharmaln), described in U.S. Patent Number 7.138,105, and U.S. publication numbers 2006/093660 and 2006/0239924, which are incorporated herein by reference. In still further embodiments, the transport of oligonucleotides into cells is facilitated W

't)y/ctoj,«gti5α.fd^f{|fnϊnlCEndocyte, Inc, West Lafayette, IN; See e.g., U.S. Patents 5,108,921, 5,416,016, 5,635,382, 6,291,673 and WO 02/085908; each of which is herein incorporated by reference). In other embodiments, oligonucleotides are conjugated to nanoparticles (e.g., NanoMed Pharmaceuticals; Kalamazoo, MI). [0265] In still other embodiments, oligonucleotides are associated with dendrimers. Dendrimers are synthetic macromolecules with highly branched molecular structures. Representative dendrimeric structures are cationic polymers such as starburst polyamidoamine (PAMAM), one of which, SuperFect^®, is available from Qiagen (Valencia, CA). Other dendrimers include polyester dentrimers described by Gillies, et al., MoI. Pharm., 2:129-38, 2005, which is incorporated herein by reference; phenylacetylene dendrimers, described in Janssen and Meijer, eds, Synthesis of Polymers, Materials science and technology series, Weinheim, Germany: Wiley- VCH Verlag GMBH, Chapter 12, 1999, which is incorporated herein by reference; poly(L-lysine) dendrimer-block-poly(ethylene glycol)-block-poly(L- lysine) dendrimers described by Choi, et al., J. Am. Chem. Soc. 122, 474-80, 2000, which is incorporated herein by reference; amphiphilic dendrimers, described by Joester, et al., Angew Chem Int. Ed. Engl, 42:1486-90, 2003, which is incorporated herein by reference; polyethylene glycol star like conjugates, described by Liu et al., Polym Chem, 37:3492-3503, 1999, which is incorporated herein by reference; cationic phosphorus-containing dendrimers described by Loup, et al., Chem Eur J, 5:3644-50, 1999, which is incorporated herein by reference; poly(L-lysine) dendrimers, described by Ohasaki, et al., Bioconjug Chem, 13:510- 17, 2002, which is incorporated herein by reference and amphipathic asymmetric dendrimers, described by Shah, et al., Int. J. Pharm, 208:41-48, 2000, which is incorporated herein by reference. Polypropylene imine dendrimers, described in Tack, et al., J. Drug Traget, 14;69- 86, 2006, which is incorporated herein by reference; and other dendrimers described above, can be chemically modified to reduce toxicity, for example, as described in Tack, et al. [0266] Dendrimers complex with nucleic acids as do other cationic polymers with high charge density. In general, the dendrimer-nucleic acid interaction is based on electrostatic interactions. Dendrimers can be conjugated with other molecules, such as cyclodextrins to increase efficiency of systemic delivery of dendrimer-nucleic acid complexes. (See Dufes, et al., Adv. Drug Del. Rev, 57, 2177-2202, 2005, and Svenson and Tomalia, Adv. Drug Del. Rev., 57, 2106-29, 2005, both of which are incorporated herein by reference.) Some dendrimers have a flexible open structure that can capture small molecules in their interior, and others have an inaccessible interior. (See Svenson and Tomalia, Adv. Drug Del. Rev., 57, 2106-29, 2005.) % 02d5jS5f Jtøh#Mi>€SϊAiiήits, oligonucleotides are sequestered in polymer vesicles. Polymer vesicles can be made from a number of different materials, but in general are formed from block copolymers, for example, polystyrene₄o-poly(isocyano-L-alanine-L-alanine)_m. (See for example, Discher, et al., Science, 297:967-73, 2002; Torchilin, Cell. MoI. Life Sci, 61:2549- 59, 2004; Taubert, et al., Curr Opin Chem Biol, 8:598-603, 2004; Lee, et al., Pharm. Res., 22:1-10, 2005; and Gaucher, et al., J. Control. ReI, 109:169-88, 2005, each of which is incorporated herein by reference.) Copolymer vesicles are formed from a number of molecules, including, without limitation, polyacrylic acid-polystyrene, nonionic polyethyleneoxide-polybutadiene, the triblock (polyethyleneoxide)₅-(poly[propyleneoxide])₆₈- (polyethyleneoxide)₅, polyethyleneoxide-poly(propylenesulfide), polyethyleneoxide- polylactide, and polyethylene glycol-polylysine. Many copolymers, particularly those of either amphiphilic or oppositely charged copolymers, including polystyrene₄o-poly(isocyano-L- alanine-L-alanine)_mi self assemble into vesicles in aqueous conditions.

[0268] Oligonucleotides can be loaded into polymer vesicles using several methods. First, the block copolymer can be dissolved along with the oligonucleotides in an aqueous solvent. This method works well with moderately hydrophobic copolymers. Second, for amphiphilic copolymers that are not readily soluble in water, and where a solvent that solubilizes both the oligonucleotides and the copolymer is available, the oligonucleotide and copolymer are dissolved in the solvent and the mixture is dialyzed against water. A third method involves dissolving both the oligonucleotides and copolymer in a water/tert-butanol mixture and subsequent lyophilization of the solvents. The oligonucleotide-loaded vesicles are formed spontaneously when the lyophilized oligonucleotide-copolymer is reconstituted in an injectable vehicle. (Dufresne, et al., in Gurny, (ed.), B.T. Gattefosse, vol. 96, Gattefosse, Saint-Priest, p. 87-102, 2003, which is incorporated herein by reference.)

[0269] Polymer vesicles can be targeted to specific cells by tethering a ligand to the outer shell of vesicles by post modification of a copolymer with a bifunctional spacer molecule or by the direct synthesis of heterobifunctional block copolymers.

[0270] In some embodiments, oligonucleotides are enclosed in lipids (e.g., liposomes or micelles) to aid in delivery (See e.g., U.S. Patents 6,458,382, 6,429,200; U.S Patent Publications 2003/0099697, 2004/0120997, 2004/0131666, 2005/0164963, and International Publication WO 06/048329, each of which is herein incorporated by reference). Liposomes include, without limitation, cardiolipin based cationic liposomes (e.g., NeoPhectin, available from NeoPharm, Forest Lake, IL) and pH sensitive liposomes. ^'fO.2i]|!SMI(tiSie^*'er|ιlidϊΛMis of the present invention, NeoPhectin is utilized as the liposomal delivery vehicle. In some embodiments, the NeoPhectin is formulated with the oligonucleotide so as to reduce free NeoPhectin. In other embodiments, NeoPhectin is present at a charge ratio 6:1 or less (e.g., 5:1, and 4:1) of NeoPhectin to oligonucleotide. [0272] In yet other embodiments, lipids, particularly phospholipids that comprise some liposomes, are conjugated to polyethylene glycol or a derivative thereof, to increase the time that the liposomes circulate in the blood after intravenous injection. (See e.g., Moghimi, S.M. and Szebeni, J, Prog. Lipid Res., 42:463-78, 2003 and Li, W., et al., J. Gene Med., 7:67-79, 2005, which are incorporated herein by reference.) Such liposomes, termed "stealth liposomes" are able to avoid the reticuloentothelial system (RES), resulting in half lives of more than 24 hours in some cases. In one embodiment, the phospholipids in liposomes are conjugated to polyethylene glycol-diorthoester molecules, as described in Li, W., et al., J. Gene Med., 7:67-79, 2005. In other embodiments, the PEG-liposomes are targeted to specific cell receptors. For example, haloperidol conjugated at the distal end of a PEG-linked phospholipids in a cationic liposome targeted sigma receptors that are overexpressed on some cancer cells as described in Mukherjee, et al., J. Biol. Chem., 280, 15619-27, 2005, which is incorporated herein by reference. Anisamide conjugated to PEG-linked phospholipids in liposomes also targets the sigma receptor. (Banerjee, et al., Int. J. Cancer, 112, 693-700, 2004, which is incorporated herein by reference.)

[0273] hi yet another embodiment, oligonucleotides can be sequestered in hybrid liposome- copolymer vesicles, as described in Ruysschaert, et.al., J. Am. Chem. Soc, 127, 6242-47, 2005, which is incorporated herein by reference. For example, an amphiphilic triblock copolymers, including poly(2-methyloxazoline)-block-poly(dimethylsiloxan)-block-poly(2- methyloxazoline) can interact with lipids, including phospholipids to form hybrid liposome- copolymer vesicles.

[0274] In still further embodiments, oligonucleotides are complexed with additional polymers to aid in delivery (See e.g., U.S. Patents 6,379,966, 6,339,067, 5,744,335; each of which is herein incorporated by reference. For example, polymers of N-2-hydroxypropyl methylacrylamide are described in U.S. patent publication number 2006/0014695, which is incorporated herein by reference. Similar cationic polymers are described in International Patent Publication number WO 03/066054 and U.S. patent publication number 2006/0051315, both of which are incorporated herein by reference. Other polymers are described by Intradigm Corp., Rockville, MD). iSdSiments, the controlled high pressure delivery system developed by Minis (Madison, WI) is utilized for delivery of oligonucleotides. The delivery system is described in U.S. patent number 6,379,966, which is incorporated herein by reference.

C. Dosages

[0276] 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. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and the delivery means, and can generally be estimated based on EC50S found to be effective in in vitro and in vivo animal models or based on the examples described herein. 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. In some embodiments, dosage is continuous {e.g., intravenously) for a period of from several hours to several days or weeks. In some embodiments, treatment is given continuously for a defined period followed by a treatment free period. In some embodiments, the pattern of continuous dosing followed by a treatment free period is repeated several times {e.g., until the disease state is diminished). [0277] The treating physician can 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 subject 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, from 1 mg to 50 mg, and from 6 mg to 30 mg per kg of body weight, once or more daily, to once every 20 years.

D. Combination Therapy

[0278] In some embodiments, the compositions of the present invention are provided in combination with existing therapies. In other embodiments, two or more compounds of the present invention are provided in combination. In some embodiments, the compounds of the present invention are provided in combination with known cancer chemotherapy agents. The present invention is not limited to a particular chemotherapy agent.

[0279] Various classes of antineoplastic {e.g., anticancer) agents are contemplated for use in certain embodiments of the present invention. Anticancer agents suitable for use with the present invention include, but are not limited to, agents that induce apoptosis, agents that inhibit adenosine deaminase function, inhibit pyrimidine biosynthesis, inhibit purine ring ^"Bø's|diιφeif Cilϊhff ilinifeMdϊide interconversions, inhibit ribonucleotide reductase, inhibit thymidine monophosphate (TMP) synthesis, inhibit dihydrofolate reduction, inhibit DNA synthesis, form adducts with DNA, damage DNA, inhibit DNA repair, intercalate with DNA, deaminate asparagines, inhibit RNA synthesis, inhibit protein synthesis or stability, inhibit microtubule synthesis or function, and the like.

[0280] Any oncolytic agent that is routinely used in a cancer therapy context finds use in the compositions and methods of the present invention. For example, the U.S. Food and Drug Administration maintains a formulary of oncolytic agents approved for use in the United States. International counterpart agencies to the U.S.F.D.A. maintain similar formularies. h Chemotherapy Agents

[0281] Chemotherapy agents of the present invention can include any suitable chemotherapy drug or combinations of chemotherapy drugs (e.g., a cocktail). Exemplary chemotherapy agents include, without limitation, alkylating agents, platinums, anti-metabolites, anthracyclines, taxanes, camptothecins, nitrosoureas, EGFR inhibitors, antibiotics, HER2/neu inhibitors, angiogenesis inhibitors, kinase inhibitors, proteaosome inhibitors, immunotherapies, hormone therapies, photodynamic therapies, cancer vaccines, histone deacetylase inhibitors, sphingolipid modulators, oligomers, other unclassified chemotherapy drugs and combinations thereof. a. Alkylating Agents

[0282] Alkylating agents are chemotherapy agents that are thought to attack the negatively charged sites on the DNA (e.g., the oxygen, nitrogen, phosphorous and sulfur atoms) and bind to the DNA thus altering replication, transcription and even base pairing. It is also believed that alkylation of the DNA also leads to DNA strand breaks and DNA strand cross-linking. By altering DNA in this manner, cellular activity is effectively stopped and the cancer cell will die. Common alkylating agents include, without limitation, Procarbazine, Ifosphamide, Cyclophosphamide, Melphalan, Chlorambucil, Decarbazine, Busulfan, Thiotepa, and the like. Alkylating agents such as those mentioned above can be used in combination with one or more other alkylating agents and/or with one or more chemotherapy agents of a different class(es). b. Platinums

[0283] Platinum chemotherapy agents are believed to inhibit DNA synthesis, transcription and function by cross-linking DNA subunits. (The cross-linking can happen either between two strands or within one strand of DNA.) Common platinum chemotherapy agents include, without limitation, Cisplatin, Carboplatin, Oxaliplatin, Eloxatin, and the like. Platinum ^■Jch6ήiDΪl^fy,.agέn|^-s|ich;|g those mentioned above can be used in combination with one or more other platinums and/or with one or more chemotherapy agents of a different class(es). c. Anti-metabolites

[0284] Anti-metabolite chemotherapy agents are believed to interfere with normal metabolic pathways, including those necessary for making new DNA. Common anti-metabolites include, without limitation, Methotraxate, 5-Fluorouracil (e.g., Capecitabine), Gemcitabine (2'-deoxy- 2',2'-difluorocytidine monohydrochloride (β-isomer), Eli Lilly), 6-mercaptopurine, 6- thioguanine, fludarabine, Cladribine, Cytarabine, tegafur, raltitrexed, cytosine arabinoside, and the like. Gallium nitrate is another anti-metabolite that inhibits ribonucleotides reductase. Anti-metabolites such as those mentioned above can be used in combination with one or more other anti-metabolites and/or with one or more chemotherapy agents of a different class(es). d. Anthracyclines

[0285] Anthracyclines are believed to promote the formation of free oxygen radicals. These radicals result in DNA strand breaks and subsequent inhibition of DNA synthesis and function. Anthracyclines are also thought to inhibit the enzyme topoisomerase by forming a complex with the enzyme and DNA. Common anthracyclines include, without limitation, Daunorubicin, Doxorubicin, Idarubicin, Epirabicin, Mitoxantrone, adriamycin, bleomycin, mitomycin-C, dactinomycin, mithramycin and the like. Anthracyclines such as those mentioned above can be used in combination with one or more other anthracyclines and/or with one or more chemotherapy agents of a different class(es). e. Taxanes

[0286] Taxanes are believed to bind with high affinity to the microtubules during the M phase of the cell cycle and inhibit their normal function. Common taxanes include, without limitation, Paclitaxel, Docetaxel, Taxotere, Taxol, taxasm, 7-epipaclitaxel, t-acetyl paclitaxel, 10-desacetyl-paclitaxel, lO-desacetyl-7-epipaclitaxel, 7-xylosylpaclitaxel, 10-desacetyl-7- epipaclitaxel, 7-N-N-dimethylglycylpaclitaxel, 7-L-alanylpaclitaxel and the like. Taxanes such as those mentioned above can be used in combination with one or more other taxanes and/or with one or more chemotherapy agents of a different class(es).

£ Camptothecins

[0287] Camptothecins are thought to complex with topoisomerase and DNA resulting in the inhibition and function of this enzyme. It is further believed that the presence of topoisomerase is required for on-going DNA synthesis. Common camptothecins include, without limitation, Mnotecan, Topotecan, Etoposide, vinca alkaloids (e.g., Vincristine, Vinblastine or Vinorelbine), amsacrine, teniposide and the like. Camptothecins such as those ,_,w

Wφ&M&<£kΕby&$$ji;0§iM4ξSttm combination with one or more other camptothecins and/or with one or more chemotherapy agents of a different class(es). g. Nitrosoureas

[0288] Nitrosoureas are believed to inhibit changes necessary for DNA repair. Common nitrosoureas include, without limitation, Carmustine (BCNU), Lomustine (CCNU), semustine and the like. Nitrosoureas such as those mentioned above can be used in combination with one or more other nitrosoureas and/or with one or more chemotherapy agents of a different class(es). h. EGFR Inhibitors

[0289] EGFR (i.e., epidermal growth factor receptor) inhibitors are thought to inhibit EGFR and interfere with cellular responses including cell proliferation and differentiation. EGFR inhibitors include molecules that inhibit the function or production of one or more EGFRs. They include small molecule inhibitors of EGFRs, antibodies to EGFRs, antisense oligomers, RNAi inhibitors and other oligomers that reduce the expression of EGFRs. Common EGFR inhibitors include, without limitation, Gefitinib, Erlotinib (Tarceva^®), Cetuximab (Erbitux^®), panitumumab (Vectibix™'Amgen) lapatinib (GlaxoSmithKline), CIl 033 or PD 183805 or Canternib (6-acrylamide-N-(3 -chloro-4-flurorphenyl)-7-(3 -morpholinopropoxy)quinazolin-4- amine, Pfizer), and the like. Other inhibitors include PKI-166 (4-[(lR)-l-phenylethylamino]- 6-(4-hydroxyphenyl)-7H-ρyrrolo[2,3-J]pyrimidine, Novartis), CL-387785 (N-[4-(3- bromoanilino)quinazolin-6-yl]but-2-ynamide), EKB-569 (4-(3-chloro-4-fluroranilino)-3- cyano-6-(4-dimethylaminobut2(E)-enamido)-7-ethozyquinoline, Wyeth), lapatinib (GW2016, GlaxoSmithKline), EKB509 (Wyeth), Panitumumab (ABX-EGF, Abgenix), matuzumab (EMD 72000, Merck) and the monoclonal antibody RH3 (New York Medical). EGFR inhibitors such as those mentioned above can be used in combination with one or more other EGFR inhibitors and/or with one or more chemotherapy agents of a different class(es). i. Antibiotics

[0290] Antibiotics are thought to promote the formation of free oxygen radicals that result in DNA breaks leading to cancer cell death. Common antibiotics include, without limitation, Bleomycin and rapamycin and the like. The macrolide fungicide rapamycin (also called RAP, Rapamune and Sirolimus) binds intracellularly to the to the immunophilin FK506 binding protein 12 (FKBP 12) and the resultant complex inhibits the serine protein kinase activity of mammalian target of rapamycin (mTOR). Rapamycin macrolides include naturally occurring forms of rapamycin as well as rapamycin analogs and derivatives that target and inhibit mTOR. Other rapamycin macrolides include, without limitation, temsirolimus (CCI-779,

^"^yέβi^ffitirplifiiS

Antibiotics such as those mentioned above can be used in combination with one or more other antibiotics and/or with one or more chemotherapy agents of a different class(es). i. HER2/neu Inhibitors

[0291] HER2/neu Inhibitors are believed to block the HER2 receptor and prevent the cascade of reactions necessary for tumor survival. Her2 inhibitors include molecules that inhibit the function or production of Her2. They include small molecule inhibitors of Her2, antibodies to Her2, antisense oligomers, RNAi inhibitors and other oligomers that reduce the expression of tyrosine kinases. Common HER2/neu inhibitors include, without limitation, Trastuzumab (Herceptin®, Genentech) and the like. Other Her2/neu inhibitors include bispecifϊc antibodies MDX-210(FCγRl-Her2/neu) and MDX-447 (Medarex), pertuzumab (rhuMAb 2C4, Genentech), HER2/neu inhibitors such as those mentioned above can be used in combination with one or more other HER2/neu inhibitors and/or with one or more chemotherapy agents of a different class(es). k. Angiogenesis Inhibitors

[0292] Angiogenesis inhibitors are believed to inhibit vascular endothelial growth factor , i.e. VEGF, thereby inhibiting the formation of new blood vessels necessary for tumor life. VEGF inhibitors include molecules that inhibit the function or production of one or more VEGFs. They include small molecule inhibitors of VEGF, antibodies to VEGF, antisense oligomers, RNAi inhibitors and other oligomers that reduce the expression of tyrosine kinases. Common angiogenesis inhibitors include, without limitation, Bevacizumab (Avastin , Genentech). Other angiogenesis inhibitors include, without limitation, ZD6474 (AstraZeneca), Bay-43-9006, sorafenib (Nexavar, Bayer), semaxamib (SU5416, Pharmacia), SU6668 (Pharmacia), ZD4190 (N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[2-( IH- 1 ,2,3-triazol- 1 - yl)ethoxy]quinazolin-4-amine, Astra Zeneca), Zactima™ (ZD6474, JV-(4-bromo-2- fluorophenyl)-6-methoxy-7-[2-(lH-l,2,3-triazol-l-yl)ethoxy]quinazolin-4-amine, Astra Zeneca), Vatalanib, (PTK787, Novartis), the monoclonal antibody IMC-ICl 1 (Imclone) and the like. Angiogenesis inhibitors such as those mentioned above can be used in combination with one or more other angiogenesis inhibitors and/or with one or more chemotherapy agents of a different class(es). h Other Kinase Inhibitors

[0293] In addition to EGFR, ΗER2 and VEGF inhibitors, other kinase inhibitors are used as chemotherapeutic agents. Aurora kinase inhibitors include, without limitation, compounds such as 4-(4-N benzoylamino)aniline)-6-methyxy-7-(3-(l-morpholino)propoxy)quinazolme }jtdIfiS@©$C J. Cell. Biol, 161:267-80 (2003)) and Hesperadin (Haaf et al., J. Cell Biol., 161 : 281-94 (2003)). Other compounds suitable for use as Aurora kinase inhibitors are described in Vankayalapati H, et al., MoI. Cancer Ther. 2:283-9 (2003). SRC/Abl kinase inhibitors include, without limitation, AZD0530 (4-(6~chloro-2,3-methylenedioxyanilino)-7-[2- (4-methylpiperazin-l-yl)ethoxy]-5-tetrahycropyran-4-yloxyquinazoline). Tyrosine kinase inhibitors include molecules that inhibit the function or production of one or more tyrosine kinases. They include small molecule inhibitors of tyrosine kinases, antibodies to tyrosine kinases and antisense oligomers, RNAi inhibitors and other oligomers that reduce the expression of tyrosine kinases. CEP-701 and CEP-751 (Cephalon) act as tyrosine kinase inhibitors. Imatmib mesylate is a tyrosine kinase inhibitor that inhibits bcr-abl by binding to the ATP binding site of bcr-abl and competitively inhibiting the enzyme activity of the protein. Although imatinib is quite selective for bcr-abl, it does also inhibit other targets such as c-kit and PDGF-R. FLT-3 inhibitors include, without limitation, tandutinib (MLN518, Millenium), Sutent (SUl 1248, 5- [5-fluoro-2-oxo-l,2- dihydroindol-(3Z)-ylidenemethyl]-2, 4-dimethyl-li/- pyrrole-3-carboxylic acid [2-diethylaminoethyl] amide, Pfizer), midostaurin (4'-N-Benzoyl Staurosporine,_Novartis), lefunomide (SUlOl) and the like. MEK inhibitors include, without limitation, 2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide) (PDl 84352/CI- 1044, Pfizer), PD198306 (Pfizer), PD98059 (2'-amino-3'-methoxyflavone), UO 126 (Promega), Ro092210 from fermented microbial extracts (Roche), the resorcyclic acid lactone, L783277, also isolated from microbial extracts (Merck) and the like. Tyrosine kinase inhibitors such as those mentioned above can be used in combination with one or more other tyrosine kinase inhibitors and/or with one or more chemotherapy agents of a different class(es). m. Proteaosome Inhibitors

[0294] Proteaosome inhibitors are believed to inhibit the breakdown of some of these proteins that have been marked for destruction. This results in growth arrest or death of the cell. Common proteaosome inhibitors include, without limitation, Bortezomib, ortezomib and the like. Proteaosome inhibitors such as those mentioned above can be used in combination with one or more other proteaosome inhibitors and/or with one or more chemotherapy agents of a different class(es).

IL Immunotherapies

[0295] Immunotherapies are thought to bind to and block specific targets, thereby disrupting the chain of events needed for tumor cell proliferation. Common immunotherapies include, without limitation, Rituximab and other antibodies directed against CD20, Campath-1H and other antibodies directed against CD-50, epratuzmab and other antibodies directed against CD-

directed atainst CD-80, apolizumab HUlDlO and other antibodies directed against HLA-DR, and the like. Radioisotopes can be conjugated to the antibody, resulting in radioimmunotherapy. Two such anti-CD20 products are tositumomab (Bexxar) and ibritumomab (Zevalin). Immunotherapies such as those mentioned above can be used in combination with one or more other immunotherapies and/or with one or more chemotherapy agents of a different class(es). o. Hormone Therapies

[0296] Hormone therapies are thought to block cellular receptors, inhibit the in vivo production of hormones, and/or eliminate or modify hormone receptors on cells, all with the end result of slowing or stopping tumor proliferation. Common hormone therapies include, without limitation, antiestrogens (e.g., tamoxifen, toremifene, fulvestrant, raloxifene, droloxifene, idoxyfene and the like), progestogens) e.g., megestrol acetate and the like) aromatase inhibitors (e.g., Anastrozole, Letrozole, Exemestane, vorazole, exemestane, fadrozole, aminoglutethimide, exemestane, 1 -methyl- l,4-androstadiene-3,17-dione and the like), anti- androgens (e.g., Bicalutimide, Nilutamide, Flutamide, cyproterone acetate, and the like), luteinizing hormone releasing hormone agonist (LHRH Agonist) (e.g., Goserelin, Leuprolide, buserelin and the like); 5-α-reductase inhibitors such as finasteride, and the like. Hormone therapies such as those mentioned above can be used in combination with one or more other hormone therapies and/or with one or more chemotherapy agents of a different class(es). p. Photodynamic Therapies

[0297] Photodynamic therapies expose a photosensitizing drug to specific wavelengths of light to kill cancer cells. Common photodynamic therapies include, for example, porfimer sodium (e.g., Photofrin ) and the like. Photodynamic therapies such as those mentioned above can be used in combination with one or more other photodynamic therapies and/or with one or more chemotherapy agents of a different class(es).

Q. Cancer Vaccines

[0298] Cancer vaccines are thought to utilize whole, inactivated tumor cells, whole proteins, peptide fragments, viral vectors and the like to generate an immune response that targets cancer cells. Common cancer vaccines include, without limitation, modified tumor cells, peptide vaccine, dendritic vaccines, viral vector vaccines, heat shock protein vaccines and the like. r. Histone Deacetylase Inhibitors

[0299] Histone deacetylase inhibitors are able to modulate transcriptional activity and consequently, can block angiogenesis and cell cycling, and promote apoptosis and ^iff^nBltiab^KillSliiydaeetylase inhibitors include, without limitation, SAHA (Suberoylanilide hydroxamic acid), depsipeptide (FK288) and analogs, Pivanex (Titan), CI994 (Pfizer), MS275 PXDlOl (CuraGen, TopoTarget) MGCD0103 (MethylGene), LBH589, NVP- LAQ824 (Novartis) and the like and have been used as chemotherapy agents. Histone deacetylase inhibitors such as those mentioned above can be used in combination with one or more other histone deacetylase inhibitors and/or with one or more chemotherapy agents of a different class(es). s. Sphingolipid Modulators

[0300] Modulators of Sphingolipid metabolism have been shown to induce apoptosis. For reviews see N.S. Radin, Biochem J, 371:243-56 (2003); D.E. Modrak, et al., MoI. Cancer Ther, 5:200-208 (2006), K. Desai, et al., Biochim Biophys Acta, 1585:188-92 (2002) and CP. Reynolds, et al. and Cancer Lett, 206, 169-80 (2004), all of which are incorporated herein by reference. Modulators and inhibitors of various enzymes involved in sphingolipid metabolism can be used as chemotherapeutic agents.

[0301] (1) Ceramide has been shown to induce apoptosis, consequently, exogenous ceramide or a short chain ceramide analog such as N-acetylsphingosine (Ca-Cer), C₆-Cer or C₈-Cer has been used. Other analogs include, without limitation, Cer 1-glucuronide, poly(ethylene glycol)-derivatized ceramides and pegylated ceramides.

[0302] (2) Modulators that stimulate ceramide synthesis have been used to increase ceramide levels. Compounds that stimulate serine palmitoyltransferase, an enzyme involved in ceramide synthesis, include, without limitation, tetrahydrocannabinol (THC) and synthetic analogs and anandamide, a naturally occurring mammalian cannabinoid. Gemcitabine, retinoic acid and a derivative, fenretinide [N-(4-hycroxyphenyl)retinamide, (4-HPR)], camptothecin, homocamptothecin, etoposide, paclitaxel, daunorubicin and fludarabine have also been shown to increase ceramide levels. In addition, valspodar (PSC833, Novartis), a non- irnmunosuppressive non-ephrotoxic analog of cyclosporin and an inhibitor of p-glycoprotein, increases ceramide levels.

[0303] (3) Modulators of sphingomyelinases can increase ceramide levels. They include compounds that lower GSH levels, as GSH inhibits sphingomyelinases. For example, betathine (β-alanyl cysteamine disulphide), oxidizes GSH, and has produced good effects in patients with myeloma, melanoma and breast cancer. COX-2 inhibitors, such as celecoxib, ketoconazole, an antifungal agent, doxorubicin, mitoxantrone, D609 (tricyclodecan-9-yl- xanthogenate), dexamethasone, and Ara-C (l-/?-D-arabinoruranosylcytosine) also stimulate sphingomyelinases. ^I'p3^'(ϊ#]f^;_!!^£αleMlls'Φ-lt-s1:ϊimulate the hydrolysis of glucosylceramide also raise cerainide levels. The enzyme, GlcCer glucosidase, which is available for use in Gaucher's disease, particularly with retinol or pentanol as glucose acceptors and/or an activator of the enzyme can be used as therapeutic agents. Saposin C and analogs thereof, as well as analogs of the antipsychotic drug, chloropromazine, may also be useful.

[0305] (5) Inhibitors of glucosylceramide synthesis include, without limitation, PDMP (N-[2- hydroxy- 1 -(4-morpholinylmethyl)-2-phenylethyldecanamide]), PMPP (D,L-threo-(l -phenyl-2- hexadecanoylamino-3-morpholino-l-propanol), P4 or PPPP (D-threo-l-phenyl-2- palmitoylamino-3 -pyrrolidino- 1 -propanol), ethyl enedioxy-P4, 2-decanoylamine-3 - morpholinoprophenone, tamixofen, raloxifene, mifepristone (RU486), N-butyl deoxynojirimycin and anti androgen chemotherapy (bicalutamide + leuprolide acetate). Zavesca, (l,5-(butylimino)-l,5-dideoxy-D-glucitol) usually used to treat Gaucher's disease, is another inhibitor of glucosylceramide synthesis.

[0306] (6) Inhibitors of ceramidase include, without limitation, N-oleoylethanolamine, a truncated form of ceramide, D-MAPP (D-erj^/ϊro-2-tetradecanoylamino-l -phenyl- 1 -propanol) and the related inhibitor B13 (p-nitro-D-MAPP).

[0307] (7) Inhibitors of sphingosine kinase also result in increased levels of ceramide. Inhibitors include, without limitation, safϊngol (L-t/zreø-dihydrosphingosine), N,N-dimethyl sphingosine, trimethylsphingosine and analogs and derivatives of sphingosine such as dihydrosphingosine, and myriocin.

[0308] (8) Fumonisins and fumonisin analogs, although they inhibit ceramide synthase, also increase levels of sphinganine due to the inhibition of de novo sphingolipid biosynthesis, resulting in apoptosis.

[0309] (9) Other molecules that increase ceramide levels include, without limitation, miltefosine (hexadecylphosphocholine).

[0310] Sphingolipid modulators such as those mentioned above, can be used in combination with one or more other sphingolipid modulators and/or with one or more chemotherapy agents of a different class(es). t Oligomers

[0311] In addition to the oligonucleotides of the present invention, other oligonucleotides have been used as cancer therapies. They include, without limitation, Genasense (oblimersen, G3139, from Genta), an antisense oligonucleotide that targets bcl-2 and G4460 (LR3001, from Genta) another antisense oligonucleotide that targets c-myb. Other oligomers include, without limitation, siRNAs, decoys, RNAi oligonucleotides and the like. Oligomers, such as those joVέf O.atf:leluied in combination with one or more other oligomers and/or with one or more chemotherapy agents of a different class(es). u. Other Chemotherapy Drugs [0312] Additional unclassified chemotherapy agents are described in Table 3 below.

Table 3 Additional unclassified chemotherapy agents.

V. Cocktails

[0313] Chemotherapy agents can include cocktails of two or more chemotherapy drugs mentioned above. In several embodiments, a chemotherapy agent is a cocktail that includes two or more alkylating agents, platinums, anti-metabolites, anthracyclines, taxanes, camptothecins, nitrosoureas, EGFR inhibitors, antibiotics, HER2/neu inhibitors, angiogenesis inhibitors, kinase inhibitors, proteaosome inhibitors, immunotherapies, hormone therapies, photodynamic therapies, cancer vaccines, sphingolipid modulators, oligomers or combinations thereof.

2. Radiation Therapy

[0314] In several embodiments of the present invention, radiation therapy is administered in addition to the administration of an oligonucleotide compound. Radiation therapy includes both external and internal radiation therapies. a. External Radiation Therapy

[0315] External radiation therapies include directing high-energy rays (e.g., x-rays, gamma rays, and the like) or particles (alpha particles, beta particles, protons, neutrons and the like) at the cancer and the normal tissue surrounding it. The radiation is produced outside the patient's body in a machine called a linear accelerator. External radiation therapies can be combined with chemotherapies, surgery or oligonucleotide compounds. b. Internal Radiation Therapy

[0316] Internal radiation therapies include placing the source of the high-energy rays inside the body, as close as possible to the cancer cells. Internal radiation therapies can be combined with external radiation therapies, chemotherapies or surgery. ^''ifOθβJϊKafflatiori-ilϊe^^'fi¹ be administered with chemotherapy simultaneously, concurrently, or separately. Moreover radiation therapy can be administered with surgery simultaneously, concurrently, or separately.

3. Surgery

[0318] In alternative embodiments, of the present invention, surgery is used to remove cancerous tissue from a patient. Cancerous tissue can be excised from a patient using any suitable surgical procedure including, for example, laparoscopy, scalpel, laser, scissors and the like. In several embodiments, surgery is combined with chemotherapy. In other embodiments, surgery is combined with radiation therapy. In still other embodiments, surgery is combined with both chemotherapy and radiation therapy.

E. Liposome Based Therapeutics

[0319] In certain embodiments, liposomes (e.g., cardiolipin based cationic liposomes) are administered with known chemotherapy agents (e.g. those disclosed herein). In some embodiments, the liposomes and the chemotherapy agents are administered separately from each other, hi some embodiments, the chemotherapy agent is not formulated with the liposome. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, it is contemplated that the administration of liposomes and chemotherapeutic agents to the same host greatly reduces the required dose of chemotherapy agent e.g. to less than 50%, less than 10%, less than 5%, or less than 1% of the minimum of the standard dosage range required in the absence of liposome.

[0320] In some embodiments, the liposomes and the chemotherapeutic agents are provided as a kit. hi some embodiments, the liposomes and the chemotherapeutic agents are provided in separate vessels. In some embodiments, the kit comprises instructions for using the components (e.g., liposomes, alone or in combination with known chemotherapeutic agents) for treating cancer. In some embodiments, the kits are approved by the Food and Drug administration a pharmaceuticals for use in the treatment of humans.

F. Customized Patient Care

[0321] In some embodiments, the present invention provides customized patient care. [0322] The compositions of the present invention are targeted to specific genes unique to a patient's disease (e.g., cancer). For example, in some embodiments, a sample of the patient's cancer or other affected tissue (e.g., a biopsy) is first obtained. The biopsy is analyzed for the presence of expression of a particular gene (e.g., oncogene). In some embodiments, the level of expression of a gene in a patient is analyzed. Expression may be detected by monitoring for

e'^^iMj>P^&' θ_'W^Α corresponding to a particular oncogene. Any suitable detection method may be utilized, including, but not limited to, those disclosed below. [0323] Following the characterization of the gene expression pattern of a patient's gene of interest, a customized therapy is generated for each patient. In some embodiments, oligonucleotide compounds specific for genes that are aberrantly expressed in the patient (e.g., in a tumor) are combined in a treatment cocktail. In some embodiments, the treatment cocktail further includes additional chemotherapeutic agents (e.g., those described above). The cocktail is then administered to the patient as described above.

[0324] In some embodiments, the analysis of cancer samples and the selection of oligonucleotides for a treatment compound are automated. For example, in some embodiments, a software program that analyses the expression levels of a series of oncogenes to arrive at the optimum selection and concentration of oligonucleotides is utilized. In some embodiments, the analysis is performed by the clinical laboratory analyzing the patient sample and is transmitted to a second provider for formulation of the treatment cocktail. In some embodiments, the information is transmitted over the Internet, thus allowing for the shortest possible time in between diagnosis and the beginning of treatment.

1. Detection of RNA

[0325] In some embodiments, detection of oncogenes (e.g., including but not limited to, those disclosed herein) is detected by measuring the expression of corresponding mRNA in a tissue sample (e.g., cancer tissue). In other embodiments, expression of mRNA is measured in bodily fluids, including, but not limited to, blood, serum, mucus, and urine, hi some embodiments, the level of mRNA expression in measured quantitatively. RNA expression may be measured by any suitable method, including but not limited to, those disclosed below. [0326] In some embodiments, RNA is detected by Northern blot analysis. Northern blot analysis involves the separation of RNA and hybridization of a complementary labeled probe. In other embodiments, RNA expression is detected by enzymatic cleavage of specific structures (INVADER assay, Third Wave Technologies; See e.g., U.S. Patent Nos. 5,846,717, 6,090,543; 6,001,567; 5,985,557; and 5,994,069; each of which is herein incorporated by reference). The INVADER assay detects specific nucleic acid (e.g., RNA) sequences by using structure-specific enzymes to cleave a complex formed by the hybridization of overlapping oligonucleotide probes.

[0327] In still further embodiments, RNA (or corresponding cDNA) is detected by hybridization to an oligonucleotide probe). A variety of hybridization assays using a variety of technologies for hybridization and detection are available. For example, in some ^"im6MfiMϊit$ TaljiffiiSalMjf. (PE Biosystems, Foster City, CA; See e.g., U.S. Patent Nos. 5,962,233 and 5,538,848, each of which is herein incorporated by reference) is utilized. The assay is performed during a PCR reaction. The TaqMan assay exploits the 5'-3' exonuclease activity of the AMPLITAQ GOLD DNA polymerase. A probe consisting of an oligonucleotide with a 5 '-reporter dye {e.g., a fluorescent dye) and a 3 '-quencher dye is included in the PCR reaction. During PCR, if the probe is bound to its target, the 5'-3' nucleolytic activity of the AMPLITAQ GOLD polymerase cleaves the probe between the reporter and the quencher dye. The separation of the reporter dye from the quencher dye results in an increase of fluorescence. The signal accumulates with each cycle of PCR and can be monitored with a fluorimeter.

[0328] In yet other embodiments, reverse-transcriptase PCR (RT-PCR) is used to detect the expression of RNA. In RT-PCR, RNA is enzymatically converted to complementary DNA or "cDNA" using a reverse transcriptase enzyme. The cDNA is then used as a template for a PCR reaction. PCR products can be detected by any suitable method, including but not limited to, gel electrophoresis and staining with a DNA specific stain or hybridization to a labeled probe. In some embodiments, the quantitative reverse transcriptase PCR with standardized mixtures of competitive templates method described in U.S. Patents 5,639,606, 5,643,765, and 5,876,978 (each of which is herein incorporated by reference) is utilized.

2. Detection of Protein

[0329] In other embodiments, gene expression of oncogenes is detected by measuring the expression of the corresponding protein or polypeptide. In some embodiments, protein expression is detected in a tissue sample. In other embodiments, protein expression is detected in bodily fluids. In some embodiments, the level of protein expression is quantitated. Protein expression may be detected by any suitable method. In some embodiments, proteins are detected by their binding to an antibody raised against the protein. The generation of antibodies is well known to those skilled in the art.

[0330] Antibody binding is detected by techniques known in the art {e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays {e.g., using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays {e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, and Immunoelectrophoresis assays, etc. 1033JJtEfMe ^rrtlfflitlMCantibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many methods are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

[0332] In some embodiments, an automated detection assay is utilized. Methods for the automation of immunoassays include those described in U.S. Patents 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which is herein incorporated by reference. In some embodiments, the analysis and presentation of results is also automated. For example, in some embodiments, software that generates an expression profile based on the presence or absence of a series of proteins corresponding to oncogenes is utilized.

[0333] In other embodiments, the immunoassay described in U.S. Patents 5,599,677 and 5,672,480; each of which is herein incorporated by reference.

V. EXAMPLES

[0334] The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

EXAMPLE 1 LNA-PNT-100 Inhibition of PC-3 Cell Proliferation [0335] The effect of LNA-PNT-100 (SEQ ID NO:1251) on cell proliferation was tested in cultured PC-3 cells. The PNT-100 used in this example was synthesized so that with the two nucleotides on the 3' and 5' ends in the LNA configuration. For LNA-PNT-100, CACGCACGCGCATCCCCGCCCGTG (SEQ ID NO:1251), the nucleotides in bold and underlined are in the locked configuration. LNA-PNT-100 and LNA-PNT-100R (SEQ ID NO:1288)were combined with NeoPhectin according to manufacturer's instructions (NeoPharm) with LNA-PNT-100. The optimum NeoPhectin:LNA-PNT100 charge ratio used is 6:1.

[0336] PC-3 cells are a widely used model of hormone refractory prostate cancer (HRPC). Low passage PC-3 cells were obtained and cultured according to ATCC recommendations. Cells were seeded in 6-well plates for 48 hrs in complete serum containing media, incubated for two hours in fresh media, and the media was replaced with serum free media. LNA-PNT- 100 and LNA-PNT100R combined with NeoPhectin were added to the cells to a final concentration of 2 μM. The drug mixture was incubated with cells for 6 hrs at 37⁰C. Cells were then returned to complete media and fed daily for 48 hours. To determine cell viability, ^'IfietSJllϊitSif^rόlMMmflMofiit cell viability assay was performed according to manufacture's instructions (Promega, Madison, WI). This assay uses a luciferase reaction to measure ATP, a global indicator of cellular metabolism.

[0337] As shown in Figure 1, the signal obtained from cells treated with LNA-PNTlOO is 63% of the signal obtained from cells treated with LNA-PNT100R, and approximately 50% of the signal obtained from cells receiving no treatment. In addition, the concentration of LNA-PNT- 100 needed to obtain these inhibition levels in PC-3 cells is five fold lower than the concentration of unmodified PNT-100 needed to obtain the same levels of inhibition. To obtain these inhibition levels with unmodified PNT-100, PC-3 cells must be treated with 10 μM PNT-100. Consequently, the LNA modification to PNT-100 increases anti-proliferation activity five fold over unmodified PNT-100, leading to significant PNTlOOR activity.

[0338] EXAMPLE 2 Thiolated PNT-100 Inhibition of PC-3 Cell Proliferation [0339] The effect of PNT-100 (SEQ ID NO: 1251) with a completely thiolated backbone on cell proliferation was tested in cultured PC-3 cells. Thiolated PNT-100 and Thiolated PNT- 10OR were combined with NeoPhectin and the resulting mixtures were added to PC-3 cells as described in Example 1.

[0340] As shown in Figure 1, the signal obtained from cells treated with thiolated-PNTIOO is 49% of the signal obtained from cells treated with thiolated PNTlOOR, and approximately 40% of the signal obtained from cells receiving no treatment. As with LNA-PNT-100, the concentration of thiolated PNT-100 needed to obtain these inhibition levels in PC-3 cells is five fold lower than the concentration of unmodified PNT-100 needed to obtain the same levels of inhibition. To obtain these inhibition levels with unmodified PNT-100, PC-3 cells must be treated with 10 μM PNT-100. Consequently, the thiolated backbone modification to PNT-100 increases anti-proliferation activity five fold over unmodified PNT-100, leading to significant PNTlOOR activity.

VI. Other Embodiments

[0341] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims. [0342] All references cited herein, are incorporated herein by reference in their entirety.

Claims

What is claimed is:

1. An oligonucleotide that hybridizes to SEQ ID No: 1249 or the complement thereof, wherein the oligonucletotide comprises from 10 to 60 nucleotides in which one or more nucleotides are LNA or LNA* nucleotides.

2. The oligonucleotide of Claim 1 wherein the oligonucleotide is comprised of 15 to 40 nucleotides wherein one or more nucleotides are LNA or LNA* nucleotides.

3. The oligonucleotide of Claim 1 wherein the oligonucleotide is comprised of 20 to 30 nucleotides wherein one or more nucleotides are LNA or LNA* nucleotides.

4. The oligonucleotide of Claim 1 comprised of 10 to 15 nucleotides wherein the number of LNA or LNA* nucleotides is from 1 to 15.

5. The oligonucleotide of Claim 1 comprised of 15 to 40 nucleotides wherein the number of LNA or LNA* nucleotides is from 1 to 16.

6. The oligonucleotide of Claim 1 comprised of 20 to 30 nucleotides wherein the number of LNA or LNA* nucleotides is from 1 to 12.

7. An oligonucleotide of Claims 1 thru 6 wherein up to 10 LNA or LNA* nucleotides are sequential.

8. An oligonucleotide according to Claim 7 wherein the LNA and LNA* nucleotides are separated by one or more DNA or RNA nucleotides.

9. An oligonucleotide according to Claim 7 wherein from 1 to 3 LNA and LNA* nucleotides are sequential.

10. An oligonucleotide according to Claim 7 comprised of 15 to 40 nucleotides wherein the number of LNA or LNA* nucleotides is 4.

11. An oligonucleotide according to Claim 7 comprised of 15 to 40 nucleotides wherein the oligomer includes 2 pair of sequential LNA or LNA* nucleotides.

12. An oligonucleotide according to Claim 11 comprised of 15 to 40 nucleotides wherein one pair is at the 3' terminus.

13. An oligonucleotide according to Claim 11 comprised of 15 to 40 nucleotides wherein one pair is at the 5' terminus.

14. An oligonucleotide according to Claim 11 comprised of 15 to 40 nucleotides wherein one pair is at the 3' terminus and the other pair is at the 5' terminus.

to any of .claims 1 through 14 wherein LNA is a compound of Formula 1

i wherein:

B constitutes a nucleobase;

Z* is selected from an internucleoside linkage and a terminal group; and

Z is selected from a bond to the internucleoside linkage of a preceding nucleotide/nucleoside and a terminal group, provided that only one of Z and Z* can be a terminal group.

16. An oligonucleotide according to any of claims 1 through 14 wherein LNA* is a compound of Formula 2

2 wherein:

B constitutes a nucleobase;

Z* is selected from an internucleoside linkage and a terminal group;

Z is selected from a bond to the internucleoside linkage of a preceding nucleotide/nucleoside and a terminal group, provided that only one of Z and Z* can be a terminal group;

X and Y are independently selected from -O-, -S-, -N(H)-, -N(R)-, -CH₂- or -C(H)=, CH₂- O-, -CH₂-S-, -CH₂-N(H)-, -CH₂-N(R)-, -CH₂-CH₂- or -CH₂-C(H)=, -CH=CH-; provided that X and Y are not both O, and R is selected from hydrogen and C_1-4-alkyl.

17. Aa3»@lg©nucfefiifHfeMd&rding to any of claims 11 through 14 wherein the LNA/LNA* pair is LNA-LNA.

18. An oligonucleotide according to any of claims 11 through 14 wherein the LNA/LNA* pair is LNA*-LNA*.

19. An oligonucleotide according to any of claims 11 through 14 wherein the LNA/LNA* pair is LNA*-LNA.

20. An oligonucleotide according to any of claims 11 through 14 wherein the LNA/LNA* pair is LNA-LNA*.

21. The oligonucleotide of any of claims 1 -20 wherein the oligomer comprises an oligomer that hybridizes under physiological conditions to SEQ ID NO: 1249 or the complement thereof.

22. The oligonucleotide of any of claims 1-20 wherein the oligomer comprises an oligomer that hybridizes under physiological conditions to nucleotides 500-2026 of SEQ ID NO: 1249 or the complement thereof.

23. The oligonucleotide of any of claims 1-20 wherein the oligonucleotide comprises an oligonucleotide that hybridizes under physiological conditions to nucleotides 500-1525 of SEQ ID NO: 1249 or the complement thereof.

24. The oligonucleotide of any of claims 1 -20 wherein the oligonucleotide comprises an oligonucleotide that hybridizes under physiological conditions to nucleotides 800-1225 of SEQ ID NO: 1249 or the complement thereof.

25. The oligonucleotide of any of claims 1 -20 wherein the oligonucleotide comprises an oligonucleotide that hybridizes under physiological conditions to nucleotides 900-1125 of SEQ ID NO: 1249 or the complement thereof.

26. The oligonucleotide of any of claims 1-20 wherein the oligonucleotide comprises an oligonucleotide that hybridizes under physiological conditions to nucleotides 950-1075 of SEQ ID NO: 1249 or the complement thereof.

27. The oligonucleotide of any of claims 1 -20 wherein the oligonucleotide comprises an oligonucleotide that hybridizes under physiological conditions to nucleotides 970-1045 of SEQ ID NO: 1249 or the complement thereof.

28.

of claims 1-20 wherein the oligonucleotide comprises an oligonucleotide selected from the group consisting of SEQ ID NOs:1250, 1251, 1252, 1253, 1267-1477 or the complements thereof.

29. The oligonucleotide of any of claims 1-20 wherein the oligonucleotide comprises an oligonucleotide selected from the group consisting of SEQ ID NOs 1250, 1251, 1267, 1268, 1276, 1277, 1285, 1286 or the complements thereof.

30. The oligonucleotide of any of claims 1-20 wherein the oligonucleotide comprises an oligonucleotide selected from the group consisting of SEQ ID NOs 1250, 1251, 1289- 1358 or the complements thereof.

31. The oligonucleotide of any of claims 1 -20, wherein the oligonucleotide comprises SEQ ID NO:1250 or l251.

32. An oligonucleotide that hybridizes to SEQ ID No: 936 or the complement thereof, wherein the oligonucletotide comprises from 10 to 60 nucleotides in which one or more nucleotides are LNA or LNA* nucleotides.

33. The oligonucleotide of claim 32, wherein the oligonucleotide hybridizes with nucleotides 1-1000 of SEQ ID NO:936.

34. The oligonucleotide of claim 32, wherein the oligonucleotide is selected from SEQ ID NO:940 and SEQ ID NO:943.

35. The oligonucleotide of any of claims 1-31, further comprising an additional oligonucleotide.

36. The oligonucleotide of claim 32, wherein the second oligomer comprises any one of SEQ ID NOs:1250-1253 and 1267-1477.

37. The oligonucleotide of claim 32, wherein the second oligomer is selected from the group consisting of SEQ ID NOs 2-281, 283-461, 463-935, 937-1080, and 1082-1248.

38. The oligonucleotide of any of claims 1 -34, wherein the oligonucleotides are between 15 and 35 base pairs in length.