WO1999009214A1 - Antisense oligonucleotide compositions and methods for the modulation of jnk proteins - Google Patents
Antisense oligonucleotide compositions and methods for the modulation of jnk proteins Download PDFInfo
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Definitions
- the present invention provides compositions and methods for detecting and modulating levels of Jun N- terminal inases (JNK proteins) , enzymes which are encoded by JNK genes.
- the invention relates to antisense oligonucleotides specifically hybridizable with nucleic acids encoding JNK proteins. It has been found that antisense oligonucleotides can modulate the expression of these and other JNK proteins, kinases which were initially discovered due to their ability to catalyze the phosphorylation of the c-Jun subunit of transcription factor AP-1 and thereby increase AP-1 activity.
- transcription factor AP-1 has been implicated in abnormal cell proliferation, oncogenic transformation, and tumor formation, development and maintenance (Vogt, Chapter 15 In : The FOS and JUN Families of Transcription Factors,
- JNK proteins are aberrantly expressed in some neoplasms and tumors with resultant increased AP-1 activity, and (2) even in abnormally proliferating cells in which a JNK gene is not aberrantly expressed, inhibition of JNK expression will result in decreased AP-1 activity and thus, inhibition of abnormal cell proliferation and tumor formation, development and maintenance.
- the invention is thus directed to diagnostic methods for detecting, and therapeutic methods for inhibiting, the hyperproliferation of cells and the formation, development and maintenance of tumors. Furthermore, this invention is directed to treatment of conditions associated with abnormal expression of JNK genes.
- This invention also relates to therapies, diagnostics, and research reagents for disease states or disorders which respond to modulation of the expression of JNK proteins. Inhibition of the hyperproliferation of cells, and corresponding prophylactic, palliative and therapeutic effects result from treatment with the oligonucleotides of the invention.
- Transcription factors play a central role in the expression of specific genes upon stimulation by extracellular signals, thereby regulating a complex array of biological processes.
- AP-1 activating protein-1
- AP-1 alter gene expression in response to growth factors, cytokines, tumor promoters, carcinogens and increased expression of certain oncogenes (Rahmsdorf, Chapter 13, and Rapp et al . , Chapter 16 In : The FOS and JUN Families of
- AP-1 denotes one member of a family of related heterodimeric transcription factor complexes found in eukaryotic cells or viruses ⁇ The FOS and JUN Families of Transcription Factors, Angel and Herrlich, Eds., CRC Press, Boca Raton, FL, 1994; Bohmann et al . , Science, 1987, 238, 1386; Angel et al . , Na ture, 1988, 332, 166) .
- Two relatively well-characterized AP-1 subunits are c-Fos and c-Jun; these two proteins are products of the c- fos and c-j un proto-oncogenes, respectively.
- Repression of the activity of either c-fos or c-j un, or of both proto- oncogenes, and the resultant inhibition of the formation of c-Fos and c-Jun proteins, is desirable for the inhibition of cell proliferation, tumor formation and tumor growth.
- Mitogen-activated protein kinases MAPKs
- enzymes which effect such phosphorylations are targets for the action of growth factors, hormones, and other agents involved in cellular metabolism, proliferation and differentiation (Cobb et al . , J. Biol . Chem . , 1995, 270, 14843).
- MAPKs also referred to as extracellular signal-regulated protein kinases, or ERKs
- ERKs extracellular signal-regulated protein kinases
- ERKs extracellular signal-regulated protein kinases
- MAPKs are themselves activated by phosphorylation catalyzed by, e.g., receptor tyrosine kinases, G protein-coupled receptors, protein kinase C (PKC) , and the apparently MAPK-dedicated kinases MEK1 and MEK2.
- PKC protein kinase C
- MEK1 and MEK2 the apparently MAPK-dedicated kinases
- MAP kinases are involved in a variety of signal transduction pathways (sometimes overlapping and sometimes parallel) that function to convey extracellular stimuli to protooncogene products to modulate cellular proliferation and/or differentiation (Seger et al . , FASEB J. , 1995, 9, 726; Cano et al .
- MAP kinase pathway In a typical MAP kinase pathway, it is thought that a first MAP kinase, called a MEKK, phosphorylates and thereby activates a second MAP kinase, called a MEK, which, in turn, phosphorylates and activates a MAPK/ERK or JNK/SAPK enzyme ("SAPK” is an abbreviation for stress- activated protein kinase) . Finally, the activated MAPK/ERK or JNK/SAPK enzyme itself phosphorylates and activates a transcription factor (such as, e . g. , AP-1) or other substrates (Cano et al . , Trends Biochem . Sci . , 1995, 20, 117) .
- This canonical cascade can be simply represented as follows :
- JNK1 Jun N-terminal kinase 1
- JNK2 Jun N- terminal kinase 2
- JNK protein shall mean a member of the JNK family of kinases, including but not limited to JNK1, JNK2 and JNK3, their isoforms (Gupta et al . , EMBO J. , 1996, 15, 2760) and other members of the JNK family of proteins whether they function as Jun N-terminal kinases per se (that is, phosphorylate Jun at a specific amino terminally located position) or not.
- At least one human leukemia oncogene has been shown to enhance Jun N-terminal kinase function (Raitano et al . , Proc . Na tl . Acad . Sci . (USA) , 1995, 92, 11746).
- Modulation of the expression of one or more JNK proteins is desirable in order to interfere with hyperproliferation of cells and with the transcription of genes stimulated by AP-1 and other JNK protein phosphorylation substrates.
- Modulation of the expression of one or more other JNK proteins is also desirable in order to interfere with hyperproliferation of cells resulting from abnormalities in specific signal transduction pathways.
- cellular hyperproliferation in an animal can have several outcomes. Internal processes may eliminate hyperproliferative cells before a tumor can form. Tumors are abnormal growths resulting from the hyperproliferation of cells. Cells that proliferate to excess but stay put form benign tumors, which can typically be removed by local surgery. In contrast, malignant tumors or cancers comprise cells that are capable of undergoing metastasis, i.e., a process by which hyperproliferative cells spread to, and secure themselves within, other parts of the body via the circulatory or lymphatic system (see, generally, Chapter 16 In : Molecular Biology of the Cell , Alberts et al . , eds., Garland Publishing, Inc., New York, 1983) .
- antisense oligonucleotides it has surprisingly been discovered that several genes encoding enzymes required for metastasis are positively regulated by AP-1, which may itself be modulated by antisense oligonucleotides targeted to one or more JNK proteins. Consequently, the invention satisfies the long-felt need for improved compositions and methods for modulating the metastasis of malignant tumors.
- oligonucleotides are provided which specifically hybridize with a nucleic acid encoding a JNK protein.
- Certain oligonucleotides of the invention are designed to bind either directly to mRNA transcribed from, or to a selected DNA portion of, a JNK gene that encodes a JNK protein, thereby modulating the expression thereof and/or the phosphorylation of one or more substrates for the JNK protein.
- Pharmaceutical compositions comprising the oligonucleotides of the invention, and various methods of using the oligonucleotides of the invention, including methods of modulating one or more metastatic events, are also herein provided.
- Oligonucleotides may comprise nucleotide sequences sufficient in identity and number to effect specific hybridization with a particular nucleic acid. Such oligonucleotides are commonly described as “antisense.” Antisense oligonucleotides are commonly used as research reagents, diagnostic aids, and therapeutic agents. It has been discovered that genes ⁇ JNK) encoding Jun N-terminal kinase (JNK proteins) are particularly amenable to this approach.
- JNK protein refers to proteins actually known to phosphorylate the amino terminal (N- terminal) portion of the Jun subunit of AP-1, as well as those that have been tentatively identified as JNK proteins based on amino acid sequence but which may in fact additionally or alternatively bind and/or phosphorylate either other transcription factors (e.g., ATF2) or kinase substrates that are not known to be involved in transcription (Derijard et al . , Cell , 1994, 76, 1025; Kallunki et al . , Genes & Development , 1994, 8, 2996; Gupta et al . , EMBO J.
- ATF2 kinase substrates
- the present invention is directed to antisense oligonucleotides that modulate the JNK1, JNK2 and JNK3 proteins.
- the present invention is directed to antisense oligonucleotides that modulate the JNK1, JNK2 and JNK3 proteins.
- JNK1, JNK2 and JNK3 proteins As a consequence of the association between cellular proliferation and activation ⁇ via phosphorylation) of AP-1, other transcription factors and/or other proteins by JNK proteins, inhibition of the expression of one or more JNK proteins leads to inhibition of the activation of AP-1 and/or other factors involved in cellular proliferation, cell cycle progression or metastatic events, and, accordingly, results in modulation of these activities.
- Such modulation is desirable for treating, alleviating or preventing various hyperproliferative disorders or diseases, such as various cancers.
- Such inhibition is further desirable for preventing or modulating the development of such diseases or disorders in an animal suspected of being, or known to be, prone to such diseases or disorders.
- modulation of the expression of one JNK protein can be combined with modulation of one or more additional JNK proteins in order to achieve a requisite level of interference with AP-1-mediated transcription .
- Methods of modulating the expression of JNK proteins comprising contacting animals with oligonucleotides specifically hybridizable with a nucleic acid encoding a JNK protein are herein provided. These methods are believed to be useful both therapeutically and diagnostically as a consequence of the association between kinase-mediated activation of AP-1 and cellular proliferation. These methods are also useful as tools, for example, in the detection and determination of the role of kinase-mediated activation of AP-1 in various cell functions and physiological processes and conditions, and for the diagnosis of conditions associated with such expression and activation.
- the present invention also comprises methods of inhibiting JNK-mediated activation using the oligonucleotides of the invention.
- Methods of treating conditions in which abnormal or excessive JNK-mediated cellular proliferation occurs are also provided.
- These methods employ the oligonucleotides of the invention and are believed to be useful both therapeutically and as clinical research and diagnostic tools.
- the oligonucleotides of the present invention may also be used for research purposes.
- the specific hybridization exhibited by the oligonucleotides of the present invention may be used for assays, purifications, cellular product preparations and in other methodologies which may be appreciated by persons of ordinary skill in the art.
- the present invention employs oligonucleotides for use in antisense modulation of the function of DNA or messenger RNA (mRNA) encoding a protein the modulation of which is desired, and ultimately to regulate the amount of such a protein.
- mRNA messenger RNA
- Hybridization of an antisense oligonucleotide with its mRNA target interferes with the normal role of mRNA and causes a modulation of its function in cells.
- the functions of mRNA to be interfered with include all vital functions such as translocation of the RNA to the site for protein translation, actual translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and possibly even independent catalytic activity which may be engaged in by the RNA.
- modulation means either an increase (stimulation) or a decrease (inhibition) in the expression of the protein.
- inhibition is the preferred form of modulation of gene expression.
- Targeting an oligonucleotide to the associated nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a foreign nucleic acid from an infectious agent. In the present invention, the target is a cellular gene associated with hyperproliferative disorders.
- the targeting process also includes determination of a site or sites within this gene for the oligonucleotide interaction to occur such that the desired effect, either detection or modulation of expression of the protein, will result. Once the target site or sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i . e . , hybridize sufficiently well and with sufficient specificity to give the desired effect.
- the 5' untranslated region hereinafter, the "5'-UTR”
- the translation initiation codon region hereinafter, the "tIR”
- the open reading frame hereinafter, the "ORF”
- the translation termination codon region hereinafter, the "tTR”
- the 3' untranslated region hereinafter, the "3'-UTR”
- these regions are arranged in a typical messenger RNA molecule in the following order (left to right, 5' to 3'): 5'-UTR, tIR, ORF, tTR, 3'-UTR.
- ORFs As is known in the art, although some eukaryotic transcripts are directly translated, many ORFs contain one or more sequences, known as "introns," which are excised from a transcript before it is translated; the expressed (unexcised) portions of the ORF are referred to as “exons" (Alberts et al . , Molecular Biology of the Cell , 1983, Garland Publishing Inc., New York, pp. 411-415). Furthermore, because many eukaryotic ORFs are a thousand nucleotides or more in length, it is often convenient to subdivide the ORF into, e.g., the 5' ORF region, the central ORF region, and the 3' ORF region.
- an ORF contains one or more sites that may be targeted due to some functional significance in vivo .
- sites include intragenic stem-loop structures (see, e.g., U.S. Patent No. 5,512,438 and, in unprocessed mRNA molecules, intron/exon splice sites .
- one preferred intragenic site is the region encompassing the translation initiation codon of the open reading frame (ORF) of the gene.
- the translation initiation codon is typically 5 ' -AUG (in transcribed mRNA molecules; 5 ' -ATG in the corresponding DNA molecule)
- the translation initiation codon is also referred to as the "AUG codon,” the “start codon” or the "AUG start codon.”
- a minority of genes have a translation initiation codon having the RNA sequence 5'-GUG, 5 ' -UUG or 5'-CUG, and 5 ' -AUA, 5 ' -ACG and 5 ' -CUG have been shown to function in vivo.
- 5 ' -UUU functions as a translation initiation codon in vitro (Brigstock et al., Growth Factors, 1990, 4, 45; Gelbert et al., Somat. Cell. Mol. Genet., 1990, 16, 173; Gold and Stormo, in:
- ransmid DNA Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Vol. 2, 1987, Neidhardt et al., eds., American Society for Microbiology, Washington, D.C., p. 1303,) .
- the terms "translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (prokaryotes) .
- eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions, in order to generate related polypeptides having different amino terminal sequences (Markussen et al., Development, 1995, 121, 3723; Gao et al., Cancer Res., 1995, 55, 743; McDermott et al., Gene, 1992, 117, 193; Perri et al., J. Biol. Chem., 1991, 266, 12536; French et al., J.
- start codon and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding a JNK protein, regardless of the sequence (s) of such codons.
- a translation termination codon (or "stop codon”) of a gene may have one of three sequences, i.e., 5 ' -UAA, 5 ' -UAG and 5'-UGA (the corresponding DNA sequences are 5 ' -TAA, 5 ' -TAG and 5' -TGA, respectively).
- start codon region and “translation initiation region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3 ' ) from a translation initiation codon.
- stop codon region and “translation termination region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3 ' ) from a translation termination codon.
- Oligonucleotides of the Invention The present invention employs oligonucleotides for use in antisense modulation of one or more JNK proteins.
- oligonucleotide refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid.
- This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent intersugar (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly.
- modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced binding to target and increased stability in the presence of nucleases.
- An oligonucleotide is a polymer of a repeating unit generically known as a nucleotide.
- the oligonucleotides in accordance with this invention preferably comprise from about 8 to about 30 nucleotides.
- An unmodified (naturally occurring) nucleotide has three components: (1) a nitrogen- containing heterocyclic ase linked by one of its nitrogen atoms to (2) a 5-pentofuranosyl sugar and (3) a phospha te esterified to one of the 5' or 3' carbon atoms of the sugar.
- the phosphate of a first nucleotide is also esterified to an adjacent sugar of a second, adjacent nucleotide via a 3 '-5' phosphate linkage.
- the "backbone" of an unmodified oligonucleotide consists of (2) and (3), that is, sugars linked together by phosphodiester linkages between the 5' carbon of the sugar of a first nucleotide and the 3' carbon of a second, adjacent nucleotide.
- a “nucleoside” is the combination of (1) a nucleobase and (2) a sugar in the absence of (3) a phosphate moiety (Kornberg, A., DNA Replica tion, W.H.
- Oligonucleotides may comprise nucleotide sequences sufficient in identity and number to effect specific hybridization with a particular nucleic acid.
- hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleotides.
- adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
- oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position.
- the oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.
- “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target.
- An oligonucleotide is specifically hybridizable to its target sequence due to the formation of base pairs between specific partner nucleobases in the interior of a nucleic acid duplex.
- guanine (G) binds to cytosine (C)
- adenine (A) binds to thymine (T) or uracil (U) .
- nucleobase equivalents include 5- methylcytosine, 5-hydroxymethylcytosine (HMC) , glycosyl HMC and gentiobiosyl HMC (C equivalents) , and 5- hydroxymethyluracil (U equivalent) .
- synthetic nucleobases which retain partner specificity include, for example, 7-deaza-Guanine, which retains partner specificity for C.
- an oligonucleotide' s capacity to specifically hybridize with its target sequence will not be altered by any chemical modification to a nucleobase in the nucleotide sequence of the oligonucleotide which does not significantly effect its specificity for the partner nucleobase in the target oligonucleotide. It is understood in the art that an oligonucleotide need not be 100% complementary to its target DNA sequence to be specifically hybridizable.
- An oligonucleotide is specifically hybridizable when there is a sufficient degree of complementarity to avoid nonspecific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i . e . , under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vi tro assays, under conditions in which the assays are performed.
- Antisense oligonucleotides are commonly used as research reagents, diagnostic aids, and therapeutic agents.
- antisense oligonucleotides which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes, for example to distinguish between the functions of various members of a biological pathway. This specific inhibitory effect has, therefore, been harnessed by those skilled in the art for research uses.
- the specificity and sensitivity of oligonucleotides is also harnessed by those of skill in the art for therapeutic uses.
- Modified Linkages Specific examples of some preferred modified oligonucleotides envisioned for this invention include those containing phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
- oligonucleotides with phosphorothioates and those with CH 2 - NH-0-CH 2 , CH 2 -N(CH 3 ) -0-CH 2 [known as a methylene (methylimino) or MMI backbone], CH 2 -0-N (CH 3 ) -CH 2 , CH 2 -N (CH 3 ) -N (CH 3 ) -CH 2 and O-N (CH 3 ) -CH 2 -CH 2 backbones, wherein the native phosphodiester backbone is represented as 0-P-0-CH 2 ) .
- oligonucleotides having morpholino backbone structures (Summerton and Weller, U.S. Patent No.
- oligonucleotides with NR-C (*) -CH 2 -CH 2 , CH 2 -NR- C(*)-CH 2/ CH 2 -CH 2 -NR-C(*) , C ( * ) -NR-CH 2 -CH 2 and CH 2 -C ( * ) -NR-CH 2 backbones, wherein "*” represents 0 or S (known as amide backbones; DeMesmaeker et al . , WO 92/20823, published November 26, 1992) .
- the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleobases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al . , Science, 1991, 254:1497; U.S. Patent No. 5,539,082).
- the oligonucleotides of the invention may additionally or alternatively include nucleobase modifications or substitutions.
- "unmodified" or “natural” nucleobases include adenine (A) , guanine (G) , thymine (T) , cytosine (C) and uracil (U) .
- Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-methylcytosine, 5- hydroxymethylcytosine (HMC) , glycosyl HMC and gentiobiosyl HMC, as well synthetic nucleobases, e.g., 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5- hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N 6 (6- aminohexyl) adenine and 2, 6-diaminopurine (Kornberg, A., DNA Replica tion, W.H.
- nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-methylcytosine, 5- hydroxymethylcytosine (HMC) ,
- oligonucleotides of the invention may additionally or alternatively comprise substitutions of the sugar portion of the individual nucleotides.
- oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
- modified oligonucleotides may contain one or more substituted sugar moieties comprising one of the following at the 2 ' position: OH, SH, SCH 3 , F, OCN, OCH 3 OCH 3 , OCH 3 0 (CH 2 ) n CH 3 , 0(CH 2 ) n NH 2 or 0(CH 2 ) n CH 3 where n is from 1 to about 10; C x to C 10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF 3 ; OCF 3 ; 0-, S-, or N- alkyl; 0-, S-, or N-alkenyl; SOCH 3 ; S0 2 CH 3 ; 0N0 2 ; N0 2 ; N 3 ; NH 2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an
- a preferred modification includes 2'- methoxyethoxy [2 ' -0-CH 2 CH 2 OCH 3 , also known as 2'-0-(2- methoxyethyl) ] (Martin et al . , Helv. Chim . Acta , 1995, 78;486) .
- Other preferred modifications include 2 ' -methoxy- (2'-0-CH 3 ), 2'-propoxy- (2 ⁇ -OCH 2 CH 2 CH 3 ) and 2 '-fluoro- (2'- F) .
- effector group is a chemical moiety that is capable of carrying out a particular chemical or biological function.
- effector groups include, but are not limited to, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties.
- a variety of chemical linkers may be used to conjugate an effector group to an oligonucleotide of the invention.
- U.S. Patent No. 5,578,718 to Cook et al discloses methods of attaching an alkylthio linker, which may be further derivatized to include additional groups, to ribofuranosyl positions, nucleosidic base positions, or on internucleoside linkages. Additional methods of conjugating oligonucleotides to various effector groups are known in the art; see, e.g., Protocols for Oligonucleotide Conj uga tes (Methods in Molecular Biology, Volume 26) Agrawal, S., ed. , Humana Press, Totowa, NJ, 1994.
- oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more lipophilic moieties which enhance the cellular uptake of the oligonucleotide.
- lipophilic moieties may be linked to an oligonucleotide at several different positions on the oligonucleotide. Some preferred positions include the 3' position of the sugar of the 3' terminal nucleotide, the 5' position of the sugar of the 5' terminal nucleotide, and the 2' position of the sugar of any nucleotide.
- the N 6 position of a purine nucleobase may also be utilized to link a lipophilic moiety to an oligonucleotide of the invention (Gebeyehu, G., et al., Nucleic Acids Res., 1987, 15:4513) .
- lipophilic moieties include but are not limited to a cholesteryl moiety (Letsinger et al., Proc. Natl. Acad. Sci. U.S.A., 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.
- a thioether e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl . Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.
- a phospholipid e.g., di- hexadecyl-rac-glycerol or triethylammonium 1,2-di-O- hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.
- Acids Res., 1990, 18:3777 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys . Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther.
- Oligonucleotides comprising lipophilic moieties, and methods for preparing such oligonucleotides, are disclosed in U.S. Patents Nos. 5,138,045, 5,218,105 and 5,459,255.
- the present invention also includes oligonucleotides that are substantially chirally pure with regard to particular positions within the oligonucleotides.
- substantially chirally pure oligonucleotides include, but are not limited to, those having phosphorothioate linkages that are at least 75% Sp or Rp (Cook et al . , U.S. Patent No. 5,587,361) and those having substantially chirally pure (Sp or Rp) alkylphosphonate, phosphoamidate or phosphotriester linkages (Cook, U.S. Patents Nos. 5,212,295 and 5,521,302).
- the present invention also includes oligonucleotides which are chimeric oligonucleotides.
- "Chimeric" oligonucleotides or “chimeras,” in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
- An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA: DNA or RNA: RNA hybrids.
- RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA: DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of antisense inhibition of gene expression. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
- such “chimeras” may be “gap ers,” i.e., oligonucleotides in which a central portion (the "gap") of the oligonucleotide serves as a substrate for, e.g., RNase H, and the 5' and 3' portions (the “wings”) are modified in such a fashion so as to have greater affinity for the target RNA molecule but are unable to support nuclease activity (e.g., 2 '-fluoro- or 2 ' -methoxyethoxy- substituted) .
- nuclease activity e.g., 2 '-fluoro- or 2 ' -methoxyethoxy- substituted
- chimeras include "wingmers,” that is, oligonucleotides in which the 5' portion of the oligonucleotide serves as a substrate for, e.g., RNase H, whereas the 3' portion is modified in such a fashion so as to have greater affinity for the target RNA molecule but is unable to support nuclease activity (e.g., 2 '-fluoro- or 2 ' -methoxyethoxy- substituted), or vice-versa .
- wingmers that is, oligonucleotides in which the 5' portion of the oligonucleotide serves as a substrate for, e.g., RNase H, whereas the 3' portion is modified in such a fashion so as to have greater affinity for the target RNA molecule but is unable to support nuclease activity (e.g., 2 '-fluoro- or 2 ' -methoxyethoxy- substituted), or vice-versa .
- the oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA) . Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives.
- 5,506,351 drawn to processes for the preparation of 2'-0-alkyl guanosine and related compounds, including 2, 6-diaminopurine compounds;
- U.S. Patent No. 5,587,469 drawn to oligonucleotides having N-2 substituted purines;
- U.S. Patent No. 5,587,470 drawn to oligonucleotides having 3-deazapurines;
- U.S. Patent Nos. 5,602,240, and 5,610,289 drawn to backbone modified oligonucleotide analogs; and
- the ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL) .
- the solution was poured into fresh ether (2.5 L) to yield a stiff gum.
- the ether was decanted and the gum was dried in a vacuum oven (60°C at 1 mm Hg for 24 h) to give a solid which was crushed to a light tan powder (57 g, 85% crude yield) .
- the material was used as is for further reactions.
- N-Benzoyl-2 ' -O-methoxyethyl-5 ' -O- dimethoxytrityl-5- ⁇ tethylcytidine-3 ' -amidite N-Benzoyl-2 ' - O-methoxyethyl-5 ' -O-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) was dissolved in CH 2 C1 2 (1 L) . Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra- (isopropyl) phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere.
- the resulting mixture was stirred for 20 hours at room temperature (tic showed the reaction to be 95% complete) .
- the reaction mixture was extracted with saturated NaHC0 3 (lx 300 mL) and saturated NaCl (3x 300 mL) .
- the aqueous washes were back- extracted with CH 2 C1 2 (300 mL) , and the extracts were combined, dried over MgS0 4 and concentrated.
- the residue obtained was chromatographed on a 1.5 kg silica column using EtOAc ⁇ Hexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g (87%) of the title compound .
- the compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to "prodrugs” and “pharmaceutically acceptable salts” of the oligonucleotides of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
- the oligonucleotides of the invention may additionally or alternatively be prepared to be delivered in a "prodrug" form.
- the term "prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
- prodrug versions of the oligonucleotides of the invention are prepared as SATE [ (S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al . , published December 9, 1993.
- compositions of the oligonucleotides of the invention refers to physiologically and pharmaceutically acceptable salts of the oligonucleotides of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
- Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like.
- Suitable amines are N,N ' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al . , "Pharmaceutical Salts," J. of Pharma Sci . , 1977, 66:1) .
- the base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner.
- the free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner.
- a "pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates.
- Suitable pharmaceutically acceptable salts include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotin
- Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.
- preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.
- salts formed with inorganic acids for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like
- salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine .
- oligonucleotides of the present invention specifically hybridize to nucleic acids (e.g., mRNAs) encoding a JNK protein.
- the oligonucleotides of the present invention can be utilized as therapeutic compounds, as diagnostic tools or research reagents that can be incorporated into kits, and in purifications and cellular product preparations, as well as other methodologies, which are appreciated by persons of ordinary skill in the art.
- the oligonucleotides of the present invention can be used to detect the presence of JNK protein-specific nucleic acids in a cell or tissue sample.
- radiolabeled oligonucleotides can be prepared by 32 P labeling at the 5' end with polynucleotide kinase. (Sambrook et al . , Molecular Cloning. A Labora tory Manual , Cold Spring Harbor Laboratory Press, 1989, Volume 2, pg.
- Radiolabeled oligonucleotides are then contacted with cell or tissue samples suspected of containing JNK protein message RNAs (and thus JNK proteins) , and the samples are washed to remove unbound oligonucleotide. Radioactivity remaining in the sample indicates the presence of bound oligonucleotide, which in turn indicates the presence of nucleic acids complementary to the oligonucleotide, and can be quantitated using a scintillation counter or other routine means. Expression of nucleic acids encoding these proteins is thus detected.
- Radiolabeled oligonucleotides of the present invention can also be used to perform autoradiography of tissues to determine the localization, distribution and quantitation of JNK proteins for research, diagnostic or therapeutic purposes.
- tissue sections are treated with radiolabeled oligonucleotide and washed as described above, then exposed to photographic emulsion according to routine autoradiography procedures.
- the emulsion when developed, yields an image of silver grains over the regions expressing a JNK protein gene. Quantitation of the silver grains permits detection of the expression of mRNA molecules encoding these proteins and permits targeting of oligonucleotides to these areas.
- Analogous assays for fluorescent detection of expression of JNK protein nucleic acids can be developed using oligonucleotides of the present invention which are conjugated with fluorescein or other fluorescent tags instead of radiolabeling. Such conjugations are routinely accomplished during solid phase synthesis using fluorescently-labeled amidites or controlled pore glass (CPG) columns. Fluorescein-labeled amidites and CPG are available from, e.g., Glen Research, Sterling VA. Other means of labeling oligonucleotides are known in the art (see, e . g. , Ruth, Chapter 6 In : Methods in Molecular Biology, Vol . 26: Protocols for Oligonucleotide Conj uga tes, Agrawal, ed. , Humana Press Inc., Totowa, NJ, 1994, pages 167-185) .
- Kits for detecting the presence or absence of expression of a JNK protein may also be prepared.
- Such kits include an oligonucleotide targeted to an appropriate gene, i.e., a gene encoding a JNK protein.
- Appropriate kit and assay formats such as, e.g., "sandwich” assays, are known in the art and can easily be adapted for use with the oligonucleotides of the invention.
- Hybridization of the oligonucleotides of the invention with a nucleic acid encoding a JNK protein can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection systems.
- the oligonucleotides of the invention are also useful for the purification of specific Jun kinase proteins from cells that normally express a set of JNK proteins which are similar to each other in terms of their polypeptide sequences and biochemical properties.
- the purification of a JNK1 protein from cells that expresses JNK1, JNK2 and JNK3 proteins can be enhanced by first treating such cells with oligonucleotides that inhibit the expression of JNK2 and JNK3 and/or with oligonucleotides that increase the expression of JNK1, because such treatments will increase the relative ratio of JNK1 relative to JNK2 and JNK3.
- the yield of JNK1 from subsequent purification steps will be improved as the amount of the biochemically similar (and thus likely to contaminate) JNK2 and JNK3 proteins in extracts prepared from cells so treated will be diminished.
- Biologically Active Oligonucleotides The invention is also drawn to the administration of oligonucleotides having biological activity to cultured cells, isolated tissues and organs and animals.
- having biological activity it is meant that the oligonucleotide functions to modulate the expression of one or more genes in cultured cells, isolated tissues or organs and/or animals.
- Such modulation can be achieved by an antisense oligonucleotide by a variety of mechanisms known in the art, including but not limited to transcriptional arrest; effects on RNA processing (capping, polyadenylation and splicing) and transportation; enhancement of cellular degradation of the target nucleic acid; and translational arrest (Crooke et al . , Exp. Opin . Ther. Pa tents, 1996, 6:855) .
- compositions and methods of the invention can be used to study the function of one or more genes in the animal.
- antisense oligonucleotides have been systemically administered to rats in order to study the role of the W-methyl-D-aspartate receptor in neuronal death, to mice in order to investigate the biological role of protein kinase C- , and to rats in order to examine the role of the neuropeptide Yl receptor in anxiety (Wahlestedt et al . , Na ture, 1993, 363:260; Dean et al . , Proc . Na tl . Acad. Sci . U. S . A.
- antisense knockouts i.e., inhibition of a gene by systemic administration of antisense oligonucleotides
- antisense oligonucleotides may represent the most accurate means for examining a specific member of the family (see, generally, Albert et al . , Trends Pharmacol . Sci . , 1994, 15:250).
- compositions and methods of the invention also have therapeutic uses in an animal, including a human, having (i.e., suffering from), or known to be or suspected of being prone to having, a disease or disorder that is treatable in whole or in part with one or more nucleic acids.
- therapeutic uses is intended to encompass prophylactic, palliative and curative uses wherein the oligonucleotides of the invention are contacted with animal cells either in vivo or ex vivo .
- a therapeutic use includes incorporating such cells into an animal after treatment with one or more oligonucleotides of the invention.
- an animal suspected of having a disease or disorder which can be treated or prevented by modulating the expression or activity of a JNK protein is, for example, treated by administering oligonucleotides in accordance with this invention.
- the oligonucleotides of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an oligonucleotide to a suitable pharmaceutically acceptable carrier such as, e.g., a diluent.
- a suitable pharmaceutically acceptable carrier such as, e.g., a diluent.
- Workers in the field have identified antisense, triplex and other oligonucleotide compositions which are capable of modulating expression of genes implicated in viral, fungal and metabolic diseases. Antisense oligonucleotides have been safely administered to humans and several clinical trials are presently underway.
- oligonucleotides can be useful therapeutic instrumentalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.
- the following U.S. patents demonstrate palliative, therapeutic and other methods utilizing antisense oligonucleotides.
- U. S. Patent No. 5,135,917 provides antisense oligonucleotides that inhibit human interleukin-1 receptor expression.
- U.S. Patent No. 5,098,890 is directed to antisense oligonucleotides complementary to the c-myb oncogene and antisense oligonucleotide therapies for certain cancerous conditions.
- U.S. Patent No. 5,087,617 provides methods for treating cancer patients with antisense oligonucleotides.
- Patent No. 5,166,195 provides oligonucleotide inhibitors of Human Immunodeficiency Virus (HIV).
- U.S. Patent No. 5,004,810 provides oligomers capable of hybridizing to herpes simplex virus Vmw65 mRNA and inhibiting replication.
- U.S. Patent No. 5,194,428 provides antisense oligonucleotides having antiviral activity against influenzavirus .
- U.S. Patent No. 4,806,463 provides antisense oligonucleotides and methods using them to inhibit HTLV-III replication.
- U.S. Patent No. 5,286,717 provides oligonucleotides having a complementary base sequence to a portion of an oncogene.
- U.S. Patent No. 5,264,423 are directed to phosphorothioate oligonucleotide analogs used to prevent replication of foreign nucleic acids in cells.
- U.S. Patent No. 4,689,320 is directed to antisense oligonucleotides as antiviral agents specific to cytomegalovirus (CMV) .
- CMV cytomegalovirus
- U.S. Patent No. 5,098,890 provides oligonucleotides complementary to at least a portion of the mRNA transcript of the human c-myb gene.
- U.S. Patent No. 5,242,906 provides antisense oligonucleotides useful in the treatment of latent Epstein-Barr virus (EBV) infections.
- EBV Epstein-Barr virus
- the term "disease or disorder” (1) includes any abnormal condition of an organism or part, especially as a consequence of infection, inherent weakness, environmental stress, that impairs normal physiological functioning; (2) excludes pregnancy per se but not autoimmune and other diseases associated with pregnancy; and (3) includes cancers and tumors.
- the term "known to be or suspected of being prone to having a disease or disorder” indicates that the subject animal has been determined to be, or is suspected of being, at increased risk, relative to the general population of such animals, of developing a particular disease or disorder as herein defined.
- a subject animal "known to be or suspected of being prone to having a disease or disorder” could have a personal and/or family medical history that includes frequent occurrences of a particular disease or disorder.
- a subject animal "known to be or suspected of being prone to having a disease or disorder" could have had such a susceptibility determined by genetic screening according to techniques known in the art (see, e . g. , U.S. Congress, Office of Technology Assessment, Chapter 5 In : Genetic Moni toring and Screening in the Workplace, OTA-BA-455, U.S. Government Printing Office, Washington, D.C., 1990, pages 75-99).
- a disease or disorder that is treatable in whole or in part with one or more nucleic acids refers to a disease or disorder, as herein defined, (1) the management, modulation or treatment thereof, and/or (2) therapeutic, curative, palliative and/or prophylactic relief therefrom, can be provided via the administration of an antisense oligonucleotide .
- Pharmaceutical Compositions The formulation of pharmaceutical compositions comprising the oligonucleotides of the invention, and their subsequent administration, are believed to be within the skill of those in the art. A.
- a patient i.e., an animal, including a human, having or predisposed to a disease or disorder
- a pharmaceutically acceptable carrier in doses ranging from 0.01 ⁇ g to 100 g per kg of body weight depending on the age of the patient and the severity of the disorder or disease state being treated.
- the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease or disorder, its severity and the overall condition of the patient, and may extend from once daily to once every 20 years.
- the term "treatment regimen” is meant to encompass therapeutic, palliative and prophylactic modalities.
- the dosage of the nucleic acid may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disorder or disease state is observed, or if the disorder or disease state has been ablated.
- Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved.
- Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC 50 s found to be effective in in vi tro and in vivo animal models.
- dosage is from 0.01 ⁇ g to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years.
- An optimal dosing schedule is used to deliver a therapeutically effective amount of the oligonucleotide being administered via a particular mode of administration.
- the term "therapeutically effective amount,” for the purposes of the invention refers to the amount of oligonucleotide-containing pharmaceutical composition which is effective to achieve an intended purpose without undesirable side effects (such as toxicity, irritation or allergic response) .
- side effects such as toxicity, irritation or allergic response
- human doses can be extrapolated from animal studies (Katocs et al .
- the dosage required to provide an effective amount of a pharmaceutical composition will vary depending on the age, health, physical condition, weight, type and extent of the disease or disorder of the recipient, frequency of treatment, the nature of concurrent therapy (if any) and the nature and scope of the desired effect (s) (Nies et al . , Chapter 3 In : Goodman & Gilman ' s The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al .
- the term "high risk individual” is meant to refer to an individual for whom it has been determined, via, e.g., individual or family history or genetic testing, has a significantly higher than normal probability of being susceptible to the onset or recurrence of a disease or disorder.
- the individual can be prophylactically treated to prevent the onset or recurrence of the disease or disorder.
- prophylactically effective amount is meant to refer to an amount of a pharmaceutical composition which produces an effect observed as the prevention of the onset or recurrence of a disease or disorder.
- Prophylactically effective amounts of a pharmaceutical composition are typically determined by the effect they have compared to the effect observed when a second pharmaceutical composition lacking the active agent is administered to a similarly situated individual.
- nucleic acid is administered in maintenance doses, ranging from 0.01 ⁇ g to 100 g per kg of body weight, once or more daily, to once every 20 years.
- maintenance doses ranging from 0.01 ⁇ g to 100 g per kg of body weight, once or more daily, to once every 20 years.
- preventative doses ranging from 0.01 ⁇ g to 100 g per kg of body weight, once or more daily, to once every 20 years.
- an individual may be made less susceptible to an inflammatory condition that is expected to occur as a result of some medical treatment, e.g., graft versus host disease resulting from the transplantation of cells, tissue or an organ into the individual.
- it may be more effective to treat a patient with an oligonucleotide of the invention in conjunction with other traditional therapeutic modalities in order to increase the efficacy of a treatment regimen.
- treatment regimen is meant to encompass therapeutic, palliative and prophylactic modalities.
- a patient may be treated with conventional chemotherapeutic agents, particularly those used for tumor and cancer treatment.
- chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis- chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6- mercaptopurine, 6-thioguanine, cytarabine (CA) , 5- azacytidine, 5-
- chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide) , or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide) .
- a first antisense oligonucleotide targeted to a first JNK protein is used in combination with a second antisense oligonucleotide targeted to a second JNK protein in order to such JNK proteins to a more extensive degree than can be achieved when either oligonucleotide is used individually.
- the first and second JNK proteins which are targeted by such oligonucleotides are identical, are different JNK proteins or are different isoforms of the same JNK protein.
- compositions for the non-parenteral administration of oligonucleotides may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
- Pharmaceutically acceptable organic or inorganic carrier substances suitable for non-parenteral administration which do not deleteriously react with oligonucleotides can be used.
- Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
- compositions 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 react with the oligonucleotide (s) of the pharmaceutical composition.
- 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 react with the oligonucleotide (s) of the pharmaceutical composition.
- Pharmaceutical compositions in the form of aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
- such suspensions may also contain stabilize
- an oligonucleotide is administered via the rectal mode.
- pharmaceutical compositions for rectal administration include foams, solutions (enemas) and suppositories. Rectal suppositories for adults are usually tapered at one or both ends and typically weigh about 2 g each, with infant rectal suppositories typically weighing about one-half as much, when the usual base, cocoa butter, is used (Block, Chapter 87 In : Remington ' ' s Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, PA, 1990) .
- one or more oligonucleotides are administered via oral delivery.
- Pharmaceutical compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, troches, tablets or SECs (soft elastic capsules or "caplets") . Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, carrier substances or binders may be desirably added to such pharmaceutical compositions.
- the use of such pharmaceutical compositions has the effect of delivering the oligonucleotide to the alimentary canal for exposure to the mucosa thereof.
- the pharmaceutical composition can comprise material effective in protecting the oligonucleotide from pH extremes of the stomach, or in releasing the oligonucleotide over time, to optimize the delivery thereof to a particular mucosal site.
- Enteric coatings for acid-resistant tablets, capsules and caplets are known in the art and typically include acetate phthalate, propylene glycol and sorbitan monoleate.
- Various methods for producing pharmaceutical compositions for alimentary delivery are well known in the art. See, generally, Nairn, Chapter 83; Block, Chapter 87; Rudnic et al . , Chapter 89; Porter, Chapter 90; and Longer et al .
- oligonucleotides of the invention can be incorporated in a known manner into customary pharmaceutical compositions, such as tablets, coated tablets, pills, granules, aerosols, syrups, emulsions, suspensions and solutions, using inert, non-toxic, pharmaceutically acceptable carriers (excipients) .
- the therapeutically active compound should in each case be present here in a concentration of about 0.5% to about 95% by weight of the total mixture, i.e., in amounts which are sufficient to achieve the stated dosage range.
- compositions are prepared, for example, by diluting the active compounds with pharmaceutically acceptable carriers, if appropriate using emulsifying agents and/or dispersing agents, and, for example, in the case where water is used as the diluent, organic solvents can be used as auxiliary solvents if appropriate.
- Pharmaceutical compositions may be formulated in a conventional manner using additional pharmaceutically acceptable carriers as appropriate.
- compositions may be prepared by conventional means with additional excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose) ; fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrates (e.g., starch or sodium starch glycolate) ; or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art.
- the preparations may also contain flavoring, coloring and/or sweetening agents as appropriate.
- compositions 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 ingredient (s) with the pharmaceutically acceptable carrier (s). In general the pharmaceutical compositions are prepared by uniformly and intimately bringing into association the active ingredient (s) with liquid excipients or finely divided solid excipients or both, and then, if necessary, shaping the product.
- compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing predetermined amounts of the active ingredients; as powders or granules; as solutions or suspensions in an aqueous liquid or a non-aqueous liquid; or as oil-in-water emulsions or water-in-oil liquid emulsions.
- a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
- Compressed tablets may be prepared by compressing in a suitable machine, the active ingredients in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent.
- Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
- the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredients therein.
- Pharmaceutical compositions for parenteral, intrathecal or intraventricular administration, or colloidal dispersion systems may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
- C. Penetration Enhancers Pharmaceutical compositions comprising the oligonucleotides of the present invention may also include penetration enhancers in order to enhance the alimentary delivery of the oligonucleotides.
- Penetration enhancers may be classified as belonging to one of five broad categories, i.e., fatty acids, bile salts, chelating agents, surfactants and non-surfactants (Lee et al . , Cri tical Reviews in Therapeutic Drug Carrier Systems, 1991, 8:91-192; Muranishi, Cri tical Reviews in Therapeutic Drug Carrier Systems, 1990, 7:1) .
- Fatty Acids Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, recinleate, monoolein (a.k.a.
- Bile Salts The physiological roles of bile include the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 In : Goodman & Gilman ' s The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al . , eds., McGraw-Hill, New York, NY, 1996, pages 934-935) .
- Various natural bile salts, and their synthetic derivatives act as penetration enhancers.
- the term "bile salt” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives.
- Chelating agents have the added advantage of also serving as DNase inhibitors and include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA) , citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate) , W-acyl derivatives of collagen, laureth- 9 and IV-amino acyl derivatives of beta-diketones (enamines) (Lee et al . , Cri tical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Cri tical Reviews in Therapeutic Drug Carrier Systems, 1990, 7:1; Buur et al . , J. Control Rel . , 1990, 14:43).
- EDTA disodium ethylenediaminetetraacetate
- citric acid citric acid
- salicylates e.g., sodium salicylate, 5-methoxysalicylate and homovanilate
- Surfactants include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Lee et al . , Cri tical Reviews in Therapeuti c Drug Carrier Systems, 1991, page 92) ; and perfluorochemical emulsions, such as FC-43 (Takahashi et al . , J. Pharm . Phama col . , 1988, 40:252). 5.
- Non-surfactants include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et a l . , Cri tical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92) ; and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al . , J. Pharm . Pharmacol . , 1987, 39:621).
- carrier compound refers to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation.
- carrier compound typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor.
- the recovery of a partially phosphorothioated oligonucleotide in hepatic tissue is reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4- acetamido-4 ' -isothiocyano-stilbene-2 , 2 ' -disulfonic acid (Miyao et al . , Antisense Res . Dev. , 1995, 5:115; Takakura et al . , Antisense & Nucl . Acid Drug Dev. , 1996, 6:177).
- a "pharmaceutically acceptable carrier” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal.
- the pharmaceutically acceptable carrier may be liquid or solid and is selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, etc. , when combined with a nucleic acid and the other components of a given pharmaceutical composition.
- Typical pharmaceutically acceptable carriers include, but are not limited to, binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc. ) ; lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.
- binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.
- fillers e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin
- disintegrates e.g., starch, sodium starch glycolate, etc.
- wetting agents e.g., sodium lauryl sulphate, etc.
- Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are described in U.S. Patents Nos. 4,704,295; 4,556,552; 4,309,406; and 4,309,404.
- compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels.
- the compositions may contain additional compatible pharmaceutically-active materials such as, e.g., antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the composition of present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
- additional compatible pharmaceutically-active materials such as, e.g., antipruritics, astringents, local anesthetics or anti-inflammatory agents
- additional materials useful in physically formulating various dosage forms of the composition of present invention such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
- such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the invention.
- colloidal Dispersion Systems may be used as delivery vehicles to enhance the in vivo stability of the oligonucleotides and/or to target the oligonucleotides to a particular organ, tissue or cell type.
- Colloidal dispersion systems include, but are not limited to, macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems including oil- in-water emulsions, micelles, mixed micelles and liposomes.
- a preferred colloidal dispersion system is a plurality of liposomes, artificial membrane vesicles which may be used as cellular delivery vehicles for bioactive agents in vi tro and in vivo (Mannino et al . , Biotechniques, 1988, 6, 682; Blume and Cevc, Biochem . et Biophys . Acta , 1990, 1029, 91; Lappalainen et al . , Antiviral Res . , 1994, 23, 119; Chonn and Cullis, Current Op. Biotech . , 1995, 6, 698).
- RNA, DNA and intact virions can be encapsulated within the aqueous interior and delivered to brain cells in a biologically active form (Fraley et al . , Trends Biochem . Sci . , 1981, 6, 77).
- the composition of the liposome is usually a combination of lipids, particularly phospholipids, in particular, high phase transition temperature phospholipids, usually in combination with one or more steroids, particularly cholesterol.
- lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, sphingolipids, phosphatidylethanolamine, cerebrosides and gangliosides .
- Particularly useful are diacyl phosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated (lacking double bonds within the 14-18 carbon atom chain) .
- Illustrative phospholipids include phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine .
- the targeting of colloidal dispersion systems can be either passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticuloendothelial system in organs that contain sinusoidal capillaries. Active targeting, by contrast, involves modification of the liposome by coupling thereto a specific ligand such as a viral protein coat (Morishita et al . , Proc . Na tl . Acad. Sci . (U. S . A.
- lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in close association with the lipid bilayer.
- Various linking groups can be used for joining the lipid chains to the targeting ligand.
- the targeting ligand which binds a specific cell surface molecule found predominantly on cells to which delivery of the oligonucleotides of the invention is desired, may be, for example, (1) a hormone, growth factor or a suitable oligopeptide fragment thereof which is bound by a specific cellular receptor predominantly expressed by cells to which delivery is desired or (2) a polyclonal or monoclonal antibody, or a suitable fragment thereof (e.g., Fab; F(ab') 2 ) which specifically binds an antigenic epitope found predominantly on targeted cells.
- Two or more bioactive agents e.g., an oligonucleotide and a conventional drug; two oligonucleotides
- compositions comprising oligonucleotides intended for administration to an animal.
- animal is meant to encompass humans as well as other mammals, as well as reptiles, amphibians, and birds.
- parenteral delivery refers to the administration of an oligonucleotide of the invention to an animal in a manner other than through the digestive canal.
- Means of preparing and administering parenteral pharmaceutical compositions are known in the art (see, e.g., Avis, Chapter 84 In : Remington ' s Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, PA, 1990, pages 1545- 1569) .
- Parenteral means of delivery include, but are not limited to, the following illustrative examples.
- Intraluminal drug administration for the direct delivery of drug to an isolated portion of a tubular organ or tissue (e.g., such as an artery, vein, ureter or urethra) , may be desired for the treatment of patients with diseases or conditions afflicting the lumen of such organs or tissues.
- a catheter or cannula is surgically introduced by appropriate means.
- a cannula is inserted thereinto via the external carotid artery.
- a composition comprising the oligonucleotides of the invention is infused through the cannula or catheter into the isolated segment.
- the infusion cannula or catheter is removed and flow within the tubular organ or tissue is restored by removal of the ligatures which effected the isolation of a segment thereof (Morishita et al . , Proc . Na tl . Acad . Sci . U. S . A . , 1993, 90, 8474).
- Antisense oligonucleotides may also be combined with a biocompatible matrix, such as a hydrogel material, and applied directly to vascular tissue in vivo (Rosenberg et al . , U.S. Patent No. 5,593,974, issued January 14, 1997).
- a biocompatible matrix such as a hydrogel material
- Intraventricular drug administration for the direct delivery of drug to the brain of a patient, may be desired for the treatment of patients with diseases or conditions afflicting the brain.
- a silicon catheter is surgically introduced into a ventricle of the brain of a human patient, and is connected to a subcutaneous infusion pump (Medtronic Inc., Minneapolis, MN) that has been surgically implanted in the abdominal region (Zimm et al . , Cancer Research, 1984, 44, 1698; Shaw, Cancer, 1993, 72 (11 Suppl . ) , 3416).
- the pump is used to inject the oligonucleotides and allows precise dosage adjustments and variation in dosage schedules with the aid of an external programming device.
- the reservoir capacity of the pump is 18-20 mL and infusion rates may range from 0.1 mL/h to 1 mL/h.
- the pump reservoir may be refilled at 3-10 week intervals. Refilling of the pump is accomplished by percutaneous puncture of the self-sealing septum of the pump. 5.
- Intrathecal drug administration for the introduction of a drug into the spinal column of a patient may be desired for the treatment of patients with diseases of the central nervous system.
- a silicon catheter is surgically implanted into the L3-4 lumbar spinal interspace of a human patient, and is connected to a subcutaneous infusion pump which has been surgically implanted in the upper abdominal region (Luer and Hatton, The Annals of Pharmacotherapy, 1993, 27, 912; Ettinger et al . , 1978, Cancer, 41 , 1270, 1978; Yaida et al . , Regul . Pept . , 1995, 59, 193) .
- the pump is used to inject the oligonucleotides and allows precise dosage adjustments and variations in dose schedules with the aid of an external programming device.
- the reservoir capacity of the pump is 18-20 mL, and infusion rates may vary from 0.1 mL/h to 1 mL/h.
- the pump reservoir may be refilled at 3-10 week intervals. Refilling of the pump is accomplished by a single percutaneous puncture to the self-sealing septum of the pump.
- the distribution, stability and pharmacokinetics of oligonucleotides within the central nervous system may be followed according to known methods (Whitesell et al . , Proc. Natl . Acad. Sci . (USA) , 1993, 90, 4665).
- the silicon catheter is configured to connect the subcutaneous infusion pump to, e . g. , the hepatic artery, for delivery to the liver (Kemeny et al . , Cancer, 1993, 71, 1964).
- Infusion pumps may also be used to effect systemic delivery of oligonucleotides (Ewel et al . , Cancer Research, 1992, 52, 3005; Rubenstein et al . , J. Surg. Oncol., 1996, 62, 194) .
- Epidermal and Transdermal Delivery in which pharmaceutical compositions containing drugs are applied topically, can be used to administer drugs to be absorbed by the local dermis or for further penetration and absorption by underlying tissues, respectively.
- Means of preparing and administering medications topically are known in the art (see, e.g., Block, Chapter 87 In : Remington ' s Pharmaceutical Sciences, 18th Ed., Gennaro, ed. , Mack Publishing Co., Easton, PA, 1990, pages 1596-1609).
- Vaginal Delivery provides local treatment and avoids first pass metabolism, degradation by digestive enzymes, and potential systemic side-effects. This mode of administration may be preferred for antisense oligonucleotides targeted to pathogenic organisms for which the vagina is the usual habitat, e.g., Trichomonas vaginalis . In another embodiment, antisense oligonucleotides to genes encoding sperm-specific antibodies can be delivered by this mode of administration in order to increase the probability of conception and subsequent pregnancy. Vaginal suppositories (Block, Chapter 87 In : Remington ' s Pharmaceutical Sciences, 18th Ed., Gennaro, ed. , Mack Publishing Co., Easton, PA, 1990, pages 1609-1614) or topical ointments can be used to effect this mode of delivery.
- Intravesical Delivery provides local treatment and avoids first pass metabolism, degradation by digestive enzymes, and potential systemic side-effects.
- the method requires urethral catheterization of the patient and a skilled staff. Nevertheless, this mode of administration may be preferred for antisense oligonucleotides targeted to pathogenic organisms, such as T. vaginalis, which may invade the urogenital tract.
- Alimentary Delivery refers to the administration, directly or otherwise, to a portion of the alimentary canal of an animal.
- the term “alimentary canal” refers to the tubular passage in an animal that functions in the digestion and absorption of food and the elimination of food residue, which runs from the mouth to the anus, and any and all of its portions or segments, e.g., the oral cavity, the esophagus, the stomach, the small and large intestines and the colon, as well as compound portions thereof such as, e.g., the gastro-intestinal tract.
- the term “alimentary delivery” encompasses several routes of administration including, but not limited to, oral, rectal, endoscopic and sublingual/buccal administration. A common requirement for these modes of administration is absorption over some portion or all of the alimentary tract and a need for efficient mucosal penetration of the nucleic acid(s) so administered .
- Delivery of a drug via the oral mucosa has several desirable features, including, in many instances, a more rapid rise in plasma concentration of the drug than via oral delivery (Harvey, Chapter 35 In : Remington ' s
- Endoscopic Administration can be used for drug delivery directly to an interior portion of the alimentary tract.
- endoscopic retrograde cystopancreatography ERCP
- ERCP endoscopic retrograde cystopancreatography
- Rectal Administration Drugs administered by the oral route can often be alternatively administered by the lower enteral route, i.e., through the anal portal into the rectum or lower intestine.
- Rectal suppositories, retention enemas or rectal catheters can be used for this purpose and may be preferred when patient • compliance might otherwise be difficult to achieve (e.g., in pediatric and geriatric applications, or when the patient is vomiting or unconscious) . Rectal administration may result in more prompt and higher blood levels than the oral route, but the converse may be true as well (Harvey, Chapter 35 In : Remington ' s Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, PA, 1990, page 711) . Because about 50% of the drug that is absorbed from the rectum will bypass the liver, administration by this route significantly reduces the potential for first- pass metabolism (Benet et al . , Chapter 1 In : Goodman & Gilman ' s The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al . , eds., McGraw-Hill, New York, NY, 1996) .
- Oral Administration The preferred method of administration is oral delivery, which is typically the most convenient route for access to the systemic circulation. Absorption from the alimentary canal is governed by factors that are generally applicable, e.g., surface area for absorption, blood flow to the site of absorption, the physical state of the drug and its concentration at the site of absorption (Benet et al . , Chapter 1 In : Goodman & Gilman ' s The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al . , eds., McGraw- Hill, New York, NY, 1996, pages 5-7) .
- factors that are generally applicable e.g., surface area for absorption, blood flow to the site of absorption, the physical state of the drug and its concentration at the site of absorption (Benet et al . , Chapter 1 In : Goodman & Gilman ' s The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al . , eds., McG
- a significant factor which may limit the oral bioavailability of a drug is the degree of "first pass effects.” For example, some substances have such a rapid hepatic uptake that only a fraction of the material absorbed enters the peripheral blood (Van Berge-Henegouwen et al . , Gastroenterology, 1977, 73:300) .
- the compositions and methods of the invention circumvent, at least partially, such first pass effects by providing improved uptake of nucleic acids and thereby, e.g., causing the hepatic uptake system to become saturated and allowing a significant portion of the nucleic acid so administered to reach the peripheral circulation. Additionally or alternatively, the hepatic uptake system is saturated with one or more inactive carrier compounds prior to administration of the active nucleic acid.
- Oligonucleotides were synthesized on an automated DNA synthesizer using standard phosphoramidite chemistry with oxidation using iodine.
- ⁇ -Cyanoethyldiisopropyl phosphoramidites were purchased from Applied Biosystems (Foster City, CA) .
- the standard oxidation bottle was replaced by a 0.2 M solution of 3H-1,2- benzodithiole-3-one-l, 1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages.
- the 2 ' -fluoro-phosphorothioate oligonucleotides of the invention are synthesized using 5 ' -dimethoxytrityl-3 ' - phosphoramidites and prepared as disclosed in U.S. patent application Serial No. 08/383,666, filed February 3, 1995, and U.S. Patent 5,459,255, which issued October 8, 1996, both of which are assigned to the same assignee as the instant application.
- the 2 ' -fluoro-oligonucleotides were prepared using phosphoramidite chemistry and a slight modification of the standard DNA synthesis protocol (i.e., deprotection was effected using methanolic ammonia at room temperature) .
- the 2 ' -methoxyethoxy oligonucleotides were synthesized essentially according to the methods of Martin et al . ⁇ Helv. Chim . Acta , 1995, 78, 486) .
- the 3' nucleotide of the 2 ' -methoxyethoxy oligonucleotides was a deoxynucleotide, and 2 ' -0-CH 2 CH 2 OCH 3 _ cytosines were 5-methyl cytosines, which were synthesized according to the procedures described below.
- PNA antisense analogs are prepared essentially as described in U.S. Patents Nos. 5,539,082 and 5,539,083, both of which (1) issued July 23, 1996, and (2) are assigned to the same assignee as the instant application.
- Example 2 Assays for Oligonucleotide-Mediated Inhibition of JNK mRNA Expression in Human Tumor Cells
- human lung carcinoma cell line A549 American Type Culture Collection, Rockville, MD No. ATCC CCL-185 cells or other cell lines as indicated in the Examples, were grown and treated with oligonucleotides or control solutions as detailed below. After harvesting, cellular extracts were prepared and examined for specific JNK mRNA levels or JNK protein levels (i.e., Northern or Western assays, respectively) .
- the blots were stripped and reprobed with a 32 P-labeled glyceraldehyde 3-phosphate dehydrogenase (G3PDH) probe (Clontech Laboratories, Inc., Palo Alto, CA) in order to confirm equal loading of RNA and to allow the levels of JNK transcripts to be normalized with regard to the G3PDH signals.
- G3PDH glyceraldehyde 3-phosphate dehydrogenase
- DMEM media
- DOPE dioleoyl phosphatidylethanolamine
- the oligonucleotides were added from a 10 ⁇ M stock solution to a final concentration of 400 nM, and the two solutions were mixed by swirling the flasks.
- As a control cells were treated with LIPOFECTINTM without oligonucleotide under the same conditions and for the same times as the oligonucleotide-treated samples. After 4 hours at 37°C, the medium was replaced with fresh DMEM containing 10% serum. The cells were allowed to recover for 18 hours.
- the primary antibodies were detected by means well known in the art (see, e.g., Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al . , eds., John Wiley & Sons, New York, 1992, pages 10-33 to 10-35; and Chapter 18 In : Molecular Cloning: A Labora tory Manual , 2nd Ed., Sambrook et al . , eds., pages 18.1-18.75 and 18.86-18.88) and quantitated using a PHOSPHORIMAGERTM essentially according to the manufacturer's instructions (Molecular Dynamics, Sunnyvale, CA) . Levels of JNK proteins can also be quantitated by measuring the level of their corresponding kinase activity.
- Such kinase assays can be done in gels in si tu (Hibi et al . , Genes & Dev. , 1993, 7, 2135) or after immunoprecipitation from cellular extracts (Derijard et al . , Cell , 1994, 76, 1025).
- Substrates and/or kits for such assays are commercially available from, for example, Upstate Biotechnology, Inc. (Lake Placid, NY) , New England Biolabs, Inc., (Beverly, MA) and Calbiochem-Novabiochem Biosciences, Inc., (La Jolla, CA) .
- Example 3 Oligonucleotide-Mediated Inhibition of JNKl Expression
- Table 1 lists the nucleotide sequences of a set of oligonucleotides designed to specifically hybridize to JNKl mRNAs and their corresponding ISIS and SEQ ID numbers. The nucleotide coordinates of the target gene, JNKl , and gene target regions are also included. The nucleotide co-ordinates are derived from GenBank accession No. L26318, locus name "HUMJNK1" (see also Figure 1(A) of Derijard et al . , Cell , 1994, 76, 1025) .
- gene target regions are as follows: 5'-UTR, 5' untranslated region; tIR, translation initiation region; ORF, open reading frame; 3'-UTR, 3' untranslated region.
- the nucleotides of the oligonucleotides whose sequences are presented in Table 1 are connected by phosphorothioate linkages and are unmodified at the 2' position (i.e., 2 ' -deoxy) . It should be noted that the oligonucleotide target co-ordinate positions and gene target regions may vary within RNAs encoding related isoforms of JNKl (see subsection G, below) .
- ISIS Nos. 12548 SEQ ID NO: 17
- 12551 SEQ ID NO: 20
- ISIS 12548 hybridizes to bases 498-517 of GenBank accession No.
- L27129, locus name "RATSAPKD, " and ISIS 12551 hybridizes to bases 803-822 of the same sequence.
- These oligonucleotides are thus preferred embodiments of the invention for investigating the role of the p54 ⁇ protein kinase in rat in vi tro, i . e . , in cultured cells or tissues derived from whole animals, or in vivo.
- JNKl-specific probes In initial screenings of a set of oligonucleotides derived from the JNKl sequence (Table 2) for biological activity, a cDNA clone of JNKl (Derijard et al . , Cell , 1994, 76, 1025) was radiolabeled and used as a JNKl-specific probe in Northern blots. Alternatively, however, one or more of the oligonucleotides of Table 1 is detectably labeled and used as a JNKl- specific probe.
- Oligonucleotides are thus preferred embodiments of the invention for modulating JNKl expression.
- Oligonucleotides showing levels of inhibition of from > about 80% to about 100% of JNKl mRNAs in this assay include ISIS Nos. 11982, 12539, 12548, 12554 and 12464 (SEQ ID NOS: 5, 16, 17 and 23, respectively) . These oligonucleotides are thus more preferred embodiments of the invention for modulating JNKl expression.
- JNKl oligonucleotides The results for JNKl-specific oligonucleotides (Table 2) indicate that one of the most active phosphorothioate oligonucleotides for modulating JNKl expression is ISIS 12539 (SEQ ID NO: 16) . As detailed in Table 4, additional oligonucleotides based on this oligonucleotide were designed to confirm and extend the findings described above.
- Oligonucleotides ISIS Nos. 14320 (SEQ ID NO: 27) and 14321 (SEQ ID NO: 28) are 2 ' -deoxy-phosphorothioate sense strand and scrambled controls for ISIS 12539 (SEQ ID NO: 16), respectively.
- ISIS Nos. 15346 and 15347 are "gapmers” corresponding to ISIS 12539; both have 2 ' -methoxyethoxy "wings” (having phosphorothioate linkages in the case of ISIS 15346 and phosphodiester linkages in the case of ISIS 15347) and a central 2 ' -deoxy "gap" designed to support RNaseH activity on the target mRNA molecule.
- ISIS Nos. 15348 to 15350 are "wingmers” corresponding to ISIS 12539 and have a 5' or 3' 2 ' -methoxyethoxy RNaseH- refractory "wing” and a 3' or 5' (respectively) 2 ' -deoxy "wing” designed to support RNaseH activity on the target JNKl mRNA.
- P 0, phosphodiester linkage
- MOE methoxyethoxy-.
- Dose- and sequence-dependent response to JNKl oligonucleotides In order to demonstrate a dose-dependent response to ISIS 12539 (SEQ ID NO: 16), different concentrations (i.e., 50, 100, 200 and 400 nM) of ISIS 12539 were tested for their effect on JNKl mRNA levels in A549 cells (Table 6) . In addition, two control oligonucleotides (ISIS 14320, SEQ ID NO: 27, sense control, and ISIS 14321, SEQ ID NO: 28, scrambled control; see also Table 4) were also applied to A549 cells in order to demonstrate the specificity of ISIS 12539. The results (Table 6) demonstrate that the response of A549 cells to ISIS 12539 is dependent on dose in an approximately linear fashion. In contrast, neither of the control oligonucleotides effect any consistent response on JNKl mRNA levels.
- oligonucleotides showing a level of mRNA % inhibition from > about 70% to about 100% include ISIS Nos. 12539 (phosphorothioate linkages), 15346 and 15347 (“gapmers"), and 15348 and 15351 (5' "wingmers") (SEQ ID NO: 16) .
- Oligonucleotides showing levels of mRNA inhibition of from > about 90% to about 100% of JNKl mRNAs in this assay include ISIS Nos. 12539, 15345 AND 15346 (SEQ ID NO: 16) .
- the oligonucleotides tested showed approximately parallel levels of JNKl protein inhibition; ISIS Nos. 12539, 15346-15348 and 15351 effected levels of protein inhibition > about 40%, and ISIS Nos. 12539, 15346 and 15347 effected levels of protein inhibition > . about
- oligonucleotides showing a level of mRNA % inhibition from > about 70% to about 100% include ISIS Nos. 12539 (phosphorothioate linkages) , 15346 and 15347 (“gapmers"), and 15348 (5' "wingmers”) (SEQ ID NO: 16). Oligonucleotides showing levels of mRNA inhibition of from > about 90% to about 100% of JNKl mRNAs at this point in the assay include ISIS Nos. 12539 and 15346 (SEQ ID NO: 16) . Overall, the oligonucleotides tested showed higher levels of JNKl protein inhibition at this point in the assay.
- JNKl- ⁇ l GenBank accession No. L26318, locus name "HUMJNKl"
- JNKl- ⁇ l GeneBank accession No. U34822, locus name "HSU34822”
- JNKl- ⁇ l GenBank accession No.
- JNKl- ⁇ 2 GenBank accession No. U35005, locus name "HSU35005" .
- the four isoforms of JNKl which probably arise from alternative mRNA splicing, may each interact with different transcription factors or sets of transcription factors
- oligonucleotides of the invention are specific for certain members or sets of these isoforms of JNKl.
- nucleotides (nt) 631-665 of JNKl/JNKl- ⁇ l (Genbank accession No. L26318) and nt 625-659 of JNKl- ⁇ 2 (Genbank accession No. U34822) have the sequence shown below as SEQ ID NO: 63
- nt 631-665 of JNKl- ⁇ l GenBank accession No. U35004
- nt 626-660 of JNKl- ⁇ 2 GenBank accession No. U35005
- SEQ ID NO: 64 For purposes of illustration, SEQ ID NOS: 63 and 64 are shown aligned with each other (vertical marks, "I,” indicate bases that are identical in both sequences) :
- antisense oligonucleotides derived from the reverse complement of SEQ ID NO: 63 can be used to modulate the expression of JNK1/JNK1- ⁇ l and JNKl- ⁇ 2 without significantly effecting the expression of JNKl- ⁇ l and JNKl- ⁇ 2.
- antisense oligonucleotides derived from the reverse complement of SEQ ID NO: 64 can be selected and used to modulate the expression of JNKl- ⁇ l and JNKl- ⁇ 2 without significantly effecting the expression of JNKl/JNKl- ⁇ l and JNKl- ⁇ 2.
- SEQ ID NO: 66 see below
- an oligonucleotide having a sequence derived from SEQ ID NO: 65 but not to SEQ ID NO: 66 is specifically hybridizable to mRNAs encoding JNKl/JNKl- ⁇ l and JNKl- ⁇ 2 but not to those encoding JNKl- ⁇ l and JNKl- ⁇ 2:
- antisense oligonucleotides derived from the reverse complement of SEQ ID NO: 67 are specifically hybridizable to mRNAs encoding, and may be selected and used to modulate the expression of, JNKl/JNKl- ⁇ l and JNKl- ⁇ 2 without significantly effecting the expression of JNKl- ⁇ l and JNKl- ⁇ 2.
- antisense oligonucleotides derived from the reverse complement of SEQ ID NO: 68 are specifically hybridizable to mRNAs encoding, and may be selected and used to modulate the expression of, can be selected and used to modulate the expression of JNKl- ⁇ l and JNKl- ⁇ 2 without significantly effecting the expression of JNKl/JNKl- ⁇ l and JNKl- ⁇ 2 :
- JNKl/JNKl- ⁇ l shares identity with JNKl- ⁇ l; similarly, JNKl- ⁇ 2 and JNKl- ⁇ 2 have identical carboxy terminal portions.
- the substantial differences in the amino acid sequences of these isoforms (5 amino acids in JNKl/JNKl- ⁇ l and JNKl- ⁇ l are replaced with 48 amino acids in JNKl- ⁇ 2 and JNKl- ⁇ 2) result from a slight difference in nucleotide sequence that shifts the reading frame.
- nt 1144-1175 of JNKl/JNKl- ⁇ l (Genbank accession No. L26318) and JNKl- ⁇ l (Genbank accession No. U35004) have the sequence shown below as SEQ ID NO: 71
- nt 1138-1164 of JNKl- ⁇ 2 (GenBank accession No. U34822)
- nt 1139-1165 of JNKl- ⁇ 2 (GenBank accession No. U35005) have the sequence shown below as SEQ ID NO: 72.
- SEQ ID NOS: 71 and 72 are shown aligned with each other (dashes, "-,” indicate bases that are absent in the indicated sequence, and emboldened bases indicate the stop codon for the JNKl/JNKl- ⁇ l and JNKl- ⁇ l ORFs) :
- antisense oligonucleotides derived from the reverse complement of SEQ ID NO: 71 are specifically hybridizable to mRNAs encoding, and may be selected and used to modulate the expression of, JNKl/JNKl- ⁇ l and JNKl- ⁇ l without significantly effecting the expression of JNK1- ⁇ 2 and JNKl- ⁇ 2.
- antisense oligonucleotides derived from the reverse complement of SEQ ID NO: 72 are specifically hybridizable to mRNAs encoding, and may be selected and used to modulate the expression of, JNKl- ⁇ 2 and JNKl- ⁇ 2 without significantly effecting the expression of JNKl/JNKl- ⁇ l and JNKl- ⁇ l: 5 ' -GATCACTGCTGCACCTGTGCTAAAGGAGAGGG SEQ ID NO: 73
- such isoform-specific oligonucleotides such as are described above are methoxyethoxy "gapmers” or “wingmers” in which the RNase H- sensitive "gap” or “wing” is positioned so as to overlap a region of nonidentity in the above antisense sequences, i.e., SEQ ID NOS: 65, 66, 69, 70, 73 and 74.
- Example 4 Oligonucleotide-Mediated Inhibition of JNK2 Expression
- Table 8 lists the nucleotide sequences of oligonucleotides designed to specifically hybridize to JNK2 mRNAs and the corresponding ISIS and SEQ ID numbers thereof.
- the target gene nucleotide co-ordinates and gene target region are also included.
- the nucleotide co-ordinates are derived from GenBank accession No. L31951, locus name "HUMJNK2" (see also Figure 1(A) of Sluss et al . , Mol . Cel . Biol . , 1994, 1 4 , 8376, and Kallunki et a l .
- gene target regions are as follows: 5 ' -UTR, 5' untranslated region; tIR, translation initiation region; ORF, open reading frame; 3' -UTR, 3' untranslated region.
- the nucleotides of the oligonucleotides whose sequences are presented in Table 8 are connected by phosphorothioate linkages and are unmodified at the 2' position (i.e., 2-deoxy) . It should be noted that the oligonucleotide target co-ordinate positions and gene target regions may vary within mRNAs encoding related isoforms of JNK2 (see subsection G, below) .
- ISIS No. 12562 hybridizes to the ORF of mRNAs from Ra ttus norvegicus that encode a stress-activated protein kinase named "p54 ⁇ 2" (Kyriakis et al . , Na ture, 1994, 369, 156). Specifically, ISIS 12562 (SEQ ID NO: 33) hybridizes to bases 649-668 of GenBank accession No.
- This oligonucleotide is thus a preferred embodiment of the invention for investigating the role of the p54 ⁇ 2 protein kinase in rat in vi tro, i . e . , in cultured cells or tissues derived from whole animals, or in vivo .
- JNK2-specific probes In initial screenings of a set of oligonucleotides derived from the JNK2 sequence (Table 9) for biological activity, a cDNA clone of JNK2 (Kallunki et al . , Genes & Development , 1994, 8, 2996) was radiolabeled and used as a JNK2-specific probe in Northern blots. Alternatively, however, one or more of the oligonucleotides of Table 8 is detectably labeled and used as a JNK2-specific probe. C.
- Oligonucleotides showing levels of inhibition of from > about 80% to about 100% of JNK2 mRNAs in this assay include ISIS Nos. 12558, 12560, 12565, 12567, 12568 and 12569 (SEQ ID NOS: 29, 31, 36, 38, 39 and 40, respectively) . These oligonucleotides are thus more preferred embodiments of the invention for modulating JNK2 expression.
- JNK2 oligonucleotides The results for JNK2-specific oligonucleotides (Table 9) indicate that one of the most active phosphorothioate oligonucleotides for modulating JNK2 expression is ISIS 12560 (SEQ ID NO: 31) . As detailed in Table 11, additional oligonucleotides based on this oligonucleotide were designed to confirm and extend the findings described above. Oligonucleotides ISIS Nos. 14318 (SEQ ID NO: 42) and 14319 (SEQ ID NO: 43) are 2 ' -deoxy-phosphorothioate sense strand and scrambled controls for ISIS 12560 (SEQ ID NO: 31), respectively.
- ISIS Nos. 15353 and 15354 are "gapmers" corresponding to ISIS 12560; both have 2 ' -methoxyethoxy "wings” (having phosphorothioate linkages in the case of ISIS 15353 and phosphodiester linkages in the case of ISIS 15354) and a central 2 ' -deoxy "gap” designed to support RNaseH activity on the target mRNA molecule.
- 15355 to 15358 are "wingmers” corresponding to ISIS 12560 and have a 5' or 3 ' 2 ' -methoxyethoxy RNaseH- refractory "wing” and a 3' or 5 ' (respectively) 2-deoxy "wing” designed to support RNaseH activity on the target JNK2 mRNA.
- Dose- and sequence-dependent response to JNK2 oligonucleotides In order to demonstrate a dose-dependent response to ISIS 12560 (SEQ ID NO: 31), different concentrations (i.e., 50, 100, 200 and 400 nM) of ISIS 12560 were tested for their effect on JNK2 mRNA levels in A549 cells (Table 13) . In addition, two control oligonucleotides (ISIS 14318, SEQ ID NO: 42, sense control, and ISIS 14319, SEQ ID NO: 43, scrambled control; see also Table 11) were also applied to A549 cells in order to demonstrate the specificity of ISIS 12560. The results (Table 12) demonstrate that the response of A549 cells to ISIS 12539 is dependent on dose in an approximately linear fashion. In contrast, neither of the control oligonucleotides effect any consistent response on JNK2 mRNA levels.
- Western Assays In order to assess the effect of oligonucleotides targeted to JNK2 mRNAs on JNK2 protein levels, Western assays are performed essentially as described above in Examples 2 and 3.
- a primary antibody that specifically binds to JNK2 is purchased from, for example, Santa Cruz Biotechnology, Inc., Santa Cruz, CA; Upstate Biotechnology, Inc., Lake Placid, NY; StressGen Biotechnologies, Inc., Victoria, BC, Canada; or Research Diagnostics, Inc., Flanders, NJ.
- JNK2- ⁇ 2 (GenBank accession No. L31951, locus name "HUMJNK2”), which encodes a polypeptide having an amino acid sequence identical to that of JNK2, the additional isoforms include JNK2- ⁇ l (GenBank accession No. U34821, locus name "HSU34821”) , JNK2- ⁇ l (GenBank accession No. U35002, locus name "HSU35002”) and JNK2- ⁇ 2 (GenBank accession No. U35003, locus name "HSU35003”) .
- the four isoforms of JNK2, which probably arise from alternative mRNA splicing, may each interact with different transcription factors or sets of transcription factors (Gupta et al .
- the oligonucleotides of the invention are specific for certain members or sets of these isoforms of JNK2.
- nucleotides (nt) 689-748 of JNK2/JNK2- ⁇ 2 GenBank accession No. L31951)
- nt 675-734 of JNK2- ⁇ l GenBank accession No.
- SEQ ID NO: 75 nt 653-712 of JNK2- ⁇ l (GenBank accession No. U35002) and nt 665-724 of JNK2- ⁇ 2 (GenBank accession No. U35003) have the sequence shown below as SEQ ID NO: 76.
- SEQ ID NOS: 75 and 76 are shown aligned with each other (vertical marks, "
- antisense oligonucleotides derived from the reverse complement of SEQ ID NO: 75 are specifically hybridizable to, and may be selected and used to modulate the expression of, JNK2/JNK2- ⁇ 2 and JNK2- ⁇ l without significantly effecting the expression of JNKl- ⁇ l and JNKl- ⁇ 2.
- antisense oligonucleotides derived from the reverse complement of SEQ ID NO: 76 are specifically hybridizable to, and may be selected and used to modulate the expression of, JNK2- ⁇ l and JNK2- ⁇ 2 without significantly effecting the expression of JNK2/JNK2- ⁇ 2 and JNK2- ⁇ l.
- SEQ ID NO: 78 an oligonucleotide having a sequence derived from SEQ ID NO: 77 but not from SEQ ID NO : 78 is specifically hybridi zable to , mRNAs encoding JNKl/JNKl- ⁇ l and JNKl- ⁇ 2 but not to those encoding JNK2- ⁇ l and JNK2- ⁇ 2 :
- JNK2/JNK2- ⁇ 2 shares identity with JNKl- ⁇ 2; similarly, JNK2- ⁇ l and JNK2- ⁇ l have identical carboxy terminal portions.
- the substantial differences in the amino acid sequences of these isoforms (5 amino acids in JNK2- ⁇ 2 and JNK2- ⁇ 2 are replaced with 47 amino acids in JNK2/JNK2- ⁇ 2 and JNK2- ⁇ 2) result from a slight difference in nucleotide sequence that shifts the reading frame.
- nt 1164-1198 of JNK2- ⁇ l (GenBank accession No.
- nt 1142-1176 of JNK2- ⁇ l (GenBank accession No. U35002) have the sequence shown below as SEQ ID NO: 79, whereas, in the ORFs of mRNAs encoding JNK2/JNK2- ⁇ 2 and JNK2- ⁇ 2, nt 1178-1207 of JNK2/JNK2- ⁇ 2 (GenBank accession No. L31951) and nt 1154-1183 of JNK2- ⁇ 2 (GenBank accession No. U35003) have the sequence shown below as SEQ ID NO: 80.
- SEQ ID NOS: 79 and 80 are shown aligned with each other (dashes, "-,” indicate bases that are absent in the indicated sequence, and emboldened bases indicate the stop codon for the JNK2- ⁇ l and JNK2- ⁇ l ORFs) : 5 ' -GATCAGCCTTCAGCACAGATGCAGCAGTAAGTAGC SEQ ID NO: 79
- antisense oligonucleotides derived from the reverse complement of SEQ ID NO: 79 are specifically hybridizable to, and may be selected and used to modulate the expression of, mRNAs encoding JNK2- ⁇ l and JNK2- ⁇ l without significantly effecting the expression of JNK2/JNK2- ⁇ 2 and JNK2- ⁇ 2.
- antisense oligonucleotides derived from the reverse complement of SEQ ID NO: 80 are specifically hybridizable to, and may be selected and used to modulate the expression of, mRNAs encoding JNK2/JNK2- ⁇ 2 and JNK2- ⁇ 2 without significantly effecting the expression of JNK2- ⁇ l and JNK2- ⁇ l.
- ISIS 12564 (SEQ ID NO: 35) corresponds to SEQ ID NO: 82 but not to SEQ ID NO: 81, and is thus specifically hybridizable to, and may be used to modulate the expression of, mRNAs encoding JNK2/JNK2- ⁇ 2 and JNK2- ⁇ 2 but not those encoding JNK2- ⁇ l and JNK2- ⁇ l:
- such isoform-specific oligonucleotides such as are described above are methoxyethoxy "gapmers” or “wingmers” in which the RNase H- sensitive "gap” or “wing” is positioned so as to overlap a region of nonidentity in the above antisense sequences, i.e., SEQ ID NOS: 77, 78, 81 and 82.
- JNK3 oligonucleotide sequences Table 14 lists the nucleotide sequences of oligonucleotides designed to specifically hybridize to JNK3 mRNAs and the corresponding ISIS and SEQ ID numbers thereof.
- the target gene nucleotide co-ordinates and gene target region are also included.
- the nucleotide co-ordinates are derived from GenBank accession No. U07620, locus name "HSU07620” see also Figure 4(A) of Mohit et al . , Neuron, 1994, 14, 67).
- gene target regions are as follows: 5' -UTR, 5' untranslated region; tIR, translation initiation region; ORF, open reading frame; 3 ' -UTR, 3' untranslated region. It should be noted that the oligonucleotide target co-ordinate positions and gene target regions may vary within mRNAs encoding related isoforms of JNK3 (see subsection D, below) .
- nucleotides of the oligonucleotides whose sequences are presented in Table 14 are connected by phosphorothioate linkages and are "gapmers.” Specifically, the six nucleotides of the 3' and 5' termini are 2'- methoxyethoxy- modified and are shown emboldened in Table 14, whereas the central eight nucleotides are unmodified at the 2' position (i.e., 2-deoxy) .
- the full oligonucleotide sequences of ISIS Nos. 16692, 16693, 16703, 16704, 16705, 16707, and 16708 specifically hybridize to mRNAs from Ra ttus norvegicus that encode a stress- activated protein kinase named "p54 ⁇ " (Kyriakis et al . , Na ture, 1994, 369, 156; GenBank accession No.
- the full oligonucleotide sequences of 16692, 16693, 16695, 16703, 16704, 16705, 16707 and 16708 (SEQ ID NOS: 46, 47, 49, 56, 57, 58, 60 and 61, respectively) specifically hybridize to mRNAs from Mus musculus that encode a mitogen activated protein (MAP) kinase stress activated protein named the "p459 3F12 SAP kinase” (Martin et al . , Brain Res . Mol . Brain Res . , 1996, 35, 47; GenBank accession No. L35236, locus name "MUSMAPK”) .
- MAP mitogen activated protein
- oligonucleotides are thus preferred embodiments of the invention for investigating the role of the p54 ⁇ and p459 3F12 SAP protein kinases in rat or mouse, respectively, in vi tro, i . e . , in cultured cells or tissues derived from whole animals or in vivo .
- the target gene nucleotide co-ordinates and gene target regions for these oligonucleotides, as defined for these GenBank entries, are detailed in Table 15.
- JNK3-specific probes In initial screenings of a set of oligonucleotides derived from the JNK3 sequence for biological activity, a cDNA clone of JNK3 (Derijard et al . , Cell , 1994, 76, 1025) was radiolabeled and used as a JNK3- specific probe in Northern blots. Alternatively, however, one or more of the oligonucleotides of Table 14 is detectably labeled and used as a JNK3-specific probe.
- Emboldened residues are 2 ' -methoxyethoxy- modified .
- JNK3- ⁇ l was initially cloned and named "p49 3F12 kinase" by Mohit et al . ⁇ Neuron, 1995, 14, 67) . Subsequently, two cDNAs encoding related isoforms of JNK3 were cloned and their nucleotide sequences determined (Gupta et al . , EMBO Journal , 1996, 15, 2760) . The isoforms are named JNK3- ⁇ l (GenBank accession No.
- JNK3- ⁇ 2 GenBank accession No. U34819, locus name "HSU34819" herein.
- the two isoforms of JNK3, which probably arise from alternative mRNA splicing, may each interact with different transcription factors or sets of transcription factors (Gupta et al . , EMBO Journal, 1996, 15, 2760).
- certain oligonucleotides of the invention are specific for each of these isoforms of JNK3.
- JNK3- ⁇ l and JNK- ⁇ 2 differ at their carboxyl terminal portions.
- the substantial differences in the amino acid sequences of these isoforms (5 amino acids in JNK3- ⁇ l are replaced with 47 amino acids in JNK3- ⁇ 2) result from a slight difference in nucleotide sequence that shifts the reading frame.
- nucleotides (nt) 1325-1362 of JNK3- ⁇ l (GenBank accession No. U34820) have the sequence shown below as SEQ ID NO: 83
- nt 1301-1333 of JNK3- ⁇ 2 GenBank accession No.
- SEQ ID NO: 84 have the sequence shown below as SEQ ID NO: 84.
- SEQ ID NOS: 83 and 202 are shown aligned with each other (vertical marks, "I,” indicate bases that are identical in both sequences; dashes, "-,” indicate bases that are absent in the indicated sequence; and emboldened bases indicate the stop codon for the JNK3- ⁇ l ORF) : 5 ' -GGACAGCCTTCTCCTTCAGCACAGGTGCAGCAGTGAAC SEQ ID NO: 83
- antisense oligonucleotides derived from the reverse complement of SEQ ID NO: 83 are specifically hybridizable to mRNAs encoding, and may be selected and used to modulate the expression of JNK3- ⁇ l without significantly effecting the expression of JNK3- ⁇ 2.
- antisense oligonucleotides derived from the reverse complement of SEQ ID NO: 84 are specifically hybridizable to mRNAs encoding, and may be selected and used to modulate the expression of JNK3- ⁇ 2 without significantly effecting the expression of JNK3-0.1:
- such isoform-specific oligonucleotides such as are described above are methoxyethoxy "gapmers” or “wingmers” in which the RNase H- sensitive "gap” or “wing” is positioned so as to overlap a region of nonidentity in the above antisense sequences, i.e., SEQ ID NOS: 85 and 86.
- E. Activities of JNK3 oligonucleotides The JNK3- specific phosphorothioate, 2 ' -methoxyethoxy "gapmer” oligonucleotides (Table 14) were screened for their ability to affect JNK3 mRNA levels in SH-SY5Y cells (Biedler et al .
- SH-SY5Y cells express a variety of mitogen-activated protein kinases (MAPKs; see, e.g., Cheng et al . , J. Biol . Chem . , 1998, 273, 14560).
- MAPKs mitogen-activated protein kinases
- Cells were grown in DMEM essentially as previously described (e.g., Singleton et al . , J. Biol . Chem . , 1996, 271, 31791; Jalava et al . , Cancer Res . , 1990, 50, 3422) and treated with oligonucleotides at a concentration of 200 nM as described in Example 2. Control cultures were treated with an aliquot of LIPOFECTINTM that contained no oligonucleotide .
- Oligonucleotides showing levels of inhibition from > . about 45% to about 100% of JNK3 mRNA levels include ISIS Nos. 16692, 16693, 16694, 16695, 16696, 16697, 16702, 16703, 16704, 16705 and 16706 (SEQ ID NOS:46, 47, 48, 49, 50, 51, 55, 56, 57, 58 and 59, respectively) . These oligonucleotides are preferred embodiments of the invention for modulating JNK3 expression.
- Oligonucleotides showing levels of inhibition of from > about 60% to about 100% of JNK3 mRNAs in this assay, wherein "about” indicates ⁇ 5%, include ISIS Nos. 16693, 16694, 16695, 16702, 16703, 16704 and 16705 (SEQ ID NOS:47, 48, 49, 55, 56, 57 and 58, respectively). These oligonucleotides are thus more preferred embodiments of the invention for modulating JNK3 expression.
- Example 6 Effect of Oligonucleotides Targeted to AP-1 Subunits on Enzymes Involved in Metastasis Patients having benign tumors, and primary malignant tumors that have been detected early in the course of their development, may often be successfully treated by the surgical removal of the benign or primary tumor. If unchecked, however, cells from malignant tumors are spread throughout a patient's body through the processes of invasion and metastasis. Invasion refers to the ability of cancer cells to detach from a primary site of attachment and penetrate, e . g. , an underlying basement membrane.
- Metastasis indicates a sequence of events wherein (1) a cancer cell detaches from its extracellular matrices, (2) the detached cancer cell migrates to another portion of the patient's body, often via the circulatory system, and (3) attaches to a distal and inappropriate extracellular matrix, thereby created a focus from which a secondary tumor can arise.
- Normal cells do not possess the ability to invade or metastasize and/or undergo apoptosis (programmed cell death) if such events occur (Ruoslahti, Sci . Amer. , 1996, 275 , 72).
- MMPs matrix metalloproteinases
- MMP-9 matrix metalloproteinase-9
- NHEKs normal human epidermal keratinocytes
- TPA (12-O-tetradecanoylphorbol 13-acetate)
- ISIS 10582 an oligonucleotide targeted to c- jun, was evaluated for its ability to modulate MMP- 9 expression (see pending application Serial No. 08/837,201, filed April 14, 1997, attorney docket No. ISPH-0209. The results (Table 16) demonstrate that ISIS 10582 is able to completely inhibit the expression of MMP- 9 after induction with TPA.
- Example 7 Treatment of Human Tumors in Mice with
- Tumor size was measured and tumor volume was calculated on days 12, 19, 26 and 33 following tumor cell inoculation.
- the antisense compounds of the invention are also tested for their ability to slow or eliminate the growth of xenografts resulting from, for example, human cervical epithelial carcinoma cells (HeLa cell line, ATCC No. ATCC CCL-2), human lung carcinoma cells (cell line A549, ATCC No. ATCC CCL-185), human adenocarcinoma cells (cell line SW480, ATCC No. ATCC CCL-228), human bladder carcinoma cells (cell line T24, ATCC No. HTB-4), human pancreatic - Ill
- carcinoma cells cell line MIA PaCa, ATCC No. CRL-1420
- human small cell carcinoma cells cell line NCI-H69, ATCC HTB-119 .
- Xenografts resulting from these and other cell lines are established using essentially the same techniques as were used for the experiments using MDA-MB 231 cells.
- oligonucleotides targeted to the genes encoding JNKl, JNK2 and JNK3 of Ra ttus norvegicus were prepared. These oligonucleotides are 2 ' -methoxyethoxy, phosphodiester / 2 '-hydroxyl, phosphorothioate / 2 ' -methoxyethoxy, phosphodiester "gapmers" in which every cytosine residue is 5-methylcytosine (m5c) . These antisense compounds were synthesized according to the methods of the disclosure. Certain of these oligonucleotides are additionally specifically hybridizable to JNK genes from other species as indicated herein.
- oligonucleotides described in this Example were tested for their ability to modulate rat JNK mRNA levels essentially according to the methods described in the preceding Examples, with the exceptions that the cell line used was rat A10 aortic smooth muscle cells (ATCC No. ATCC CRL-1476) and the probes used were specific for rat JNKl, JNK2 or JNK3 (see infra ) .
- A10 cells were grown and treated with oligonucleotides essentially as described by Cioffi et al . ⁇ Mol . Pharmacol . , 1997, 51 , 383) .
- JNKl Table 19 describes the sequences and structures of a set of oligonucleotides, ISIS Nos. 21857 to 21870 (SEQ ID NOS: 111 to 124, respectively) that were designed to be specifically hybridizable to nucleic acids from Ra ttus norvegicus that encode a stress-activated protein kinase named "p54 ⁇ ” or "SAPKy” that is homologous to the human protein JNKl (Kyriakis et al . , Na ture, 1994, 369, 156; GenBank accession No. L27129, locus name "RATSAPKD”) .
- emboldened residues are 2'- methoxyethoxy-residues (others are 2 '-deoxy-); "C” residues are 2 ' -methoxyethoxy-5-methyl-cytosines and “C” residues are 5-methyl-cytosines; "o” indicates a phosphodiester linkage; and "s” indicates a phosphorothioate linkage.
- the target gene co-ordinates are from GenBank Accession No. L27129, locus name "RATSAPKD.” TABLE 19: Nucleotide Sequences of Rat JNKl Oligonucleotides
- Oligonucleotides showing levels of inhibition from > . about 75% to about 100% of rat JNKl mRNA levels include ISIS Nos. 21857 to 21870 (SEQ ID NOS: 111 to 124, respectively). These oligonucleotides are preferred embodiments of the invention for modulating rat JNKl expression. Oligonucleotides showing levels of inhibition of from > . about 90% to about 100% of rat JNKl mRNAs in this assay include ISIS Nos. 21858, 21859, 21860, 21861, 21862, 21865,
- Pan JNK oligonucleotides demonstrated a capacity to modulate both JNKl and JNK2.
- Such oligonucleotides are referred to herein as "Pan JNK” antisense compounds because the term "Pan” is used in immunological literature to refer to an antibody that recognizes, e.g., all isoforms of a protein or subtypes of a cell type.
- Pan JNK oligonucleotides are discussed in more detail infra .
- ISIS 21859 (SEQ ID NO: 113) is complementary to bases 4 to 23 of cDNAs encoding human JNKl ⁇ l and JNKl ⁇ l (i.e., GenBank accession Nos. L26318 and U35004, respectively).
- ISIS 21862 (SEQ ID NO:116) is complementary to bases 294 to 313 of the human JNKl ⁇ l and JNKl ⁇ l cDNAs (GenBank accession Nos.
- ISIS 18257 is specifically hybridizable to nucleic acids encoding p54 ⁇ 2 (i.e., GenBank accession No. L27112, locus name "RATSAPKB”) .
- emboldened residues are 2 ' -methoxyethoxy-residues (others are 2 '-deoxy-);
- C residues are 2 ' -methoxyethoxy-5-methyl-cytosines and "C” residues are 5-methyl-cytosines; "o” indicates a phosphodiester linkage; and "s” indicates a phosphorothioate linkage.
- the target gene co-ordinates are from GenBank Accession No. L27112, locus name "RATSAPKB.”
- Oligonucleotides showing levels of inhibition from > . about 60% to about 100% of rat JNK2 mRNA levels include ISIS Nos. 18254, 18255, 18257, 18258, 18259, 18260 and 18264 (SEQ ID NOS:125, 126, 128, 129, 130, 131 and 135, respectively). These oligonucleotides are preferred embodiments of the invention for modulating rat JNK2 expression. Oligonucleotides showing levels of inhibition of from > .
- rat JNKl mRNAs in this assay include ISIS Nos. 18254, 18255, 18258 and 18259 (SEQ ID NOS:125, 126, 129 and 130, respectively). These oligonucleotides are thus more preferred embodiments of the invention for modulating rat JNK2 expression.
- ISIS 18259 SEQ ID NO: 130 was chosen for use in further studies ⁇ infra ) .
- C. Dose Response A dose response study was conducted using oligonucleotides targeted to rat JNKl (ISIS 21859; SEQ ID NO: 113) and JNK2 (ISIS 18259; SEQ ID NO: 130) and Northern assays.
- JNK3 Table 24 describes the sequences and structures of a set of oligonucleotides, ISIS Nos. 21899 to 21912 (SEQ ID NOS: 142 to 155, respectively) that were designed to be specifically hybridizable to nucleic acids from Ra ttus norvegicus that encode a stress-activated protein kinase named "p54 ⁇ " that is homologous to the human protein JNK3 (Kyriakis et al . , Na ture, 1994, 369, 156; GenBank accession No. L27128, locus name "RATSAPKC”) .
- emboldened residues are 2 ' -methoxyethoxy-residues (others are 2 '-deoxy-); "C” residues are 2 ' -methoxyethoxy- 5-methyl-cytosines and “C” residues are 5-methyl-cytosines; "o” indicates a phosphodiester linkage; and "s” indicates a phosphorothioate linkage.
- the target gene co-ordinates are from GenBank Accession No. L27128, locus name "RATSAPKC.”
- the oligonucleotides are tested for their ability to modulate rat JNK3 mRNA levels essentially according to the methods described in the preceding Examples.
- oligonucleotides described in Table 24 are also specifically hybridizable with JNK3-encoding nucleic acids from humans and Mus musculus (mouse) .
- Table 25 sets out these relationships. These oligonucleotides are tested for their ability to modulate mRNA levels of the human JNK genes according to the methods described in Example 5.
- GenBank accession No. U34819 locus name "HSU34819” (see also Gupta et al . , EMBO Journal , 1996, 15, 2760).
- Pan JNK Oligonucleotides Certain of the oligonucleotides of the invention are capable of modulating two or more JNK proteins and are referred to herein as "Pan JNK" oligonucleotides.
- ISIS Nos. Nos. 21861 and 21867 demonstrated a capacity to modulate both JNKl and JNK2 (Table 20) .
- Such oligonucleotides are useful when the concomitant modulation of several JNK proteins is desired.
- Human Pan JNK oligonucleotides are described in Table 26.
- oligonucleotides are designed to be complementary to sequences that are identically conserved in (i.e., SEQ ID NOS:156, 158, 159, 160 and 161), or which occur with no more than a one-base mismatch (SEQ ID NO: 157), in nucleic acids encoding human JNKl ⁇ l, JNKl ⁇ 2, JNK2 ⁇ l and JNK2 ⁇ 2.
- SEQ ID NOS:156, 158, 159, 160 and 161 or which occur with no more than a one-base mismatch (SEQ ID NO: 157), in nucleic acids encoding human JNKl ⁇ l, JNKl ⁇ 2, JNK2 ⁇ l and JNK2 ⁇ 2.
- SEQ ID NO: 157 one-base mismatch
- hypoxanthine inosine (I) is capable of hydrogen bonding with a variety of nucleobases and thus serves as a "universal" base for hybridization purposes.
- an oligonucleotide having a sequence that is a derivative of SEQ ID NO: 157 having one inosine substitution (TAGGAIATTCTTTCATGATC, SEQ ID NO: 162) is predicted to bind to nucleic acids encoding human JNKl ⁇ l, JNKl ⁇ 2, JNK2 ⁇ l and JNK2 ⁇ 2 with no mismatched bases.
- an oligonucleotide having a sequence that is a derivative of SEQ ID NO: 161 having one inosine substitution is predicted to bind with no mismatched bases to nucleic acids encoding human JNK3 ⁇ l and JNK3 ⁇ 2 in addition to JNKl ⁇ l, JNKl ⁇ 2, JNK2 ⁇ l and JNK2 ⁇ 2.
- Such oligonucleotides are evaluated for their ability to modulate JNKl and JNK2 mRNA levels in A549 cells, and JNK3 mRNA levels in SH-SY5Y cells, using the methods and assays described in Examples 3, 4 and 5.
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WO2002033064A1 (en) * | 2000-10-17 | 2002-04-25 | UNIVERSITé LAVAL | Zipper protein kinase and uses thereof |
US7422883B2 (en) | 2000-12-22 | 2008-09-09 | Inter-K Pty Limited | Map kinase integrin-binding domain |
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WO2009024763A3 (en) * | 2007-08-17 | 2010-04-29 | The University Of York | Combination therapy |
WO2009044392A2 (en) | 2007-10-03 | 2009-04-09 | Quark Pharmaceuticals, Inc. | Novel sirna structures |
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EP1003916A1 (en) | 2000-05-31 |
US6221850B1 (en) | 2001-04-24 |
US5877309A (en) | 1999-03-02 |
JP3527200B2 (en) | 2004-05-17 |
AU8775098A (en) | 1999-03-08 |
AU730916B2 (en) | 2001-03-22 |
CA2301139A1 (en) | 1999-02-25 |
JP2001514905A (en) | 2001-09-18 |
EP1003916A4 (en) | 2002-01-09 |
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