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Definitions

• the present invention relates to novel oligoribonucleotide derivatives which have a 2′5′-linked oligoribonucleotide residue without a 5′-phosphate residue on the 3′ end and to the use thereof for specific inhibition of gene expression.
• oligonucleotides are antisense oligonucleotides, ribozymes, DNA enzymes and external guide sequences (EGS).
• Antisense oligonucleotides are short single-stranded nucleic acid derivatives which bind via Watson-Crick base pairing to a complementary messenger ribonucleic acid (mRNA) whose translation into the corresponding protein is to be inhibited.
• mRNA complementary messenger ribonucleic acid
• antisense oligonucleotides exhibit their action according to a mechanism which is supported by cellular ribonuclease H (RNase H); numerous studies have shown evidence for this.
• RNase H which is present in all cells recognizes a double strand of DNA and RNA and cuts the mRNA complementary to said oligonucleotide via hydrolysis of one or in most cases more phosphodiester bonds.
• the way in which the oligonucleotides have to be modified in order for activation of RNase H to take place is known and is described, for example, in Uhlmann (2000) Curr. Opin. Drug Discov. Dev. 3, 203-213. Synthetic ribozymes carry this nuclease activity in their sequence.
• ribozyme The most common type of ribozyme is the “hammerhead” ribozyme in which the consensus sequence GAAAC which is derived from naturally occurring ribozymes forms the RNase part and the flanking sequences form the antisense oligonucleotide part.
• DNA enzymes which, however, are not derived from naturally occurring ribozyme motifs but have been found by in-vitro selection, act in a similar way.
• EGS are synthetic RNA analogs which activate the cellular RNase P and bind via appropriate flanking sequences to the target mRNA and induce a specific mRNA degradation.
• a common problem of the inhibition of gene expression with the aid of synthetic oligonucleotides is that it is always necessary to assay a relatively large number of oligonucleotides against various regions of the target nucleic acid, in order to identify an efficient sequence.
• antisense oligonucleotides often inhibit gene expression only inefficiently or incompletely.
• sequence-unspecific side effects were observed, which may be caused by the fact that even relatively short part sequences of about five bases in length activate RNase H. This is shown, for example, by “Woolf et al. (1992). Proc. Natl. Acad. Sci. U.S.A. 89, 7305-7309)”.
• there are also side effects which are caused by interaction of the antisense oligonucleotides with proteins.
• Double-stranded RNA is a signal for particular cells and organisms to induce a sequence-specific degradation of mRNA according to a process which is known as RNA interference (RNAi).
• RNAi RNA interference
• the RNAi phenomenon was observed in a number of different organisms such as, for example, C. elegans , flies, fungi, plants and mouse embryos. RNAi is believed to be very similar or identical to post-transcriptional gene silencing (PTGS) found in plants.
• PTGS post-transcriptional gene silencing
• RNAi is a post-transcriptional process in which the dsRNA is first cleaved into relatively small fragments which are then probably used for sequence-specific degradation of the target mRNA.
• RNAi with long dsRNA is the fact that only particular organisms such as C. elegans , zebra fish, plants, particular types of fungi, Drosophila, oocytes and embryos of mice allow sequence-specific inhibition by dsRNA, while most animal cells when treated with dsRNA cause apoptosis. Long dsRNA still inhibits gene expression when the sequence homology is from 70 to 90%. For this reason, it is possible in the case of gene families with high sequence homology for misinterpretations of the phenotype to occur by simultaneous inhibition of the expression of a plurality of not completely homologous genes.
• dsRNA for example with dsRNA viruses
• the treatment of cells with dsRNA generally leads to an apoptotic process or to the sequence-unspecific degradation of the mRNA due to induction of a 2′5′-oligoadenylate-synthase activity.
• the infected cell synthesizes in response to the viral dsRNA trimeric or tetrameric adenylate (2′5′-A) with the unusual 2′5′-phosphodiester-internucleoside bond.
• 2′5′-A is phosphorylated by cellular kinases on its 5′ end and then activates a nuclease called RNase L.
• 2′5′-A may also be chemically synthesized and be introduced into the cell (Torrence et al. (1994) Curr. Med. Chem 1, 176-191). However, synthetic 2′5′-A activates RNase L only if it has been converted to the 5′-phosphate or 5′-triphosphate form. RNase L activated by 5′-p-2′5′-A (p is phosphate, diphosphate or triphosphate) then degrades the entire RNA of the cell in a sequence-unspecific manner. In addition, it was shown that it is possible to inhibit gene expression sequence-specifically with the aid of antisense oligonucleotide conjugates with a 5′-p-2′5′-A residue.
• the 5′ end of the 2′5′-A residue is not linked to the oligonucleotide but is present as phosphate, thiophosphate or triphosphate (Torrence et al. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1300-4).
• the target RNA-recognizing oligonucleotide part (antisense part) must be in single-stranded form.
• oligonucleotides having on their 3′ ends 2′5′-A residues which consequently have no free 5′-phosphate or triphosphate function have not been described previously as inhibitors of gene expression.
• the inhibition of gene expression by the single-stranded, 5′-phosphorylated 5′-p-2′5′-A antisense oligonucleotide conjugates is a variation of the antisense principle and is therefore also subject to the limitations of the antisense-oligonucleotide approach.
• the 2′5′-A residue is attached to the 5′ end of the oligonucleotide via a spacer (linker) so that the 2′ or 3′ end of the 2′5′-A residue is present in bound form.
• the RNA-binding portion preferably comprises DNA (FIGS. 4 to 6 in Torence, Curr. Opin. Mol. Ther. (1999) 1, 307).
• oligonucleotides have been used increasingly as tools for studying the function of new genes (functional genomics).
• the use of antisense oligonucleotides and ribozymes for sequence-specific inhibition of gene expression of new genes coding for proteins with unknown function is made more difficult by the fact that generally a large variety of oligonucleotides of different sequences have to be assayed, and this is a disadvantage in particular for a high-throughput process.
• novel oligonucleotide derivatives which have a 2′5′-linked oligonucleotide residue on the 3′ end, which carries no phosphate, thiophosphate or triphosphate group.
• the sequence of the novel oligonucleotide derivatives is complementary to the RNA sequence whose translation is to be inhibited.
• the invention accordingly provides oligonucleotide derivatives of the formula I,
• N is naturally or not naturally occurring nucleotides, preferably ribonucleotides, which are at least partly complementary to a target RNA,
• x is independently 10 to 100, preferably 15 to 45 and particularly preferably 16 to 25,
• n is 2 to 20, preferably 3 to 10, particularly preferably 3 to 6,
• Z is naturally or not naturally occurring nucleotides which are linked via a 2′5′internucleoside bond
• v and w independently of one another are 2 to 20, preferably 2 to 10, particularly preferably 2 to 6 and
• z is 15 to 25, preferably 16 to 23 and particularly preferably 19 to 21 and
• U is uridine
• N is adenosine (A), guanosine (G), cytidine (C) or U
• X is A, G or C, preferably A.
• N may be a ribonucleotide.
• the gene whose expression is to be inhibited contains, for example, the following DNA sequence
• the target RNA has the following sequence pattern
• oligonucleotides of the formula I in which one or more phosphodiester bonds have been replaced, for example by phosphorothioate bonds or N3′,P5′-phosphoramidate bonds.
• Particular preference is given to oligonucleotides of the formula I in which one or more phosphodiester bonds have been replaced by phosphorothioate residues.
• the phosphorothioate residues are preferably introduced on the 3′ ends, the 5′ ends and on the internal pyrimidine nucleotides C and U, in particular if several pyrimidine nucleotides succeed one another in the sequence.
• a particular embodiment of the invention comprises the use of a mixture of two or more oligonucleotide derivatives in accordance with formula 1 for inhibiting gene expression.
• the oligonucleotide derivatives in this case may be directed against different regions of an RNA or against the RNA of different genes.
• the single-stranded oligonucleotides of the fomula I were originally employed as control oligonucleotides for RNAi experiments using short dsRNA. Thus, owing to the single-stranded character, inhibition of gene expression was not expected. Surprisingly, however, particular single-stranded oligonucleotides inhibited gene expression, too, in particular when sufficiently stable toward nucleases. Another surprise was that the oligonucleotides of the formula I in which the 2′5′-linked oligoadenylate residue has no free 5′-phosphate, 5′-thiophosphate or 5′-triphosphate residue inhibited gene expression in a sequence-specific manner.
• the 2′5′-linked oligoadenylate residue can be bound to the 3′5′-linked RNA directly via the 5′ function. It has been a valid dogma up until now that the 2′5′-linked oligoadenylate residue must have a free phosphate, thiophosphate or triphosphate residue on the 5′ end in order to inhibit gene expression. Moreover, a 2′5′ oligoadenylate-mediated inhibition had previously always been asscociated with an unspecific, i.e. sequence-independent, effect (Bass, Nature (2001) 411, 428).
• oligonucleotides of the formula I not only deviate in their structure from the oligonucleotide conjugates described by Torrence (Curr. Opin. Mol. Ther. (1999) 1, 307) but also exhibit a much better inhibitory action which consequently is based on a different mechanism.
• the oligonucleotides of the invention also had an inhibitory sequence-specific effect on human primary cells. As far as we know, the inhibition of gene expression by oligonucleotides having 2′5′-linked nucleotides in human primary cells has not been observed previously.
• inventive oligonucleotides of the formula I may also be used for inhibiting gene expression in cells which express only a small amount of, a defective or no 2′5′-oligoadenylate synthase.
• oligonucleotides of the formula I for treating patients having a deficiency or defect in 2′5′-oligoadenylate synthase.
• Patients with CFS chronic fatigue syndrome
• sequences of the oligonucleotides of the formula I which are used for inhibiting the gene expression of particular targets are selected on the basis of the corresponding gene sequences.
• the sequences of said genes are obtained by sequencing or from gene databases.
• An example which may be illustrated here is the inhibition of luciferase (firefly) by double-stranded nucleic acids.
• the accession number for this gene is U47298.
• the coding region of firefly luciferase comprises 1 653 nucleotides. The following four regions may be selected, inter alia, as target sequences for the inhibition by double-stranded nucleic acids.
• RNA for these regions then has the following sequence.
• (Seq ID No.7) GCUUUUACAGAUGCACAUAUCGAGGUGGACAUCACUUACG (Seq ID No.8) CCGCGAACGACAUUUAUAAUGAACGUGAAUUGCUCAACAG (Seq ID No.9) GCGGUCGGUAAAGUUGUUCCAUUUUUUGAAGCGAAGGUUG (Seq ID No.10) AUUUUUUGAAGCGAAGGUUGUGGAUCUGGAUACCGGGAAA
• inventive complementary oligonucleotides of the formula I derived therefrom have the following sequences and are characterized in that two or more nucleotides (indicated here by lower-case letters) are linked via a 2′5′-internucleoside bond. Preference is given to 2′5′-linked adenylate residues.
• 3′ aaaaAUGUCUACGUGUAUAGCUCCAC Seq ID No.11 3′ aaaaAUAUUACUUGCACUUAACGAG Seq ID No.12 3′ aaaCCAUUUCAACAAGGUAAAAAA Seq ID No.13
• oligonucleotides for example as phosphorothioates (asterisks). Stabilization by phosphorothioates is preferably carried out on the ends and internal pyrimidine nucleotides.
• the specificity of the inhibition of luciferase expression can be checked on the basis of control oligonucleotides which are not completely complementary to the target RNA and have, for example, 4 base mismatches.
• B is a naturally or not naturally occurring nucleobase
• U, V and W independently of one another are O, S, NH or CH 2 , preferably O or S,
• R 1 is independently of one another OH, SH, CH 3 or BH 3 , preferably OH or SH, or physiologically tolerated salts thereof,
• R 2 is independently of one another OH, H, O—C 1 to C 12 -alkyl , preferably OH (ribonucleotide), where C 1 to C 12 -alkyl preferably is CH 3 or CH 3 —O—CH 2 CH 2 ,
• R 3 is independently of one another OH, H, O—C 1 to C 12 -Alkyl , preferably OH or H, where C 1 to C 12 -alkyl preferably is CH 3 or CH 3 —O—CH 2 CH 2 ,
• x is independently 10 to 100, preferably 15 to 45, and particularly preferably 16 to 25,
• n is 2 to 20, preferably 3 to 10, particularly preferably 3 to 6,
• A is adenine or an adenine derivative, for example 8-bromoadenine, 8-methyladenine, or hypoxanthine.
• oligonucleotides of the invention are directed, for example, against a human gene or the corresponding RNA thereof and assayed in human cells (HUVEC, human umbilical vein endothelial cells).
• human cells human umbilical vein endothelial cells
• Edg-1 DNA accession number M31210
• the gene database may be transcribed into the corresponding messenger RNA and the following two regions (175 and 725) could be selected for synthesizing appropriate oligonucleotides.
• Edg-1 RNA ′′175′′ GACCUCGGUGGUGUUCAUUCUCAUCUGCUGCU (Seq ID No.16) UUAUCAUCCUGGAGAACAUCUUUGUCUU ′′725′′ AUUUCCAAGGCCAGCCGCAGCUCUGAGAAUGU (Seq ID No.17) GGCGCUGCUCAAGACCGUAAUUAUCGUC
• the mismatch control differs in 5 nucleotides (underlined as mismatch) from the edg-1 RNA.
• oligonucleotides directed against edg-1 were prepared, which have improved nuclease stability and increased inhibitory activity and are derived from the above edg-1 sequences.
• an oligonucleotide may be synthesized completely from the nucleotides adenosine phosphate, guanosine phosphate, inosine phosphate, cytidine phosphate, uridine phosphate and thymidine phosphate. Preference is given to oligonucleotides which are synthesized from ribonucleotides, the “oligoribonucleotides”.
• an oligonucleotide may contain, where appropriate, one or more modifications, for example chemical modifications.
• An oligonucleotide may have a plurality of identical and/or different modifications.
• the 2′5′-linked residue may contain, for example, adenosine, 3′-deoxyadenosine (cordycepin), inosine, 8-bromoadenosine, 8-methyladenosine and other 8-substituted adenosine derivatives.
• the ribose residue may also be derivatized as 3′-O-methyladenosine.
• the internucleoside bonds in the 2′5′-linked portion are preferably phosphodiester and phosphorothioate bonds. Common derivatives of 2′5′-adenylate, their synthesis and activation of RNase L are described in the literature (Player et al. (1998) Pharmacol. Ther. 78, 55).
• the chemical modification of an oligonucleotide may include, for example,
• R 1 and R 1′ independently of one another are hydrogen, (C 1 -C 18 )alkyl, (C 6 -C 20 )aryl, (C 6 -C 14 )aryl-(C 1 -C 8 )alkyl, preferably hydrogen, (C 1 -C 8 )alkyl and/or methoxyethyl, particularly preferably hydrogen, (C 1 -C 4 )alkyl and/or methoxyethyl, or
• R 1 and R 1′ together with the nitrogen atom to which they are bound, form a 5-6-membered heterocycle which may additionally contain another heteroatom selected from the group consisting of O, S, N;
• Heterocyclic base modifications are described, for example, in Herdewijn, Antisense & Nucl. Acid Drug Dev. (2000) 297.
• the chemical modification of the oligonucleotide furthermore comprises conjugating an oligonucleotide with one or more molecules which influence advantageously the properties (e.g. nuclease stability, affinity for target sequence, pharmacokinetics) of said oligonucleotide and/or, during hybridization of the modified oligonucleotide to the target sequence, attack said target sequence with binding and/or crosslinking (oligonucleotide conjugates).
• properties e.g. nuclease stability, affinity for target sequence, pharmacokinetics
• Examples thereof are conjugates with polylysine, with intercalators such as pyrene, acridine, phenazine, phenanthridine, with fluorescent compounds such as fluorescein, with crosslinkers such as psoralen, azidoproflavin, with lipophilic molecules such as (C 12 -C 20 )alkyl, with lipids such as 1,2-dihexadecyl-rac-glycerol, with steroids such as cholesterol or testosterone, with vitamins such as vitamin E, with poly- or oligoethylene glycol, with (C 12 -C 18 )alkyl phosphate diesters and/or with —O—CH 2 —CH(OH)—O—(C 12 -C 18 )alkyl.
• intercalators such as pyrene, acridine, phenazine, phenanthridine
• fluorescent compounds such as fluorescein
• crosslinkers such as psoralen, azidoproflavin
• Such molecules may be conjugated at the 5′ and/or 3′ end and/or within the sequence, for example at a nucleobase.
• oligonucleotide conjugates known to the skilled worker are described in Manoharan (2001) Conjugated Oligonucleotides in Antisense technology. In: Crooke (Editor) Antisense Technology. Marcel Dekker, New York.
• a specific embodiment of the chemical modification relates to conjugation of the oligonucleotide a) with lipophilic molecules, for example (C 12 -C 20 )alkyl, b) with steroids such as cholesterol and/or testosterone, c) with poly- and/or oligoethylene glycol, d) with vitamin E, e) with intercalators such as pyrene, f) with (C 14 -C 18 )alkyl phosphate diesters and/or g) with —O—CH 2 —CH(OH)—O—(C 12 -C 16 )alkyl.
• lipophilic molecules for example (C 12 -C 20 )alkyl
• steroids such as cholesterol and/or testosterone
• poly- and/or oligoethylene glycol d) with vitamin E
• intercalators such as pyrene
• Another specific embodiment of the chemical modification relates to derivatization of the oligonucleotide, as described in HMR 99/L045, as aryl ester conjugate, for example as FDA conjugate, which derivatization benefits the cellular uptake of said oligonucleotides.
• the oligonucleotide may have on its 5′ end a 5′-5′ inversion.
• This type of chemical modification is known to the skilled worker and described, for example, in M. Koga et al., J. Org. Chem. 56 (1991) 3757.
• the 5′ end is a preferred position for conjugating the oligonucleotide with one or more molecules which have a beneficial effect on the properties (for example stability against nucleases, cellular uptake, affinity for the target sequence, pharmacokinetics) of the oligonucleotide.
• the invention further provides methods for preparing the oligonucleotides.
• the oligonucleotides described may be prepared with the aid of various known chemical methods, as described, for example, in Eckstein, F. (1991) “Oligonucleotides and Analogues, A Practical Approach”, IRL Press, Oxford.
• the oligonucleotides may also be prepared by methods which, where appropriate, contain one or more enzymic steps.
• the invention furthermore provides the use of the oligonucleotides for modulating and for completely or partially inhibiting the expression of particular target genes, for example for completely or partially inhibiting translation.
• the invention furthermore relates to the use of said oligonucleotides for modulating and for completely or partially inhibiting expression in cells which have only a small amount of, a defective or no 2′5′-oligoadenylate synthase.
• the invention furthermore provides the use of said oligonucleotides as pharmaceuticals or to the use of said oligonucleotides for the production of pharmaceuticals.
• said oligonucleotides in pharmaceuticals which are suitable for the prevention and/or treatment of diseases which accompany the expression or overexpression of particular genes.
• the invention further provides the use of said oligonucleotides or of pharmaceuticals containing said oligonucleotides for the treatment of diseases in which specific genes are the cause or are involved, due to overexpression.
• the pharmaceuticals of the present invention may be used, for example, for the treatment of disorders caused by viruses, for example by CMV, HIV, HSV-1, HSV-2, hepatitis B, hepatitis C viruses, or papillomaviruses.
• Pharmaceuticals of the present invention are particularly suitable for the treatment of RNA viruses such as, for example, polio viruses, VSV or Influenza virus, in particular also of double-stranded RNA viruses such as reoviruses, for example.
• the pharmaceuticals of the present invention are also suitable, for example, for cancer treatment.
• nuclear oncoproteins such as, for example, c-myc, N-myc, c-myb, c-fos, c-fos/jun, PCNA, p120,
• cytoplasmic/membrane-associated oncoproteins such as, for example, EJ-ras, c-Ha-ras, N-ras, rrg, bcl-2, cdc-2, c-raf-1, c-mos, c-src, c-abl, c-ets,
• cellular receptors such as, for example, EGF receptor, Her-2, c-erbA, VEGF receptor (KDR-1), retinoid receptors, protein kinase regulatory subunit, c-fms, Tie-2, c-raf-1 kinase, PKC-alpha, protein kinase A (R1 alpha),
• cytokines cytokines, growth factors, extracellular matrix such as, for example, CSF-1, IL-6, IL-1 a, IL-1b, IL-2, IL-4, IL-6, IL-8, bFGF, VEGF, myeloblastin, fibronectin,
• extracellular matrix such as, for example, CSF-1, IL-6, IL-1 a, IL-1b, IL-2, IL-4, IL-6, IL-8, bFGF, VEGF, myeloblastin, fibronectin,
• the pharmaceuticals of the present invention are further suitable, for example, for the treatment of disorders which are influenced by integrins or cell-cell adhesion receptors, for example by VLA-4, VLA-2, ICAM, VCAM or ELAM.
• the pharmaceuticals of the present invention are also suitable, for example, for preventing restenosis.
• nuclear transactivator proteins and cyclins such as, for example, c-myc, c-myb, c-fos, c-fos/jun, cyclins and cdc2 kinase,
• mitogens or growth factors such as, for example, PDGF, bFGF, VEGF, EGF, HB-EGF and TGF- ⁇
• cellular receptors such as, for example, bFGF receptor, EGF receptor and PDGF receptor.
• the invention further relates to oligonucleotides for the treatment of asthma, with expression of the adenosine-A1 receptor, adenosine-A3 receptor, Bradikinin receptor or of IL-13 being inhibited with the aid of suitable oligonucleotides.
• the invention also relates to oligonucleotides, for example, for the treatment of cardiovascular diseases, with, for example, expression of the ⁇ 1-adrenergic receptor or of a protein from the EDG family such as, for example, Edg-1 being inhibited.
• the invention also relates to oligonucleotides, for example, for the treatment of diabetes, with expression of PTP-1 B being inhibited, for example.
• the pharmaceuticals may be used, for example, in the form of pharmaceutical preparations which may be administered orally, for example in the form of tablets, coated tablets, hard or soft gelatin capsules, solutions, emulsions or suspensions. They may also be administered rectally, for example in the form of suppositories, or parenterally, for example in the form of injection solutions.
• Pharmaceutical preparations may be produced by processing said compounds in therapeutically inert organic and inorganic carriers. Examples of such carriers for tablets, coated tablets and hard gelatin capsules are lactose, corn starch or derivatives thereof, talc and stearic acid or salts thereof.
• Carriers suitable for the preparation of solutions are water, polyols, sucrose, invert sugar and glucose.
• Carriers suitable for injection solutions are water, alcohols, polyols, glycerol and vegetable oils.
• Carriers suitable for suppositories are vegetable and hardened oils, waxes, fats and semisolid polyols.
• the pharmaceutical preparations may also contain preservatives, solvents, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorings, salts for modifying the osmotic pressure, buffers, coating agents, antioxidants and, where appropriate, other therapeutically active substances.
• Preferred administration forms are topical administrations, local administrations such as, for example, with the aid of a catheter or by inhalation, injections or infusions, and oral administration.
• the oligonucleotide derivatives are formulated in a liquid solution, preferably in a physiologically acceptable buffer such as, for example, Hank's solution or Ringer's solution.
• the oligonucleotides may also be formulated in solid form and be dissolved or suspended prior to use.
• the dosages preferred for systematic administration are from approx. 0.01 mg/kg to approx. 50 mg/kg body weight and day.
• the invention furthermore relates to pharmaceutical preparations which contain oligonucleotides and/or physiologically tolerated salts thereof in addition to pharmaceutically suitable carriers and/or additives.
• the oligonucleotides and/or physiologically tolerated salts thereof may be administered to animals, preferably to mammals, and in particular to humans as pharmaceuticals on their own, in mixtures with one another or in the form of pharmaceutical preparations which permit topical, percutaneous, parenteral or enteral application and which contain as active ingredient an active dose of at least one oligonucleotide in addition to common pharmaceutically suitable carriers and additives.
• the preparations normally contain about from 0.1 to 90% by weight of the therapeutically active compound.
• a topical application for example in the form of ointments, lotions or tinctures, emulsions, or suspensions is preferred.
• the pharmaceutical preparations are produced in a manner known per se (e.g. Remingtons Pharmaceutical Sciences, Mack Publ. Co., Easton, Pa.), with pharmaceutically inert inorganic and/or organic carriers being used.
• pharmaceutically inert inorganic and/or organic carriers are used, for example.
• carriers for soft gelatin capsules and/or suppositories are fats, waxes, semisolid and liquid polyols, natural and/or hardened oils, etc.
• Examples of carriers suitable for the preparation of solutions and/or syrups are water, sucrose, invert sugar, glucose, polyols, etc.
• Carriers suitable for the preparation of injection solutions are water, alcohols, glycerol, polyols, vegetable oils, etc.
• Carriers suitable for microcapsules, implants and/or rods are mixed polymers of glycolic acid and lactic acid. Liposome formulations which are known to the skilled worker (N. Weiner, Drug Develop Ind Pharm 15 (1989) 1523; “Liposome Dermatics, Springer Verlag 1992), for example HVJ liposomes (Hayashi, Gene Therapy 3 (1996) 878), are also suitable.
• Dermal administration may also be carried out, for example, with the aid of ionophoretic methods and/or with the aid of electroporation.
• lipofectins and other carrier systems for example those which are used in gene therapy.
• Particularly suitable systems are those which can be used to introduce oligonucleotides into eukaryotic cells with great efficiency.
• a pharmaceutical preparation may also contain additives such as, for example, fillers, extenders, disintegrants, binding agents, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colorants, flavorings or aromatizers, thickening agents, diluents, buffer substances, furthermore solvents and/or solubilizers and/or agents for achieving a depot effect, and also salts for modifying the osmotic pressure, coating agents and/or antioxidants. They may also contain two or more different oligonucleotides and/or their physiologically tolerated salts and furthermore, in addition to at least one oligonucleotide, one or more other therapeutically active substances.
• additives such as, for example, fillers, extenders, disintegrants, binding agents, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colorants, flavorings or aromatizers, thickening agents,
• the dose may vary within wide limits and, in each individual case, has to be adjusted to the individual circumstances.
• the 2′5′-linked oligonucleotide part was synthesized by five condensations with 5′-O-dimethoxytrityl-N-6-benzoyl-3′-O-tertbutyldimethylsilyladenosine-2′-O-phosphoramidite (ANP-5681, Chemgenes).
• the cells were washed with 3 ml of serum-free medium and 800 ⁇ l of SF 900II SFM and the nucleic acid/lipofectin mixture were successively added to the cells, followed by incubation at 25 degrees overnight. On the next day, 1 ml of medium and serum (Gibco BRL 10122-166; final concentration 2%) is added.
• Dual-luciferase reporter (DLR; Promega E 1960) assay system [0112] Dual-luciferase reporter (DLR; Promega E 1960) assay system:
• the Promega DLR assay allows the sequential determination of the firefly luciferase and Renilla luciferase activities having different nucleic acid sequences from a single sample.
• the oligonucleotides according to the formula I, which were to be measured, were directed against firefly luciferase. Thus, only firefly luciferase activity but not Renilla luciferase activity should be inhibited. Thus, apart from the inhibitory action, the specificity may also be tested for.
• the passive lysis of the cells in the well plates was carried out by first removing the medium and washing the cells with PBS (phosphate-buffered saline (Gibco BRL 14200-067). The medium was completely removed by suction and then the PLB (passive lysis buffer, diluted 1:5 with water; 500 ⁇ l of PLB (1 ⁇ ) to be introduced into one well of a 6-well plate) was added thereto. This was followed by a 15-minute incubation with shaking at room temperature.
• PBS phosphate-buffered saline
• the luciferase assay reagent II (LAR II) was prepared by resuspending the luciferase assay substrate (LAS) in 10 ml of luciferase assay buffer II (LAB II).
• the Stop & Glo reagent was prepared by adding 200 ⁇ l of the Stop & Glo substrate (solution) into the bottle containing dry Stop & Glo substrate and mixing the solution for 10 seconds using a vortexer. In order to produce a 1 ⁇ Stop & Glo solution, 20 ⁇ l of the 50 ⁇ Stop & Glo substrate and 1 ml of the Stop & Glo buffer are combined. This is sufficient for 10 assays.
• DLR-assay 100 ⁇ l of LAR II were introduced together with 20 ⁇ l of cell lysate into a well and mixed by pipetting up and down for 2-3 seconds. After luminometric measurement of firefly luciferase activity, 100 ⁇ l of Stop & Glo reagent were added, the solution was mixed and then the Renilla-luciferase activity was determined. The luminescence was determined using the Fluoroskan Ascent FL luminometer (Thermo Labsystems, Frankfurt, Germany).
• Oligonucleotide % Inhibition* a) 3′ aaaaaaCUUCGCUUCCAACACCUAGAC 43 (RNA in antisense orientation, with 2′5′ A) b) 3′ a*a*a a-C*U*U*C G C*U*U C*C A A*C A C*C*U A G A*C 43 (RNA in antisense orientation, with 2′5′ A) c) 3′ aaaaTTTTTTACCTTGTTGAAATGG 12 (not complementary to target RNA; sense orientation) d) 3′ a*a*a a- C*U*U*C G C*U*U C*C A A*C A C*C*U A G A*C 7 (antisense orientation, underlined 2′-O-methyl) 5′-G A A G*C G A A G G*U*U G*U G G A U*C*U*G-teg 0 (Seq ID No.20; sense
• the stabilization of the oligonucleotide by phosphorothioate residues (oligonucleotide b) at particular positions on the oligomer resulted in a markedly improved action.
• the entire 3′5′-linked complementary sequence was derivatized as 2′-O-methyl derivative, virtually no activity was detectable (oligonucleotide d).
• oligonucleotides of the invention were also directed against a human gene or the corresponding RNA and tested on human cells (HUVEC, human umbilical vein endothelial cells).
• control oligonucleotides used were the complementary sequences (sense orientation) without 2′5′-oligoadenylate,
• * is phosphorothioate
• a*a*a a is a 2′5′-linked adenylate (partially modified with *)
• teg is triethylene glycol phosphate
• oligoribonucleotide analogs which had been modified with phosphothioate at particular positions were used in human primary cells as follows, in order to inhibit gene expression of Edg-1 in human cells (HUVEC, human umbilical vein endothelial cells).
• the cell lawn was washed again with PBS and then overlaid with serum-containing EGM medium (CellSystems, # CC-3024+EGM supplements # CC-3124) and incubated for a further 24 or 48 h.
• serum-containing EGM medium CellSystems, # CC-3024+EGM supplements # CC-3124
• the cells were incubated for 4 hours, then fixed with 5% paraformaldehyde (in PBS, pH 7.4) and directly photographed in an inverted fluorescence microscope (Zeiss Axiovert 135M) with its 200-fold magnification using a cooled CCD camera (ORCA-1, Bfi optilas) and excitation through an FITC filter (excitation: 490 nm, emission: 510 nm) and processed via AQM2000 software (Kinetic Imaging).
• the gel was run in 1 ⁇ Tris/glycine/SDS buffer (Bio-Rad # 161-0732).
• the gel was transferred with the aid of the Bio-Rad criterion Western blot apparatus (#170-4070) to a nitrocellulose (NC) membrane (Amersham # RPN 2020D) in 1 ⁇ Tris/glycine buffer (Bio-Rad #161-0732, +10% methanol).
• NC membrane was then saturated at room temperature for 1 hour using 1 ⁇ TBS buffer (Bio-Rad # 170-6435), which contained 5% milk powder (“Blotto”, Bio-Rad #170-6404) and 0.1% Tween 20 (Bio-Rad # 170-6531).
• the membrane was incubated with the anti-hEDG-1 primary antibody (polyclonal rabbit serum obtained by immunization with the EDG-1-specific peptide sequence CKAHRSSVSDYVNYD, coupled to KLH and affinity-purified against the abovementioned peptide sequence) in a 1:50 dilution in TBST-Blotto at 4° C. overnight.
• the secondary antibody anti-rabbit, alkaline phosphatase-coupled, Dianova # 111-055-045 was incubated in a 1:2000 dilution in TBST-Blotto at room temperature for one hour.
• the ECF (“enhanced chemifluorescence”) detection reaction (Amersham #RPN5785) was carried out, and the NC membrane which was covered with clingfilm was incubated with 1 ml of ECF substrate (Amersham Pharmacia #RPN5785) at room temperature for 5 minutes and then detected using a Fluor-imager 595 scanner (Amersham Pharmacia).
• the signal was quantified using the ImageQuant software (Amersham Pharmacia) and normalized to the ⁇ -tubulin signal which was obtained after destaining (Alpha Diagnostic Kit # 90100) the NC membrane once and incubating the ⁇ -tubulin-specific primary antibody (affinity-purified rabbit antibody, Santa Cruz # sc-9104) according to the above-described method.
• EDG-1 protein (% of control) Concentration Oligo #2 Oligo #3 Oligo #5 Oligo #6 Oligo #7 Oligo #8 ( ⁇ M) region “175” region “175” region “725” region “725” mismath mismath 0 100.0 100.0 100.0 100.0 100.0 0.01 87.7 51.4 98.6 47.2 89.4 128.3 0.05 100.8 44.2 129.3 35.5 109.7 107.5 0.1 103.0 35.5 109.4 25.1 121.8 103.6 0.5 119.2 40.3 107.2 27.1 95.7 85.6 1.0 104.4 34.0 96.2 22.6 100.1 83.5
• the inhibition proved to be also sequence-specific with regard to the oligoribonucleotides used, since only the edg-1-homologous oligoribonucleotides #3 and #6 inhibited edg-1 expression, while the oligoribonucleotide #8 with antisense orientation, which differs from the edg-1 sequence by 5 nucleotides, did not inhibit edg-1 expression.

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