HK1012005B - G-cap stabilized oligonucleotides - Google Patents

Description

Antisense oligonucleotides (AO), triple helix forming oligonucleotides (TFO) and sense oligonucleotides have been shown to be specific inhibitors of gene expression in a wide variety of systems, both in vitro and in vivo [Uhlmann & Peyman, Chem. Rev. 1990, 90, 543; Milligan et al., J. Med. Chem. 1993, 36, 1923; Stein & Cheng, Science 1993, 261, 1004; Bielinski et al., Science 1990, 250, 997].

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Blackburne [Nature 350:569, 1991] describes structures that can be taken up by G-rich oligonucleotides. G-rich oligonucleotides with at least four short sections with G-residue are able to form compact intramolecular structures in the presence of Na+ or K+ containing as a stabilizing element so-called G-quartets, which are four guanine residues linked in a plane above highest base pair. Several such elements are arranged one above the other.

It has now been found that there is a very simple way of significantly improving unmodified or modified oligonucleotides in terms of their nuclease resistance and cell penetration, namely by lengthening the oligonucleotide at the 3' and/or 5' end by one to 10 guanines, thus significantly improving efficacy.

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Err1:Expecting ',' delimiter: line 1 column 262 (char 261)

In cases where the oligonucleotide (oligog) ends at the 5' or 3' end with one or more guanines, CAP may be advantageous, wherein m is defined as above and n is the number of naturally occurring guanines at the 5' or 3' end of the oligonucleotide (oligog) and m is preferred for two to six, particularly preferred for three to five, and very particularly preferred for four.

The oligonucleotides thus converted may be unmodified or modified, the following variants being authorised: (a) complete or partial replacement of the 3'- and/or 5'-phosphoric acid diester bridges, for example by a phosphorothioate, phosphorodiethyoate, (NR1R2) phosphoramidate, boron phosphate, phosphate- (((C1-C21) -O-alkylated ester, phosphate- ((C6-C12) -aryl- ((C1-C21) -O-alkylated ester, 2,2,2-trichlorodimethylethylphosphate, (C1-C8) -alkylphosphonate, (C6-C12) -aryl-phosphate bridges. The substitution of a phosphorothioate, phosphorodiethoate, NR1R2 phosphoramidate, phosphate-O-methylester, phosphate-O-ethylester, phosphate-O-isopropylester, methylphosphonate, phenylphosphonate bridge is preferred and the substitution of a phosphorothioate, phosphorodithiotheate, methylphosphonate bridge is particularly preferred. The substitution of a phosphorothiate bridge is particularly desirable. R1 and R2 stand for hydrogen or (C1-C18) alkyl, (C6-C20) aryl, (C6-C14) aryl- ((C1-C8) alkyl, - ((CH2) c-[NH(CH2) c]d-NR3R3 where c is an integer from 2 to 6 and d is an integer from 0 to 6 and R3 is an independent hydrogen,Err1:Expecting ',' delimiter: line 1 column 791 (char 790)Err1:Expecting ',' delimiter: line 1 column 53 (char 52)(d) a complete or partial substitution of the β-D-2'-deoxyribose units, for example by α-D-2'-deoxyribose, L-2'-deoxyribose, 2'-F-2'-deoxyribose, 2'-O-(C1-C6) alkyl ribose, 2'-O-(C2-C6) alkenyl ribose, 2'-NH2-2'-deoxyribose, β-D-xylofuranose, α-Arabinofuranose, 2,4-Dideoxy-β-D-pytherohexocyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyclic tetracyc The preferred substitution is 2'-F-2'-deoxyribose, 2'-O- ((C1-C6) alkyl ribose, 2'-O- ((C2-C6) alkenyl ribose, 2'-NH2-2'-deoxyribose. The substitution of 2'-F-2'-deoxyribose, 2'-O- ((C1-C4) alkyl-ribose, 2'-O- ((C2-C4) alkenyl-ribose, 2'-NH2-2'-deoxyribose is particularly preferred. Preferably one, two or three units of ribose should be replaced at the 5' and/or 3' ends, preferably at the 5' and 3' ends.(e) complete or partial replacement of the natural nucleoside bases, such as by 5-hydroxymethyluracil, 5-aminouracil, pseudouracil, dihydrouracil, 5-C1-C6-alkyluracil, 5-C2-C6-alkenyluracil, 5-C2-C6-alkyluracil, 5-C1-C6-alkenyl-cytosine, 5-C2-C6-alkenyl-cytosine, 5-C2-C6-alkenyl-cytosine, 5-C2-C6-alkyllucytosine, 5-Foruracil, 5-Foruracil, 5-Chloruracil, 5-Chloruroracil, 5-Cytosine, 5-Boracytosine, 5-Promzacil, 7-Dea substitute. The substitution of 5-C1-C6 alkyl uracil, 5-C2-C6 alkenyl uracil is preferred. The test chemical is a chemical that is used to determine the concentration of a substance in a solution. The following are to be classified in the same heading as the additive: 5-chlorcytosine, 5-bromuracil, 5-bromcytosine, 7-Deaza-7-alkynyl, preferably hexinyl-substituted purines, 7-Deaza-7-methyl-substituted purines, 7-Deaza-7-brom-substituted purines. The substitution of 5-C3-C6-alkyl-uracil, 5-C2-C6-alkenyl-uracil, 5-C2-C6-alkinyl-uracil, 5-C1-C6-alkenyl-cytosine, 5-C2-C6-alkenyl-cytosine, 5-C2-C6-alkinyl-cytosine is particularly preferred. The substitution of 5-hexinylcytosine, 5-hexinyluracil, 5-hexinylcytosine, 5-propinyluracil, 5-propinylcytosine is particularly preferred. The substitution of nucleoside bases is not to be carried out in the CAP areas.

Of the above modifications, the modifications from groups a), b), c) and d) are preferred.

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Furthermore, the oligonucleotides of the invention may be carried at the 3' and/or 5' end in 3'-3' and 5'-5' inversions (described e.g. in M. Koga et al., J. Org. Chem. 56:3757, 1991).

The invention also relates to processes for the production of the compounds of the invention by means of processes known to the professional, in particular chemical synthesis, the use of the compounds of the invention for the manufacture of a medicinal product and a process for the manufacture of a medicinal product characterized by mixing the oligonucleotides of the invention with a physiologically acceptable carrier and, where appropriate, suitable additives and/or excipients.

In general, the present invention also covers the use of therapeutically active oligonucleotides to produce a medicinal product in which at least one incomplete pyrimidine nucleoside is modified.

In addition, another subject of the present invention is the use of oligonucleotides with at least one non-terminal and modified pyrimidine nucleoside as a diagnostic agent, for example to detect the presence or absence or amount of a specific double-stranded or single-stranded nucleic acid molecule in a biological sample.

The oligonucleotides have a length of 10 to 40 for the use of the invention, in particular from about 12 to 31 nucleotides.

The medicinal products of the present invention may be used, for example, to treat diseases caused by viruses, such as HIV, HSV-1, HSV-2, influenza, VSV, hepatitis B or papilloma viruses.

Antisense oligonucleotides of the invention that are effective against such targets include: (a) against HIV, for example (b) against HSV-1, for example

The Arabic numerals given here refer to the Roman numerals given above; the oligonucleotides given here are additionally given the CAPs of the invention.

The phosphorothiocyanate bonds replaced by a phosphorothiocyanate bridge (P=S) are marked with an asterisk (*) in the sequences.

The drugs of the present invention are also suitable for the treatment of cancer or restenosis, for example by using oligonucleotide sequences directed against targets responsible for the development or growth of cancer, such as: 1) Nuclear oncoproteins such as c-myc, N-myc, c-myb, c-fos, c-fos/jun, PCNA, p1202) Cytoplasmic/membrane-associated oncoproteins such as EJ-ras, c-Ha-ras, N-ras, rrg, bcl-2, cdc-2, c-raf-1, c-mos, c-src, c-abl3) Cellular receptors such as EGF receptor, c-erbA, retinoid receptor, protein kinase regulatory subunit, c-fms4) Cytokines, growth factors, extracellular matrix such as ILF-1, ILF-1, CSF-1, ILB-2, ILF-4, ILF-4, Blastin, Fibroblastin, VEGF, Myelothelial growth factor (EGF).

Antisense oligonucleotides of the invention, which are effective against such targets, are, for example: (a) against c-ha-ras, e.g. b) c-myc, e.g. c-myb, e.g. c-fos, e.g. p120, e.g. f) EGF receptor, e.g. p53, tumor suppressor, e.g. h) bFGF, e.g. i) VEGF, e.g.

For example, the drugs of the present invention are also suitable for the treatment of diseases affected by integrins or cell-cell adhesion receptors, such as VLA-4, VLA-2, ICAM or ELAM.

Antisense oligonucleotides of the invention, which are effective against such targets, are, for example: (a) VLA-4, for example (b) ICAM, for example (c) ELAM-1, for example

The medicinal products of the present invention are also suitable, for example, for the treatment of diseases caused by factors such as TNF alpha.

Antisense oligonucleotides of the invention, which are effective against such targets, are, for example: (a) TNF-alpha, for example

The above applies to the numbering of the sample oligonucleotides and the asterisk symbol.

The medicinal products may be administered orally, e.g. in the form of tablets, drogues, hard or soft gelatine capsules, solutions, emulsions or suspensions, or rectally, e.g. in the form of suppositories or parenterally, e.g. in the form of solutions for injection. For the manufacture of pharmaceutical products, these compounds may be processed into therapeutically active organic and inorganic media. Examples of such media for tablets, drogues and hard gelatine capsules are lard, cornstarch or derivatives thereof, tallow and stearic acid or their derivatives.Alcohols, polyoles, glycerol and vegetable oils. Suitable suppository media are vegetable and hardened oils, waxes, fats and semi-liquid polyoles. Pharmaceutical preparations may also contain preservatives, solvents, stabilizers, net agents, emulsifiers, sweeteners, dyes, flavourings, salts to alter osmotic pressure, buffers, surfactants, antioxidants, and other therapeutic agents. For injection, the antisense oligonucleotides are formulated in a liquid solution, preferably in a physiologically acceptable buffer such as Hank's solution or Ringer's solution, but the antisense oligonucleotides can also be formulated in solid form and dissolved or suspended before use.

The doses preferred for systemic administration are approximately 0.01 mg/ kg body weight to approximately 50 mg/ kg body weight per day.

The following examples are intended to illustrate the invention in more detail. Table 1 shows oligonucleotides tested for their in vitro activity against HSV-1. Oligonucleotide No. 4 is described as in Mann et al. (Bioconj. Chem. 3: 554, 1992) by introducing a modified [4- ((1-pyrenyl) butanyl]phosphodiester at the 5'-end. The oligonucleotides of the invention work at a minimum inhibitory concentration of 9 μM (e.g. 1 and 4) or even as low as 3 μM (e.g. 2 and 3).

Example 1 Oligonucleotide synthesis

Modified oligonucleotides were synthesised on an automatic DNA synthesizer (Applied Biosystems Model 380B or 394) using standard phosphoramidite chemistry and iodine oxidation. For introduction of phosphorothiate bridges into mixed phosphorothioates and phosphodiester oligonucleotides were oxidised with TETD (tetraethylthidisulfide) instead of iodine (Applied Biosystems User Bulletin 65). After cleavage from the solid carrier (CPG or tentagel) and removal of the protective groups with NH3 at 55°C for 18h, the oligonucleotides were obtained first by precipitation of butanol (Saadogo, Vanke, Dyucr. vol. 19: 4, 1991), then by purification with a solution of 2.5 ml of naphthal ethanol.

The analysis of oligonucleotides was carried out by: (a) Analytical gel electrophoresis in 20% acrylamide, 8M urea, 45 mM trisborate buffer, pH 7.0 and/ or (b) HPLC analysis: Waters GenPak FAX, Gradient CH3CN (400 ml), H2O (1.6 l), NaH2PO4 (3.1 g), NaCl (11.7 g), pH6.8 (0.1 M in NaCl) to CH3CN (400 ml), H2O (1.6 l), NaH2PO4 (3.1 g), NaCl (175.3 g), pH6.8 (1.5 M in NaCl) and/ or (c) Capillary gel electrophoresis or Beckmann capillary ECAPTM, U100P Column, 65 cm length, 100 I.D., 15 cm from one end, buffer window 140 μm Trism, 360 M Boresay, 7 mm/ m in the case of gel electrophoresis and microscopy

Analysis of the oligonucleotides showed that they were each present at a purity of more than 90%.

Example 2 Testing of antiviral activity of test substances against herpes viruses in vitro

The antiviral activity of the test substances against various human pathogenic herpes viruses is investigated in the cell culture test system.

For the test, monkey kidney cells (Vero, 2x105/ml) are sown in serum Dulbecco's MEM (5% fetal calf serum (FCS)) in 96-pot microtiter plates and incubated for 24 h at 37°C and 5% CO2.

The test substances are pre-diluted in H2O to a concentration of 600 μM and stored at -18 °C. Further dilution steps are performed in Dulbecco's Minimal Essential Medium (MEM) for the test. Each 100 μl of the individual test substance dilutions is added to the rinsed cells together with 100 μl of serum-free Dulbecco's MEM (-FCS).

After 3 h incubation at 37°C and 5% CO2, the cells are infected with herpes simplex virus type 1 (ATCC VR733, HSV-1 F strain) or herpes simplex virus type 2 (ATCC VR734, HSV-2 G strain) at concentrations where the cell walls are completely destroyed within 3 days. In HSV-1, the infection rate is 500 plaque-forming units (PFU) per nap, in HSV-2, 350 PFU/nap. The test pieces then contain test substance at concentrations of 80 μM to 0.04 μM in MEM, supplemented by 100 U/ml penicillin G and 100 mg/l streptomycin. All tests are performed as a double determination with eight controls, except for the one panel.

The test pieces are incubated for 17 h at 37°C and 5% CO2.The cytotoxicity of the test substances is determined after 20 h total incubation time by microscopic examination of the cell cultures.The maximum tolerated dose (DTM) is the highest concentration of the product which under the specified test conditions does not cause microscopically detectable cell damage.

The untreated infection controls then show a complete cytopath effect (CPE). After microscopic examination of the cell cultures, they are then stained with neutral red according to the Finter (1966) vitalising method. The antiviral activity of a test substance is defined as the minimum inhibition concentration (MHK) needed to protect 30-60% of the cells from the virus-induced cytopathogenic effect.

Table 1: Activity of various modified antisense oligonucleotides against HSV-1 in cell culture. The phosphodiester bonds replaced by a phosphorothioate bridge (P=S) were marked in the sequence with ; HMK = minimum inhibitory concentration; DTM = dose tolerated maxima; Py = pyrene.

The following shall be added:

(1) GENERAL INFORMATION: The following is the list of products: (i) The applicant: (A) NAME: Hoechst Aktiengesellschaft (B) BRIDGE: - (C) LOCATION: Frankfurt (D) LAND: - (E) COUNTRY: Germany (F) POST CALL: 65926 (G) TELEPHONE: 069-305-3005 (H) TELEFACS: 069-357175 (I) TELEX: - (II) REPORT TITLE: G-Cap Stabilized Oligonucleotides (G) NUMBER OF SEQUENCES: 34 (IV) COMPUTER-LESBARE FORM: The name of the person who is to be notified of the presence of the substance in the product is the person who is to be notified of the presence of the substance in the product. (a) Data carrier: floppy disk (b) computer: IBM PC compatible (c) operating system: PC-DOS/MS-DOS (d) software: patent in release #1.0, version #1.25 (EPA) (e) information about the SEQ (i) Sequence characteristics: (a) LENGTH: 20 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: (c) (2) INFORMATION about the SEQ ID NO: 2: (i) Sequence characteristics: (a) LENGTH: 20 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (A) NAME/CLEAR: exon (B) LOCATION: 1. The name of the manufacturer and the address of the manufacturer..20(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:(2) Information to be provided to the SEQ ID NO: 3: (i) Sequence characteristics: (a) LENGTH: 28 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..28 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: (c) (2) INFORMATION about the SEQ ID NO: 4: (i) Sequence characteristics: (a) LENGTH: 25 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: (c) (2) INFORMATION about the SEQ ID NO: 5: (i) Sequence characteristics: (a) LENGTH: 26 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..26 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: (c) (2) INFORMATION about the SEQ ID NO: 6: (i) Sequence characteristics: (a) LENGTH: 27 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristic: (A) NAME/CLEAR: exon (B) LOCATION: 1. The name of the manufacturer and the address of the manufacturer..27 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: (xi) (2) Information to be provided by the SEQ ID NO: 7: (i) Sequence characteristics: (a) LENGTH: 20 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: (c) (2) INFORMATION about the SEQ ID NO: 8: (i) Sequence characteristics: (a) LENGTH: 15 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: (c) (2) INFORMATION about the SEQ ID NO: 9: (i) Sequence characteristics: (a) LENGTH: 21 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: (c) (2) INFORMATION about the SEQ ID NO: 10: (i) Sequence characteristics: (a) LENGTH: 15 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (A) NAME/CLEAR: exon (B) LOCATION: 1. The name of the manufacturer and the address of the manufacturer..15 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 10: (xi) (2) Information to be provided to the SEQ ID No: 11: (i) Sequence characteristics: (a) LENGTH: 18 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..18 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: (c) (2) INFORMATION about the SEQ ID NO: 12: (i) Sequence characteristics: (a) LENGTH: 21 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: (c) (2) INFORMATION about the SEQ ID NO: 13: (i) Sequence characteristics: (a) LENGTH: 22 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..22 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: (c) (2) INFORMATION about the SEQ ID No: 14: (i) Sequence characteristics: (a) LENGTH: 20 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (A) NAME/CLEAR: exon (B) LOCATION: 1. The name of the manufacturer and the address of the manufacturer..20 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 14: (xi) (2) Information to be provided by the SEQ ID No: 15: (i) Sequence characteristics: (a) LENGTH: 20 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..20 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 15: (c) (2) INFORMATION about the SEQ ID No: 16: (i) Sequence characteristics: (a) LENGTH: 18 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..18 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: (c) (2) INFORMATION about the SEQ ID NO: 17: (i) Sequence characteristics: (a) LENGTH: 20 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: (c) (2) INFORMATION about the SEQ ID NO: 18: (i) Sequence characteristics: (a) LENGTH: 19 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (A) NAME/CLEAR: exon (B) LOCATION: 1. The name of the manufacturer and the address of the manufacturer..19 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 18: (xi) (2) Information to be provided to the SEQ ID No: 19: (i) Sequence characteristics: (a) LENGTH: 21 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: (c) (2) INFORMATION about the SEQ ID NO: 20: (i) Sequence characteristics: (a) LENGTH: 18 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..18 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: (c) (2) INFORMATION about the SEQ ID NO: 21: (i) Sequence characteristics: (a) LENGTH: 20 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: (c) (2) INFORMATION about the SEQ ID NO: 22: (i) Sequence characteristics: (a) LENGTH: 20 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (A) NAME/CLEAR: exon (B) LOCATION: 1. The name of the manufacturer and the address of the manufacturer..20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: (xi) (2) Information to be provided by the SEQ ID NO: 23: (i) Sequence characteristics: (a) LENGTH: 19 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..19 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: (c) (2) INFORMATION about the SEQ ID NO: 24: (i) Sequence characteristics: (a) LENGTH: 21 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: (c) (2) INFORMATION about the SEQ ID NO: 25: (i) Sequence characteristics: (a) LENGTH: 22 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..22 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: (c) (2) INFORMATION about the SEQ ID NO: 26: (i) Sequence characteristics: (a) LENGTH: 19 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (A) NAME/CLEAR: exon (B) LOCATION: 1. The name of the manufacturer and the address of the manufacturer..19 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: (xi) (2) Information to be provided by the SEQ ID NO: 27: (i) Sequence characteristics: (a) LENGTH: 17 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..17 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: (c) (2) INFORMATION about the SEQ ID NO: 28: (i) Sequence characteristics: (a) LENGTH: 17 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..17 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: (c) (2) INFORMATION about the SEQ ID NO: 29: (i) Sequence characteristics: (a) LENGTH: 14 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: The following information is provided for the purpose of the analysis: (i) Sequence characteristics: (a) LENGTH: 19 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (A) NAME/CLEAR: exon (B) LOCATION: 1. The name of the manufacturer and the address of the manufacturer..19 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 30: (xi) (2) Information to be provided by the SEQ ID No: 31: (i) Sequence characteristics: (a) LENGTH: 15 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..15 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 31: (c) (2) INFORMATION about the SEQ ID No: 32: (i) Sequence characteristics: (a) LENGTH: 20 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: (c) (2) INFORMATION about the SEQ ID NO: 33: (i) Sequence characteristics: (a) LENGTH: 18 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (a) NAME/CLEW: exon (b) LOCATION: 1..18 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: (c) (2) INFORMATION about the SEQ ID NO: 34: The name of the person who is responsible for the operation of the vehicle is the person who is responsible for the operation of the vehicle. (i) Sequence characteristics: (a) LENGTH: 16 base pairs (b) TYPE: nucleic acid (c) String form: single (d) Topology: linear (ii) TYPE of molecule: DNA (genomic) (ix) Characteristics: (A) NAME/CLEAR: exon (B) LOCATION: 1. The name of the manufacturer and the address of the manufacturer.- I 'm not .

Claims (32)

1. An oligonucleotide of the formula         5'-(CAP)-(oligo)-(CAP)-3' wherein (oligo) is a nucleotide sequence of from 10 to 40 nucleotides in length and wherein the nucleotide sequence of the "(oligo)" moiety is derived from those regions of genes which are of importance for the transcription of the DNA or the translation of the corresponding mRNA; and CAP is G_m, wherein m is a whole number from zero to ten, and wherein the two CAPs can be defined independently of each other and, if m is zero at the 5' end or 3' end, the CAPs have to be different, and wherein the end of the "oligo" sequence is not formed by a guanine.
2. An oligonucleotide as claimed in claim 1, wherein m is a whole number from two to six.
3. An oligonucleotide as claimed in claim 1, wherein m is from three to five.
4. An oligonucleotide as claimed in claim 1, wherein m is four.
5. An oligonucleotide of the formula         5'-(CAP)-(oligo)-(CAP)-3' wherein (oligo) is a nucleotide sequence of from 10 to 40 nucleotides in length and wherein the nucleotide sequence of the "(oligo)" moiety is derived from those regions of genes which are of importance for the transcription of the DNA or the translation of the corresponding mRNA and wherein the (oligo) ends at the 5' end and/or 3' end with one or more guanines; CAP is G_(m-n), wherein m is defined as in claim 1 and n is the number of the guanines which are naturally present at the 5' end or 3' end of the oligonucleotide (oligo), and wherein (m-n) is from two to six, and wherein the two CAPs which are present in the molecule can be defined independently of each other.
6. An oligonucleotide as claimed in claim 5, wherein (m - n) is from three to five.
7. An oligonucleotide as claimed in claim 5, wherein (m - n) is four.
8. An oligonucleotide as claimed in one or more of claims 1 to 7, wherein the oligonucleotide is modified.
9. An oligonucleotide as claimed in one or more of claims 1 to 8, wherein from 2 to 10 non-terminal pyrimidine nucleosides are modified.
10. An oligonucleotide as claimed in one or more of claims 1 to 9, wherein the oligonucleotide is modified at the 5'-terminal and/or the 3'-terminal nucleotide.
11. An oligonucleotide as claimed in one or more of claims 1 to 10, wherein one or more groups which have at least 1 to 4 nucleotides which are linked together are not modified.
12. An oligonucleotide as claimed in one or more of claims 1 to 11, wherein the modification is defined as

    (a) complete or partial replacement of the 3'- and/or 5'-phosphoric acid diester bridges and/or

    (b) complete or partial replacement of the 3'- or 5'-phosphoric acid diester bridges with "dephospho" bridges and/or

    (c) complete or partial replacement of the sugar phosphate backbone and/or

    (d) complete or partial replacement of the β-D-2'-deoxyribose units and/or

    (e) complete or partial replacement of the natural nucleoside bases.
13. An oligonucleotide as claimed in one or more of claims 1 to 12, wherein the modification is defined as

    (a) complete or partial replacement of the 3'- and/or 5'-phosphoric acid diester bridges with phosphorothioate bridges, phosphorodithioate bridges, (NR¹R²)-phosphoramidate bridges, boranophosphate bridges, phosphate-(C₁-C₂₁)-O-alkyl ester bridges, phosphate-[(C₆-C₁₂)aryl-(C₁-C₂₁)-O-alkyl] ester bridges, 2,2,2-trichlorodimethylethylphosphonate bridges, (C₁-C₆)alkylphosphonate bridges or (C₆-C₁₂)arylphosphonate bridges,    wherein    R¹ and R² are, independently of each other, hydrogen or (C₁-C₁₈)-alkyl, (C₆-C₂₀)-aryl, (C₆-C₁₄)-aryl-(C₁-C₈)-alkyl or -(CH₂)_c-[NH(CH₂)_c]_d-NR³R³, in which c is a whole number from 2 to 6 and d is a whole number from 0 to 6, and R³ is, independently of each other, hydrogen, (C₁-C₆)-alkyl or (C₁-C₄)-alkoxy-C₁-C₆-alkyl, or R¹ and R² form, together with the nitrogen atom carrying them, a 5-6-membered heterocyclic ring which can additionally contain a further heteroatom from the series O, S and N; and/or

    (b) complete or partial replacement of the 3'- and/or 5'-phosphoric acid diester bridges with formacetal, 3'-thioformacetal, methylhydroxylamine, oxime, methylenedimethylhydrazo, dimethylenesulfone or silyl groups, and/or

    (c) complete or partial replacement of the sugar phosphate backbone with "morpholinonucleoside" oligomers, PNAs or PNA-DNA hybrids, and/or

    (d) complete or partial replacement of the β-D-2'-deoxyribose units with α-D-2'-deoxyribose, L-2'-deoxyribose, 2'-F-2'-deoxyribose, 2'-O- (C₁-C₆)alkyl-ribose, 2'-O-(C₂-C₆)alkenyl-ribose, 2'-NH₂-2'-deoxyribose, β-D-xylofuranose, α-arabinofuranose, 2,4-dideoxy-β-D-erythrohexopyranose, or carbocyclic, open-chain or bicyclo sugar analogs, and/or

    (e) complete or partial replacement of the natural nucleoside bases with 5-(hydroxymethyl)uracil, 5-aminouracil, pseudouracil, dihydrouracil, 5-(C₁-C₆)-alkyl-uracil, 5-(C₂-C₆)-alkenyl-uracil, 5-(C₂-C₆)-alkynyl-uracil, 5-(C₁-C₆)-alkyl-cytosine, 5-(C₂-C₆)-alkenyl-cytosine, 5-(C₂-C₆)-alkynyl-cytosine, 5-fluorouracil, 5-fluorocytosine, 5-chlorouracil, 5-chlorocytosine, 5-bromouracil or 5-bromocytosine,    wherein the nucleoside bases are not to be replaced in the CAP regions.
14. An oligonucleotide as claimed in one or more of claims 1 to 13, wherein the modification is defined as

    (a) the partial or complete replacement of the 3'-and/or 5'-phosphoric acid diester bridges with phosphate-O-methyl ester bridges, phosphate-O-ethyl ester bridges, phosphate-O-isopropyl ester bridges, methylphosphonate bridges and/or phenylphosphonate bridges, and/or

    (b) the partial or complete replacement of the β-D-2'-deoxyribose units with 2'-O-methyl-, 2'-O-allyl-and/or 2'-O-butylribose, and/or

    (c) the partial or complete replacement of the natural nucleoside bases with 5-pentynylcytosine, 5-hexynyluracil and/or 5-hexynylcytosine, wherein the nucleoside bases are not to be replaced in the CAP regions.
15. An oligonucleotide as claimed in one or more of claims 1 to 14, wherein two or three phosphoric acid diester bridges are replaced at the 5' end and/or 3' end of the oligonucleotide.
16. An oligonucleotide as claimed in one or more of claims 1 to 12, wherein phosphoric acid diester bridges are replaced at pyrimidine positions.
17. An oligonucleotide as claimed in one or both of claims 15 and 16, wherein the phosphoric acid diester bridges are replaced with phosphorothioate bridges.
18. An oligonucleotide as claimed in one or more of claims 1 to 17, wherein two or three ribose units are replaced at the 5' end and/or the 3' end.
19. An oligonucleotide as claimed in one or more of claims 1 to 18, wherein ribose units are replaced at pyrimidine positions.
20. An oligonucleotide as claimed in one or more of claims 1 to 19, wherein the oligonucleotide at the 3' end and/or 5' end is linked to molecules which promote cell penetration, nuclease resistance, affinity for the target RNA/DNA or pharmacokinetics.
21. An oligonucleotide as claimed in one or more of claims 1 to 20, wherein the oligonucleotide at the 5' end and/or 3' end is linked to molecules which are selected from polylysine, intercalators, fluorescent compounds, linkers, lipophilic molecules, lipids, steroids, vitamins, polyethylene glycol or oligoethylene glycol, (C₁₂-C₁₈)-alkyl-phosphate diesters or -O-CH₂-CH(OH)-O-(C₁₂-C₁₈)-alkyl radicals.
22. An oligonucleotide as claimed in one or more of claims 1 to 21, wherein the oligonucleotide at the 5' end and/or 3' end is linked to molecules which are selected from pyrene, acridine, phenazine, phenanthridine, fluorescein, psoralen, azidoproflavine, (C₁₂-C₂₀)-alkyl, 1,2-dihexadecyl-rac-glycerol, cholesterol, testosterone or vitamin E.
23. An oligonucleotide as claimed in one or more of claims 1 to 22, wherein the oligonucleotide carries 3'-3' and/or 5'-5' inversions at the 3' end and/or 5' end.
24. An oligonucleotide as claimed in one or more of claims 1 to 23, wherein (oligo) is one of the sequences
25. An oligonucleotide having one of the sequences or wherein "*" is a phosphorothioate bridge and "PY" is pyrene.
26. An oligonucleotide as claimed in one or more of claims 1 to 24, wherein the oligonucleotide has a sequence which is directed against a target which is responsible for the origin of cancer and/or the growth of cancer.
27. A process for preparing an oligonucleotide as claimed in one or more of claims 1 to 26, wherein the oligonucleotide is synthesized chemically on a solid support.
28. A drug which comprises one or more oligonucleotides as claimed in one or more of claims 1 to 26 and a physiologically acceptable excipient and also, where appropriate, suitable additives and/or adjuvants.
29. The use of an oligonucleotide as claimed in one or more of claims 1 to 26 for producing a drug.
30. The use of an oligonucleotide as claimed in one or more of claims 1 to 26 for producing a drug for treating diseases which are caused by viruses and for treating cancer, restenosis or diseases which are influenced by integrins or cell-cell adhesion receptors or which are triggered by diffusible factors.
31. The use of an oligonucleotide as claimed in one or more of claims 1 to 26 for preparing a diagnostic agent.
32. The use of an oligonucleotide as claimed in one or more of claims 1 to 26 for preparing a diagnostic agent for diseases which are caused by viruses, for cancer, for restenosis or for diseases which are influenced by integrins or cell-cell adhesion receptors or triggered by diffusible factors.