EP1030914A2 - Antisense oligonucleotides against tenascin for treating vitiligo - Google Patents

Antisense oligonucleotides against tenascin for treating vitiligo

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Publication number
EP1030914A2
EP1030914A2 EP98954464A EP98954464A EP1030914A2 EP 1030914 A2 EP1030914 A2 EP 1030914A2 EP 98954464 A EP98954464 A EP 98954464A EP 98954464 A EP98954464 A EP 98954464A EP 1030914 A2 EP1030914 A2 EP 1030914A2
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Prior art keywords
seq
oligonucleotide
modified
alkyl
bridge
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EP98954464A
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German (de)
French (fr)
Inventor
Anuschirwan Peyman
Eugen Uhlmann
Caroline Weiser
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Sanofi Aventis Deutschland GmbH
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Aventis Pharma Deutschland GmbH
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Publication of EP1030914A2 publication Critical patent/EP1030914A2/en
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
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    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate

Definitions

  • the invention relates to specific, optionally modified oligonucleotides with a length of up to 18 nucleotides, preferably with a length of 7-15 nucleotides, which correspond to sections of tenascin-coding sequences and which can bind to these sequences, their preparation and their use, for example for specific inhibition of the expression of tenascin and for the manufacture of medicinal products which can be used for the treatment of vitiligo.
  • Vitiligo is understood to mean an acquired lack of melanocytes, which creates hypopigmented areas of the skin, which are usually sharply defined and often symmetrically arranged, form one or two spots or cover almost the entire skin.
  • the hair in hypopigmented areas is usually white and also appears white in wood light.
  • the affected skin areas are susceptible to sunburn. The cause of the disease is unknown.
  • vitiligo is considered to be a disease acquired over the course of a lifetime, there is occasionally a family cluster (autosomal dominant, with incomplete penetrance and variable expression). It can also follow unusual physical trauma, especially a skull injury.
  • the available therapies include photochemotherapy (PUVA), for example with methoxypsoralen, phenylalanine, or khellin, the transplantation of cultivated Melanocytes, epidermal grafting, and treatment with steroids or placenta extracts.
  • PUVA photochemotherapy
  • Treatment with pseudocatalase has recently been reported (Schallreuter et al., Dermatology 190 (1995) 223).
  • Small stoves can also be covered with cosmetic make-up or tanning solutions.
  • Tenascin (Crossin, J. Cell. Biol. 61 (1996) 592) is an extracellular matrix glycoprotein which consists of six identical subunits which are linked at the amino terminus via disulfide bridges.
  • the Tenascin subunits have a characteristic domain structure: a cysteine-rich sequence at the amino-terminal end is followed by three sequence sections, each made up of repeating units, from units homologous to EGF, from units homologous to fibronectin (type III) and from units homologous to fibrinogen.
  • tenascin isoforms There are several isoforms of the tenascin subunits (hereinafter referred to as tenascin isoforms) which differ in the number of repeating units which are homologous to fibronectin type III. These isoforms are formed by alternative splicing of the Tenascin pre-mRNA and subsequent translation of the different Spliceva anten (A. Leprini et al., Perspectives on Developmental Neurobiology 2 (1994) 117-123). A human tenascin cDNA was developed by A. Siri et al. (Nucl. Acids Res. 19 (1991) 525-531) (sequence in Table 1).
  • This cDNA is stored in gene databases under the accession number X56160 and can be obtained under this number, for example under EMBIJGenbank / DDBJ / NBRF-PIR.
  • This cDNA contains a sequence section which codes for 12 repeating units which are homologous to fibrinogen type III.
  • the cDNAs of the other isoforms of human tenascins are truncated in this sequence section and code for less than 12 of these repeating units.
  • the expression of tenascin is limited in space and time and is assigned importance during the development of an organism and in the case of pathological changes (Crossin, vide supra). Such pathological changes include vitiligo, tumors and inflammation.
  • Antisense oligonucleotides (E. Uhlmann and A. Peyman, Chemical Reviews 90, 543 (1990); S. Agrawal, TIBTECH 1996, 376) offer a possibility for regulating gene expression.
  • WO 94/21664 (L. Denner et al.) Describes antisense oligonucleotides against tenascin, which are used to inhibit the proliferation of smooth cell muscles.
  • the oligonucleotides described there have a length of at least 18 nucleotides.
  • An object of the present invention was to provide new oligonucleotides which have advantageous properties and which can be used for the complete and / or partial inhibition of the gene expression of tenascin.
  • oligonucleotides that are up to 18 nucleotides in length can effectively influence the expression of tenascin.
  • the present invention relates to oligonucleotides with 7-17 nucleotide units, which are optionally modified.
  • the oligonucleotide has a length of 17, 16, 15, 14, 13, 12, 11, 10, 9, 8 or 7 nucleotides.
  • the oligonucleotide corresponds to sections of tenascin-coding sequences (ie the oligonucleotide has a sequence which is complementary to the corresponding section of a tenascin-coding sequence) and the oligonucleotide specifically binds to this tenascin-coding sequence (nucleic acid), for example to the tenascin gene and / or tenascin mRNA and / or tenascin cDNA, the tenascin coding sequence preferably being of human origin (eg human tenascin gene, human tenascin mRNA, human tenascin cDNA).
  • the section of the Tenascin coding sequence to which the oligonucleotide corresponds or to which the oligonucleotide is complementary has preferably a length of 17, 16, 15, 14, 13, 12, 11, 10, 9, 8 or 7 nucleotide units (this applies in particular to the determination of the length of a modified and / or chimeric oligonucleotide or of oligonucleotide analogs).
  • a particular embodiment of the invention relates to an oligonucleotide which binds to a nucleic acid which codes for one of the isoforms human Tenascins or parts thereof and inhibits their expression, the oligonucleotide having a length of 7 to 15 nucleotides and possibly being modified and which physiologically acceptable salts of the oligonucleotide.
  • a particular embodiment of the invention relates to an oligonucleotide which is directed against one or more specific regions of a tenascin-coding sequence, for example the start of transformation, the 5'-untranslated region, the coding region and / or the 3'-non-coding region .
  • the oligonucleotide can also be directed against one or more regions of a tenascin-coding sequence, which e.g. encodes for certain domains of tenascin, for example against the cysteine-rich domain, against a domain homologous to EGF, against a domain homologous to fibronectin type III and / or against a domain homologous to fibrinogen.
  • One embodiment of the invention relates to an oligonucleotide which binds to a nucleic acid which codes for one of the isoforms human Tenascins or parts thereof and inhibits their expression, the oligonucleotide being able to bind to a region of the nucleic acid which a) a part of the 5 ' non-coding region and / or the translation start or b) the translation start and / or a part of the coding region or C) comprises part of the coding region and / or part of the 3 ' non-coding region.
  • the invention relates in particular to an oligonucleotide that a sequence section of the human cDNA according to SEQ ID NO. 1 (Table 1) corresponds.
  • the invention further relates to an oligonucleotide which corresponds to a sequence section of the cDNA which is stored in gene databases under the accession number X56160.
  • an oligonucleotide can have, for example, one of the following sequences or parts thereof:
  • SEQ ID NO. . 9 3'- AAAGGAACGGGAGCG -5 '
  • SEQ ID NO. 19 3'- CCCGGTACTGA -5 'and SEQ ID NO. 20: 3'- CCACAGAAAGAAC -5 '.
  • sequences SEQ ID NO. 2 to SEQ ID NO. 20 correspond to sections of the tenascin-encoding cDNA as shown in Table 1.
  • An oligonucleotide that is one of the sequences SEQ ID NO. 2 to SEQ ID NO. 20 is complementary to a corresponding portion of a tenascin-encoding nucleic acid, e.g. a human Tenascin cDNA and can bind to this nucleic acid.
  • Sequences SEQ ID NO.3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 7 and SEQ ID NO. 18 are examples of oligonucleotides that have a sequence that is directed against the translation start of the tenascin coding sequences.
  • the invention also relates to derivatives of an oligonucleotide, for example its salts, in particular its physiologically tolerable salts.
  • Physiologically acceptable salts are understood as meaning compounds which are readily soluble, soluble and slightly soluble in water, for example as defined in the "German Pharmacopoeia” (9th edition 1986, official edition, German Pharmacist Verlag Stuttgart), page 19.
  • a special embodiment of the invention relates to the sodium salt of the oligonucleotide according to the invention.
  • Derivatives are also modified oligonucleotides.
  • An oligonucleotide can, for example, be composed entirely or partially of the natural nucleotides adenosine phosphate, guanosine phosphate, inosine phosphate, cytidine phosphate, uridine phosphate and thymidine phosphate.
  • One embodiment of the invention relates to an oligonucleotide which is composed of the natural nucleosides adenosine, guanosine, inosine, cytidine, uridine and thymidine and in which the nucleosides are linked to one another via phosphoric diester internucleoside bridges (“phosphoric diester bridges”).
  • an oligonucleotide can optionally contain one or more modifications, for example chemical modifications.
  • An oligonucleotide can be several of the same and / or have various modifications. Modifications can be located at certain nucleoside positions (nucleobase and / or ⁇ -D-2'-deoxyribose unit) and / or certain internucleoside bridges.
  • the chemical modification of an oligonucleotide can mean, for example, a) the complete or partial replacement of the phosphoric diester bridges (internucleoside bridge) by modified phosphorus bridges, phosphorothioate, phosphorus dithioate, NR 1 R 1 ' phosphoramidate, boranophosphate, phosphate (C ⁇ -C 2 ⁇ ) -O-alkyl esters, phosphate - [(C 6 -C ⁇ 2 ) aryl- (C ⁇ -C 2 ⁇ ) -O-alkyl] esters, (Ci- C 8 ) alkyl phosphonate and / or (C 6 -C ⁇ 2 ) arylphosphonate bridges are examples of modified phosphobridges, wherein
  • R 1 and R 1 ' independently of one another for hydrogen, (-C 8 -C 8 ) alkyl, (C 6 -C 20 ) aryl,
  • Methoxyethyl particularly preferably for hydrogen, (-CC 4 ) alkyl and / or
  • R 1 and R 1 together with the nitrogen atom carrying them form a 5-6-membered heterocyclic ring, which is also a further heteroatom from the series
  • phosphoric diester bridges May contain O, S, N; and / or b) the complete or partial replacement of the 3'- and / or 5'-phosphoric diester internucleoside bridges (“phosphoric diester bridges”) by “dephospho” bridges (described, for example, in Uhlmann, E. and Peyman, A. in “Methods in Molecular Biology “, vol. 20,” Protocols for Oligonucleotides and Analogs ", S.
  • PNA polyamide nucleic acid
  • PHONA phosphomono acid ester nucleic acid
  • nucleoside bases are examples of modified sugar units; and / or e) the modification or the complete or partial replacement of the natural nucleoside bases by modified (nucleoside) bases (“nucleobases”), where 5- (hydroxymethyl) uracil, 5-aminouracil, pseudouracil, dihydrouracil, 5- (C ⁇ - C 6) -alkyl-uracil, 5- (C 2 -C 6) -alkenyl-uracil, 5- (C 2 - C 6) alkynyl-uracil, 5- (C ⁇ -C6) alkyl-cytosine, 5 - (C 2 -C 6 ) alkenyl cytosine, 5- (C 2 -C 6 ) alkynyl cytosine, 5-fluorouracil, 5-fluorocytosine, 5-chlorouracil, 5-chlorocytosine, 5-bromouracil, 5-bromocytosine , 7-deaza-7-substit
  • oligonucleotide eg antisense oligonucleotide, triple helix-forming oligonucleotide
  • affinity for the tenascin-coding target sequence, pharmacokinetics) of the oligonucleotide favorably influence and / or when hybridizing the oligonucleotide to the target sequence can attack it with binding and / or crosslinking or can mean, where poly-lysine, intercalators such as pyrene, acridine, phenazine, phenanthridine, fluorescent compounds such as fluorescein, cross-linkers such as psoralen, azidoproflavin, lipophilic molecules such as (-C 2 -C 20 ) alkyl, lipids such as 1 , 2-Di-hexadecyl-rac-glycerol, steroids such as cholesterol, testosterone, vitamins such as vitamin E, poly- or oligo-ethylene glycol, (C ⁇ 2 -C ⁇ 8
  • the oligonucleotide has one or more chemical modifications which are selected independently of one another from a) the complete or partial replacement of the phosphoric diester bridges by phosphorothioate and / or (C 1 -C 8 ) alkylphosphonate bridges, b) the complete one or partial replacement of the sugar phosphate backbone by PNA units and / or PHONA units, c) the complete or partial replacement of the ⁇ -D-2'-deoxyribose units by 2'-F-2'-deoxyribose, 2'-O- (-CC 6 ) alkyl ribose and / or 2 '- [O- (-C 6 ) alkyl-O- (-C 6 ) alkyl] -ribose, d) the complete or partial replacement of the natural nucleoside Bases by 5- (C 2 -C 6 ) alkynyl uracii and / or 5- (C 2 -C 6 )
  • the oligonucleotide has one or more chemical modifications which can be selected independently of one another from the series comprising a) the Complete or partial replacement of the phosphoric diester bridges (phosphodiester bridges) by phosphorothioate bridges, b) Complete or partial replacement of the ß-D-2'-deoxyhbose units by 2'-F-2'-deoxyhbose, 2'-O- (C ⁇ - C 6 ) alkyl-ribose and / or 2 '- [O- (-C-C 6 ) alkyl-O- (-C-C ⁇ ) alkyl] -ribos ⁇ , c) the conjugation with lipophilic molecules, for example (C ⁇ 2 -C 2 o) -alkyl, with lipids, for example 1, 2-di-hexadecyl-rac-glycerol, with (-C 2 -C 8 ) -alkyl phosphate die
  • an oligonucleotide which can have one or more modifications and which has one of the sequences SEQ ID NO. 2 - SEQ ID NO. 20 or one of the sequences SEQ ID NO. 2 to SEQ ID NO. 20 corresponds to or corresponds to the corresponding sequence sections of a tenascin coding sequence and can bind to this section of the tenascin coding sequence.
  • oligonucleotide is provided, in the sequence of which each nucleotide (base and / or sugar and / or internucleoside bridge) is modified.
  • the oligonucleotide is entirely composed of phosphorothioates constructed (consistently modified phosphothioate, all internucleoside bridges modified).
  • an oligonucleotide is provided which has one of the sequences SEQ ID NO. 2 - SEQ ID NO.
  • an oligonucleotide is provided in that only a part of the phosphodiester bridges is replaced by phosphothioate bridges.
  • the invention includes oligonucleotides that are only minimally (or partially) modified. The principle of minimally modified oligonucleotides is described in A. Peyman, E. Uhlmann, Biol. Chem. Hoppe-Seyler, 377 (1996) 67-70.
  • oligonucleotides preferably 1-3 terminal nucleotide units (or preferably the corresponding internucleoside bridges) at the 5 'and / or at the 3' end and, if appropriate, additionally selected internal pyrimidine positions or preferably the corresponding ones Internucleoside bridges which are located at the 3 ' and / or 5 ' end of the corresponding pyrimidine nucleoside are modified or replaced, preferably internucleoside bridges being replaced by phosphorothioate bridges.
  • minimally modified oligonucleotides have particularly advantageous properties, for example they show particular nuclease stability with minimal modification.
  • a particular embodiment of the invention relates to an oligonucleotide in which selected internucleoside bridges are replaced by modified internucleoside bridges, preferably by phosphorothioate bridges.
  • the invention relates to an oligonucleotide in which either a) only certain phosphodiester internucleoside bridges or b) all phosphodiester internucleoside bridges are modified.
  • the invention further relates to a terminal in the 1-5
  • Internucleoside bridges at the 5 ' and / or at the 3 ' end of the oligonucleotide are modified.
  • the invention also relates to an oligonucleotide in which the 3 ' and / or 5 ' ends of non-terminal nucleosides, the one
  • Internucleoside bridges are modified.
  • oligonucleotide that is one of the sequences selected from the series of sequences SEQ ID NO. 21 to SEQ ID NO. 39, wherein
  • sequences SEQ ID NO. 21 to SEQ ID NO. 39 correspond to the sequences SEQ ID NO. 2 - SEQ ID NO. 20, i.e. they can bind to the same regions of a tenascin-coding sequence, but in contrast to SEQ ID NO. 2-20 some of the phosphodiester bridges are replaced by modified phosphodiester bridges or dephosphobridges, preferably by phosphothioate bridges (identified by an "s" in the sequence).
  • a chimeric oligonucleotide is constructed from at least two different sequence segments, for example a DNA segment and a modified segment, e.g. a PNA section and / or a PHONA section. These different sections give the entire oligonucleotide special properties.
  • a special form of chimeric oligonucleotides is described, for example, in Matteucci and Wagner, Nature 384 SUPP (1996) 20-22.
  • a chimeric oligonucleotide can e.g.
  • core sequence which consists of about seven nucleotides and which can activate RNase H and
  • flanking sequences which increase the affinity, specificity and / or nuclease stability of the oligonucleotide.
  • the "core sequence” can have modified internucleoside bridges at certain positions, for example the "core sequence” can contain phosphorothioate and / or phosphodiester bridges.
  • Suitable flanking sequences are, for example, sequences in which the sugar phosphate backbone (replacement of one or more sugar phosphate units) ) and / or ß-D-2 ' - Deoxyribose units are replaced.
  • Suitable flanking sequences are, for example, PNAs and / or 2'-O-alkyl derivatives such as 2'-O-methyl and / or 2'-O-propyl and / or 2'-methoxyethoxy derivatives.
  • a particular embodiment of the invention relates to a chimeric oligonucleotide which has one of the sequences SEQ ID NO. 40 - SEQ ID NO. 58, where x independently of one another stands for an unmodified or a modified phosphodiester internucleoside bridge or a dephospho bridge, preferably for phosphorothioate and / or phosphorus diester, and y independently of one another for the replacement of a sugar phosphate unit or a ⁇ -D-2 ' deoxyribose unit, preferably represents 2'-O-methyl-, 2'-O-propyl- and / or 2'-methoxyethoxyribose or a PNA building block, where
  • SEQ ID NO. 56 3'- GyGyTxGxGxTxAxCxCyCyC -5 '
  • SEQ ID NO. 57 3'- CyCxCxGxGxTxAxCyTyGyA -5 '
  • SEQ ID NO. 58 3'- CyCyAxCxAxGxAxAxAxGyAyAyC -5 '.
  • sequences SEQ ID NO. 40 - SEQ ID NO. 58 correspond to the sequences SEQ ID NO. 2 to SEQ ID NO. 20, i.e. they bind to the corresponding sequence sections of a tenascin-coding sequence, although the modifications mentioned are included.
  • the invention relates to methods for producing the oligonucleotides.
  • the oligonucleotides described can be made using various known chemical methods, e.g. using the standard phosphoramidite chemistry using iodine or TED (tetraethythiuram disulfide) as an oxidizing agent. This method is e.g. in Eckstein, F. (1991) "Oligonucleotides and Analogues, A Practical Approach", IRL Press, Oxford.
  • the oligonucleotides can also be prepared by methods that optionally contain one or more enzymatic steps.
  • the invention relates to the use of the oligonucleotides.
  • the oligonucleotides can be used for hybridization or binding to tenascin-encoding (single-stranded and / or double-stranded) nucleic acids, for example DNA (e.g. genes, cDNA) and / or RNA (e.g. pre-mRNA, mRNA).
  • DNA e.g. genes, cDNA
  • RNA e.g. pre-mRNA, mRNA
  • this relates to the use of the oligonucleotides for hybridization with or binding to nucleic acids which have the sequence SEQ ID NO.
  • nucleic acids which have parts of this sequence for example sequences which code for tenascin isoforms
  • nucleic acids whose sequence deviates slightly from these sequences which have, for example, one or more point mutations.
  • the invention further relates to the use of the oligonucleotides for modulation and for the total or partial inhibition of the expression of tenascin or different tenascin isoforms or of mutants thereof, for example for the total or partial inhibition of transcription and / or translation.
  • the invention relates, for example, to the use of the oligonucleotides as antisense oligonucleotides.
  • the oligonucleotides can be used as aids in molecular biology.
  • the invention further relates to the use of the oligonucleotides as pharmaceuticals and / or diagnostic agents or the use of the oligonucleotides for the production of pharmaceuticals and / or diagnostic agents.
  • the oligonucleotides can be used in medicaments which are used for the prevention and / or treatment of diseases which are associated with the expression or overexpression of tenascin. Since the expression of tenascin is usually, i.e. e.g. in healthy people is limited in space and time, a deviation from this normal spatial and temporal expression can be regarded as overexpression.
  • the oligonucleotides can be used in diagnostic methods. Such diagnostic methods can e.g. used for the diagnosis or early detection of diseases that will be associated with an abnormal expression (e.g. overexpression) of Tenascin.
  • the invention also relates to a test kit which contains one or more oligonucleotides according to the invention and optionally other components.
  • a test kit which contains one or more oligonucleotides according to the invention and optionally other components.
  • Such a test kit can be used, for example, in diagnostics and for prevention, for example of skin cancer.
  • the invention further relates to the use of the oligonucleotides or of medicaments which contain these oligonucleotides for the treatment of diseases in which tenascin or an overexpression of tenacsin is causal or involved.
  • the invention relates in particular to the use of the oligonucleotides or of medicaments which contain these oligonucleotides for the treatment and / or prevention of diseases in which there is a malfunction or disruption of the immigration or the presence or storage of melanocytes in epithelial cell layers, for example in the epithelial cell layer of the epidermis, the choroid of the eye or the substantia nigra, underlies or is involved and of Addison's disease, diabetes mellitus, pernicious anemia and / or thyroid dysfunction.
  • the invention relates in particular to the use of the oligonucleotides or of medicaments which contain these oligonucleotides for the treatment and / or prevention of vitiligo and other depigmentation diseases or depigmentation disorders (for example the skin, hair, eyes), for example albinism and / or for the treatment of psoriasis and / or for the treatment of cancer, e.g. for inhibitors of tumor growth and tumor metastasis, for example in the case of melanoma and / or for the treatment of inflammation, in particular as an anti-inflammatory agent and / or for the treatment and / or prophylaxis of cardiovascular diseases, for example restenosis.
  • vitiligo and other depigmentation diseases or depigmentation disorders for example the skin, hair, eyes
  • albinism and / or for the treatment of psoriasis and / or for the treatment of cancer e.g. for inhibitors of tumor growth and tumor metastasis, for example in the case
  • the invention relates to the use of the oligonucleotides for the treatment of vitiligo or for the production of medicaments which can be used for the treatment of vitiligo.
  • the invention also relates generally (i.e. also oligonucleotides with a length of greater than or equal to 18 nucleotides) to the use of oligonucleotides for the treatment of vitiligo or the production of medicaments which can be used for the treatment of vitiligo.
  • the invention further relates to the use for the treatment of vitiligo in combination with known therapeutic methods, for example in combination a) with photochemotherapy (PUVA), e.g. using methoxypsoralen, phenylalanine and / or khellin and / or b) with the transplantation of cultured melanocytes ("epidermal grafting") and / or c) with a steroid treatment and / or d) with a treatment with placenta extracts and / or e) treatment with pseudocataiase.
  • PUVA photochemotherapy
  • the invention further relates to methods for the production of pharmaceuticals (pharmaceutical preparations).
  • pharmaceuticals pharmaceutical preparations
  • One or more different oligonucleotides or their physiologically compatible ones are used for the production of pharmaceuticals Salts mixed, where appropriate further pharmaceutical carriers and / or additives can be added.
  • the invention further relates to pharmaceutical preparations (medicaments) which contain one or more different oligonucleotides and / or their physiologically tolerable salts and, if appropriate, pharmaceutical carriers and / or additives.
  • the oligonucleotide (s) and / or their physiologically tolerable salts can be administered to animals, preferably to mammals, in particular to humans, as a medicament on their own, in mixtures with one another or in the form of pharmaceutical preparations.
  • the drugs can allow topical, percutaneous, parenteral and / or enteral use.
  • the preferred form of application depends on the particular circumstances.
  • a topical application e.g. preferred in the form of ointments, lotions or tinctures, emulsions, suspensions.
  • the frequency of application also depends on the individual circumstances.
  • a topical composition can be applied to the depigmented skin area once or twice a day.
  • Medicaments or pharmaceutical preparations can contain as active ingredient an effective dose of at least one oligonucleotide and / or a mixture of several oligonucleotides and, if necessary, additional, pharmaceutically perfect carriers and / or additives.
  • a pharmaceutical preparation can contain about 0.1% (percent by weight) or less to about 90% (percent by weight) or more of the therapeutically active oligonucleotide or the pharmaceutically active oligonucleotides.
  • the pharmaceutically effective dose of the respective oligonucleotide or an oligonucleotide which is part of a mixture of different oligonucleotides can vary within wide limits and must be adapted to the individual circumstances in each individual case.
  • the preparation of the pharmaceutical preparations can be carried out in a manner known per se, e.g. B. described in Remingtons Pharmaceutical Sciences (1985), Mack Publ. Co., Easton, PA. be carried out, where appropriate pharmaceutically inert inorganic and / or organic carriers can be used.
  • suitable pharmaceutically inert inorganic and / or organic carriers can be used.
  • pills tablets, dragees and / or hard gelatin capsules e.g. Lactose, corn starch and / or derivatives thereof, talc, stearic acid and / or their salts can be used.
  • As carriers for soft gelatin capsules and / or suppositories e.g. Fats, waxes, semi-solid and / or liquid polyols, natural and / or hardened oils can be used.
  • ⁇ As carriers for the preparation of solutions and / or syrups z. B. water, sucrose, invert sugar, glucose and / or polyols can be used. As carriers for the production of injection solutions, e.g. Water, alcohols, glycerin, polyols and / or vegetable oils can be used. As a carrier for microcapsules, implants and / or rods, for example, copolymers, e.g. from glycolic acid and lactic acid can be used. In addition, liposome formulations which are known to the person skilled in the art (N.
  • the dermal application can also be carried out, for example, with the aid of ionophoretic methods and / or with the aid of electroporation, and lipofectins and / or other (nucleic acid or DNA) carrier systems, for example those used in gene therapy, can also be used Systems are particularly suitable with the aid of which oligonucleotides can be introduced into eukaryotic cells or the nuclei of eukaryotic cells with great efficiency.
  • a pharmaceutical preparation can also contain additives, such as fillers, extenders, explosives, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colors, flavors or Flavoring agents, thickeners, diluents, buffer substances, furthermore solvents and / or solubilizers and / or agents for achieving a depot effect, and salts for changing the osmotic pressure, coating agents and / or antioxidants. They can also contain two or more different oligonucleotides and / or their physiologically tolerable salts and, in addition to at least one oligonucleotide, one or more other therapeutically active substances.
  • additives such as fillers, extenders, explosives, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colors, flavors or Flavoring agents, thickeners, diluents, buffer substances, furthermore solvents
  • the oligonucleotide was synthesized on an automatic DNA synthesizer (Applied Biosystems Model 380B or 394) using standard phosphoramidite chemistry and oxidation with iodine (F. Eckstein, Ed “Oligonucleotides and Analogues, A Practical Approach", IRL Press, Oxford, 1991 ).
  • TETD tetraethylthiuram disulfide
  • the oligonucleotide was first purified by butanol precipitation (Sawadogo, Van Dyke, Nucl. Acids Res. 19 (1991) 674). The sodium salt was then obtained by precipitation from a 0.5 M NaCl solution with 2.5 parts by volume of ethanol.
  • the oligonucleotide was determined using the
  • oligonucleotide showed that it was in each case in a purity of greater than 90%.
  • the methods for analyzing oligonucleotides are e.g. in Schweiber and Engler "Analysis of oligonucleotides” (in “Antisense - from technology to therapy", a laboratory manual and textbook, Schlingensiepen et al. eds., Biol. Science, Vol. 6 (1997) p. 78-103) .
  • ODN1 (Sequence SEQ ID NO. 24): 3'- GsGsAGGTsGGTsACsCsCsC -5 '
  • ODN 1 from Example 1 can e.g. closely mixed with 1g Dermatop® (Hoechst Aktiengesellschaft, Frankfurt am Main, Germany) base cream and the mixture stored at temperatures ⁇ 10 ° C.
  • Dermatop® Hoechst Aktiengesellschaft, Frankfurt am Main, Germany
  • the cream from Example 2 can then be applied, for example, twice a day (in the morning and in the afternoon or in the evening) to a depigmented skin area of a Vitiligo patient.
  • Table 1 Sequence SEQ ID NO. 1 :
  • TTCACAGGCC TGGACTGTGG CCAGCACTCC TGCCCCAGTG ACTGCAACAA CTTAGGACAA 1860
  • CAACAAGCCA CAACCAAAAC CACACTCACA GGTCTGAGGC CGGGAACTGA ATATGGGATT 2940
  • CAAGGGCATC AAACCAAGCC CTTGAGGGCT GAGATTGTTA CAGAAGCCGA ACCGGAAGTT 4980
  • ATCACAGCCC AGGGGCAGTA CGAGCTCCGG GTGGACCTGC GGGACCATGG GGAGACAGCC 6360

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Abstract

The invention relates to specific, optionally modified oligonucleotides with a length of up to 18 nucleotides. Said oligonucleotides correspond to segments of tenascin-coding sequences or can bind to these sequences. The invention also relates to the production and use of the oligonucleotides, for example for the specific inhibition of the expression of tenascin and for producing medicaments used to treat vitiligo.

Description

Antisense Oligonukleotide gegen Tenascin zur Behandlung von VitiligoAntisense oligonucleotides against tenascin for the treatment of vitiligo
Die Erfindung betrifft spezifische, gegebenenfalls modifizierte Oligonukleotide mit einer Länge von bis zu 18 Nukleotiden, vorzugsweise einer Länge von 7-15 Nukleotiden, die Abschnitten Tenascin-kodierender Sequenzen entsprechenden und die an diese Sequenzen binden können, deren Herstellung sowie die Verwendung derselben, beispielsweise zur spezifischen Inhibition der Expression von Tenascin und zur Herstellung von Arzneimitteln, die zur Behandlung von Vitiligo verwendet werden können.The invention relates to specific, optionally modified oligonucleotides with a length of up to 18 nucleotides, preferably with a length of 7-15 nucleotides, which correspond to sections of tenascin-coding sequences and which can bind to these sequences, their preparation and their use, for example for specific inhibition of the expression of tenascin and for the manufacture of medicinal products which can be used for the treatment of vitiligo.
Unter Vitiligo wird ein erworbenes Fehlen von Melanozyten verstanden, wodurch hypopigmentierte Hautbereiche entstehen, die in der Regel scharf begrenzt und häufig symmetrisch angeordnet sind, einen oder zwei Flecke bilden oder fast die ganze Haut erfassen. Das Haar in hypopigmentierten Bezirken ist normalerweise weiß und erscheint auch im Wood-Licht weiß. Die betroffenen Hautstellen sind anfällig gegen Sonnenbrand. Die Ursache der Erkrankung ist unbekannt. Obwohl die Vitiligo als eine im Laufe des Lebens erworbene Krankheit gilt, findet sich gelegentlich eine familiäre Häufung (autosomal-dominant, mit inkompletter Penetranz und variabler Ausprägung). Sie kann auch einem ungewöhnlichen physischen Trauma, insbesondere einer Schädelverletzung, folgen. Die Assoziation von Vitiligo mit einem Morbus Addison, Diabetes mellitus, perniziöser Anämie oder Schilddrüsendysfunktion wie auch das gehäufte Vorkommen von Antikörpern gegen Thyreoglobulin, Zellen der Nebenniere und Belegzellen des Magens im Serum haben dazu geführt, eine immunologische oder neurochemische Ursache zu vermuten. Antikörper gegen Melanin wurden bei einigen Patienten gefunden.Vitiligo is understood to mean an acquired lack of melanocytes, which creates hypopigmented areas of the skin, which are usually sharply defined and often symmetrically arranged, form one or two spots or cover almost the entire skin. The hair in hypopigmented areas is usually white and also appears white in wood light. The affected skin areas are susceptible to sunburn. The cause of the disease is unknown. Although vitiligo is considered to be a disease acquired over the course of a lifetime, there is occasionally a family cluster (autosomal dominant, with incomplete penetrance and variable expression). It can also follow unusual physical trauma, especially a skull injury. The association of vitiligo with Addison's disease, diabetes mellitus, pernicious anemia or thyroid dysfunction as well as the increased presence of antibodies against thyroglobulin, cells of the adrenal gland and gastric parietal cells in the serum have led to the assumption of an immunological or neurochemical cause. Antibodies to melanin have been found in some patients.
Alle verfügbaren Therapiemethoden führen nur bei einem Teil der Patienten zu befriedigenden Therapieerfolgen (F. Wach et al., H+G 71 (1996) 206). Zu den vorhandenen Therapien ( S. P. W. Kumarasinghe, Ceylon Medical Journal 40 (1995) 94) gehören Photochemotherapien (PUVA), beispielsweise mit Methoxypsoralen, Phenylalanin, oder Khellin, die Transplantation von kultivierten Melanocyten, "epidermal grafting", und die Behandlung mit Steroiden oder Plazenta-Extrakten. Kürzlich wurde über die Behandlung mit Pseudokatalase berichtet (Schallreuter et al., Dermatology 190 (1995) 223). Kleine Herde können auch mit kosmetischer Schminke oder Gerblösungen abgedeckt werden.All available therapy methods only lead to satisfactory therapeutic success in some of the patients (F. Wach et al., H + G 71 (1996) 206). The available therapies (SPW Kumarasinghe, Ceylon Medical Journal 40 (1995) 94) include photochemotherapy (PUVA), for example with methoxypsoralen, phenylalanine, or khellin, the transplantation of cultivated Melanocytes, epidermal grafting, and treatment with steroids or placenta extracts. Treatment with pseudocatalase has recently been reported (Schallreuter et al., Dermatology 190 (1995) 223). Small stoves can also be covered with cosmetic make-up or tanning solutions.
Poole et al. (British Journal of Dermatol. 137 (1997) 171 ) konnten zeigen, daß die Vitiligo befallene Haut im Vergleich zu normaler Haut einen hohen Gehalt an Tenascin aufweist. Der hohe Tenascin-Gehalt kann zum Verlust der Pigmentierung beitragen und die Repigmentierung verhindern. Tenascin (Crossin, J. Cell. Biol. 61 (1996) 592) ist ein extrazelluläres Matrix Glykoprotein, das aus sechs identischen Untereinheiten besteht, welche am Amino-Terminus über Disulfid-Brücken verknüpft sind. Die Tenascin Untereinheiten weisen eine charakteristische Domänenstruktur auf: Auf eine Cystein-reiche Sequenz am aminotermialen Ende folgen drei, jeweils aus sich wiederholenden Einheiten aufgebaute Sequenzabschnitte aus zum EGF homologen Einheiten, aus zum Fibronektin (Typ III) homologen Einheiten und aus zum Fibrinogen homologen Einheiten.Poole et al. (British Journal of Dermatol. 137 (1997) 171) were able to show that the vitiligo-affected skin has a high content of tenascin compared to normal skin. The high tenascin content can contribute to the loss of pigmentation and prevent repigmentation. Tenascin (Crossin, J. Cell. Biol. 61 (1996) 592) is an extracellular matrix glycoprotein which consists of six identical subunits which are linked at the amino terminus via disulfide bridges. The Tenascin subunits have a characteristic domain structure: a cysteine-rich sequence at the amino-terminal end is followed by three sequence sections, each made up of repeating units, from units homologous to EGF, from units homologous to fibronectin (type III) and from units homologous to fibrinogen.
Es existieren mehrere Isoformen der Tenascin Untereinheiten (im folgenden als Tenascin Isoformen bezeichnet), die sich in der Anzahl der sich wiederholenden Einheiten, die zum Fibronektin Typ III homolog sind, unterscheiden. Diese Isoformen werden durch alternatives splicing der Tenascin pre-mRNA und anschließende Translation der verschiedenen Spliceva anten gebildet (A. Leprini et al., Perspectives on Developmental Neurobiology 2 (1994) 117-123). Eine cDNA von humanem Tenascin wurde von A. Siri et al. (Nucl. Acids Res. 19 (1991 ) 525- 531 ) beschrieben (Sequenz in Tabelle 1 ). Diese cDNA ist unter der Zugangsnummer X56160 in Gen-Datenbanken gespeichert und kann unter dieser Nummer beispielsweise unter EMBIJGenbank/DDBJ/NBRF-PIR erhalten werden. Diese cDNA enthält einen Sequenzabschnitt, der für 12 sich wiederholende Einheiten, die zum Fibrinogen Typ III homolog sind, kodiert. Die cDNAs der anderen Isoformen humanen Tenascins sind in diesem Sequenzabschnitt verkürzt und kodieren für weniger als 12 dieser sich wiederholenden Einheiten. Die Expression von Tenascin ist räumlich und zeitlich begrenzt und ihm wird eine Bedeutung während der Entwicklung eines Organismus sowie bei pathologischen Veränderungen zugeschrieben (Crossin, vide supra). Solche pathologischen Veränderungen sind beispielsweise Vitiligo, Tumore und Entzündungen.There are several isoforms of the tenascin subunits (hereinafter referred to as tenascin isoforms) which differ in the number of repeating units which are homologous to fibronectin type III. These isoforms are formed by alternative splicing of the Tenascin pre-mRNA and subsequent translation of the different Spliceva anten (A. Leprini et al., Perspectives on Developmental Neurobiology 2 (1994) 117-123). A human tenascin cDNA was developed by A. Siri et al. (Nucl. Acids Res. 19 (1991) 525-531) (sequence in Table 1). This cDNA is stored in gene databases under the accession number X56160 and can be obtained under this number, for example under EMBIJGenbank / DDBJ / NBRF-PIR. This cDNA contains a sequence section which codes for 12 repeating units which are homologous to fibrinogen type III. The cDNAs of the other isoforms of human tenascins are truncated in this sequence section and code for less than 12 of these repeating units. The expression of tenascin is limited in space and time and is assigned importance during the development of an organism and in the case of pathological changes (Crossin, vide supra). Such pathological changes include vitiligo, tumors and inflammation.
Eine Möglichkeit zur Regulation der Genexpression bieten Antisense- Oligonukleotide (E. Uhlmann and A. Peyman, Chemical Reviews 90, 543 (1990); S. Agrawal, TIBTECH 1996, 376). In WO 94/21664 (L. Denner et al.) werden Antisense Oligonukleotide gegen Tenascin, die zur Inhibition der Proliferation der glatten Zellmuskulatur eingesetzt werden, beschrieben. Die dort beschriebenen Oligonukleotide haben eine Länge von mindestens 18 Nukleotiden.Antisense oligonucleotides (E. Uhlmann and A. Peyman, Chemical Reviews 90, 543 (1990); S. Agrawal, TIBTECH 1996, 376) offer a possibility for regulating gene expression. WO 94/21664 (L. Denner et al.) Describes antisense oligonucleotides against tenascin, which are used to inhibit the proliferation of smooth cell muscles. The oligonucleotides described there have a length of at least 18 nucleotides.
Eine Aufgabe der vorliegenden Erfindung war es, neue Oligonukleotide, die vorteilhafte Eigenschaften aufweisen und die zur vollständigen und/oder teilweisen Inhibition der Genexpression von Tenascin verwendet werden können, bereitzustellen.An object of the present invention was to provide new oligonucleotides which have advantageous properties and which can be used for the complete and / or partial inhibition of the gene expression of tenascin.
Überraschenderweise wurde gefunden, daß Oligonukleotide, die ein Länge von bis zu 18 Nukleotiden aufweisen, die Expression von Tenascin effektiv beeinflussen können. Gegenstand der vorliegenden Erfindung sind Oligonukleotide mit 7 - 17 Nukleotid-Einheiten, die gegebenenfalls modifiziert sind. In besonderen Ausführungsformen der Erfindung weist das Oligonukleotid eine Länge von 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8 oder 7 Nukleotiden auf. Das Oligonukleotid entspricht Abschnitten Tenascin-kodierender Sequenzen (d.h. das Oligonukleotid hat eine Sequenz, die zu dem entsprechenden Abschnitt einer Tenascin-kodierenden Sequenz komplementär ist) und das Oligonukleotid bindet spezifisch an diese Tenascin-kodierende Sequenz (Nukleinsäure), beispielsweise an das Tenascin-Gen und/oder Tenascin mRNA und/oder Tenascin cDNA, wobei die Tenascin-kodierende Sequenz vorzugsweise humanen Ursprungs ist (z.B. humanes Tenascin Gen, humane Tenascin mRNA, humane Tenascin cDNA). Der Abschnitt der Tenascin- kodierenden Sequenz, dem das Oligonukleotid entspricht bzw. zu dem das Oligonukleotid komplementär ist, hat vorzugsweise eine Länge von 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8 oder 7 Nukleotid-Einheiten (dies gilt insbesondere für die Bestimmung der Länge eines modifizierten und/oder Chimären Oligonukleotids bzw. von Oligonukleotid-Analoga).Surprisingly, it was found that oligonucleotides that are up to 18 nucleotides in length can effectively influence the expression of tenascin. The present invention relates to oligonucleotides with 7-17 nucleotide units, which are optionally modified. In particular embodiments of the invention, the oligonucleotide has a length of 17, 16, 15, 14, 13, 12, 11, 10, 9, 8 or 7 nucleotides. The oligonucleotide corresponds to sections of tenascin-coding sequences (ie the oligonucleotide has a sequence which is complementary to the corresponding section of a tenascin-coding sequence) and the oligonucleotide specifically binds to this tenascin-coding sequence (nucleic acid), for example to the tenascin gene and / or tenascin mRNA and / or tenascin cDNA, the tenascin coding sequence preferably being of human origin (eg human tenascin gene, human tenascin mRNA, human tenascin cDNA). The section of the Tenascin coding sequence to which the oligonucleotide corresponds or to which the oligonucleotide is complementary has preferably a length of 17, 16, 15, 14, 13, 12, 11, 10, 9, 8 or 7 nucleotide units (this applies in particular to the determination of the length of a modified and / or chimeric oligonucleotide or of oligonucleotide analogs).
Eine besondere Ausführungsform der Erfindung betrifft ein Oligonukleotid, das an eine Nukleinsäure, die für eine der Isoformen humanen Tenascins oder Teile derselben kodiert, bindet und deren Expression inhibiert, wobei das Oligonukleotid eine Länge von 7 bis 15 Nukleotiden aufweist und gegebenenfalls modifiziert sein kann sowie die physiologisch verträglichen Salze des Oligonukleotids.A particular embodiment of the invention relates to an oligonucleotide which binds to a nucleic acid which codes for one of the isoforms human Tenascins or parts thereof and inhibits their expression, the oligonucleotide having a length of 7 to 15 nucleotides and possibly being modified and which physiologically acceptable salts of the oligonucleotide.
Eine besondere Ausführungsform der Erfindung betrifft ein Oligonukleotid, das gegen einen oder mehrere bestimmte Bereiche einer Tenascin-kodierenden Sequenz gerichtet ist, beispielsweise den Transiationsstart, den 5'- nicht translatierten Bereich, den kodierenden Bereich und/oder den 3'-nicht-kodierenden Bereich. In einer besonderen Ausführungsform der Erfindung kann das Oligonukleotid auch gegen einen oder mehrere Bereiche einer Tenascin- kodierenden Sequenz gerichtet sein, die z.B. für bestimmte Domänen des Tenascins kodiert, beispielsweise gegen die Cystein-reiche Domäne, gegen eine zum EGF homologe Domäne, gegen eine zum Fibronektin Typ III homologe Domäne und/oder gegen eine zum Fibrinogen homologe Domäne.A particular embodiment of the invention relates to an oligonucleotide which is directed against one or more specific regions of a tenascin-coding sequence, for example the start of transformation, the 5'-untranslated region, the coding region and / or the 3'-non-coding region . In a special embodiment of the invention, the oligonucleotide can also be directed against one or more regions of a tenascin-coding sequence, which e.g. encodes for certain domains of tenascin, for example against the cysteine-rich domain, against a domain homologous to EGF, against a domain homologous to fibronectin type III and / or against a domain homologous to fibrinogen.
Eine Ausführungsform der Erfindung betrifft ein Oligonukleotid, das an eine Nukleinsäure, die für eine der Isoformen humanen Tenascins oder Teile derselben kodiert, bindet und deren Expression inhibiert, wobei das Oligonukleotid an einen Bereich der Nukleinsäure binden kann, der a) einen Teil des 5'-nichtkodierenden Bereichs und/oder den Translationsstart oder b) den Translationsstart und/oder einen Teil des kodierenden Bereichs oder C) einen Teil des kodierenden Bereichs und/oder einen Teil des 3'-nichtkodierenden Bereichs umfaßt.One embodiment of the invention relates to an oligonucleotide which binds to a nucleic acid which codes for one of the isoforms human Tenascins or parts thereof and inhibits their expression, the oligonucleotide being able to bind to a region of the nucleic acid which a) a part of the 5 ' non-coding region and / or the translation start or b) the translation start and / or a part of the coding region or C) comprises part of the coding region and / or part of the 3 ' non-coding region.
Gegenstand der Erfindung ist insbesondere ein Oligonukleotid, das einem Sequenzabschnitt der humanen cDNA gemäß SEQ ID NO. 1 (Tabelle 1 ) entspricht. Gegenstand der Erfindung ist weiterhin ein Oligonukleotid, das einem Sequenzabschnitt der cDNA, die in Gendatenbanken unter der Zugangsnummer X56160 gespeichert ist, entspricht.The invention relates in particular to an oligonucleotide that a sequence section of the human cDNA according to SEQ ID NO. 1 (Table 1) corresponds. The invention further relates to an oligonucleotide which corresponds to a sequence section of the cDNA which is stored in gene databases under the accession number X56160.
In speziellen Ausführungsformen der Erfindung kann ein Oligonukleotid beispielsweise eine der folgenden Sequenzen oder Teile derselben haben:In specific embodiments of the invention, an oligonucleotide can have, for example, one of the following sequences or parts thereof:
SEQ ID NO. 2: 3'- GGTTTGGGTGGAGGTGG -5'SEQ ID NO. 2: 3'- GGTTTGGGTGGAGGTGG -5 '
SEQ ID NO. 3: 3'- GGAGGTGGTACCCCCGG -5'SEQ ID NO. 3: 3'- GGAGGTGGTACCCCCGG -5 '
SEQ ID NO. 4: 3'- GGTGGTACCCCCGG -5'SEQ ID NO. 4: 3'- GGTGGTACCCCCGG -5 '
SEQ ID NO. 5: 3'- GGAGGTGGTACCCC -5'SEQ ID NO. 5: 3'- GGAGGTGGTACCCC -5 '
SEQ ID NO. 6: 3'- AGAAAGAACGAAAGGAA -5'SEQ ID NO. 6: 3'- AGAAAGAACGAAAGGAA -5 '
SEQ ID NO. 7: 3'- GGAGGTGGTACC -5'SEQ ID NO. 7: 3'- GGAGGTGGTACC -5 '
SEQ ID NO. 8: 3'- GGAGCGATGGCTTCCA -5'SEQ ID NO. 8: 3'- GGAGCGATGGCTTCCA -5 '
SEQ ID NO. . 9: 3'- AAAGGAACGGGAGCG -5'SEQ ID NO. . 9: 3'- AAAGGAACGGGAGCG -5 '
SEQ ID NO. 10 3'- GGTCGGTTTGGGTGG -5'SEQ ID NO. 10 3'- GGTCGGTTTGGGTGG -5 '
SEQ ID NO. 11 3'- CTTACAGGTCCGTTGA -5'SEQ ID NO. 11 3'- CTTACAGGTCCGTTGA -5 '
SEQ ID NO. 12 3'- GGCCGTGTTCGCTGT -5'SEQ ID NO. 12 3'- GGCCGTGTTCGCTGT -5 '
SEQ ID NO. 13 3'- TCACCCCTCTTTCTGG -5'SEQ ID NO. 13 3'- TCACCCCTCTTTCTGG -5 '
SEQ ID NO. 14 3*- GGACACCGACACGG -5'SEQ ID NO. 14 3 * - GGACACCGACACGG -5 '
SEQ ID NO. 15 3'- AACGGGAGCGATGG -5'SEQ ID NO. 15 3'- AACGGGAGCGATGG -5 '
SEQ ID NO. 16 3'- ATCTCGGGGTCGTC -5'SEQ ID NO. 16 3'- ATCTCGGGGTCGTC -5 '
SEQ ID NO. 17 3'- AAAGAACGAAAGGAA -5'SEQ ID NO. 17 3'- AAAGAACGAAAGGAA -5 '
SEQ ID NO. 18 3'- GGTGGTACCCC -5'SEQ ID NO. 18 3'- GGTGGTACCCC -5 '
SEQ ID NO. 19 3'- CCCGGTACTGA -5' und SEQ ID NO. 20: 3'- CCACAGAAAGAAC -5'.SEQ ID NO. 19 3'- CCCGGTACTGA -5 'and SEQ ID NO. 20: 3'- CCACAGAAAGAAC -5 '.
Die Sequenzen SEQ ID NO. 2 bis SEQ ID NO. 20 entsprechen Abschnitten der Tenascin-kodierenden cDNA, wie sie in Tabelle 1 dargestellt ist. Ein Oligonukleotid, das eine der Sequenzen SEQ ID NO. 2 bis SEQ ID NO. 20 hat, ist komplementär zu einem entsprechenden Abschnitt einer Tenascin-kodierenden Nukleinsäure, z.B. einer humanen Tenascin cDNA und kann an diese Nukleinsäure binden. Sequenzen SEQ ID NO.3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 7 und SEQ ID NO. 18 sind Beispiele für Oligonukleotide, die eine Sequenz aufweisen, die gegen den Translationsstart der Tenascin-kodierenden Sequenzen gerichtet ist.The sequences SEQ ID NO. 2 to SEQ ID NO. 20 correspond to sections of the tenascin-encoding cDNA as shown in Table 1. An oligonucleotide that is one of the sequences SEQ ID NO. 2 to SEQ ID NO. 20 is complementary to a corresponding portion of a tenascin-encoding nucleic acid, e.g. a human Tenascin cDNA and can bind to this nucleic acid. Sequences SEQ ID NO.3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 7 and SEQ ID NO. 18 are examples of oligonucleotides that have a sequence that is directed against the translation start of the tenascin coding sequences.
Gegenstand der Erfindung sind auch Derivate eines Oligonukleotids, beispielsweise dessen Salze, insbesondere dessen physiologisch verträglichen Salze. Unter physiologisch verträglichen Salzen werden in Wasser leicht lösliche, lösliche und wenig lösliche Verbindungen, beispielsweise gemäß der Definition im "Deutschen Arzneibuch" (9. Ausgabe 1986, Amtliche Ausgabe, Deutscher Apotheker Verlag Stuttgart), Seite 19, verstanden. Eine spezielle Ausführungsform der Erfindung betrifft das Natriumsalz des erfindungsgemäßen Oligonukleotids. Derivate sind auch modifizierte Oligonukleotide.The invention also relates to derivatives of an oligonucleotide, for example its salts, in particular its physiologically tolerable salts. Physiologically acceptable salts are understood as meaning compounds which are readily soluble, soluble and slightly soluble in water, for example as defined in the "German Pharmacopoeia" (9th edition 1986, official edition, German Pharmacist Verlag Stuttgart), page 19. A special embodiment of the invention relates to the sodium salt of the oligonucleotide according to the invention. Derivatives are also modified oligonucleotides.
Ein Oligonukleotid kann beispielsweise vollständig oder teilweise aus den natürlichen Nukleotiden Adenosinphosphat, Guanosinphosphat, Inosinphosphat, Cytidinphosphat, Uridinphosphat und Thymidinphosphat aufgebaut sein. Eine Ausführungsform der Erfindung betrifft ein Oligonukleotid, das aus den natürlichen Nukleosiden Adenosin, Guanosin, Inosin, Cytidin, Uridin und Thymidin aufgebaut ist und in welchem die Nukleoside über Phosphorsäurediester Internukleosid-Brücken („Phosphorsäurediester-Brücken") miteinander verknüpft sind.An oligonucleotide can, for example, be composed entirely or partially of the natural nucleotides adenosine phosphate, guanosine phosphate, inosine phosphate, cytidine phosphate, uridine phosphate and thymidine phosphate. One embodiment of the invention relates to an oligonucleotide which is composed of the natural nucleosides adenosine, guanosine, inosine, cytidine, uridine and thymidine and in which the nucleosides are linked to one another via phosphoric diester internucleoside bridges (“phosphoric diester bridges”).
In anderen Ausführungsformen der Erfindung kann ein Oligonukleotid gegebenenfalls ein oder mehrere Modifikationen, beispielsweise chemische Modifikationen, enthalten. Ein Oligonukleotid kann mehrere gleiche und/oder verschiedene Modifikationen aufweisen. Modifikationen können an bestimmten Nukleosid Positionen (Nukleobase und/oder ß-D-2'-Deoxyribose Einheit) und/oder bestimmten Internukleosid-Brücken lokalisiert sein.In other embodiments of the invention, an oligonucleotide can optionally contain one or more modifications, for example chemical modifications. An oligonucleotide can be several of the same and / or have various modifications. Modifications can be located at certain nucleoside positions (nucleobase and / or β-D-2'-deoxyribose unit) and / or certain internucleoside bridges.
Beispiele für chemische Modifikationen sind dem Fachmann bekannt und beispielsweise in E. Uhlmann and A. Peyman, Chemical Reviews 90 (1990) 543 und "Protocols for Oligonukleotides and Anaiogs" Synthesis and Properties & Synthesis and Analytical Techniques, S. Agrawal, Ed, Humana Press, Totowa, USA 1993 , S. T. Crooke, F. Bennet, Ann. Rev. Pharmacol. Toxicol. 36 (1996) 107-129 und J. Hunziber and C. Leumann (1995) Mod. Synt. Methods, 7, 331-417 beschrieben.Examples of chemical modifications are known to the person skilled in the art and are described, for example, in E. Uhlmann and A. Peyman, Chemical Reviews 90 (1990) 543 and "Protocols for Oligonucleotides and Anaiogs" Synthesis and Properties & Synthesis and Analytical Techniques, S. Agrawal, Ed, Humana Press, Totowa, USA 1993, ST Crooke, F. Bennet, Ann. Rev. Pharmacol. Toxicol. 36 (1996) 107-129 and J. Hunziber and C. Leumann (1995) Mod. Synt. Methods, 7, 331-417.
Die chemische Modifikation eines Oligonukleotids kann beispielsweise a) den vollständigen oder teilweisen Ersatz der Phosphorsäurediester-Brücken (Internukleosid-Brücke) durch modifizierte Phosphobrücken bedeuten, wobei Phosphorothioat-, Phoshorodithioat-, NR1R1 'Phosphoramidat-, Boranophosphat-, Phosphat-(Cι-C2ι)-O-Alkylester, Phosphat-[(C6-Cι2)Aryl-(Cι-C2ι)-O-Alkyl]ester, (Ci- C8)Alkylphosphonat- und/oder (C6-Cι2)-Arylphosphonat-Brücken Beispiele für modifizierte Phosphobrücken sind, wobeiThe chemical modification of an oligonucleotide can mean, for example, a) the complete or partial replacement of the phosphoric diester bridges (internucleoside bridge) by modified phosphorus bridges, phosphorothioate, phosphorus dithioate, NR 1 R 1 ' phosphoramidate, boranophosphate, phosphate (Cι -C 2 ι) -O-alkyl esters, phosphate - [(C 6 -Cι 2 ) aryl- (Cι-C 2 ι) -O-alkyl] esters, (Ci- C 8 ) alkyl phosphonate and / or (C 6 -Cι 2 ) arylphosphonate bridges are examples of modified phosphobridges, wherein
R1 und R1' unabhängig voneinander für Wasserstoff, (Cι-Cι8)-Alkyl, (C6-C20)-Aryl,R 1 and R 1 ' independently of one another for hydrogen, (-C 8 -C 8 ) alkyl, (C 6 -C 20 ) aryl,
(C6-Cι )-Aryl-(C -C8)-alkyl, bevorzugt für Wasserstoff, (C1-C8)-Alkyl und/oder(C 6 -C) aryl- (C -C 8 ) alkyl, preferably for hydrogen, (C 1 -C 8 ) alkyl and / or
Methoxyethyl, besonders bevorzugt für Wasserstoff, (Cι-C4)-Alkyl und/oderMethoxyethyl, particularly preferably for hydrogen, (-CC 4 ) alkyl and / or
Methoxyethyl stehen oderStand methoxyethyl or
R1 und R1 zusammen mit dem sie tragenden Stickstoffatom einen 5-6-gliedrigen heterocyclischen Ring bilden, der zusätzlich ein weiteres Heteroatom aus der ReiheR 1 and R 1 together with the nitrogen atom carrying them form a 5-6-membered heterocyclic ring, which is also a further heteroatom from the series
O, S, N enthalten kann; und/oder b) den vollständigen oder teilweisen Ersatz der 3'- und/oder 5'- Phosphorsäurediester Internukleosid Brücken („Phosphorsäurediesterbrücken") durch "Dephospho"-Brücken (beschrieben beispielsweise in Uhlmann, E. und Peyman, A. in "Methods in Molecular Biology", Vol. 20, "Protocols for Oligonukleotides and Analogs", S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16, 355ff) bedeuten, wobei Formacetal-, 3'-Thioformacetal-, Methylhydroxylamin-, Oxim-, Methylendimethylhydrazo-, Dimethylensulfon- und/oder Silylgruppen Beispiele für "Dephospho"-Brücken sind; und/oder c) den vollständigen oder teilweisen Ersatz des Zuckerphosphat-Rückgrats (Ersatz von Zucker-Phosphat-Einheiten) durch andere Einheiten bedeuten, wobei die andere Einheit beispielsweise geeignet, ist ein "Morpholin-Derivaf'-Oligomer (beispielweise in E. P. Stirchak et al., Nucleic Acids Res. 17 (1989) 6129 beschrieben) aufzubauen (d.h. Ersatz durch eine Morpholino-De vat Einheit) und/oder geeignet ist eine Polyamid Nucleinsäure ("PNA") (beispielsweise beschrieben in P. E. Nielsen et al, Bioconj. Chem. 5 (1994) 3 (EP 0 672 677)) aufzubauen (d.h. Ersatz durch eine PNA Einheit, beispielsweise 2-Amino- ethylgiycin) und/oder geeignet ist, eine Phosphomonosäureester Nukleinsäure ("PHONA", „PMENA") (beschrieben beispielsweise in Peyman et al., Angew. Chem. Int. Ed. Engl. 35 (1996) 2632-2638, EP 0 739 898) aufzubauen (d.h. Ersatz durch eine PHONA Einheit); und/oder d) den vollständigen oder teiiweisen Ersatz der ß-D-2'-Desoxyribose (ß-D-2'- Desoxyribose-Einheiten) durch modifizierte Zuckereinheiten bedeuten, wobei α-D- 2'-Desoxyribose, L-2'-Desoxyribose, 2'-F-2'-Desoxyribose, 2'-O-(Cι-C6)Alkyl-Ribose, vorzugsweise 2'-O-Methylribose, 2'-O-(C2-C6)Alkenyl-Ribose, 2'-[O-(d-C6)Alkyl-O- (Ci-Ce Alkyll-Ribose, 2'-NH2-2'-desoxyribose, ß-D-Xylofuranose, a-Arabinofuranose, 2,4-Dideoxy-ß-D-erythro-hexo-pyranose, carbocyclische Zuckeranlaoga (beschrieben beispielsweise in Froehler, J.Am.Chem.Soc. 114 (1992) 8320), offenkettige Zuckeranaloga (beschrieben beispielsweise in Vandendriessche et al., Tetrahedron 49 (1993) 7223) und Bicyclo-Zuckeranaloga (beschrieben beispielsweise in M. Tarkov et al., Helv. Chim. Acta 76 (1993) 481 ) Beispiele für modifizierte Zuckereinheiten sind; und/oder e) die Modifikation beziehungsweise den vollständigen oder teilweisen Ersatz der natürlichen Nukleosid-Basen durch modifizierte (Nukleosid-) Basen („Nukleobasen") bedeuten, wobei 5-(Hydroxymethyl)uracil, 5-Aminouracil, Pseudouracil, Dihydrouracil, 5-(Cι-C6)-Alkyl-uracil, 5-(C2-C6)-Alkenyl-uracil, 5-(C2- C6)-Alkinyl-uracil, 5-(Cι-C6)-Alkyl-cytosin, 5-(C2-C6)-Alkenyl-cytosin, 5-(C2-C6)- Alkinyl-cytosin, 5-Fluoruracil, 5-Fluorcytosin, 5-Chloruracil, 5-Chlorcytosin, 5- Bromuracil, 5-Bromcytosin, 7-Deaza-7-substituierte Purine, 7-Deaza-8-substituierte Purine, 8-Azapuhne, 2,4-Diamino-purine, 5-Bromcytosin, 5-Bromuracil, 5- Chlorcytosin, 5-Chloruracil, 5-Fluorcytosin, 5-Fluoruracil, Hypoxanthin und Uracil Beispiele für modifizierte Basen sind; und/oder f) die Konjugation mit einem oder mehreren Molekülen (Oligonukleotid- Konjugate), die die Eigenschaft(en) des Oligonukleotids an spezielle Anforderungen anpassen bzw. die Eigenschaften (z.B. Zellpenetration, Nukleasestablilität, Affinität zur Tenascin-kodierenden Target-Sequenz, Pharmakokinetik) des Oligonukleotids (z.B. Antisense-Oligonukleotid, Tripelhelix- bildendes Oligonukleotid) günstig beeinflußen und/oder bei der Hybridisierung des Oligonukleotids an die Target-Sequenz diese unter Bindung und/oder Quervernetzung angreifen kann bzw. können, bedeuten, wobei Poly-Lysin, Interkalatoren wie Pyren, Acridin, Phenazin, Phenanthridin, fluoreszierende Verbindungen wie Fluorescein, Cross-Linker wie Psoralen, Azidoproflavin, lipophile Moleküle wie (Cι2-C20)-Alkyl, Lipide wie 1 ,2-Di-hexadecyl-rac-glycerin, Steroide wie Cholesterin, Testosteron, Vitamine wie Vitamin E, Poly- bzw. Oligo-ethylengylcol, (Cι2-Cι8)-Alkyl-Phosphatdiester und -O-CH2-CH(OH)-O-(Cι2-C18)-Alkyl Beispiele für Moleküle sind, die an ein Oligonukleotid konjugiert werden können, wobei solche Moleküle am 5'- und/oder am 3'-Ende und/oder innerhalb der Sequenz, z.B. über eine Nukleobase an das Oligonukleotid konjugiert sein können; und/oder g) die Konjugation an ein 2'5'-verbundenes Oligoadenylat oder ein Derivat desselben bedeutet, wobei ein 2'5'-verbundenes Triodenylat, ein 2'5' -verbundenes Tetraadenylat, ein 2'5'-verbundenes Pentaadenylat u.s.w. Beispiele für 2'5'- verbundene Oligoadenylate sind und Cordycepin (2'5'-verbundenes 3'- Deoxyadenylat) ein Beispiel für ein Derivat eines 2'5'-verbundenen Oligoadenylats ist, wobei die Konjugation vorzugsweise über einen Linker erfolgt, wobei das 5'- Ende des 2'5'-verbundenen Oligoadenylats vorzugsweise eine Phosphat-, Diphosphat- oder Triphosphatgruppe sein kann, wobei der Linker beispielsweise ein Oligoethylenglykole sein kann, wobei Triethylenglykol, Tetraethylenglykol und Hexaethylenglykol Beispiele für Oligoethylenglykol Linker sind; und/oder h) die Einführung einer 3'-3'- und/oder 5'-5'-lnversion am 3' und/oder am 5'- Ende des Oligonukleotids bedeuten, wobei diese Art der chemischen Modifikation dem Fachmann bekannt und beispielsweise in M. Koga et al., J. Org. Chem. 56 (1991 ) 3757 beschrieben ist.May contain O, S, N; and / or b) the complete or partial replacement of the 3'- and / or 5'-phosphoric diester internucleoside bridges (“phosphoric diester bridges”) by “dephospho” bridges (described, for example, in Uhlmann, E. and Peyman, A. in “Methods in Molecular Biology ", vol. 20," Protocols for Oligonucleotides and Analogs ", S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16, 355ff) mean, where formacetal, 3'-thioformacetal, methylhydroxylamine, oxime, methylenedimethylhydrazo, dimethylene sulfone and / or silyl groups Examples of "dephospho" bridges are: and / or c) the complete or partial replacement of the sugar phosphate backbone (replacement of sugar-phosphate units) by other units, the other unit being suitable, for example, is a "morpholine derivative"'Oligomer (e.g. described in EP Stirchak et al., Nucleic Acids Res. 17 (1989) 6129) (ie replacement by a morpholino-devat unit) and / or a polyamide nucleic acid ("PNA") (e.g. described in PE Nielsen et al, Bioconj. Chem. 5 (1994) 3 (EP 0 672 677)) (ie replacement by a PNA unit, for example 2-aminoethylglycine) and / or a phosphomono acid ester nucleic acid (" PHONA "," PMENA ") (described be is, for example, in Peyman et al., Angew. Chem. Int. Ed. Engl. 35 (1996) 2632-2638, EP 0 739 898) (ie replacement by a PHONA unit); and / or d) mean the complete or partial replacement of the β-D-2'-deoxyribose (β-D-2'-deoxyribose units) by modified sugar units, where α-D- 2'-deoxyribose, L-2 ' -Desoxyribose, 2'-F-2'-deoxyribose, 2'-O- (-C-C 6 ) alkyl-ribose, preferably 2'-O-methylribose, 2'-O- (C 2 -C 6 ) alkenyl- Ribose, 2 '- [O- (dC 6 ) alkyl-O- (Ci-Ce alkyl-ribose, 2'-NH 2 -2'-deoxyribose, ß-D-xylofuranose, a-arabinofuranose, 2,4-dideoxy -ß-D-erythro-hexo-pyranose, carbocyclic sugar analogues (described for example in Froehler, J.Am.Chem.Soc. 114 (1992) 8320), open-chain sugar analogs (described for example in Vandendriessche et al., Tetrahedron 49 (1993) 7223) and bicyclo sugar analogs (described for example in M. Tarkov et al., Helv. Chim. Acta 76 (1993) 481) are examples of modified sugar units; and / or e) the modification or the complete or partial replacement of the natural nucleoside bases by modified (nucleoside) bases (“nucleobases”), where 5- (hydroxymethyl) uracil, 5-aminouracil, pseudouracil, dihydrouracil, 5- (Cι- C 6) -alkyl-uracil, 5- (C 2 -C 6) -alkenyl-uracil, 5- (C 2 - C 6) alkynyl-uracil, 5- (Cι-C6) alkyl-cytosine, 5 - (C 2 -C 6 ) alkenyl cytosine, 5- (C 2 -C 6 ) alkynyl cytosine, 5-fluorouracil, 5-fluorocytosine, 5-chlorouracil, 5-chlorocytosine, 5-bromouracil, 5-bromocytosine , 7-deaza-7-substituted purines, 7-deaza-8-substituted purines, 8-azapuhne, 2,4-diamino-purine, 5-bromocytosine, 5-bromouracil, 5-chlorocytosine, 5-chlorouracil, 5-fluorocytosine , 5-fluorouracil, hypoxanthine and uracil are examples of modified bases; and / or f) conjugation with one or more molecules (oligonucleotide conjugates) that adapt the property (s) of the oligonucleotide to special requirements or the properties (e.g. Cell penetration, nuclease test Ability, affinity for the tenascin-coding target sequence, pharmacokinetics) of the oligonucleotide (eg antisense oligonucleotide, triple helix-forming oligonucleotide) favorably influence and / or when hybridizing the oligonucleotide to the target sequence can attack it with binding and / or crosslinking or can mean, where poly-lysine, intercalators such as pyrene, acridine, phenazine, phenanthridine, fluorescent compounds such as fluorescein, cross-linkers such as psoralen, azidoproflavin, lipophilic molecules such as (-C 2 -C 20 ) alkyl, lipids such as 1 , 2-Di-hexadecyl-rac-glycerol, steroids such as cholesterol, testosterone, vitamins such as vitamin E, poly- or oligo-ethylene glycol, (Cι 2 -Cι 8 ) alkyl phosphate diester and -O-CH 2 -CH ( OH) -O- (-C 2 -C 18 ) alkyl are examples of molecules that can be conjugated to an oligonucleotide, such molecules at the 5 'and / or at the 3' end and / or within the sequence, for example conjugated to the oligonucleotide via a nucleobase can be; and / or g) means conjugation to a 2'5'-linked oligoadenylate or a derivative thereof, wherein a 2'5'-linked triodenylate, a 2'5 '-linked tetraadenylate, a 2'5'-linked pentaadenylate, etc Examples for 2'5'- are linked oligoadenylates and cordycepin (2'5'-linked 3'-deoxyadenylate) is an example of a derivative of a 2'5'-linked oligoadenylate, the conjugation preferably taking place via a linker, the 5'-end of the 2 '5'-linked oligoadenylate may preferably be a phosphate, diphosphate or triphosphate group, the linker being, for example, an oligoethylene glycol, triethylene glycol, tetraethylene glycol and hexaethylene glycol being examples of oligoethylene glycol linkers; and / or h) mean the introduction of a 3'-3 'and / or 5'-5' inversion at the 3 'and / or at the 5' end of the oligonucleotide, this type of chemical modification being known to the person skilled in the art and for example in M. Koga et al., J. Org. Chem. 56 (1991) 3757.
In bevorzugten Ausführungsformen der Erfindung weist das Oligonukleotid eine oder mehrere chemische Modifikationen auf, die unabhängig voneinander ausgewählt werden aus a) dem vollständigen oder teilweisen Ersatz der Phosphorsäurediesterbrücken durch Phosphorothioat- und/oder (Cι-C8)Alkylphosphonat-Brücken, b) dem vollständigen oder teilweisen Ersatz des Zuckerphosphat-Rückgrats durch PNA-Einheiten und/oder PHONA-Einheiten, c) den vollständigen oder teilweisen Ersatz der ß-D-2'-Desoxyriboseeinheiten durch 2'-F-2'-Desoxyribose, 2'-O-(Cι-C6)Alkyl-Ribose und/oder 2'-[O-(Cι-C6)Alkyl-O- (Cι-C6)Alkyl]-Ribose, d) den vollständigen oder teilweisen Ersatz der natürlichen Nucleosid-Basen durch 5-(C2-C6)-Alkinyl-uracii und/oder 5-(C2-C6)-Alkinyl-cytosin, e) die Konjugation des Oligonukleotids mit einem oder mehreren Molekülen, die unabhängig voneinander ausgewählt werden können aus der Reihe enthaltend lipophile Moleküle, z.B. (Cι2-C20)-Alkyl, Lipide, z.B. 1 ,2-Di-hexadecyl-rac-glycerin, Steroide, z.B. Cholesterin und/oder Testosteron, Vitamine, z.B. Vitamin E, Poly- bzw. Oligo-ethylengylcol, (Cι2-Cι8)-Alkyl-Phosphatdiestem und -O-CH2-CH(OH)-O- (C12-C 8)-Alkyl und f) ein oder mehreren 3'-3'- Inversionen am 3'-Ende des Oligonucleotids, In einer anderen bevorzugten Ausführungsform der Erfindung weist das Oligonukleotid eine oder mehrere chemische Modifikationen auf, die unabhängig voneinander ausgewählt werden können aus der Reihe enthaltend a) den vollständigen oder teilweisen Ersatz der Phosphorsäurediesterbrücken (Phosphodiester Brücken) durch Phosphorothioat-Brücken, b) den vollständigen oder teilweisen Ersatz der ß-D-2'-Desoxyhboseeinheiten durch 2'-F-2'-Desoxyhbose, 2'-O-(Cι-C6)Alkyl-Ribose und/oder 2'-[O-(Cι-C6)Alkyl-O- (Cι-Cβ)Alkyl]-Ribosθ, c) die Konjugation mit lipophilen Molekülen, z.B. (Cι2-C2o)-Alkyl, mit Lipiden, z.B. 1 ,2-Di-hexadecyl-rac-glycerin, mit (Cι2-Cι8)-Alkyl-Phosphatdiestern und/oder mit -O- CH2-CH(OH)-O-(Cι2-C18)-Alkyl.In preferred embodiments of the invention, the oligonucleotide has one or more chemical modifications which are selected independently of one another from a) the complete or partial replacement of the phosphoric diester bridges by phosphorothioate and / or (C 1 -C 8 ) alkylphosphonate bridges, b) the complete one or partial replacement of the sugar phosphate backbone by PNA units and / or PHONA units, c) the complete or partial replacement of the β-D-2'-deoxyribose units by 2'-F-2'-deoxyribose, 2'-O- (-CC 6 ) alkyl ribose and / or 2 '- [O- (-C 6 ) alkyl-O- (-C 6 ) alkyl] -ribose, d) the complete or partial replacement of the natural nucleoside Bases by 5- (C 2 -C 6 ) alkynyl uracii and / or 5- (C 2 -C 6 ) alkynyl cytosine, e) the conjugation of the oligonucleotide with one or more molecules which can be selected independently of one another from the series containing lipophilic molecules, for example (-C 2 -C 20 ) alkyl, lipids, for example 1 , 2-di-hexadecyl-rac-glycerol, steroids, for example cholesterol and / or testosterone, vitamins, for example vitamin E, poly- or oligo-ethylene glycol, (-C 2 -C 8 ) -alkyl phosphate diester and -O-CH 2 -CH (OH) -O- (C 12 -C 8 ) alkyl and f) one or more 3'-3 'inversions at the 3' end of the oligonucleotide. In another preferred embodiment of the invention, the oligonucleotide has one or more chemical modifications which can be selected independently of one another from the series comprising a) the Complete or partial replacement of the phosphoric diester bridges (phosphodiester bridges) by phosphorothioate bridges, b) Complete or partial replacement of the ß-D-2'-deoxyhbose units by 2'-F-2'-deoxyhbose, 2'-O- (Cι- C 6 ) alkyl-ribose and / or 2 '- [O- (-C-C 6 ) alkyl-O- (-C-C β ) alkyl] -ribosθ, c) the conjugation with lipophilic molecules, for example (Cι 2 -C 2 o) -alkyl, with lipids, for example 1, 2-di-hexadecyl-rac-glycerol, with (-C 2 -C 8 ) -alkyl phosphate diesters and / or with -O- CH 2 -CH (OH) -O - (-C 2 -C 18 ) alkyl.
Verfahren zur Herstellung eines Oligonukleotid-Konjugats sind dem Fachmann bekannt und z.B. in Uhlmann, E. & Peyman, A., Chem. Rev. 90 (1990) 543 und/oder M. Manoharan in "Antisense Research and Applications", Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993, Chapter 17, S.303ff. und/oder EP-A 0 552 766 beschrieben.Methods for making an oligonucleotide conjugate are known to those skilled in the art and e.g. in Uhlmann, E. & Peyman, A., Chem. Rev. 90 (1990) 543 and / or M. Manoharan in "Antisense Research and Applications", Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993, Chapter 17, pp. 303ff. and / or EP-A 0 552 766.
In einer besonderen Ausführungsform der Erfindung wird ein Oligonukleotid bereitgestellt, das eine oder mehrere Modifikationen aufweisen kann und das eine der Sequenzen SEQ ID NO. 2 - SEQ ID NO. 20 aufweist bzw. das einer der Sequenzen SEQ ID NO. 2 bis SEQ ID NO. 20 entspricht bzw. das den entsprechenden Sequenz-Abschnitten einer Tenascin-kodierenden Sequenz entspricht und an diesen Abschnitt der Tenascin-kodierenden Sequenz binden kann.In a particular embodiment of the invention, an oligonucleotide is provided which can have one or more modifications and which has one of the sequences SEQ ID NO. 2 - SEQ ID NO. 20 or one of the sequences SEQ ID NO. 2 to SEQ ID NO. 20 corresponds to or corresponds to the corresponding sequence sections of a tenascin coding sequence and can bind to this section of the tenascin coding sequence.
In einer besonderen Ausführungsform der Erfindung wird Oligonukleotid bereitgestellt, in dessen Sequenz jedes Nukleotid (Base und/oder Zucker und/oder Internukleosid Brücke) modifiziert ist . In einer besonderen Ausführungsform der Erfindung ist beispielsweise das Oligonukleotid vollständig aus Phosphorothioaten aufgebaut (durchgängig modifiziertes Phosphothioat, alle Internukleosid Brücken modifiziert). In einer weiteren speziellen Ausführungsform der Erfindung wird ein Oligonukleotid bereitgestellt, das einer der Sequenzen SEQ ID NO. 2 - SEQ ID NO. 20 entspricht, wobei aber die Phosphodiester Brücken zwischen den einzelnen Nukleosiden (d.h. die Internukleosid-Brücken zwischen den einzelnen Nukleosiden) vollständig durch Phosphothioat Brücken (d.h. Phosphothioatgruppen zwischen den Nukleosiden) ersetzt sind.In a particular embodiment of the invention, oligonucleotide is provided, in the sequence of which each nucleotide (base and / or sugar and / or internucleoside bridge) is modified. In a special embodiment of the invention, for example, the oligonucleotide is entirely composed of phosphorothioates constructed (consistently modified phosphothioate, all internucleoside bridges modified). In a further special embodiment of the invention, an oligonucleotide is provided which has one of the sequences SEQ ID NO. 2 - SEQ ID NO. 20 corresponds, but the phosphodiester bridges between the individual nucleosides (ie the internucleoside bridges between the individual nucleosides) are completely replaced by phosphothioate bridges (ie phosphothioate groups between the nucleosides).
In einer weiteren besonderen Ausführungsform der Erfindung wird ein Oligonukleotid bereitgestellt, indem nur ein Teil der Phosphodiester Brücken durch Phosphothioat Brücken ersetzt ist. Insbesondere beinhaltet die Erfindung Oligonukleotide die nur minimal (bzw. partiell) modifiziert sind. Das Prinzip der minimal modifizierten Oligonukleotide ist beschrieben in A. Peyman, E. Uhlmann, Biol. Chem. Hoppe-Seyler, 377 (1996) 67-70. Dabei werden 1-5, vorzugsweise 1-3 endständige Nukleotid-Einheiten (bzw. vorzugsweise die entsprechenden Internukleosid-Brücken) am 5'- und/oder am 3'-Ende und ggf. zusätzlich ausgewählte interne Pyrimidin-Positionen bzw. vorzugsweise die entsprechenden Internukleosid Brücken, die am 3'- und/oder 5 '-Ende des entsprechenden Pyrimidin Nukleosids lokalisierte sind, modifiziert bzw. ersetzt, wobei vorzugsweise Internukleosid Brücken durch Phosphorothioat Brücken ersetzt werden. Auf diese Weise minimal modifizierte Oligonukleotide weisen besonders vorteilhafte Eigenschaften auf, beispielsweise zeigen sie besondere Nukleasestabilität bei minimaler Modifikation.In a further particular embodiment of the invention, an oligonucleotide is provided in that only a part of the phosphodiester bridges is replaced by phosphothioate bridges. In particular, the invention includes oligonucleotides that are only minimally (or partially) modified. The principle of minimally modified oligonucleotides is described in A. Peyman, E. Uhlmann, Biol. Chem. Hoppe-Seyler, 377 (1996) 67-70. Here 1-5, preferably 1-3 terminal nucleotide units (or preferably the corresponding internucleoside bridges) at the 5 'and / or at the 3' end and, if appropriate, additionally selected internal pyrimidine positions or preferably the corresponding ones Internucleoside bridges which are located at the 3 ' and / or 5 ' end of the corresponding pyrimidine nucleoside are modified or replaced, preferably internucleoside bridges being replaced by phosphorothioate bridges. In this way, minimally modified oligonucleotides have particularly advantageous properties, for example they show particular nuclease stability with minimal modification.
Eine besondere Ausführungsform der Erfindung betrifft ein Oligonukleotid, bei dem ausgewählte Internukleosid Brücken durch modifizierte Internukleosid Brücken ersetzt wird, vorzugsweise durch Phosphorothioat Brücken.A particular embodiment of the invention relates to an oligonucleotide in which selected internucleoside bridges are replaced by modified internucleoside bridges, preferably by phosphorothioate bridges.
Gegenstand der Erfindung ist ein Oligonukleotid bei dem entweder a) nur bestimmte Phosphodiester Internukleosid-Brücken oder b) alle Phosphodiester Internukleosid-Brücken modifiziert sind.The invention relates to an oligonucleotide in which either a) only certain phosphodiester internucleoside bridges or b) all phosphodiester internucleoside bridges are modified.
Gegenstand der Erfindung ist weiterhin ein bei dem 1 - 5 endständigeThe invention further relates to a terminal in the 1-5
Internukleosid-Brücken am 5 '-und/oder am 3 '-Ende des Oligonukleotids modifiziert sind. Gegenstand der Erfindung ist auch ein Oligonukleotid, bei dem die am 3'- und/oder 5 '-Ende von nicht endständigen Nukleosiden, die eineInternucleoside bridges at the 5 ' and / or at the 3 ' end of the oligonucleotide are modified. The invention also relates to an oligonucleotide in which the 3 ' and / or 5 ' ends of non-terminal nucleosides, the one
Pyrimidin Base enthalten (interne Pyrimidin Nukleoside), lokalisiertenContain pyrimidine base (internal pyrimidine nucleosides), localized
Internukleosid-Brücken modifiziert sind.Internucleoside bridges are modified.
Spezielle Ausführungsformen der Erfindung beinhalten ein minimal modifiziertes Oligonukleotid, das ist eine der Sequenzen, ausgewählt aus der Reihe der Sequenzen SEQ ID NO. 21 bis SEQ ID NO. 39, aufweist, wobeiSpecific embodiments of the invention include a minimally modified oligonucleotide, that is one of the sequences selected from the series of sequences SEQ ID NO. 21 to SEQ ID NO. 39, wherein
SEQ ID NO. 21 3'- GsGsTsTsTGGGTsGGAGGsTsGsG -5',SEQ ID NO. 21 3'- GsGsTsTsTGGGTsGGAGGsTsGsG -5 ',
SEQ ID NO. 22 3'- GsGsAsGGTsGGTsACsCCsCCsGsG -5',SEQ ID NO. 22 3'- GsGsAsGGTsGGTsACsCCsCCsGsG -5 ',
SEQ ID NO. 23 3'- GsGsTGGTsACsCsCCsCsGsG -5',SEQ ID NO. 23 3'- GsGsTGGTsACsCsCCsCsGsG -5 ',
SEQ ID NO. 24 3'- GsGsAGGTsGGTsACsCsCsC -5',SEQ ID NO. 24 3'- GsGsAGGTsGGTsACsCsCsC -5 ',
SEQ ID NO. 25 3'- AsGsAAAGAAsCsGAAAGGsAsA -5',SEQ ID NO. 25 3'- AsGsAAAGAAsCsGAAAGGsAsA -5 ',
SEQ ID NO. 26 3'- GsGsAGGTsGGTsAsCsC -5*,SEQ ID NO. 26 3'- GsGsAGGTsGGTsAsCsC -5 * ,
SEQ ID NO. 27 3'- GsGsAGCsGATsGGCsTsTsCsCsA -5',SEQ ID NO. 27 3'- GsGsAGCsGATsGGCsTsTsCsCsA -5 ',
SEQ ID NO. 28 3'- AsAsAGGAACsGGGAGsCsG -5',SEQ ID NO. 28 3'- AsAsAGGAACsGGGAGsCsG -5 ',
SEQ ID NO. 29 3'- GsGsTCGGTsTsTGGGTsGsG -5',SEQ ID NO. 29 3'- GsGsTCGGTsTsTGGGTsGsG -5 ',
SEQ ID NO. 30 3'- CsTsTACAGGTsCsCGTsTsGsA -5',SEQ ID NO. 30 3'- CsTsTACAGGTsCsCGTsTsGsA -5 ',
SEQ ID NO. 31 3'- GsGsCsCGsTGTsTCGCsTsGsT -5',SEQ ID NO. 31 3'- GsGsCsCGsTGTsTCGCsTsGsT -5 ',
SEQ ID NO. 32 3'- TsCsACsCCsCTsCsTTsTsCsTsGsG -5',SEQ ID NO. 32 3'- TsCsACsCCsCTsCsTTsTsCsTsGsG -5 ',
SEQ ID NO. 33 3'- GsGsAsCACsCGACsACsGsG -5',SEQ ID NO. 33 3'- GsGsAsCACsCGACsACsGsG -5 ',
SEQ ID NO. 34 3'- AsAsCsGGGAGCGATsGsG -5',SEQ ID NO. 34 3'- AsAsCsGGGAGCGATsGsG -5 ',
SEQ ID NO. 35 3'- AsTsCsTCGGGGTsCsGsTsC -5',SEQ ID NO. 35 3'- AsTsCsTCGGGGTsCsGsTsC -5 ',
SEQ ID NO. 36 3'- AsAsAGAACsGAAAGGsAsA -5',SEQ ID NO. 36 3'- AsAsAGAACsGAAAGGsAsA -5 ',
SEQ ID NO. 37 3'- GsGsTGGTsACsCsCsC -5',SEQ ID NO. 37 3'- GsGsTGGTsACsCsCsC -5 ',
SEQ ID NO. 38 3'- CsCsCsGGTsACsTsGsA -5', SEQ ID NO. 39: 3'- CsCsAsCAGAAAGsAsAsC -5' ist undSEQ ID NO. 38 3'- CsCsCsGGTsACsTsGsA -5 ', SEQ ID NO. 39: 3'- CsCsAsCAGAAAGsAsAsC -5 'and
wobei "s" die Position einer modifzierten Internukleosid-Brücke bzw. Dephosphobrücke angibt, wobei „s" vorzugsweise die Position einer Phosphorothioat Brücke angibt .where "s" indicates the position of a modified internucleoside bridge or dephospho bridge, "s" preferably indicating the position of a phosphorothioate bridge.
Die Sequenzen SEQ ID NO. 21 bis SEQ ID NO. 39 entsprechen den Sequenzen SEQ ID NO. 2 - SEQ ID NO. 20, d.h. sie können an die gleichen Bereiche einer Tenascin-kodierenden Sequenz binden, wobei allerdings im Gegensatz den SEQ ID NO. 2-20 ein Teil der Phosphodiester Brücken durch modifizierte Phosphodiester-Brücken bzw. Dephosphobrücken, vorzugsweise durch Phosphothioat-Brücken (in der Sequenz durch ein "s" gekennzeichnet) ersetzt ist.The sequences SEQ ID NO. 21 to SEQ ID NO. 39 correspond to the sequences SEQ ID NO. 2 - SEQ ID NO. 20, i.e. they can bind to the same regions of a tenascin-coding sequence, but in contrast to SEQ ID NO. 2-20 some of the phosphodiester bridges are replaced by modified phosphodiester bridges or dephosphobridges, preferably by phosphothioate bridges (identified by an "s" in the sequence).
Eine weitere Ausführungsform der Erfindung betrifft Chimäre Oligonukleotide. Ein chimäres Oligonukleotid ist aus mindestens zwei verschiedenen Sequenzabschnitten aufgebaut, beispielsweise aus einem DNA-Abschnitt und einem modifizierten Abschnitt, z.B. einem PNA-Abschnitt und/oder einem PHONA- Abschnitt. Diese unterschiedlichen Abschnitte verleihen dem gesamten Oligonukleotid besondere Eigenschaften.Another embodiment of the invention relates to chimeric oligonucleotides. A chimeric oligonucleotide is constructed from at least two different sequence segments, for example a DNA segment and a modified segment, e.g. a PNA section and / or a PHONA section. These different sections give the entire oligonucleotide special properties.
Eine besondere Form chimärer Oligonukleotide ist beispielsweise in Matteucci und Wagner, Nature 384 SUPP (1996) 20-22 beschrieben. Ein chimäres Oligonukleotid kann z.B.A special form of chimeric oligonucleotides is described, for example, in Matteucci and Wagner, Nature 384 SUPP (1996) 20-22. A chimeric oligonucleotide can e.g.
1. eine sogenannte "Core Sequenz", die aus etwa sieben Nukleotiden besteht und die die RNase H aktivieren kann sowie1. a so-called "core sequence", which consists of about seven nucleotides and which can activate RNase H and
2. eine oder mehrere flankierende Sequenzen, welche die Affinität, Spezifität und/oder Nuklease-Stabilität des Oligonukleotids erhöhen, enthalten.2. contain one or more flanking sequences which increase the affinity, specificity and / or nuclease stability of the oligonucleotide.
Beispielsweise kann die "Core Sequenz" an bestimmten Positionen modifizierte Internukleosid Brücken aufweisen, beispielsweise kann die „Core Sequenz" Phosphorothioat und/oder Phosphodiester Brücken enthalten. Als flankierende Sequenzen eignen sich beispielsweise Sequenzen, bei denen das Zuckerphosphat Rückgrat (Ersatz einer oder mehrerer Zuckerphosphat Einheiten) und/oder ß-D-2'- Deoxyriboseeinheiten ersetzt sind. Als flankierende Sequenzen eignen sich beispielsweise PNAs und/oder 2'-O-Alkyl-Derivate wie etwa 2'-O-Methyl- und/oder 2'-O-Propyl- und/oder 2'-Methoxyethoxy-Derivate.For example, the "core sequence" can have modified internucleoside bridges at certain positions, for example the "core sequence" can contain phosphorothioate and / or phosphodiester bridges. Suitable flanking sequences are, for example, sequences in which the sugar phosphate backbone (replacement of one or more sugar phosphate units) ) and / or ß-D-2 ' - Deoxyribose units are replaced. Suitable flanking sequences are, for example, PNAs and / or 2'-O-alkyl derivatives such as 2'-O-methyl and / or 2'-O-propyl and / or 2'-methoxyethoxy derivatives.
Eine besondere Ausführungsform der Erfindung betrifft ein chimäres Oligonukleotid, das eine der Sequenzen SEQ ID NO. 40 - SEQ ID NO. 58 aufweist, wobei x unabhängig voneinander für eine unmodifizierte oder eine modifizierte Phosphodiester Internukleosid Brücke oder eine Dephosphobrücke, vorzugsweise für Phosphorothioat und/oder Phosphordiester steht und y unabhängig voneinander für den Ersatz einer Zuckerphosphat Einheit oder einer ß-D-2 '-Deoxyriboseeinheit, vorzugsweise für 2'-O-Methyl-, 2'-O-Propyl- und/oder 2'-Methoxyethoxyribose oder einen PNA-Baustein steht, wobeiA particular embodiment of the invention relates to a chimeric oligonucleotide which has one of the sequences SEQ ID NO. 40 - SEQ ID NO. 58, where x independently of one another stands for an unmodified or a modified phosphodiester internucleoside bridge or a dephospho bridge, preferably for phosphorothioate and / or phosphorus diester, and y independently of one another for the replacement of a sugar phosphate unit or a β-D-2 ' deoxyribose unit, preferably represents 2'-O-methyl-, 2'-O-propyl- and / or 2'-methoxyethoxyribose or a PNA building block, where
SEQ ID NO. 40 3'- GyGyTyTyTyGxGxGxTxGxGxAxGyGyTyGyG -5',SEQ ID NO. 40 3'- GyGyTyTyTyGxGxGxTxGxGxAxGyGyTyGyG -5 ',
SEQ ID NO. 41 3'- GyGyAyGyGyTxGxGxTxAxCxCxCyCyCyGyG -5',SEQ ID NO. 41 3'- GyGyAyGyGyTxGxGxTxAxCxCxCyCyCyGyG -5 ',
SEQ ID NO. 42 3'- GyGyTxGxGxTxAxCxCxCxCyCyGyG -5',SEQ ID NO. 42 3'- GyGyTxGxGxTxAxCxCxCxCyCyGyG -5 ',
SEQ ID NO. 43 3'- GyGyAyGyGxTxGxGxTxAxCyCyCyC -5*,SEQ ID NO. 43 3'- GyGyAyGyGxTxGxGxTxAxCyCyCyC -5 * ,
SEQ ID NO. 44 3'- AyGyAyAxAxGxAxAxCxGxAxAxAyGyGyAyA -5',SEQ ID NO. 44 3'- AyGyAyAxAxGxAxAxCxGxAxAxAyGyGyAyA -5 ',
SEQ ID NO. 45 3'- GyGyAxGxGxTxGxGxTxAyCyC -5',SEQ ID NO. 45 3'- GyGyAxGxGxTxGxGxTxAyCyC -5 ',
SEQ ID NO. 46 3'- GyGyAxGxCxGxAxTxGyGyCyTyTyCyCyA -5',SEQ ID NO. 46 3'- GyGyAxGxCxGxAxTxGyGyCyTyTyCyCyA -5 ',
SEQ ID NO. 47 3'- AyAyAyGxGxAxAxCxGxGyGyAyGyCyG -5',SEQ ID NO. 47 3'- AyAyAyGxGxAxAxCxGxGyGyAyGyCyG -5 ',
SEQ ID NO. 48 3'- GyGyTyCxGxGxTxTxTxGxGyGyTyGyG -5',SEQ ID NO. 48 3'- GyGyTyCxGxGxTxTxTxGxGyGyTyGyG -5 ',
SEQ ID NO. 49 3'- CyTyTyAxCxAxGxGxTxCxCxGyTyTyGyA -5',SEQ ID NO. 49 3'- CyTyTyAxCxAxGxGxTxCxCxGyTyTyGyA -5 ',
SEQ ID NO. 50 3'- GyGyCyCxGxTxGxTxTxCxGyCyTyGyT -5',SEQ ID NO. 50 3'- GyGyCyCxGxTxGxTxTxCxGyCyTyGyT -5 ',
SEQ ID NO. 51 3'- TyCyAyCxCxCxCxTxCxTxTyTyCyTyGyG -5',SEQ ID NO. 51 3'- TyCyAyCxCxCxCxTxCxTxTyTyCyTyGyG -5 ',
SEQ ID NO. 52 3'- GyGyAyCxAxCxCxGxAxCxAyCyGyG -5',SEQ ID NO. 52 3'- GyGyAyCxAxCxCxGxAxCxAyCyGyG -5 ',
SEQ ID NO. 53 3'- AyAyCyGxGxGxAxGxCxGxAyTyGyG -5',SEQ ID NO. 53 3'- AyAyCyGxGxGxAxGxCxGxAyTyGyG -5 ',
SEQ ID NO. 54 3'- AyTyCyTxCxGxGxGxGxTxCxGyTyC -5',SEQ ID NO. 54 3'- AyTyCyTxCxGxGxGxGxTxCxGyTyC -5 ',
SEQ ID NO. 55 3'- AyAyAyGxAxAxCxGxAxAxAxGyGyAyA -5',SEQ ID NO. 55 3'- AyAyAyGxAxAxCxGxAxAxAxGyGyAyA -5 ',
SEQ ID NO. 56 3'- GyGyTxGxGxTxAxCxCyCyC -5', SEQ ID NO. 57: 3'- CyCxCxGxGxTxAxCyTyGyA -5',SEQ ID NO. 56 3'- GyGyTxGxGxTxAxCxCyCyC -5 ', SEQ ID NO. 57: 3'- CyCxCxGxGxTxAxCyTyGyA -5 ',
SEQ ID NO. 58: 3'- CyCyAxCxAxGxAxAxAxGyAyAyC -5' ist.SEQ ID NO. 58: 3'- CyCyAxCxAxGxAxAxAxGyAyAyC -5 '.
Die Sequenzen SEQ ID NO. 40 - SEQ ID NO. 58 entsprechen den oben genannten Sequenzen SEQ ID NO. 2 bis SEQ ID NO. 20, d.h. sie binden an die entsprechenden Sequenzabschnitte einer Tenascin-kodierenden Sequenz, wobei allerdings die genannten Modifikationen enthalten sind.The sequences SEQ ID NO. 40 - SEQ ID NO. 58 correspond to the sequences SEQ ID NO. 2 to SEQ ID NO. 20, i.e. they bind to the corresponding sequence sections of a tenascin-coding sequence, although the modifications mentioned are included.
Die Erfindung betrifft Verfahren zur Herstellung der Oligonukleotide. Die beschriebenen Oligonukleotide können mit Hilfe verschiedener bekannter, chemischer Verfahren, z.B. unter Anwendung der Standard Phosphoramidit-Chemie unter Verwendung von Jod bzw. TED (Tetraethythiuramdisulfid) als Oxidationsmittel, hergestellt werde. Dieses Verfahren ist z.B. in Eckstein, F. (1991) "Oligonukleotides and Analogues, A Practical Approach", IRL Press, Oxford beschrieben. Die Oligonukleotide können auch durch Verfahren hergestellt werden, die gegebenenfalls einen oder mehrere enzymatische Schritte enthalten.The invention relates to methods for producing the oligonucleotides. The oligonucleotides described can be made using various known chemical methods, e.g. using the standard phosphoramidite chemistry using iodine or TED (tetraethythiuram disulfide) as an oxidizing agent. This method is e.g. in Eckstein, F. (1991) "Oligonucleotides and Analogues, A Practical Approach", IRL Press, Oxford. The oligonucleotides can also be prepared by methods that optionally contain one or more enzymatic steps.
Die Erfindung betrifft die Verwendung der Oligonukleotide. Die Oligonukleotide können zur Hybridisierung bzw. Bindung an Tenascin-kodierende (einzelsträngige und/oder doppelsträngige) Nukleinsäuren, beispielsweise DNA (z.B. Gene, cDNA) und/oder RNA (z.B. pre-mRNA, mRNA) verwendet werden. Insbesondere betrifft dies die Verwendung der Oligonukleotide zur Hybridisierung mit bzw. Bindung an Nukleinsäuren, die die Sequenz SEQ ID NO. 1 gemäß Tabelle 1 aufweisen bzw. mit Nukleinsäuren, die Teile diese Sequenz aufweisen (beispielsweise Sequenzen, die für Tenascin Isoformen kodieren) bzw. mit Nukleinsäuren, deren Sequenz geringfügig von diesen Sequenzen abweicht (die z.B. eine oder mehrere Punktmutationen aufweisen).The invention relates to the use of the oligonucleotides. The oligonucleotides can be used for hybridization or binding to tenascin-encoding (single-stranded and / or double-stranded) nucleic acids, for example DNA (e.g. genes, cDNA) and / or RNA (e.g. pre-mRNA, mRNA). In particular, this relates to the use of the oligonucleotides for hybridization with or binding to nucleic acids which have the sequence SEQ ID NO. 1 according to Table 1 or with nucleic acids which have parts of this sequence (for example sequences which code for tenascin isoforms) or with nucleic acids whose sequence deviates slightly from these sequences (which have, for example, one or more point mutations).
Die Erfindung betrifft weiterhin die Verwendung der Oligonukleotide zur Modulation sowie zur ganzen oder teilweisen Inhibition der Expression von Tenascin bzw. verschiedener Tenascin Isoformen bzw. von Mutanten derselben, beispielsweise zur ganzen oder teilweisen Inhibition der Transkription und/oder der Translation. Die Erfindung betrifft beispielsweise die Verwendung der Oligonukleotide als Antisense Oligonukleotide. Darüber hinaus können die Oligonukleotide als Hilfsmittel in der Molekularbiologie verwendet werden.The invention further relates to the use of the oligonucleotides for modulation and for the total or partial inhibition of the expression of tenascin or different tenascin isoforms or of mutants thereof, for example for the total or partial inhibition of transcription and / or translation. The invention relates, for example, to the use of the oligonucleotides as antisense oligonucleotides. In addition, the oligonucleotides can be used as aids in molecular biology.
Die Erfindung betrifft weiterhin die Verwendung der Oligonukleotide als Arzneimittel und/oder Diagnostikum bzw. die Verwendung der Oligonukleotide zur Herstellung von Arzneimitteln und/oder Diagnostika. Insbesondere können die Oligonukleotide in Arzneimitteln, die zur Prävention und/oder Behandlung von Krankheiten, die mit der Expression bzw. einer Überexpression von Tenascin einhergehen, eingesetzt werden. Da die Expression von Tenascin normalerweise, d.h. z.B. beim gesunden Menschen räumlich und zeitlich begrenzt ist, kann ein Abweichen von dieser normalen räumlichen und zeitlichen Expression, als Überexpression angesehen werden. Weiterhin können die Oligonukleotide für in Diagnostischen Verfahren eingesetzt werden. Solche Diagnostischen Verfahren können z.B. zur Diagnose bzw. Früherkennung von Krankheiten eingesetzt, die mit einer abnormalen Expression (z.B. Überexpression) von Tenascin einhergehen werden.The invention further relates to the use of the oligonucleotides as pharmaceuticals and / or diagnostic agents or the use of the oligonucleotides for the production of pharmaceuticals and / or diagnostic agents. In particular, the oligonucleotides can be used in medicaments which are used for the prevention and / or treatment of diseases which are associated with the expression or overexpression of tenascin. Since the expression of tenascin is usually, i.e. e.g. in healthy people is limited in space and time, a deviation from this normal spatial and temporal expression can be regarded as overexpression. Furthermore, the oligonucleotides can be used in diagnostic methods. Such diagnostic methods can e.g. used for the diagnosis or early detection of diseases that will be associated with an abnormal expression (e.g. overexpression) of Tenascin.
Die Erfindung betrifft auch einen Testkit, der ein oder mehrere erfindungsgemäße Oligonukleotide und gegenenfalls weitere Komponenten enthält. Solch ein Testkit kann beispielsweise in der Diagnostik und zur Vorsorge, beispielsweise von Hautkrebserkrankungen, eingesetzt werden.The invention also relates to a test kit which contains one or more oligonucleotides according to the invention and optionally other components. Such a test kit can be used, for example, in diagnostics and for prevention, for example of skin cancer.
Die Erfindung betrifft weiterhin die Verwendung der Oligonukleotide bzw. von Arzneimitteln, die diese Oligonukleotide enthalten, zur Behandlung von Krankheiten, bei denen Tenascin bzw. eine Überexpression von Tenacsin ursächlich bzw. beteiligt ist.The invention further relates to the use of the oligonucleotides or of medicaments which contain these oligonucleotides for the treatment of diseases in which tenascin or an overexpression of tenacsin is causal or involved.
Die Erfindung betrifft insbesondere die Verwendung der Oligonukleotide bzw. von Arzneimitteln, die diese Oligonukleotide enthalten, zur Behandlung und/oder Prävention von Krankheiten, bei denen eine Fehlsteuerung bzw. Störung der Einwanderung bzw. des Vorhandenseins bzw. der Einlagerung von Melanocyten in Epithelzellschichten, beispielsweise in Epithelzellschicht der Epidermis, der Aderhaut des Auges oder der Substantia nigra, zugrunde liegt bzw. beteiligt ist und von Morbus Addison, Diabetes mellitus, pernizi öser Anämie und/oder Schilddrüsendysfunktionen.The invention relates in particular to the use of the oligonucleotides or of medicaments which contain these oligonucleotides for the treatment and / or prevention of diseases in which there is a malfunction or disruption of the immigration or the presence or storage of melanocytes in epithelial cell layers, for example in the epithelial cell layer of the epidermis, the choroid of the eye or the substantia nigra, underlies or is involved and of Addison's disease, diabetes mellitus, pernicious anemia and / or thyroid dysfunction.
Die Erfindung betrifft insbesondere die Verwendung der Oligonukleotide bzw. von Arzneimitteln, die diese Oligonukleotide enthalten zur Behandlung und/oder Prävention von Vitiligo und anderen Depigmentierungskrankheiten bzw. Depigmentierungsstörungen (z.B. der Haut, Haare, Augen) beispielsweise Albinismus und/oder zur Behandlung von Psoriasis und/oder zur Behandlung von Krebs, z.B. zur Inhibitoren von Tumorwachstum und Tumormetastasierung, beispielsweise bei Melanomen und/oder zur Behandlung von Entzündungen, insbesondere als Entzündungshemmer und/oder zur Behandlung und/oder Prophylaxe cardiovaskulärer Erkrankungen, beispielsweise der Restenose.The invention relates in particular to the use of the oligonucleotides or of medicaments which contain these oligonucleotides for the treatment and / or prevention of vitiligo and other depigmentation diseases or depigmentation disorders (for example the skin, hair, eyes), for example albinism and / or for the treatment of psoriasis and / or for the treatment of cancer, e.g. for inhibitors of tumor growth and tumor metastasis, for example in the case of melanoma and / or for the treatment of inflammation, in particular as an anti-inflammatory agent and / or for the treatment and / or prophylaxis of cardiovascular diseases, for example restenosis.
Insbesondere betrifft die Erfindung die Verwendung der Oligonukleotide zur Behandlung von Vitiligo bzw. zur Herstellung von Arzneimitteln, die zur Behandlung von Vitiligo verwendet werden können. Die Erfindung betrifft darüber hinaus ganz allgemein (d.h. auch Oligonukleotide mit einer Länge von größer oder gleich 18 Nukleotiden) die Verwendung von Oligonukleotiden zur Behandlung von Vitiligo bzw. die Herstellung von Arzneimitteln, die zur Behandlung von Vitiligo verwendet werden können.In particular, the invention relates to the use of the oligonucleotides for the treatment of vitiligo or for the production of medicaments which can be used for the treatment of vitiligo. The invention also relates generally (i.e. also oligonucleotides with a length of greater than or equal to 18 nucleotides) to the use of oligonucleotides for the treatment of vitiligo or the production of medicaments which can be used for the treatment of vitiligo.
Die Erfindung betrifft weiterhin die Verwendung zur Behandlung von Vitiligo in Kombination mit bekannten therapeutischen Verfahren, beispielsweise in Kombination a) mit Photochemotherapie (PUVA), z.B. unter Verwendung von Methoxypsoralen, Phenylalanin und/oder Khellin und/oder b) mit der Transplantation von kultivierten Melanozyten ("epidermal grafting") und/oder c) mit einer Steroid-Behandlung und/oder d) mit einer Behandlung mit Plazenta-Extrakten und/oder e) mit einer Behandlung mit Pseudokataiase.The invention further relates to the use for the treatment of vitiligo in combination with known therapeutic methods, for example in combination a) with photochemotherapy (PUVA), e.g. using methoxypsoralen, phenylalanine and / or khellin and / or b) with the transplantation of cultured melanocytes ("epidermal grafting") and / or c) with a steroid treatment and / or d) with a treatment with placenta extracts and / or e) treatment with pseudocataiase.
Die Erfindung betrifft weiterhin Verfahren zur Herstellung von Arzneimitteln (pharmazeutischen Zubereitungen). Zur Herstellung von Arzneimitteln werden ein oder mehrere verschiedene Oligonukleotide bzw. deren physiologisch verträgliche Salze vermischt, wobei gegebenfalls weitere pharmazeutische Träger- und/oder Zusatzstoffe zugegeben werden können.The invention further relates to methods for the production of pharmaceuticals (pharmaceutical preparations). One or more different oligonucleotides or their physiologically compatible ones are used for the production of pharmaceuticals Salts mixed, where appropriate further pharmaceutical carriers and / or additives can be added.
Die Erfindung betrifft weiterhin pharmazeutische Zubereitungen (Arzneimittel), die ein oder mehrere verschiedene Oligonukleotide und/oder deren physiologisch verträgliche Salze sowie gegebenfalls pharmazeutische Träger- und/oder Zusatzstoffe enthalten.The invention further relates to pharmaceutical preparations (medicaments) which contain one or more different oligonucleotides and / or their physiologically tolerable salts and, if appropriate, pharmaceutical carriers and / or additives.
Das bzw. die Oligonukleotid(e) und/oder deren physiologisch verträgliche Salze können am Tier, bevorzugt am Säugetier, insbesondere am Menschen als Arzneimittel für sich allein, in Mischungen untereinander oder in Form von pharmazeutischen Zubereitungen verabreicht werden. Die Arzneimittel können eine topische, perkutane, parenterale und/oder enterale Anwendung gestatten. Die jeweils bevorzugte Anwendungsfrom hängt von den jeweils speziellen Gegebenheiten ab. Zur Behandlung von Vitiligo beispielsweise wird eine topische Anwendung, z.B. in Form von Salben, Lotionen oder Tinkturen, Emulsionen, Suspensionen bevorzugt. Ebenso hängt die Häufigkeit der Applikation von den individuellen Gegebenheiten ab. Zur Behandlung von Vitiligo kann beispielsweise eine topische Komposition ein bis zweimal am Tag auf die depigmentierte Hautstelle aufgetragen werden.The oligonucleotide (s) and / or their physiologically tolerable salts can be administered to animals, preferably to mammals, in particular to humans, as a medicament on their own, in mixtures with one another or in the form of pharmaceutical preparations. The drugs can allow topical, percutaneous, parenteral and / or enteral use. The preferred form of application depends on the particular circumstances. For the treatment of vitiligo, for example, a topical application, e.g. preferred in the form of ointments, lotions or tinctures, emulsions, suspensions. The frequency of application also depends on the individual circumstances. For the treatment of vitiligo, for example, a topical composition can be applied to the depigmented skin area once or twice a day.
Arzneimittel bzw. pharmazeutische Zubereitungen können als aktiven Bestandteil eine wirksame Dosis mindestens eines Oligonukleotids und/oder eine Mischung mehrerer Oligonukleotide und gegebenfalls zusätzliche, pharmazeutisch einwandfreie Träger- und/oder Zusatzstoffe enthalten. Eine pharmazeutische Zubereitung kann etwa 0,1 % (Gewichtsprozent) oder weniger bis etwa 90 % (Gewichtsprozent) oder mehr des therapeutisch wirksamen Oligonukleotids bzw. der pharmazeutisch wirksamen Oligonukleotide enthalten.Medicaments or pharmaceutical preparations can contain as active ingredient an effective dose of at least one oligonucleotide and / or a mixture of several oligonucleotides and, if necessary, additional, pharmaceutically perfect carriers and / or additives. A pharmaceutical preparation can contain about 0.1% (percent by weight) or less to about 90% (percent by weight) or more of the therapeutically active oligonucleotide or the pharmaceutically active oligonucleotides.
Die pharmazeutisch wirksame Dosis des jeweiligen Oligonukleotids bzw. eines Oligonukleotids, welches Bestandteil einer Mischung verschiedener Oligonukleotide ist, kann innerhalb weiter Grenzen variieren und ist in jedem einzelnen Fall den individuellen Gegebenheiten anzupassen.The pharmaceutically effective dose of the respective oligonucleotide or an oligonucleotide which is part of a mixture of different oligonucleotides can vary within wide limits and must be adapted to the individual circumstances in each individual case.
Die Herstellung der pharmazeutischen Zubereitungen kann in an sich bekannter Weise, z. B. beschrieben in Remingtons Pharmaceutical Sciences (1985), Mack Publ. Co., Easton, PA. durchgeführt werden, wobei gegebenfalls pharmazeutisch inerte anorganische und/oder organische Trägerstoffe verwendet werden können. Für die Herstellung von Pillen, Tabletten, Dragees und/oder Hartgelatinekapseln können z.B. Lactose, Maisstärke und/oder Derivate derselben, Talk, Stearinsäure und/oder deren Salze verwendet werden. Als Trägerstoffe für Weichgelatinekapseln und/oder Suppositorien können z.B. Fette, Wachse, halbfeste und/oder flüssige Polyole, natürliche und/oder gehärtete öle verwendet werden. Als Trägerstoffe für die Herstellung von Lösungen und/oder Sirupen können z. B.Wasser, Saccharose, Invertzucker, Glukose und/oder Polyole verwendet werden. Als Trägerstoffe für die Herstellung von Injektionslösungen können z.B. Wasser, Alkohole, Glycerin, Polyole und/oder pflanzliche öle verwendet werden. Als Trägerstoffe für Mikrokapseln, Implantate und/oder Rods können beispielsweise Mischpolymerisate, z.B. aus Glykolsäure und Milchsäure verwendet werden. Darüber hinaus sind Liposomen- formulierungen, die dem Fachmann bekannt sind (N. Weiner, Drug Develop Ind Pharm 15 (1989) 1523; "Liposome Dermatics, Springer Verlag 1992), beispielsweise HVJ-Liposomen (Hayashi, Gene Therapy 3 (1996) 878) geeignet. Die dermale Applikation kann beispielsweise auch auch unter Zuhilfenahme ionophoretischer Methoden und/oder mit Hilfe der Elektroporation erfolgen. Darüber hinaus können Lipofektine und/oder andere (Nukleinsäure- bzw. DNA-)Carriersysteme, beispielsweise solche, die in der Gentherapie Anwendung finden, verwendet werden. Insbesondere sind Systeme geeignet, mit deren Hilfe Oligonukleotide mit großer Effizienz in eukaryotische Zellen bzw. die Kerne eukaryotischer Zellen eingebracht werden können.The preparation of the pharmaceutical preparations can be carried out in a manner known per se, e.g. B. described in Remingtons Pharmaceutical Sciences (1985), Mack Publ. Co., Easton, PA. be carried out, where appropriate pharmaceutically inert inorganic and / or organic carriers can be used. For the production of pills, tablets, dragees and / or hard gelatin capsules e.g. Lactose, corn starch and / or derivatives thereof, talc, stearic acid and / or their salts can be used. As carriers for soft gelatin capsules and / or suppositories, e.g. Fats, waxes, semi-solid and / or liquid polyols, natural and / or hardened oils can be used. As carriers for the preparation of solutions and / or syrups z. B. water, sucrose, invert sugar, glucose and / or polyols can be used. As carriers for the production of injection solutions, e.g. Water, alcohols, glycerin, polyols and / or vegetable oils can be used. As a carrier for microcapsules, implants and / or rods, for example, copolymers, e.g. from glycolic acid and lactic acid can be used. In addition, liposome formulations which are known to the person skilled in the art (N. Weiner, Drug Develop Ind Pharm 15 (1989) 1523; "Liposome Dermatics, Springer Verlag 1992), for example HVJ liposomes (Hayashi, Gene Therapy 3 (1996) 878 The dermal application can also be carried out, for example, with the aid of ionophoretic methods and / or with the aid of electroporation, and lipofectins and / or other (nucleic acid or DNA) carrier systems, for example those used in gene therapy, can also be used Systems are particularly suitable with the aid of which oligonucleotides can be introduced into eukaryotic cells or the nuclei of eukaryotic cells with great efficiency.
Eine pharmazeutische Zubereitung kann neben den Wirk- und Trägerstoffen noch Zusatzstoffe, wie z.B. Füllstoffe, Streck-, Spreng-, Binde-, Gleit-, Netz-, Stabilisierungs-, Emulgier-, Konservierungs-, Süß-, Färbe-, Geschmacks- oder Aromatisierungs-, Dickungs-, Verdünnungsmittel, Puffersubstanzen, ferner Lösungsmittel und/oder Lösungsvermittler und/oder Mittel zur Erzielung eines Depoteffekts, sowie Salze zur Veränderung des osmotischen Drucks, Überzugsmittel und/oder Antioxidantien enthalten. Sie können auch zwei oder mehrere verschiedene Oligonukleotide und/oder deren physiologisch verträgliche Salze enthalten sowie ferner neben mindestens einem Oligonukleotid einen oder mehrere andere therapeutisch wirksame Stoffe.In addition to the active ingredients and carriers, a pharmaceutical preparation can also contain additives, such as fillers, extenders, explosives, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colors, flavors or Flavoring agents, thickeners, diluents, buffer substances, furthermore solvents and / or solubilizers and / or agents for achieving a depot effect, and salts for changing the osmotic pressure, coating agents and / or antioxidants. They can also contain two or more different oligonucleotides and / or their physiologically tolerable salts and, in addition to at least one oligonucleotide, one or more other therapeutically active substances.
BeispieleExamples
Beispiel 1 : OligonukleotidsyntheseExample 1: Oligonucleotide Synthesis
Das Oligonukleotid wurde auf einem automatischen DNA Synthesizer (Applied Biosystems Model 380B oder 394) unter Anwendung der Standard Phosphoramidit- Chemie und Oxidation mit Jod synthetisiert (F. Eckstein, Ed "Oligonucleotides and Analogues, A Practical Approach", IRL Press, Oxford, 1991 ). Zur Einführung von Phosphorthioat-Brücken in gemischten Phosphorothioaten und Phosphodiester Oligonukleotid wurde anstelle von Jod mit TETD (Tetraethylthiuramdisulfid) oxidiert (Applied Biosystems User Bulletin 65). Nach Abspaltung vom festen Träger (CPG oder Tentagel) und Entfernung der Schutzgruppen mit konz. NH3 bei 55°C (18h) wurde das Oligonukleotid zunächst durch Butanol-Fällung (Sawadogo, Van Dyke, Nucl. Acids Res. 19 (1991 ) 674 ) gereinigt. Das Natriumsalz wurde dann durch Ausfällung aus einer 0.5 M NaCI Lösung mit 2.5 Volumenteilen Ethanol erhalten.The oligonucleotide was synthesized on an automatic DNA synthesizer (Applied Biosystems Model 380B or 394) using standard phosphoramidite chemistry and oxidation with iodine (F. Eckstein, Ed "Oligonucleotides and Analogues, A Practical Approach", IRL Press, Oxford, 1991 ). To introduce phosphorothioate bridges in mixed phosphorothioates and phosphodiester oligonucleotides, TETD (tetraethylthiuram disulfide) was used instead of iodine (Applied Biosystems User Bulletin 65). After separation from the solid support (CPG or tentagel) and removal of the protective groups with conc. NH3 at 55 ° C (18h) the oligonucleotide was first purified by butanol precipitation (Sawadogo, Van Dyke, Nucl. Acids Res. 19 (1991) 674). The sodium salt was then obtained by precipitation from a 0.5 M NaCl solution with 2.5 parts by volume of ethanol.
Das Oligonukleotid wurde mit Hilfe derThe oligonucleotide was determined using the
a) Analytischen Gelelektrophorese (Gel: 20% Acrylamid, 8M Harnstoff; Laufpuffer: 454M Tris-borat Puffer, pH 7.0) und/odera) Analytical gel electrophoresis (gel: 20% acrylamide, 8M urea; running buffer: 454M tris-borate buffer, pH 7.0) and / or
b) HPLC-Analyse (Säulenmaterial: Waters GenPak FAX; Gradient: CH3CN (400ml), H2O (1.61), NaH2PO (3.1g), NaCI (11.7g), pH6.8 (0.1M an NaCI) nach CH3CN (400ml), H2O (1.61), NaH2PO4 (3.1 g), NaCI (175.3g), pH6.8 (1.5M an NaCI)) und/oderb) HPLC analysis (column material: Waters GenPak FAX; gradient: CH3CN (400ml), H 2 O (1.61), NaH 2 PO (3.1g), NaCI (11.7g), pH6.8 (0.1M on NaCI) CH3CN (400ml), H 2 O (1.61), NaH 2 PO 4 (3.1 g), NaCI (175.3g), pH6.8 (1.5M on NaCI)) and / or
c) Kapillargelelektrophorese (Beckmann Kapillare eCAP™, U100P Gel Column, 65 cm length, 100 mm I.D., window 15 cm from one end; Puffer: 140 μM Tris, 360mM Borsäure, 7M Harnstoff) und/oderc) Capillary gel electrophoresis (Beckmann capillary eCAP ™, U100P Gel Column, 65 cm length, 100 mm I.D., window 15 cm from one end; buffer: 140 μM Tris, 360mM boric acid, 7M urea) and / or
d) Elektrospray Massenspektroskopied) electrospray mass spectrometry
analysiert.analyzed.
Die Analyse des Oligonukleotids ergab, daß dieses jeweils in einer Reinheit von größer 90% vorlag. Die Methoden zur Analyse von Oligonukleotiden sind z.B. in Schweiber und Engler "Analysis of oligonucleotides" (in "Antisense - from technology to therapy", a laboratory manual and textbook, Schlingensiepen et al. eds., Biol. Science, Vol. 6 (1997) p. 78-103) beschrieben.The analysis of the oligonucleotide showed that it was in each case in a purity of greater than 90%. The methods for analyzing oligonucleotides are e.g. in Schweiber and Engler "Analysis of oligonucleotides" (in "Antisense - from technology to therapy", a laboratory manual and textbook, Schlingensiepen et al. eds., Biol. Science, Vol. 6 (1997) p. 78-103) .
Synthetisiertes Oligonukleotid:Synthesized oligonucleotide:
ODN1 (Sequenz SEQ ID NO. 24): 3'- GsGsAGGTsGGTsACsCsCsC -5'ODN1 (Sequence SEQ ID NO. 24): 3'- GsGsAGGTsGGTsACsCsCsC -5 '
Beispiel 2: Herstellung einer pharmazeutischen ZubereitungExample 2: Preparation of a pharmaceutical preparation
50 mg ODN 1 aus Beispiel 1 können z.B. mit 1g Dermatop® (Hoechst Aktiengesellschaft, Frankfurt am Main, Germany) Basiscreme eng vermischt und die Mischung bei Temperaturen < 10 °C aufbewahrt.50 mg ODN 1 from Example 1 can e.g. closely mixed with 1g Dermatop® (Hoechst Aktiengesellschaft, Frankfurt am Main, Germany) base cream and the mixture stored at temperatures <10 ° C.
Beispiel 3:Example 3:
Die Creme aus Beispiel 2 kann dann beispielsweise zweimal täglich (morgens und nachmittags bzw. abends) auf eine depigmentierte Hautstelle eines Vitiligo- Patienten aufgetragen werden. Tabelle 1 : Sequenz SEQ ID NO. 1 :The cream from Example 2 can then be applied, for example, twice a day (in the morning and in the afternoon or in the evening) to a depigmented skin area of a Vitiligo patient. Table 1: Sequence SEQ ID NO. 1 :
Sequenz der humanen Tenascins cDNA nach A. Siri et al. Nucl. Acids Res. 19 (1991 ) 525-531.Sequence of the human Tenascins cDNA according to A. Siri et al. Nucl. Acids Res. 19 (1991) 525-531.
GAATTCGCTA GAGCCCTAGA GCCCCAGCAG CACCCAGCCA AACCCACCTC CACCATGGGG 60GAATTCGCTA GAGCCCTAGA GCCCCAGCAG CACCCAGCCA AACCCACCTC CACCATGGGG 60
GCCATGACTC AGCTGTTGGC AGGTGTCTTT CTTGCTTTCC TTGCCCTCGC TACCGAAGGT 120GCCATGACTC AGCTGTTGGC AGGTGTCTTT CTTGCTTTCC TTGCCCTCGC TACCGAAGGT 120
GGGGTCCTCA AGAAAGTCAT CCGGCACAAG CGACAGAGTG GGGTGAACGC CÄCCCTGCCA 180GGGGTCCTCA AGAAAGTCAT CCGGCACAAG CGACAGAGTG GGGTGAACGC CÄCCCTGCCA 180
GAAGAGAACC AGCCAGTGGT GTTTAACCAC GTTTACAACA TCAAGCTGCC AGTGGGATCC 240GAAGAGAACC AGCCAGTGGT GTTTAACCAC GTTTACAACA TCAAGCTGCC AGTGGGATCC 240
CAGTGTTCGG TGGATCTGGA GTCAGCCAGT GGGGAGAAAG ACCTGGCACC GCCTTCAGAG 300CAGTGTTCGG TGGATCTGGA GTCAGCCAGT GGGGAGAAAG ACCTGGCACC GCCTTCAGAG 300
CCCAGCGAAA GCTTTCAGGA GCACACAGTA GATGGGGAAA ACCAGATTGT CTTCACACAT 360CCCAGCGAAA GCTTTCAGGA GCACACAGTA GATGGGGAAA ACCAGATTGT CTTCACACAT 360
CGCATCAACA TCCCCCGCCG GGCCTGTGGC TGTGCCGCAG CCCCTGATGT TAAGGAGCTG 420CGCATCAACA TCCCCCGCCG GGCCTGTGGC TGTGCCGCAG CCCCTGATGT TAAGGAGCTG 420
CTGAGCAGAC TGGAGGAGCT GGAGAACCTG GTGTCTTCCC TGAGGGAGCA ATGTACTGCA 480CTGAGCAGAC TGGAGGAGCT GGAGAACCTG GTGTCTTCCC TGAGGGAGCA ATGTACTGCA 480
GGAGCAGGCT GCTGTCTCCA GCCTGCCACA GGCCGCTTGG ACACCAGGCC CTTCTGTAGC 540GGAGCAGGCT GCTGTCTCCA GCCTGCCACA GGCCGCTTGG ACACCAGGCC CTTCTGTAGC 540
GGTCGGGGCA ACTTCAGCAC TGAAGGATGT GGCTGTGTCT GCGAACCTGG CTGGAAAGGC 600GGTCGGGGCA ACTTCAGCAC TGAAGGATGT GGCTGTGTCT GCGAACCTGG CTGGAAAGGC 600
CCCAACTGCT CTGAGCCCGA ATGTCCAGGC AACTGTCACC TTCGAGGCCG GTGCATTGAT 660CCCAACTGCT CTGAGCCCGA ATGTCCAGGC AACTGTCACC TTCGAGGCCG GTGCATTGAT 660
GGGCAGTGCA TCTGTGACGA CGGCTTCACG GGCGAGGACT GCAGCCAGCT GGCTTGCCCC 720GGGCAGTGCA TCTGTGACGA CGGCTTCACG GGCGAGGACT GCAGCCAGCT GGCTTGCCCC 720
AGCGACTGCA ATGACCAGGG CAAGTGCGTG AATGGAGTCT GCATCTGTTT CGAAGGCTAC 780AGCGACTGCA ATGACCAGGG CAAGTGCGTG AATGGAGTCT GCATCTGTTT CGAAGGCTAC 780
GCGGCTGACT GCAGCCGTGA AATCTGCCCA GTGCCCTGCA GTGAGGAGCA CGGCACATGT 840GCGGCTGACT GCAGCCGTGA AATCTGCCCA GTGCCCTGCA GTGAGGAGCA CGGCACATGT 840
GTAGATGGCT TGTGTGTGTG CCACGATGGC TTTGCAGGCG ATGACTGCAA CAAGCCTCTG 900GTAGATGGCT TGTGTGTGTG CCACGATGGC TTTGCAGGCG ATGACTGCAA CAAGCCTCTG 900
TGTCTCAACA ATTGCTACAA CCGTGGACGA TGCGTGGAGA ATGAGTGCGT GTGTGATGAG 960TGTCTCAACA ATTGCTACAA CCGTGGACGA TGCGTGGAGA ATGAGTGCGT GTGTGATGAG 960
GGTTTCACGG GCGAAGACTG CAGTGAGCTC ATCTGCCCCA ATGACTGCTT CGACCGGGGC 1020GGTTTCACGG GCGAAGACTG CAGTGAGCTC ATCTGCCCCA ATGACTGCTT CGACCGGGGC 1020
CGCTGCATCA ATGGCACCTG CTACTGCGAA GAAGGCTTCA CAGGTGAAGA CTGCGGGAAA 1080CGCTGCATCA ATGGCACCTG CTACTGCGAA GAAGGCTTCA CAGGTGAAGA CTGCGGGAAA 1080
CCCACCTGCC CACATGCCTG CCACACCCAG GGCCGGTGTG AGGAGGGGCA GTGTGTATGT 1140 GATGAGGGCT TTGCCGGTGT GGACTGCAGC GAGAAGAGGT GTCCTGCTGA CTGTCACAAT 1200CCCACCTGCC CACATGCCTG CCACACCCAG GGCCGGTGTG AGGAGGGGCA GTGTGTATGT 1140 GATGAGGGCT TTGCCGGTGT GGACTGCAGC GAGAAGAGGT GTCCTGCTGA CTGTCACAAT 1200
CGTGGCCGCT GTGTAGACGG GCGGTGTGAG TGTGATGATG GTTTCACTGG AGCTGACTGT 1260CGTGGCCGCT GTGTAGACGG GCGGTGTGAG TGTGATGATG GTTTCACTGG AGCTGACTGT 1260
GGGGAGCTCA AGTGTCCCAA TGGCTGCAGT GGCCATGGCC GCTGTGTCAA TGGGCÄGTGT 1320GGGGAGCTCA AGTGTCCCAA TGGCTGCAGT GGCCATGGCC GCTGTGTCAA TGGGCÄGTGT 1320
GTGTGTGATG AGGGCTATAC TGGGGAGGAC TGCAGCCAGC TACGGTGCCC CAATGACTGT 1380GTGTGTGATG AGGGCTATAC TGGGGAGGAC TGCAGCCAGC TACGGTGCCC CAATGACTGT 1380
CACAGTCGGG GCCGCTGTGT CGAGGGCAAA TGTGTATGTG AGCAAGGCTT CAAGGGCTAT 1440CACAGTCGGG GCCGCTGTGT CGAGGGCAAA TGTGTATGTG AGCAAGGCTT CAAGGGCTAT 1440
GACTGCAGTG ACATGAGCTG CCCTAATGAC TGTCACCAGC ACGGCCGCTG TGTGAATGGC 1500GACTGCAGTG ACATGAGCTG CCCTAATGAC TGTCACCAGC ACGGCCGCTG TGTGAATGGC 1500
ATGTGTGTTT GTGATGACGG CTACACAGGG GAAGACTGCC GGGATCGCCA ATGCCCCAGG 1560ATGTGTGTTT GTGATGACGG CTACACAGGG GAAGACTGCC GGGATCGCCA ATGCCCCAGG 1560
GACTGCAGCA ACAGGGGCCT CTGTGTGGAC GGACAGTGCG TCTGTGAGGA CGGCTTCACC 1620GACTGCAGCA ACAGGGGCCT CTGTGTGGAC GGACAGTGCG TCTGTGAGGA CGGCTTCACC 1620
GGCCCTGACT GTGCAGAACT CTCCTGTCCA AATGACTGCC ATGGCCAGGG TCGCTGTGTG 1680GGCCCTGACT GTGCAGAACT CTCCTGTCCA AATGACTGCC ATGGCCAGGG TCGCTGTGTG 1680
AATGGGCAGT GCGTGTGCCA TGAAGGATTT ATGGGCAAAG ACTGCAAGGA GCAAAGATGT 1740AATGGGCAGT GCGTGTGCCA TGAAGGATTT ATGGGCAAAG ACTGCAAGGA GCAAAGATGT 1740
CCCAGTGACT GTCATGGCCA GGGCCGCTGC GTGGACGGCC AGTGCATCTG CCACGAGGGC 1800CCCAGTGACT GTCATGGCCA GGGCCGCTGC GTGGACGGCC AGTGCATCTG CCACGAGGGC 1800
TTCACAGGCC TGGACTGTGG CCAGCACTCC TGCCCCAGTG ACTGCAACAA CTTAGGACAA 1860TTCACAGGCC TGGACTGTGG CCAGCACTCC TGCCCCAGTG ACTGCAACAA CTTAGGACAA 1860
TGCGTCTCGG GCCGCTGCAT CTGCAACGAG GGCTACAGCG GAGAAGACTG CTCAGAGGTG 1920TGCGTCTCGG GCCGCTGCAT CTGCAACGAG GGCTACAGCG GAGAAGACTG CTCAGAGGTG 1920
TCTCCTCCCA AAGACCTCGT TGTGACAGAA GTGACGGAAG AGACGGTCAA CCTGGCCTGG 1980TCTCCTCCCA AAGACCTCGT TGTGACAGAA GTGACGGAAG AGACGGTCAA CCTGGCCTGG 1980
GACAATGAGA TGCGGGTCAC AGAGTACCTT GTCGTGTACA CGCCCACCCA CGAGGGTGGT 2040GACAATGAGA TGCGGGTCAC AGAGTACCTT GTCGTGTACA CGCCCACCCA CGAGGGTGGT 2040
CTGGAAATGC AGTTCCGTGT GCCTGGGGAC CAGACGTCCA CCATCATCCG GGAGCTGGAG 2100CTGGAAATGC AGTTCCGTGT GCCTGGGGAC CAGACGTCCA CCATCATCCG GGAGCTGGAG 2100
CCTGGTGTGG AGTACTTTAT CCGTGTATTT GCCATCCTGG AGAACAAGAA GAGCATTCCT 2160CCTGGTGTGG AGTACTTTAT CCGTGTATTT GCCATCCTGG AGAACAAGAA GAGCATTCCT 2160
GTCAGCGCCA GGGTGGCCAC GTACTTACCT GCACCTGAAG GCCTGAAATT CAAGTCCATC 2220GTCAGCGCCA GGGTGGCCAC GTACTTACCT GCACCTGAAG GCCTGAAATT CAAGTCCATC 2220
AAGGAGACAT CTGTGGAAGT GGAGTGGGAT CCTCTAGACA TTGCTTTTGA AACCTGGGAG 2280AAGGAGACAT CTGTGGAAGT GGAGTGGGAT CCTCTAGACA TTGCTTTTGA AACCTGGGAG 2280
ATCATCTTCC GGAATATGAA TAAAGAAGAT GAGGGAGAGA TCACCAAAAG CCTGAGGAGG 2340ATCATCTTCC GGAATATGAA TAAAGAAGAT GAGGGAGAGA TCACCAAAAG CCTGAGGAGG 2340
CCAGAGACCT CTTACCGGCA AACTGGTCTA GCTCCTGGGC AAGAGTATGA GATATCTCTG 2400CCAGAGACCT CTTACCGGCA AACTGGTCTA GCTCCTGGGC AAGAGTATGA GATATCTCTG 2400
CACATAGTGA AAAACAATAC CCGGGGCCCT GGCCTGAAGA GGGTGACCAC CACACGCTTG 2460 GATGCCCCCA GCCAGATCGA GGTGAAAGAT GTCACAGACA CCACTGCCTT GATCACCTGG 2520CACATAGTGA AAAACAATAC CCGGGGCCCT GGCCTGAAGA GGGTGACCAC CACACGCTTG 2460 GATGCCCCCA GCCAGATCGA GGTGAAAGAT GTCACAGACA CCACTGCCTT GATCACCTGG 2520
TTCAAGCCCC TGGCTGÄGAT CGATGGCATT GAGCTGACCT ACGGCATCAA AGACGTGCCA 2580TTCAAGCCCC TGGCTGÄGAT CGATGGCATT GAGCTGACCT ACGGCATCAA AGACGTGCCA 2580
GGAGACCGTA CCACCATCGA TCTCACAGAG GACGAGAACC AGTACTCCAT CGGGAACCTG 2640GGAGACCGTA CCACCATCGA TCTCACAGAG GACGAGAACC AGTACTCCAT CGGGAACCTG 2640
AAGCCTGACA CTGAGTACGA GGTGTCCCTC ATCTCCCGCA GAGGTGACAT GTCAAGCAAC 2700AAGCCTGACA CTGAGTACGA GGTGTCCCTC ATCTCCCGCA GAGGTGACAT GTCAAGCAAC 2700
CCAGCCAAAG AGACCTTCAC AACAGGCCTC GATGCTCCCA GGAATCTTCG ACGTGTTTCC 2760CCAGCCAAAG AGACCTTCAC AACAGGCCTC GATGCTCCCA GGAATCTTCG ACGTGTTTCC 2760
CAGACAGATA ACAGCATCAC CCTGGAATGG AGGAATGGCA AGGCAGCTAT TGACAGTTAC 2820CAGACAGATA ACAGCATCAC CCTGGAATGG AGGAATGGCA AGGCAGCTAT TGACAGTTAC 2820
AGAATTAAGT ATGCCCCCAT CTCTGGAGGG GACCACGCTG AGGTTGATGT TCCAAAGAGC 2880AGAATTAAGT ATGCCCCCAT CTCTGGAGGG GACCACGCTG AGGTTGATGT TCCAAAGAGC 2880
CAACAAGCCA CAACCAAAAC CACACTCACA GGTCTGAGGC CGGGAACTGA ATATGGGATT 2940CAACAAGCCA CAACCAAAAC CACACTCACA GGTCTGAGGC CGGGAACTGA ATATGGGATT 2940
GGAGTTTCTG CTGTGAAGGA AGACAAGGAG AGCAATCCAG CGACCATCAA CGCAGCCACA 3000GGAGTTTCTG CTGTGAAGGA AGACAAGGAG AGCAATCCAG CGACCATCAA CGCAGCCACA 3000
GAGTTGGACA CGCCCAAGGA CCTTCAGGTT TCTGAAACTG CAGAGACCAG CCTGACCCTG 3060GAGTTGGACA CGCCCAAGGA CCTTCAGGTT TCTGAAACTG CAGAGACCAG CCTGACCCTG 3060
CTCTGGAAGA CACCGTTGGC CAAATTTGAC CGCTACCGCC TCAATTACAG TCTCCCCACA 3120CTCTGGAAGA CACCGTTGGC CAAATTTGAC CGCTACCGCC TCAATTACAG TCTCCCCACA 3120
GGCCAGTGGG TGGGAGTGCA GCTTCCAAGA AACACCACTT CCTATGTCCT GAGAGGCCTG 3180GGCCAGTGGG TGGGAGTGCA GCTTCCAAGA AACACCACTT CCTATGTCCT GAGAGGCCTG 3180
GAACCAGGAC AGGAGTACAA TGTCCTCCTG ACAGCCGAGA AAGGCAGACA CAAGAGCAAG 3240GAACCAGGAC AGGAGTACAA TGTCCTCCTG ACAGCCGAGA AAGGCAGACA CAAGAGCAAG 3240
CCCGCACGTG TGAAGGCATC CACTGAACAA GCCCCTGAGC TGGAAAACCT CACCGTGACT 3300CCCGCACGTG TGAAGGCATC CACTGAACAA GCCCCTGAGC TGGAAAACCT CACCGTGACT 3300
GAGGTTGGCT GGGATGGCCT CAGACTCAAC TGGACCGCGG CTGACCAGGC CTATGAGCAC 3360GAGGTTGGCT GGGATGGCCT CAGACTCAAC TGGACCGCGG CTGACCAGGC CTATGAGCAC 3360
TTTATCATTC AGGTGCAGGA GGCCAACAAG GTGGAGGCAG CTCGGAACCT CACCGTGCCT 3420TTTATCATTC AGGTGCAGGA GGCCAACAAG GTGGAGGCAG CTCGGAACCT CACCGTGCCT 3420
GGCAGCCTTC GGGCTGTGGA CATACCGGGC CTCAAGGCTG CTACGCCTTA TACAGTCTCC 3480GGCAGCCTTC GGGCTGTGGA CATACCGGGC CTCAAGGCTG CTACGCCTTA TACAGTCTCC 3480
ATCTATGGGG TGATCCAGGG CTATAGAACA CCAGTGCTCT CTGCTGAGGC CTCCACAGGG 3540ATCTATGGGG TGATCCAGGG CTATAGAACA CCAGTGCTCT CTGCTGAGGC CTCCACAGGG 3540
GAAACTCCCA ATTTGGGAGA GGTCGTGGTG GCCGAGGTGG GCTGGGATGC CCTCAAACTC 3600GAAACTCCCA ATTTGGGAGA GGTCGTGGTG GCCGAGGTGG GCTGGGATGC CCTCAAACTC 3600
AACTGGACTG CTCCAGAAGG GGCCTATGAG TACTTTTTCA TTCAGGTGCA GGAGGCTGAC 3660AACTGGACTG CTCCAGAAGG GGCCTATGAG TACTTTTTCA TTCAGGTGCA GGAGGCTGAC 3660
ACAGTAGAGG CAGCCCAGAA CCTCACCGTC CCAGGAGGAC TGAGGTCCAC AGACCTGCCT 3720ACAGTAGAGG CAGCCCAGAA CCTCACCGTC CCAGGAGGAC TGAGGTCCAC AGACCTGCCT 3720
GGGCTCAAAG CAGCCACTCA TTATACCATC ACCATCCGCG GGGTCACTCA GGACTTCAGC 3780 ACAACCCCTC TCTCTGTTGA AGTCTTGACA GAGGAGGTTC CAGATATGGG AAACCTCACA 3840GGGCTCAAAG CAGCCACTCA TTATACCATC ACCATCCGCG GGGTCACTCA GGACTTCAGC 3780 ACAACCCCTC TCTCTGTTGA AGTCTTGACA GAGGAGGTTC CAGATATGGG AAACCTCACA 3840
GTGACCGAGG TTAGCTGGGA TGCTCTCAGA CTGAACTGGA CCACGCCAGA TGGAACCTAT 3900GTGACCGAGG TTAGCTGGGA TGCTCTCAGA CTGAACTGGA CCACGCCAGA TGGAACCTAT 3900
GACCAGTTTA CTATTCAGGT CCAGGAGGCT GACCAGGTGG AAGAGGCTCA CAATCTCACG 3960GACCAGTTTA CTATTCAGGT CCAGGAGGCT GACCAGGTGG AAGAGGCTCA CAATCTCACG 3960
GTTCCTGGCA GCCTGCGTTC CATGGAAATC CCAGGCCTCA GGGCTGGCAC TCCTTACACA 4020GTTCCTGGCA GCCTGCGTTC CATGGAAATC CCAGGCCTCA GGGCTGGCAC TCCTTACACA 4020
GTCACCCTGC ACGGCGAGGT CAGGGGCCAC AGCACTCGAC CCCTTGCTGT AGAGGTCGTC 4080GTCACCCTGC ACGGCGAGGT CAGGGGCCAC AGCACTCGAC CCCTTGCTGT AGAGGTCGTC 4080
CAGTGGGACG TGCCGCTCCA GTCCCCGGTG TCGTGAGCTG GGGAACGACA TCTCCAGCAG 4140CAGTGGGACG TGCCGCTCCA GTCCCCGGTG TCGTGAGCTG GGGAACGACA TCTCCAGCAG 4140
ACAGAGGATC TCCCACAGCT GGGAGATTTA GCCGTGTCTG AGGTTGGCTG GGATGGCCTC 4200ACAGAGGATC TCCCACAGCT GGGAGATTTA GCCGTGTCTG AGGTTGGCTG GGATGGCCTC 4200
AGACTCAACT GGACCGCAGC TGACAATGCC TATGAGCACT TTGTCATTCA GGTGCAGGAG 4260AGACTCAACT GGACCGCAGC TGACAATGCC TATGAGCACT TTGTCATTCA GGTGCAGGAG 4260
GTCAACAAAG TGGAGGCAGC CCAGAACCTC ACGTTGCCTG GCAGCCTCAG GGCTGTGGAC 4320GTCAACAAAG TGGAGGCAGC CCAGAACCTC ACGTTGCCTG GCAGCCTCAG GGCTGTGGAC 4320
ATCCCGGGCC TCGAGGCTGC CACGCCTTAT AGAGTCTCCA TCTATGGGGT GATCCGGGGC "380ATCCCGGGCC TCGAGGCTGC CACGCCTTAT AGAGTCTCCA TCTATGGGGT GATCCGGGGC "380
TATAGAACAC CAGTACTCTC TGCTGAGGCC TCCACAGCCA AAGAACCTGA AATTGGAAAC 4440TATAGAACAC CAGTACTCTC TGCTGAGGCC TCCACAGCCA AAGAACCTGA AATTGGAAAC 4440
TTAAATGTTT CTGACATAAC TCCCGAGAGC TTCAATCTCT CCTGGATGGC TACCGATGGG 4500TTAAATGTTT CTGACATAAC TCCCGAGAGC TTCAATCTCT CCTGGATGGC TACCGATGGG 4500
ATCTTCGAGA CCTTTACCAT TGAAATTATT GATTCCAATA GGTTGCTGGA GACTGTGGAA 4560ATCTTCGAGA CCTTTACCAT TGAAATTATT GATTCCAATA GGTTGCTGGA GACTGTGGAA 4560
TATAATATCT CTGGTGCTGA ACGAACTGCC CATATCTCAG GGCTACCCCC TAGTACTGAT 4620TATAATATCT CTGGTGCTGA ACGAACTGCC CATATCTCAG GGCTACCCCC TAGTACTGAT 4620
TTTATTGTCT ACCTCTCTGG ACTTGCTCCC AGCATCCGGA CCAAAACCAT CAGTGCCACA 4680TTTATTGTCT ACCTCTCTGG ACTTGCTCCC AGCATCCGGA CCAAAACCAT CAGTGCCACA 4680
GCCACGACAG AGGCCCTGCC CCTTCTGGAA AACCTAACCA TTTCCGACAT TAATCCCTAC 4740GCCACGACAG AGGCCCTGCC CCTTCTGGAA AACCTAACCA TTTCCGACAT TAATCCCTAC 4740
GGGTTCACAG TTTCCTGGAT GGCATCGGAG AATGCCTTTG ACAGCTTTCT AGTAACGGTG 4800GGGTTCACAG TTTCCTGGAT GGCATCGGAG AATGCCTTTG ACAGCTTTCT AGTAACGGTG 4800
GTGGATTCTG GGAAGCTGCT GGACCCCCAG GAATTCACAC TTTCAGGAAC CCAGAGGAAG 4860GTGGATTCTG GGAAGCTGCT GGACCCCCAG GAATTCACAC TTTCAGGAAC CCAGAGGAAG 4860
CTGGAGCTTA GAGGCCTCAT AACTGGCATT GGCTATGAGG TTATGGTCTC TGGCTTCACC 4920CTGGAGCTTA GAGGCCTCAT AACTGGCATT GGCTATGAGG TTATGGTCTC TGGCTTCACC 4920
CAAGGGCATC AAACCAAGCC CTTGAGGGCT GAGATTGTTA CAGAAGCCGA ACCGGAAGTT 4980CAAGGGCATC AAACCAAGCC CTTGAGGGCT GAGATTGTTA CAGAAGCCGA ACCGGAAGTT 4980
GACAACCTTC TGGTTTCAGA TGCCACCCCA GACGGTTTCC GTCTGTCCTG GACAGCTGAT 5040GACAACCTTC TGGTTTCAGA TGCCACCCCA GACGGTTTCC GTCTGTCCTG GACAGCTGAT 5040
GAAGGGGTCT TCGACAATTT TGTTCTCAAA ATCAGAGATA CCAAAAAGCA GTCTGAGCCA 5100 CTGGAAATAA CCCTACTTGC CCCCGAACGT ACCAGGGACA TAACAGGTCT CAGAGAGGCT 5160GAAGGGGTCT TCGACAATTT TGTTCTCAAA ATCAGAGATA CCAAAAAGCA GTCTGAGCCA 5100 CTGGAAATAA CCCTACTTGC CCCCGAACGT ACCAGGGACA TAACAGGTCT CAGAGAGGCT 5160
ACTGAATACG AAATTGAACT CTATGGAATA AGCAAAGGAA GGCGATCCCA GACAGTCAGT 5220ACTGAATACG AAATTGAACT CTATGGAATA AGCAAAGGAA GGCGATCCCA GACAGTCAGT 5220
GCTATAGCAA CAACAGCCAT GGGCTCCCCA AAGGAAGTCA TTTTCTCAGA CATCACTGAA 5280GCTATAGCAA CAACAGCCAT GGGCTCCCCA AAGGAAGTCA TTTTCTCAGA CATCACTGAA 5280
AATTCGGCTA CTGTCAGCTG GAGGGCACCC ACGGCCCAAG TGGAGAGCTT CCGGATTACC 5340AATTCGGCTA CTGTCAGCTG GAGGGCACCC ACGGCCCAAG TGGAGAGCTT CCGGATTACC 5340
TATGTGCCCA TTACAGGAGG TACACCCTCC ATGGTAACTG TGGACGGAAC CAAGACTCAG 5400TATGTGCCCA TTACAGGAGG TACACCCTCC ATGGTAACTG TGGACGGAAC CAAGACTCAG 5400
ACCAGGCTGG TGAAACTCAT ACCTGGCGTG GAGTACCTTG TCAGCATCAT CGCCATGAAG 5460ACCAGGCTGG TGAAACTCAT ACCTGGCGTG GAGTACCTTG TCAGCATCAT CGCCATGAAG 5460
GGCTTTGAGG AAAGTGAACC TGTCTCAGGG TCATTCACCA CAGCTCTGGA TGGCCCATCT 5520GGCTTTGAGG AAAGTGAACC TGTCTCAGGG TCATTCACCA CAGCTCTGGA TGGCCCATCT 5520
GGCCTGGTGA CAGCCAACAT CACTGACTCA GAAGCCTTGG CCAGGTGGCA GCCAGCCATT 5580GGCCTGGTGA CAGCCAACAT CACTGACTCA GAAGCCTTGG CCAGGTGGCA GCCAGCCATT 5580
GCCACTGTGG ACAGTTATGT CATCTCCTAC ACAGGCGAGA AAGTGCCAGA AATTACACGC 5640GCCACTGTGG ACAGTTATGT CATCTCCTAC ACAGGCGAGA AAGTGCCAGA AATTACACGC 5640
ACGGTGTCCG GGAACACAGT GGAGTATGCT CTGACCGACC TCGAGCCTGC CACGGAATAC 5700ACGGTGTCCG GGAACACAGT GGAGTATGCT CTGACCGACC TCGAGCCTGC CACGGAATAC 5700
ACACTGAGAA TCTTTGCAGA GAAAGGGCCC CAGAAGAGCT CAACCATCAC TGCCAAGTTC 5760ACACTGAGAA TCTTTGCAGA GAAAGGGCCC CAGAAGAGCT CAACCATCAC TGCCAAGTTC 5760
ACAACAGACC TCGATTCTCC AAGAGACTTG ACTGCTACTG AGGTTCAGTC GGAAACTGCC 5820ACAACAGACC TCGATTCTCC AAGAGACTTG ACTGCTACTG AGGTTCAGTC GGAAACTGCC 5820
CTCCTTACCT GGCGACCCCC CCGGGCATCA GTCACCGGTT ACCTGCTGGT CTATGAATCA 5880CTCCTTACCT GGCGACCCCC CCGGGCATCA GTCACCGGTT ACCTGCTGGT CTATGAATCA 5880
GTGGATGGCA CAGTCAAGGA AGTCATTGTG GGTCCAGATA CCACCTCCTA CAGCCTGGCA 5940GTGGATGGCA CAGTCAAGGA AGTCATTGTG GGTCCAGATA CCACCTCCTA CAGCCTGGCA 5940
GACCTGAGCC CATCCACCCA CTACACAGCC AAGATCCAGG CACTCAATGG GCCCCTGAGG 6000GACCTGAGCC CATCCACCCA CTACACAGCC AAGATCCAGG CACTCAATGG GCCCCTGAGG 6000
AGCAATATGA TCCAGACCAT CTTCACCACA ATTGGACTCC TGTACCCCTT CCCCAAGGAC 6060AGCAATATGA TCCAGACCAT CTTCACCACA ATTGGACTCC TGTACCCCTT CCCCAAGGAC 6060
TGCTCCCAAG CAATGCTGAA TGGAGACACG ACCTCTGGCC TCTACACCAT TTATCTGAAT 6120TGCTCCCAAG CAATGCTGAA TGGAGACACG ACCTCTGGCC TCTACACCAT TTATCTGAAT 6120
GGTGATAAGG CTCAGGCGCT GGAAGTCTTC TGTGACATGA CCTCTGATGG GGGTGGATGG 6180GGTGATAAGG CTCAGGCGCT GGAAGTCTTC TGTGACATGA CCTCTGATGG GGGTGGATGG 6180
ATTGTGTTCC TGAGACGCAA AAACGGACGC GAGAACTTCT ACCAAAACTG GAAGGCATAT 6240ATTGTGTTCC TGAGACGCAA AAACGGACGC GAGAACTTCT ACCAAAACTG GAAGGCATAT 6240
GCTGCTGGAT TTGGGGACCG CAGAGAAGAA TTCTGGCTTG GGCTGGACAA CCTGAACAAA 6300GCTGCTGGAT TTGGGGACCG CAGAGAAGAA TTCTGGCTTG GGCTGGACAA CCTGAACAAA 6300
ATCACAGCCC AGGGGCAGTA CGAGCTCCGG GTGGACCTGC GGGACCATGG GGAGACAGCC 6360ATCACAGCCC AGGGGCAGTA CGAGCTCCGG GTGGACCTGC GGGACCATGG GGAGACAGCC 6360
TTTGCTGTCT ATGACAAGTT CAGCGTGGGA GATGCCAAGA CTCGCTACAA GCTGAAGGTG 6420 GAGGGGTACA GTGGGACAGC AGGTGACTCC ATGGCCTACC ACAATGGCAG ATCCTTCTCC 6480TTTGCTGTCT ATGACAAGTT CAGCGTGGGA GATGCCAAGA CTCGCTACAA GCTGAAGGTG 6420 GAGGGGTACA GTGGGACAGC AGGTGACTCC ATGGCCTACC ACAATGGCAG ATCCTTCTCC 6480
ACCTTTGÄCA AGGACACAGA TTCAGCCATC ACCAACTGTG CTCTGTCTAC AAGGGGCTTC 6540ACCTTTGÄCA AGGACACAGA TTCAGCCATC ACCAACTGTG CTCTGTCTAC AAGGGGCTTC 6540
TGGTACAGGA ACTGTCACCG TGTCAACCTG ATGGGGAGAT ATGGGGACAA TAACCACAGT 6600TGGTACAGGA ACTGTCACCG TGTCAACCTG ATGGGGAGAT ATGGGGACAA TAACCACAGT 6600
CÄGGGCGTTA ACTGGTTCCA CTGGAAGGGC CACGAACACT CAATCCAGTT TGCTGAGATG 6660CÄGGGCGTTA ACTGGTTCCA CTGGAAGGGC CACGAACACT CAATCCAGTT TGCTGAGATG 6660
AAGCTGAGAC CAAGCAACTT CAGAAATCTT GAAGGCAGGC GCAAACGGGC ATAAATTGGA 6720AAGCTGAGAC CAAGCAACTT CAGAAATCTT GAAGGCAGGC GCAAACGGGC ATAAATTGGA 6720
GGGACCACTG GGTGAGAGAG GAATAAGGCG GCCCAGAGCG AGGAAAGGAT TTTACCAAAG 6780GGGACCACTG GGTGAGAGAG GAATAAGGCG GCCCAGAGCG AGGAAAGGAT TTTACCAAAG 6780
CATCAATACA ACCAGCCCAA CCATCGGTCC ACACCTGGGC ATTTGGTGAG AATCAAAGCT 6840CATCAATACA ACCAGCCCAA CCATCGGTCC ACACCTGGGC ATTTGGTGAG AATCAAAGCT 6840
GACCATGGAT CCCTGGGGCC AACGGCAACA GCATGGGCCT CACCTCCTCT GTGATTTCTT 6900GACCATGGAT CCCTGGGGCC AACGGCAACA GCATGGGCCT CACCTCCTCT GTGATTTCTT 6900
TCTTTGCACC AAAGACATCA GTCTCCAACA TGTTTCTGTT TTGTTGTTTG ATTCAGCAAA 6960TCTTTGCACC AAAGACATCA GTCTCCAACA TGTTTCTGTT TTGTTGTTTG ATTCAGCAAA 6960
AATCTCCCAG TGACAACATC GCAATAGTTT TTTACTTCTC TTAGGTGGCT CTGGGATGGG 7020AATCTCCCAG TGACAACATC GCAATAGTTT TTTACTTCTC TTAGGTGGCT CTGGGATGGG 7020
AGAGGGGTAG GATGTACAGG GGTAGTTTGT TTTAGAACCA GCCGTATTTT ACATGAAGCT 7080AGAGGGGTAG GATGTACAGG GGTAGTTTGT TTTAGAACCA GCCGTATTTT ACATGAAGCT 7080
GTATAATTAA TTGTCATTAT TTTTGTTAGC AAAGATTAAA TGTGTCATTG GAAGCCATCC 7140GTATAATTAA TTGTCATTAT TTTTGTTAGC AAAGATTAAA TGTGTCATTG GAAGCCATCC 7140
CTTTTTTTAC ATTTCATACA ACAGAAACCA GAAAAGCAAT ACTGTTTCCA TTTTAAGGAT 7200CTTTTTTTAC ATTTCATACA ACAGAAACCA GAAAAGCAAT ACTGTTTCCA TTTTAAGGAT 7200
ATGATTAATA TTATTAATAT AATAATGATG ATGATGATGA TGAAAACTAA GGATTTTTCA 7260ATGATTAATA TTATTAATAT AATAATGATG ATGATGATGA TGAAAACTAA GGATTTTTCA 7260
AGAGATCTTT CTTTCCAAAA CATTTCTGGA CAGTACCTGA TTGTATTTTT TTTTTAAATA 7320AGAGATCTTT CTTTCCAAAA CATTTCTGGA CAGTACCTGA TTGTATTTTT TTTTTAAATA 7320
AAAGCACAAG TACTTTTGAA AAAAAA 7346 AAAGCACAAG TACTTTTGAA AAAAAA 7346

Claims

Patentansprüche: Claims:
1. Oligonukleotid, das an eine Nukleinsäure, die für eine der Isoformen humanen Tenascins oder Teile derselben kodiert, bindet und deren Expression inhibiert, wobei das Oligonukleotid eine Länge von 7 bis 15 Nukleotideinheiten aufweist und wobei das Oligonukleotid gegebenfalls modifiziert sein kann sowie die physiologisch verträglichen Salze des Oligonukleotids.1. oligonucleotide which binds to a nucleic acid which codes for one of the isoforms human Tenascins or parts thereof and inhibits their expression, the oligonucleotide having a length of 7 to 15 nucleotide units and where the oligonucleotide may be modified and the physiologically tolerable Salts of the oligonucleotide.
2. Oligonukleotid nach Anspruch 1 , wobei das Oligonukleotid an einen Bereich der Nukleinsäure bindet, der a) einen Teil des 5'-nichtkodierenden Bereichs und/oder den Translationsstart oder b) den Translationsstart und/oder einen Teil des kodierenden Bereichs oder c) einen Teil des kodierenden Bereichs und/oder einen Teil des 3'-nichtkodierenden Bereichs umfaßt.2. The oligonucleotide according to claim 1, wherein the oligonucleotide binds to a region of the nucleic acid which a) a part of the 5 ' non-coding region and / or the translation start or b) the translation start and / or part of the coding region or c) one Part of the coding area and / or part of the 3 ' non-coding area.
3. Oligonukleotid nach Anspruch 1 , wobei das Oligonukleotid eine der Sequenzen SEQ ID NO. 2 bis SEQ ID NO. 20 hat, wobei SEQ ID NO. 2 bis SEQ ID NO. 20 folgende Bedeutung haben:3. The oligonucleotide according to claim 1, wherein the oligonucleotide is one of the sequences SEQ ID NO. 2 to SEQ ID NO. 20, with SEQ ID NO. 2 to SEQ ID NO. 20 have the following meaning:
SEQ. ID NO. 2 3'- GGTTTGGGTGGAGGTGG -5'SEQ. ID NO. 2 3'- GGTTTGGGTGGAGGTGG -5 '
SEQ. ID NO. 3 3'- GGAGGTGGTACCCCCGG -5'SEQ. ID NO. 3 3'- GGAGGTGGTACCCCCGG -5 '
SEQ. ID NO. 4 3'- GGTGGTACCCCCGG -5'SEQ. ID NO. 4 3'- GGTGGTACCCCCGG -5 '
SEQ. ID NO. 5 3'- GGAGGTGGTACCCC -5'SEQ. ID NO. 5 3'- GGAGGTGGTACCCC -5 '
SEQ. ID NO. 6 3'- AGAAAGAACGAAAGGAA -5'SEQ. ID NO. 6 3'- AGAAAGAACGAAAGGAA -5 '
SEQ. ID NO. 7 3'- GGAGGTGGTACC -5'SEQ. ID NO. 7 3'- GGAGGTGGTACC -5 '
SEQ. ID NO. 8 3'- GGAGCGATGGCTTCCA -5'SEQ. ID NO. 8 3'- GGAGCGATGGCTTCCA -5 '
SEQ. ID NO. 9 3'- AAAGGAACGGGAGCG -5'SEQ. ID NO. 9 3'- AAAGGAACGGGAGCG -5 '
SEQ. ID NO. 10: 3'- GGTCGGTTTGGGTGG -5' SEQ. ID NO. 11 : 3'- CTTACAGGTCCGTTGA -5'SEQ. ID NO. 10: 3'- GGTCGGTTTGGGTGG -5 ' SEQ. ID NO. 11: 3'- CTTACAGGTCCGTTGA -5 '
SEQ. ID NO. 12: 3'- GGCCGTGTTCGCTGT -5'SEQ. ID NO. 12: 3'- GGCCGTGTTCGCTGT -5 '
SEQ. ID NO. 13: 3'- TCACCCCTCTTTCTGG -5'SEQ. ID NO. 13: 3'- TCACCCCTCTTTCTGG -5 '
SEQ. ID NO. 14: 3'- GGACACCGACACGG -51 SEQ. ID NO. 14: 3'- GGACACCGACACGG -5 1
SEQ. ID NO. 15: 3'- AACGGGAGCGATGG -5'SEQ. ID NO. 15: 3'- AACGGGAGCGATGG -5 '
SEQ. ID NO. 16: 3'- ATCTCGGGGTCGTC -5'SEQ. ID NO. 16: 3'- ATCTCGGGGTCGTC -5 '
SEQ. ID NO. 17: 3'- AAAGAACGAAAGGAA -5'SEQ. ID NO. 17: 3'- AAAGAACGAAAGGAA -5 '
SEQ. ID NO. 18: 3'- GGTGGTACCCC -5'SEQ. ID NO. 18: 3'- GGTGGTACCCC -5 '
SEQ. ID NO. 19: 3'- CCCGGTACTGA -5'SEQ. ID NO. 19: 3'- CCCGGTACTGA -5 '
SEQ. ID NO. 20: 3'- CCACAGAAAGAAC -5'SEQ. ID NO. 20: 3'- CCACAGAAAGAAC -5 '
4. Oligonukleotid nach einem oder mehreren der Ansprüche 1 bis 3, wobei das Oligonukleotid eine oder mehrere Modifikationen aufweist, die an bestimmten Nukleosid Positionen und/oder Internukleosid Brücken lokalisiert sind.4. Oligonucleotide according to one or more of claims 1 to 3, wherein the oligonucleotide has one or more modifications which are located at certain nucleoside positions and / or internucleoside bridges.
5. Oligonukleotid nach einem oder mehreren der Ansprüche 1 bis 4, wobei die chemischen Modifikationen unabhängig voneinander ausgewählt werden können aus der Reihe der chemischen Modifikationen a) bis h), wobei a) den Ersatz einer Phosphorsäurediester Internukleosid-Brücke lokalisiert ist durch eine modifizierte Phospho-Brücke, b) den Ersatz einer Phosphorsäurediester Internukleosid-Brücke durch eine "Dephospho"-Brücke, c) den Ersatz einer Zuckerphosphat-Einheit durch eine andere Einheit, d) den Ersatz einer ß-D-2'-Desoxyribose-Einheit durch eine modifizierte Zuckereinheit, e) die Modifikation beziehungsweise den Ersatz einer natürlichen Nukleosid-Base, durch eine modifizierte Nukleosid-Base, f) die Konjugation des Oligonukleotids mit einem Molekül, welches die Eigenschaften des Oligonukleotids an eine spezielle Anforderung anpaßt, g) die Konjugation des Oligonukleotids an ein 2'5'-verbundenes Oligoadenylat oder ein Derivat davon, wobei die Konjugation des 2'5'-verbundenen Oligoadenylats oder eines Derivats davon gegebenenfalls über einen Linker erfolgt, und h) die Einführung einer 3 '-3 '-Inversion und/oder 5 '-5 '-Inversion am 3'- beziehungsweise 5'- Ende des Oligonukleotids, bedeuten.5. Oligonucleotide according to one or more of claims 1 to 4, wherein the chemical modifications can be selected independently of one another from the series of chemical modifications a) to h), wherein a) the replacement of a phosphoric acid diester internucleoside bridge is localized by a modified phospho Bridge, b) the replacement of a phosphoric acid diester internucleoside bridge by a "dephospho" bridge, c) the replacement of a sugar phosphate unit by another unit, d) the replacement of a β-D-2'-deoxyribose unit by one modified sugar unit, e) the modification or replacement of a natural nucleoside base by a modified nucleoside base, f) the conjugation of the oligonucleotide with a molecule which adapts the properties of the oligonucleotide to a special requirement, g) the conjugation of the oligonucleotide to a 2'5'-linked oligoadenylate or a derivative thereof, the conjugation of the 2'5'-linked oligoadenylate or a derivative thereof optionally via a linker, and h) the introduction of a 3 '- 3 'inversion and / or 5' -5 'inversion at the 3 ' or 5 ' end of the oligonucleotide.
6. Oligonukleotid nach einem oder mehreren der Ansprüche 1 bis 5, wobei das Oligonukleotid eine oder mehrere chemische Modifikationen enthält, die unabhängig voneinander ausgewählt werden können aus der Reihe der chemischen Modifikationen a) bis h), wobei a) den Ersatz einer Phosphorsäurediester Internukleosid-Brücke durch eine modifizierte Phosphobrücke bedeutet, wobei eine modifizierte Phosphobrücke eine Phosphorothioat-,6. Oligonucleotide according to one or more of claims 1 to 5, wherein the oligonucleotide contains one or more chemical modifications which can be selected independently of one another from the series of chemical modifications a) to h), where a) the replacement of a phosphoric diester internucleoside Bridge through a modified phosphorus bridge means that a modified phosphorus bridge is a phosphorothioate,
Phoshorodithioat-, NR1R1 "Phosphoramidat-, Boranophosphat-, Phosphat-(Cι-Phosphorodithioate, NR 1 R 1 " phosphoramidate, boranophosphate, phosphate (Cι-
C2ι)-O-Alkylester-, Phosphat-[(C6-C12)Aryl-(Cι-C2ι)-O-Alkyl]ester-, (Ci-C 2 ι -O-alkyl ester, phosphate - [(C 6 -C 12 ) aryl- (Cι-C 2 ι) -O-alkyl] ester-, (Ci-
C8)Alkylphosphonat- oder (C6-Cι2)-Arylphosphonat-Brücke ist, wobeiC 8 ) is alkylphosphonate or (C 6 -C 2 ) arylphosphonate bridge, wherein
R1 und R1 unabhängig voneinander ausgewählt werden aus der Reihe enthaltend Wasserstoff, (Cι-Cι8)-Alkyl, (C6-C2o)-Aryl, (C6-Cι4)-Aryl-(Cι-C8)- alkyl oderR 1 and R 1 are selected independently of one another from the series comprising hydrogen, (-CC 8 ) -alkyl, (C 6 -C 2 o) -aryl, (C 6 -C 4 ) -aryl- (-C- 8 ) - alkyl or
R1 und R1 zusammen mit dem sie tragenden Stickstoffatom einen 5-6- gliedrigen heterocyclischen Ring bilden, der zusätzlich ein weiteresR 1 and R 1 together with the nitrogen atom carrying them form a 5-6-membered heterocyclic ring, which in addition is another
Heteroatom aus der Reihe O, S, N enthalten kann, b) den Ersatz einer Phosphorsäurediester Internukleosid-Brücke durch eine "Dephospho"-Brücke bedeutet, wobei eine "Dephospho-Brücken" eine Formacetal-, 3'-Thioformacetal-, Methylhydroxylamin-, Oxim-, Methylendimethylhydrazo-, Dimethylensulfon- oder Silyl-Brücke ist, c) den vollständigen oder teilweisen Ersatz des Zuckerphosphat-Rückgrats (Ersatz von Zucker-Phosphat-Einheiten) durch andere Einheiten bedeutet, wobei eine andere Einheit geeignet ist, ein "Morphoiin-Derivat"-Oligomer, eine Polyamid Nukleinsäure ("PNA") oder eine Phosphomonosäureester Nukleinsäure aufzubauen , c) den Ersatz einer ß-D-2'-Desoxyribose-Einheit durch eine modifizierte Zuckereinheit bedeutet, wobei eine modifizierte Zuckereinheit eine α-D-2'-Desoxyribose, L-2'- Desoxyribose, 2'-F-2'-Desoxyribose, 2'-O-(Cι-C6)Alkyl-Ribose, 2'-O-(C - C6)Alkenyl-Ribose, 2'-[O-(Cι-C6)Alkyl-O-(Cι-C6)Alkyl]-Ribose, 2'-NH2-2'- desoxyribose, ß-D-Xylofuranose, a-Arabinofuranose, 2,4-Dideoxy-ß-D- erythro-hexo-pyranose, ein carbozyklisches Zuckeranalogon, ein offenkettiges Zuckeranalogon oder ein Bicyclo-Zuckeranalogon ist, d) den Ersatz einer natürlichen Nukleosid-Base durch eine modifizierte Nukleosid- Base bedeutet, wobei eine modifizierte Nukleosid-Base 5-(Hydroxymethyl)uracil, 5- Aminouracil, Pseudouracil, Dihydrouracil, 5-(Cι-C6)-Alkyl-uracil, 5-(C2-C6)- Alkenyl-uracil, 5-(C2-C6)-Alkinyl-uracil, δ^d-CeJ-Alkyl-cytosin, 5-(C2-C6)- Alkenyl-cytosin, 5-(C2-C6)-Alkinyl-cytosin, 5-Fluoruracil, 5-Fluorcytosin, 5- Chloruracil, 5-Chlorcytosin, 5-Bromuracil, 5-Bromcytosin, ein 7-Deaza-7- substituiertes Purin .oder ein 7-Deaza-8-substituiertes Purin ist, e) die Konjugation mit einem Molekül bedeutet, wobei das Molekül ein Poly-Lysin, Interkalator, fluoreszierendes Molekül, Cross-Linker, lipophiles Molekül, Lipid, Steroid, Vitamin, Polyethlyenglykol, Oligoethylenglykol, (C12-Cι8)-Alkyl-Phosphatdiester oder -O-CH2-CH(OH)-O- (C12-Cιs)-Alkyl-Gruppe ist, f) die Konjugation an ein 2'5'-verbundenes Oligoadenylat oder ein Derivat desselben bedeutet, wobei ein 2'5' -verbundenes Oligoadenylat oder ein Derivat desselben ein 2'5'-verbundenes Triadenylat, 2'5'-verbundenes Tetraadenylat, 2'5'- verbundenes Pentaadenylat oder Cordycepin (2'5'-verbundenes 3'-Heteroatom from the series O, S, N may contain, b) means the replacement of a phosphoric acid diester internucleoside bridge by a "dephospho" bridge, a "dephospho bridge" being a formacetal, 3'-thioformacetal, methylhydroxylamine, Is oxime, methylene dimethyl hydrazo, dimethylene sulfone or silyl bridge, c) means the complete or partial replacement of the sugar phosphate backbone (replacement of sugar-phosphate units) by other units, where another unit is suitable, a "morphoiin derivative" oligomer, a polyamide nucleic acid ("PNA") or to build up a phosphomonic acid ester nucleic acid, c) means the replacement of a β-D-2'-deoxyribose unit by a modified sugar unit, a modified sugar unit being an α-D-2'-deoxyribose, L-2'-deoxyribose, 2'- F-2'-deoxyribose, 2'-O- (-C-C 6 ) alkyl-ribose, 2'-O- (C-C 6 ) alkenyl-ribose, 2 '- [O- (Cι-C 6 ) alkyl -O- (-CC 6 ) alkyl] -ribose, 2'-NH 2 -2'- deoxyribose, ß-D-xylofuranose, a-arabinofuranose, 2,4-dideoxy-ß-D-erythro-hexo-pyranose , is a carbocyclic sugar analog, an open chain sugar analog or a bicyclo sugar analog, d) means the replacement of a natural nucleoside base by a modified nucleoside base, a modified nucleoside base being 5- (hydroxymethyl) urac il, 5- aminouracil, pseudouracil, dihydrouracil, 5- (-C-C 6 ) alkyl uracil, 5- (C 2 -C 6 ) alkenyl uracil, 5- (C 2 -C 6 ) alkynyl uracil , δ ^ d-CeJ-alkyl cytosine, 5- (C 2 -C 6 ) - alkenyl cytosine, 5- (C 2 -C 6 ) alkynyl cytosine, 5-fluorouracil, 5-fluorocytosine, 5-chlorouracil , 5-chlorocytosine, 5-bromouracil, 5-bromocytosine, a 7-deaza-7-substituted purine. Or a 7-deaza-8-substituted purine, e) means conjugation with a molecule, the molecule being a poly- Lysine, intercalator, fluorescent molecule, cross-linker, lipophilic molecule, lipid, steroid, vitamin, polyethylene glycol, oligoethylene glycol, (C 12 -Cι 8 ) alkyl phosphate diester or -O-CH 2 -CH (OH) -O- ( Is C 12 -Cιs) alkyl group, f) is the conjugation to a 2'5'-linked oligoadenylate or a derivative thereof, wherein a 2'5 '-linked oligoadenylate or a derivative thereof is a 2'5'-linked Triadenylate, 2'5'-linked tetraadenylate, 2'5'- linked pentaadenylate or cordycepin (2'5'-linked 3'-
Deoxyadenylat) ist, wobei die Konjugation gegebenenfalls über einen Linker erfolgt und wobei das 5'-Ende des 2'5'-verbundenen Oligoadenylats gegebenenfalls eineDeoxyadenylate), the conjugation optionally taking place via a linker and the 5'-end of the 2'5'-linked oligoadenylate optionally being one
Phosphat-, Diphosphat- oder Triphosphatgruppe enthält und g) die Einführung einer 3'-3'- und/oder 5'-5'-lnversion am 3' und/oder am 5'-Ende des Oligonukleotids bedeutet.Contains phosphate, diphosphate or triphosphate group and g) means the introduction of a 3'-3 'and / or 5'-5'inversion at the 3' and / or at the 5 'end of the oligonucleotide.
7. Oligonukleotid nach einem oder mehreren der Ansprüche 1 bis 6, wobei entweder a) nur bestimmte Phosphodiester Internukleosid-Brücken oder b) alle Phosphodiester Internukleosid-Brücken durch modifiziert sind.7. Oligonucleotide according to one or more of claims 1 to 6, wherein either a) only certain phosphodiester internucleoside bridges or b) all phosphodiester internucleoside bridges are modified by.
8. Oligonukleotid nach einem oder mehreren der Ansprüche 1 bis 7, wobei 1 - 5 endständige Internukleosid-Brücken am 5 '-und/oder am 3 '-Ende des Oligonukleotids modifiziert sind.8. Oligonucleotide according to one or more of claims 1 to 7, wherein 1-5 terminal internucleoside bridges at the 5 'and / or at the 3 ' end of the oligonucleotide are modified.
9. Oligonukleotid nach einem oder mehreren der Ansprüche 1 bis 8, wobei die am 3'- und/oder 5 '-Ende von nicht endständigen Nukleosiden, die eine Pyrimidin Base enthalten, lokalisierten Internukleosid-Brücken modifiziert sind.9. The oligonucleotide according to one or more of claims 1 to 8, wherein the internucleoside bridges located at the 3 ' and / or 5 ' ends of non-terminal nucleosides containing a pyrimidine base are modified.
10. Oligonukleotid nach einem oder mehreren der Ansprüche 1 bis 9, wobei das Oligonukleotid eine Sequenz ausgewählt aus der Reihe der Sequenzen SEQ ID NO. 21 bis SEQ ID NO. 39 hat, wobei die Sequenzen SEQ ID NO. 21 bis SEQ ID NO. 39 folgende Bedeutung haben:10. Oligonucleotide according to one or more of claims 1 to 9, wherein the oligonucleotide is a sequence selected from the series of sequences SEQ ID NO. 21 to SEQ ID NO. 39, the sequences SEQ ID NO. 21 to SEQ ID NO. 39 have the following meaning:
SEQ ID NO. 21 3'- GsGsTsTsTGGGTsGGAGGsTsGsG -5', SEQ ID NO. 22 3'- GsGsAsGGTsGGTsACsCCsCCsGsG -5' SEQ ID NO. 23 3'- GsGsTGGTsACsCsCCsCsGsG -5', SEQ ID NO. 24 3'- GsGsAGGTsGGTsACsCsCsC -5', SEQ ID NO. 25 3'- AsGsAAAGAAsCsGAAAGGsAsA -5', SEQ ID NO. 26: 3'- GsGsAGGTsGGTsAsCsC -5',SEQ ID NO. 21 3'- GsGsTsTsTGGGTsGGAGGsTsGsG -5 ', SEQ ID NO. 22 3'- GsGsAsGGTsGGTsACsCCsCCsGsG -5 'SEQ ID NO. 23 3'- GsGsTGGTsACsCsCCsCsGsG -5 ', SEQ ID NO. 24 3'- GsGsAGGTsGGTsACsCsCsC -5 ', SEQ ID NO. 25 3'- AsGsAAAGAAsCsGAAAGGsAsA -5 ', SEQ ID NO. 26: 3'- GsGsAGGTsGGTsAsCsC -5 ',
SEQ ID NO. 27: 3'- GsGsAGCsGATsGGCsTsTsCsCsA -5',SEQ ID NO. 27: 3'- GsGsAGCsGATsGGCsTsTsCsCsA -5 ',
SEQ ID NO. 28: 3'- AsAsAGGAACsGGGAGsCsG -5',SEQ ID NO. 28: 3'- AsAsAGGAACsGGGAGsCsG -5 ',
SEQ ID NO. 29: 3'- GsGsTCGGTsTsTGGGTsGsG -5',SEQ ID NO. 29: 3'- GsGsTCGGTsTsTGGGTsGsG -5 ',
SEQ ID NO. 30: 3'- CsTsTACAGGTsCsCGTsTsGsA -5',SEQ ID NO. 30: 3'- CsTsTACAGGTsCsCGTsTsGsA -5 ',
SEQ ID NO. 31 : 31- GsGsCsCGsTGTsTCGCsTsGsT -5',SEQ ID NO. 31: 3 1 - GsGsCsCGsTGTsTCGCsTsGsT -5 ',
SEQ ID NO. 32: 3'- TsCsACsCCsCTsCsTTsTsCsTsGsG -5',SEQ ID NO. 32: 3'- TsCsACsCCsCTsCsTTsTsCsTsGsG -5 ',
SEQ ID NO. 33: 3'- GsGsAsCACsCGACsACsGsG -5',SEQ ID NO. 33: 3'- GsGsAsCACsCGACsACsGsG -5 ',
SEQ ID NO. 34: 3'- AsAsCsGGGAGCGATsGsG -5',SEQ ID NO. 34: 3'- AsAsCsGGGAGCGATsGsG -5 ',
SEQ ID NO. 35: 3'- AsTsCsTCGGGGTsCsGsTsC -5',SEQ ID NO. 35: 3'- AsTsCsTCGGGGTsCsGsTsC -5 ',
SEQ ID NO. 36: 3'- AsAsAGAACsGAAAGGsAsA -5',SEQ ID NO. 36: 3'- AsAsAGAACsGAAAGGsAsA -5 ',
SEQ ID NO. 37: 3'- GsGsTGGTsACsCsCsC -5',SEQ ID NO. 37: 3'- GsGsTGGTsACsCsCsC -5 ',
SEQ ID NO. 38: 3'- CsCsCsGGTsACsTsGsA -5' undSEQ ID NO. 38: 3'- CsCsCsGGTsACsTsGsA -5 'and
SEQ ID NO. 39: 3'- CsCsAsCAGAAAGsAsAsC -5',SEQ ID NO. 39: 3'- CsCsAsCAGAAAGsAsAsC -5 ',
wobei "s" die Position einer modifizierten Internukleosid-Brücke angibt.where "s" indicates the position of a modified internucleoside bridge.
11. Oligonukleotid nach einem oder mehreren der Ansprüche 1 bis 8, wobei das Oligonukleotid eine der Sequenzen SEQ ID NO. 40 bis SEQ ID NO. 58 hat, wobei die Sequenzen SEQ ID NO. 40 bis SEQ ID NO. 58 folgende Bedeutung haben11. Oligonucleotide according to one or more of claims 1 to 8, wherein the oligonucleotide is one of the sequences SEQ ID NO. 40 to SEQ ID NO. 58, the sequences SEQ ID NO. 40 to SEQ ID NO. 58 have the following meaning
SEQ ID NO. 40 3'- GyGyTyTyTyGxGxGxTxGxGxAxGyGyTyGyG -5',SEQ ID NO. 40 3'- GyGyTyTyTyGxGxGxTxGxGxAxGyGyTyGyG -5 ',
SEQ ID NO. 41 3'- GyGyAyGyGyTxGxGxTxAxCxCxCyCyCyGyG -5',SEQ ID NO. 41 3'- GyGyAyGyGyTxGxGxTxAxCxCxCyCyCyGyG -5 ',
SEQ ID NO. 42 3'- GyGyTxGxGxTxAxCxCxCxCyCyGyG -5',SEQ ID NO. 42 3'- GyGyTxGxGxTxAxCxCxCxCyCyGyG -5 ',
SEQ ID NO. 43 3'- GyGyAyGyGxTxGxGxTxAxCyCyCyC -5',SEQ ID NO. 43 3'- GyGyAyGyGxTxGxGxTxAxCyCyCyC -5 ',
SEQ ID NO. 44 3'- AyGyAyAxAxGxAxAxCxGxAxAxAyGyGyAyA -5',SEQ ID NO. 44 3'- AyGyAyAxAxGxAxAxCxGxAxAxAyGyGyAyA -5 ',
SEQ ID NO. 45 3'- GyGyAxGxGxTxGxGxTxAyCyC -5',SEQ ID NO. 45 3'- GyGyAxGxGxTxGxGxTxAyCyC -5 ',
SEQ ID NO. 46 3'- GyGyAxGxCxGxAxTxGyGyCyTyTyCyCyA -5',SEQ ID NO. 46 3'- GyGyAxGxCxGxAxTxGyGyCyTyTyCyCyA -5 ',
SEQ ID NO. 47 3'- AyAyAyGxGxAxAxCxGxGyGyAyGyCyG -5',SEQ ID NO. 47 3'- AyAyAyGxGxAxAxCxGxGyGyAyGyCyG -5 ',
SEQ ID NO. 48 3'- GyGyTyCxGxGxTxTxTxGxGyGyTyGyG -5',SEQ ID NO. 48 3'- GyGyTyCxGxGxTxTxTxGxGyGyTyGyG -5 ',
SEQ ID NO. 49 3'- CyTyTyAxCxAxGxGxTxCxCxGyTyTyGyA -5', SEQ ID NO. 50 3'- GyGyCyCxGxTxGxTxTxCxGyCyTyGyT -5',SEQ ID NO. 49 3'- CyTyTyAxCxAxGxGxTxCxCxGyTyTyGyA -5 ', SEQ ID NO. 50 3'- GyGyCyCxGxTxGxTxTxCxGyCyTyGyT -5 ',
SEQ ID NO. 51 3'- TyCyAyCxCxCxCxTxCxTxTyTyCyTyGyG -5',SEQ ID NO. 51 3'- TyCyAyCxCxCxCxTxCxTxTyTyCyTyGyG -5 ',
SEQ ID NO. 52 3'- GyGyAyCxAxCxCxGxAxCxAyCyGyG -5',SEQ ID NO. 52 3'- GyGyAyCxAxCxCxGxAxCxAyCyGyG -5 ',
SEQ ID NO. 53 3'- AyAyCyGxGxGxAxGxCxGxAyTyGyG -5',SEQ ID NO. 53 3'- AyAyCyGxGxGxAxGxCxGxAyTyGyG -5 ',
SEQ ID NO. 54 3'- AyTyCyTxCxGxGxGxGxTxCxGyTyC -5',SEQ ID NO. 54 3'- AyTyCyTxCxGxGxGxGxTxCxGyTyC -5 ',
SEQ ID NO. 55 3'- AyAyAyGxAxAxCxGxAxAxAxGyGyAyA -5',SEQ ID NO. 55 3'- AyAyAyGxAxAxCxGxAxAxAxGyGyAyA -5 ',
SEQ ID NO. 56 3'- GyGyTxGxGxTxAxCxCyCyC -5',SEQ ID NO. 56 3'- GyGyTxGxGxTxAxCxCyCyC -5 ',
SEQ ID NO. 57 3'- CyCxCxGxGxTxAxCyTyGyA -5' undSEQ ID NO. 57 3'- CyCxCxGxGxTxAxCyTyGyA -5 'and
SEQ ID NO. 58 3'- CyCyAxCxAxGxAxAxAxGyAyAyC -5', wobeiSEQ ID NO. 58 3'- CyCyAxCxAxGxAxAxAxGyAyAyC -5 ', where
"X" unabhängig voneinander für eine Phosphodiester Internukleosid-Brücke oder eine modifizierte Internukleosid-Brücke steht und unabhängig voneinander für den Ersatz einer Zuckerphosphat Einheit oder einer ß-D-2 '-Deoxyriboseeinheit steht, wobei die modifizierte ß-D-2 '-"X" independently of one another stands for a phosphodiester internucleoside bridge or a modified internucleoside bridge and, independently of one another, stands for the replacement of a sugar phosphate unit or a β-D-2 ' deoxyribose unit, the modified ß-D-2' -
Deoxyriboseeinheit am 3 '-Ende von „y" lokalisierte ist.Deoxyribose unit is located at the 3 'end of "y".
12. Oligonukleotid nach Anspruch 11 , wobei „y" für 2'-O-Methyl-, 2'-O-Propyl-, 2'-Methoxyethoxy-ribose oder eine PNA Einheit steht.12. The oligonucleotide of claim 11, wherein "y" for 2'-O-methyl, 2 '-O-propyl, 2' methoxyethoxy-ribose or is a PNA unit.
13. Verwendung eines Oligonukleotids nach einem oder mehreren der Ansprüche 1 bis 12 zur Inhibition der Expression von Tenascin.13. Use of an oligonucleotide according to one or more of claims 1 to 12 for inhibiting the expression of tenascin.
14. Verwendung eines Oligonukleotids nach einem oder mehreren der Ansprüche 1 bis 12 als Werkzeug in der Molekularbiologie.14. Use of an oligonucleotide according to one or more of claims 1 to 12 as a tool in molecular biology.
15. Verwendung eines Oligonukleotids nach einem oder mehreren der Ansprüche 1 bis 12 als Diagnostikum.15. Use of an oligonucleotide according to one or more of claims 1 to 12 as a diagnostic agent.
16. Verwendung eines Oligonukleotids nach einem oder mehreren der Ansprüche 1 bis 12 als Arzneimittel. 16. Use of an oligonucleotide according to one or more of claims 1 to 12 as a medicament.
17. Verwendung eines Oligonukleotids nach einem oder mehreren der Ansprüche 1 bis 12 zur Herstellung eines Arzneimittels.17. Use of an oligonucleotide according to one or more of claims 1 to 12 for the manufacture of a medicament.
18. Arzneimittel enthaltend eines oder mehrere Oligonukleotide nach einem oder mehreren der Ansprüche 1 bis 12 sowie gegebenenfalls einen oder mehrere pharmazeutische Träger- und/oder Zusatzstoffe.18. Medicament containing one or more oligonucleotides according to one or more of claims 1 to 12 and optionally one or more pharmaceutical carriers and / or additives.
19. Verwendung eines Arzneimittels nach Anspruch 18 in Kombination mit Photochemotherapie und/oder der Transplantation von kultivierten Melanocyten und/oder der Behandlung mit Steroiden und/oder der Behandlung mit Plazenta- Extrakten.19. Use of a medicament according to claim 18 in combination with photochemotherapy and / or the transplantation of cultured melanocytes and / or the treatment with steroids and / or the treatment with placenta extracts.
20. Verfahren zur Herstellung eines Arzneimittels, wobei eine wirksame Dosis eines oder mehrerer Oligonukleotide nach einem oder mehreren der Ansprüche 1 bis 12 mit einem oder mehreren pharmazeutischen Träger- und/oder Zusatzstoffen gemischt wird.20. A method for producing a medicament, wherein an effective dose of one or more oligonucleotides according to one or more of claims 1 to 12 is mixed with one or more pharmaceutical carriers and / or additives.
21. Verfahren zur Herstellung eines Oligonukleotids nach einem oder mehreren der Ansprüche 1 bis 12, wobei das Oligonukleotid chemisch an einer Festphase synthetisiert wird.21. A method for producing an oligonucleotide according to one or more of claims 1 to 12, wherein the oligonucleotide is chemically synthesized on a solid phase.
22. Diagnostikum enthaltend ein oder mehrere Oligonukleotide nach einem oder mehreren der Ansprüche 1 bis 12.22. Diagnostic agent containing one or more oligonucleotides according to one or more of claims 1 to 12.
23. Testkit enthalten ein oder mehrere Oligonukleotide nach einem oder mehreren der Ansprüche 1 bis 12. 23. Test kit contain one or more oligonucleotides according to one or more of claims 1 to 12.
EP98954464A 1997-11-15 1998-10-29 Antisense oligonucleotides against tenascin for treating vitiligo Withdrawn EP1030914A2 (en)

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