EP1877069A2 - Neue verwendung von spiegelmeren - Google Patents

Neue verwendung von spiegelmeren

Info

Publication number
EP1877069A2
EP1877069A2 EP06742799A EP06742799A EP1877069A2 EP 1877069 A2 EP1877069 A2 EP 1877069A2 EP 06742799 A EP06742799 A EP 06742799A EP 06742799 A EP06742799 A EP 06742799A EP 1877069 A2 EP1877069 A2 EP 1877069A2
Authority
EP
European Patent Office
Prior art keywords
helix
nucleic acid
section
hmga
nucleotides
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP06742799A
Other languages
German (de)
English (en)
French (fr)
Inventor
Florian Jarosch
Christian Maasch
Sven Klussmann
Axel Vater
Dirk Eulberg
Werner Purschke
Klaus Buchner
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
TME Pharma AG
Original Assignee
Noxxon Pharma AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Noxxon Pharma AG filed Critical Noxxon Pharma AG
Priority to EP09016125A priority Critical patent/EP2206501A3/de
Publication of EP1877069A2 publication Critical patent/EP1877069A2/de
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates in one aspect to a new use of aptmers.
  • the present invention relates to HMG protein binding aptmers.
  • the target molecules can basically be divided into two groups.
  • the first group comprises target molecules which are present extracellularly and thus in principle be brought into contact with it by administering an active substance into a body fluid or a body cavity containing the target molecule.
  • the first group of target molecules are also referred to herein as extracellular target molecules.
  • the second group of target molecules includes target molecules that are present in cells, which cells are related to the disease to be treated or the disposition for the disease. It is not necessary that the target molecule is directly responsible for the disease state or is directly related to the disposition for the disease.
  • the second group of target molecules are also referred to herein as intracellular target molecules.
  • target molecule ie, extracellular or intracellular target molecule
  • target molecule basically determines the class of compounds that can be used to provide the therapeutic or preventive effect necessary interaction between the drug, typically the pharmaceutical agent, and the target molecule.
  • small molecules can be used almost ubiquitously, ie chemical compounds with a molecular weight of typically 1000 or less daltons. These molecules can interact directly with extracellular but also with intracellular target molecules as desired.
  • new classes of drugs have been developed by the biotechnology industry, such as antibodies, in particular monoclonal antibodies, antisense molecules, siRNA molecules, aptamers and aptmers.
  • aptamers and spiegelmers d. H. functional nucleic acids with a defined three-dimensional structure, which allows the specific interaction with the respective target molecules.
  • the use of aptamers to target intracellular target molecules uses genetic engineering methods, more specifically, gene therapy.
  • the aptamers directed against an intracellular target molecule, also called intramers are introduced into the respective target cells by means of genetic engineering methods.
  • intracellular expression of intracellular expression of an intracellular nucleic acid coding for the respective aptamer is in principle closed to the Spiegelmers since no biological system exists, which Spiegelmers, d. H. L-nucleotide aptamers would be able to build up.
  • the object is achieved in a first aspect by the use of an L-nucleic acid as intracellular active agent.
  • the L-nucleic acid is a spiegelmer.
  • the L-nucleic acid interacts with an intracellular receptor.
  • the intracellular receptor is selected from the group comprising molecular receptors, enzymes, chaperone molecules, signal peptides, intracellular structures and metabolic intermediates.
  • the intracellular receptor is selected from the group comprising polypeptides, carbohydrates, nucleic acids, lipids and combinations thereof.
  • the L-nucleic acid interacts with an intracellular receptor within a cell.
  • the intracellular receptor is selected from the group comprising transcription factors and an AT hook-binding DNA-binding protein.
  • the intracellular receptor is selected from the group comprising the HMG proteins, preferably from the group comprising HMGA1, HMGA1a, HMGA1b, and HMGA2.
  • the object is achieved in a second aspect by a method for binding an intracellular receptor comprising:
  • the incubation is carried out under conditions such that the L-nucleic acid binds to the intracellular receptor in the cell.
  • the L-nucleic acid is a spiegelmer.
  • a binding, in particular an intracellular binding, of the L-nucleic acid takes place at the intracellular receptor is determined after incubation of the cell with the L-nucleic acid, whether a binding, in particular an intracellular binding, of the L-nucleic acid takes place at the intracellular receptor is.
  • the intracellular receptor is selected from the group comprising molecular receptors, metabolic intermediates and enzymes.
  • the intracellular receptor is selected from the group comprising polypeptides, carbohydrates, nucleic acid, lipids and combinations thereof.
  • the intracellular receptor is selected from the group comprising transcription factors and an AT hook-binding DNA-binding protein.
  • the intracellular receptor is selected from the group comprising HMG proteins, preferably selected from the group comprising HMGA1, HMGA1a, HMGA1b and HMGA2.
  • the object is achieved in a third aspect by the use of an L-nucleic acid for the production of a medicament for the treatment and / or prevention of a disease, wherein the target molecule of the medicament is an intracellular target molecule.
  • the intracellular receptor is selected from the group comprising molecular receptors, enzymes, chaperone molecules, signal peptides, intracellular structures and metabolic intermediates.
  • the intracellular receptor is selected from the group comprising polypeptides, carbohydrates, nucleic acids, lipids, and combinations thereof.
  • the target molecule is selected from the group comprising transcription factors and an AT hook-binding DNA-binding protein.
  • the target molecule is selected from the group comprising HMG proteins, preferably selected from the group comprising HMGA1, HMGA1a, HMGA1b and HMGA2.
  • the disease is selected from the group comprising tumor diseases, viral infections and arteriosclerosis.
  • the tumor disease is selected from the group comprising mesenchymal tumors, epithelial tumors, benign tumors, malignant tumors and metastatic tumors.
  • the target molecule HMGA and the disease are selected from the group comprising carcinomas of the prostate, pancreas, thyroid, cervix, stomach, breast, colon / Rectum, ovaries; neuroblastomas; Lymphomas, uterine leiomyomas; lipomas; endometrial polyps; chondroid hamartomas of the lung; pleomorphic adenomas of the salivary glands; Hämangiopericytome; chondromatous tumors; aggressive angiomyomas; diffuse astrocytomas; osteoclastomas; Skin cancer; Burkitts lymphoma; Lewis lung cancer; Leukemia; non-small cell lung carcinoma; and in each case metastases and / or metastatic forms thereof.
  • arteriosclerosis is triggered or caused by HMGA1, HMGA1a, HMGIb and / or HMGA2 mediated formation of atherosclerotic plaques.
  • the intracellular target molecule is intracellular.
  • the object is achieved in a fourth aspect by the use of an L-nucleic acid for the production of a diagnostic agent for diagnosis, wherein the target molecule of the diagnostic agent is an intracellular target molecule.
  • the intracellular receptor is selected from the group comprising molecular receptors, enzymes, chaperone molecules, signal peptides, intracellular structures and metabolic intermediates.
  • the intracellular receptor is selected from the group comprising polypeptides, carbohydrates, nucleic acids, lipids and combinations thereof.
  • the target molecule is selected from the group comprising transcription factors and AT hook-binding DNA-binding proteins.
  • the target molecule is selected from the group comprising HMG proteins, preferably selected from the group comprising HMGA, HMGAIa, HMGAIb and HMGA2.
  • the disease is selected from the group comprising tumor diseases, viral infections and arteriosclerosis.
  • the tumor disease is selected from the group comprising mesenchymal tumors, epithelial tumors, benign tumors, malignant tumors, and metastatic tumors.
  • the target molecule is HMGA and the disease is selected from the group comprising carcinomas of the prostate, pancreas, thyroid, cervix, stomach, breast, Colon / rectum, ovaries; neuroblastomas; Lymphomas, uterine leiomyomas; lipomas; endometrial polyps; chondroid hamartomas of the lung; pleomorphic adenomas of the salivary glands; Hämangiopericytome; chondromatous tumors; aggressive angiomyomas; diffuse astrocytomas; osteoclastomas; Skin cancer; Burkitts lymphoma; Lewis lung cancer; Leukemia; non-small cell lung carcinoma; and in each case metastases and / or metastatic forms thereof.
  • arteriosclerosis is triggered by HMGA1, HMGA1a
  • the intracellular target molecule is intracellular.
  • the object is achieved in a fifth aspect by a composition comprising an L-nucleic acid binding to an intracellular target molecule and a delivery vehicle.
  • the delivery vehicle is a delivery vehicle suitable for intracellular delivery of the L-nucleic acid.
  • the delivery vehicle is selected from the group comprising vehicles, conjugates and physical measures.
  • the delivery vehicle is a vehicle, wherein the vehicle is selected from the group comprising liposomes, nanoparticles, microparticles, cyclodextrins or dendrimers, or a vesicle consisting of Polypeptides, polyethylenimine and / or amphipathic molecules.
  • the delivery vehicle is a conjugate, wherein the conjugate is a receptor-mediated endocytosis conjugate, a fusogenic peptide conjugate, a signal peptide conjugate, a conjugate with a nucleic acid, preferably a conjugate with a aptmer or a lipophilic conjugate.
  • the conjugate is a receptor-mediated endocytosis conjugate, a fusogenic peptide conjugate, a signal peptide conjugate, a conjugate with a nucleic acid, preferably a conjugate with a aptmer or a lipophilic conjugate.
  • the delivery vehicle is a physical measure, wherein the dimensional preferably is selected from the group comprising electroporation, iontophoresis, pressure, ultrasound and shock waves.
  • the delivery vehicle comprises polyethyleneimine.
  • the polyethyleneimine is a branched polyethyleneimine having a molecular weight of about 25 kDa.
  • the polyethyleneimine forms a micelle or a micelle-like structure.
  • the L-nucleic acid is a spiegelmer.
  • the aptmer carries a modification, wherein the modification is selected from the group comprising PEG residues.
  • the PEG moiety has a molecular weight of about 1,000 to 10,000 Da, preferably a molecular weight of about 1,500 to 2,500 Da, and most preferably a molecular weight of about 2,000 Da ,
  • the modification is bound to the 5 'terminus or the 3' terminus of the L-nucleic acid.
  • the ratio of the total number of the nitrogen groups of the polyethyleneimine and the total number of the phosphate groups of the nucleic acid contained in the composition is about 1 to 20, before- usually about 1.5 to 10, more preferably about 2 to 5, and most preferably about 2 to 3.
  • the composition provides the L-nucleic acid intracellularly.
  • the object is achieved in a sixth aspect by the pharmaceutical composition
  • the pharmaceutical composition comprising a composition according to the fifth aspect, and a pharmaceutically acceptable carrier.
  • the L-nucleic acid is a composition according to the fifth aspect.
  • the L-nucleic acid is a composition according to the fifth aspect.
  • the L-nucleic acid is a composition according to the fifth aspect.
  • the L-nucleic acid is a composition according to the fifth aspect.
  • an HMGA-binding nucleic acid characterized in that the nucleic acid comprises a section box Al and a section box A2, wherein the section box Al and the section box A2 are interconnected by an intermediate section and wherein Box Al and Box A2 are individually and independently selected from the group comprising GGGCG, GGGUG and GGGAG.
  • the intermediate section consists either of an intermediate section Z1 comprising six or seven nucleotides or of an intermediate section Z2 comprising 12 to 25 nucleotides.
  • the nucleic acid at the 5 'end of the section Box Al has a first portion and at the 3' end of the portion Box A2 a second portion, preferably both Sections independently comprise four to eight nucleotides each.
  • the two sections are at least partially or completely hybridized with each other, wherein the hybridization extends over four to eight nucleotide pairs.
  • the nucleic acid has a section Helix A1 at the 5 'end of the section Box Al and a section Helix A2 at the 3' end of the section Box A2 , wherein preferably the portion Helix Al comprises four to eight nucleotides, and preferably the portion Helix A2 comprises four to eight nucleotides, and wherein preferably the portion helix Al represents the first portion at the 5 'end of the portion box Al or a part thereof, and wherein preferably the section Helix A2 represents the second section at the 3 'end of the section Box A2 or a part thereof, wherein the length of the section Helix Al is independent of the length of the section Helix A2.
  • the sections Helix Al and Helix A2 are at least partially or completely hybridized with each other, the hybridization extending over four to eight nucleotide pairs.
  • a section Helix B2 is arranged, wherein preferably the length of the section Helix Bl and Helix B2 individually and independently comprises a length of four to eight nucleotides.
  • the sections Helix Bl and Helix B2 are at least partially or hybridized completely to each other, wherein the hybridization extends over four to eight nucleotide pairs.
  • nucleotides are arranged between the 3 'end of the section Helix Al and the 5' end of the section Helix Bl.
  • nucleotides are located between the 3 'end of the Helix Al section and the 5' end of the Helix Bl section.
  • nucleotides are arranged between the 3 'end of the section Helix B2 and the 5' end of the section Helix A2.
  • a nucleotide is between the 3 'end of the helix B2 portion and the 5' end of the helix A2 portion arranged.
  • the sum of nucleotides of section Helix A1 and section Helix Bl is 10 to 12 nucleotides and the sum of nucleotides from section Helix A2 and from section Helix B2 10 to 12 nucleotides.
  • the sum of the hybridized nucleotides resulting from the hybridization of section Helix A1 with section Helix A2 and section Helix Bl with section Helix B2 is 10 to 12 nucleotide pairs.
  • the nucleic acid does not comprise a section Helix Al and Helix A2, which at the 5 'end of the nucleic acid portion Helix Bl and at the 3' end the helix B2 is arranged, wherein preferably the length of the section Helix Bl and Helix B2 each individually and independently comprises a length of four to eight nucleotides.
  • the sections Helix Bl and Helix B2 are at least partially or completely hybridized with each other, wherein the hybridization extends over four to eight nucleotide pairs.
  • between the 3 'end of the section Helix Al and the 5' end of the section Box Al are one to five nucleotides and between the 3 ' End of the section box A2 and the 5 'end of the section Helix A2 one to three nucleotides arranged.
  • a seventeenth embodiment of the seventh aspect which is also an embodiment of the sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth and fifteenth embodiment, are between the 3 'end of the section Helix Bl and the 5' - End of the section box Al two nucleotides and arranged between the 3 'end of the section box A2 and the 5' end of the section helix B2 one to seven nucleotides.
  • an eighteenth embodiment of the seventh aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth and tenth embodiments insofar as it is an embodiment of the sixth, seventh and eighth embodiments, the twelfth and thirteenth embodiments Embodiment, as far as these embodiments of the sixth, seventh, eighth and tenth embodiments, the fourteenth and fifteenth embodiment, as far as these embodiments of the sixth, seventh, eighth, tenth, twelfth and thirteenth embodiment, or the seventeenth embodiment, as far as these embodiments of the sixth , eighth, tenth, twelfth, thirteenth and fifteenth embodiments, each within the scope limited herein, includes the intermediate section Zl six or seven nucleotides.
  • the intermediate portion Zl has the sequence NiN 2 GNgN 3 N 4 Ns, wherein
  • the nucleic acid comprises a section Box Al and a section Box A2, with the 3 'end of the section Box Al directly connected to the 5' end of the intermediate section Zl is connected, and the 3 'end of the intermediate portion Zl is connected directly to the 5' end of the section box A2.
  • the nucleic acid comprises a section Helix Bl and a section Helix B2.
  • the sections Helix Bl and Helix B2 each individually and independently of each other comprise four to eight nucleotides, which are preferably fully or partially hybridized with each other.
  • helix is provided between the 3 'end of the section box A2 and the 5' end of the section B2 no nucleotide, or arranged in the 5'-3 'direction, the nucleotide sequence GN y , wherein N y comprises zero to six nucleotides, preferably 0 or 6 nucleotides.
  • the nucleic acid comprises a section Helix A1 and Helix A2.
  • the sections Helix A1 and Helix A2 each independently and independently of each other comprise four to eight nucleotides, and preferably the sections Helix A1 and Helix A2 are completely or partially hybridized with each other.
  • a nucleotide sequence N x is disposed between the 3 'end of the section Helix Al and the 5' end of the section Helix Bl, where N x zero to five nucleotides.
  • a nucleotide sequence N z is disposed between the 3 'end of the helix B2 portion and the 5' end of the helix A2 portion N 2 zero to six nucleotides.
  • a twenty-ninth embodiment of the seventh aspect which is also an embodiment of the twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh and twenty-eighth embodiments, the sum of the hybridized nucleotides from the hybridization of section Helix A1 with section Helix A2 and of Section Helix Bl with section Helix B2 10 to 12 nucleotide pairs.
  • a thirtieth embodiment of the seventh aspect which is also an embodiment of the twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth and twenty-ninth embodiments, between the 3 'end of the section Box A2 and the 5' end of the section helix B2 in FIG '-3' direction the nucleus nucleotide GN y , where N y comprises zero to six nucleotides, preferably 0 or 6 nucleotides.
  • the HMGA-binding nucleic acid has the following structure
  • N 1 U, C, A or G;
  • N 2 G or U
  • N 3 U or C
  • N 4 U or A
  • N 5 G or A
  • N 6 G, A or U
  • N 7 G or U
  • N 8 U or no nucleotide
  • N x zero to five nucleotides
  • N 2 zero to six nucleotides
  • section Box Al and section box A2 are each individually and independently selected from the group of nucleotide sequences comprising GGGCG, GGGUG and GGGAG;
  • section Helix A1 and the section Helix A2 each individually and independently comprise four to eight nucleotides, wherein preferably the sections Helix Al and Helix A2 are completely or partially hybridized with each other, and
  • the sections Helix Bl and Helix B2 each individually and independently comprise four to eight nucleotides, wherein preferably the sections Helix Bl and Helix B2 are completely or partially hybridized with each other and the hybridizing region four to eight Nucleotides, and wherein the sum of the hybridized nucleotides from the hybridization of section Helix Al with section Helix A2 and of section Helix Bl with section Helix B2 is 10 to 12 nucleotide pairs.
  • the HMGA-binding nucleic acid comprises a sequence selected from the group comprising SEQ. ID. No. 1, SEQ. ID. No. 2, SEQ. ID. No. 3, SEQ. ID. No. 5, SEQ. ID. No. 6, SEQ. ID. No. 7 and SEQ. ID. No. 13th
  • the 3 'end of the section Box A2 immediately adjoins the 5' end of the section Helix B2 ,
  • the HMGA-binding nucleic acid has the following structure
  • N 2 G or U
  • N 3 U or C
  • N 4 U or A
  • N 5 G or A
  • N 6 G, A or U
  • N 7 G or U
  • Ng U or no nucleotide
  • N x zero to five nucleotides
  • N z zero to six nucleotides;
  • the section Box Al and section box A2 are each individually and independently selected from the group of nucleotide sequences comprising GGGCG, GGGUG and GGGAG;
  • section Helix A1 and the section Helix A2 each individually and independently comprise four to eight nucleotides, wherein preferably the sections Helix Al and Helix A2 are completely or partially hybridized with each other, and
  • the sections Helix Bl and Helix B2 each individually and independently comprise four to eight nucleotides, wherein preferably the sections Helix Bl and Helix B2 are fully or partially hybridized with each other and the hybridizing region comprises four to eight nucleotides, and wherein the sum of the hybridized nucleotides from the hybridization of section Helix Al with section Helix A2 and section Helix Bl with section Helix B2 is 10 to 12 nucleotide pairs.
  • the HMGA-binding nucleic acid comprises a sequence comprising SEQ. ID. No. Third
  • the HMGA-binding nucleic acid has the following structure
  • the HMGA-binding nucleic acid has the following structure
  • the HMGA comprises binding nucleic acid a sequence selected from the group comprising SEQ. ID. No. 15 and SEQ. ID. No. 16th
  • the HGMA-binding nucleic acid comprises an intermediate section Z 2 of 12 to 25 nucleotides.
  • the HGMA binding nucleic acid comprises an intermediate portion Z2, a portion Helix C1, and a portion Helix C2.
  • a central portion N c is disposed between the helix Cl portion and the helix C2 portion of the HMGA-binding nucleic acid.
  • the length of the helix C1 and helix C2 portion of the HGMA-binding nucleic acid is the same length.
  • the length of the helix Cl and helix C2 portion of the HGMA-binding nucleic acid is individually and independently three to six nucleotides.
  • helix Cl and helix C2 portions of the HMGA-binding nucleic acid are fully or partially hybridized with each other.
  • the central portion N c of the HMGA-binding nucleic acid comprises three to five nucleotides.
  • the HMGA-binding nucleic acid comprises a section Box Al and a section box A2, wherein between the 3 ' End of the section Box Al and the 5 'end of the section Helix Cl is a nucleotide sequence N b is arranged and comprises three nucleotides.
  • the HMGA-binding nucleic acid comprises a section Box Al and a section box A2, wherein between the 3'-end of the section Helix C2 and the 5 'end of the section Box A2 a nucleotide sequence N d is arranged and comprises two to five nucleotides.
  • the HMGA-binding nucleic acid comprises a section Helix A1 and a section Helix A2.
  • the sections Helix A1 and Helix A2 of the HMGA-binding nucleic acid each individually and independently of each other comprise five to six nucleotides, preferably the section Helix A1 and the section Helix A2 completely or partially hybridized with each other.
  • a nucleotide sequence N a is arranged between the 3 'end of the section Helix Al and the 5' end of the section Box Al of the HMGA-binding nucleic acid, wherein N 3 comprises one to five nucleotides.
  • a nucleotide sequence GN e between the 3 'end of the box A2 section and the 5' end of the helix A2 section of the HMGA binding nucleic acid in FIG '-3' direction a nucleotide sequence GN e , where N e comprises one to two nucleotides, preferably A or UU.
  • the helix Cl and helix C2 portions of the HMGA-binding nucleic acid are each independently and independently of one another five or six nucleotides in length , wherein preferably the sections Helix Cl and Helix C2 are completely or partially hybridized with each other.
  • the HMGA-binding nucleic acid has the following structure:
  • N 3 one to five nucleotides
  • N b three nucleotides
  • N c three to five nucleotides
  • N d two to five nucleotides
  • N e one to two nucleotides, preferably A or UU;
  • section Box Al and the section box A2 are each individually and independently selected from the group comprising GGGCG, GGGUG and GGGAG,
  • the sections Helix A1 and Helix A2 each individually and independently comprise five or six nucleotides, and ⁇
  • the sections helix Cl and helix C2 each comprise five or six nucleotides, which are preferably fully or partially hybridized with each other.
  • the HMGA-binding nucleic acid comprises a sequence selected from the group comprising SEQ. ID. No. 8, SEQ. ID. No. 9, SEQ. ID. No. 10, SEQ. ID. No. 11, SEQ. ID. No. 14, SEQ. ED. No. 22 and SEQ. ID. No. 24th
  • the nucleic acid comprises a portion of box Al and a portion of helix Cl of the HMGA-binding nucleic acid, wherein between the 3 ' End of the section box Al and the 5 'end of the section Helix Cl is a nucleotide A.
  • the HMGA-binding nucleic acid comprises a section helix C2 and a section box A2, wherein between the 3 'end of the section helix C2 and the 5' end of the Section box A2 a nucleotide G is arranged.
  • the central portion N c of the HMGA-binding nucleic acid comprises four nucleotides, wherein N c is preferably GAUG.
  • the HMGA-binding nucleic acid comprises a section Helix Bl and a section Helix B2.
  • the sections Helix Bl and Helix B2 of the HMGA-binding nucleic acid individually and independently each five Nucleotides, wherein preferably the section Helix Bl is hybridized with the section Helix B2.
  • a sixtieth embodiment of the seventh aspect which is also an embodiment of the fifty-eighth or fifty-ninth embodiment, between the 3 'end of section Helix Bl and the 5' end of section Box Al of the HMGA-binding nucleic acid is a nucleotide sequence comprising two nucleotides N j , where N j is preferably AG.
  • a nucleotide G is located between the 3 'end of the box A2 section and the 5' end of helix B2 of the HMGA binding nucleic acid.
  • the HGMA-binding nucleic acid comprises a section Helix A1 and a section Helix A2.
  • the sections Helix A1 and Helix A2 of the HMGA-binding nucleic acid individually and independently each six nucleotides and are preferably the section Helix Al and the section Helix A2 hybridized with each other ,
  • a nucleotide sequence comprising two nucleotides Nj is located between the 3 'end of section Helix Al and the 5' end of section Helix Bl, where Nj preferably CA is.
  • a nucleotide A is located between the 3 'end of the helix B2 section and the 5' end of the helix A2 section.
  • the sections helix Cl and helix C2 each comprise three nucleotides, wherein preferably the sections helix Cl and helix C2 are hybridized with each other.
  • the HMGA-binding nucleic acid has the following structure:
  • the sections Box Al and Box A2 are each individually and independently selected from the group comprising the sequences GGGCG, GGGUG and GGGAG;
  • the sections Helix A1 and Helix A2 each individually and independently comprise six nucleotides, which are preferably hybridized with each other;
  • the sections Helix Bl and Helix B2 individually and independently comprise five nucleotides, whereby preferably the section Helix Bl and the section Helix B2 are hybridized with each other;
  • the sections helix Cl and helix C2 each individually and independently comprise three nucleotides, wherein preferably the sections helix Cl and helix C2 are hybridized with each other.
  • the HMGA-binding nucleic acid comprises a sequence selected from the group comprising SEQ. ID. No. 12th
  • the nucleic acid is one which binds to transcription factors, particularly transcription factors having an AT-hook.
  • the object is achieved in an eighth aspect by a nucleic acid which binds to a transcription factor comprising an AT hook, wherein the nucleic acid comprises a construction according to the seventh aspect.
  • the L-nucleic acid is a nucleic acid according to the seventh aspect.
  • the L-nucleic acid is a nucleic acid according to the seventh aspect.
  • the L-nucleic acid is a nucleic acid according to the seventh aspect.
  • the L-nucleic acid is a nucleic acid according to the seventh aspect.
  • the L-nucleic acid is a nucleic acid according to the seventh aspect.
  • the object is achieved in a ninth aspect by a method for screening an HMGA antagonist or HMGA agonist comprising the following steps:
  • the object is achieved in a tenth aspect by a method for screening an HMGA agonist and / or an HMGA antagonist comprising the following steps:
  • HMGA immobilized on one phase preferably a solid phase
  • nucleic acid according to the seventh aspect preferably a nucleic acid according to the seventh aspect, which is labeled
  • the determination is made to test whether the nucleic acid is being replaced by the candidate HMGA agonist or by the candidate HMGA antagonist.
  • the object is achieved in an eleventh aspect by a kit for the detection of HMGA comprising a nucleic acid according to the seventh aspect.
  • the object is achieved in a twelfth aspect by an HMGA antagonist obtainable by a method according to the tenth aspect.
  • the object is achieved in a thirteenth aspect by a HMGA agonist obtainable by a method according to the tenth aspect.
  • the object is achieved in a fourteenth aspect by a complex comprising an HMGA protein and a nucleic acid according to the seventh aspect.
  • the present invention is based on the surprising finding that contrary to the prior art it is possible to use L-nucleic acids and in particular aptmers to address intracellular target molecules.
  • the intracellular target molecules are preferably target molecules present in a cell. It allows the functional L-nucleic acids by their structure of L-nucleotides inherent properties such as high specificity of the interaction with their target molecules with high stability and lack of toxic or immunologically active degradation products when applied in biological systems and in particular in the animal and human body not to use the cellular mechanisms to encode, as in the case of intramers, L-nucleic acids from a plasmid or, more generally, a vector and thus to provide the actual functional nucleic acid through the intracellular process of transcription.
  • Functional L-nucleic acids and in particular aptmers can be transported while maintaining their specificity with respect to their binding to their target molecule and their activity via a cytoplasmic membrane.
  • This patency of the functional L-nucleic acids is inherent in the Spiegelmers and may be exacerbated by the use of delivery vehicles or delivery techniques.
  • the present inventors assume that functional L-nucleic acids per se can overcome the cytoplasmic membrane or, in the case of involvement of endosomal transport mechanisms in overcoming the cytoplasmic membrane, they can become involved forming vesicle structures capable of releasing a two- or three-dimensional structure that allows the specific interaction of the functional nucleic acid with its target molecule.
  • the present teachings deliberately turn away from the aptamer-based principle of utilizing intracellular transcription mechanisms to produce aptamers in the cell and, for the first time, provide means for the application of functional L-nucleic acids, and in particular aptmers, to cells.
  • functional nucleic acids refers to those nucleic acids other than structural, especially naturally occurring, structural nucleic acids such as rRNAs or coding nucleic acids such as mRNAs.
  • functional nucleic acids are nucleic acids which, due to their two- and / or three-dimensional structure, are capable of binding to target molecules.
  • binding to the target molecule is not by hybridization or base pairing based on Watson-Crick base pairings or Hoogsteen base pairing.
  • Particularly preferred functional nucleic acids are aptamers and aptmers.
  • an L-nucleic acid is a nucleic acid which is completely, substantially or partially constructed of L-nucleotides. It is particularly preferred if the L-nucleic acid consists entirely of L-nucleotides.
  • the term "essentially” designates an embodiment in which that part of the L-nucleic acid which is responsible for the interaction with the target molecule or the part which mediates the binding to the target molecule consists of L-nucleotides or these is constructed.
  • a functional L-nucleic acid is a functional nucleic acid that is wholly, substantially or partially constructed of L-nucleotides.
  • L-nucleic acids The synthesis of L-nucleic acids is known to those skilled in the art and described, for example, in Nolte et al., Nat. Biotech, 14, 1116-1119, 1996; and Klussmann et al, Nat. Biotechnol, 14, 1112-1115, 1996.
  • aptamers consisting of L nucleotides rather than D nucleotides.
  • an intracellularly active agent is a chemical compound capable of binding to a molecule in a cell. It is particularly preferred if the cell is a cell isolated in a tissue or organ, but more preferably not in a human or animal body. If the intracellularly active agent is a aptmer, then it is preferably an intracellularly active agent if it is capable of binding to an intracellular target molecule.
  • the aptmer is an intracellularly active agent if it is capable of binding to its target molecule under conditions such as are present in a cell.
  • Assays to determine these properties are known to those of skill in the art and include, for example, equilibrium binding assays under buffer conditions as they are intracellular (ionic strength and parent compound, pH, temperature) as disclosed in Example 1.
  • the target molecule of the L-nucleic acid, in particular of the functional L-nucleic acid is an intracellular receptor.
  • An intracellular receptor as used herein is preferably a chemical compound or a chemical structure, or a portion thereof, with which the functional L-nucleic acid interacts, and preferably one to which the functional L-nucleic acid binds the intracellular receptor, ie the chemical compound or the chemical structure or a part thereof, intracellular, d. H. is present in a cell, as it is preferably described in the preceding paragraph, is present. It is within the scope of the present invention that the intracellular receptor is the target molecule in the production of the functional nucleic acid, in particular of the functional L-nucleic acid.
  • the term receptor refers to any interaction partner, preferably a specifically binding interaction partner of the functional nucleic acid, ie an interaction partner interacting with the functional nucleic acid, which have a specific spatial structure, charge distribution, hydrophobicity distribution, etc.
  • the interaction partner corresponds to the target molecule of the functional nucleic acid as used in the production of the functional nucleic acid. It is within the scope of the present invention that a receptor can also be different from the target molecule used in the production of the functional nucleic acid, but the specific interaction by a Cross-reactivity of the functional nucleic acid between the interaction partner and the target molecule used in the production of the functional nucleic acid is caused.
  • the term "intracellular receptor” refers to a receptor that is present in a cell, or a receptor that may be present in a cell, naturally occurring in a cell, or present among such in a cell
  • the term "intracellular receptor” also refers to a receptor that is in conditions such as they are in a cell.
  • the term cell refers to a cell selected from the group comprising prokaryotic and eukaryotic cells.
  • the eukaryotic cell is selected from the group comprising fungal cells, plant cells, animal cells and human cells.
  • the term cell herein generally refers to a compartment bounded by a phospholipid double membrane, which in a preferred embodiment corresponds to a cytoplasmic membrane, which is separated from the environment by the membrane. The separation from the environment is not a complete separation, but allows an energy exchange and a mass transfer between the cell and the environment. Preferably, the mass transfer is limited.
  • the restriction of mass transfer is defined in the case of delimitation of the cell from the environment by a cytoplasmic membrane or by a membrane similar to a cytoplasmic membrane by the transport properties of the membrane.
  • the term "cell” herein thus also encompasses vesicles and / or compartments of a prokaryotic or eukaryotic cell as defined herein, which in turn may be present or present within a prokaryotic or eukaryotic cell as well as outside such a prokaryotic or eukaryotic cell
  • a vesicle or parts of a prokaryotic or eukaryotic cell surrounded by a cytoplasmic membrane which in one embodiment may be in a body fluid.
  • the conditions within such a cell substantially correspond to those which are present in a prokaryotic or eukaryotic cell, in particular the factors which affect the binding of the functional nucleic acid to it Influence the target molecule.
  • the body fluid is selected from the group consisting of blood, urine, cerebrospinal fluid, lymphatic fluid, serum, plasma, vaginal secretions, saliva and semen.
  • the receptor is defined by its function in a cell. Accordingly, the receptor may be selected from the group consisting of molecular receptors, enzymes, metabolic intermediates, signal peptides, chaperone molecules, and intracellular structures such as ribosomes, mitochondria, cytoskeletal elements such as cytoskeletal cells. Tubulin and actin filaments, endosomal particles, lysosomes, other intracellular structures such as vesicles, in particular intracellular vesicles comprises.
  • the term molecular receptor refers to a molecule that receives and propagates information within a cell, tissue, organ, or organism. The information is typically mediated by a molecule that interacts with the molecular receptor.
  • the molecular receptor is able to produce a signal.
  • a signal may be due to or manifest in the change in conformation and / or activity of the receptor.
  • the signal itself is able to relay the recorded or represented information in a different form.
  • the signal may preferably be a chemical, biochemical or electrical signal.
  • the molecular receptor is part of a reaction cascade, more preferably a signal cascade.
  • the information transmitted by a molecular receptor may be quantitative and / or qualitative information, for example regarding the presence of a compound and / or its concentration.
  • metabolite intermediates refers to all those compounds which occur as a component of catabolism as well as anabolism through metabolic activities in a cell.
  • the receptor is defined by its chemical nature.
  • the receptor is selected from the group comprising polypeptides, carbohydrates, nucleic acid, lipids, and combinations thereof.
  • polypeptide preferably refers to any polymer of two or more amino acids.
  • the amino acids are L-amino acids, but also D-amino acids can be used in the context of the embodiment.
  • nucleic acids preferably refers to a polymer of two or more nucleotides or nucleotide analogs known to those skilled in the art, which are either D nucleotides or L nucleotides or mixtures thereof.
  • Preferred combinations include glycosylated polypeptides and glycosylated lipids
  • a particular group of intracellular receptors are transcription factors and DNA-binding proteins that bind to an AT hook.
  • Exemplary transcription factors are given in the following Table 1:
  • AhR Arnt AML3b ATBF 1 -A
  • ER-alpha ER-beta EZH 1 FOXJ2 (long isoform)
  • FXR RXR-alpha GLI3 HEB1-p94
  • IA-1 IRF-1 C / EBPbeta KLF15 iASPP IRF-2 KLF7 iASPP-RAI IRF-3 Kox1
  • LXR-alpha RXR-alpha MEF-2C / delta8.32 MTF-1
  • Nrf1 MafG p160MBP Pax-4a
  • Nrf1 MafK p28 Modulator Pax-5
  • Nrf2 MafG p38erg Pax-6 / Pd-5a
  • Nrf2 MafK p40x; HTLV-I, T-cell Pax-7
  • Nrf3 MafK p49erg Pax-8c
  • Pbx1A HoxA5 PITX2A: Nkx2.5 POU3F4
  • Pbx1a Hoxb8 PITX2B: Nkx2.5 POU4F1 (s)
  • Pbx1A HoxC8 PITX2C: Nkx2.5 POU4F3
  • Pbx1A HoxD4 PITX3 POU5F1
  • Pbx1 a IPF1 PKN0X1 POU5F1A
  • Pbx1 B HoxA5 PLAGL1 POU5F1C
  • Pbx1 B HoxB7 PLAGL2 POU6F1
  • Pbx1B HoxB8 PLZF PPAR-alpha
  • Pbx1B HoxC8 PML PPAR-alpha: RXR-
  • Pbx1 B HoxD4 PML-3 alpha
  • Pbx1b PKN0X1 Pmx2a PPAR-beta
  • RAR-alpha RXR-alpha RORalphal Six-3
  • RAR-alpha RXR-beta RORalpha2 Six-4
  • RAR-alpha RXR-RORalpha3 Six-5 gamma RORbeta Six-6
  • RAR-beta RXR-alpha RPF1 Smad2
  • Sox14 STAT1 STAT3 TAF (II) 31
  • Sox7 STAT5B STAT5B TaMbeta Tal-2 TCF-1D TFIID
  • TCF-1 E TFIIE did; HIV-1, immunode- TCF-1F TFIIE-alpha efficiency virus type 1 TCF-1G TFIIE-beta
  • TR2-5 VITF VITF
  • Intracellular target molecules Another group of intracellular receptors are the intracellular target molecules shown in Table 2 below. Table 2: Intracellular target molecules
  • Acetyl-CoA-carboxylase amyloid ⁇ I A4-protein catalase Catechol dihydrouracil glycerol phosphate
  • Hemoglobin M Iwate Hydroxysteroid N-Acetylglucuronidase Hemoglobin M Saskatoon Dehydrogenase NAD-dependent Sterol-4 Hemoglobin Nancy Hypoxanthine Guanine Carboxylase Hemoglobin Philly Phosphoribosyltransferase NADase Hemoglobin Quong Sze IMP Dehydrogenase NADPH-dependent 3-Hemoglobin Raleigh Indollyase Oxosteroid Reductase Hemoglobin Ranier Inositol Phosphate Nexin Hemoglobin S Phosphatase N-ras Oncogene protein Hemoglobin Sealy int-1 Oncogene protein Nuclear protein B23 Hemoglobin Seattle isocitrate lyase Nucleoside diphosphate hemoglobin St.
  • HMGA proteins preferably denotes, in summary, the following proteins: HMGA1, HMGA1a, HMGA1b, and HMGA2.
  • HMGA proteins are modular and each have three DNA-binding domains, called “AT-hooks” or “AT-hooks”, represented as DBD1 to DBD3 in Figure 2, and a very acidic C- terminal area. It is obvious to those skilled in the art that antagonists that bind to one of the "AT-hooks” not only recognize the HMGA1 proteins and thereby the two splice variants HMGAIa and HMGAIb (see FIG. 2), but also cross-reactivity with similar ones In addition to HMGA2, many other proteins have sequences similar to "AT-hooks" and are in any case other receptors. Such proteins include those listed in Table 3:
  • the present invention also relates to L-nucleic acids and in particular Spiegelmers which are directed against any of the target molecules mentioned in Tables 1 to 3.
  • L-nucleic acid as an intracellularly active agent, particularly within a cell, to bind to an intracellular receptor, intracellularly various forms of interactions between the intracellular receptor and its interaction partners are influenced.
  • the intracellular use of L-nucleic acids thus makes it possible to influence interactions of proteins, nucleic acids, lipids, carbohydrates or combinations of proteins, nucleic acids, lipids, carbohydrates with one another and with one another.
  • an L-nucleic acid in particular a Spiegeimers
  • an intracellular agent or the method for binding an intracellular receptor is noteworthy that it is preferably an in vitro application or an in vitro method.
  • the target molecule is an intracellular target molecule.
  • the intracellular target molecule is one which is causally or non-causally involved in the disease or disease to be prevented, treated or diagnosed, but in each case results in its binding to an L-nucleic acid specifically binding thereto; that in the case of a drug, the disease is alleviated, prevented, or cured and / or in the case of a diagnostic agent, the disease or a disposition can be detected or diagnosed.
  • diagnosis includes a first diagnosis as well as subsequent diagnoses, particularly diagnoses or examinations, to, for example, track or determine the course of the disease or the stages of the disease.
  • the target molecule is an intracellular receptor as described herein, in particular a transcription factor, an intracellular target molecule or an HMG protein. It is very particularly preferred in the context of the present invention that the target molecule is present intracellularly, ie within a cell and the interaction influencing the disease and / or diagnosis between the L-nucleic acid and in particular the spiegelmer and the target molecule, ie the receptor, intracellularly.
  • the target molecule is outside of a cell and the interaction between the L- Nucleic acid and especially the Spiegeimer and the target molecule, ie the receptor, extracellular takes place.
  • HMGA proteins are known to be associated with carcinomas (including the breast, lung, skin, thyroid), as well as leukemias and lymphomas, as well as other malignant tumors such as the like.
  • Sarcomas rhabdomyosarcoma, osteosarcoma
  • mesenchymal tumors i.a.
  • HMGA Hamartomas (breast and lung), adipose tissue tumors (lipomas), pleomorphic adenomas of the salivary glands, uterine leiomyomas, angiomyxomas, breast fibroadenomas, polyps of the endometrium, and atherosclerotic plaques.
  • HMGA is an interesting therapeutic target.
  • Blockade of HMGA may be a suitable starting point for the control of cancer and prevent its metastatic spread.
  • L-nucleic acids directed against HMGA proteins are also useful for the diagnosis and / or therapy of viral diseases and arteriosclerosis due to the involvement of the HMGA proteins in the regulation of transcription of a variety of viral genes or the high expression of HMGA and in particular HMGA1 in the atherosclerosis affected tissue associated with neointimal vascular smooth muscle cells, macrophages and new blood vessels
  • nucleic acids preferably L-nucleic acids and preferably spiegelmers are capable of penetrating as such a phospholipid double membrane as a cytoplasmic membrane and then being intracellularly functional in the sense of - specific - interaction with the intracellular receptor
  • the efficacy of the introduction of L-nucleic acid can be influenced and in particular increased by the use of different techniques. These techniques include the use of chemical compounds or molecules as well as the use of physical measures. Whatever the nature, these techniques are generally referred to herein as delivery vehicles.
  • aptamers also have this property and, like the aptamers thus also together with the erfmdungsge- According to the composition can be used or used for the basically same purposes, applications and uses.
  • nucleic acid When using chemical compounds or molecules, it is further possible to distinguish whether the nucleic acid has to be modified for delivery or whether it does not have to be modified.
  • Modification for purposes of using a delivery vehicle is typically not required when the delivery vehicle is or comprises a vesicle, such as in liposomes, polypeptide vehicles, cyclodextrins, dendrimers, nano- and microparticles, and polyethyleneimine.
  • modification for purposes of using a delivery vehicle is typically required when the delivery vehicle uses receptor-mediated endocytosis, fusogenic peptides, signal peptides, or biopsy conjugates.
  • the group of physical techniques includes in particular electroporation and iontophoresis.
  • a phospholipid double membrane such as a cytoplasmic membrane
  • a functional nucleic acid such as an aptamer and / or a gelator
  • Liposomes consist of artificial cationic lipids such as N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA) and N- [1- (2,3-dioleoyloxy) propyl] - N, N, N-trimethylammonium sulfate (DOTAP), in which the cationic groups interact with the negatively charged nucleic acids and neutralize their anionic charge. Transport is via endocytosis (PNAS, 93: 11493-11498, 1996).
  • DOTMA N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride
  • DOTAP N- [1- (2,3-dioleoyloxy) propyl] - N, N, N-trimethylammonium sulfate
  • Transport is via endocytosis (PNAS, 93:
  • cationic liposomes are cytotoxic, especially at higher concentrations, limiting their use in vitro and in vivo (Biochem Biophys Res Commun, 197: 818, 1993, Biochem Biophys Res Commun, 1372: 55-68, 1998).
  • a non-toxic formulation is the amphiphilic pyridinium-based lipid SAINT-2 (Nucleic Acids Res, 29: 2079-2087, 2001).
  • An alternative are also pH-sensitive liposomes consisting of amphipatic molecules such as cholesteryl hemisuccinate (CHEMS) and dioleoylphosphatidylethanolamine (DOPE) (J Pharmacol Exp Ther, 297: 1129-1136, 2001).
  • CHEMS cholesteryl hemisuccinate
  • DOPE dioleoylphosphatidylethanolamine
  • Receptor-mediated endocytosis utilizes transport mechanisms that are already present in the cell membrane.
  • the nucleic acid is covalently linked to a transporter protein ("carrier" protein) via a poly-L-lysine (PPL) linker, the selection of the transporter protein being dependent on the ability to particular receptors of the cell membrane
  • PPL poly-L-lysine
  • Cell-specific transport can be achieved, for example, by targeting an antisense phosphorothioate directed against c-myc into M-14 human melanoma cells (Anticancer Res, 17: 29-35
  • effective delivery by RME depends not only on the affinity of the receptor for the ligand but also on the limitation of the receptor selected on the cells, especially in vivo, and the inactivated or amplified ligand must be obtained be on the therapeutic result to avoid possible toxicity of the transport vehicle.
  • Fusogenic peptides have been used to allow peptide-oligonucleotide conjugates to fuse with the cell membrane and thus to facilitate transport into the cell (Bioconjug Chem., 9: 466-475, 1998; Bioconjug Chem., 6: 43-53 , Nucleic Acids Research, 25: 2730-2736, 1997).
  • NLS nuclear localization signals
  • IP signal import peptides
  • Blocks of polypeptides allow vesicles to be formed analogously to viral capsids that serve as potential delivery vehicles for intracellular transport (Nat Materials, 3 (4): 244-8, 2004).
  • lipophilic conjugates provide a way to increase the ability of oligonucleotides to bind to lipoproteins and thereby improve intracellular delivery.
  • the best studied conjugate is cholesterol (Antisense and Nucleic Acid Drug Development, 12: 103-128, 2002).
  • Cyclodextrins are cyclic oligosaccharides that have a central hydrophobic cavity and multiple hydroxyl groups on the outer surface. Cyclodextrins have thus already been used for the transport of antisense oligonucleotides into human T cell lines (Antisense Res Dev, 5: 185-192, 1995) and also in vivo for intracellular transport or intracellular delivery or delivery of immunogenic CpG sequences (Biochem Pharmacol, 52: 1537-1544, 1996). Various formulations of cyclodextrins can be found in the review by Davis and Brewster (Nature Reviews Drug Discovery 3: 1023-1035, 2004).
  • Dendrimers are highly branched macromolecules composed of repetitive units of - typically - polyamides.
  • the molecules carry on their surface functional groups such as primary amino groups, which interact with other molecules through electrostatic interaction.
  • surface functional groups such as primary amino groups, which interact with other molecules through electrostatic interaction.
  • complex, rapid, and highly reproducible complex formation occurs, resulting in complexes with weak cytotoxicity (Nucleic Acids Research, 28: 4225-4231, 2000, Clin Cancer Res, 7: 3606-3612, 2001;
  • Cyanoacrylate nanoparticles have been tested since the early 1990's for delivery or delivery of oligonucleotides.
  • the interaction of oligonucleotides with the nano- Particles are formed by ion pairs of the anionic charge of the oligonucleotides with different hydrophobic cations, primarily with charged nanoparticles.
  • PIHCA polyisohexyl
  • PIBCA polyisobutyl
  • PHCA polyhexycyanoacrylates
  • Nanoparticles have also been used for in vivo use (Biochem Biophys Res Commun, 279: 401-406, 2000, Pharm Res, 13: 38-43, 1996).
  • Microparticles or so-called microspheres are typically biodegradable polymers poly (d, l-lactide-co-glycolide [P (LA-GA)] and are used for the sustained release of oligonucleotides (J Pharm Sei, 91: 790-799, 2000; J Controlled Release, 69: 197-207, 2000; J Drug Traget, 5: 291-302, 1998).
  • poly d, l-lactide-co-glycolide [P (LA-GA)
  • Electroporation is a transport technology that uses a strong electric field to destabilize the lipid double membrane, thereby permeabilizing the cell membrane and thus allowing transport of the substance to be administered, which may also be ionized (iontophoresis), into the cell. Electroporation has been used successfully to facilitate transdermal delivery of oligonucleotides ex vivo and in vivo (Int J Pharm, 184: 147-156, 1999, J Drug Traget, 5: 275-289, 1998, Pharm Res, 15: 1596 Int J Cancer, 85: 260-266, 2000; Biochem Biophys Res Commun, 212: 286-292, 1995; Blood, 88: 731-741, 1996).
  • shock waves high pressure acoustic pulses
  • Ultrasound is also an acoustic technology comparable to the shock waves, but uses higher frequency ranges (MHz instead of Hz) and shorter use times (seconds to minutes) and has already been supportive for gene therapy. See (Gene Gene Ther, 7: 1339-1346, 1996; Invest Radiol, 32: 723-727, 1997; Ultrasound Med Bio, 25: 1451-1457, 1999).
  • a novel delivery vehicle which is particularly suitable for the transport of functional nucleic acids such as aptamers, preferably functional L-nucleic acids and most preferably Spiegelmeren.
  • the delivery vehicle is a micelle-like or liposomal-like structure based on polyethyleneimine.
  • polyethyleneimine may, in principle, be present as a linear or branched polyethylenimine and also be used, with branched polyethylenimine in the branched form being particularly preferred.
  • polyethyleneimine can be present as a high molecular weight or low molecular weight polyethyleneimine and also be used.
  • high molecular weight polyethyleneimine has a molecular weight of about 800 kDa and low molecular weight polyethyleneimine has a molecular weight of about 3 kDa.
  • a polyethyleneimine having an average molecular weight of about 25 kDa is preferred, which is more preferably a branched polyethylenimine having a molecular weight of about 25 kDa.
  • the nucleic acid to be delivered itself also carries a modification. It is preferred if the modification is selected from the group comprising PEG radicals. It is further preferred that the PEG moiety has a molecular weight of about 1,000 to 10,000 Da, more preferably about 1,200 to 5,000 Da, and more preferably about 1,500 to 2,500 Da, and most preferably about 2,000 Da.
  • a quotient of the total number of nitrogen groups of the polyethyleneimine and the total number of phosphate groups of the nucleic acid to be delivered or packaged with or by the delivery vehicle is from about 1 to 20, preferably Example of about 1.5 to 10, more preferably from 2 to 5 and most preferably adjusted from about 2 to 3 or realized.
  • the delivery vehicle according to the invention thus also makes it possible to use the mechanism of the intracellular transport of nucleic acids via condensation or packaging with charged particles or reagents and associated change in the charge of the overall complex, also for functional nucleic acids such as aptamers and in particular L-nucleic acids such as Spiegelmers.
  • This complex is easily absorbed via endocytosis and thus enters the cytosol of the cell.
  • a disadvantage of this method is the stability of the DNA / RNA or the release of the nucleic acid from the endosomal compartment.
  • a lysosome is rapidly formed from the constricted endosome by the introduction of proteases or nucleases and by protonation of the compartment.
  • Nucleases digest the nucleic acids there. However, this does not apply to aptmers, as they are nuclease-stable due to their unnatural configuration. In addition, nucleic acids are not stable in the acidic milieu of the lysosome. However, this applies more to nucleic acids from DNA, less to nucleic acids made up of RNA. Through exocytosis or degradation in the Golgi apparatus, the entire complex is quickly transported back out of the cell and thus only a few nucleic acids enter the cell. One of the challenges posed by a suitable transfection system is thus the stabilization as well as the release of the nucleic acid from the endosomes into the cytosol. In terms of stability, RNA aptmers have ideal properties for transfection of eukaryotic cells since they are not cleaved by enzymes as enantiomers.
  • the transfection system according to the invention provided or designed with the delivery vehicles according to the invention is based on the formation of micelles from nucleic acids and branched polyethyleneimine (PEI).
  • PEI polyethyleneimine
  • the phosphodiester backbone of the nucleic acids interacts with the free nitrogen positions of the PEI and cross-branches to form small micelles, which have a positive charge due to the PEI.
  • These micelles are easily taken up by constricting the plasma membrane as endosomes from a cell.
  • the PEI now buffers incoming protons, whereupon many chloride ions inside the endosome lead to a swelling of the compartment due to the osmotic pressure.
  • composition according to the invention is administered as an aerosol.
  • spiegelmers can be derivatized with signal peptides for intracellular as well as intranuclear delivery, as well as for organ specific delivery Coupling of signal peptides directly to the polyethyleneimine can be done for a localized localization to organs or within the cell can be used.
  • the present invention relates to L-nucleic acids, particularly aptmers, and more preferably RNA aptmers directed against HMGA proteins.
  • the disclosed herein Spiegelmers against HMGA proteins are particularly examples of the present application also underlying finding that L-nucleic acids and aptmers in particular are able to overcome a phospholipid double membrane or a cytoplasmic membrane of a cell and intracellularly with the intracellular To bind receptor on the specific binding of which they have been selected.
  • HMGA high mobility group
  • HMGB HMGB
  • HMGN HMGN
  • HMGA HMGA
  • AT-hook HMGA
  • the HMGA family includes two genes, HMGA1 and HMGA2. From HMGA1, three different proteins can be expressed by alternative splicing (HMGAIa [formerly HMG-I], HMGAIb [formerly HMG-Y], HMGAIc [formerly HMG-I / R]), HMGA2 only one (HMGA2 [earlier : HMGI-C]).
  • HMGA1a, HMGA1b and HMGA2 are polypeptides of approximately 100 amino acids in length and have a modular sequence organization: they have three strongly basic regions ("AT-hooks") which bind the narrow minor groove of double-stranded AT-rich DNA (Reeves & Nissen 1990)
  • the C-terminus contains many acidic amino acids, which have no stable secondary structure in solution and do not adopt a defined conformation until complexed with DNA or other proteins (Huth et al 1997) to the most highly modified proteins in the mammalian cell nucleus and are phosphorylated, acetylated, and methylated (Reeves & Beckerbauer 2001).
  • HMGA proteins do not have any transcriptional activity per se, but as so-called architectural transcription factors, through their protein-protein and protein-DNA interactions, they organize the formation of the nucleoprotein-DNA transcription complex (Wolffe 1994). They thus have an activating or inhibitory influence on the expression of a multiplicity of genes.
  • the most prominent example of a positive regulation is the involvement of HMGA1 in the regulation of IFN- / 3 (Thanos & Maniatis, 1992).
  • the HMGAIb stimulates the binding of NF- ⁇ B and ATF-2 to the DNA double helix and at the same time alters the DNA structure such that NF- ⁇ B and ATF-2 interact with each other and presumably also with the rest of the transcription machinery (Thanos & Maniatis 1992, Du et al 1993).
  • Another transcriptional activating effect associated with arteriosclerotic pathogenesis is HMGA1-induced CD44 gene regulation (Foster et al 1998).
  • CD44 is a cell surface glycoprotein and involved in the migration and proliferation of smooth muscle cells after endothelial injury (Jain et al 1996, Cuff et al 2001).
  • CD44 Transcriptional regulation of CD44 is induced by the binding of c-Fos and c-Jun to the AP-I binding site in the CZW promoter and enhanced by the binding of HMGA1.
  • Studies in rats have shown that CD44 overexpression leads to an increased recruitment of smooth muscle cells and has a direct influence on the development of arteriosclerotic lesions (Pellacani et al 1999, Foster et al., 1998, 2000).
  • HMGA1 gene located in the chromosomal band 6 ⁇ 21.3 and the HMGA2 gene localized in the region 12ql4-15 revealed that they are mainly active in processes of cellular differentiation. Accordingly, strong expression of these genes is found during embryonic development and in undifferentiated cells (Chiappetta et al 1996) as well as in growth factor-stimulated cells (Friedman et al 1993, Johnson et al 1990, Ogram et al 1995, Holth et al 1997).
  • HMGA1 In the adult, differentiated tissue HMGA1 is strongly expressed only in the retina, while in the other tissues HMGA2 is not found at all and HMGA1 is found only in very low concentrations (Bussemakers et al 1991, Chiappetta et al 1996, Rogalla et al 1996 Zhou et al 1995; Chau et al 2000).
  • HMGA proteins in differentiated normal tissue is thereby associated with the growth and differentiation of adipocytes (Zhou et al 1995, Anand & Chada 2000, Melillo et al 2001), the proliferation of smooth muscle cells in the blood vessels after vascular injury (Chin et al 1999), the immune response in inflammatory reactions (Pellacani et al 1999) and apoptotic processes (Diana et al 2001, Sgarra et al 2003).
  • the amount of HMGA1 varies depending on the proliferation rate of the cells (Johnson et al 1990).
  • HMGA1 expression is concentrated on specific organs of ecto-, meso- or endodermal origin, while HMGA2 is restricted to mesenchymal tissues. So far, there is no information on the phenotype of HMGA1 knockout mice, possibly because the lack of this factor damages embryonic development too much.
  • HMG ⁇ 2-knockout mice are dwarf and have low levels of adipose tissue (Zhou et al 1995) and are also resistant to diet-induced obesity (Anand & Chada 2000).
  • HMGA2 or HMGAlb expression is undetectable in adipose tissue of normal mice, but dramatically increased in fat of fat or diabetic mice (Chada et al., 2004), suggesting an association between obesity and HMGA expression
  • HMGA1 overexpression of HMGA1 influences (Reeves et al 2001):
  • Intermediary filament markers such as cytokeratin, type 1
  • apoptosis regulators such as TRARl 5
  • Oncogenic and tumor suppressor genes such as MET
  • Angiogenesis regulators such as FGFR2
  • cell-cell interaction genes such as cadherin 12
  • HMGA1 Abnormal regulation of HMGA1 could therefore lead to general alterations in gene expression, thereby significantly contributing to the formation of transformed and / or metastatic phenotypes.
  • HMGA proteins appear to play different roles in mesenchymal or epithelial tumors: in malignant epithelial tumors, HMGA expression is more associated with later stages of carcinogenesis, while benign - rarely-converting mesenchymal tumors - already express HMGA in early hyperplasia. This indicates that HMGA proteins in tissues of different embryonic origins perform different functions, which also directly affects the corresponding applications. fertilize the L-nucleic acids according to the invention in the diagnosis and / or treatment of - corresponding - diseases, as shown in more detail below darg Dead.
  • HMGA 1 expression of the tumor correlates with its metastatic potential and thus is a characteristic feature of a malignant transformed cell (Giancotti et al 1987).
  • HMGAl-associated - in this case benign, mesenchymal tumors - are characterized by chromosomal alterations in the chromosomal HMGL4 / region 6p21.3. Such aberrations have so far been among others for
  • HMGA proteins Aberrations in the genetic mechanisms that control growth and proliferation are the primary cause of carcinogenesis.
  • the expression of HMGA proteins is strongly associated with tumor development, as shown in a number of papers (Giancotti et al., 1987, 1989, 1993).
  • HMGA2 expression was found in chemically or virally generated as well as in spontaneously obtained tumors, whereas in non-transformed cells or healthy tissue this protein could not be detected (Giancotti et al., 1989).
  • oncogenic retrovirus-infected cells in which the synthesis of HMGA2 expression had been specifically blocked lacked various phenotypic markers for transformation (Berlingieri et al., 1995).
  • HMGA2 knockout MMse show dwarfism, d. H. the animals are about 60% smaller than wild-type mice. However, these dwarf mice have a high resistance to chemically induced skin tumors.
  • the main cause of mortality and morbidity of cancer patients is the metastatic spread of the primary neoplasm in the body. Metastasis is not a simple process, as successful colonization of distant organs by scattered neoplastic cells must pass through many stages. Neoplastic cells must detach from the primary neoplasm, enter the bloodstream, extravagate at distant locations, and finally proliferate again in the parenchyma of the corresponding organ. Many genes are involved in the different stages of this highly complex metastatic cascade express proteins such as proteases, adhesion molecules, motility factors and angiogenic factors.
  • HMGA1 HMGA1 gene as one of the most important factors controlling this process is a valid candidate for this.
  • the gene products of HMGA1 affect the transcription of many genes that are important for successful metastasis. For example, other metastasis-associated genes have been shown to be less expressed when suppressed by HMGA1 expression (Battista 1998, Vallone 1997).
  • HMGA1 is therefore an interesting therapeutic target molecule.
  • the blockade of HMGA1 is therefore in principle suitable for controlling the cancer and preventing its metastatic spread (Evans 2004, Sgarra 2004).
  • antisense RNAs directed against HMGA transcripts in cancer cells in vitro, it was possible to reduce cell proliferation or even to induce apoptosis (Masciullo 2003, Scala 2000, Chau 2003).
  • the animal model has shown that gene therapy (adoenoviral expression of antisense RNAs directed against HMGA transcripts) drastically reduces the growth of various pancreatic cancer xenografts (Trapasso et al 2004).
  • HMGA1 could continue to be used as a prognostic diagnostic marker to determine which patients would benefit from more aggressive cancer therapy. There is a close correlation between the degree of malignant transformation and the amount of HMGA1 expressed. This in turn can be correlated with a poor prognosis in many human cancers, such as prostate (Tamimi 1996, Bussemakers 1991) and colorectal carcinoma (Abe 1999) or neuroblastoma (Giannini 2000).
  • HMGA proteins are considered by many viruses to be control factors for the expression of viral genes or cofactors provided by the host cell, among others
  • herpes simplex virus Panagiotidis 1999, French et al 1996)
  • HIV-1 virus (Henderson et al 2000) used.
  • HMGA proteins are implicated in the regulation of transcription of a variety of viral genes in a host cell. Examples include the regulation of the expression of the early and late expressed genes of the human papovavirus JC (Leger et al., 1995), regulation of the EBNA1 (Epstein-Barr virus nuclear antigen 1) gene of the Ebstein-Bar virus (EBV), which is responsible for the control of viral latency is, (Schaefer et al., 1997), regulation of the IE-3 (immediate early) gene of herpes simplex virus-1 (HSV-I), which encodes the early expressed protein ICP4 (Panagiotidis et al., 1999 ), Regulation of the latent phase active promoter 2 of HSV-I (French et al., 1996) and regulation of the LTR (long terminal repeats) promoter of the human HIV-1 virus (Henderson et al., 2000).
  • EBNA1 Epstein-Barr virus nuclear antigen 1
  • HSV-I
  • HMGA1 also appears to play a decided role as an architectural cofactor in integrating the viral DNA of the HIV-I virus, the Moloney murine leukemia virus (MoMuLv) and sarcoma poultry flu virus (ASV) into the human genome and thus appears to be an interesting therapeutic approach of antiviral Therapy (Van Maele et al 2006, Li et al 1998, Hindmarsh et al., 1999)
  • inhibitors of HMGA proteins are also suitable for the treatment and diagnosis of viral infections (Reeves & Beckerbauer 2002).
  • HMGA proteins in various diseases or their suitability as diagnostic markers
  • directed L-nucleic acid and in particular aptmers can be used for the prevention, therapy and diagnosis of precisely these diseases.
  • Particularly preferred aptmers are the aptmer described herein.
  • the individual aptmers have been generated for a given HMGA protein, as a result of the domain approach illustrated in Example 2, these also allow cross-reactivity with other HMGA proteins, as can be seen in US Pat in Fig. 2 shows alignment.
  • nucleic acids of the invention have a number of structural motifs that define a class of aptmers that bind to HMGA proteins as intracellular receptors. The various structural motifs are further detailed in Example 1.
  • the nucleic acids according to the invention also comprise those nucleic acids which are substantially homologous to the sequences specifically disclosed herein.
  • the term "substantially homologous” herein is preferably understood to mean that the homology is at least 75%, preferably 85%, more preferably 90%, and most preferably more than 95, 96, 97, 98 or 99%.
  • nucleic acids or nucleic acids according to the present invention should furthermore also include those nucleic acids which comprise nucleic acid sequences as disclosed herein or parts thereof, preferably to the extent that the nucleic acids or the said parts thereof are involved in binding to HMGAs. Proteins are involved.
  • nucleic acid can be derived from those disclosed herein, e.g. B. by shortening or truncation.
  • a truncation may refer to either or both ends of the nucleic acids as disclosed herein.
  • a truncation may also involve the inner sequence of nucleotides, i. H. it may involve nucleotide (s) between the 5 'and 3' terminal nucleotides, respectively.
  • truncation should also refer to the deletion of as little as a single nucleotide from the sequence of the nucleic acids disclosed herein. Shortening may also refer to more than one region of the nucleic acid (s) of the invention, wherein each of these regions may be as small as one nucleotide in length.
  • the nucleic acids according to the present invention may furthermore be either D-nucleic acids or L-nucleic acids.
  • the nucleic acids according to the invention are L-nucleic acids.
  • one or more parts of the nucleic acid are present as D-nucleic acids, or at least one or more parts of the nucleic acids are L-nucleic acids.
  • the term "part" of the nucleic acids is not intended to refer to any nucleotide, and such nucleic acids are generally referred to herein as D- or L-nucleic acids.
  • the nucleic acids L-acids according to the present invention from L-nucleotides and comprise at least one D-nucleotide.
  • a D-nucleotide is preferably attached to a part other than the region (s) defining the nucleic acids according to the present invention, preferably those parts thereof which interact with other parts of the nucleic acids.
  • such a D-nucleotide is attached to the end of each region or nucleic acid according to the present invention.
  • such D-nucleotides may function as a spacer or a linker which preferably binds modifications such as PEG and HES to the nucleic acids of the present invention.
  • the nucleic acids according to the invention also include those which are part of a longer nucleic acid, wherein these longer nucleic acids can comprise several parts, wherein at least one part is a nucleic acid according to the present invention or a part thereof.
  • the other part or parts of these longer nucleic acids may be either a D-nucleic acid or an L-nucleic acid. Any combination may be used in connection with the present invention and for the purposes or uses as disclosed for the nucleic acids of the invention herein.
  • This other part or parts of the longer nucleic acid may have a function other than binding and in particular binding to HMGA protein.
  • One possible function is to interact with other molecules, e.g. B. for purposes of immobilization, cross-linking, detection, amplification or modification or molecular weight increase to allow.
  • L-nucleic acids as used herein are nucleic acids consisting of L-nucleotides, preferably consisting entirely of L-nucleotides.
  • D-nucleic acids are nucleic acids consisting of D-nucleotides, preferably consisting entirely of D-nucleotides.
  • the nucleic acid according to the invention consists of D-nucleotides, L-nucleotides or a combination thereof of both, wherein the combination, for. B., is a random combination or sequence of regions consisting of at least one L-nucleotide and at least one D-nucleic acid
  • the nucleic acid may consist of one or more deoxyribonucleotides, ribonucleotides, and combinations thereof.
  • the present invention relates to a pharmaceutical composition which consists of at least one of the nucleic acids according to the invention in combination with one or more other nucleic acids, wherein the other nucleic acids preferably bind / bind to target molecules other than HMGA protein or one of performs / exercise various functions on the nucleic acids of the invention.
  • L-nucleic acids are enantiomers of naturally occurring nucleic acids.
  • D-nucleic acids are not very stable in aqueous solutions and especially in biological systems and in biological samples due to the extensive presence of nucleases.
  • Naturally occurring nucleases, especially animal cell nucleases are unable to degrade L-nucleic acids.
  • the biological half-life of L-nucleic acid in such a system, including the human and animal body is significantly increased. Due to the lack of degradability of L-nucleic acids no nuclease degradation products are produced and thus no side effects are observed.
  • This aspect effectively delimits the L-nucleic acid from any other compounds used in the therapy of diseases and / or disorders, and the presence of HMGA or its causal involvement comprises L-nucleic acids that specifically bind to a target molecule through a mechanism, which is different from the Watso ⁇ -Crick base pairing, or aptamers which are partially or fully composed of L-nucleotides, especially those portions of the aptamer involved in the binding of the aptamer to the target molecule are referred to as Spiegelmers.
  • nucleic acids according to the invention are available as single-stranded or double-stranded nucleic acids. pelstrlindige nucleic acids may be present.
  • the nucleic acids according to the invention are single-stranded nucleic acids which, as a consequence of the primary sequence, have defined secondary structures and can thus also form tertiary structures.
  • the nucleic acids according to the invention can also be double-stranded in the sense that two strands which are complementary or partially complementary to one another are hybridized with one another. This confers stability to the nucleic acids, which will be particularly advantageous when the nucleic acid is present in the naturally occurring D-form rather than the L-form.
  • nucleic acids according to the invention can be modified. Such modifications may involve single nucleotides of the nucleic acid and are well known in the art. Examples of such modification include, inter alia, described in Venkatesan N. et al. (2003) Curr Med Chem. Oct; 10 (19): 1973-91; Kusser, W. (2000) J. Biotechnol. 74: 27-38; Aurup, H. et al. (1994) Nucleic Acids Res, 22, 20-4; Cummins, LL et al, (1995) Nucleic Acids Res, 23, 2019-24; Eaton, BE et al. (1995) Chem Biol, 2, 633-8; Green, LS.
  • nucleic acid according to the present invention may comprise at least one LNA nucleotide.
  • the nucleic acid according to the present invention consists of LNA nucleotides, preferably entirely of LN A nucleotides.
  • the nucleic acids according to the present invention may be a multi-part nucleic acid.
  • a multi-part nucleic acid as used herein is a nucleic acid consisting of at least two nucleic acid strands. These at least two nucleic acid strands form a functional unit, wherein the functional unit is a ligand for a target molecule.
  • the at least two strands of nucleic acid can be derived from one of the nucleic acids of the invention either by cleaving the nucleic acid to generate two strands, or by synthesizing one nucleic acid corresponding to a first part of the invention, ie total nucleic acid, and another nucleic acid corresponding to the second part of the total nucleic acid , It is recognized that Both cleavage and synthesis can be used to generate a multi-part nucleic acid where more than the two strands may be present as described above.
  • the at least two strands of nucleic acid are preferably different from two strands that are complementary to each other and hybridize to each other, although there may be some degree of complementarity between the different nucleic acid parts.
  • the present inventors have found that the nucleic acids according to the present invention have a very advantageous K D value range or dissociation value range and thus a very advantageous binding constant.
  • An example to determine the binding constant is the use of an equilibrium binding assay as described in Example 1.
  • the K D value of the nucleic acids according to the invention is preferably less than 1 ⁇ M.
  • a K D value of about one ⁇ M should be characteristic of non-specific binding of a nucleic acid to a target.
  • the KD value of a group of compounds such as the nucleic acids according to the present invention will vary within a particular range.
  • the above-mentioned K D of about 1 ⁇ M is a more preferred upper limit of the K D value.
  • the preferred lower limit for the KD of the target molecule-binding nucleic acids may be about picomolar or lower. It is within the scope of the present invention that the K D values of individual nucleic acids which bind to HMGA are preferably within this range.
  • Preferred ranges can be selected by selecting a first number within this range and a second number within that range.
  • Preferred upper values are 0.25 ⁇ M, 0.1 ⁇ M, preferred lower values are 100 nM, 10 nM, 1 nM and 0.05 nM.
  • the nucleic acids according to the invention preferably bind to HMGAIb at 37 ° C. in solution with a dissociation constant K D ⁇ 20 nM, as shown in Example 2.
  • the nucleic acids according to the present invention may be of any length provided that they are still capable of binding to the target molecule. It will be appreciated in the art that certain lengths of the nucleic acids according to the present invention are preferred. Typically, the length is between 15 and 120 nucleotides. It will be further appreciated by those skilled in the art that any integer between 15 and 120 is a preferred length of potential for the nucleic acids of the present invention. Preferred ranges for the length of the nucleic acids according to the present invention are lengths of about 20 to 100 nucleotides, about 20 to 80 nucleotides, about 20 to 60 nucleotides, about 20 to 50 nucleotides and about 30 to 50 nucleotides.
  • ⁇ nucleic acids of the invention can be modified.
  • a particularly preferred form of modification is pegylation. This is the modification of the nucleic acids according to the invention by coupling with polyethylene glycol (PEG) or other groups.
  • PEG polyethylene glycol
  • the nucleic acids according to the invention are provided as a physiological solution for local or systemic administration.
  • the pharmaceutical composition according to the present invention thus comprises at least one of the nucleic acids according to the present invention and preferably a pharmaceutically acceptable binder.
  • a binder may be any known or known binder in the art.
  • such a binder is any binder as described in connection with the preparation of the medicament as disclosed herein.
  • the pharmaceutical composition comprises another pharmaceutically active agent. It is within the scope of the present invention that the medicament described herein is the pharmaceutical composition as described herein.
  • the pharmaceutical composition is for intravenous administration.
  • compositions can be administered intramuscularly, intraperitoneally, or subcutaneously.
  • Other routes of administration are oral or intranasal, with the preferred mode of administration being the least invasive, while retaining the efficacy of the pharmaceutical composition or pharmaceutically active agent.
  • the nucleic acids of the invention are preferably contained or dissolved as such or in association with the pharmaceutical composition of the invention in a pharmaceutically acceptable solvent.
  • solvents are especially those selected from the group consisting of water, physiological saline, PBS, or a glucose solution, especially a 5% glucose solution.
  • a carrier can, for. B., water, buffer, PBS, glucose solution, preferably a 5% glucose solution (iso-osmotic), starch, sugar, gelatin or any other acceptable vehicle.
  • Such carriers are generally known to those skilled in the art.
  • the pharmaceutical composition contains at least one of the nucleic acids according to the invention in their various embodiments, including, but not limited to, the nucleic acid as a co-conjugate as described herein.
  • the medicament comprises a further pharmaceutically active agent.
  • Such further pharmaceutically active agents are, for example, protease inhibitors, proliferation inhibitors and angiogenesis inhibitors and / or agents which have a cystostatic effect.
  • another pharmaceutically active agent is another nucleic acid according to the present invention.
  • the medicament comprises at least one or more nucleic acids that bind to a target molecule other than HMGA or having a function different from any of the nucleic acids of the present invention.
  • the pharmaceutical composition according to the present invention may be used for the treatment, diagnosis and / or prevention of any of the diseases or disorders described herein.
  • the present invention relates to a method of treating a subject in need of such treatment, the method comprising administering a pharmaceutically active amount of at least one of the nucleic acids according to the present invention.
  • the subject is suffering from or at risk of contracting such a disorder, the disorder being any of those disclosed herein, in particular, any disorder associated with the use of any of the nucleic acids according to the present invention present invention for the preparation of a medicament is described.
  • HMGA proteins and their genes have been increasingly associated with the diagnosis and prognosis of neoplastic diseases and have been proposed as potential biomarkers.
  • the level of expression of HMGA la / b proteins is very low, if detectable at all.
  • Increased HMGA la / b protein expression is characteristic of the phenotype of a variety of tumors and metastases of various cancers (Sarhadi et al., 2006, Balcercak et al., 2005, Bliese et al., 2006, Chang et al., 2005, Peters et al.
  • HMGA1a / b expression level in biopsies and their histological characterization represents a diagnostic approach for the early detection, prognosis and identification of neoplastic diseases, in particular the diseases and conditions described above. Furthermore, an association of HMGA1 proteins with arteriosclerotic plaques is described in the literature (Schlueter et al., 2005).
  • HMGA1 regulates CD44, one of the major target genes for plaque formation. In comparison with the surrounding tissue, the affected regions, such as neointimal, vascular smooth muscle cells, macrophages and new blood vessels, show a high expression of HMGA1.
  • HMGA1 seems to be one of the mediators in the development of plaques and thus represents a target molecule for the diagnosis.
  • biotinylated HMGA1 a / b recognizes baclimeric 5 '-bio-NOX-A50 binding bacteria in the Western blot HMGA1 a / b as a single band in cancer cell lines. Furthermore, as described in Example 2, recombinantly expressed HMGAIb protein could be detected.
  • the detection of the biotinylated aptmer is carried out, for example, via an anti-biotin antibody conjugated by means of Horse Raddish Peroxidase (HRP).
  • HRP Horse Raddish Peroxidase
  • HMGA la / b Another approach is the in vivo diagnosis of HMGA la / b, in which the nucleic acids according to the invention can be used. Tumors and metastases are often embedded by necrotic tumor cells, which release HMGA1 a / b into the surrounding tissue. The detection of extracellular HMGA la / b represents an approach to the diagnosis of tumors and metastases embedded in healthy tissue.
  • a diagnostic or diagnostic agent or diagnostic agent is capable of detecting, either directly or indirectly, an HMGA protein, preferably HMGA la / b as described herein, and more preferably HMGAla / b as described herein in connection with U.S. Patent Nos. 4,646,974; different disorders and Diseases.
  • the diagnostic agent is suitable for detection and / or aftercare for any of the diseases or diseases described herein. Such detection is possible by the binding of the nucleic acids according to the present invention to HMGA1 a / b. Such binding can be detected either directly or indirectly.
  • the corresponding methods and means are known to those skilled in the art.
  • the nucleic acids according to the present invention may comprise a label which allows the detection of the nucleic acids according to the present invention, preferably the nucleic acid which is bound or capable of binding to HMGA protein and preferably HMGA1 a / b.
  • a label is preferably selected from the group consisting of radioactive, enzymatic and fluorescent labels.
  • any known antibody-engineered assays can be adapted for the nucleic acids of the present invention with the target-binding antibody replaced by a target-molecule-binding nucleic acid.
  • detection is preferably by a secondary antibody modified with radioactive, enzymatic or fluorescent labels and binding to the target-binding antibody on its Fc fragment.
  • the nucleic acid is modified with such a label, whereby preferably such a label is selected from the group consisting of biotin, CY-3 and CY-5, and such a label is detected by an antibody directed against such label, e.g. As an anti-biotin antibody, an anti-CY-3 antibody or an anti-CY5 antibody or in the case that the label is biotin, the label is detected by streptavidin or avidin, which binds naturally to biotin.
  • an antibody, streptavidin or avidin is in turn preferably modified with a corresponding label, e.g. As a radioactive, enzymatic or fluorescent label, analogous to a secondary antibody.
  • the nucleic acids according to the present invention are detected or analyzed by a second detection means, wherein the detection means is a molecular beacon
  • the detection means is a molecular beacon
  • molecular beacons are nucleic acid probes that have a reverse complement to the are the nucleic acid sample, and therefore hybridize with a portion of the nucleic acid sample to be detected which is to be detected.
  • the fluorophore groups of the molecular beacon are separated from each other, resulting in the change of the fluorescence signal, preferably a change in intensity. This change correlates with the amount of nucleic acid sample present.
  • nucleic acids according to the invention can be used correspondingly as L-nucleic acids within the scope of the various aspects disclosed herein.
  • the nucleic acids according to the invention can furthermore be used as starting material for the design of pharmaceutical active ingredients (English, drug design). Basically, there are two possible approaches. One approach is to screen libraries of compounds, such libraries of compounds being preferably low molecular weight (small molecule) libraries. Such libraries are known to those skilled in the art. In one embodiment, the screening is high throughput screening. Preferably, high-level screening is fast, efficient, and is performed as a trial-and-error evaluation of drugs in a target-molecule-based assay.
  • the nucleic acids can be used for the rational design of drugs.
  • the rational design of drugs is the design of a pharmaceutical drug candidate.
  • the target molecule which is usually determined by methods such as X-ray analysis or nuclear magnetic resonance spectroscopy (NMR)
  • NMR nuclear magnetic resonance spectroscopy
  • computer programs are used to search databases with structures of a variety of different chemical compounds. The selection is carried out by the computer. The selected compounds are additionally tested in the laboratory.
  • the rational design of active ingredients may here be based on any of the nucleic acids according to the present invention and comprises a structure, in particular a three-dimensional structure, which is similar to the structure of the nucleated nucleic acid according to the invention.
  • Liche acid (s) is or is identical to that part of the structure of the nucleic acid (s) of the invention, which mediates the binding to HMG proteins. In any case, such a structure still shows the same or at least a similar binding behavior as the nucleic acid (s) according to the invention.
  • the preferably three-dimensional structure of those parts of the HMG protein-binding nucleic acids is mimicked by chemical groups, which are preferably different from nucleotides and nucleic acids.
  • a compound can be constructed which is different from the nucleic acid or nucleic acids used as starting materials for the rational design of the active ingredient.
  • Such a compound or drug is preferably a small molecule (Engl., Small molecule) or a peptide.
  • HMG analogs In the case of screening of compound libraries using competitive assays known to those skilled in the art, appropriate HMG analogs, HMG agonists, and HMG antagonists can be found.
  • Such competitive assays can be constructed as follows.
  • the nucleic acid according to the invention preferably a spiegelmer, d. H. an L-nucleic acid binding the target molecule is coupled to a preferably solid phase.
  • labeled HMG protein is added to the test system.
  • the HMG protein could also be coupled to a solid phase and the nucleic acid according to the invention could be labeled.
  • a potential analogue or potential agonist or antagonist would compete with the HMG molecules that bind to the aptmer, which would be accompanied by a decrease in the signal obtained from the corresponding label. Screening for agonists or antagonists may involve the use of a cell culture assay system known to those skilled in the art.
  • the nucleic acids according to the invention can be used owing to their characteristic binding behavior to HMG protein for the target validation ("target” is English for "target molecule”).
  • the nucleic acids of the invention can be used in an ex vivo organ model to study the function of HMG protein. Basically ex vivo models exist in which HMG agonists / antagonists can be tested.
  • a kit according to the present invention may comprise at least one or more of the nucleic acids of the invention.
  • the kit may include at least one or more positive or negative controls. As positive controls can z.
  • HMG protein against which the nucleic acid of the invention has been screened or to which it binds, preferably in liquid form may be used.
  • a peptide can be used, which behaves in terms of its biophysical properties similar to HMG protein, which is not recognized by the nucleic acids of the invention or a peptide with the same amino acid composition but of HMG protein of different sequence.
  • the kit may comprise one or more buffers.
  • the various ingredients may be present in the kit in dry or lyophilized form, or dissolved in a liquid.
  • the kit may include one or more containers, which in turn may contain one or more of the components of the kit.
  • the vessels contain reaction mixtures as required for a one-time experiment using one or more components of the kit.
  • hybridizing the two sections is understood herein to mean that the sections can hybridize in vitro due to general base pairing rules, or that the sections can hybridize or hybridize under the conditions of use, but not necessarily together are hybridized or hybridized under the conditions of use.
  • nucleic acids can be freely selected according to the technical teaching disclosed herein, d. H. can be chosen so that they show the required binding behavior to the respective target molecule and / or the structures described herein, in particular secondary structures are able to form.
  • FIG. 1A shows aptamers generated by in vitro selection against D-21AS-HMGAla / b, which bind the 21 AS-HMGA1a / b domain;
  • Figure 1B is a representation of the identified recurring sequence regions of the aptamers produced by in vitro selection against D-2 IAS -HMGA la / b, which bind the 2 IAS-HMGAla / b domain;
  • FIG. 2 shows a sequence comparison of HMGA1 a / b and HMGA2;
  • FIG. 3 shows a truncation of HMGA 1 a / b-binding aptamer NOX-f
  • Figure 4 shows the binding properties of truncated HMGA1 a / b binding aptamer NOX-f; 5 shows a competition assay for measuring the binding of HMGA to the double-stranded natural target DNA in the multiwell plate assay; the binding of the aptmer competes with the binding of the recombinant HMGAIb to the biotinylated dsDNA (AT-hook motif).
  • Figure 6 is a comparison of aptmer NOX-A and aptmer NOX-f (48nt; 33nt) in the competitive multiwell plate assay; Spiegelmer NOX-A, as well as Spiegeimer NOX-f and its shortened version Spiegeimer NOX-D3 in the plate assay prevent the binding of recombinant HMGAIb to its natural occurring binding partner in the low nanomolar range.
  • Figure 7 shows the activity of 2kDa PEG-coupled aptamer NOX-A and non-functional control mirror mer in the competitive multiwell plate assay; the PEGylated aptmer NOX-A competes with the binding of recombinant HMGAIb to the AT-hook motif of the dsDNA with an IC50 of 15nM; the inverse control aptmer of NOX-A exhibits a nonspecific interaction with HMGAIb at high aptmer concentrations;
  • Fig. 8 is a Western blot; Detection of immobilized HMGAIb by biotinylated aptmer; recombinant HMGAIb migrates in the electrophoretic field like a 2OkDa protein and can be detected at low concentrations (3nM) by the biotinylated aptmer (here by the example of NOX-A); an inverse control aptmer could not detect HGMGAIb;
  • Figure 9 shows the activity of free and PEGylated aptamer NOX-A in the competitive multiwell plate assay
  • FIG. 10 an examination of the packaging of PEGylated aptmer in micelles in the "RiboGreen exclusion assay";
  • FIG. 11 shows the stability of PEI aptmer micelles in the "RiboGreen exclusion assay"
  • FIG. 14 shows a proliferation assay with a "naked” aptmer
  • FIG. 15 shows the proliferation of non-small cell lung cancer (H1299) cells after treatment with PEI-packed NOX-A-2kDa PEG; Inhibition of proliferation of H-1299 cells with 1 ⁇ M aptmer administered as PEI aptmer micelles (N / P 2.5); NOX-A showed a slight inhibition of proliferation compared to the control aptmer;
  • FIG. 16 shows the inhibition of HMGA1a / b-induced cdc25a gene expression as detected by quantitative RT-PCR; Determination of specific inhibition of cdc25a mRNA expression in H-1299 cells by l ⁇ M NOX-A Spiegelmer micelles (N / P 2.5) by means of RT-PCR;
  • NOX-A Quantification of the dose-dependent inhibition of cdc25a mRNA expression in HI 299 cells by RT-PCR; NOX-A Spiegelmer micelles (N / P 2.5) showed a specific inhibition of cdc25a mRNA expression after 25OnM; at a concentration> 4 ⁇ M, an unspecific effect of the control aptmer and toxic effects of polyethylenimine (PEI) at> 10 ⁇ M were shown (data not shown);
  • PEI polyethylenimine
  • FIG. 18 shows the inhibition of tumor growth in the xenograft model in nude mice by the aptamer NOX-A; Inhibition of tumor growth after subcutaneous injection of PSN-I cells by 2 mg / kg aptmer micelles (N / P 2.5). Spiegelmer NOX-A showed a significant reduction of tumor growth; FIG. 19 shows the statistical analysis of the data from the xenograft experiment; FIG. Inhibition of the
  • FIG. 20 the tissue distribution of aptmer NOX-A in the xenograft experiment; FIG. quantitative analysis of the distribution of aptmer NOX-A in plasma and tissues; In the tumor tissue, a high concentration of aptmer NOX-A was detectable compared to the other tissues and plasma.
  • Fig. 21 Tissue distribution of micellar packaged and unpacked aptamer
  • Fig. 22 Distribution of micropacked and unpacked aptmer in
  • the HMGAI a / b-binding RNA tikmers were generated by in vitro selection against D-21 AS-HMGAl a / b and subsequent truncation steps.
  • the generated aptameres that bind the 21 AS-HMGA la / b domain are shown in FIG. 1A.
  • the different clones were prepared as aptamers (D-RNA) by standard phosphoramidite synthesis and radioactively labeled at the 5 'end by kinasing (see below). The clones were then analyzed for their affinity and activity at two concentrations of D-bio-21aa HMGA1 a / b by equilibrium binding assay.
  • the reaction was carried out for one hour at 37 0 C and was then stopped by heating (10 min at 65 ° C).
  • the separation of radioactive nucleotides from labeled oligonucleotides was carried out by analytical polyacrylamide gel electrophoresis (PAGE) (see below). Then a crush-and-soak gel elution with ammonium acetate and an ethanol precipitation (see below) .
  • the amount of purified RNA was estimated by the radioactivity of the pellet (after precipitation) relative to the radioactivity of the excised band.
  • oligonucleotides from PAA gels 500 ⁇ l ammonium acetate (2 M) was added after chopping the excised PAA gel bands with a pipette tip or spatula.
  • the "crush-and-soak" elution was 2 x 1.5 h at 68 0 C in a thermal shaker (1000 U / min) is performed.
  • the supernatants were "Ultrafree-MC-small columns (Millipore / Amicon, Schwalbach , Germany) in a tabletop centrifuge (16,100 xg) of gel residues. The thus eluted RNA was then desalted by means of an ethanol precipitation.
  • oligonucleotides were 30 min at -80 0 C and precipitated for 30 min at 16,100 xg, 4 ° C by centrifugation. The pellet was washed Ix with 70% ethanol and 5 min at 16,100 xg, 4 ° C centrifuged.
  • each of the 5 'radiolabeled aptamers were prepared in Biotinyl-D-HMGAla / b-21mer (EPSEVPTPKRPRGRPKGSKNK [SEQ ID: 17], see Figure 2), prepared by Bachern (Weil am Rhein, Germany). Solutions in the concentration range of 1 - 3000 nM (or for two-point measurement with 30O nM and 30 nM or 10O nM and 10 nM peptide) for one hour at 37 ° C in selection buffer (10 mM Tris HCl pH 7.4, 5 mM KCl, 0.8mM MgC12, 0.1% Tween).
  • the background control was a solution without biotinylated D-HMGA1 a / b-21mer. Peptide and complexes were then immobilized with 10 ⁇ l Streptavidin Ultra-Link gel within 30 min at 37 ° C. The radioactivity of the suspension was decided. The supernatant was removed. Then the matrix was washed once with 100 ⁇ l of selection buffer and then filled up with selection buffer. By measuring the radioactivity, the aptamer content present in the complex with biotinyl-D-HMGAla / b-21mer was determined for each peptide concentration. Graphical application and Fit (Gra-Fit, version 4.0.10, Erithacus Software) were used to determine the dissociation constant of the active species and the proportion of active molecules.
  • FIG. 3 shows by way of example on the candidate NOX-f (132-B3) the shortening of the aptamer based on the stabilizing Stem structure which is shown in an extended form in the candidates 132-C3, 132-B3, 132-C4, 132-E2, 132-A2, 132-H1, 132-F1, 122-G2, 122-E2 (see FIG. 1A). Shortening to a 32-nucleotide aptamer variant of NOX-f with a 6-nucleotide star (NOX-f 32nt) did not result in loss of binding properties to the 21aa HMGAla / b fragment ( Figures 3 and 4).
  • sequences 132-G2, 122-A1, 122-C1, 122-B2 and 122-B4 have a markedly shorter stem at the 5'-end and 3'-end of the repetitive sequence motif (GGGCG and GGGAG, respectively) Structure on. Shortening them led to a loss of binding. A possible shortening of the longer for these sequences, central region between the repetitive sequence motif (GGGCG or GGGUG or GGGAG) was not made.
  • the SEQ IDs of the aptamer sequences of the HMGA-binding nucleic acids disclosed herein are as follows:
  • the L-nucleotide enantiomer of an aptamer ie, a D-nucleic acid generated against a D-peptide
  • This L-nucleic acid is also referred to herein as a spiegelmer and otherwise has basically the same binding properties as the aptamer.
  • Characteristic of all Spiegelmers that bind to HMGA la / b is a repetitive sequence element of the sequence GGGCG or GGGUG or GGGAG. This sequence element appears twice in HMGA1 a / b-binding Spiegelmers ( Figures IA and IB).
  • the sequence element closer to the 5 'end of the aptmer is referred to herein as Box Al.
  • the sequence element closer to the 3 'end of the aptmer is referred to herein as box A2.
  • Box Al and Box A2 and their arrangement to each other probably represent the decisive feature of HMGAl a / b-binding Spiegelmers.
  • box A1 and box A2 are either a sequence section of six to seven nucleotides in length or 12 to 22 nucleotides in length ( Figures IA and IB). Since these sequence sections differ not only in their length, they are dealt with separately.
  • sequence section lying between box A1 and box A2 has a length of six nucleotides, then the sequence section has the sequence UGGUUG, UGGCUG, CGGUUG, AGGUUG or GUGUAA. Insertion of one nucleotide (uracil) into the sequence CGGUUG, resulting in the sequence CGGUUUG, has neither a negative nor a positive influence on the binding properties of the Spiegelmers.
  • the sequence section lying between box Al and box A2 has a length of 12 to 22
  • Nucleotides this sequence section has two sequence regions of equal length, the optionally hybridize with each other (Helix C). The Hybidleiter is accomplished thereby in each case three to six nucleotides. Between the nucleotides forming the helix C are three to five unpaired nucleotides. Between the 3 'end of Box Al and the 5' end of Helix C, one to three nucleotides are unpaired. Between the 3 'end of Helix C and the 5' end of Box A2, one to five nucleotides can be unpaired.

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Families Citing this family (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9492400B2 (en) 2004-11-04 2016-11-15 Massachusetts Institute Of Technology Coated controlled release polymer particles as efficient oral delivery vehicles for biopharmaceuticals
US9267937B2 (en) 2005-12-15 2016-02-23 Massachusetts Institute Of Technology System for screening particles
WO2008105773A2 (en) 2006-03-31 2008-09-04 Massachusetts Institute Of Technology System for targeted delivery of therapeutic agents
JP5630998B2 (ja) 2006-05-15 2014-11-26 マサチューセッツ インスティテュート オブ テクノロジー 機能的粒子のためのポリマー
WO2007150030A2 (en) 2006-06-23 2007-12-27 Massachusetts Institute Of Technology Microfluidic synthesis of organic nanoparticles
US9217129B2 (en) 2007-02-09 2015-12-22 Massachusetts Institute Of Technology Oscillating cell culture bioreactor
WO2008124639A2 (en) 2007-04-04 2008-10-16 Massachusetts Institute Of Technology Poly (amino acid) targeting moieties
BRPI0817664A2 (pt) 2007-10-12 2015-03-24 Massachusetts Inst Technology Nanopartículas, método para preparar nanopartículas e método para tratar terapeuticamente ou profilaticamente um indivíduo
US8277812B2 (en) 2008-10-12 2012-10-02 Massachusetts Institute Of Technology Immunonanotherapeutics that provide IgG humoral response without T-cell antigen
US8343497B2 (en) 2008-10-12 2013-01-01 The Brigham And Women's Hospital, Inc. Targeting of antigen presenting cells with immunonanotherapeutics
US8343498B2 (en) 2008-10-12 2013-01-01 Massachusetts Institute Of Technology Adjuvant incorporation in immunonanotherapeutics
US8591905B2 (en) 2008-10-12 2013-11-26 The Brigham And Women's Hospital, Inc. Nicotine immunonanotherapeutics
WO2010138193A2 (en) 2009-05-27 2010-12-02 Selecta Biosciences, Inc. Targeted synthetic nanocarriers with ph sensitive release of immunomodulatory agents
US20130149314A1 (en) * 2010-02-09 2013-06-13 Jörn Bullerdiek p19Arf, HMGA2 and MDM2 For Use in the Diagnosis and Treatment of Aberrant Cell Growth
WO2011131371A1 (en) 2010-04-21 2011-10-27 Noxxon Pharma Ag Lipid binding nucleic acids
US20110293700A1 (en) 2010-05-26 2011-12-01 Selecta Biosciences, Inc. Nanocarrier compositions with uncoupled adjuvant
CN102375064A (zh) * 2010-08-26 2012-03-14 杭州华得森生物技术有限公司 用酶联免疫法检测人体中hmga2含量的体外诊断试剂盒
DE102010056610A1 (de) * 2010-12-31 2012-07-05 Volker A. Erdmann Pharmazeutische Zusammensetzung enthaltend L-DNA
CN109172819A (zh) 2011-07-29 2019-01-11 西莱克塔生物科技公司 产生体液和细胞毒性t淋巴细胞（ctl）免疫应答的合成纳米载体
JP2014533098A (ja) * 2011-10-21 2014-12-11 ノクソン・ファルマ・アクチエンゲゼルシャフト グルカゴン結合核酸
SG11201403769YA (en) * 2012-01-10 2014-07-30 Noxxon Pharma Ag Nucleic acids specifically binding cgrp
CN103566377A (zh) 2012-07-18 2014-02-12 上海博笛生物科技有限公司 癌症的靶向免疫治疗
US9233119B2 (en) * 2012-09-27 2016-01-12 Miami University Use of HGMA-targeted phosphorothioate DNA aptamers to suppress carcinogenic activity and increase sensitivity to chemotherapy agents in human cancer cells
WO2014193964A2 (en) * 2013-05-28 2014-12-04 Marsha Rosner Prognostic and predictive breast cancer signature
US10679730B2 (en) 2013-05-28 2020-06-09 The University Of Chicago Prognostic and predictive breast cancer signature
CA2936377A1 (en) 2014-01-10 2015-07-16 Shanghai Birdie Biotech, Inc. Compounds and compositions for treating egfr expressing tumors
EP4001311B1 (en) 2014-07-09 2025-11-05 Birdie Biopharmaceuticals Inc. Anti-pd-l1/pd-1 combinations for treating tumors
CN112546231A (zh) 2014-07-09 2021-03-26 博笛生物科技有限公司 用于治疗癌症的联合治疗组合物和联合治疗方法
KR101755617B1 (ko) 2014-07-23 2017-07-10 한국과학기술연구원 삼차원 자기조립 핵산 나노입자 구조를 갖는 약물전달체
CN112546238A (zh) 2014-09-01 2021-03-26 博笛生物科技有限公司 用于治疗肿瘤的抗-pd-l1结合物
EP3233174B1 (en) * 2014-12-19 2021-03-31 Kemin Industries, Inc. Intraocular delivery of bioactive molecules using iontophoresis
CA2985818A1 (en) 2015-05-31 2016-12-08 Curegenix Corporation Combination compositions comprising an antagonist of porcupine and a pd-l/pd-1 axis antagonist for immunotherapy
EP3347051A4 (en) 2015-09-10 2019-04-24 Affigen, Inc. SEQUENCING SELECTING TUMOR THEROSTICAS
DE17829597T1 (de) * 2016-11-30 2019-12-05 Noxxon Pharma Ag Verfahren zur polyalkoxylierung von nukleinsäuren zur rückgewinnung und wiederverwendung einer überschüssigen polyalkoxylierungsreagenz
US20220033813A1 (en) * 2018-09-11 2022-02-03 The Texas A&M University System L-oligonucleotide inhibitors of polycomb repressive complex 2 (prc2)
CN115997124A (zh) * 2020-04-30 2023-04-21 维兰尼迪科技有限责任公司 用于检测生物或化学实体或事件的分子导线
CN112725438B (zh) * 2021-02-05 2023-08-22 深圳市宝安区妇幼保健院 子宫内膜息肉甲基化标志物组合、检测试剂盒及应用
CN114807183B (zh) * 2021-12-06 2023-06-02 西藏自治区农牧科学院农业研究所 一种青稞矢车菊素氧甲基转移酶基因的新用途
CN118311277B (zh) * 2024-06-11 2024-12-31 北京卫未科学技术有限公司 一种用于癌症早筛和辅助诊断的试剂盒及其应用

Family Cites Families (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5622828A (en) * 1990-06-11 1997-04-22 Nexstar Pharmaceuticals, Inc. High-affinity oligonucleotide ligands to secretory phospholipase A2 (sPLA2)
EP1793006A3 (en) * 1993-09-08 2007-08-22 Gilead Sciences, Inc. Nucleic acid ligands and improved methods for producing the same
DE69638099D1 (de) * 1995-05-04 2010-01-28 Gilead Sciences Inc Nukleinsäureligand-komplexe
US5780611A (en) * 1995-09-15 1998-07-14 Ramareddy Venkata Guntaka Oligomers which inhibit expression of collagen genes
US6171779B1 (en) 1996-07-12 2001-01-09 University Of Medicine & Dentistry Of New Jersey HMGI proteins in cancer
JP2001504448A (ja) * 1996-08-30 2001-04-03 フュルステ，イェンス，ペーター 核酸の鏡面対称選択および進化
DE19726186A1 (de) * 1997-06-20 1998-12-24 Boehringer Ingelheim Int Komplexe für den Transport von Nukleinsäure in höhere eukaryotische Zellen
JP2004510829A (ja) * 2000-10-09 2004-04-08 バイエル　アクチェンゲゼルシャフト 核酸を細胞内に転移させるための複合体
DE10118452A1 (de) * 2001-04-12 2002-10-31 Joern Bullerdiek Nukleinsäuresequenzen von Hyperplasien und Tumoren der Schilddrüse
ES2346640T4 (es) * 2001-10-26 2011-04-26 Noxxon Pharma Ag Ácido l-nucleico modificado.
WO2003051383A2 (de) * 2001-12-19 2003-06-26 Alcedo Biotech Gmbh Verwendung von hmgb-proteinen und dafür codierenden nukleinsäuren
DE10211558A1 (de) * 2002-03-15 2003-10-09 Noxxon Pharma Ag Neue Formen RNAi
WO2003094973A1 (de) * 2002-05-08 2003-11-20 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Mittel und verfahren zum transport von biologisch aktiven molekülen in zellen
US7250496B2 (en) * 2002-11-14 2007-07-31 Rosetta Genomics Ltd. Bioinformatically detectable group of novel regulatory genes and uses thereof
WO2004061456A2 (de) * 2003-01-03 2004-07-22 Alcedo Biotech Gmbh Verwendungen von hmgb, hmgn, hmga proteinen
KR20060015505A (ko) * 2003-04-13 2006-02-17 엔존 파마슈티컬즈, 인코포레이티드 올리고뉴클레오타이드 전구약물
EP1628636A1 (de) * 2003-05-09 2006-03-01 Novosom AG Injizierbare liposomale depots zum wirkstoffdelivery
CA2526853A1 (en) * 2003-05-23 2005-03-03 Board Of Regents - The University Of Texas System Structure based and combinatorially selected oligonucleoside phosphorothioate and phosphorodithioate aptamer targeting ap-1 transcription factors
US7727969B2 (en) * 2003-06-06 2010-06-01 Massachusetts Institute Of Technology Controlled release nanoparticle having bound oligonucleotide for targeted delivery
WO2005072703A2 (en) * 2004-01-26 2005-08-11 Control Delivery Systems, Inc. Controlled and sustained delivery of nucleic acid-based therapeutic agents
WO2006069782A2 (en) * 2004-12-27 2006-07-06 Silence Therapeutics Ag. Lipid complexes coated with peg and their use

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2006117217A2 *

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