Classifications

• • 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
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • 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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • 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
  • C12N2310/30—Chemical structure
  • C12N2310/31—Chemical structure of the backbone
  • C12N2310/315—Phosphorothioates

Definitions

• the present invention relates to nucleic acid constructs for inhibiting the expression of ⁇ V integrins in mammalian cells, as well as to pharmaceutical compositions containing said antisense constructs.
• Integrins are a family of cell surface proteins that facilitate cell adhesion and play a major role in a number of biological processes including platelet aggregation, immune fuctions, tissue repair, cell proliferation, tumor invasion, inflammation, and inherited diseases. They are composed of two subunits ⁇ and ⁇ . So far, more than 11 ⁇ subunits and more than 6 ⁇ subunits are known.
• the subunit isotype ⁇ V is part of a number of ⁇ - ⁇ heterodimers including ⁇ 1 , ⁇ 3, ⁇ 5, ⁇ 6 and ⁇ 8. In the following, the integrin ⁇ V ⁇ 3 will be described as an example in more detail.
• the integrin V ⁇ 3 (also known as vitronectin receptor) is a heterodimeric protein which is expressed on the surface of various cell types and which functions as an adhesion molecule. Integrin ⁇ V ⁇ 3 consists of two subunits: The ⁇ 3 chain which is present in all integrins of the ⁇ 3-type and the specific ⁇ V chain. Integrin ⁇ V ⁇ 3 serves as a receptor for several extracellular matrix proteins such as vitronectin, fibronectin, laminin and collagen. This adhesion molecule is found, e.g. on activated endothelial and vascular muscle cells.
• this receptor is expressed on activated leucocytes, macrophages, osteoclasts and on cells of several types of tumors such as metastatic malignant melanoma, glioblastoma, urothelial carcinoma, breast cancer, ovarian carcinoma, prostatic carcinoma, astrocytoma, lung cancer, colon carcinoma, pancreatic carcinoma and osteosarcoma.
• ⁇ V integrins in particular integrin ⁇ V ⁇ 3, play a central role in angiogenesis since their upregulation on endothelial cells promotes migration and sustains survival of vascular cells in angiogenic blood vessels.
• Neoangiogenesis is, in turn, important for many physiological processes such as wound healing, but also for pathological disorders such as growth of solid tumors or hematological neoplasias, restenosis after angioplasty, stenosis of vein bypasses, chronic inflammatory diseases and diabetic retinopathy. Therefore, the inhibition of neoangiogenesis represents a promising approach for the treatment of the above-mentioned pathological conditions.
• cancer diseases were treated by surgery, radiation therapy or chemotherapy. Recently, treatments using growth factors or immunological therapeutic approaches have been developed. The success thereof is highly dependent on the type of disease. However, concerning restenosis after angioplasty there are no successful therapeutic regimens available. Recent approaches using gene therapy are based on the local production of vasodilators. However, the effectiveness of such approaches remains to be shown. Chronic inflammatory diseases are commonly treated with non-steroidal antiphlogistics and corticosteroids. As a molecular therapeutic approach, antisense oligonucleotides directed against the adhesion molecule ICAM-1 have been developed recently (Yacyshyn et al., 1998, Gastroenterology 114: 1133-1142).
• integrin ⁇ V ⁇ 3 has been developed recently. This inhibitor is based on a peptide which binds to the ⁇ V subunit, thereby blocking the receptor.
• the inhibitor is suggested to be used in the treatment of malignant, tumors (malignant melanoma, colon carcinoma, neuroblastoma); cf. Lode et al., 1999, Proc. Natl. Acad. Sci. USA 96: 1591-1596.
• the technical problem underlying the present invention is to provide a novel system for the specific inhibition of the expression of ⁇ V integrins, e.g. in order to inhibit neoangiogenesis.
• the present invention provides a nucleic acid, e.g. an antisense nucleic acid, comprising a nucleotide sequence which is capable of binding to a transcription product of the gene coding for the ⁇ V integrin chain thereby inhibiting the expression of ⁇ V integrins in mammalian cells by at least 50%.
• a nucleic acid e.g. an antisense nucleic acid
• the nucleic acid according to the present invention is capable of inhibiting the expression of the ⁇ V integrins selected from a group including ⁇ V ⁇ l , ⁇ V ⁇ 3, ⁇ V ⁇ , ⁇ V ⁇ 6 and ⁇ V ⁇ 8.
• ⁇ V integrins selected from a group including ⁇ V ⁇ l , ⁇ V ⁇ 3, ⁇ V ⁇ , ⁇ V ⁇ 6 and ⁇ V ⁇ 8.
• a more preferred example is the inhibition of expression of integrin ⁇ V ⁇ 3.
• nucleic acid and “nucleotide sequence” refer to endogenously expressed, semisynthetic or chemically modified nucleic acid molecules of deoxyribonucleotides and/or ribonucleotides.
• transcriptional product comprises all nucleic acid molecules which are primarily produced through transcription of the corresponding gene by the trans- cription machinery of the cell, or which are processed to mature mRNA by the splice apparatus and the further enzymatic systems for the formation of the 5'-cap and the 3'-poly-A + tail.
• binding means, that the nucleic acid according to the present invention and the transcription product of the gene coding the ⁇ V integrin chain are capable of interacting specifically which may comprise the formation of covalent chemical bonds, electrostatic interactions based on salt bridges, or bonds based on hydrogen bridges and/or hydrophobic interactions as well as binding due to base stacking.
• hydrogen bonds and hydrophobic interactions are the driving forces for binding through hybridization between the nucleic acid according to the present invention and the transcription product of the gene coding for the ⁇ V integrin chain.
• a hybridization between the above-defined nucleic acid and the ⁇ V integrin transcription product may, for example, occur in case at least a part of the nucleotide sequence of the nucleic acid according to the present invention is, at least in part, complementary to at least a part of the mRNA coding for the ⁇ V integrin chain.
• the mammalian cells are human cells which are preferably selected from endothelial cells, vascular muscle cells, activated leukocytes, macrophages, osteoclasts and tumor cells such as cells from the metastating malignant melanoma, glioblastoma, urothelial carcinoma, breast cancer, ovarian carcinoma, prostatic carcinoma, astrocytoma, lung cancer, colon carcinoma, pancreatic carcinoma and osteosarcoma.
• the nucleic acid according to the present invention may contain deoxyribonucleotides and/or ribonucleotides. Preferably, it contains modified nucleotides such as phosphorothiate nucleotides, aminonucleotides, [ ⁇ -S]- trisphosphate nucleotides, pseudouridine nucleotides and nucleotides having bases containing chemical modifications such as methyl, acetyl or isopentenyl groups. Furthermore, the nucleic acid may be modified by gap-mer technology or may be a locked nucleic acid (LNA).
• LNA locked nucleic acid
• the nucleic acid according to the present invention is an antisense nucleic acid, wherein the 3'-end thereof is directed against the loop and/or the 5'-stem part of a local structure of the ⁇ V integrin chain mRNA; i.e. the 3'-end is capable of binding within the region of nucleotides 3871 to 3992 of the ⁇ V integrin chain mRNA (cf. also Fig. 7A). It is further preferred that the nucleic acid of the present invention has a length of 16 to 24 nucleotides.
• the nucleic acid according to the present invention is an antisense nucleic acid, wherein the nucleotide sequence contains one of the following sequences:
• antisense nucleic acid means that at least a part of the nucleotide sequence of the nucleic acid is at least in part complementary to at least a part of a nucleotide sequence of the transcription product of the gene coding for the ⁇ V integrin chain.
• a further embodiment of the present invention relates to a vector comprising the above-defined nucleic acid.
• the term "vector” refers to a DNA and/or RNA replicon that can be used for the amplification of one or more heterologuous or homologuous nucleotide sequences.
• the vector according to the present invention may contain regulatory elements known in the art.
• DNA or RNA vectors which upon transcription provide the nucleic acid according to the present invention.
• An example is a DNA vector encoding the nucleic acid according to the present invention downstream of the 3'-end of a suitable promoter.
• a further embodiment of the present invention relates to a host organism contai-ning the nucleic acid or the vector as defined above.
• the term "host organism" according to the present invention relates to any suitable prokaryotic or eukaryotic cell or may be a virus.
• a further embodiment of the present invention relates to a method for the production of the above-defined nucleic acid or the vector, comprising cultivating the above- defined host organism under suitable conditions such as culture medium temperature etc.
• the method further comprises isolating the desired products from the culture (cells and/or culture medium) according to procedures known in the art.
• the present invention provides a pharmaceutical composition containing the above-defined nucleic acid or the vector, optionally in combination with a pharmaceutically acceptable carrier and/or diluent.
• a preferred embodiment of the pharmaceutical composition according to the present invention relates to the treatment of pathological disorders by modulation of cell adhesion. More preferably, the pharmaceutical composition according to the present invention affects platelet aggregation, immune functions, tissue repair, cell proliferation, tumor invasion, inflammation and inherited diseases. In case of the inhibition of the expression of integrin ⁇ V ⁇ 3, the pharmaceutical composition according to the present invention is particularly useful for the treatment of pathological disorders by inhibition of neoangiogenesis.
• disorders which can be treated with the pharmaceutical composition according to the present invention comprise the group of cancer diseases such as the growth of solid tumors such as metastating malignant melanoma, glioblastoma, urothelial carcinoma, breast cancer, ovarian carcinoma, prostatic carcinoma, astrocytoma, lung cancer, colon carcinoma, pancreatic carcinoma and osteosarcoma or hematologic neoplasias such as multiple myeloma, acute and chronic leukemias, myelodysplastic syndrome and malignant lymphomas, restenosis after angioplasty, stenosis of vein bypasses, chronic inflammatory diseases such as Crohn's disease, ulcerative colitis, rheumatiod arthritis, ocular neovascular diseases such as diabetic retinopathy, disorders associated with excessive bone resorption such as osteoporosis, the above-mentioned multiple myeloma and bone metastasis, disorders of mammalian oral cavity which are related to inflammatory processes,
• gingival mucosa such as gingivitis and peritonitis
• inflammatory skin disorders such as psoriasis, allergies, neurodermitis and akne.
• Further examples are acute or chronic inflammatory disorders of the cavity of the mouth, particularly inflammatory disorders of the gums.
• a further embodiment of the present invention relates to a cosmetological composition containing the above-defined nucleic acid or the vector, optionally in combination with a cosmetologically acceptable carrier and/or diluent.
• An application of the nucleic acid, the vector, the host organism or the pharmaceutical composition according to the present invention relates, for example, to a method for the inhibition of the expression of ⁇ V integrins such as integrin ⁇ V ⁇ 3 in mammals comprising introducing the nucleic acid, the vector, the host organism or the pharmaceutical composition according to the present invention in the mammalian organism.
• the method may be used, e.g. for the treatment of pathological disorders by modulation of cell adhesion, e.g. in order to inhibit neoangiogenesis, preferably for the treatment of the diseases as mentioned above.
• mammalian organism comprises any type of single cells derived from mammals such as culture cells, as well as the mammal perse.
• Preferred mammalian organisms include man, dog, canine, cat, cow, pig and horse.
• introducing into a mammalian organism may, e.g. in the case of single cells such as culture cells of mammalian cell lines, comprise a chemical transfection such as the Ca-phoshphate procedure or lipofection, or an electroporation, in the case of the whole animal or the human a subcutaneous, percutaneous, intramuscular, oral, intratracheal, peritoneal or intravenous and intraarterial administration of the nucleic acid, the vector, the host organism or the pharmaceutical composition according to the present invention.
• An intravenous or intraarterial administration may comprise, for example, a bolus injection and/or a continuous infusion.
• Another embodiment of the present invention relates to a diagnostic kit containing the above-defined nucleic acid and/or the vector and/or the host organism.
• the diagnostic kit according to the present invention may, e.g. be used for the detection of expression levels of ⁇ V integrins.
• the above-defined nucleic acid contained in the diagnostic kit according to the present invention may be used for PCR for the detection of ⁇ V integrin mRNA.
• Fig. 1 is a graphical representation showing the relative ⁇ V expression after stimulation with phorbol-12-myristate-13-acetate (PMA) in human umbilical vein endothelial cells (HUVEC) which have been transfected with antisense oligodeoxyribonucleotides (ODN) according to the present invention (filled bars) or with control ODN (unfilled bars).
• PMA phorbol-12-myristate-13-acetate
• UUVEC human umbilical vein endothelial cells
• ODN antisense oligodeoxyribonucleotides
• Fig. 2 is a graphical representation showing the relative proliferation rate of HUVEC cells transfected with antisense ODN according to the present invention (filled bars) or with control ODN (unfilled bar). The proliferation rate of untransfected stimulated cells was set to 100%.
• Fig. 3 is a graphical representation showing the annexin binding as a measure of apoptosis rate (expressed as mean fluorescence intensity) of HUVEC cells transfected with an ODN according to the present invention (av32, filled bar) or with a control ODN (avSC32, unfilled bar).
• Fig. 4 is a graphical representation showing the concentration dependence of ⁇ V-directed asON on protein expression in HUVEC. asON were transfected in HUVEC at indicated concentrations (10-100 nM) following stimulation of ⁇ V expression with PMA. Effects of asON on ⁇ V protein expression were determined by FACS analysis. The basis ⁇ V-expression of unstimulated cells was set to 0% and the expression following stimulation by PMA was set to 100%.
• Fig. 5 is a graphical representation showing the effect of ⁇ V-directed asON on mRNA levels in HUVEC after stimulation with PMA. ⁇ V mRNA levels are strongly decreased in the presence of 30 nM asON (left hand side), whereas levels of control mRNA 18S are not affected indicating specificity of asON-mediated inhibiton (right hand side).
• Fig. 7 is a graphical representation showing positions (A) and efficacy (B) of ⁇ V- directed asON against target region pos. 3865 - pos. 3910. Effects of asON (50 nM) on ⁇ V protein expression were determined by FACS analysis. The basis ⁇ V-expression of unstimulated cells was set to 0% and the expression following stimulation by PMA was set to 100%. The present invention is further illustrated by the following non-limiting example.
• the secondary structures of the ⁇ V mRNA were calculated with the computer program mfold 2.0 of the Heidelberg Unix Sequence Analysis Resources (HUSAR). The secondary structures were systematically analysed by the method of Patzel et al. (Nucl. Acids Res. 27: 4823-4334, 1999). On the basis of these analyses, suitable local target sequences were identified.
• Selected antisense ODN and control ODN were all phosphorothioate-modified and were synthesized by a commercial supplier.
• the antisense oligonucleotides which showed the best performance in the inhibition of ⁇ V expression had the following sequences:
• av3871 5' -GCTTGAAGGATTTGACCTAA-3 (pos. 3871-3890, SEQ ID NO 1)
• av3878 5' -TAGGTTGGCTTGAAGGATTT-3 (pos. 3878-3897, SEQ ID NO 2)
• av 3880 5' - ATAGGTTGGCTTGAAGGAT-3 (pos. 3880-3899, SEQ ID NO 3)
• av3882 5' -AGTATAGGTTGGCTTGAAGG-3 (pos. 3882-3901 , SEQ ID NO 4)
• av3884 5' -TTTAGTATAGGTTGGCTTGA-3 (pos.
• the activity of antisense ODN was tested on human umbilical vein endothelial cells (HUVEC).
• the oligonucleotides (0,05 ⁇ M final concentration) were transfected in HUVEC using lipofectin as carrier.
• the cells were stimulated with PMA after 8 hours. After further 48 hours, the expression of the ⁇ V integrin chain, the proliferation and the apoptosis rate were studied.
• Expression of the ⁇ V integrin chain was determined by flowcytometry. Using the mean fluorescence intensity of the cells in each sample, the inhibition of the PMA- induced expression of the ⁇ V integrin chain by the ODN was calculated.
• the proliferation after transfection with ODN was determined by photometrical measurement of the BrdU incorporation.
• the apoptosis rate was determined by flowcytometry by the binding of annexin.
• pretreatment of HUVEC cells with an ⁇ V-directed antisense oligonucleotide inhibited the formation of tubular structures in an endothelial cell tube assay using Matrigel-coated cell culture plates.
• antisense nucleic acids according to the present invention display a significantly stronger inhibiton of ⁇ V expression than prior art asODN (aV40: 5 ' - GCGGCGGAAAAGCCATCG-3 ' disclosed in EP-A-0 950 709; aV4: 5 ' - ACGCCAAGCCGGGAGCGGTA-3 ' disclosed in Treasurywala et al. (1998) GLIA 24, 236- 243); aV136: 5 ' -GACTGTCCACGTCTAGGT- 3 ⁇ disclosed in Nip et al. (1995) J. Clin. Invest. 95, 2096-2103). Furthermore, the 3'-end of preferred asODN is directed against a common structure, i.e.
• the present invention provides for the first time a highly specific inhibitor of ⁇ V- expression, e.g. in endothelial cells, on the basis of antisense oligonucleotides which do not act by interfering with the extracellular protein-protein interaction but which act intracellularly by downregulation of the expression of the receptor.
• a highly specific inhibitor of ⁇ V- expression e.g. in endothelial cells
• the nucleic acid inhibitors of the present invention can be prepared easily in large amounts and in good manufacturing practice (GMP) quality by simple chemical synthesis.
• the antisense-based inhibitors of the present invention can be used successfully in inhibition of neoangiogenesis, e.g. for the treatment or prevention of restenosis following angioplasty or for the treatment of malignant tumors or hematological malignancies such as multiple myeloma.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Pharmacology & Pharmacy (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Animal Behavior & Ethology (AREA)
• Medicinal Chemistry (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Genetics & Genomics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Biomedical Technology (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Biotechnology (AREA)
• Molecular Biology (AREA)
• General Engineering & Computer Science (AREA)
• Pain & Pain Management (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Plant Pathology (AREA)
• Microbiology (AREA)
• Dermatology (AREA)
• Rheumatology (AREA)
• Biochemistry (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Priority Applications (1)

Applications Claiming Priority (4)

Publications (1)

Family

ID=8169980

Family Applications (2)

Family Applications Before (1)

Country Status (3)

Cited By (1)

Families Citing this family (2)

Family Cites Families (3)

• 2000
  • 2000-10-12 EP EP00121394A patent/EP1197553A1/de not_active Withdrawn
• 2001
  • 2001-10-11 WO PCT/EP2001/011790 patent/WO2002031142A2/en not_active Application Discontinuation
  • 2001-10-11 EP EP01978406A patent/EP1409668A2/de not_active Withdrawn
  • 2001-10-11 AU AU2002210530A patent/AU2002210530A1/en not_active Abandoned

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events