EP1590457A2 - Oligonucleotides diriges contre un gene de survivine et utilisation de ceux-ci - Google Patents

Oligonucleotides diriges contre un gene de survivine et utilisation de ceux-ci

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Publication number
EP1590457A2
EP1590457A2 EP04709196A EP04709196A EP1590457A2 EP 1590457 A2 EP1590457 A2 EP 1590457A2 EP 04709196 A EP04709196 A EP 04709196A EP 04709196 A EP04709196 A EP 04709196A EP 1590457 A2 EP1590457 A2 EP 1590457A2
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EP
European Patent Office
Prior art keywords
oligonucleotide
tumor
survivin
carcinoma
therapy
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.)
Withdrawn
Application number
EP04709196A
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German (de)
English (en)
Inventor
Axel Meye
Susanne FÜSSEL
Manfred P. Wirth
Uta Schmidt
Bernd KÜPPERS
Helge Taubert
Matthias Kappler
Matthias Bache
Frank Bartel
Peter WÜRL
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.)
Technische Universitaet Dresden
Martin Luther Universitaet Halle Wittenberg
Original Assignee
Technische Universitaet Dresden
Martin Luther Universitaet Halle Wittenberg
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Publication date
Application filed by Technische Universitaet Dresden, Martin Luther Universitaet Halle Wittenberg filed Critical Technische Universitaet Dresden
Publication of EP1590457A2 publication Critical patent/EP1590457A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • 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
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • 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
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]

Definitions

  • Oligonucleotides directed against a survivin gene Oligonucleotides directed against a survivin gene
  • the present invention relates to oligonucleotides which are directed against a survivin gene and the use of these oligonucleotides for the diagnosis, prophylaxis, reduction, follow-up control of diseases associated with cell growth, differentiation and / or division, such as, for example, tumor diseases.
  • Survivin which belongs to the gene family of inhibitors of apoptosis (IAP), is a recently discovered link ("interface molecule") between cell cycle progression and apoptosis control. It is a 142 amino acid long protein with a molecular weight of approx. 16.5 kDa. The gene is located on the long arm of chromosome 17 (17q25) (Ambrosini et al., 1997; Adida et al. 1998). Survivin is usually expressed during embryonic and fetal development, but only to a very limited extent in adult tissues, the former being the Tissue homeostasis and differentiation contributes (Adida et al. 1998). Interestingly, survivin is primarily expressed during the G2 / M phase.
  • survivin in tumor cells can override the apoptosis checkpoint in the G2 / M phase and allow progression of transformed cells through mitosis (Li et al. 1998).
  • Survivin is overexposed in numerous malignant tumors, such as lung, colon, stomach, breast, pancreas, prostate, bladder carcinoma, large cell non-Hodgkin's lymphoma, melanoma and in neuroblastoma and plays a part in the genesis and in particular the progression of these tumors play an essential role
  • a disadvantage of the previous disclosures is that the person skilled in the art has no effective and specific therapeutic approaches that could be used in organisms, especially higher organisms, such as mammals, especially humans.
  • Numerous previous disclosures describe very large constructs that interact with the survivin gene. With such large constructs, effects can be achieved in certain cell suspensions or other cultures.
  • higher organisms which have an effective immune defense and numerous enzymatic control mechanisms, such large constructs or substances interacting with Survivin are immunologically attacked, destroyed or disadvantageously modified before they interact with the actual target molecule and thus develop a specific effect in the organism can.
  • the stability of relatively large constructs or substances is limited when used in humans.
  • the accessibility and target-specific movement of the constructs and substances in the body is limited. Such negative side effects are unpredictable, difficult controllable and therefore represent a relatively high safety risk in the context of treatment in humans, for example against a malignant tumor disease.
  • the object of the invention was therefore to provide alternative molecules which interact simply, safely and effectively with specific secondary structure motifs of the mRNA of the survivin gene.
  • the invention solves this technical problem by ⁇ the provision of oligonucleotides directed against the mRNA of survivin gene (database entry NM_001168) and / or its gene and ⁇ transcript, wherein the oligonucleotides with mRNA target designs of the Survivin gene interact specifically in a sequence range from 30 to 1350.
  • the numbers in the further text refer to the corresponding nucleotide positions within the survivin mRNA (total length 1619 nucleotides).
  • the invention thus relates to the surprising teaching that a highly specific and very efficient interaction with the survivin mRNA is possible with the oligonucleotides according to the invention.
  • the person skilled in the art can, by disclosing the teaching according to the invention, specific oligonucleotides such as antisense constructs, ribozymes, DNAzymes or siRNA constructs generate, which interact with 'to the target sequence region that survivin expression inhibited, reduced and / or inhibited.
  • other molecules that are able to interact with the corresponding sequence regions can of course also be selected; such as B. ' Antibodies, affilines, lectins or aptamers.
  • the oligonucleotides according to the invention can be used in vivo and in vitro, for example to attack the target mRNA of survivin temporarily and intracellularly in a specifically efficient manner and thus to inhibit the oncogenic function of tumor-associated abnormal survivin expression.
  • the oligonucleotides according to the invention can thus be used, for example, in a diagnostic or therapeutic method or can be used in a kit for therapeutic purposes.
  • Another method can be, for example, additive therapy for humans to treat human tumors locally and / or systemically, e.g. with other nucleic acid-based constructs, immunotherapeutics, chemotherapeutic agents, radiation and other methods for tumor treatment in combination with the use of oligonucleotides according to the invention.
  • Target sequence ranges from 33 - 52, 41 - 60, 49 - 68, 92 - 114, 261 - 280, 264 - 283, 278 - 297, 282 - 301, 283 -
  • the Survivin gene effectively inhibit, in particular by the oligonucleotides specifically interact with the mRNA secondary structure.
  • the oligonucleotides preferably interact with the target sequence region of 92-114, 261-280, 264-283, 283-305, 286-305, 504-523, 532-551 and / or 743-762 of the mRNA of the survivin gene.
  • the siRNA very particularly preferably interact with the areas 92-114, 262-270-IVS2 1-14, 262-270 / 389-401, 283-305.
  • the sequence region and / or the oligonucleotide is changed by addition, amplification, inversion, missense mutation, nonsense mutation, point mutation, deletion and / or substitution.
  • parts of the target sequences mentioned are used with changes within or with changed edge areas or different derivatizations / modifications / fusions / complexations.
  • oligonucleotides according to the invention with molecules which support the directed transport to the target site, the uptake into and / or the distribution within a target cell; such molecules. are known to the person skilled in the art or in Kappler et al. (2004).
  • the mutations in the sequence region of the survivin gene and its transcript variants can, for example, be inheritable or non-inheritable changes in the sense of the invention.
  • mutations in connection with a Count gene and / or chromosome mutations associated with changes in the survivin gene and its transcript variants can arise from the fact that parts of the chromosome are lost, doubled, have the opposite orientation or are transferred to other chromosomes.
  • the mutation affects only one or a few neighboring base pairs, as is the case for example with the point mutation.
  • a codon can be converted into a synonymous codon
  • the mutation ends the translation at a specific point, and the survivin fragments formed can be inactive or active.
  • the oligonucleotide is a nucleic acid construct.
  • Nucleic acid constructs in the sense of the invention can be all structures which are essentially based on nucleic acids or whose active center is essentially based on nucleic acids. It can of course be possible that part of the oligonucleotide / construct from lipids, carbohydrates. or proteins or peptides - for example in the form of a nanocapsule - and this construct comprises a region which contains nucleic acids which in particular have the sequence region of 33 -
  • Oligonucleotide an antisense oligonucleotide (AS-ON)
  • DNAzym DNAzym, a ' ribozyme, a peptide nucleic acid (PNA), a so-called “locked nucleic acid” (LNA) and / or an siRNA.
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • oligonucleotides ON
  • peptide nucleic acids peptide nucleic acids
  • PNAs ribozymes
  • DNAzymes DNAzymes
  • Tab. 1 AS effects and their mechanisms of action ss - "single stranded" (single strand)
  • AS-ON as therapeutic substances is one of several other fields of application in particular also represents a new promising therapy concept for oncological diseases. While conventional chemotherapy leads to non-specific inhibition of cell proliferation, antisense therapy can specifically inactivate those mRNAs that form the molecular basis for the degenerate, deregulated growth and the Represent tumor progression and may be responsible for the inhibition of the body's immune defense.
  • AS-ON differ from other therapeutic agents, such as antibodies, toxins or immunotoxins, in that they are relatively small molecules with a molecular weight of usually about 5 kDa.
  • the small size of the AS-ON enables good tissue penetration.
  • tumor blood vessels are permeable to substances in a size range between 4-10 kDa. This means that therapeutic AS-ON tumor blood vessels can penetrate better.
  • Another advantage of these substances for example compared to antibodies that are only effective against extracellular proteins, is that, in addition to the membrane-bound proteins, both cytoplasmic and nucleus-localized proteins can be attacked using the respective target mRNA by means of antisense technology.
  • non-antisense effects when using the phosphothioate oligonucleotides (PS-ON), in addition to the above-mentioned target-specific AS effects, so-called “non-antisense” effects also advantageously occur, which in particular lead to a non-specific Inhibition of cell growth- lead.
  • These effects are strongly dependent on the oligo sequence or of 'certain sequence motifs and occur because of the strong polyanionic charge of the PS-ON, which may have a binding of the PS-ON of vital proteins result.
  • the effects mentioned could be overcome by partially phosphothioate-modified AS-ON or by further modifications, for example the incorporation of ribonucleotides instead of deoxyribonucleotides.
  • a terminal modification of ON constructs offers in particular improved stability when applied in vivo and in the extra- and intracellular environment of the target cells, such as in particular the protection against degradation by exonucleases.
  • a positive effect when using PS-ON is its immunostimulatory effect, which can support possible therapeutic success in some tumor applications.
  • Ribozymes are 'as catalytically active RNA molecules capable of cellular RNA structures to recognize as substrates and sequence-specifically cleave phosphodiester bond at a specific sequence NUX. The detection takes place via AS arms, which enable hybridization with the target mRNA due to complementary sequences.
  • AS-ON ribozymes have the fundamental advantage that a ribozyme molecule as a real catalyst can convert a large number of identical substrate molecules. Therefore, ribozymes are already effective in a much lower concentration than ON and also lead to irreversible RNA degradation due to substrate cleavage [Sun et. al.].
  • ribozymes Compared to antisense-ON, ribozymes have the advantage that a ribozyme molecule can act as a real catalyst in a multiple-turnover reaction and convert a large number of identical substrate molecules. Therefore, ribozymes are advantageously effective at a lower concentration than AS-ON and, moreover, lead to irreversible inhibition of the RNA by the substrate cleavage.
  • the hammerhead ribozyme (review: Birikh et al. 1997; Tanner 1999) is particularly advantageous for such applications because it can be catalytically active as a comparatively small molecule (approx. 30-50 nucleotides).
  • a very 'effective trans-cleaving hammerhead ribozyme is composed for example of only 14 conserved nucleotides in the catalytic domain and two variable stem sequences - advantageously of each 6 - 8 nucleotides -, by Watson-Crick base pairing - analogous to the AS-ON - the Realize sequence-specific recognition of the substrate and then inactivate it by cleaving a phosphoric diester bond.
  • RNA interference as a method of gene inhibition is advantageously mediated by small synthetically produced RNA oligonucleotides ("small interfering RNAs", siRNA), which enable selective inhibition of the intracellular synthesis of survivin.
  • siRNAs small synthetically produced RNA oligonucleotides
  • dsRNA double-stranded RNA
  • mRNA single-stranded target RNA
  • siRNAs short interfering dsRNAs
  • siRNAs short RNA duplexes
  • siRNAs have a characteristic length of 21-23 nt with a 2 nt long single strand overhang at the 3 'terminus of both chains.
  • the sequence of this siRNA now determines the recognition of homologous mRNA regions and their degradation by activating cell-specific RNases. In this way, the protein synthesis of the repressed target gene can be influenced directly, and thus switching off of the target protein can be induced.
  • siRNA blocks the flow of information in the cell by inhibiting the translation of survivin and its variants.
  • siRNAs can advantageously inhibit other RNA molecules that are not translated and thus only act on the RNA level.
  • the oligonucleotide or the oligonucleotides are immobilized.
  • immobilization means different methods and techniques for fixing the oligonucleotides on specific supports.
  • the immobilization can serve, for example, to stabilize the oligonucleotides, as a result of which their activity, in particular during storage or in the case of a single batch approach, is not modified or reduced or disadvantageously modified by biological, chemical or physical effects.
  • the immobilization of the oligonucleotides enables repeated use under routine technical or clinical conditions. Furthermore, the sample can be continuously reacted with the oligonucleotides.
  • crosslinking In the crosslinking, the oligonucleotides are fixed to one another without their activity being adversely affected; they are advantageously no longer soluble.
  • Binding to a carrier takes place, for example, by adsorption, ion binding or covalent binding. This can also take place within microbial cells or liposomes or other membrane-containing closed or open structures.
  • the oligonucleotide is advantageously not influenced in its specificity / activity by the fixation. It can advantageously be used multiple times or continuously, for example in the clinic, for diagnosis or therapy in a vehicle-bound manner.
  • (iii) Inclusion takes place in the sense of the invention in particular on a semipermeable membrane in the form of gels, fibrils or fibers.
  • Encapsulated oligonucleotides, such as antisense constructs are separated by a semipermeable membrane from the surrounding sample solution in such a way that they advantageously still have the sequence range from 33-52, 41-60, 49-68, 92-114, 261-280 , 264 - 283, 278 - 297, 282 - 301, 283 - 305, 286 - 305, 501 - 520, 504 - 523, 516 - 535, 519 - 538, 526 - 545, 532 - 551, 716 - 735, 719 - 738, 724 - 743, 740 - 759, 743 - 762, 1126 - 1145, 1128 - 1147, 1302 - 1321, 1304 - 1323, 1317 - 13
  • Various methods are available for immobilization, such as adsorption onto an inert or electrically charged inorganic or organic carrier.
  • Such carriers can be, for example, porous gels, aluminum oxide, concrete, agarose, starch, nylon or polyacrylamide.
  • the immobilization takes place through physical / chemical binding forces, often with the participation of hydrophobic interactions and ionic bonds.
  • Such methods are advantageously easy to use and do not influence the conformation of the oligonucleotides, or only to a minor extent.
  • the binding can advantageously be improved by electrostatic binding forces between the charged groups of the oligonucleotides and the carrier.
  • the surface of microscopic porous glass particles can be activated by treatment with silanes and then reacted with oligonucleotides.
  • oligonucleotides With polyacrylamide resins, numerous oligonucleotides can advantageously form direct covalent bonds. When enclosed in three-dimensional networks, the oligonucleotides are enclosed in ionotrophic gels or other structures known to the person skilled in the art. The pores of the matrix are special.
  • oligonucleotides are retained and an interaction with the target molecules or with the sequence range from 33-52, 41-60, 49-68, 92-114, 261-280, 264-283, 278-297, 282- 301, 283 - 305, 286 - 305, 501 - 520, 504 - 523, 516 - 535, 519 - 538, 526 - 545, 532 - 551, 716 - 735, 719 - 738, 724 - 743, 740 - 759, 743 - 762, 1126 - 1145, 1128 - 1147, 1302 - 1321, 1304 - 1323, 1317 - 1336, 1325 - 1344 and / or 1327 - 1346.
  • the reaction space of the oligonucleotides is limited with the help of membranes.
  • the microencapsulation can be carried out, for example, as an interfacial polymerization. Immobilization during microencapsulation makes the oligonucleotides insoluble and therefore reusable.
  • immobilized oligonucleotides are all oligonucleotides which are in a state which permits their reuse. The restriction of the mobility and the solubility of the oligonucleotides by chemical, biological or physical means advantageously leads to low process costs.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the oligonucleotides according to the invention, optionally in a combination with a pharmaceutically acceptable carrier.
  • This pharmaceutical carrier can include, in particular, additional substances and substances, such as medical and / or pharmaceutical auxiliary substances.
  • Medical auxiliaries are, for example, those substances which are used for 'production as ingredients of pharmaceutical compositions.
  • Pharmaceutical-technical auxiliaries serve the appropriate formulation of the pharmaceutical composition or the drug and can even - if they are only required during the production process - be subsequently removed or can be used as pharmaceutically acceptable carriers are part of the pharmaceutical composition.
  • the pharmaceutical composition is optionally in combination with a pharmaceutically acceptable diluent.
  • the pharmaceutical composition can be administered, for example, in connection with gene therapy, for example also via suitable vectors, such as, for example, viral vectors.
  • suitable vectors such as, for example, viral vectors.
  • the type of dosage and route of administration can be determined by the attending physician according to the clinical factors. It is known to the person skilled in the art that the type of dosage depends on various factors, such as, for example, the size, the body surface, the age, the sex or the general health of the patient, but also on the specific agent which is administered Duration and type of administration and of other medications that may be administered in parallel, especially in combination therapy.
  • the invention also relates to a kit comprising the oligonucleotides and / or the pharmaceutical composition. Furthermore, the invention also relates to an array comprising the oligonucleotides and / or the pharmaceutical composition.
  • the kit and array can be used to diagnose and / or treat diseases associated with the activity of the survivin gene.
  • the invention also relates to the use of the oligonucleotides, the kit, the array for diagnosis, prophylaxis, reduction, therapy, monitoring and / or post-treatment of diseases associated with cell growth, differentiation and / or division, preferably benign and malignant tumor diseases (Neoplasms), other hyper- and / or dysproliferative diseases.
  • Neoplasms benign and malignant tumor diseases
  • the disease associated with cell growth, differentiation and / or division is a tumor.
  • the tumor is particularly preferably a solid tumor and / or a blood or lymph gland cancer.
  • the tumors which may be of epithelial or mesodermal origin, can be benign or malignant cancers of the organs of the lungs, prostate, bladder, kidney, esophagus, stomach, pancreas (pancreas ), the brain, the ovary, the skeletal system, in particular the adenocarcinoma of the breast, prostate, lungs and intestine; Bone marrow cancer, melanoma, hepatoma, the head and neck tumors can be treated explicitly as representatives of malignant (so-called malignant) tumors.
  • the group of blood and lymph gland cancers includes all forms of leukemia (e.g.
  • B-cell leukemia in connection with B-cell leukemia, mixed-cell leukemia, zero-cell leukemia, T-cell leukemia, chronic T-cell leukemia, HTLV-II- associated leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, mast cell leukemia and yeloid leukemia) and lymphoma.
  • mesenchymal malignant tumors (so-called bone and soft tissue sarcomas): fibrosarcoma; the malignant histiocytoma; liposarcoma; hemangiosarcoma; chondrosarcoma and osteosarcoma; Ewing's sarcoma; leio and rhabdomyosarcoma, synovial sarcoma; Carcinosarcoma.
  • Neoplasms Bone neoplasms, breast neoplasms, neoplasms of the digestive system, colorectal neoplasms, liver neoplasms, pancreatic neoplasms, neoplasms of the brain, testicular neoplasms, orbital neoplasms, in particular, count as further types of tumors, which are also summarized under the term “neoplasms”.
  • the cancer or the tumor that is treated or prevented is selected from the group: tumors of the ear, nose and throat area including tumors of the inner nose, the sinuses, the nasopharynx, the lips, the Oral cavity, the oropharynx, the larynx, the hypopharynx, the ear, " the salivary glands and paragangli ⁇ me, tumors of the lungs including non-small cell bronchial carcinomas, small cell bronchial carcinomas, tumors of the mediastinum, tumors of the gastrointestinal tract, despas pas, des pas the liver, the gallbladder and biliary tract, the small intestine, colon and rectal carcinomas and anal carcinomas, urogenital tumors including tumors of the kidneys, ureters, bladder, prostate, urethra, penis and testes, gynecological tumors including tumors of the cervix, vagina, vulva, carcinoma of the body, malignant tropho
  • Carcinoid tumors and carcinoid syndrome multiple endocrine neoplasias, bone and soft tissue sarcomas, mesotheliomas, skin tumors, melanomas including cutaneous and intraocular melanomas, tumors of the central nervous system, childhood tumors including retinoblastoma, Wilms tumor, neurofibromatosis, neurarklastoma, lymphoma, lymphoma comprising non-Hodgkin's lymphomas, cutaneous T-cell lymphomas, primary lymphomas of the central nervous system, Hodgkin's disease, leukemias including acute leukemias, chronic myeloid and lymphatic leukemias, plasma-line neoplasms, myelodysplastic syndromes, paraneoplastic syndromes, primary malignancies, metastases without metastases, metastases without metastases, metastases without metastases, metastases without metastases, metastases without metastases Carcinom
  • the tumor is a colon carcinoma, a gastric carcinoma, a pancreatic carcinoma, a colon cancer, a small bowel cancer, an ovarian carcinoma, a cervical carcinoma, a lung cancer, a renal cell carcinoma, a brain tumor, a head and neck tumor , a liver carcinoma and / or a metastasis of these tumors / carcinomas.
  • the solid tumor is a breast, bronchial, colorectal and / or prostate carcinoma.
  • the tumor of the urogenital tract is a bladder carcinoma.
  • Bladder carcinoma (BCa) is the fourth most common form of cancer and the seventh most common cause of cancer death in men in the Federal Republic of Germany.
  • TUR-B as a general primary therapy for BCa enables organ-preserving removal of superficial tumors. Despite this histopathologically defined complete removal of the tumor, 50-70% of the patients are relatively high
  • Treatment problem is the synchronous or metachronous multifocal appearance of tumor foci, which may result in the occurrence of recurrences remote from the resected primary tumor location [Sidransky et. al.].
  • recurrence or tumors primarily classified as superficial, long-term prophylaxis is usually carried out after TUR-B with an immune (Bacillus Calmette-Guerin - BCG) or chemotherapeutic agent (e.g. Mitomycin-C, Taxol, Gemcitabine / Cisplatin ).
  • bladder carcinoma Because of health policy. Significance of bladder carcinoma (especially in the western industrialized countries), the lack of tumor-specific markers and the known tumor biological and cellular heterogeneity of the tumor, there is an intensive search in the clinical research area of bladder carcinoma, which aims in particular to identify new or / and complementary therapy options.
  • the oligonucleotide In a particular embodiment of the invention, the oligonucleotide, the pharmaceutical composition,
  • the kit and / or the array is used for a follow-up, which is essentially monitoring the
  • the oligonucleotide is used in a combination therapy, in particular for the treatment of Tumors. It is particularly preferred here that the combination therapy comprises chemotherapy, cytostatic treatment and / or radiation therapy.
  • the combination therapy is an adjuvant, biologically specified form of therapy. It is very particularly preferred that this form of therapy is an immunotherapy. Furthermore, it is particularly preferred that the combination therapy comprises a gene therapy and / or a therapy with an oligonucleotide of the same or a different target molecule.
  • a gene therapy in the sense of the invention is a form of treatment using natural or recombinantly modified nucleic acid constructs, individual gene sequences or entire gene or chromosome sections or coded transcript areas, their derivatives / modifications with the aim of a biologically-based and selective inhibition or Reverting the disease symptoms and / or their causal causes, which in the special case means the inhibition of a target molecule overexpressed in the course of a disease at the nucleic acid level, in particular at the transcript level.
  • combination therapies in particular for the treatment of tumors, are known to the person skilled in the art. It can be provided, for example, that cytostatic treatment takes place within a combination therapy or, for example, irradiation of a specific area of the tumor, this treatment using gene therapy is combined, the oligonucleotide according to the invention being used as an anti-cancer agent.
  • the oligonucleotide according to the invention can also be used in combination with other oligonucleotides which are directed against the same target molecule or against a completely different structure. Accordingly, the oligonucleotide can very particularly preferably be used to increase the sensitivity of tumor cells to cytostatics and / or radiation. It is further preferred that the oligonucleotide is used to inhibit viability, the proliferation rate of cells and / or to induce apoptosis and a cell cycle arrest.
  • Antisense-ON Survivin-directed antisense constructs (Antisense-ON) and their effectiveness
  • Fig. 1 AS-ON-specific viability inhibition of EJ28 cells 48 h after a single transfection with 250 nM ON (complexed with lipofectin in the w / w ratio 1: 3). The values are mean values of a parallel quadruple determination (with standard deviations). It should be noted that a control ON (NS-1) comes from the literature (Chen et al. 2000) was carried out as a control in the test series.
  • Fig. 2 AS-specific reduction in survivin mRNA expression: EJ28 cells were ON for 4 h with 250 nM each
  • GPDH Dehydrogenase
  • Fig.4 AS-specific reduction in survivin protein expression: In the survivin ELISA (R&D), the survivm content was determined 24h after the transfection with 250nM ON (duration 4 h) in 2 ⁇ 10 4 EJ28 cells - Protein quantified. The survivin concentration (pg / ml) was related to the total protein concentration (mg / ml). The absolute mean values (pg survivin / ⁇ g protein) were normalized to NS-1. * The specified control ON and AS-ON 1099 come from a published study and serve as a comparison (Chen et al. 2000)
  • tumor cell lines there are five tumor cell lines (sarcoma cell line US 8/93 [Taubert et al. 1997], two rhabdomyosarcoma cell lines (RD: CCL-136 and A204: HTB-82), a leiomyosarcoma cell line SK-LMS: HTB-88 and an osteosarcoma cell line Saos- 2: HTB 85) in relation to their behavior after treatment with siRNA
  • Fig. 5 siRNA-specific reduction in survivin mRNA expression: WTS cell lines (A204, US8 / 93, RD, SK-LMS, Saos-2) were 6 h with 300 nM siRNA 1-2 (complexed with oligofectamine in the w / w ratio 1: 1) transfected and harvested 24 h after the start of the transfection. After the RNA extraction and cDNA synthesis, the survivin transcript amount per cell line was compared to the cells treated with the nonsense siRNA 3-4. The nonsense control siRNA 3-4 treated cells were set equal to 100% and the result was that of 300 nM each siRNA 1-2 treated cells received a relative amount of transcription.
  • Fig. 6 Representation of the time-dependent repression of the survivin mRNA in the cell lines SK-LMS after siRNAi application (6 h with 300 nM siRNA 1-2 or nonsense siRNA 3-4 (complexed with oligofectamine in the w / w ratio 1: 1) transfected). The cells were harvested 24 h, 48 h and 72 h after the transfection. After the RNA extraction and cDNA synthesis, the survivin transcript amount was determined (zmol survivin / amol GAPDH).
  • the main cell biological effect of the specific survivin inhibition was a significant increase in apoptosis. This was determined by determining the cells floating in the culture flask, with 70-90% of all cells found in the supernatant being apoptotic were (determination of the nucleus morphology after DAPI staining). A time-progressive apoptosis rate of the cells treated with anti-survivin siRNA was found, with up to 19% of all cells becoming apoptotic after 3 days. This reaction could be verified by demonstrating reduced survival of the cells treated in this way. Cell colony formation tests showed a reduced cell viability of approx. 50% compared to the nonsense control (Fig. 8).
  • Fig. 7 siRNA-specific reduction in survivin protein expression: WTS cell lines (A204, US8 / 93, RD, SK-LMS, Saos-2) were 6 h with 300 nM siRNA 1-2 (complexed with oligofectamine in the w / w ratio 1: 1) transfected and harvested 24 h after the start of the transfection.
  • the survivin protein content was quantified in the Survivin ELISA (R&D) 24 hours after the transfection.
  • the survivin concentration (pg / ml) was related to the total protein concentration (mg / ml).
  • the absolute mean values pg survivin / ⁇ g protein
  • the nonsense control siRNA 3-4 treated cells were set equal to 100% and the result was that of 300 nM each siRNA 1-2 treated cells with a relative amount of transcription (with standard deviations).
  • Fig. 8 Representation of the plating license of the WTS cell line (A204, US8 / 93, RD, SK-LMS, Saos-2)) these were 6 h with 300 nM siRNA 1-2 (complexed with oligofectamine in a w / w ratio) 1: 1) transfected and 24 h after the start of the transfection in a defined manner in cell culture bottles. After 14 days of growth, the grown-up cell colonies were counted and placed in relation to the cell-specific nonsense-siRNA 3-4 treated control.
  • the named constructs were combined with various chemotherapeutic agents.
  • the BCa-relevant cytostatics cisplatin, gemcitabine and mitomycin-C would be used.
  • the chemotherapy the previous transfection with 'og oligonucleotides resulted in remarkable and unexpected enhancement effects in terms of anticancer activity compared to the combination with the control constructs.
  • the combined treatment serves to specify chemotherapy and in this way allows a dose minimization of conventional forms of therapy.
  • some of the tumor cell lines, which are considered resistant per se were " sensitized to individual chemotherapeutic agents.
  • Figure 9 Representation of the relative plating efficiency of the cell line A 204 as a function of the radiation dose. References used
  • Boiziau C Kur requirementsst R, Cazenave C, Roig V, Thuong NT, Toulme JJ: Inhibition of translation Initiation by antisense oligonucleotides via an RNase-H independent mechanism. Nucleic Acids Res (1991) 19: 1113-9.
  • Crooke ST Molecular mechanisms of action of antisense drugs. Biochim Biophys Acta (1999) 1489: 31-44. Kole R, Sazani P: Antisense effects in the cell nucleus: modification of splicing. Curr Opin Mol Ther (2001) 3: 229-34.
  • Olie RA Simoes-Wust AP, Baumann B, Leech SH, Fabbro D, Stahel RA, Zangemeister-Wittke U.
  • a novel antisense oligonucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy.
  • Catalytic nucleic acids from lab to applications.

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Abstract

La présente invention concerne des oligonucléotides qui sont dirigés contre un gène de survivine et des variants de celui-ci, ainsi que l'utilisation de ces oligonucléotides pour le diagnostic, la prévention, l'atténuation ou le contrôle de l'évolution de maladies liées à la croissance, la différenciation et/ou la division cellulaires, telles que des maladies tumorales.
EP04709196A 2003-02-07 2004-02-09 Oligonucleotides diriges contre un gene de survivine et utilisation de ceux-ci Withdrawn EP1590457A2 (fr)

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DE10306083 2003-02-07
DE10306083A DE10306083A1 (de) 2003-02-07 2003-02-07 Erkennungsmoleküle gerichtet gegen ein Survivin-Gen und die Verwendung dieser
PCT/DE2004/000263 WO2004070034A2 (fr) 2003-02-07 2004-02-09 Oligonucleotides diriges contre un gene de survivine et utilisation de ceux-ci

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WO2009114476A1 (fr) * 2008-03-12 2009-09-17 Intradigm Corporation Compositions comportant un arnsi de survivine et leurs procédés d’utilisation
DE102011100581A1 (de) * 2011-05-04 2012-11-08 Eberhard-Karls-Universität Tübingen Universitätsklinikum Behandlung von hyperproliferativen Erkrankungen des Urogenitaltraktes
CN105177000A (zh) * 2015-08-13 2015-12-23 吉林大学 一种抑制Survivin基因表达的siRNA及其应用

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WO1999050440A2 (fr) * 1998-04-01 1999-10-07 Yale University Methode de modulation selective des interactions entre survivine et tubuline
US6335194B1 (en) * 1998-09-29 2002-01-01 Isis Pharmaceuticals, Inc. Antisense modulation of survivin expression

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