EP2665816A1 - Molécules nucléotidiques biologiquement actives pour la destruction ciblée de cellules, leur utilisation ainsi que kit d'application - Google Patents

Molécules nucléotidiques biologiquement actives pour la destruction ciblée de cellules, leur utilisation ainsi que kit d'application

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Publication number
EP2665816A1
EP2665816A1 EP12704718.1A EP12704718A EP2665816A1 EP 2665816 A1 EP2665816 A1 EP 2665816A1 EP 12704718 A EP12704718 A EP 12704718A EP 2665816 A1 EP2665816 A1 EP 2665816A1
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EP
European Patent Office
Prior art keywords
biologically active
cells
cell
nucleotide molecules
mrna
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Withdrawn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
EP12704718.1A
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German (de)
English (en)
Inventor
Tobias PÖHLMANN
Rolf Günther
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.)
Friedrich Schiller Universtaet Jena FSU
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Friedrich Schiller Universtaet Jena FSU
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Application filed by Friedrich Schiller Universtaet Jena FSU filed Critical Friedrich Schiller Universtaet Jena FSU
Publication of EP2665816A1 publication Critical patent/EP2665816A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • 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
    • 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
    • A61P31/04—Antibacterial agents
    • 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
    • A61P31/10—Antimycotics
    • 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
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    • 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
    • C12N15/1137—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 enzymes
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    • 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
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    • 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
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    • 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
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    • 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]
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/18—Type of nucleic acid acting by a non-sequence specific mechanism
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3181—Peptide nucleic acid, PNA
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA

Definitions

  • the invention relates to biologically active molecules based on nucleotides, with which targeted cells can be killed, the use of the biologically active molecules and an application kit for use.
  • RNA-induced silencing complex RNA-induced silencing complex
  • RNA molecules double-stranded RNA molecules
  • siRNA double-stranded RNA molecules
  • siRNA With the help of such molecules, the reading of a gene and the production of an mRNA is not prevented, but it is initiated by the siRNA, a cell-specific mechanism that degrades the target mRNA. Finally, as described above, the formation of a specific protein is suppressed without affecting the expression of other genes (post-transcriptional gene silencing). Current applications of siRNA often seek to suppress expression of only one gene in a cell. Effects in which several genes are switched off simultaneously or nonspecifically are therefore undesirable, which is why the sequences of the siRNA are designed so that these effects are suppressed.
  • the invention is based on the object of killing cells in a wide range of applications, effectively, reliably and as effectively as possible in the organism, without the aforementioned disadvantages of known chemical, physical, biochemical or molecular biological methods occur.
  • the biologically active nucleotide molecules based for example on the basis of RNA, siRNA, PNA, DNA or LNA, with their nucleotide sequence for binding to the mRNA of several genes are oriented thereto, by binding to these genes a plurality, in particular a plurality, "Off". Target "effects for cell-killing stress situations.
  • biologically active nucleotide molecules encompasses nucleotide molecules according to the invention which function under all conditions and applications described herein.
  • the biologically active nucleotide molecules according to the invention display their activity by triggering so-called “off-target” effects .
  • off-target effects that cause cell-killing stress situations in the context of the invention, biological activities and processes to understand in which a nucleotide sequence has multiple target mRNA sequences and potentially affects the expression of multiple genes or triggers cell stress independently of influencing the expression of genes.
  • the cell is so massively influenced by the non-specific nucleotide sequence that the cell dies or the programmed cell death in the cell ( Apoptosis) is initiated.
  • nucleotide molecules for example based on siRNA
  • this nucleotide sequence is there in each case specifically aligned for the mRNA of one or fewer genes, with selective binding to the intended target gene a defined gene manipulation in the cell and in this way to perform a gene-oriented cell influencing.
  • the nucleotide sequence is deliberately designed to be able to dock on several, in particular a variety, mRNAs of genes, possibly also regardless of whether these mRNAs of the genes that are possible for binding are actually present in the cell or not .
  • the ostensible aim of the proposed mRNA binding is therefore not the aforementioned gene activity targeting cell activity, but in particular a large number of (actually any) mRNA bonds of the nucleotide molecules should trigger as many "off-target” effects as possible have been so far as possible to avoid or reduce the targeted gene influence.
  • an excessive stress situation should be generated for the cell, which the target cell can not handle and through which the said target cell (not by targeted manipulation of gene expression, but by general stress) is deliberately killed.
  • the genetic influence inevitably and known to occur with the gene binding and acting on the cell activity is a side effect and could Depending on the effect of the gene influence, the cell impact (in addition to the said intended according to the invention stress situation) further support if necessary.
  • the selection of the binding genes by the nucleotide sequence target genes is thus not or at least not superficially the target effect of an intended gene manipulation for cell influencing, but of the intended effect of achievable by the gene binding off-target effects and the same in the Cell-induced stress situation.
  • the nucleotide sequences are chosen so that they do not coincide, as is conventionally, only with a target gene, but with as much as possible target genes of the cells, resulting in a toxic effect on a large number of genes Generates nucleotide interference and massively affects the physiology of the cell.
  • This proposed application can be used in combination with known mechanisms for achieving cell specificity and with known ways of stabilizing, for example, siRNA and for improved uptake of nucleotide molecules into cells.
  • the proposed nucleotide sequences are not limited to use as classical siRNA; also short (10-20 bp) double-stranded or single-stranded RNA, long (20-3 OObp) double- or single-stranded RNA, DNA or chemical analogs, such as PNA, can be used with the proposed nucleotide sequences.
  • the cell damaging effect of the biologically active nucleotide molecules can be assisted by known stress-inducing nucleotide sequence sequences (FEDOROV Y et al., Off-target effects by siRNA can induce toxic phenotype. RNA (2006), 12: 1188-1196.)
  • the active substance molecules can be introduced into the cells in a manner known per se.
  • the molecular constructs can also be bound to further substances (for example nanoparticles as a carrier system or fluorochromes) for better transport into or onto the cells and for their stabilization or for their detection.
  • the biologically active nucleotide molecules are suitable for the targeted killing of eukaryotic cells, in particular animal, plant or fungal cells, as well as virus-infected and prokaryotic cells. When using the biologically active nucleotide molecules, these can also be used in combination with protease inhibitors.
  • An application kit for the application and administration of the biologically active nucleotide molecules, comprising at least one of them, is advantageous
  • ampoule A which contains and may further contain the biologically active molecule:
  • At least one further ampoule (ampule B) with a transfection system for example nanoparticles, polyethyleneimines or lipids,
  • At least one further ampoule which contains further constituents for binding to the biologically active molecules or the transfection system,
  • Fig.l Schematic representation of a known siRNA, which is introduced into a cell, is specific for an mRNA and suppresses the expression of a target gene
  • Fig.2 Schematic representation of an siRNA according to the invention, which in a
  • RNAi effects off-target effects
  • Figure 3 Schematic representation of a siRNA which is introduced into a cell and there is no reduction in the expression of genes and the degradation of mRNAs, but the cell death by induced in the cell by specific sequence sections of the siRNA stress reactions.
  • FIG. 1 shows the mechanism of a conventional and known siRNA 1 which is introduced into a cell 2 (see symbolized arrow representation) and has a specific nucleotide sequence (not explicitly shown) for binding to a first gene-specific mRNA. Subsequently, the siRNA 1 is incorporated into the RNA Induced Silencing Complex (RISC) (also not explicitly shown), which divides the siRNA 1 into its two single strands and the antisense strand of the siRNA 1 together with the RISC to the first mRNA 3 attached. Thereupon, the gene-specific first mRNA 3 is cut and fragmented, whereby the expression of a target gene based on the first mRNA 3 is suppressed (cf degraded first mRNA 7 in Fig.
  • RISC RNA Induced Silencing Complex
  • siRNA 1 which has become free and has been integrated into the RISC, then attaches itself to the next specific first mRNA 3 present in cell 2 and also degrades it. It is intended that each siRNA 1 binds to and degrades only one specific first mRNA 3. Further second mRNA 4, third mRNA 5 and fourth mRNA 6, which are also present in cell 2, in each case remain unaffected by siRNA 1 or its nucleotide sequence, which is not explicitly shown, so that genes can be delivered to mRNA 4. 6 did not experience any altered expression. This method is well known. FIG.
  • FIG. 2 shows a comparison of the mechanism of an siRNA 8 according to the invention, which is introduced into the cell 2 (see also symbolized arrow representation), in which Again, for example, the first mRNA 3, the second mRNA 4, the third mRNA 5 and the fourth mRNA 6 are located.
  • the proposed siRNA 8 contains (for reasons of clarity not explicitly shown) a chain of one or more of the nucleotide sequences GGUA, CGUC, CGUU, CCAA, AAGG, GGUG, CUCG, CUCC, CUCU, CUUA, GGUC, GGUU, AAAG, AAAC, AAAU, AAGA, AAGC, AAGU, AACA, AACG, AACC, AACU, AAUA, CUUU, AAUG, AAUC, AAUU, AGGA, AGUG, AGUC, AGUU, ACAA, ACAG, ACAC, ACAU, ACGA, ACGG, ACGC, ACGU, ACCA, CAUU, CGAA, ACCG, ACCC, ACCU, ACUA, ACUG, ACUC, ACUU, AUAA, GGAG, GGAC, GGAU, GGGA, GGGC, GGGU, GGCA, GGCG, GGCC, GGCU, GCAA
  • chain-linked nucleotide sequences not only have a degrading effect on mRNA 3-6, but also bind to a plurality or a multiplicity and thus all mRNA molecules (mRNA 3-6) shown in FIG. 2. It is possible that at least one selected nucleotide sequence binds to several or all of the mRNA molecules (mRNA 3-6) shown, or in each case a selected nucleotide sequence selectively acts in each case on a specific mRNA 3-6. It is important that as many as possible (in the best case all) of the mRNA 3-6 are bound and degraded by the chain (total of all nucleotide sequences) of the siRNA 8 (compare degraded first to fourth mRNA 7, 9, 10, 11 in FIG 2).
  • siRNA 8 As a result of the degradation of said plurality of mRNA molecules (simplified in the present example, only four mRNA molecules shown) several to numerous nonspecific RNAi effects (off-target effects) are triggered by the siRNA 8 with (at best) only one nucleotide sequence expression several to many genes suppressed (see degraded mRNA 7, 9, 10, 1 1 in Fig. 2) with the aim to kill in this way the cell 2, which dies by the massive action of siRNA 8.
  • siRNA 8 with a nucleotide sequence (5 '-3') UUAACUGUAUCUGGAGCtt (SEQ ID NO: 3) the mRNA of the genes Suppressor Of Cytokine Signaling-1 (SOCS1, NM_003745.1), N-acetylneuraminic acid phosphatase (NANP , NMJ52667.2), transmembrane protein 215 (TMEM215, NM_212558.2) and the CD81 molecule (CD81, NM_004356.3).
  • SOCS1 Suppressor Of Cytokine Signaling-1
  • NANP N-acetylneuraminic acid phosphatase
  • TMEM215 transmembrane protein 215
  • CD81 CD81, NM_004356.3
  • a nucleotide sequence of the siRNA 8 AACUGUAUCUGGAGCtt (SEQ ID NO: 4) is specifically active for the mRNAs of the genes suppressor of cytokine signaling 1 (SOCS1, NM_003745.1) and N-acetylneuraminic acid phosphatase (NANP, NM_152667.2).
  • a nucleotide sequence GGCUGAACAAAGGAGAtt specifically acts on the major histocompatibility complex, class I, G (HLA-G, NM_002127.4), glycerol kinase 5 (putative) (GK5, NM_001039547.1) and DIP2 disco- interacting protein 2 homolog C (NM_014974.2).
  • the siRNA 8 with the sequence GCUCACCAAUGGAGAtt (SEQ ID NO: 5) acts specifically on the complement component (3b / 4b) receptor 1 (Knops blood group) (CR1, NM_000651.4), transcript variant S, complement component (3b / 4b) Receptor 1 (Knops blood group) (CR1, NM_000573.3), transcript variant F and glutathione S-transferase alpha 4 (GSTA4, NM_001512.3).
  • the sequence UGGCUGGCUGGCUGGCtt (SEQ ID NO: 7), advantageously against the pyroglutamyl peptidase I (PGPEP1, NM_017712.2), Rap guanine nucleotide exchange factor (GEF) 3 (RAPGEF3, NM_006105.
  • transcript variant 2 and the retinoid X receptor, alpha (RXRA, NM_002957.4) and the sequence GUCUAUCAGCACAAUtt (SEQ ID NO: l) acute-phase response factor (STAT3, NM_213662.1), transcript variant 3, signal transducer and activator of transcription 3 (STAT3, NM_003150.3), transcript variant 2, signal transducers and activators of transcription 3 (STAT3, NM_139276.2), transcript variant 1, protocadherin alpha 9 (PCDHA9, NM_014005.3) and secernine 3 (SCRN3, NM_024583.3) called.
  • nucleotide sequence which has no homology to a human mRNA and thus has no direct target gene.
  • corresponding sequences can be used, of which it is known in the prior art that these trigger cell stress.
  • Such a nucleotide sequence may have the sequence GCUUAACUGUAUCUGGAGCtt (SEQ ID NO: 2).
  • nucleotide sequences listed above these nucleotides are modified at the 3 'end, where "t” in the context of the invention is 2'-deoxythymidine
  • tt two 2'-deoxynucleotides are added at the 3' end and these terminal nucleotides are labeled "tt".
  • the structure of the overhangs is not limited to the "tt" overhangs referred to here, since the nature of the overhangs is not essential even for the inventive action of the siRNAs described herein, so other overhangs known to those skilled in the art may be used
  • the biologically active nucleotide molecules according to the invention can also be used as medicaments
  • cells can be killed directly by means of the siRNA molecules according to the invention for therapeutic applications, for example tumor cells or virus-infected cells can be specifically killed in a targeted manner proposed nucleotide sequences, ie the biologically active nucleotide molecules described above for use in the treatment and / or prevention of tumor diseases or virus-induced diseases are virus-induced diseases in the context of the invention include diseases that are caused for example by herpes viruses, papilloma viruses or HIV viruses.
  • the virus-elicited diseases include diseases such as hepatitis, cervical carcinoma or AIDS.
  • the present invention encompasses the biologically active nucleotide molecules according to the invention for use in the treatment and / or prevention of tumor diseases.
  • Tumor diseases treated with the drug of the present invention include breast cancers, ovarian cancers, bronchial carcinomas, colon carcinomas, melanomas, bladder carcinomas, gastric carcinomas, head / neck tumors, brain tumors, cervix tumors, prostate carcinomas, testicular cancers, bone tumors, renal carcinomas, pancreatic tumors, esophageal tumors, malignant lymphomas, non-Hodgkin Lymphomas, Hodgkin's lymphomas and thyroid lymphomas.
  • the biologically active nucleotide molecules, nucleotides or nucleotide analogs according to the invention can, as already mentioned above, in another preferred embodiment optionally be used in combination with protease inhibitors.
  • protease inhibitors are known to the person skilled in the art.
  • Proteiaseinhibitoren inhibitors of hepatitis C protease or inhibitors of HIV protease may be mentioned, but the present invention is not limited to these.
  • the biologically active nucleotide molecules, nucleotides or nucleotide analogs according to the invention may optionally be formulated in combination with a "pharmacologically acceptable carrier" and / or solvent
  • pharmacologically acceptable carriers include buffered saline solutions, water , Emulsions such as oil / water emulsions, various types of detergents, sterile solutions, etc.
  • Medicaments according to the invention comprising the pharmacologically acceptable carriers listed above can be prepared by known conventional methods be formulated. These drugs can be administered to an individual in a suitable dose. Administration may be oral or parenteral, eg, intravenous, intraperitoneal, subcutaneous, intramuscular, local, intranasal, intrabronchial or intradermal, or via a catheter at a site in an artery. The type of dosage is determined by the attending physician according to the clinical factors.
  • the type of dosage depends on various factors, such as the body size or the weight, the body surface, the age, sex or general health of the patient, but also on the specific means to be administered, the duration and route of administration, and other medications that may be administered in parallel.
  • a typical dose may range between 0.01 and 10000 ⁇ g, with doses below or above this exemplary range being conceivable, especially considering the factors mentioned above.
  • the dose should be in a range between 10 ng and 10 mg units per day or per application interval. If the composition is administered intravenously, the dose should be in the range of 1 ng to 0.1 mg units per kilogram of body weight per minute.
  • compositions of the invention may be administered locally or systemically.
  • Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic ester compounds such as ethyl oleate, which are suitable for injections.
  • Aqueous carriers include water, alcoholic-aqueous solutions, emulsions, suspensions, saline solutions and buffered media.
  • Parenteral carriers include sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate and bound oils.
  • Intravenous carriers include, for example, liquid, nutrient and electrolyte supplements (such as those based on Ringer dextrose).
  • the pharmaceutical compositions of the invention may further comprise preservatives and other additives such as antimicrobial compounds, antioxidants, chelating agents and inert gases.
  • preservatives and other additives such as antimicrobial compounds, antioxidants, chelating agents and inert gases.
  • compounds such as interleukins, growth factors, differentiation factors, Interferons, chemotactic proteins or a non-specific immunomodulatory agent may be included.
  • the individual sequences of the siRNA 8 can also be administered in combination at the same time or at different times and used in the same or different concentrations in order to efficiently eliminate a multiplicity of genes or to degrade mRNAs.
  • FIG. 3 shows an additional effect which can further support the toxic effect of the above-described siRNA 8 according to the invention.
  • one or more nucleotide sequences such as AAA (SEQ ID NO: 8), UUU (SEQ ID NO: 9), GCCA (SEQ ID NO: 10), UGGC (SEQ ID NO: II), GUCCUUCAA (SEQ ID NO: 12), UGUGU (SEQ ID NO: 13), AUUUG (SEQ ID NO: 14), GUUUU (SEQ ID NO: 15), AUUUU (SEQ ID NO: 16), CUUUU (SEQ ID NO: 17), UUUUU (SEQ ID NO: 18) or GUUUG (SEQ ID NO: 19)) are involved in siRNA 12, which are known to cause such stress reactions in cell 2 that are not due to binding of siRNA 8 to one or more mRNA.
  • these nucleotide sequences of siRNA 12 with this effect do not reduce the expression of genes and the breakdown of mRNAs after introduction of siRNA 12 into cell 2 (compare in FIG. 3 the mRNA 3-6 shown and not degraded with these nucleotide sequences). but induce nonspecific stress reactions in the cell, which occur in addition to the effect described in FIG. 2 and thus even more lead to the death of cell 2.

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Abstract

L'invention concerne des molécules nucléotidiques biologiquement actives pour la destruction ciblée de cellules, leur utilisation ainsi qu'un kit d'application. Le but était de détruire de manière active, fiable et la plus efficace possible dans l'organisme des cellules dans un large domaine d'application, sans les inconvénients susmentionnés des méthodes chimiques, physiques, biochimiques ou de biologie moléculaire connues en tant que telles. Selon l'invention, les molécules nucléotidiques biologiquement actives (8) avec leur séquence nucléotidique pour permettre la liaison aux ARNm (7, 9, 10, 11) de plusieurs gènes sont conçues pour déclencher par liaison plusieurs, en particulier une pluralité d'effets « off-target » pour des situations de stress détruisant les cellules, au moyen desquels, indépendamment de l'utilisation classique des molécules pour la réduction de l'expression d'un gène unique, la cellule (2) est si fortement influencée que celle-ci meurt ou bien que la mort cellulaire programmée (apoptose) est provoquée dans la cellule (2). Ces molécules trouvent en particulier une application pour la destruction ciblée de cellules infectées par une tumeur et un virus, ainsi que de cellules végétales et fongiques.
EP12704718.1A 2011-01-21 2012-01-20 Molécules nucléotidiques biologiquement actives pour la destruction ciblée de cellules, leur utilisation ainsi que kit d'application Withdrawn EP2665816A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102011009470A DE102011009470A1 (de) 2011-01-21 2011-01-21 Biologisch wirksame Nukleotid-Moleküle zur gezielten Abtötung von Zellen, Verwendung derselben sowie Applikationskit
PCT/EP2012/050879 WO2012098234A1 (fr) 2011-01-21 2012-01-20 Molécules nucléotidiques biologiquement actives pour la destruction ciblée de cellules, leur utilisation ainsi que kit d'application

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EP2665816A1 true EP2665816A1 (fr) 2013-11-27

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US (1) US20170233760A1 (fr)
EP (1) EP2665816A1 (fr)
JP (1) JP2014511173A (fr)
CN (1) CN103597075A (fr)
DE (1) DE102011009470A1 (fr)
WO (1) WO2012098234A1 (fr)

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DE102013003869B4 (de) 2013-02-27 2016-11-24 Friedrich-Schiller-Universität Jena Verfahren zur gezielten Abtötung von Zellen durch zur mRNA-Anbindung ausgerichtete Nukleotid-Moleküle sowie Nukleotid-Moleküle und Applikationskit für solche Verwendung
US20210301020A1 (en) 2018-07-24 2021-09-30 Amgen Inc. Combination of lilrb1/2 pathway inhibitors and pd-1 pathway inhibitors
DE102019000490A1 (de) 2019-01-23 2020-07-23 HAEMES Verwaltungsgesellschaft mbH Verwendung von Oligonukleotiden für die Behandlung von Tumoren

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CA2429814C (fr) 2000-12-01 2014-02-18 Thomas Tuschl Petites molecules d'arn mediant l'interference arn
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KR100793505B1 (ko) * 2006-05-30 2008-01-14 울산대학교 산학협력단 복수의 표적 ｍＲＮＡ에 적용 가능한 ｓｉＲＮＡ염기서열을 추출하는 방법
WO2008094516A2 (fr) * 2007-01-29 2008-08-07 City Of Hope Arn à interférence courte multicibles
DE102007008596B4 (de) 2007-02-15 2010-09-02 Friedrich-Schiller-Universität Jena Biologisch wirksame Moleküle auf Grundlage von PNA und siRNA, Verfahren zu deren zellspezifischen Aktivierung sowie Applikationskit zur Verabreichung
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US20100298409A1 (en) * 2007-09-17 2010-11-25 Intradigm Corporation Compositions comprising stat3 sirna and methods of use thereof
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CN103597075A (zh) 2014-02-19
WO2012098234A1 (fr) 2012-07-26
DE102011009470A1 (de) 2012-08-09
JP2014511173A (ja) 2014-05-15
US20170233760A1 (en) 2017-08-17

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