BIOLOGICALLY ACTIVE NUCLEOTIDE MOLECULES FOR SELECTIVELY KILLING OFF CELLS, USE THEREOF, AND APPLICATION KIT

Abstract

Biologically active nucleotide molecules are configured, with the nucleotide sequence thereof, to be able to trigger several, in particular a plurality of “off-target” effects to cause cell-killing stress by means of binding of same, by means of which off-target effects cells are so massively influenced that the cells die off or programmed cell death (apoptosis) is induced in the cells.

Claims

1. Biologically active nucleotide molecules, comprising at least one nucleotide sequence targeting mRNA binding for selectively influencing cells, wherein said at least one nucleotide sequence of the nucleotide molecules is configured to bind to mRNA of a plurality of genes of the cells, thereby triggering a plurality of off-target effects which have a toxic effect on the cells by subjecting the cells to cell-killing stress.

2. The biologically active nucleotide molecules according to claim 1, comprising RNA, siRNA, PNA, DNA or LNA having a size of 10-300 bp.

3. The biologically active nucleotide molecules according to claim 1, wherein the nucleotide molecules further comprise sequences triggering, per se and without binding to an mRNA, stress reactions in the cells.

4. The biologically active nucleotide molecules according to 1, wherein the nucleotide molecules, to facilitate their introduction into the cells, are bound to cell-penetrating molecules or integrated in reagents.

5. The biologically active nucleotide molecules according to claim 1, wherein the nucleotide molecules contain at least one of the nucleotide sequences TABLE-US-00003 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, GCAG, GCAC, GCAU, AUAG, AUAC, AUAU, AUGA, AUGG, AUGC, AUGU, AUCA, CGCG, CGCC, CGCU, AUCG, AUCC, AUCU, AUUA, AUUG, AUUC, AUUU, GAAA, GAAG, GAAC, GAAU, GAGA, GAGG, GAGC, GAGU, GACA, GACG, GACC, GACU, GAUA, GAUG, GAUC, GAUU, GGAA, GCGA, GCGG, GCGC, GCGU, GCCA, GCCG, GCCC, GCCU, GCUA, GCUG, GCUC, GCUU, GUAA, GUAG, GUAC, GUAU, GUGA, GUGG, GUGC, GUGU, GUCA, GUCG, GUCC, GUCU, GUUA, GUUG, GUUC, GUUU, CAAA, CAAG, CAAC, CAAU, CAGA, CAGG, CAGC, CAGU, CACA, CACG, CACC, CACU, CAUA, CAUG, CAUC, CGAG, CGAC, CGAU, CGGA, CGGG, CGGC, CGGU, CGCA, CGUA, CGUG, CCAG, CCAC, CCAU, CCGA, CCGG, CCGC, CCGU, CCCA, AGAA, AGAG, AGAC, AGAU, CCCG, CCCU, AGGG, AGGC, AGGU, AGCA, CCUA, CCUG, CCUC, CCUU, CUAA, CUAG, CUAC, CUAU, AGCG, AGUA, CUGA, CUGG, CUGC, CUGU, CUCA, CUUG, CUUC, AGCC, AGCU.

6. The biologically active molecules according to claim 1, selected from the group consisting of TABLE-US-00004 GUCUAUCAGCACAAUtt, (SEQ ID NO: 1) GCUUAACUGUAUCUGGAGCtt, (SEQ ID NO: 2) UUAACUGUAUCUGGAGCtt, (SEQ ID NO: 3) AACUGUAUCUGGAGCtt, (SEQ ID NO: 4) GCUCACCAAUGGAGAtt, (SEQ ID NO: 5) GGCUGAACAAAGGAGAtt (SEQ ID NO: 6) and UGGCUGGCUGGCUGGCtt. (SEQ ID NO: 7)

7. Pharmaceutical composition, comprising biologically active nucleotide molecules according to claim 1.

8. Method of treatment or prophylaxis of tumour diseases or virus-induced diseases, comprising administering the nucleotide molecules of claim 1.

9. Method of selectively killing eukaryotic cells, comprising transfecting the cells with the nucleotide molecules according to claim 1.

10. Method of selectively killing virus-infected cells, comprising transfecting the cells with the nucleotide molecules of claim 1.

11. Method of selectively killing prokaryotic cells, comprising transfecting the cells with the nucleotide molecules of claim 1.

12. The method of claim 2, further comprising administering the biologically active nucleotide molecules in combination with protease inhibitors.

13. Application kit for administration to target cells of the biologically active nucleotide molecules according to claim 1, comprising: at least one ampoule (ampoule A) which contains the biologically active molecule; at least one further ampoule (ampoule B) containing a transfection system; at least one further ampoule (ampoule C) containing further components for binding to the biologically active molecules or the transfection system; dilution and reaction buffers for the contents of ampoules A, B; one or more probes and syringes with cannulas for injecting a mixture of the ampoule contents into a medium containing the target cells; and instructions for use of said kit.

14. The biologically active nucleotide molecules according to claim 2, wherein the sequences triggering, per se and without binding to an mRNA, stress reactions in the cells comprise TABLE-US-00005 AAA, UUU, GCCA, UGGC, GUCCUUCAA, UGUGU AUUUG, GUUUU, AUUUU, CUUUU, UUUUU or GUUUG.

15. Method of treatment or prophylaxis of tumour diseases or virus induced diseases, comprising administering the pharmaceutical composition of claim 7.

16. The method of claim 9, wherein the eukaryotic cells are animal, plant or fungal cells.

17. The application kit according to claim 13, wherein the transfection system comprises cell-penetrating peptides, nanoparticles, polyethylenimines or lipids.

Description

[0034] In the following, the invention is exemplified in detail by embodiments illustrated in the Figures.

[0035] The Figures show:

[0036] FIG. 1: Schematic diagram of a known siRNA which is introduced into a cell, is specific for an mRNA and suppresses the expression of a target gene.

[0037] FIG. 2: Schematic diagram of a siRNA of the invention which is introduced into a cell and triggers as many unspecific RNAi effects (off-target effects) as possible in the cell.

[0038] FIG. 3: Schematic diagram of a siRNA which is introduced into a cell and is not to reduce the expression of genes and the degradation of mRNAs in the cell but is to cause cell death by means of stress reactions in the cell which are triggered by specific sequence segments of the siRNA.

[0039] FIG. 1 shows the mechanism of a conventional and known siRNA 1 which is introduced into a cell 2 (as illustrated by the arrow) and has a specific nucleotide sequence (not explicitly shown) for binding to a first gene-specific mRNA 3 nucleotide sequence. Subsequently, the siRNA 1 is introduced into the RNA-induced silencing complex (RISC) (not explicitly shown either) which separates the siRNA 1 in its two single strands and the antisense strand of the siRNA 1 together with the RISC anneals to the first mRNA 3. Then, the gene-specific first mRNA 3 is cleaved and fragmented whereby the expression of a target gene, based on the first mRNA 3, is suppressed (cf. degraded first mRNA 7 in FIG. 1). The siRNA which is thus set free and integrated into the RISC anneals to the next specific first mRNA 3 present in the cell 2 and causes its degradation. The aim is that each siRNA 1 binds to one specific first mRNA 3 only and causes its degradation. In contrast to the first mRNA 3, any second further mRNA 4, third further mRNA 5 and fourth further mRNA 6 present in the cell 2 are not affected by the siRNA 1 and its nucleotide sequence (not explicitly shown), respectively, so that the expression of the genes corresponding to mRNA 4-6 is not modified. This method is well known.

[0040] FIG. 2 shows, for comparison, the mechanism of a siRNA 8 of the invention which is introduced into the cell 2 (also illustrated by an arrow) which, in an exemplary manner, contains the first mRNA 3, the second mRNA 4, the third mRNA 5 and the fourth mRNA 6.

[0041] The suggested siRNA 8 contains a chain of one or more of the following nucleotide sequences (not explicitly illustrated for the sake of clarity)

TABLE-US-00001 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, GCAG, GCAC, GCAU, AUAG, AUAC, AUAU, AUGA, AUGG, AUGC, AUGU, AUCA, CGCG, CGCC, CGCU, AUCG, AUCC, AUCU, AUUA, AUUG, AUUC, AUUU, GAAA, GAAG, GAAC, GAAU, GAGA, GAGG, GAGC, GAGU, GACA, GACG, GACC, GACU, GAUA, GAUG, GAUC, GAUU, GGAA, GCGA, GCGG, GCGC, GCGU, GCCA, GCCG, GCCC, GCCU, GCUA, GCUG, GCUC, GCUU, GUAA, GUAG, GUAC, GUAU, GUGA, GUGG, GUGC, GUGU, GUCA, GUCG, GUCC, GUCU, GUUA, GUUG, GUUC, GUUU, CAAA, CAAG, CAAC, CAAU, CAGA, CAGG, CAGC, CAGU, CACA, CACG, CACC, CACU, CAUA, CAUG, CAUC, CGAG, CGAC, CGAU, CGGA, CGGG, CGGC, CGGU, CGCA, CGUA, CGUG, CCAG, CCAC, CCAU, CCGA, CCGG, CCGC, CCGU, CCCA, AGAA, AGAG, AGAC, AGAU, CCCG, CCCU, AGGG, AGGC, AGGU, AGCA, CCUA, CCUG, CCUC, CCUU, CUAA, CUAG, CUAC, CUAU, AGCG, AGUA, CUGA, CUGG, CUGC, CUGU, CUCA, CUUG, CUUC, AGCC, AGCU
so that, in contrast to FIG. 1, the nucleotide sequences, which are bound in the chain, as a whole do not have a degrading effect on only one of the mRNAs 3-6 but on several or a plurality of the mRNA molecules, and thus bind all the mRNA set forth in FIG. 2 (mRNA 3-6). It is possible that at least one selected nucleotide sequence binds to several or all of the shown mRNA molecules (mRNA 3-6) or that one of the selected nucleotide sequences selectively acts on one specific mRNA 3-6. It is crucial that as many of the mRNA 3-6 as possible (preferably all) are bound and degraded by the chain (total number of all nucleotide sequences) of siRNA 8 (cf. degraded first to fourth mRNA 7, 9, 10, 11 in FIG. 2).

[0042] Due to the degradation of said plurality of mRNA molecules (in a simplifying manner, the present Example shows only four mRNA molecules), several to numerous unspecific RNAi effects (off-target effects) are triggered in that the siRNA 8 suppresses the expression of several to numerous genes (cf. degraded mRNA 7, 9, 10 in FIG. 2) by means of (ideally) only one nucleotide sequence with the aim to kill the cell 2 in this way, wherein the cell dies due to the massive effect of the siRNA 8.

[0043] For example, by means of the siRNA 8 having the nucleotide sequence (5′-3′) UUAACUGUAUCUGGAGCtt (SEQ ID NO:3), it is possible to degrade the mRNA of the genes suppressor of cytokine signaling-1 (SOCS1, NM_003745.1), N-acetylneuraminic acid phosphatase (NANP, NM_152667.2) transmembrane protein 215 (TMEM215, NM_212558.2) and of the CD81 molecule (CD81, NM_004356.3).

[0044] A nucleotide sequence AACUGUAUCUGGAGCtt (SEQ ID NO:4) of the siRNA 8 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 (SEQ ID NO:6) acts specifically 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).

[0045] In analogy, 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).

[0046] As further examples for the nucleotide sequence of siRNA 8 sequence UGGCUGGCUGGCUGGCtt (SEQ ID NO:7) advantageous against pyroglutamyl peptidase I (PGPEP1, NM_017712.2), rap guanine nucleotide exchange factor (GEF) 3 (RAPGEF3, NM_006105.5), transcript variant 2 and against the retinoid X receptor, alpha (RXRA, NM_002957.4) and sequence GUCUAUCAGCACAAUtt (SEQ ID NO:1) against the signal transducer and activator of transcription 3 (acute-phase response factor) (STAT3, NM_213662.1), transcript variant 3, signal transducer and activator of transcription 3 (acute-phase response factor) (STAT3, NM 003150.3), transcript variant 2, the signal transducer and activator of transcription 3 (acute-phase response factor) (STAT3, NM_139276.2), transcript variant 1, protocadherin alpha 9 (PCDHA9, NM_014005.3) and secernin 3 (SCRN3, NM_024583.3) are mentioned.

[0047] As an alternative to the above-mentioned examples of nucleotide sequences of siRNA 8 which are directed against concrete genes, it is also possible to use a nucleotide sequence which has no homology to a human mRNA and, thus, has no direct target gene. In this case, sequences which are known in the state of the art to trigger cell stress can be used. Such nucleotide sequence can have the sequence GCUUAACUGUAUCUGGAGCtt (SEQ ID NO:2).

[0048] As can be taken from the nucleotide sequences listed above, modified nucleotides are added to the 3′ end of said sequences, with “t” being 2′-deoxythymidine according to the invention. Into the nucleotide sequences shown above, two 2′-deoxynucleotides are added at the 3′ end and these terminal nucleotides are designated “tt”. However, the structure of these overhangs is not limited to the “tt” overhangs mentioned herein since the type of overhangs per se is not crucial for the effect of the siRNAs as described herein according to the invention. It is also possible to use other overhangs known to the person skilled in the art.

[0049] The biologically active nucleotide molecules of the invention can also be used as a pharmaceutical composition. It is for example possible to directly kill cells using the siRNA molecules of the invention for therapeutic applications. Thus, it is possible to selectively kill specific tumour cells or virus-infected cells. For this reason, the nucleotide sequences suggested, i.e. the biologically active nucleotide molecules described above, can be used in the treatment and/or prophylaxis of tumour diseases or virus-induced diseases. Virus-induced diseases within the meaning of the invention comprise diseases which are, for example, caused by herpes viruses, papillomaviruses or HIV viruses. Thus, the virus-induced diseases comprise diseases such as hepatitis, cervical cancer or AIDS.

[0050] The present invention also comprises the biologically active nucleotide molecules of the invention for use in the treatment and/or prophylaxis of tumour diseases. Tumour diseases which are treated with the pharmaceutical composition of the invention comprise mamma carcinomas, ovary carcinomas, bronchial carcinomas, colon carcinomas, melanomas, urinary bladder carcinomas, gastric carcinomas, head and neck carcinomas, brain tumours, cervical tumours, prostate carcinomas, testicular carcinomas, bone tumours, renal carcinomas, pancreatic carcinomas, esophageal carcinomas, malignant lymphomas, non-Hodgkin lymphomas, Hodgkin lymphomas and thyroid lymphomas.

[0051] In a further preferred embodiment, the biologically active nucleotide molecules, nucleotides or nucleotide analogs can optionally be used in combination with protease inhibitors, as already mentioned above. Corresponding protease inhibitors are known to the skilled person from the state of the art. Inhibitors of hepatitis C protease or inhibitors of HIV protease are mentioned as examples of the protease inhibitors while the present invention is not limited to these.

[0052] Moreover, in another preferred embodiment of the invention, the biologically active nucleotide molecules, nucleotides or nucleotide analogs of the invention can optionally be formulated in combination with a “pharmacologically acceptable” carrier and/or solvent. Examples of particularly suited pharmacologically acceptable carriers are known to the person skilled in the art and comprise buffered saline, water, emulsions such as e.g. oil/water emulsions, different types of detergents, sterile solutions etc.

[0053] Pharmaceutical compositions within the meaning of the invention comprising the pharmacologically acceptable carriers listed above can be formulated using conventional methods that are known. These pharmaceutical compositions can be administered to a subject in a suited dose. The administration can be oral or parenteral, e.g. intravenous, intraperitoneal, subcutaneous, intramuscular, local, intranasal, intrabronchial or intradermal or via a catheter inserted at a site in an artery. The kind of dosage is determined by the attending physician according to the clinical factors. The person skilled in the art knows that the kind of dosage depends on different factors, such as body height and weight, body surface, age, gender or the general health condition of the patient, however, it also depends on the composition to be administered in the particular case, the time and kind of administration and on other medicaments which are possibly administered at the same time. A typical dose can, for example, be within a range between 0.01 and 10000 μg, with doses below or above this exemplary range being possible, in particular in consideration of the factors mentioned above. In general, with regular administration of the pharmaceutical preparation, the dose should be in a range between 10 ng units and 10 mg units per day and/or application interval. If the composition is administered intravenously, the dose should be within a range between 1 ng units and 0.1 mg units per kilogram body weight/minute.

[0054] The pharmaceutical composition of the invention can be administered locally or systemically. Preparations for parenteral administration comprise sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, plant oils, such as e.g. olive oil, and organic ester compounds, such as e.g. ethyolate, which are suited for injection. Aqueous carriers comprise water, alcoholic/aqueous solutions, emulsions, suspensions, saline solutions and buffered media. Parenteral carriers include sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's lactate and fixed oils. Intravenous carriers include e.g. fluid, nutrient and electrolyte replenishers (such as e.g. based on Ringer's dextrose). The pharmaceutical composition can also include preservatives and other additives, such as e.g. antimicrobial compounds, anti-oxidants, chelating agents and inert gases. Furthermore, depending on the intended use, compounds such as e.g. interleukins, growth factors, differentiation factors, interferons, chemotaxis proteins or an unspecific agent may be present.

[0055] Furthermore, the individual sequences of the siRNA 8 can also be administered in combination simultaneously or sequentially as well as in identical or different concentrations in order to silence a plurality of genes or degrade mRNAs efficiently.

[0056] FIG. 3 illustrates an additional effect which can further support the toxic effect of the siRNA 8 of the invention as described above. In this case, one or more nucleotide sequences (such as

TABLE-US-00002 AAA, (SEQ ID NO: 8) UUU, (SEQ ID NO: 9) GCCA, (SEQ ID NO: 10) UGGC, (SEQ ID NO: 11) 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))
which are known to trigger stress reactions in cell 2 that cannot be attributed to the binding of the siRNA 8 to one or more mRNAs, are additionally introduced into a siRNA 12. After the siRNA 12 has been introduced into the cell 2, the nucleotide sequences of the siRNA 12 having this effect do not reduce the expression of genes and the degradation of mRNAs (cf. the mRNA 3-6 shown in FIG. 3 that are not degraded by these nucleotide sequences) but induce unspecific stress reactions in the cell which occur in addition to the effect described in FIG. 2 and, thus, additionally contribute to the death of the cell 2.

LIST OF REFERENCES USED

[0057] 1, 8, 12—siRNA [0058] 2—cell [0059] 3, 4, 5, 6—gene-specific mRNA [0060] 7, 9, 10, 11—degraded gene-specific mRNA