EP2668275A2 - Composition pharmaceutique contenant de l'adn-l - Google Patents

Composition pharmaceutique contenant de l'adn-l

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Publication number
EP2668275A2
EP2668275A2 EP12716194.1A EP12716194A EP2668275A2 EP 2668275 A2 EP2668275 A2 EP 2668275A2 EP 12716194 A EP12716194 A EP 12716194A EP 2668275 A2 EP2668275 A2 EP 2668275A2
Authority
EP
European Patent Office
Prior art keywords
rna
dna
target
sequence
pharmaceutical composition
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Withdrawn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
EP12716194.1A
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German (de)
English (en)
Inventor
Volker A. Erdmann
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Individual
Original Assignee
Individual
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Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP2668275A2 publication Critical patent/EP2668275A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • 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
    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • 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
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/127—DNAzymes
    • 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
    • C12N2310/32—Chemical structure of the sugar
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • 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
    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised

Definitions

  • the invention relates to a pharmaceutical
  • a composition containing an L-DNA the use of an L-DNA for the preparation of a pharmaceutical composition, and a process for producing such a pharmaceutical composition.
  • aptamers are isolated from nucleic acid libraries by the SELEX method
  • Aptamers have the purpose in the therapeutic field, i.a. to bind and thereby inhibit unwanted metabolites. Only for example, oncogenic gene products are mentioned for this purpose. In the
  • aptamers have unfavorable pharmacokinetics, i.e. be broken down very quickly
  • aptamers are relatively small molecules anyway, which are therefore excreted relatively quickly on the kidney.
  • Spiegelmers are aptamers in the core, but differ in that they are formed from L-nucleotides. Spiegelmers can be single or double-stranded.
  • the use of the L-nucleotides prevents degradation by endogenous nucleases and thus greatly improves the pharmacokinetics, i.e. the residence time in the serum is prolonged.
  • Boisgard, R et al. Eur Journal of Nuclear Medicine and Molecular Imaging 32: 470-477 (2005) that non-functional aptmers are metabolically completely stable even over a period of 2 hours.
  • the diagnostic use of Spiegelmeren is described, wherein the Spiegelmer with a
  • radioactive reporter substance is coupled.
  • Target molecule specific Spiegelmeren can
  • physiological side reaction for example an immune reaction and / or an undesired one
  • mRNA or other non-coding nucleic acids known. These include, but are not limited to, antisense nucleic acids, siRNA, miR A, piRNA, aptamers, etc.
  • Inhibition of such endogenous nucleic acids can be controlled, inhibited or redirected metabolic processes, which is relevant for example in connection with tumor-associated RNA molecules, but also in other medical fields.
  • a tumor-associated gene for example, the H19 gene may be mentioned.
  • An example of a non-tumor-associated gene is the gene coding for phospholamban, which plays an important role in connection with heart failure.
  • L-DNA is known per se, for example from the
  • Nucleic acids are described as molecular tags.
  • the invention is based on the technical problem to provide improved means for cutting Spiegelmeren and endogenous nucleic acids.
  • the invention teaches the use of an L-DNA for the preparation of a pharmaceutical composition, wherein the ILDNA is preferably for binding to an L-RNA,
  • an antisense reaction Watson-Crick inhibitory reaction
  • optionally capable of cleaving the L-RNA in the region of a target sequence of L-RNA in particular for the preparation of a pharmaceutical composition for the treatment of undesired physiological side reactions, in particular immune reactions and / or undesired enzymatic or antisense reactions of L-RNA with endogenous RNA (including a regulatory RNA) due to the administration of a therapeutic molecule containing the L-RNA.
  • endogenous RNA including a regulatory RNA
  • Target D-RNA for example in an antisense
  • Target sequence is capable.
  • the invention is based initially on the recognition that Contrary to previous assumptions, aptmers are not necessarily free from side reactions, but rather may be able to
  • the invention is based on the further finding that L-DNA is surprisingly able to cut or bind to endogenous D-nucleic acids, RNA, such as DNA. This is not to be expected automatically.
  • L-DNAs are particularly stable against
  • a surprising advantage of L-DNA over L-RNA is that the activity of L-DNA against L-RNA is higher in cells, including the
  • L-DNA ie L-DNzyme, which specifically cut an administered Spiegelmer or thereto bind inhibiting and so their physiological
  • Spiegelmers are: Spiegelmer, NOXC89, NOXA42, NOXA50, NOXB11, NOXA12, NOXE36, NOXF37 (all NOXXON AG),
  • L-DNA unwanted side reaction from the metabolism can be quickly, effectively and highly selectively removed, again with extremely low risk of side effects of the administration of L-DNA.
  • the latter is not only based on the construction of L-DNA from L-DNA.
  • a specific L-DNA be constructed, which cuts a target sequence of the RNA molecule and this cleaves (action as a ribozyme), or binds thereto inhibiting (antisense reaction).
  • An essential feature of such an L-DNA is thus the sequence-specific binding to the target sequence.
  • a partial sequence of an L-DNA can be created by hybridizing the partial sequence of the L-DNA containing, for example, a cleavage site, with the target sequence. Therefore, in the context of the invention, it is not appropriate to use only certain L-DNA partial sequences with respect to certain
  • target sequences and L-DNA partial sequences given in the examples are therefore only examples and those skilled in the art can easily determine the appropriate, namely hybridizing L-DNA partial sequence for each given target sequence of a spiegelmer and the L-DNA on the basis of information for Synthesize L-DNA partial sequence by conventional means.
  • the therapeutic molecule may be a spiegelmer, or the L-RNA may be covalently bound to an aptamer.
  • the therapeutic molecule may also contain an L-DNA (in addition to a
  • Aptamer for example or consist thereof.
  • a combination aptamer / aptamer can be present, for example, in the case of a nuclease-stabilized aptamer. Then the therapeutic benefit of the invention is that by cutting the L-RNA or L-DNA, the aptamer is made available for nucleases, which then ultimately a possibly causing side effects aptamer from the serum can be eliminated comparatively short term.
  • the L-DNA is covalently bound to an aptamer, an antibody or a peptide or protein.
  • the aptamer or the antibody may for example be selected so that due to the interactions of the aptamer or the antibody with cell surfaces, the entire construct of L-DNA and aptamer or antibody is introduced into the cell.
  • a suitable L-DNA may be directed against a conserved cleavage site in the substrate sequence and may itself contain conserved nucleotides, as shown in Figure 1, in particular Fig. 1a.
  • the pharmaceutical composition contains the L-DNA in at least the dose corresponding to the dose of the administration of the L-RNA, preferably at a dose of 2 to 10 times the number of moles of the dose of the dose corresponds to the L-RNA.
  • An overdose, compared to the dose of L-RNA, will be recommended to ensure that all L-RNA to be eliminated is abreacted.
  • the pharmaceutical composition additionally contains a nucleic acid, in particular a 5- to 100-mer, preferably a 5- to 25-mer, which belongs to the
  • the invention relates to a
  • L-DNA according to the invention can also be used for the cleavage of (endogenous or else exogenous, for example from viral or bacterial sources in the course of an infection) nucleic acids, essentially RNA, but also DNA, are used, or to inhibit the same by antisense
  • RNA or DNA can be used to treat diseases associated with a particular RNA or DNA, or the overexpression of a coded thereby
  • the L-DNA is thus used as an inhibitor with regard to this
  • binding RNA or DNA within the scope of the invention insofar irrelevant as to allow arbitrary targets to be inhibited. It is only necessary, the L-DNA or its sequence of the sequence of
  • any indications can be detected, provided that the disease to be treated causes the relevant target sequence
  • the invention relates to a method for
  • composition wherein a sequence of L-
  • Deoxyribonucleotides are generated and synthesized, which bind to a given sequence of L-ribonucleotides or a predetermined sequence of D-ribonucleotides or D-deoxyribonucleotides,
  • the L-DNA is typically with galenic auxiliary and / or
  • L-DNA which promote endocytosis
  • one or more physiologically acceptable auxiliaries and / or excipients can be mixed with the L-DNA and the mixture galenically for local or systemic administration, in particular orally, parenterally, for infusion or infusion into a target organ, for injection (zBiV, im, intracapsular or intralumbar), for application in tooth pockets (space between tooth root and gums) and / or prepared for inhalation.
  • zBiV im, intracapsular or intralumbar
  • the choice of additives and / or adjuvants will depend on the chosen dosage form.
  • Composition can be carried out in the usual way. Come as counterions for ionic compounds
  • Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, dragees, tablets, (icro) capsules, suppositories, syrups, juices, suspensions, emulsions, drops or solutions for injection (iv, ip, im, sc) or nebulization (aerosols ), Formulations for dry powder inhalation, transdermal systems, and
  • nanoparticles of D and L nucleic acids for example for the cell targeting of Spiegelzymes and Spiegelmers or aptamers (Seeman, NC, Nano Lett. 10 (6): 1971-1978 (2010); , Nature 478 (7368): 225-228 (2011); Sigh, W. Synapse 65 (12): 1289-97 (2011) Zhao, W. et al., Nanotechnology 22 (49): 490201 (2011); JZ, Adv Drug Deliv. Rev. 56 (11): 1533-1536 (2004); Lin, C, et al., Biochemistry 48 (8): 1663-1674 (2007))
  • excipients for example, sodium carbonates
  • a pharmaceutical composition according to the invention can be prepared by at least one substance combination used according to the invention in a defined dose with a pharmaceutically suitable and physiological
  • the diluents are polyglycols, water,
  • Dimethyl sulfoxide (for example for preparations for the treatment of skin diseases or rheumatism) and
  • Buffer solutions in question are, for example, N, N'-dibenzylethylenediamine, diethanolamine, ethylenediamine, N-methylglucamine, N-benzylphenethylamine, diethylamine, phosphate, sodium bicarbonate, or
  • Physiologically acceptable salts are salts with inorganic or organic acids, e.g. Lactic acid, hydrochloric acid, sulfuric acid, acetic acid,
  • Citric acid Citric acid, p-toluenesulfonic acid, or with inorganic or organic bases, such as NaOH, KOH, Mg (OH) 2 ,
  • Diethanolamine, ethylenediamine, or with amino acids such as arginine, lysine, glutamic acid, etc. or with inorganic salts such as CaCl 2 , NaCl or their free ions, such as Ca 2+ , Na + , Pb ++ , Cl " , S0 4 2 ⁇ or corresponding salts and free ions of the Mg ++ or Mn ++ , or combinations thereof, are prepared by standard methods, preferably a pH in the range between 5 and 9, in particular between 6 and 8, set.
  • Target sequence of a gene encoding endogenous target D-RNA or target D-DNA, in particular for antisense reaction, and optionally capable of cleaving said target sequence comprises.
  • the L-DNA is capable of cleaving a (or for binding, in particular antisense reaction, to a) target sequence of a coding for a gene of the microorganism target D-RNA or target D-DNA.
  • Suitable microorganisms include viruses, bacteria and fungi.
  • the L-DNA can bind to or cleave nucleic acids of any microorganism at least partially known gene sequences are used, wherein areas of the gene sequences for the purpose
  • Cleavage can be selected which, for example, the activity of the microorganism and / or its
  • the L-DNA also for inhibition by antisense reaction with or cleavage of D-RNA, in particular mRNA or
  • Regulatory RNA for example, but not exclusively siRNA, microRNA, shRNA, ncRNA, tRNA, rRNA, etc., can be used, but also for the cleavage of D-DNA or binding thereto. As a result, genes or proteins encoded thereby or coding or noncoding RNAs can be inhibited. This is from
  • this variant has the advantage that the cleavage of the target sequence or the binding thereto takes place with very high specificity and thus also no other interference with the regulatory system takes place.
  • side effects like them
  • inhibitory D-nucleic acids such as siRNA
  • L-RNA ribozymes and L-DNAzymes do not require helper molecules, unlike the siRNAs (Risc factors, etc.), which causes unwanted Side reactions are drastically reduced.
  • an L-DNA can be used not only for binding to another nucleic acid, but in principle analogous to the known fields of application of aptamers from D-nucleic acids. This means that in principle any target molecule can be bound in an organism and thus inhibited by means of an L-DNA according to the invention.
  • a target binding L-DNA is available in a following procedure.
  • a selected target molecule is attached to an immobile (resp.
  • Solid phase for example, also magnetic beads
  • the screening assay comprises in addition to the immobile phase a mobile phase (usually an aqueous solution in which
  • Nucleic acids and the target molecule are stable and can bind), which with the immobile phase in
  • the mobile phase contains an L-DNA library, ie Polynucleotides, typically 10 to 500 nucleotides in length, whose sequences vary, are usually randomized.
  • L-DNA libraries can be prepared by conventional and from the generation of D-DNA libraries known synthesis method. With the contacting, such L-DNA molecules bind to the target molecule, which by their sequence are capable of stable Van der Waals
  • Bindings to the target molecule to enter The
  • Bond strengths are typically included
  • Dissociation constants with values below 100 nmol, better below 100 pmol, often even below 1 pmol.
  • the mobile phase (with the non-binding or only weakly binding nucleic acids) is separated from the immobile phase, for example by means of one or more washing process stages. This is followed by immobile phase with target molecules
  • D-DNA molecules contacted with a D-DNA library D-DNA hybridizing to the L-DNA bound to the target molecule binds to the L-DNA, resulting in a complex of target molecule / L-DNA / D-DNA. Unbound D-DNA is removed again with the mobile phase. From the complex thus obtained, the D-DNA is then eluted again in the usual manner, i. transferred to a mobile phase.
  • the D-DNA molecules thus obtained can now optionally
  • amplified for example by PCR
  • the complementary L-DNA can then be determined and synthesized.
  • the L-DNA can be eluted from the target molecule and labeled with a elsewhere in this description Sequencing methods are sequenced. The procedure described in this section can
  • the target molecule instead of being bound to the immobile phase, bound to a marker molecule.
  • the separation of unbound L-DNA from the complex target / bound L-DNA is then carried out by conventional methods by binding of the marker molecule and separation of molecules which this
  • Bind affinity to the target molecule can then be used in the context of an inventive
  • Indications are used. The indication will then depend on which target molecule has been causally recognized as causally related to a disease and is to be inhibited for the treatment or prophylaxis thereof.
  • an L-RNA binding to a target can also be isolated or determined.
  • an L-RNA library is used instead of an L-DNA library.
  • a target molecule for example a target molecule, a virus, a bacterium, a fungus or a other cell (or fragments thereof) binding L-DNA or L-RNA isolated or determined and produced.
  • the L-nucleic acid library for example a target molecule, a virus, a bacterium, a fungus or a other cell (or fragments thereof) binding L-DNA or L-RNA isolated or determined and produced.
  • the L-nucleic acid library for example a target molecule, a virus, a bacterium, a fungus or a other cell (or fragments thereof) binding L-DNA or L-RNA isolated or determined and produced
  • nucleic acids optionally a coupling molecule or marker molecule is bound, for example, biotin to the 5 'end.
  • Target molecule is attached to a solid phase
  • Nucleic acid library contacted. In the process, those nucleic acids which bind to the target molecule and have a high binding affinity thereto.
  • the solid phase becomes one or more washing steps
  • L-DNAs can also be used for testing for potential side effects of L-DNA.
  • L-DNAs can also be used for testing for potential side effects of L-DNA.
  • Test procedure is the L-DNA (or a larger
  • the L-nucleic acid is bound to a solid phase. This can be done, for example, in that the nucleic acid is a coupling or
  • the solid phase carries a molecule complementary to the coupling molecule and this binding molecule, such as avidin or streptavidin.
  • This binding molecule such as avidin or streptavidin.
  • the solid phase is washed in one or more washing steps, removing the unbound D-DNA. Then the bound D-DNA is released from the L-nucleic acid.
  • the complementary sequence of the L-nucleic acid can then be determined from the D-DNA sequence thus obtained.
  • an L-nucleic acid in particular L-nucleic acid RNA
  • the nucleic acid carries at one end, for example at the 5 "end, a coupling or marker molecule (as described above), for example biotin
  • the nucleic acid is then broken down into a" ladder ", ie by hydrolysis different fragments of the nucleic acid are obtained ideally, from 1 base to the base number of the complete nucleic acid.
  • L-RNA this can be done in the alkaline, pH typically> 8, usually 8.5-10.
  • a commercially available hydrolysis buffer with KOH or sodium bicarbonate can be used.
  • the fragments thus obtained, which still carry the coupling molecule are then bound to a solid phase.
  • the solid phase contributes to the coupling molecule complementary and this binding molecule, such as avidin or streptavidin.
  • the solid phase then becomes one or more
  • nucleic acid fragments are removed, which do not carry the coupling molecule. The result is the "ladder" of labeled nucleic acid fragments, which are then eluted from the solid phase and
  • L-DNA or Spiegelzyme can also be used to determine accessible mRNA sequences. Because the Determination of such mRNA sequences that are accessible to siRNAs, miroRNAs and also Spiegelzymen very difficult, since a secondary structure prediction using computer technology is very uncertain. This is due to the fact that too little is known about the rules of three-dimensional folding of RNA molecules, so that the
  • Scissors of the prior art such as Ribozymes, siRNAs, etc., are for stability reasons or due to the fact that these scissors helper molecules
  • L-DNA can also be used for non-pharmaceutical purposes.
  • One application is the marking of
  • the L-DNA is applied to the subject or person and to her
  • the first group includes the so-called
  • Security and / or valuable documents such as passports, identity cards, driver's licenses,
  • the second group includes items which represent a material value and are to be secured against theft, such as jewelry, watches, miscellaneous
  • the marking of persons can, for example be desirable in case of attack.
  • Spraying devices for example, be provided for marking unauthorized premises entering or leaving the premises. This is a spray device, which with an alarm system
  • Alarm system in an alarm state of the alarm system the presence of a person within reach of the
  • Spray device detected. Then the person is sprayed with the solution contained in the spraying device, which in turn contains the L-DNA, and the
  • D-nucleic acids for such purposes is known, however, they have the disadvantage that they can be degraded by an unauthorized person, for example by means of nuclease. This disturbs in particular in such cases, where an object against theft is to be secured, or where a person has been marked, as by the employment of a
  • L-nucleic acids destroys the mark and so removed.
  • the use of L-nucleic acids also has the great advantage that they can not (as opposed to D-nucleic acids), be incorporated into the human genome, so the risk of a possible induction of a disease, in particular
  • the invention described herein in this respect, relates to a method for marking an object or a person, wherein the object or the person or their clothing is provided with an L-DNA and wherein the L-DNA is fixed on or in this object, the person or their clothing becomes. she
  • Article or a person wherein the article or the person or their clothing is subjected to an analysis for the presence of an L-DNA, optionally in addition to its sequencing.
  • marking here means one
  • Marking an object or person by attaching a feature on or to that object or person who was not previously on or on the object or person.
  • the feature is one of a given structure and which can not be affected by circumstances other than (intentional) marking on the subject or person.
  • a solution in particular aqueous solution, containing the L-DNA on the object or the person or their
  • the L-DNA in a preparation which additionally contains a dissolved or dispersed binder.
  • Base preparations are basically all not yet cured liquid or pasty paint binder preparations, adhesive preparations or the like, provided that their pH is less than 9, better less than 8.
  • All solvents in paint technology or as solvents also come as solvents
  • Adhesive technology usual solvents in question. This also applies to the usable binders and customary additives.
  • This preparation is applied to the object to be marked or to the person to be marked.
  • the solution or preparation to be administered per ml preferably contains between 1 or 10 A 1 and 10 A 16, for example between 10 A 3 and 10 A 12, in particular between 10 A 3 and 10 A 9 molecules of L-DNA.
  • the L-DNA carries at the 5 ' or at the 3 ' end a covalently bonded photoluminescent reporter molecule group, which is further preferably selected with the proviso that the luminescence, in particular fluorescence, upon excitation with UV radiation he follows.
  • Reporter molecule groups which can be employed are all photoluminescent molecular groups customary in biochemistry. Is a marked according to the invention
  • the L-DNA can be on
  • the L-DNA may contain in development of this invention at least one invariant sequence block and / or a variable sequence block.
  • the invariant sequence block is then the same for all or at least one set of labels with the L-DNA, i. all labels contain a partial sequence with the sequence of this sequence block.
  • Sequence block can then be individualizing. This is useful if the detection by illumination, for example, with UV should also be dependent on the presence of an L-DNA with said sequence block. This distinguishes you from one
  • Luminescent regardless of the presence of an L-DNA according to the invention, can occur.
  • Structure of a molecular beacon has. This is a single-stranded nucleic acid with hairpin structure or stem-loop structure, wherein the ends forming the stem on the one hand Luminescent molecule and, opposite one
  • Quencher such as dabcyl wear. At least one of the ends is an invariant sequence (sequence block) (typically 5 to 20 base pairs long). In the non-hybridized state of L-DNA is the
  • the L-DNA of the label is not present as a single strand, but an end (the reference to an end always means either 3 or 5 ') of one
  • Luminescent molecule is arranged to the luminescence to suppress.
  • the length of this complementary L-DNA is at least 2, more preferably at least 5 bases shorter than the length of the invariant sequence block.
  • the luminescent molecule lights up and the marking is visible or detectable by apparatus. Thereafter, as above eluted and sequenced again, to determine a possible variable sequence.
  • the invention thus also comprises a register system comprising a database, wherein in the database variable sequence blocks of different L-DNA
  • Marks are recorded and assigned to a person, company or authority.
  • Register system can be one of a marker
  • FIG. 3 Comparison of MgCl 2 -
  • FIG. 6 Quantification of the results from FIG.
  • Figure 7 direct comparison of the activities of L-DNAzyme and L-ribozyme at different
  • FIG. 8 Quantification of the results from FIG.
  • Figure 9 subject of Figure 5, but after 48h
  • RNA or DNA were incubated with 10 ⁇ reaction mixture in the presence of 2 ⁇ DNAzym or RNAzym in 50 mM Tris. HCL buffer, pH 7.5, incubated at 20 ° C. for 2 hours ⁇ ratio of DNAzyme or RNAzyme / target consequently 10: 1).
  • target RNA or DNA and DNAzyme or RNAzyme were denatured for 2 minutes at 72 ° C and slowly (1 ° C / min) in the heating block to 25 ° C
  • the synthesis products had a purity of more than 90%.
  • AI DNAzym- or RNAzym sequences were selected in accordance with the target sequences, the variable regions of the DNAzyme or RNAzyme to the interface triplet and synthesized the RNAzyme or DNAzyme sequences.
  • the synthesis products had a purity of over 85%.
  • L-DNAzyme is able to cut both the L-target sequence and the D-target sequence.
  • Target intersects, but not D-DNAzyme, the L-target.
  • FIG. 4 once again shows measurements corresponding to FIG. 3, wherein additionally the interface at
  • RNAzymes in cells In FIG. 5, HeLa cells were transfected with EGFP plasmid, thus containing a D target. In particular, it can be seen that L-DNAzyme inhibits flourescence more strongly than L-RNAzyme, or else as DR-SFAzyme or D-DNAzyme. This finding is shown in FIG. 6
  • FIGS. 7 and 8 show that L-DNAzyme is also active at 48 h
  • RNAi RNAi is used.
  • Corresponding L-DNAzymes can be easily constructed on the basis of the known sequence information on human phospholamban and it follows compared to the known therapy methods, the advantages of better stability of the L-DNAzyme
  • Another target in the human organism is, for example, the noncoding H19 RNA.
  • This gene becomes differential, for example in cancer cells
  • L-DNAzyme molecule can be selected, which known and suitable sites of H19

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EP12716194.1A 2010-12-31 2012-01-02 Composition pharmaceutique contenant de l'adn-l Withdrawn EP2668275A2 (fr)

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DE102010056610A DE102010056610A1 (de) 2010-12-31 2010-12-31 Pharmazeutische Zusammensetzung enthaltend L-DNA
PCT/DE2012/000008 WO2012089207A2 (fr) 2010-12-31 2012-01-02 Composition pharmaceutique contenant de l'adn-l

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BR (1) BR112013017001A2 (fr)
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US9983565B2 (en) * 2015-03-27 2018-05-29 Intel Corporation Technologies for bio-chemically controlling operation of a machine
CN105274196A (zh) * 2015-04-01 2016-01-27 湖南大学 一种基于l型脱氧核酶生物体系中金属离子的检测试剂盒、检测方法和应用

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DE19800899A1 (de) * 1998-01-13 1999-07-15 Biotecon Ges Fuer Biotechnologische Entwicklung & Consulting Mbh Oligoliganden mit Bindungsvermögen etc.
DE10020275A1 (de) * 2000-04-25 2001-10-31 Manfred Schneider Enantiomere Bausteine der 2-Deoxy-L-Ribose und 2-Deoxy-D-Ribose, Verfahren zu ihrer Herstellung sowie Verwendung zur Synthese von natürlichen und nicht natürliche L- und D-Nucleinsäuren, L- und D- konfigurierten Oligonucleotiden, L- und D-konfigurierter DNA und davon abgeleiteten pharmazeutischen Wirkstoffen
DK1264603T3 (da) * 2001-06-10 2010-04-26 Noxxon Pharma Ag Anvendelse af L-polynukleotider til in vivo-billeddannelse
US20030219422A1 (en) * 2001-11-15 2003-11-27 Noxxon Pharma Ag Allosteric ribozymes and uses thereof
DE60332363D1 (de) * 2002-10-02 2010-06-10 Univ British Columbia Oligonucleotide zur behandlung von prostatakrebs und anderen krebserkrankungen
DE10346487A1 (de) * 2003-10-02 2005-05-12 Transmit Technologietransfer Verfahren zur Herstellung eines Zell- und/oder Gewebe- und/oder Krankheitsphasen-spezifischen Arzneimittels
SG161311A1 (en) * 2005-05-04 2010-05-27 Noxxon Pharma Ag Novel use of spiegelmers
EP2111449B1 (fr) 2007-01-16 2012-03-07 Yissum Research Development Company of the Hebrew University of Jerusalem Agents nucléotidiques de silençage de h19 destinés au traitement de l'arthrite rhumatoïde
EP2393504B1 (fr) 2009-02-06 2013-06-05 Freie Universität Berlin Compositions pharmaceutiques renfermant un ribozyme pour le traitement des effets secondaires de spiegelmers

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JP2014504589A (ja) 2014-02-24
WO2012089207A2 (fr) 2012-07-05
MX2013007543A (es) 2014-04-30
DE102010056610A1 (de) 2012-07-05
KR20140043052A (ko) 2014-04-08
RU2013135647A (ru) 2015-02-10
CN103492571A (zh) 2014-01-01
AU2012203994A1 (en) 2013-08-22
WO2012089207A3 (fr) 2012-10-04
CA2850863A1 (fr) 2012-05-07
US20130345290A1 (en) 2013-12-26

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