EP2668275A2 - Pharmaceutical composition containing l-dna - Google Patents

Abstract

The invention relates to the use of an L-DNA which is capable of binding to an L-RNA, especially in an antisense reaction, and which is optionally capable of cleaving the L-RNA in the region of a target sequence of the L-RNA, for producing a pharmaceutical composition for the treatment of undesired physiological side reactions caused by the administration of a therapeutic molecule containing said L-RNA. The L-DNA can alternatively be used to cleave an endogenous target RNA or DNA.

Description

 Pharmaceutical composition containing L-DNA

Technical field of the invention

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.

Background of the Invention and Related Art Aptamers are mostly double-stranded D-nucleic acids that bind specifically to any target molecule, analogous to an antibody / antigen reaction

(Ellington, A. D. et al., Nature 346: 818-822 (1990)). Specific for a given target molecule

For example, aptamers are isolated from nucleic acid libraries by the SELEX method

{Tuerk, C. et al., Science 249: 505-510 (1990)].

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

therapeutic use of aptamers is disadvantageous in that they have unfavorable pharmacokinetics, i.e. be broken down very quickly,

for example, by endogenous nucleases. Regardless, 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. Thus, in the reference Boisgard, R et al. , Eur Journal of Nuclear Medicine and Molecular Imaging 32: 470-477 (2005) that non-functional spiegelmers are metabolically completely stable even over a period of 2 hours. In this reference, the diagnostic use of Spiegelmeren is described, wherein the Spiegelmer with a

For example, radioactive reporter substance is coupled.

The identification of for a given

Target molecule specific Spiegelmeren can

for example as in the reference Klussmann, S. et al., Nat Biotechnol 14: 1112-1115 (1996)

described described. Regarding the Spiegelmers and their therapeutic applications, reference is also made to the reference Vater, A. et al., Curr Opin Drug Discov Devel 6: 253-261 (2003). In the therapeutic use of Spiegelmeren it has been assumed that Spiegelmers are not immunogenic (Wlotzka et al., Proc Natl Acad See USA 99: 8898-8902 (2002)). However, show

Investigations presented in the present specification, that L-nucleic acids in an organism are not necessarily

are free of side effects. It follows that with the use of Spiegelmeren quite a not

negligible risk of an unwanted

physiological side reaction, for example an immune reaction and / or an undesired one

enzymatic reaction with endogenous RNA

 {including a regulatory RNA), or an antisense inhibition (Watson-Crick reaction) of an endogenous nucleic acid, when administered to a patient. Especially in the light of the negative experiences with the monoclonal antibody TGN1412 made in the clinical phase 1 and against the background that the residence time of spiegelmers is comparatively very high due to the above-mentioned relationships, it would be desirable to have an antidote for a spiegelmer to be used in the administration of the Spiegelmers ready to hold, thus on occurrence of an unwanted physiological

Side reaction immediately administered the antidote and so the (biologically active) Spiegelmer level in the serum can be reduced in the short term.

From other contexts, namely the ribozyme-catalyzed stereoselective Diels-Alder reaction L-ribozymes are known, to which the references Seelig, B. et al. , Angew.Chem. Int., 39: 4576-4579

(2000) and Seelig, B. et al. , Angew. Chem. 112: 4764-4768 (2000). Furthermore, the most varied approaches to the inhibition of endogenous nucleic acids,

For example, 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. As an example of 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.

From the reference WO 2010/088899 A2 it is known L-ribozymes for the cleavage of Spiegelzymen (as antidote) and / or endogenous RNA molecules

use.

L-DNA is known per se, for example from the

Reference G. Hayashi et al. , Nucleic Acid Symp Ser 49 (1); 261-262 (2005), in which such

Nucleic acids are described as molecular tags.

Technical problem of the invention.

The invention is based on the technical problem to provide improved means for cutting Spiegelmeren and endogenous nucleic acids.

Broad features of the invention

To solve this technical problem, 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,

in particular an antisense reaction (Watson-Crick inhibitory reaction), and 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. Furthermore, the use of an L-DNA for the preparation of a pharmaceutical composition for the treatment or prophylaxis of diseases associated with overexpression

at least one endogenous gene, wherein the L-DNA for binding to a target sequence of a coding for the gene endogenous target D-DNA or

Target D-RNA, for example in an antisense

Reaction, and optional to split said

Target sequence is capable.

The invention is based initially on the recognition that Contrary to previous assumptions, spiegelmers are not necessarily free from side reactions, but rather may be able to

naturally occurring in an organism

Cut nucleic acids to produce unpredictable side effects. Likewise, undesirable antisense reactions, i.e. Inhibition of endogenous nucleic acid by Watson-Crick base bonds

between the spiegelmer and the endogenous

Nucleic acid, possible, independent of enzymatic reactions of the bound Spiegelmer.

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. In addition, L-DNAs are particularly stable against

enzymatic degradation, so that no (usually voluminous) Schu zgruppen must be attached to the molecules, which on the one hand advantageous

pharmacokinetic properties are maintained and on the other hand, the uptake is favored in cells.

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

Reference is made to exemplary embodiments.

The invention is based on these findings

building on the technical teaching to provide L-DNA, ie L-DNzyme, which specifically cut an administered Spiegelmer or thereto bind inhibiting and so their physiological

Effectiveness, especially with regard to undesirable side reactions to destroy. examples for

Spiegelmers are: Spiegelmer, NOXC89, NOXA42, NOXA50, NOXB11, NOXA12, NOXE36, NOXF37 (all NOXXON AG),

Spiegelmers from Eli Lilly & Co., NU172 from ARCA biopharm Inc., ARCHEMIX, ARC1905, ARC1779,

ARC183, ARC184, E10030, NU172, REG2, REG1 (all

Archemix Corp.), AS1411, AS1405 (both Antisoma

Research Ltd.), DsiRNA from Dicerna Pharmaceuticals Inc. , RNA Aptamer BEXCORE from BexCore Inc. , ELAN of Elan Corp Plc. , or Macugen, by the administration of such an L-DNA in pursuance of the observation of an unwanted side reaction with administration of a

Spiegelmeres can therefore be the cause of

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.

Deoxyribonucleotides, but in addition to the high selectivity of L-DNA, namely directed to the target sequence of the Spiegelmeres. As a result, a highly effective and highly selective antidote is obtained against a therapeutically used Spiegelmer and unwanted side reactions of Spiegelmeres can be countered effectively, quickly and without side effects. Basically, against every RNA molecule,

including aptamers, whether made up of D or L nucleotides, 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. However, this also means that, for any desired 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

Specify target sequences structurally. The 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.

In principle, 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 spiegelmer / 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. However, it is also possible that the L-DNA is covalently bound to an aptamer, an antibody or a peptide or protein. In this case, 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. One

concrete example of this is in the figure lc

also shown.

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

According to the invention provided absolute doses are doing strictly in the specified relative

Proportions at the prescribed doses of the RNA can therefore be easily determined and established by the skilled person with knowledge of the prescribed doses for L-RNA. In a preferred embodiment of the invention, 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

Is capable of melting a double-stranded L-RNA in the region of its target sequence. These are sequences which hybridize with partial sequences adjacent to the target sequence. This ensures that cleavable regions of L-RNA, which are usually not steric reasons

are accessible due to the tertiary structure of the L-RNA, for which L-DNA is made accessible. In addition, it is further achieved that the desired interfaces are cleaved very specifically, and not corresponding doublets or triplets with other adjacent sequences.

Furthermore, the invention relates to a

pharmaceutical composition containing an L-DNA for the treatment of undesired physiological

Side reactions, in particular immune reactions, due to the administration of a therapeutic molecule containing the L-RNA.

According to the invention, however, 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

Reaction to the endogenous nucleic acids. In terms of cutting DNA is complementary to the

References Lu, Y., et al. , Current Opinions in Biotechnology 17: 580-588 (2006), or Jiang, D., et al., FEBS 277 (11): 2543-2549 (2010).

In particular, it can be used to treat diseases associated with a particular RNA or DNA, or the overexpression of a coded thereby

 Accompanying the expression product. The L-DNA is thus used as an inhibitor with regard to this

Expression products work, namely by the expression is suppressed or reduced by

Cleavage of the coding RNA or DNA or by antisense binding thereto. Here is the specific target sequence {that is to be split or to

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

Adjust target sequence in the region of the selected cleavage site in the manner described above.

In principle, any indications can be detected, provided that the disease to be treated causes the relevant target sequence

related. In the following, only examples are mentioned, but in no way limit the applicability of the invention.

With regard to the pharmaceutical composition, all preceding and following statements apply analogous .

Finally, the invention relates to a method for

Production of such a pharmaceutical

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,

in particular for antisense reaction, and optionally capable of cleaving said sequence, wherein the L-DNA thus obtained is prepared in a pharmacologically effective dose for administration. The L-DNA is typically with galenic auxiliary and / or

Carriers mixed. It is in the context of

 Preparation also possible, customary substances,

which promote endocytosis (the L-DNA), either to covalently couple to the L-DNA, or the

Add composition separately.

In principle, 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. The choice of additives and / or adjuvants will depend on the chosen dosage form. The galenic

Preparation of the pharmaceutical according to the invention

Composition can be carried out in the usual way. Come as counterions for ionic compounds

for example, Mg ⁺⁺ , Pb ⁺⁺ , Mn ⁺⁺ , Ca ⁺⁺ , CaCl ⁺ , Na +, +, Li + or cyclohexylammonium, or Cl ^" , Br ^" , acetate,

Trifluoroacetate, propionate, lactate, oxalate, malonate,

Maleinate, citrate, benzoate, salicylate, putrecine, cadaverine, spermidine, spermine, etc. in question. 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

Preparations with protracted release of active ingredient, in the production of which customary auxiliaries such as carriers, disintegrants, binders, coating substances, swelling agents, lubricants or lubricants, flavorings, sweeteners and solubilizers are used. It is also possible to use the active ingredient in preferably biodegradable cancans

encapsulate or in pores of porous nanoparticles, biodegradable or stable, for example, for the preparation of a preparation for inhalation. However, it is also possible to use 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)) As excipients, for example, sodium carbonates,

Magnesium carbonate, magnesium bicarbonate, titanium dioxide,

Lactose, mannitol and other sugars or cyclodextrins, Talc, milk protein, gelatin, starch, cellulose and its derivatives, animal and vegetable oils such as cod liver oil, sunflower, peanut or sesame oil, polyethylene glycols and solvents such as sterile water and monohydric or polyhydric alcohols, for example glycerol. 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

compatible carrier and optionally other suitable active ingredients, additives or excipients mixed with a defined dose and prepared for the desired dosage form. The diluents are polyglycols, water,

Dimethyl sulfoxide (for example for preparations for the treatment of skin diseases or rheumatism) and

 Buffer solutions in question. Suitable buffer substances are, for example, N, N'-dibenzylethylenediamine, diethanolamine, ethylenediamine, N-methylglucamine, N-benzylphenethylamine, diethylamine, phosphate, sodium bicarbonate, or

Sodium . But it can also be worked without Verdünnungsmi tel. Physiologically acceptable salts are salts with inorganic or organic acids, e.g. Lactic acid, hydrochloric acid, sulfuric acid, acetic 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.

Of independent importance is already

abovementioned variant of the invention, which comprises the use of an L-DNA for the preparation of a pharmaceutical composition for the treatment or prophylaxis of diseases associated with a

Overexpression of at least one endogenous gene

associated with the L-DNA for binding to a

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. A treatment or prophylaxis of viral or bacterial

Infections are possible if the L-DNA binds to a target sequence of that virus or

Bacterium is set up. Otherwise, the above statements apply analogously. In this

Context of importance is another variant of the above aspect of the invention with the

Use of an L-DNA to make a

pharmaceutical composition for the treatment or prophylaxis of diseases associated with a

Infection of a mammal associated with a microorganism, wherein 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. Basically, 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

Inactivating or inhibiting replication activity and / or attenuating or inhibiting binding to cell surfaces.

In this variant, it is used that 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

therapeutic benefit for all diseases associated with the overexpression of certain genes compared to the expression of the non-diseased organism,

accompanied.

On the one hand, 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. In addition, side effects, like them

for example, associated with the use of inhibitory D-nucleic acids, such as siRNA, sure

be avoided. 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.

Finally, it is possible to use two L-DNA or L-DNAzymes in combination, the sequences being selected with the proviso that double-stranded DMA or RNA can also be cut.

In the context of the inventive use of an L-DNA for the preparation of a pharmaceutical composition, further variants are possible.

First, 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. The entire the

One skilled in the art of aptamer technology is accordingly transferable to L-DNA aptamers.

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) phase of a screening assay bound. 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

Contact is or is brought into contact with this. The mobile phase contains an L-DNA library, ie Polynucleotides, typically 10 to 500 nucleotides in length, whose sequences vary, are usually randomized. Such 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. Following this, 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

bound L-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) and

in any case be sequenced. From the sequence of D-DNA thus obtained, the complementary L-DNA can then be determined and synthesized. Alternatively, 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

In principle, even without an immobile phase to be carried out, then 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

Do not wear marker molecule. Otherwise, this alternative works quite analogous to the above

Description .

The result is an L-DNA molecule species or a mixture of such species, which with high

 Bind affinity to the target molecule. These can then be used in the context of an inventive

pharmaceutical composition with any

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. In a corresponding manner, an L-RNA binding to a target can also be isolated or determined. In this case, instead of an L-DNA library, an L-RNA library is used. In a further alternative method, to a (random) target, 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. For this purpose, the L-nucleic acid library

provided, wherein the nucleic acids optionally a coupling molecule or marker molecule is bound, for example, biotin to the 5 'end. The

Target molecule is attached to a solid phase,

For example, magnetic beads, bound. The

Solid phase is then with the

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

subjected to removal of the non-binding L-nucleic acids. Then the L-nucleic acids are eluted again and thus separated from the target molecules in the usual way. Finally, sequencing of the L-nucleic acids thus obtained is then carried out as described elsewhere in this description.

In the context of uses according to the invention, L-DNAs can also be used for testing for potential side effects of L-DNA. As part of such

Test procedure is the L-DNA (or a larger

 Number of identical L-DNA molecules) selected as a prospective drug in contact with a cell extract, for example, one

and it is measured by conventional means, whether only the target molecule in the cell extract binds to the L-DNA, or other molecules. In the former case would be the prospective Drug most likely to be side-effect free in the sense that no cell components other than the target molecule are bound or inhibited. In the latter case, side effects would be expected.

The sequencing of L-nucleic acids can be independent of the other aspects of the invention

different ways are done. In a first

For sequencing an L-RNA or L-DNA, 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

Marker molecule such as biotin (as described above). Then, the solid phase carries a molecule complementary to the coupling molecule and this binding molecule, such as avidin or streptavidin. The thus bound to the solid phase L-nucleic acid is then contacted with a D-DNA library.

In this case, hybridize such D-DNA molecules from the library with the L-nucleic acid, which

contain or consist of complementary sequences. Then 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.

 Subsequently, an amplification, for example by means of PCR, take place. Then the D-DNA

sequenced. The complementary sequence of the L-nucleic acid can then be determined from the D-DNA sequence thus obtained.

Alternatively, an L-nucleic acid, in particular L-nucleic acid RNA, in a sequencing method, as follows. Here, 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. In the case of L-RNA, this can be done in the alkaline, pH typically> 8, usually 8.5-10. For this purpose, 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

Subjected to washing steps, wherein

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

investigated mass spectrometry, in which case on the basis of the found masses and their distribution, the

original sequence of the L-nucleic acid can be determined. In the case of L-DNA, the procedure is the same, except that the "ladder" is produced by hydrolysis in acid, pH <6, better <5.

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

Computer programs are inadequate. On the other hand one can assume that a mRNA in the cell differently

folded is as free in solution. Therefore, there is a great need for the use of siRNAs, microRNAs and

 To optimize mirror enzymes. This could be e.g. with an in vitro protein biosynthesis, in which in pro- or eukaryotic in vitro systems (for example, available from RiNA GmbH, Germany) a certain

 (given) mRNA is translated and the yield of the protein is tested depending on the different Spiegelzyme (prospective agents). molecular

Scissors of the prior art, such as Ribozymes, siRNAs, etc., are for stability reasons or due to the fact that these scissors helper molecules

need, not suitable.

Regardless of the contexts described herein and representing an independent invention, L-DNA can also be used for non-pharmaceutical purposes. One application is the marking of

Objects or persons with L-DNA for security and / or authentication purposes and / or for

Identification of a person. In doing so, the L-DNA is applied to the subject or person and to her

Presence and / or its sequence tested by appropriate methods. As objects, in principle, all objects in question, whose authenticity is to be checked, which are to be marked for theft prevention purposes, or in which an assignment to an owner is desirable. The first group includes the so-called

Security and / or valuable documents, such as passports, identity cards, driver's licenses,

Motor vehicle papers, visas, other identity and / or access documents, such as access cards,

 Membership cards, banknotes, tickets, tax stamps, postage stamps, credit cards, or adhesive labels (for example, for product assurance). The second group includes items which represent a material value and are to be secured against theft, such as jewelry, watches, miscellaneous

Valuables, technical equipment, vehicles, etc. With the use of an L-DNA objects can also be individualized, namely by an object with an L-DNA is marked, which one for the object and only for this object

having characteristic sequence. If such a sequence is assigned to a person or to an owner, then by determining the sequence of the L-DNA applied to the article, it is also possible to assign the object to the person or the owner

carry out. Preferred application are also the marking of pharmaceutical products or their packaging or exhibits in a museum or the like.

The marking of persons can, for example be desirable in case of attack. The

Attacked person can then attack the attacker

For example, spray by means of a spray containing an L-DNA, whereby the person by detection of L-DNA on her or her clothing

is identifiable. Also can be automatic

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

is coupled, actuated as soon as a sensor of

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

Identification can then be made as above.

Although the use of 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

 Nuclease 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

Tumor development, not given. 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

further relates to an article having an L-DNA fixed thereto or therein. It also relates to a method for identifying a

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.

The term 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. In any case, 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.

The fixation may be with all known in connection with the labeling by means of D-nucleic acids

Technologies are made. In the simplest case, a solution, in particular aqueous solution, containing the L-DNA on the object or the person or their

Clothes applied and dried. However, it will be preferable if the L-DNA in a preparation which additionally contains a dissolved or dispersed binder. When

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. In addition to water, 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. Preferably, at least 10%, preferably at least 50%, of the L-DNA molecules have an identical nucleotide sequence.

In a preferred variant of this invention, 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

Illuminated object or a marked person with UV light, the mark is apparent from the thereby stimulated luminescence with the eye or apparatus detectable. The L-DNA can be on

opposite end a marker group,

for example biotin, covalently bound. Then, for positive detection of a label with L-DNA, detachment thereof and sequencing can be carried out according to one of the methods described above. If necessary, the sequence can then be assigned to a person or business unit registered under the measured sequence.

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. The variable

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

Luminous effect, which of any

 Luminescent, regardless of the presence of an L-DNA according to the invention, can occur.

In this development, it is also advantageous if the inventively used L-DNA the

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

Flourescence suppressed by Förster resonance energy transfer to the quencher, upon irradiation with UV no glowing effect can be seen. A proof of the L-RNA takes place then in that the marked area first with a solution of the invariant

 Sequence block of this L-DNA complementary nucleic acid, in particular L-DNA, sprayed. This hybridizes with the invariant sequence block and thereby the luminescent molecule and the quencher are removed from each other. If now the marker is irradiated with UV light, fluorescence is now to be observed or detected by apparatus. An elution and

Sequencing of a possible variable

Sequence blocks then take place as above

described.

In a variant of the above preferred

 Embodiment, 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

 (longer) single strand carries a luminescent molecule. This end forms an invariant sequence block of a given number of bases. Hybridized to this is a complementary L-DNA carrying a quencher at one end, with the proviso that the quencher is sufficiently close to the quencher

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. Now, if a solution with an L-DNA which is complementary to the invariant sequence block and whose length is at least 2, better at least 5, bases longer than the L-DNA carrying the Quentcher, so this is the L-DNA with the Displace quencher and hybridize with the invariant sequence block. Now takes one

Irradiation with UV, 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. By means of this

 Register system can be one of a marker

determined variable sequence block easily associated with the assigned authority, museum, company or person.

Basically, it should be noted that the uses of L-DNA described above are also without

Further analogous with L-KNA can be performed.

The invention will be explained in more detail below with reference to figures and examples. Show it:

Figure 1: General structures of inventive

 L-DNA (a) and hammerhead ribozyme (b), each listed with

 Target sequence specificities and conserved structural elements as well as comparison of

 Binding of a specific L-DNA (c) and a specific hammerhead ribozyme (d) after binding to the same target sequence of the green fluorescent protein GFP (b), representation of secondary structures (see also Zaborowska, Z., et al., The Journal of Biological Chemistry 277 (43): 40617-40622 (2002), Kim, K., et al., Bull. Korean Chem. Sco. 27 (5): 657ff (2006), and Hertel, KJ, et al., Nucleic Acids Research 20 (12): 3252ff (1992)),

Figure 2: Analysis of the cleavage of an L- and D-GFP

Target Sequence by L- and D-DNAzyme and Hammerhead Ribozyme FIG. 3: Comparison of MgCl ₂ -

Concentration dependence of cleavage of the D-GFP target sequence by L-DNAzyme (L-Dz) and the L-GFP target sequence by D-DNAzyme (D-Dz),

Figure 4: MgCl ₂ concentration dependence of

Cleavage of the D-GFP target sequence by L- DNAzyme (L-Dz) with Determination of

 Cleavage site,

Figure 5: Comparison of the activities of various

 DNAzyme and RNAzyme in GFP-transfected

Cells at different enzyme concentrations and 24h incubation,

FIG. 6: Quantification of the results from FIG

 5 by indicating the fluorescence intensities,

Figure 7: direct comparison of the activities of L-DNAzyme and L-ribozyme at different

 Enzyme concentrations and 24h incubation,

FIG. 8: Quantification of the results from FIG

 7 by indicating the fluorescence intensities,

Figure 9: subject of Figure 5, but after 48h

 Incubation, and

Quantification of the results from the figure by indicating the fluorescence intensities

Example 1: Cleavage assay

The activities of L-ribozymes and D-ribozymes were measured under different conditions. The

Basic conditions were as follows. 0.2 μΜ of target RNA or DNA were incubated with 10 μΐ reaction mixture in the presence of 2 μΜ 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). Prior to the reaction, 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

cooled. The influence of Mg ²⁺ ions in concentrations of 0.1 to 10 tnM was investigated.

Cleavage products were based on 20% polyacrylamide

Gel electrophoresis in the presence of 7% urea in 0.09 M Tris-borate buffer, pH 8.3. Fluorescence analysis was performed on Phosphoimager Fuji Film FLA 5100. Data were obtained with the Fuji Analysis Program. Charts were created with Excel.

Example 2: Preparation of the Target Sequences and the

 Ribozymes All target sequences were analyzed by chemical

Synthesis produced. 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%.

All synthesis products were labeled with fluorescein at the 5 'end. Example 3: Measurement of Activities in Cells HeLa cells were resuspended with 1 g of EGFP plasmid

 Regulation transfected. This was followed by incubation with 25, 50 or 100 nM solution of the DNAzyme or RNAzyme to be used. The cells were analyzed after 24h or 48h with a Leica microscope, or the fluorescence intensity (RFU) was determined by multi-mode

 Microplate Reader Synergy-2 measured to specification.

Comparison of the interaction

 various DNAzyme with

 target sequences

In FIG. 2 {10 mM MgCl ₂ ) it can be seen that a

L-DNAzyme is able to cut both the L-target sequence and the D-target sequence. The figure

3 shows the MgCl ₂ concentration dependencies.

From this figure one also deduces that L-DNAzyme

Target intersects, but not D-DNAzyme, the L-target.

FIG. 4 once again shows measurements corresponding to FIG. 3, wherein additionally the interface at

 Target is apparent according to the figure la.

Example 5: Activities of Various DNAzymes and

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

significantly confirmed. This demonstrates in particular the superior effect of L-DNAzyme on L-RNAzyme in the cell. In FIGS. 7 and 8, these results are also confirmed for 24 hours incubation times. Finally, FIGS. 9 and 10 show that L-DNAzyme is also active at 48 h

Incubation times compared to the other enzymes shows a significantly better inhibition.

Example 6: Possible Pharmaceutical Applications

In addition to the use as an antidote in the therapy with Spiegelmeren the invention is also in others

can be used in general contexts. These include in principle all indications in which a disease is correlated with the undesired expression of a gene product. An example is heart failure or hypertrophy. From the reference L. S ckau et al. , Circulation 119: 1241-1252 (2009), it is known that a therapy which inhibits phospholamban is suitable for this purpose. In this reference 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

Active substance against enzymatic degradation with simultaneously good activity with respect to inhibition of the target RNA coding for Phos holamban.

Another target in the human organism is, for example, the noncoding H19 RNA. This gene becomes differential, for example in cancer cells

expressed. Among others, from the document US 2010/0086526 A it is known to inhibit H19 RNA by siRNA. In an analogous way, a

According to the invention L-DNAzyme molecule can be selected, which known and suitable sites of H19

Nucleic acids cut so that its transcription is reduced or inhibited. It turns out

Advantages, as already explained above.

Claims

Claims;

Use of an L-DNA for the preparation of a pharmaceutical composition.

Use according to claim 1, wherein the L-DNA is suitable for binding to an L-RNA or D-RNA, in particular in an antisense reaction, and optionally to the

 Cleavage of L-RNA or D-RNA in the region of a target sequence of L-RNA or D-RNA is capable.

Use of an L-DNA, which binds to an L-RNA or D-RNA, in particular in an antisense reaction, and optionally to cleave the L-RNA or D-RNA in the region of a target sequence of the L-RNA or D-RNA. RNA is capable of producing a pharmaceutical composition for

 Treatment of adverse physiological side reactions, due to the administration of a

 therapeutic molecule containing the L-RNA or D-RNA.

Use of an L-DNA for the preparation of a pharmaceutical composition for the treatment or prophylaxis of diseases associated with overexpression of at least one endogenous gene, wherein the L-DNA binds to an endogenous target D-DNA or target-D coding for the gene RNA, especially in one

antisense reaction, and optionally cleavage of a target sequence encoding the gene endogenous target D-DNA or target D-RNA.

Use according to claim 3, wherein the

therapeutic molecule consists of the L-RNA, in particular a double-stranded L-RNA,

for example, a spiegelmer is.

Use according to claim 3, wherein the

therapeutic molecule containing a covalently bound to the L-RNA aptamer or with the L-RNA covalently bound antibody.

Use according to any one of claims 3 and 5 to 6, wherein the pharmaceutical composition contains the L-DNA in at least the dose corresponding to the dose of the L-RNA, preferably at a dose of 2 to 100-fold , preferably 2 to 20 times, corresponds to the dose of the administration of L-RNA.

Use according to any one of claims 3 to 7, wherein the pharmaceutical composition additionally contains a nucleic acid,

in particular a 5- to 100-mer, which is capable of melting a double-stranded D-RNA or L-RNA in the region of the target sequence.

Pharmaceutical composition containing an L-DNA for the treatment of unwanted

physiological side reactions, due to the administration of a therapeutic molecule containing an L-RNA or a D-RNA.

Pharmaceutical composition containing an L-DNA for the treatment or prophylaxis of

Diseases which are associated with an overexpression of at least one endogenous gene, wherein the L-DNA is capable of binding to an endogenous target D-RNA or target D-DNA coding for the gene, in particular for an antisense reaction, and optionally for cleavage one

Target sequence of the gene coding endogenous target D-RNA or target D-DNA is capable.

A process for producing a pharmaceutical composition according to claim 10 or 11, wherein a sequence of L-deoxyribonucleotides capable of binding to a given sequence of L-ribonucleotides or a predetermined sequence of D-ribonucleotides or D-deoxyribonucleotides is prepared and synthesized, and optionally capable of cleaving said given sequence, and wherein the L-DNA thus obtained is prepared in a pharmacologically effective dose for administration.

The method of claim 12, wherein the L-DNA with galenic excipients and / or carriers

is mixed.