Methods for altering the functional state of mRNA to enable its selective and specific recognition

Abstract

The present invention relates to a method for altering the functional state of any mRNA, allowing its selective and specific recognition and subsequent selective manipulation. [Solution] The present invention describes a general principle for increasing the specificity and selectivity of molecular target recognition at the nucleic acid level. The principle of specific and selective recognition of nucleic acids is based on the simultaneous recognition of two or more sequences of target nucleic acids that must be separated from each other by a certain, defined distance. This method of nucleic acid recognition by specifically recognizing clearly defined sequences of nucleic acids separated from each other by a defined distance minimizes the possibility of stable binding of an interfering construct to an unexpected nucleic acid, thereby dramatically increasing the selectivity of target nucleic acid recognition. Specific recognition of defined sequences of nucleic acids located at a certain, defined distance from each other is achieved by the simultaneous complementary interference of short, sequence-specific oligonucleotides interconnected by size-specific polymer linking moieties.

Description

[Technical field]

[0001] The present invention is directed to changing the functional state of any nucleic acid, allowing its specific and selective recognition and subsequent selective manipulation. This solution is completely universal and applicable in fields such as biotechnology, molecular biology, virology, medicine, etc. The present invention is most preferably directly applicable and has a wide therapeutic potential in the field of oncology, but is not limited to this particular area. [Background technology]

[0002] In view of the current state of the art, the solution described in this invention is different from other chemically similar constructs and is designed for a completely opposite and different purpose.

[0003] The bivalent system described in WO 2011 / 117353 (Moeller, Udesen, 2011) presents a multifunctional construct for the simultaneous interaction of linked oligonucleotides with two separate target nucleic acids to saturate their binding sites and modulate their biological function. Since each oligonucleotide recognizes a different target nucleic acid molecule, the construct itself in principle functions in the same way as a standard antisense oligonucleotide (with respect to the individual target nucleic acid molecule) and does not in any way increase the selectivity of recognition of the target nucleic acid molecule.

[0004] Similarly, the conjugate of antisense oligonucleotide described in WO2011 / 031520 (Agrawal et al., 2011) is designed to recognize two (identical or different) nucleic acid molecules, and similarly to WO2011 / 117353 (Moeller, Udesen, 2011), it recognizes the target nucleic acid molecule individually (i.e., via a single antisense oligonucleotide).Therefore, the above documents do not address the issue of selective recognition of target nucleic acid molecules.

[0005] Other chemically similar constructs described in WO 2009 / 023819 (Kandimalla et al., 2009), WO 2005 / 111238 (Epstein et al., 2005), WO 2005 / 078096 (Zamore et al., 2005), WO 2004 / 064782 (Agrawal et al., 2004) were designed for immunomodulation to enhance their cellular uptake, miRNA recruitment and immune stimulatory purposes, respectively. However, none of them solve the problem of selective recognition of target nucleic acid molecules.

[0006] In each case, the described constructs represent only one kind of "good" multivalent form of the interacting molecules, and moreover, they are designed to solve a different, completely opposite problem unrelated to the present solution. Thus, the fundamental difference lies in the fact that existing constructs do not solve the promiscuity of antisense oligonucleotides, whereas the present invention provides a solution to this problem.

[0007] Although the current scientific knowledge in this field is directed towards the treatment of oncological diseases, it is important to note that fusion genes, which have unique characteristics in tumor cells, are the cause of many tumor diseases (leukemia, lymphoma, sarcoma, etc.) and can certainly be considered as promising targets for anticancer therapy. The unique sequence of the fusion nucleic acid allows specific and selective targeting of tumor cells without therapeutic intervention on healthy cells. Therefore, anticancer therapy based on the interference of therapeutic agents with nucleic acids can be directed against the causative fusion nucleic acid, thereby achieving a therapeutic effect only on tumor cells. From the point of view of antisense strategies, it is refers to the targeted silencing of the fusion gene via interference with the fusion mRNA, thereby preventing the synthesis of the responsible fusion protein.

[0008] The problem of insufficient specificity (in terms of binding specificity of therapeutic agents to the target sequence of mRNA, their binding affinity to the target mRNA and binding energy with the target mRNA) of these antisense strategies has been mainly solved by chemical modification of the therapeutic agents, such as by chemical modification of the ribose and / or phosphodiester backbone (Pirollo, Rait et al., 2003; Stahel, Zangmeister-Wittke, 2003; Jansen, Zangemeister-Wittke, 2002). Despite this, a final solution has yet to be identified, as modified therapeutic agents are not specific and selective enough, leading to silencing of genes outside the therapeutic target (Burnett, Rossi, 2012). As a result, this affects the expression of non-target proteins, resulting in severe clinical side effects and negatively affecting the patient's quality of life.

[0009] Regarding the principle of target mRNA recognition, the application of a single therapeutic sequence-specific oligonucleotide that binds to a specific region of the target mRNA is the commonly used standard of antisense strategies. In the case of fusion mRNA, this specific region is most commonly the site of direct fusion of two individual fusion partners (Diakos et al., 2007; Rangatia, Bonnet, 2006; Rapozzi et al., 2006; Scherr et al., 2005; Scherr et al., 2003; Tanaka et al., 1997). However, published data clearly demonstrate the fact that, despite the significant improvement of the physicochemical properties of antisense therapeutics, the application of a single interfering oligonucleotide has not yet resolved the necessary progress against the problem of non-specific binding of antisense oligonucleotides to unanticipated mRNA molecules (Summerton et al., 2007).

[0010] For this reason, the current state of the art still faces the fundamental challenge of specificity and selectivity of therapeutic effect only on the primary target.Accordingly, the real progress in anticancer strategies does not lie in the development of essentially new therapeutic agents, but in the development of systems that allow selective therapeutic action.

[0011] The present invention, which represents a universal solution for any therapeutic strategy based on interference with nucleic acid, overcomes the limitations of insufficient specificity and selectivity of anti-cancer strategies.Directly introducing the present invention into anti-cancer antisense strategies, the principle of specific and selective recognition of target nucleic acid is described and explained together with the principle of selective action via targeted interference with the causative fusion gene.

[0012] In both of the cited patents and publications, the term "specificity" strictly refers to the complementary base pairing of the oligonucleotide with the target sequence of the nucleic acid, i.e. specificity = complementarity, whereas in the present invention, the term "specificity" refers to the complementary recognition and binding to a unique, defined target nucleic acid, i.e. specificity = selective recognition of the target nucleic acid.

[0013] Thus, the present invention is clearly different from existing antisense systems designed for the controlled intervention of nucleic acids, in other words, the present invention represents a largely innovative solution. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] International Publication No. 2011 / 117353 [Patent Document 2] International Publication No. 2011 / 031520 [Patent Document 3] International Publication No. 2009 / 023819 [Patent Document 4] International Publication No. 2005 / 111238 [Patent Document 5] International Publication No. 2005 / 078096 [Patent Document 6] International Publication No. 2004 / 064782 Summary of the Invention

[0015] The insufficient specificity caused by sequence homology to target nucleic acid, for example target mRNA, is effectively addressed by changing the functional state of target mRNA to allow its selective and specific recognition and subsequent selective intervention, manipulation, detection, quantification, labeling, pre-targeting and selection, while mRNA is targeted by a construct comprising at least two sequence-specific oligonucleotides interconnected by size-specific polymeric moieties whose length defines their mutual distance, each sequence-specific oligonucleotide targets a predefined target sequence of mRNA to generate a stable heteroduplex, and this change allows this mRNA to be selectively and specifically recognized.The specific and selective recognition of nucleic acid can then be used for selective therapeutic intervention when the transmission of genetic information encoded by nucleic acid is interrupted, or for diagnostic and research purposes.

[0016] This change allows specific recognition of defined sequences of nucleic acid, but these sequences must be at a precisely defined distance from each other.When simultaneously recognizing defined sequences at a defined distance from each other, the interference system designed for the specific spatial distribution of target sequences forms thermodynamically favorable and energetically stable bonds with target nucleic acid.This recognition principle minimizes the possibility of non-specific interactions and stable binding to unexpected nucleic acids, thus dramatically reducing undesirable interference with nucleic acids other than the primary target.

[0017] The described nucleic acid recognition principle dramatically increases the specificity and selectivity of interference with a single, specific, selected and defined nucleic acid. The described principle is completely universal and is not limited to the interconnection of two sequence-specific oligonucleotides, i.e. allows the purposeful interconnection of any number of oligonucleotides (n≧2) with a corresponding number of non-interfering polymer linking moieties (n≧1), both of which are relevant to the final application intent.

[0018] In a preferred embodiment of the present invention, when the target mRNA is a fusion mRNA, each of the specified sequences is located on each fusion partner, and the interference system is targeted only to the fusion nucleic acid, which directly guarantees selective therapeutic action only on tumor cells.Furthermore, since the antisense system can exert its effect on any cell that contains the target mRNA, the present invention allows selective therapeutic targeting of tumor stem cells by its implementation, thus bringing the prospect of curative therapy.In other words, the described technological innovation brings the hope of complete and permanent cure of oncological disorders.

[0019] From the perspective of anticancer antisense strategies aimed at the intervention of fusion genes, this principle also allows stable interference with only target fusion mRNA only when both complementary sequences of individual fusion partners are recognized, which are at precisely defined distances from each other.By this means, the possibility of non-specific stable interaction, especially with homologous sequences, is eliminated, while the specificity and selectivity of interference is significantly increased.In the absence of simultaneous recognition of both complementary sequences, i.e., in the case of undesired interaction with unexpected mRNA, the partial interaction of such a system with non-target mRNA is energetically unstable, leading to spontaneous cleavage (Dias, Stein, 2002).

[0020] The application of the present invention to medicine is demonstrated by the antisense system designed for anticancer therapy, which has the primary objective of selectively intervening in causative fusion genes.In the context of oncological diseases characterized by the presence of fusion genes, this means selectively targeting only the diseased cells in a completely revolutionary way, maximizing therapeutic intervention against the primary target.In the field of oncology, the present invention represents a revolutionary tool that only intervenes in tumor cells (i.e., without intervening in healthy cells). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The solution of the invention, being a method for changing the functional state of any mRNA, allowing its selective and specific recognition and subsequent selective intervention, manipulation, detection, quantification, labeling, pre-targeting and selection of this mRNA, is proposed to increase the specificity and selectivity of the targeting of a particular nucleic acid, and together with the specific fields of application (e.g. controlled inhibition of causative fusion genes, detection and quantification of the presence of nucleic acids, labeling of nucleic acids, pre-targeting and selection of nucleic acids) forms an integral and inseparable part of the invention, which clearly defines the subject of the invention. The subject of the invention thus comprises the specific and selective targeting of any particular nucleic acid by the simultaneous recognition of its two (or more) specified sequences (which must further be at a specific predefined distance from each other).

[0022] From an innovation standpoint, the present invention provides innovative tools that increase the selectivity and specificity of target nucleic acid recognition, allowing its selective intervention, manipulation, detection, quantification, labeling, pre-targeting and sorting.

[0023] The selective recognition of a particular nucleic acid (preferably mRNA) in the described manner can subsequently be exploited for selective therapeutic intervention of its natural biological function when its functional state is altered to prevent the transmission of the encoded genetic information. In this way, the mechanism of transmission of genetic information from nucleic acid to protein is interrupted, allowing in the case of fusion mRNAs to directly suppress the expression of the responsible fusion oncoprotein.

[0024] Similarly, the methods described can selectively recognize specific nucleic acids (preferably mRNA) resulting in a change in their functional state, which can then be used for a variety of diagnostic or research purposes. - FITC, RITC, and isotope P are added to the primary structure of sequence-specific oligonucleotides 32The intentional incorporation of detectable moieties such as , allows for direct detection, visualization, localization and quantification of the recognized nucleic acid (preferably mRNA). The use of labeled sequence-specific oligonucleotides allows for the quantification of the recognized mRNA in samples analyzed in situ, in vitro, in vivo and ex vivo. Purify and separate the recognized mRNA from other nucleic acids present in the treated sample. · Functional analysis of specific genes by deliberately incorporating photo-labile functional groups into the primary structure of sequence-specific oligonucleotides to allow reversible changes in the functional state of recognized mRNAs.

[0025] Terms and Definitions Antisense systems. Unlike the antisense systems currently used for controlled inhibition of target mRNA (Guo et al., 2013; Burnett, Rossi, 2012; Vaishnaw et al., 2010; Missalidis, 2008; Wang et al., 2003; Clark, 2000), the present invention eliminates their most important drawback: insufficient specificity and selectivity of primary target recognition (i.e., promiscuity of antisense oligonucleotides). The promiscuity of standard antisense systems, which is closely related to the length of the antisense oligonucleotide, is effectively solved by the simultaneous interference of two (or more) antisense oligonucleotides that complementarily recognize target sequences localized at a precisely defined distance from each other. In the field of anticancer therapy, especially for controlled inhibition of causative fusion genes, the present invention represents an experimental (not previously applied) antisense system through its design and mechanism of target mRNA recognition.

[0026] Constructs. The described constructs are interconnected with each other via size-specific polymer linking moieties. The nucleic acid sequence comprises two (or more) sequence-specific oligonucleotides.

[0027] Sequence-specific oligonucleotides. The oligonucleotides represent a sequence complementary to a target nucleic acid, preferably an mRNA, or a nucleic acid derivative. The target nucleic acid is preferably of human origin.

[0028] Oligonucleotides may be formed by any nucleotide, optionally chemical derivatives / analogs, such as DNA, RNA, 2'-O-(2-methoxyethyl)-RNA, 2'-O-methyl-RNA, 2'-fluoro-RNA, LNA, PNA, morpholino, INA, FANA, ANA, UNA, HNA, etc., which may be combined with each other within the scope of the present invention as blocks of oligonucleotides with specific chemical modifications or as individual oligonucleotides containing different modified nucleotides. Both the nucleobase and the sugar phosphate backbone may be chemically modified. [ka]

[0029] Examples of RNA derivatives A: Modification of the sugar moiety, B: Modification of the phosphate bond, C: Modification of the sugar phosphate backbone.

[0030] In the present invention, the first oligonucleotide, the second oligonucleotide, and optionally other oligonucleotides comprise a contiguous sequence of at least three nucleotides capable of base pairing with a complementary sequence of a target nucleic acid, preferably an mRNA, wherein: the nucleotides may be A, I, U, T, C, G or derivatives thereof; An oligonucleotide may contain any mutual combination of nucleotides or their derivatives.

[0031] Other preferred sequences of oligonucleotides include at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 26 nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least 29 nucleotides, at least 30 nucleotides, at least 31 nucleotides, at least 32 nucleotides, at least 33 nucleotides, at least 34 nucleotides, at least 35 nucleotides, at least 36 nucleotides, at least 37 nucleotides, at least 38 nucleotides, at least 39 nucleotides, at least 40 nucleotides, at least 41 nucleotides, at least 42 nucleotides, at least 43 nucleotides, at least 44 nucleotides, at least 45 nucleotides, at least 46 nucleotides, at least 47 nucleotides, at least 48 nucleotides, at least 49 nucleotides, at least 50 nucleotides, at least 51 nucleotides, at least 52 nucleotides, at least 53 nucleotides, at least 54 nucleotides, at least 55 nucleotides, at least 56 nucleotides, at least 57 nucleotides, at least 58 nucleotides, at least 59 nucleotides, at least 60 nucleotides or less, 29 nucleotides or less, 28 nucleotides or less, 27 nucleotides or less, 26 nucleotides or less, 25 nucleotides or less, 24 nucleotides or less, 23 nucleotides or less, 22 nucleotides or less, 21 nucleotides or less, 20 nucleotides or less, 19 nucleotides or less, 18 nucleotides or less, 17 nucleotides or less, 16 nucleotides or less, 15 nucleotides or less, 14 nucleotides or less, 13 nucleotides or less, 12 nucleotides or less, 11 nucleotides or less, 10 nucleotides or less, 9 nucleotides or less, 8 nucleotides or less, 7 nucleotides or less, 6 nucleotides or less, 5 nucleotides or less, 4 nucleotides or less, 3 nucleotides or less in length.

[0032] The preferred length of the oligonucleotides (individually) is 10 to 25 nucleotides.

[0033] Each of the oligonucleotides within a single construct recognizes a different complementary sequence of an arbitrary but single defined target nucleic acid, preferably mRNA.

[0034] The length of the oligonucleotides in one of the present constructs may vary for a particular application. By extending the length of the oligonucleotides, stronger interaction with the target nucleic acid may be achieved. On the other hand, the extension of oligonucleotides may impair their bioavailability and cellular internalization. However, since the present invention involves two (or more) oligonucleotides in combination (jointly and simultaneously binding to the target nucleic acid), it is possible to use relatively short oligonucleotides, preferably consisting of less than 17 nucleotides.

[0035] Size-specific polymer linking moieties. The sequence-specific oligonucleotides are covalently interconnected via size-specific polymer linking moieties, maintaining the 5' to 3' orientation of every consecutively interconnected oligonucleotide. The polymer linking moieties are attached to the 5' end of the first oligonucleotide and the 3' end of the second oligonucleotide, and the oligonucleotides are linked by said polymer linking moieties.

[0036] The polymer linking moiety may be comprised of any sequence and number of nucleotides (or nucleotide derivatives / analogs), preferably 3-50 nucleotides (in any combination thereof). The polymer linking moiety may be comprised of an abasic unit, in which case the linking moiety is a polymeric sugar phosphate or a chemically modified backbone.

[0037] The polymer linking moiety may be covalently attached to the nucleobase or sugar phosphate backbone of the oligonucleotide.

[0038] The polymer linking moiety may be, for example, a polypeptide, a polysaccharide, a saturated or unsaturated hydrocarbon (C2-C40), preferably a water-soluble natural or synthetic polymer.

[0039] In preferred embodiments, the polymer linking moiety comprises a non-nucleotide polymer, such as poly(meth)acrylate or modified poly(meth)acrylate (preferably poly(ethyleneoxy) and 2(N,N-dimethylamino)ethyl (meth)acrylate), poly(vinyl alcohol), poly(vinylpyrrolidone), poly(ethylene glycol), poly(acrylamide), poly(oxazoline), poly(ethyleneimine), poly(alkylene oxide), lactone-based polymers, poly(acrylic acid), poly(lactide acid), poly(glycolic acid), poly(propylene), poly(styrene), poly(olefin), poly(amide), poly(cyanoacrylate), poly(imide), poly(ethylene terephthalate), poly(tetramethylene glycol), poly(urethane), as well as combinations of these with each other or other natural or synthetic polymers (Kadajji, Betageri, 2011).

[0040] The length of the polymer linking moiety may be adjusted according to the particular use of the construct. The length of the polymer linking moiety is preferably adjusted according to the mutual distance between the target sequences of the target nucleic acid, preferably mRNA. If the complementary sequences are separated from each other by up to 20 nucleotides, the final length of the size-specific polymer linking moiety can be 10-100 angstroms based on the distance between the nucleotides in the linear fully extended nucleic acid. The length of the polymer linking moiety is not limited and can be freely adjusted according to the final use. It is generally preferred that the polymer linking moiety is 1000 angstroms or less in length, 900, 800, 700, 600, 500, 400, 300, 200 or 100 angstroms or less in length. It is also preferred that the polymer linking moiety is at least 5 angstroms long, for example 10, 15, 20, 25, 30, 35, 40 or 45 angstroms long.

[0041] Preferred ranges for size specific polymer linking moieties are 5-1000 angstroms, 10-800 angstroms, 20-500 angstroms, 20-200 angstroms, 10-1000 angstroms, and 20-80 angstroms.

[0042] synthesis Sequence-specific oligonucleotides. Chemical synthesis of sequence-specific oligonucleotides is carried out on solid supports using the phosphoramidite method, i.e., through sequential oligomerization of individual monomers derived from protected 2'-deoxynucleosides (dA, dC, dG, dT), ribonucleosides (A, C, G, U) or other chemically modified nucleosides, the specific selection and sequence order of which depends on the final application. The synthesis cycle involves the stepwise conjugation of individual nucleosides to a growing oligomer in an order corresponding to the sequence complementary to the target nucleic acid (preferably mRNA). Upon completion, the sequence-specific oligonucleotide is released from the solid support into solution and prepared for conjugation with a polymeric linking moiety.

[0043] Synthesis of RNA and DNA oligonucleotides. Synthesis cycles. The synthesis of size-specific oligonucleotides proceeds stepwise in the 3' to 5' direction as one nucleotide is added to the growing oligomer per synthesis cycle until the desired sequence is obtained (Greco, Tor, 2007). [ka]

[0044] Step 1: Detritylation (removal of protecting groups) The dimethoxytrityl (DMT) protecting group is removed by a solution of an acid, such as 2% trichloroacetic acid or 3% dichloroacetic acid, in an inert solvent (dichloromethane or toluene).

[0045] Steps 2 and 3: Activation and Coupling (Conjugation of Oligonucleosides) A 0.02-0.2 M solution of nucleoside phosphoramidites in acetonitrile is activated by a 0.2-0.7 M solution of an acidic azole catalyst, i.e., 1H-tetrazole, 2-ethylthiotetrazole, 2-benzylthiotetrazole, 4,5-dicyanoimidazole or other similar compound (Wei, 2013). A 1.5-2.0-fold excess of the activated phosphoramidite relative to the support-bound oligonucleotide is then reacted with the starting solid support (first coupling) or the support-bound oligonucleotide precursor (after coupling). The 5'-hydroxy group reacts with the activated phosphoramidite moiety of the incoming nucleoside phosphoramidite to form a phosphite triester bond. Upon completion of the coupling reaction, unbound reagents and by-products are removed by washing.

[0046] Step 4: Oxidation The newly formed three-coordinate phosphite triester bond is then oxidized to a four-coordinate phosphate triester using iodine and water in the presence of a weak base (pyridine, lutidine or collidine). The oxidation may be carried out under anhydrous conditions using tert-butyl hydroperoxide (Alul et al., 1991) or finally using (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (Manoharan et al., 2000).

[0047] Step 5: Capping A mixture of acetic anhydride and 1-methylimidazole is used to treat the solid support bound material.

[0048] After completion of the coupling and oxidation reactions, a small amount of solid support-bound 5'-OH groups (0.1-1%) remain unreacted and need to be permanently blocked, for example by acetylation, to prevent the formation of oligonucleotides with internal base deletions, commonly referred to as (n-1) shortmers.

[0049] Step 6: Cutting Cleavage of the oligonucleotide from the solid support and deprotection is accomplished with concentrated ammonium hydroxide.

[0050] Synthesis of RNA and DNA oligonucleotides with phosphorothioate backbones Similar to common RNA and DNA oligonucleotides, modified oligonucleotides are synthesized in which one of the oxygen atoms in the phosphodiester backbone is replaced by a sulfur atom (see the synthesis cycle for details). The difference is that the oxidation step is replaced by a sulfuration step.

[0051] Synthesis of LNA oligonucleotides Modified oligonucleotides are synthesized according to Obika et al. (1997) and Koshkin et al. (1998) in which the ribose is modified by a covalent bridge between the 2'-O and 4'-C atoms.

[0052] Synthesis of PNA oligonucleotides Modified oligonucleotides are synthesized according to Nielsen et al. (1991) in which the sugar-phosphate backbone is formed by repeating N-(2-aminoethyl)-glycine units linked together via peptide-like bonds.

[0053] Synthesis of morpholino oligonucleotides According to Summerton and Weller (1991, 1993b, 1997), modified oligonucleotides are synthesized in which the nucleobases are linked to morpholine rings and interconnected via phosphorodiamidate bonds.

[0054] Synthesis of GNA oligonucleotides Modified oligonucleotides are synthesized according to Ueda et al. (1971) and Cook et al. (1995, 1999) in which the sugar phosphate backbone is made up of repeating glycol moieties interconnected via phosphodiester bonds.

[0055] Synthesis of TNA oligonucleotides Modified oligonucleotides in which the sugar phosphate backbone is composed of threose instead of ribose are synthesized according to Chaput and Szostak (2003).

[0056] Size-specific polymer linking moieties. The chemical synthesis of size-specific polymer linking moieties is carried out using organocatalysts. This is done using common methods of chemical and polymer chemistry, which allows control of the final molecular weight and therefore the final length of the polymer chain, e.g., atom transfer radical polymerization (Matyjaszewski, Xia, 2001). The size-specific polymer linking moiety may be bifunctional (i.e., functionally modified at both ends) to allow for the subsequent attachment of both sequence-specific oligonucleotides to the final 5'→3' oriented construct.

[0057] The bifunctional size-specific polymer linking moiety is preferably formed by a non-nucleotide polymer such as polyethylene glycol (PEG), although the resulting construct is not limited to this particular type of polymer moiety (see the Detailed Description of the Invention section for a detailed description of possible structural variations).PEGylation of nucleotides and methods for preparing PEGylated nucleotides have been previously described by Jaschke et al. (1994) and Fischer et al. (2008).

[0058] Alternatively, if the size-specific polymer linking moiety is not directly synthesized, commercially available polymer moieties such as 17-O-DMT-hexaethyleneoxide-1-O-phosphoramidite, 8-DMT-O-triethyleneoxide-1-O-phosphoramidite, 6-DMT-hexanediol-1-O-DMT-phosphoramidite or 1,3-propanediol, phosphoramidite can be used.

[0059] Constructs. The synthesis of the final construct is carried out by sequential conjugation of a size-specific polymer linking moiety to a first sequence-specific oligonucleotide, followed by attachment of the resulting precursor to a second sequence-specific oligonucleotide, for example via "click" chemistry (Presolski et al., 2011; Besanceney-webler et al., 2011; Bowman, Hoyle, 2010). [Brief description of the drawings]

[0060] [Figure 1] We present a general principle of highly specific interaction of an interference system with a target sequence of a nucleic acid via two sequence-specific oligonucleotides interconnected by a size-specific polymer linking moiety. The proposed principle is universal and adjustable to allow the purposeful interconnection of any number of sequence-specific oligonucleotides (n≧2) via a corresponding number of non-interfering size-specific polymer linking moieties (n≧1). [Diagram 2] The present invention shows a highly specific interaction of an interference system with a target sequence of a fusion mRNA via two sequence-specific oligonucleotides interconnected by a size-specific polymer linker, each of which binds to a corresponding sequence of the fusion partner. EXAMPLES

[0061] The solution described in the present invention can be considered universally applicable to the selective and specific recognition of any target nucleic acid, preferably fusion mRNA.In the context of antisense systems, when the target nucleic acid is fusion mRNA, the presence of which clearly indicates the characteristics of tumor cells from healthy cells and distinguishes tumor cells from healthy cells, the present invention allows the selective recognition and targeting of tumor cells only.The innovation of the form of improved selectivity and specificity of the recognition of target nucleic acid, preferably fusion mRNA only, allows the controlled intervention of target fusion genes.

[0062] Example 1. Selective and specific recognition of BCR-ABL fusion mRNA using the present invention in chronic myeloid leukemia or Ph+ acute lymphoblastic leukemia or other tumors in which BCR-ABL fusion mRNA is present.

[0063] The first sequence-specific oligonucleotide targets a sequence in the BCR and the second oligonucleotide In the case where the otide targets the sequence of ABL, the proposed construct selectively and specifically recognizes BCR-ABL mRNA, whereas both oligonucleotides are interconnected by a size-specific polymer linking moiety. In the case where each of the sequence-specific oligonucleotides targets either BCR or ABL, it is also possible to apply two or more sequence-specific oligonucleotides, where each of the fusion partners is targeted by at least one oligonucleotide. The sequence-specific oligonucleotides are interconnected by a corresponding number of polymer linking moieties. Only when each of the target sequences is fully recognized and further separated from each other by a certain distance defined by the size-specific polymer linking moiety, a stable thermodynamically and energetically favorable complementary interaction is formed between the construct and the targeted BCR-ABL mRNA. By this means, the possibility of stable binding of the construct to partially homologous mRNA is minimized, preventing stable intervention by unanticipated mRNA molecules. With regard to the fact that the fusion BCR-ABL mRNA is only present in tumor cells, the described principle of BCR-ABL mRNA recognition leads to preferential and stable intervention only in tumor cells, thereby selectively recognizing and targeting tumor cells.

[0064] The sequence of each sequence-specific oligonucleotide in the construct is complementary to the target sequence of each fusion partner BCR, ABL. The target sequence of BCR, ABL can be any sequence with respect to the primary sequence of the fusion BCR-ABL mRNA, but in a preferred embodiment, the distance of the target sequence from the fusion breakpoint site is 100 nucleotides or less, regardless of the particular fusion BCR-ABL mRNA variant. TIFF0007657756000003.tif62170

[0065] As with Example 1, a simplified version of Examples 2-7 as well as other fusion mRNAs are listed below.

[0066] Example 2. Selective and specific recognition of AML1-ETO fusion mRNA using the present invention against acute myeloid leukemia M2 or other tumors in which AML1-ETO fusion mRNA is present. TIFF0007657756000004.tif61170

[0067] Example 3. Selective and specific recognition of CBFB-MYH11 fusion mRNA using the present invention against acute myeloid leukemia M4 or other tumors in which CBFB-MYH11 fusion mRNA is present. TIFF0007657756000005.tif57170

[0068] Example 4. Selective and specific recognition of RBM15-MKL1 fusion mRNA using the present invention in acute myeloid leukemia or other tumors in which RBM15-MKL1 fusion mRNA is present. TIFF0007657756000006.tif59170

[0069] Example 5. Selective and specific recognition of MOZ-CBP fusion mRNA using the present invention in acute myeloid leukemia or other tumors in which the MOZ-CBP fusion mRNA is present. TIFF0007657756000007.tif59170

[0070] Example 6. Selective and specific recognition of TAF2N-TEC fusion mRNA using the present invention in myxoid chondrosarcoma or other tumors in which TAF2N-TEC fusion mRNA is present. TIFF0007657756000008.tif58170

[0071] Example 7. Selective and specific recognition of BRD4-NUT fusion mRNA using the present invention in mediastinal carcinoma or other tumors in which BRD4-NUT fusion mRNA is present. TIFF0007657756000009.tif64170

[0072] Similarly, it is possible to selectively and specifically recognize the following fusion mRNAs: Fusion PML-RARA mRNA Fusion BCM-IL2 mRNA Fusion TEL-AML1 mRNA Fusion CEV14-PDGFRB mRNA Fusion TCR-RBTN2 mRNA Fusion RBM15-MKL mRNA Fusion TMPRSS2-ETS mRNA Fusion ETV6-NTRK3 mRNA Fusion NPM-ALK mRNA Fusion TFE3-PRCC mRNA Fusion PLZF-RARA mRNA Fusion TFE3-ASPSCR1 mRNA Fusion MLL-AF9 mRNA Fusion PAX8-PPARG mRNA Fusion DEK-CAN mRNA Fusion TET1-TP53 mRNA Fusion FUS-ERG mRNA Fusion TFEB-ALPHA mRNA Fusion AML1-MTG mRNA Fusion TFE3-PSF mRNA Fusion AML1-EAP mRNA Fusion CHOP-EWS mRNA Fusion NUP98-PMX1 mRNA Fusion PAX3-FKHR mRNA MLL-AFP1 mRNA fusion JAZF1-JJAZ1 mRNA fusion Fusion EA2-HLF mRNA Fusion FUS-CREB312 mRNA Fusion MOZ-P300 mRNA Fusion TPM3-ALK mRNA Fusion TEL-PDGFRB mRNA Fusion CLTC-ALK mRNA MLL-AFX1 mRNA fusion RPN1-EVI1 mRNA fusion Fusion E2A-PBX1 mRNA Fusion EWS-FLI1 mRNA Fusion MLL-AF6 mRNA Fusion AML1-EVI-1 mRNA Fusion NUP98-HOXA9 mRNA Fusion ETV6-MN1 mRNA Fusion MLL-AF4 mRNA Fusion MLL-ENL mRNA Fusion NUP98-RAP1GDS1 mRNA Fusion CALM-AF10 mRNA Fusion FUS-CHOP mRNA Fusion PAX7-FKHR mRNA Fusion SYT-SSX mRNA Fusion EWS-CHN mRNA Fusion TCF12-TEC mRNA Fusion EWS-WT1 mRNA Fusion ASPL-TFE3 mRNA Fusion COL1A1-PDGFB mRNA Fusion TPM4-ALK mRNA

[0073] Other applications of the present invention are demonstrated by Examples 8-11.

[0074] Example 8. Selective and specific therapeutic intervention of the natural biological function of any mRNA when the functional state of the recognized mRNA is altered and the transmission of the genetic information encoded by this mRNA is prevented, by applying the present invention, which leads to the interruption of the translation mechanism of the genetic information from the mRNA to a protein and, in the case of fusion mRNAs as in examples 1 to 7, to the direct suppression of the expression of the responsible fusion oncoprotein.

[0075] Example 9. Application of the present invention to FITC, RITC, and P, which emit detectable signals corresponding to stable heteroduplexes after the functional state of the recognized mRNA is changed. 32 Selective and specific detection of any mRNAs and their subsequent quantification when sequence-specific oligonucleotides contain an incorporated detectable label, such as an isotope.

[0076] Example 10. Purification and sorting of selectively and specifically recognized mRNA from other nucleic acids present in the analyzed sample, when application of the present invention leads to a change in the functional state of the recognized mRNA, i.e. when a stable heteroduplex with a different electrophoretic mobility is formed. As a result, it becomes possible to separate the recognized mRNA from other nucleic acids by applying an external electric field. Similarly, deliberate modification of a sequence-specific oligonucleotide by a primary antibody makes it possible to sort the recognized mRNA on the basis of its interaction with a secondary antibody after application of the present invention.

[0077] Example 11. Functional analysis of individual genes when the primary structure of sequence-specific oligonucleotides contains an incorporated photolabile functional group, allowing a reversible change in the functional state of the recognized mRNA. The present invention can be applied to reverse this effect after the functional state of the recognized mRNA has changed, by irradiation with radiation of the required wavelength. In this way, it is possible to selectively and specifically study the effect of suppression of gene expression under in situ and in vivo conditions.

Claims

1. A construct that binds a target nucleic acid to form a heteroduplex, comprising: the target nucleic acid is an mRNA; The construct comprises two sequence-specific single-stranded oligonucleotides interconnected via a corresponding number of size-specific linking moieties, each of the sequence-specific single-stranded oligonucleotides binds to a target sequence of the target nucleic acid to form a heteroduplex; the sequence-specific single-stranded oligonucleotide comprises 10 to 25 nucleotides; The construct specifically binds to the target nucleic acid, the linking portion is of a length corresponding to the number of nucleotides separating the target sequences in the target nucleic acid, which allows the sequence-specific single-stranded oligonucleotides to bind to their target sequences in the target nucleic acid to form a heteroduplex, and the linking portion is attached to the 5' end of one sequence-specific single-stranded oligonucleotide and the 3' end of another sequence-specific single-stranded oligonucleotide such that the sequence-specific single-stranded oligonucleotides are positioned in such a way that all of the sequence-specific single-stranded oligonucleotides can simultaneously bind to their respective target sequences in the target nucleic acid to form a heteroduplex; the length of the linking moiety is in the range of 20 to 80 angstroms; and the linking moiety is selected from a) an abasic sugar phosphate backbone, an abasic chemically modified sugar phosphate backbone, or a combination thereof; b) a polypeptide; c) polysaccharide; d) saturated or unsaturated hydrocarbons having 2 to 40 carbon atoms; e) non-nucleotide polymers including poly(meth)acrylates, modified poly(meth)acrylates, poly(vinyl alcohol), poly(vinylpyrrolidone), poly(ethylene glycol), poly(acrylamide), poly(oxazoline), poly(ethyleneimine), poly(alkylene oxide), lactone-based polymers, poly(acrylic acid), poly(lactide acid), poly(glycolic acid), poly(propylene), poly(styrene), poly(olefin), poly(amide), poly(cyanoacrylate), poly(imide), poly(ethylene terephthalate), poly(tetramethylene glycol), poly(urethane), or combinations thereof; or f) any combination of a) to e).

2. The construct of claim 1 , wherein the target nucleic acid encodes a fusion protein and is composed of at least two fusion partners and two or more target sequences, each located on a different fusion partner nucleic acid.

3. The construct of claim 2 , wherein the target sequence is located no more than 100 nucleotides from the fusion breakpoint site.

4. The construct of claim 1, wherein the construct comprises at least three sequence-specific single-stranded oligonucleotides interconnected via linking moieties, and each of the sequence-specific single-stranded oligonucleotides binds to a target sequence of the target nucleic acid.

5. The construct of claim 1 , wherein the linking moiety is a polymeric linking moiety.

6. The construct of claim 1 , wherein the modified poly(meth)acrylate comprises poly(ethyleneoxy), 2(N,N-dimethylamino)ethyl (meth)acrylate, or a combination thereof.

7. The construct described in claim 1, wherein the sequence-specific single-stranded oligonucleotide comprises DNA, RNA, a nucleotide derivative, a nucleotide analog, or a combination of two or more types of DNA, RNA, nucleotide derivative, or nucleotide analog.

8. The construct of claim 7, wherein the DNA, RNA, nucleotide derivatives, nucleotide analogs, or combinations of two or more DNAs, RNA, nucleotide derivatives, or nucleotide analogs are combined with each other as blocks of oligonucleotides having specific chemical modifications or as individual oligonucleotides composed of differently modified nucleotides.

9. 8. The construct of claim 7, wherein the sequence-specific single-stranded oligonucleotide comprises DNA, RNA, 2'-O-(2-methoxyethyl)-RNA, 2'-O-methyl-RNA, 2'-fluoro-RNA, LNA, PNA, morpholino, INA, FANA, ANA, UNA, HNA, or a combination thereof.

10. A composition comprising the construct of claim 1 and an inert solvent.

11. A composition comprising the construct of claim 2 and an inert solvent.

12. A pharmaceutical composition comprising the construct of claim 2, wherein the fusion protein is a fusion oncoprotein.

13. A pharmaceutical composition for treating a cancer patient, comprising administering the pharmaceutical composition of claim 12 to a patient in need thereof.

14. The pharmaceutical composition of claim 13, wherein the patient has leukemia.

15. 15. The pharmaceutical composition of claim 14, wherein the leukemia is chronic myelogenous leukemia, acute myelogenous leukemia, or acute lymphoblastic leukemia.

16. The pharmaceutical composition according to claim 12 for killing tumor cells.

17. The pharmaceutical composition of claim 16 , wherein the tumor cells are present in a patient.

18. The pharmaceutical composition of claim 17 , wherein the patient has cancer.

19. The at least two sequence-specific single-stranded oligonucleotides i) SEQ ID NO:8 (cttccttatt gatggtc) and SEQ ID NO:9 (gatgctactg gccgctg); ii) SEQ ID NO:10 (gcccatccac tgtgat) and SEQ ID NO:11 (ggagtgcttc tcagtacg); iii) SEQ ID NO:12 (ccgatgagac ctgtctctat) and SEQ ID NO:13 (gctctcaact tcatt); iv) SEQ ID NO:14 (tcttcagatt tggc), SEQ ID NO:15 (gacaatgatc atgacca), and SEQ ID NO:16 (gaaatgcggc tggact); v) SEQ ID NO:17 (catcatctga ctcttcat), SEQ ID NO:18 (atggacttgt gttcccagt), and SEQ ID NO:19 (cctccagctt gacta); vi) SEQ ID NO:20 (cttgtgtgtg gtggct), SEQ ID NO:21 (atattgggct tggacgc), and SEQ ID NO:22 (gcataactgg aacctg); or vii) SEQ ID NO:23 (tggactcact ggagctct), SEQ ID NO:24 (tcggagtctt cgctgtca), SEQ ID NO:25 (cggtcccggc aatgcaga), and SEQ ID NO:26 (gcggcactag gtttcatg); The construct of claim 1 , comprising:

20. BCR-ABL fusion mRNA in chronic myeloid leukemia, Ph+ acute lymphoblastic leukemia or other tumors in which BCR-ABL fusion mRNA is present; AML1-ETO fusion mRNA in acute myeloid leukemia M2 or other tumors in which AML1-ETO fusion mRNA is present, CBFB-MYH11 fusion mRNA in acute myeloid leukemia M4 or other tumors in which CBFB-MYH11 fusion mRNA is present; RBM15-MKL1 fusion mRNA in acute myeloid leukemia or other tumors in which RBM15-MKL1 fusion mRNA is present; MOZ-CBP fusion mRNA in acute myeloid leukemia or other tumors in which MOZ-CBP fusion mRNA is present; TAF2N-TEC fusion mRNA in myxoid chondrosarcoma or other tumors in which TAF2N-TEC fusion mRNA is present, and The construct according to claim 1 or claim 2, which selectively and specifically recognizes any one selected from the group consisting of mediastinal carcinoma or BRD4-NUT fusion mRNA of other tumors in which BRD4-NUT fusion mRNA is present.

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