RNA-targeting composition and use thereof

Abstract

A composition comprising a first RNA ligand and a second RNA ligand, the first RNA ligand comprising an oligonucleotide capable of specifically targeting a double-stranded section of a target RNA element, and the second RNA ligand comprising an oligonucleotide capable of specifically targeting a single-stranded section of the target RNA element, so that the composition can specifically target a double-strand / single-strand junction of the target RNA. The composition is used for targeting a complex RNA structure, and is expected to become a third generation novel nucleic acid ligand with great prospects for use.

Description

RNA-targeting compositions and uses thereof

[0001] This application claims priority to the Chinese patent application filed with the State Intellectual Property Office on September 1, 2023, with application number 202311137198.3 and application name “Compositions targeting RNA and their uses”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention belongs to the field of biomedicine, and specifically relates to a composition targeting RNA. The present invention also relates to a composition comprising the composition for regulating the function of RNA elements in an organism and / or regulating the expression of RNA elements in an organism, as well as the use of the composition for treating, preventing and / or treating diseases. Background Art

[0003] RNA molecules play a crucial role in all biological processes. The self-folding secondary and tertiary structures of RNA fundamentally regulate gene coding, decoding, splicing, and expression. Recent advances in RNA structure, folding thermodynamics, dynamics, and function have provided a foundation for the development of nucleic acid-based diagnostics, therapeutics, and biotechnologies.

[0004] A variety of RNA molecules play an important regulatory role in both healthy and diseased cells. Although only 1-2% of the human genome encodes proteins, it is now known that most of the genome is transcribed (Carninci et al., Science 309: 1559-1563; 2005). Therefore, non-coding transcripts (non-coding transcriptome) represent a large group of new therapeutic targets. Non-coding RNAs such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) regulate transcription, splicing, mRNA stability / decay and translation. In addition, the non-coding regions of mRNAs, such as 5' untranslated regions (5'UTRs), 3'UTRs and introns, can play a regulatory role in affecting mRNA expression levels, alternative splicing, translation efficiency and mRNA and protein subcellular localization. RNA secondary and tertiary structures are critical for these regulatory activities.

[0005] Notably, GWAS studies have shown that there are many more single nucleotide polymorphisms (SNPs) associated with human diseases in the non-coding transcriptome relative to coding transcripts (Maurano et al., Science 337:1190-1195; 2012). Therefore, therapeutic targeting of non-coding RNAs and non-coding regions of mRNAs may generate novel agents for treating previously intractable human diseases.

[0006] Current therapeutic approaches to block mRNA require methods such as gene therapy, genome editing, or a wide range of oligonucleotide technologies (antisense, RNAi, etc.). Oligonucleotides modulate the action of RNA through typical base / base hybridization. The appeal of this approach is that the basic pharmacophore of the oligonucleotide can be defined in a simple and clear manner by the sequence to be blocked. Many RNA ligands, such as peptide nucleic acids (PNA), antisense oligonucleotides, and interfering RNA have been used in therapy.

[0007] PNA is a synthetic nucleotide analog composed of a polypeptide chain consisting of amino acid residues and nucleotides linked by phosphodiester bonds. The PNA backbone is neutral and unrecognizable by proteases and nucleases, resulting in high stability. Traditional antisense PNA (asPNA) can reverse complementarity with corresponding RNA sequences, forming a PNA-RNA duplex and interfering with the function of the corresponding RNA.

[0008] The major groove of double-stranded RNA (dsRNA) can form a triplex with a third strand (triplex-forming oligonucleotide, TFO) through base pairing. Therefore, the triplex formation strategy is expected to be applied to the development of programmable ligands that bind specifically and tightly to RNA structures. However, due to electrostatic repulsion, the triplex structure formed by traditional TFOs with a negatively charged phosphate backbone is usually unstable, and C + The formation of GC triplexes is strongly pH-dependent. Furthermore, the natural bases C and T can recognize GC and AU base pairs, respectively, but no natural base can recognize CG and UA base pairs. Nucleic acid chemists have conducted extensive chemical modifications of the sugar-phosphate backbone and bases to improve the properties of TFOs. Existing technologies focus on designing dbPNA (double-stranded RNA binding PNA) oligomers that target stable RNA secondary structures and form dbPNA-RNA2 triplexes, thereby affecting the original RNA tertiary structure or protein binding, thereby interfering with its function.

[0009] However, many RNA structures in organisms are composed of both double-stranded RNA and single-stranded RNA (ssRNA), forming complex tertiary structures. Alternatively, double-stranded regions of RNA are unwound by proteins with helicase activity during life, forming complex dsRNA-ssRNA regions. This makes dbPNAs, which specifically bind to double-stranded RNA, unsuitable for design. Furthermore, traditional asPNAs, which require complete base pairing with the corresponding RNA to bind, are unable to fully penetrate and unwrap the complex tertiary structure of RNA, resulting in weak binding between asPNAs and their corresponding sequences, severely limiting their application.

[0010] Therefore, there is a current demand for the development of nucleic acid drugs with better binding capabilities.

[0011] Summary of the Invention

[0012] Therefore, the purpose of the present invention is to provide a composition that can bind to RNA double strands and RNA single strands on the basis of the existing technology. The composition of the present invention can specifically target the double-stranded-single-stranded junction region of the target RNA and has better binding force to complex RNA structures, and therefore has broad application prospects.

[0013] The purpose of the present invention is achieved through the following technical solutions:

[0014] The first aspect of the present invention provides a composition, which comprises a first RNA ligand and a second RNA ligand, wherein the first RNA ligand comprises an oligonucleotide capable of specifically targeting a double-stranded segment of a target RNA element, and the second RNA ligand comprises an oligonucleotide capable of specifically targeting a single-stranded segment of a target RNA element, so that the composition can specifically target the double-stranded-single-stranded interface region of the target RNA element.

[0015] In a preferred embodiment, the first RNA ligand forms a triplex with the double-stranded segment in the RNA structure through non-Watson-Crick pairing.

[0016] In a preferred embodiment, the second RNA ligand forms a duplex with the single-stranded segment in the RNA structure through Watson-Crick pairing.

[0017] In a preferred embodiment, the composition can specifically target the double-stranded-single-stranded interface of the target RNA element, thereby overcoming the defect that the nucleic acid drugs in the prior art cannot target complex RNA structures.

[0018] In a preferred embodiment, the first RNA ligand comprises a chemically modified oligonucleotide, wherein the chemical modification enables the first RNA ligand to selectively bind to double-stranded RNA, and / or

[0019] The chemical modification weakens the binding of the first RNA ligand to the single-stranded RNA.

[0020] In a preferred embodiment, the first RNA ligand and / or the second RNA ligand comprises at least one nucleic acid selected from the group consisting of:

[0021] Peptide nucleic acid (PNA), ribonucleic acid (RNA), locked nucleic acid (LNA), phosphorodiamidate morpholino oligomer (PMO), glycol nucleic acid (GNA), threose nucleic acid (TNA), bridged nucleic acid (BNA).

[0022] In a preferred embodiment, the first RNA ligand and / or the second RNA ligand can be linked to functional molecules with different functions;

[0023] Preferably, the functional molecule is selected from one or more of fluorescent molecules, binding-induced luminescence molecules, PET imaging probes, membrane-penetrating small molecules, targeted delivery molecules, nanocarriers, additional RNA ligands, nanoantibodies and / or protein-binding ligands.

[0024] In a preferred embodiment, the first RNA ligand comprises a chemically modified peptide nucleic acid; and / or

[0025] The second RNA ligand comprises an antisense peptide nucleic acid.

[0026] In a preferred embodiment, the first RNA ligand comprises no less than 3 bases, and / or,

[0027] The second RNA ligand contains no less than 3 bases, and / or,

[0028] The composition comprises no more than 20 bases; and / or

[0029] A linker may or may not be included between the first RNA ligand and the second RNA ligand;

[0030] Preferably, the linker is one or more aminoacetic acid or diaminobutyric acid.

[0031] In a preferred embodiment, the chemically modified peptide nucleic acid is selected from one or more of the following peptide nucleic acids:

[0032] i) A peptide nucleic acid that forms a triplex with GC base pairs as shown below:

[0033] ii) a peptide nucleic acid that forms a triplex with AU base pairs as shown below;

[0034] iii) a peptide nucleic acid that forms a triplex with CG base pairs as shown below; and

[0035] iv) Peptide nucleic acid that forms a triplex with UA base pair as shown below

[0036] In a preferred embodiment, the target RNA is selected from: the target RNA element is selected from one or more of nuclear RNA, cytoplasmic RNA, membrane RNA and extracellular RNA, and the target RNA element comprises a double-stranded segment and a single-stranded segment;

[0037] Preferably, the nuclear RNA or cytoplasmic RNA is selected from non-coding RNA, mRNA, miRNA, enhancer RNA, nascent RNA, chromosome-enriched RNA, ribosomal RNA, membrane-enriched RNA or mitochondrial RNA.

[0038] In a preferred embodiment, the target RNA element is associated with a disease or disorder.

[0039] In a preferred embodiment, the target RNA element comprises an RNA sequence selected from miRNA, novel coronavirus, or human immunodeficiency syndrome virus HIV;

[0040] In a preferred embodiment, the novel coronavirus comprises the pseudoknot structure of the novel coronavirus SARS-CoV-2.

[0041] In a preferred embodiment, the miRNA is miRNA-21.

[0042] In a preferred embodiment, the composition is a peptide nucleic acid oligomer targeting the double-stranded-single-stranded junction region of the SARS-CoV-2 virus pseudoknot structure and has the following structure, wherein the base TTL is the first RNA ligand, the base ACGGGC2 is the second RNA ligand (where 2 is s 2 U):

[0043] In a preferred embodiment, the composition is a composition targeting the double-stranded-single-stranded interface of miRNA-21 and has the following structure, wherein the base TQTTQT is the first RNA ligand and the base CAACAG is the second RNA ligand:

[0044] The second aspect of the present invention provides a nucleic acid drug comprising the aforementioned composition.

[0045] The third aspect of the present invention provides a pharmaceutical composition comprising the aforementioned composition, or the aforementioned nucleic acid drug, and a pharmaceutically acceptable carrier.

[0046] The fourth aspect of the present invention provides the use of the aforementioned composition, the aforementioned nucleic acid drug or the aforementioned pharmaceutical composition in the preparation of a reagent for regulating the function of RNA elements in an organism, wherein

[0047] When the composition, the nucleic acid drug or the pharmaceutical composition is contacted with an organism, the structure and / or expression of the RNA element is changed compared to that observed under reference conditions, thereby causing a change in the biological function of the RNA element;

[0048] The reference condition is selected from the group consisting of: the absence of the composition, the presence of a reference composition, and combinations thereof.

[0049] The fourth aspect of the present invention provides the use of the aforementioned composition, the aforementioned nucleic acid drug or the aforementioned pharmaceutical composition in the preparation of a drug for treating a disease.

[0050] Among them, the disease is associated with functional changes of RNA elements in cells.

[0051] In some preferred embodiments, when the composition, the nucleic acid drug or the pharmaceutical composition is contacted with an organism, the structure and / or expression of the RNA element is altered compared to that observed under reference conditions, thereby causing a change in the biological function of the RNA element;

[0052] The reference condition is selected from the group consisting of: the absence of the composition, the presence of a reference composition, and combinations thereof.

[0053] In some preferred embodiments, the disease is selected from cancer; in some preferred embodiments, the disease is selected from neurodegenerative diseases; in some preferred embodiments, the disease is selected from metabolic disorders; in some preferred embodiments, the disease is selected from inflammatory disorders; in some preferred embodiments, the disease is selected from autoimmune diseases; in some preferred embodiments, the disease is selected from infectious diseases; in some preferred embodiments, the disease is selected from genetic diseases.

[0054] A fifth aspect of the present invention provides a method for regulating the function of an RNA element in an organism and / or regulating the expression of an RNA element in an organism, the method comprising contacting the organism with the aforementioned composition, the aforementioned nucleic acid drug or the aforementioned pharmaceutical composition, wherein

[0055] When the composition, the nucleic acid drug or the pharmaceutical composition is contacted with an organism, the structure and / or expression of the RNA element is changed compared to that observed under reference conditions, thereby causing a change in the biological function of the RNA element;

[0056] The reference condition is selected from the group consisting of: the absence of the composition, the presence of a reference composition, and combinations thereof.

[0057] The sixth aspect of the present invention provides a method for treating a disease in a subject, comprising administering a therapeutically effective amount of the aforementioned composition, the aforementioned nucleic acid drug or the aforementioned pharmaceutical composition to a subject in need thereof.

[0058] In some preferred embodiments, the disease is selected from cancer; in some preferred embodiments, the disease is selected from neurodegenerative diseases; in some preferred embodiments, the disease is selected from metabolic disorders; in some preferred embodiments, the disease is selected from inflammatory disorders; in some preferred embodiments, the disease is selected from autoimmune diseases; in some preferred embodiments, the disease is selected from infectious diseases; in some preferred embodiments, the disease is selected from genetic diseases.

[0059] Prior art focuses on the study of dbPNA-RNA2 triplexes. Building on this foundation, the present invention provides a composition wherein the first RNA ligand of the composition forms a triplex with a double-stranded region of an RNA structure through non-Watson-Crick pairing, while the second RNA ligand can form a duplex with a single-stranded region through Watson-Crick pairing, greatly enhancing the application of the composition in targeting RNA functional elements. More importantly, by overcoming the shortcomings of dbPNA and asPNA and organically combining the advantages of both, the composition of the present invention is expected to become a third-generation novel nucleic acid ligand with enormous application prospects for targeting complex RNA structures.

[0060] The present invention is illustrated by the accompanying drawings and examples, which are not intended to limit the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] In order to more clearly illustrate the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to the accompanying drawings:

[0062] FIG1 is a schematic diagram showing RNA bases that are paired for the modified peptide nucleic acid of the present invention.

[0063] FIG2 shows the compositions daPNA H1S2 and dbPNA P5 designed based on the RNA hairpin structure of rHP1-5t according to an embodiment of the present invention, and the binding results of the compositions with the rHP1-5t hairpin structure and its variants.

[0064] Among them, (a) daPNA H1S2 and dbPNA P5 designed based on rHP1-5t; (b) RNA hairpin structure (5t-Cy3); (c) binding of RNA hairpin and PNA; (d) non-denaturing PAGE data.

[0065] The gels shown are representative gels; the errors given are standard errors.

[0066] FIG3 shows the efficiency of stimulating ribosome frameshifting (PRF) using the composition daPNA H1S2 in combination with the rHP1-5t complex according to an embodiment of the present invention.

[0067] (a) daPNA H1S2, dbPNA P5, and NC designed based on rHP1-5t and their binding to RNA; (b) PRF efficiency of daPNA-H1S2 bound to rHP1-5t.

[0068] FIG. 4 shows the fluorescence intensity results of mCherry and eGFP in a dual fluorescent protein reporter system using the daPNA H1S2 composition combined with the rHP1-5t complex according to an embodiment of the present invention.

[0069] Figure 5 shows a result diagram of the composition daPNA-SFSE-1E-2 targeting the pseudoknot structure of the new coronavirus and its regulation efficiency according to another embodiment of the present invention.

[0070] Among them, (a) a schematic diagram of the designed daPNA binding to the pseudoknot structure of the new coronavirus; (b) the result of daPNA regulating ribosome frameshifting efficiency outside the cell; (c) the antiviral effect of the composition daPNA-SFSE-1E-2 targeting the pseudoknot structure of the new coronavirus.

[0071] The mRNA copy number was calculated based on the CT value using the standard curve formula (Y = -3.7823X + 44.339). Significance testing was performed compared with the liposome control group. ns P > 0.05; *P < 0.1; **P < 0.01; ***P < 0.001. daPNA-SFSE-1E-2 inhibited viral replication in intracellular experiments, with an effective concentration range of 0.1-20 μM.

[0072] FIG6 shows a diagram showing that a composition targeting a microRNA (miRNA) precursor can inhibit biological functions according to another embodiment of the present invention.

[0073] Among them, (a) native acrylamide gel electrophoresis (PAGE) showed that daPNA-1 had a high binding affinity for Cy3-pre-miR-21-59nt; (b) the Kd value of daPNA-1 binding to miRNA precursor (Cy3-pre-miR-21-59nt) was 0.52±0.05μM.

[0074] Incubation buffer: 200 mM NaCl, 0.5 mM EDTA, 20 mM HEPES, pH 7.5. The Cy3-pre-miR-21-59nt concentration in all samples was 80 nM. The Cy3-pre-miR-21-59nt was snap-cooled to form a hairpin structure, which was then slowly cooled to room temperature with PNA at 65°C and incubated overnight at 4°C.

[0075] FIG7 shows a dual luciferase assay result of miRNA-21 precursor and daPNA-1 according to another embodiment of the present invention, showing that daPNA-1 can inhibit the biological function of miRNA-21 in vivo.

[0076] The full-length miRNA-21 precursor (pre-miR-21-59nt) and daPNA-1 were co-transfected into HEK-293T cells (anti-miR-21 was used as a positive control). P values ​​were calculated using a t-test. P values ​​< 0.05 were considered statistically significant. DETAILED DESCRIPTION

[0077] The technical solution of the present invention is described below with reference to specific test examples, but the protection scope of the present invention is not limited thereto.

[0078] The contents described in the test examples of this specification are merely an enumeration of the implementation forms of the inventive concept. The scope of protection of the present invention should not be deemed to be limited to the specific forms described in the test examples. The scope of protection of the present invention also belongs to the equivalent technical means that those skilled in the art can think of based on the inventive concept. Although the embodiments of the present invention are described below, the present invention is not limited to the above-mentioned specific embodiments and application fields. The following specific embodiments are merely illustrative and instructive, and not restrictive. Under the guidance of this specification and without departing from the scope protected by the claims of the present invention, those of ordinary skill in the art can also make many forms, which all fall within the scope of protection of the present invention.

[0079] The practice of the present invention employs many of the conventional techniques of molecular biology. Such techniques are well known and described in, for example, Current Protocols in Molecular Biology, Vols. I, II, and III, 1997 (FM Ausubel, ed.); Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; DNA Cloning: A Practical Approach, Vols. I and II, 1985 (DN Glover, ed.); Oligonucleotide Synthesis, 1984 (ML Gait, ed.); Nucleic Acid Hybridization, 1985, (Hames and Higgins); Transcription and Translation, 1984 (Hames and Higgins, eds.); Animal Cell Culture, 1986 (RI Freshney, ed.); Immobilized Cells and Enzymes, 1986 (IRL Press); Perbal, 1984, A Practical Guide to Molecular Cloning; Methods in Enzymology series (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells, 1987 (JH Miller and MP Calos, eds., Cold Spring Harbor Laboratory); and Methods in Enzymology, Vol. 154 and Vol. 155 (edited by Wu and Grossman, and Wu, respectively).

[0080] PNA: peptide nucleic acid

[0081] asPNA: antisense peptide nucleic acid

[0082] dbPNA: double-stranded peptide nucleic acid

[0083] daPNA: double-stranded binding peptide nucleic acid

[0084] dsRNA: double-stranded RNA

[0085] ssRNA: single-stranded RNA

[0086] miRNA:miRNA

[0087] TFO: triplex forming oligonucleotide

[0088] SARS-CoV-2: The new coronavirus

[0089] daPNA H1S2: a compound designed based on rHP1-5t targeting its double-single strand junction region H1S2

[0090] dbPNA P5: a combination targeting both strands of rHP1-5t designed based on its

[0091] daPNA-SFSE-1E-2: daPNA oligomer targeting the pseudosegment structures stem-1 and stem2 of the SARS-CoV-2 RNA sequence.

[0092] rHP1-5t-Cy3: rHP1-5t RNA labeled with Cy3

[0093] rHP1-5t: RNA hairpin structure, with partial sequence at the 5' end deleted

[0094] rHP1-5m: RNA hairpin structure with partial sequence mutation at the 5' end

[0095] rHP1-5t-U29C-Cy3: Based on rHP1-5t, the base at position 29 was mutated to C

[0096] pre-miR-21-59nt: Precursor microRNA-21, 59nt is the sequence length.

[0097] Cy3-pre-miR-21-59nt: Precursor microRNA-21 labeled with Cy3

[0098] NC: No binding negative control

[0099] mCherry: red fluorescent protein

[0100] eGFP: enhanced green fluorescent protein

[0101] dbPNA: double-stranded RNA-binding peptide nucleic acid

[0102] PRF: programmed ribosomal frameshifting at position 1

[0103] K D :Binding strength parameters

[0104] Target RNA-mRNA

[0105] Within mRNA, noncoding regions can affect the levels of mRNA and protein expression. Briefly, these noncoding regions contain IRES and upstream open reading frames (uORFs) that affect translation efficiency; intron sequences that affect splicing efficiency and alternative splicing patterns; 3′UTR sequences that affect mRNA and protein localization; and particles that control mRNA decay and half-life. Therapeutic modulation of these RNA particles can have beneficial effects.

[0106] The expression of mRNA and its translation product may be affected by the targeted non-coding sequences and structures in 5' and 3' UTR. For example, the RNA structure in 5' UTR can affect translation efficiency. The RNA structure such as hairpin in 5' UTR has been shown to affect translation. In general, RNA structure is considered to play a key role in the translation of mRNA. Two examples of these RNA structures are internal ribosome entry sites (IRES) and upstream open reading frames (uORFs) that can affect the translation level of the main open reading frame. For example, almost half of all human mRNAs have uORFs, and many of them reduce the translation of the main ORF. The compositions of the present invention targeting these RNAs can be used to regulate specific protein levels to obtain therapeutic benefits.

[0107] Target RNA-noncoding RNA

[0108] The largest group of RNA targets is RNA that is transcribed but not translated into protein, referred to as “non-coding RNA”. Non-coding RNA is highly conserved and many types of non-coding RNA play a wide range of regulatory functions. As used herein, the term “non-coding RNA” includes but is not limited to microRNA (miRNA), long non-coding RNA (lncRNA), long intergenic non-coding RNA (lincRNA), Piwi-interacting RNA (piRNA), competitive endogenous RNA (ceRNA) and pseudogenes. These subcategories of non-coding RNA provide a large number of RNA targets with significant therapeutic potential. Therefore, in some embodiments, the present invention provides methods for treating diseases mediated by non-coding RNA. In some embodiments, the disease is caused by miRNA, lncRNA, lincRNA, piRNA, ceRNA or pseudogene. In another aspect, the present invention provides a method for producing a composition of the present invention that regulates the activity of a target RNA-non-coding RNA to treat a disease or condition, comprising the steps of: screening one or more disclosed compositions that bind to the target RNA-non-coding RNA; and analyzing the results by RNA binding analysis disclosed herein. In some embodiments, the target RNA - non-coding RNA is a miRNA, lncRNA, lincRNA, piRNA, ceRNA, or a pseudogene.

[0109] MiRNA is a short double-stranded RNA that regulates gene expression. Each miRNA can affect the expression of multiple human genes. There are nearly 2,000 miRNAs in the human body. These RNAs regulate many biological processes, including cell differentiation, cell fate, movement, survival and function. MiRNA expression levels are different between different tissues, cell types and disease backgrounds. Compared with normal tissues, it is often abnormally expressed in tumors, and its activity can play an important role in cancer. MiRNA has been shown to regulate oncogenes and tumor suppressors, and it itself can act as an oncogene or tumor suppressor. Some miRNAs have been shown to promote epithelial-mesenchymal transition (EMT) and cancer cell invasion and metastasis. In the case of oncogenic miRNA, its inhibition can be an effective anticancer treatment.

[0110] Thus, in one aspect, the present invention provides a method for producing a composition of the present invention that modulates the activity of a target miRNA to treat a disease or condition, comprising the steps of: screening one or more disclosed compositions that bind to the target miRNA precursor; and analyzing the results by the target RNA binding assay disclosed herein. In some embodiments, the miRNA modulates or acts as an oncogene or tumor suppressor. In some embodiments, the disease is cancer. In some embodiments, the cancer is a solid tumor.

[0111] There are multiple oncogenic miRNAs that can be targeted therapeutically, including miR-155, miR-17-92, miR-19, miR-21, and miR-10b. MiR-155 plays a pathological role in inflammation, hypertension, heart failure, and cancer. In cancer, miR-155 triggers oncogenic cascades and resistance to apoptosis, as well as increases cancer cell invasiveness. Altered expression of miR-155 has been described in a variety of cancers, reflecting stage, progression, and treatment outcome. Cancers reported to overexpress miR-155 are breast, thyroid, colon, cervix, and lung cancer. It has been reported to play a role in drug resistance in breast cancer. MiR-17-92 (also known as Oncomir-1) is a polycistronic 1 kb primary transcript that includes miR-17, 20a, 18a, 19a, 92-1, and 19b-1. It is activated by MYC. MiR-19 alters gene expression and signal transduction pathways in a variety of hematopoietic cells, and it triggers leukemogenesis and lymphomagenesis. It is involved in a wide variety of human solid tumors and blood cancers. MiR-21 is an oncogenic miRNA that reduces the expression of multiple tumor suppressors. It stimulates cancer cell invasion and is associated with a wide variety of human cancers, including breast cancer, ovarian cancer, cervical cancer, colon cancer, lung cancer, liver cancer, brain cancer, esophageal cancer, prostate cancer, pancreatic cancer, and thyroid cancer. Therefore, in some embodiments of the methods described above, the target miRNA is selected from miR-155, miR-17-92, miR-19, miR-21, or miR-10b. In some embodiments, the disease or condition is a cancer selected from the following: breast cancer, ovarian cancer, cervical cancer, thyroid cancer, colon cancer, liver cancer, brain cancer, esophageal cancer, prostate cancer, lung cancer, leukemia, or lymph node cancer. In some embodiments, the cancer is a solid tumor.

[0112] In addition to oncology, miRNAs play a role in many other diseases, including cardiovascular and metabolic diseases.

[0113] lncRNAs are RNAs with more than 200 bases (nt) that do not encode proteins. They can affect the expression of protein-coding mRNAs at the levels of transcription, splicing, and mRNA decay. Numerous studies have shown that lncRNAs can regulate transcription by recruiting epigenetic regulators that increase or decrease transcription by altering chromatin structure (e.g., Holoch and Moazed, Nature Reviews Genetics 16:71-84, 2015). lncRNAs have been implicated in human diseases including cancer, inflammatory diseases, neurological diseases, and cardiovascular diseases (e.g., Presner and Chinnaiyan, Cancer Discovery 1:391–407, 2011; Johnson, Neurobiology of Disease 46:245–254, 2012; Gutscher and Diederichs, RNA Biology 9:703–719, 2012; Kumar et al., PLoS Genetics 9:e1003201, 2013; van de Vondervoort et al., Frontiers in Molecular Neuroscience 9:e1003201, 2014). Neuroscience, 2013; Li et al., International Journal of Molecular Sciences (Int. J. Mol. Sci.) 14: 18790-18808, 2013). lncRNAs can be targeted to upregulate or downregulate the expression of specific genes and proteins for therapeutic benefit (e.g., Wahlestedt, Nature Reviews Drug Discovery 12: 433-446, 2013; Guil and Esteller, Nature Structural & Molecular Biology 19: 1068-1075, 2012). In general, lncRNAs are expressed at lower levels relative to mRNAs. Many lncRNAs are physically associated with chromatin (Werner et al., Cell Reports 12, 1-10, 2015) and are transcribed very close to protein-coding genes. They often remain physically associated at their transcription sites and act locally in cis to regulate the expression of adjacent mRNAs. Mutations and dysregulation of lncRNAs are associated with human diseases; therefore, there are numerous lncRNAs that could be therapeutic targets. Thus, in some embodiments of the methods described above, the target RNA—noncoding RNA—is a lncRNA.In some embodiments, the lncRNA is associated with cancer, inflammatory disease, neurological disease, or cardiovascular disease.

[0114] lncRNAs regulate the expression of protein-coding genes, acting at various levels to influence transcription, alternative splicing, and mRNA decay. For example, lncRNAs have been shown to bind to the epigenetic regulator PRC2 to promote its recruitment to genes, where it subsequently represses transcription through chromatin modifications. lncRNAs can form complex structures that mediate their association with various regulatory proteins. Compositions of the present invention that bind to these lncRNA structures can be used to modulate the expression of genes that are normally regulated by individual lncRNAs.

[0115] An exemplary target lncRNA is HOTAIR, a lncRNA expressed by the HoxC locus on human chromosome 12. Its expression level is low (about 100 RNA copies / cell). Unlike many lncRNAs, HOTAIR can act in trans to affect the expression of distal genes. It binds to the epigenetic repressor PRC2 and the LSD1 / CoREST / REST complex, another inhibitory epigenetic regulator (Tsai et al., Science 329, 689-693, 2010). HOTAIR is a highly structured RNA with more than 50% of its nucleotides involved in base pairing. It is often dysregulated (often upregulated) in various types of cancer (Yao et al., International Journal of Molecular Sciences 15: 18985-18999, 2014; Deng et al., Public Library of Science Comprehensive (PLOS One) 9: e110059, 2014). Compared with those patients with low expression levels, cancer patients with high HOTAIR expression levels have significantly worse prognosis. HOTAIR is reported to be involved in the control of apoptosis, proliferation, metastasis, angiogenesis, DNA repair, chemoresistance and tumor cell metabolism. It is highly expressed in metastatic breast cancer. High expression levels in primary breast tumors are a significant predictor of subsequent metastasis and death. HOTAIR has also been reported to be associated with esophageal squamous cell carcinoma and is a prognostic factor in colorectal cancer, cervical cancer, gastric cancer and endometrial cancer. Therefore, the composition of the present invention combined with HOTAIR is a novel anticancer drug candidate. Therefore, in some embodiments of the method described above, the target RNA-noncoding RNA is HOTAIR. In some embodiments, the disease or condition is breast cancer, esophageal squamous cell carcinoma, colorectal cancer, cervical cancer, gastric cancer or endometrial cancer.

[0116] Another potential cancer target in lncRNA is MALAT-1 (metastasis-associated lung adenocarcinoma transcript 1), also known as NEAT2 (nucleus-enriched abundant transcript 2) (Gutschner et al., Cancer Research 73:1180-1189, 2013; Brown et al., Nature Structure & Mol Biol 21:633-640, 2014). It is a highly conserved 7kb nuclear lncRNA located in nuclear speckles. It is widely expressed in normal tissues but is upregulated in many cancers. MALAT-1 is a predictive marker for metastasis development in a variety of cancers including lung cancer. It appears to act as a regulatory factor for gene expression, potentially affecting transcription and / or splicing. MALAT-1 gene knockout mice do not have a phenotype, indicating that it has limited normal function. However, cancer cell migration lacking MALAT-1 is weakened and forms fewer tumors in mouse xenograft tumor models. Block the antisense oligonucleotide (ASO) of MALAT-1 and stop metastasis formation after tumor implantation in mice.Some mouse xenograft tumor model data show that the MALAT-1 gene knockout carried out by ASO can suppress both primary tumor growth and metastasis.Therefore, it is expected that the compositions of the present invention of targeting MALAT-1 can effectively suppress tumor growth and metastasis.Therefore, in some embodiments of method described above, target RNA-noncoding RNA is MALAT-1.In certain embodiments, disease or illness are the cancer that MALAT-1 raises, as lung cancer.

[0117] In some embodiments, the present invention provides a method for treating a disease or condition mediated by a non-coding RNA (e.g., HOTAIR or MALAT-1), comprising the steps of administering a composition of the present invention to a patient in need thereof. Such compositions are described in detail herein.

[0118] Target RNA-toxic RNA (repetitive sequence RNA)

[0119] Simple repeats in mRNA are often associated with human diseases. They are usually (but not exclusively) repeats with three bases, such as CAG ("triplet repeats"). Triplet repeats are abundant in the human genome, and they tend to undergo several generations of amplification. Roughly 40 human diseases are associated with the amplification of repeats. Diseases caused by triple amplification are called triplet repeat expansion diseases (TRED). Healthy individuals have a variable number of triplet repeats, but there is a threshold, and the number of higher repeats exceeding the threshold can cause disease. The threshold is different for different diseases. Triplet repeats may be unstable. When a gene is inherited, the number of repeats may increase, and the symptoms may become more severe or occur earlier from one generation to the next. When an individual has a variety of repeats in the normal range, it is expected that they will not amplify when passed to the next generation. When the number of repeats is in the premutation range (normal but unstable number of repeats), then the repeats may or may not amplify when passed to the next generation. Normal individuals with a premutation do not have the disease, but are at risk of having children who inherit a triplet repeat within the full mutation range and who will be affected. TREDs can be autosomal dominant, autosomal recessive, or X-linked. The more common triplet repeat disorders are autosomal dominant.

[0120] The repetitive sequence may be in the coding or non-coding portion of the mRNA. In the case where the repetitive sequence is within the non-coding region, the repetitive sequence may be located in the 5'UTR, intron or 3'UTR sequence.

[0121] The toxicity produced by the repetitive sequence can be a direct result of the effect of the toxic RNA itself, or in the case where the repetitive sequence expansion is in the coding sequence, it is due to the toxicity of RNA and / or abnormal proteins. Repetitive sequence expansion RNA can work by sequestering key RNA binding proteins (RBPs) into the lesion. An example of a sequestered RBP is the muscleblind family protein MBNL1. Sequestration of RBP leads to splicing defects and nucleus-cytoplasm transport defects of RNA and protein. Sequestration of RBP can also affect miRNA biosynthesis. These disturbances in RNA biology can greatly affect neuronal function and survival, leading to various neurological diseases.

[0122] The present invention encompasses a method for treating a disease or condition in which the abnormal RNA itself causes the pathogenic effect, rather than acting through mechanisms of protein expression or regulation of protein expression. In some embodiments, the disease or condition is mediated by repetitive sequence RNA. In some embodiments, the disease or condition is a repetitive sequence expansion disease in which the repetitive sequence is present in the coding region of an mRNA.

[0123] In some embodiments, the present invention provides a method for treating a disease or condition mediated by repetitive sequence RNA, comprising the steps of administering a composition of the present invention to a patient in need thereof. Such compositions are described in detail herein.

[0124] Also provided is a method for producing a small molecule that modulates the activity of a target repeat expansion RNA to treat a disease or condition, comprising the steps of: screening one or more disclosed compositions for binding to the target repeat expansion RNA; and analyzing the results using an RNA binding assay disclosed herein. In some embodiments, the repeat expansion RNA causes a disease or condition selected from HD, DRPLA, SBMA, SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17. In some embodiments, the disease or condition is selected from Fragile X syndrome, DM1, FRDA, SCA8, SCA10, SCA12, or C9FTD.

[0125] triple helical polynucleotide

[0126] The major groove of the double-stranded RNA (dsRNA) structure can form a triplex through base pairing with the third strand (triplex-forming oligonucleotide, TFO), that is, "triple helix" base pairing, which can inhibit gene expression.

[0127] Preparation method

[0128] All polynucleotides of the present invention can be prepared using any known technology for synthesizing nucleic acid molecules in the art, including antisense molecules, triple helix DNA, RNA ligands, and ribozymes. They include the technology of chemically synthesizing oligonucleotides, such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be prepared by transcribing the DNA sequence encoding the polypeptide gene described herein in vitro and in vivo. Such DNA sequence can be incorporated into a variety of vectors with an appropriate RNA polymerase promoter (such as T7 or SP6). Alternatively, a cDNA construct of a constitutive or inducible synthetic antisense RNA can be introduced into a cell line, cell, or tissue.

[0129] These compositions can be produced by those skilled in the art using conventional techniques without undue effort or experimentation. All regions of a gene can be used to design antisense molecules to produce antisense molecules with the strongest hybridization to mRNA, and such suitable antisense oligonucleotides can be prepared and identified by standard experimental methods well known to those skilled in the art.

[0130] PNA

[0131] As used herein, the term "peptide nucleic acid" or "PNA" refers to a nucleic acid mimetic, such as a DNA mimetic, in which the deoxyribonucleic acid backbone is replaced with a pseudopeptide backbone in which 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbones, retaining only the four natural nucleobases. The neutral backbone of PNA has been shown to hybridize specifically to DNA and RNA under conditions of low ionic strength. Synthesis of PNA oligomers can be achieved using standard solid-phase peptide synthesis protocols, as described in Hyrup et al. (1996); Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670-675, supra.

[0132] PNAs can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents to sequence-specifically modulate gene expression by, for example, inducing transcriptional or translational arrest or inhibiting replication.

[0133] Modified PNA

[0134] The present invention provides modified PNAs. In some embodiments, the bases described herein are typically chemically modified on the surface binding to the major groove. Exemplary and non-limiting modifications include amino, sulfhydryl, alkyl, halogen groups, or any modification described herein. The modified bases can be synthesized by any available method as described herein (e.g., by chemical, enzymatic, or recombinant methods to include one or more modified or non-natural bases).

[0135] Modified base pairing includes not only standard adenosine-thymine, adenosine-uracil or guanosine-cytosine base pairs, but also base pairs formed between nucleotides containing non-standard or modified bases and / or modified nucleotides, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures. An example of such non-standard base pairing is base pairing between the modified nucleotides inosine and adenine, cytosine or uracil.

[0136] The base and basic acid of modification can comprise the core base of modification.The example of the core base found in RNA includes but is not limited to adenine, guanine, cytosine and uracil.The example of the core base found in DNA includes but is not limited to adenine, guanine, cytosine and thymine.These core bases can be modified or replaced completely, to provide the polynucleotide molecule with enhanced properties (for example, resistance to nuclease, stability), and these properties can be shown by the combination of interfering major groove binding partners.For example, the described base can be chemically modified on the major groove binding surface.In some embodiments, the chemical modification of major groove can include amino, sulfhydryl, alkyl or halogen groups.

[0137] In some embodiments, the chemically modified peptide nucleic acid is selected from one or more of the following peptide nucleic acids:

[0138] i) A peptide nucleic acid that forms a triplex with GC base pairs as shown below:

[0139] Among them, C + : Protonated cytosine

[0140] M + :Protonated 2-aminopyridine

[0141] J: pseudoisocytosine

[0142] L: thiopseudoisocytosine

[0143] R:simple guanidinium moiety

[0144] M1: 2-aminopyridine-1

[0145] M2: 2-aminopyridine-2

[0146] M3: 2-aminopyridine-3

[0147] M4: 2-aminopyridine-4

[0148] ii) a peptide nucleic acid that forms a triplex with AU base pairs as shown below;

[0149] T: thymine

[0150] x U: Halouracil

[0151] s2U: 2-thiouracil

[0152] bt U:5-Benzothiopheneuracil

[0153] T1: Thymine-1

[0154] Tr:thymine-R

[0155] iii) a peptide nucleic acid that forms a triplex with CG base pairs as shown below; and

[0156] iC: methylated cytosine

[0157] P:2-pyrimidinone

[0158] Q: Guanidine-modified cytosine

[0159] M2:2-aminopyridine-2

[0160] M3R:2-aminopyridine-3R

[0161] iv) Peptide nucleic acid that forms a triplex with UA base pair as shown below

[0162] E:3-oxo-2,3-hydropyridine

[0163] S: Sulfur.

[0164] hairpin

[0165] The term "hairpin" is used to describe a polynucleotide sequence in which two regions of the same chain are reverse complementary to each other in terms of nucleotide sequence, resulting in intramolecular base pairing to form a double-stranded region and an unpaired loop. The term is used herein to describe DNA sequences encoding such structures, although DNA is typically double-stranded through intermolecular base pairing. The term is also used to refer to RNA sequences that adopt a hairpin structure.

[0166] The terms "polybasic acid," "oligonucleotide," "nucleic acid," and "nucleic acid molecule" are used interchangeably to refer to a polymeric form of nucleotides of any length and may include ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. The term refers only to the primary structure of the molecule. Thus, the term includes triple-stranded, double-stranded, and single-stranded deoxyribonucleic acids ("DNA"), and triple-stranded, double-stranded, and single-stranded ribonucleic acids ("RNA"). It also includes modified, for example, by alkylation and / or by capping, as well as unmodified forms of polynucleotides. More specifically, the terms "polynucleotide," "oligonucleotide," "nucleic acid," and "nucleic acid molecule" include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA, and mRNA, whether spliced ​​or unspliced, any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing non-nucleotide backbones, such as polyamides [e.g., peptide nucleic acids ("PNA")] and polymorpholines (commercially available as Neugene from Anti-Virals, Inc., Corvallis, Oreg.), as well as other synthetic, sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in a configuration that allows base pairing and base stacking, such as found in DNA and RNA.

[0167] Pharmaceutical composition

[0168] The pharmaceutical compositions disclosed herein are administered to patients at a therapeutically effective dose for the prevention, treatment, or amelioration of pathological diseases associated with functional changes in RNA elements. A therapeutically effective dose refers to an amount of the compound sufficient to cause substantial inhibition, treatment, or amelioration of the occurrence of the disease. Administration "in combination" with one or more other therapeutic agents includes simultaneous (coexisting) and sequential administration in any order.

[0169] The compositions or nucleic acid drugs of the present invention comprising a first RNA ligand and a second RNA ligand are referred to herein as “active compounds.” These compositions or nucleic acid drugs can be incorporated into pharmaceutical compositions suitable for administration to a patient in need thereof.

[0170] As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in textbooks such as Remington's Pharmaceutical Sciences, Gennaro AR ed. 20th ed. (2000) Williams & Wilkins PA, USA and Wilson and Gisvold's Textbook of Organic Medicinal and Pharmaceutical Chemistry, Delgado and Remers, Lippincott-Raven, each of which is incorporated herein by reference. Preferred examples of components that can be used in these carriers or diluents include, but are not limited to, water, saline, phosphates, carboxylates, amino acid solutions, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers such as fixed oils may also be used.

[0171] The compositions of the present invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can include antisense polynucleotides and inhibitory polynucleotides, including microRNA (miRNA), modified miRNA, small inhibitory RNA (siRNA), and modified siRNA, wherein modifications are introduced as described herein to at least confer stability to the molecules.

[0172] The gene therapy vector can be delivered to a subject by any of a number of routes, such as described in U.S. Patent No. 5,703,055, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it enters the cell, such as by infection with a defective or attenuated retrovirus or other expression vector (see, e.g., U.S. Patent No. 4,980,286 and other patents mentioned below), or by direct injection of naked DNA, or by using microparticle bombardment (e.g., a gene gun; Biolistics, Dupont), or by coating with lipids or cell surface receptors or transfection agents, encapsulation in liposomes, microparticles or microcapsules, or by administering it in conjunction with a peptide known to enter the nucleus, by administering it in conjunction with a ligand that undergoes receptor-mediated endocytosis (see, e.g., U.S. Patent Nos. 5,166,320; 5,728,399; 5,874,297 and 6,030,954, all of which are incorporated herein by reference in their entireties) (which can be used to target cell types that specifically express the receptor), etc. Thus, delivery can include, for example, intravenous injection, topical administration (see U.S. Pat. No. 5,328,470), or stereotactic injection (see Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3054-3057). Pharmaceutical preparations of gene therapy vectors can include the gene therapy vector in an acceptable diluent, or can comprise a sustained-release matrix in which the gene delivery vector is embedded. Alternatively, when recombinant cells can produce the complete gene delivery vector (e.g., retroviral vectors), the pharmaceutical preparation can comprise one or more cells that produce the gene delivery system.

[0173] In an alternative embodiment, retroviral vectors can be used (see, e.g., U.S. Patent Nos. 5,219,740, 5,604,090, and 5,834,182). These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging the viral genome and integrating into host cell DNA. The TORC nucleic acid to be used for gene therapy is cloned into a vector that facilitates gene delivery to the patient.

[0174] Adenovirus is another viral vector that can be used in gene therapy. Adenovirus is a particularly advantageous vector for delivering genes to the respiratory epithelium. Adenovirus naturally infects the respiratory epithelium, causing mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenovirus has the advantage of being able to infect non-dividing cells. Methods for performing adenovirus-based gene therapy are described, for example, in U.S. Patent Nos. 5,824,544, 5,868,040, 5,871,722, 5,880,102, 5,882,877, 5,885,808, 5,932,210, 5,981,225, 5,994,106, 5,994,132, 5,994,134, 6,001,557, and 6,033,8843, all of which are incorporated herein by reference in their entirety.

[0175] Adeno-associated virus (AAV) has also been proposed for use in gene therapy. Methods for producing and using AAV are described in, for example, U.S. Patent Nos. 5,173,414, 5,252,479, 5,552,311, 5,658,785, 5,763,416, 5,773,289, 5,843,742, 5,869,040, 5,942,496, and 5,948,675, all of which are incorporated herein by reference in their entirety.

[0176] Another approach to gene therapy involves transferring genes into cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated transfection, or viral infection. The transfer method typically involves transferring a selectable marker into the cells. The cells are then placed under selective pressure to isolate cells that have taken up and expressed the transferred gene. These cells are then delivered to the patient. In a preferred embodiment, the cells used for gene therapy are the patient's own cells.

[0177] The dosage and desired drug concentration of the pharmaceutical compositions of the present invention may vary depending on the specific application. Determining the appropriate dosage or route of administration is within the capabilities of the ordinary physician. Animal studies provide reliable guidance for determining effective dosages for human therapy. Interspecies scaling of effective dosages can be performed according to the principles set forth in Mordenti, J. and Chappell, W., "The use of interspecies scaling in toxicokinetics," Toxicokinetics and New Drug Development, ed. Yacobi et al., Pergamon Press, New York, 1989, pp. 42-96.

[0178] Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. Determination of an effective amount is within the capabilities of those skilled in the art.

[0179] For any compound, the therapeutically effective dose can first be estimated in a cell culture assay (such as cancer cells) or an animal model (typically mice, rabbits, dogs or pigs). Animal models can also be used to determine appropriate concentration ranges and routes of administration. Doses can be formulated in animal models to obtain a circulating plasma concentration range that includes the IC50 (i.e., the concentration at which the test compound achieves half-maximal inhibition of symptoms). This information can then be used to determine the dosage and route of administration for human administration.

[0180] A therapeutically effective dose refers to the amount of the active ingredient used to prevent, treat, or ameliorate a specific pathological disease of interest. Effective doses and toxicity, such as ED50 (the dose that has therapeutic effect in 50% of the population) and LD50 (the dose that is lethal to 50% of the population), can be determined by standard procedures in cell cultures or experimental animals. The dose ratio between toxic effects and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Pharmaceutical compositions that exhibit a high therapeutic index are preferred. Data from cell culture assays and animal studies are used to formulate dosage ranges for use in humans. The dosage contained in these compositions is preferably within a circulating concentration range that includes the ED50 with little or no toxicity. Depending on the dosage form used, the patient's sensitivity, and the route of administration, the dosage can vary within this range.

[0181] The specific dosage can be determined by the practitioner taking into account the relevant factors of the subject in need of treatment. The dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors that may be considered include the severity of the disease state, the overall health of the subject, the subject's age, weight and sex, diet, administration time and frequency, drug combination, reaction sensitivity and tolerance / responsiveness to therapy. The long-acting pharmaceutical composition can be administered every 3 to 4 days, weekly or once every two weeks, depending on the half-life and clearance rate of the specific formulation.

[0182] Depending on the route of administration, normal dosages may vary from 0.1 to 100,000 micrograms, up to about 1 g. Guidance on specific dosages and delivery methods is provided in the literature and is generally available to practitioners in the field. For bases, one skilled in the art will use different dosage forms than for proteins or their inhibitors. Similarly, delivery of polybases or polypeptides will require specific considerations for specific cells, conditions, locations, etc.

[0183] Therapeutically effective dose

[0184] As used herein, "therapeutically effective amount" refers to an amount of a drug sufficient to treat or ameliorate the pathological effects of a relevant disorder. For example, a therapeutically effective amount of a composition sufficient to treat or ameliorate the pathological effects of a relevant disorder is an amount sufficient to produce the desired change in the function of the target RNA element.

[0185] Example

[0186] The experimental methods in the following examples without specifying specific conditions were carried out according to conventional methods and conditions, or according to the product instructions.

[0187] The reagents, cell lines, and instruments used in the present invention are shown in the following table.

[0188] 1. Preparation of the composition of the present invention

[0189] In this embodiment, the composition daPNA targets the double-stranded-single-stranded junction of the target RNA element, and its preparation and detection methods include the following steps:

[0190] 1) Based on the dbPNA and asPNA monomers, daPNA RNA-binding functional elements were designed. Each monomer was linked to a glycine backbone via solid-phase synthesis to form a daPNA oligomer. The dbPNA monomers that can be used are shown in Figure 1.

[0191] 2) After HPLC purification and mass spectrometry identification, the binding affinity between daPNA and target RNA was determined by native polyacrylamide gel electrophoresis.

[0192] 3) Using the viral ribosome frameshift mechanism as a model, the biological activity of daPNA, namely its promoting effect on ribosome frameshift efficiency, was analyzed using a cell-free translation system.

[0193] 4) Deliver daPNA into cells via liposome delivery, and evaluate the biological function of daPNA in cells using dual fluorescent proteins.

[0194] According to the above method, P5 and H1S1 were designed based on the sequence and hairpin structure of rHP1, as shown in Figure 2(a). The sequence of rHP1 is shown in SEQ ID NO: 1.

[0195] rHP15'-3' (SEQ ID NO: 1)

[0196] Based on the sequence of the pseudoknot structure of SARS-CoV-2 (as shown in Figure 5(a)), daPNA-SFSE-1E-2 was designed. Partial sequences of SARS-CoV-2 are shown in SEQ ID NOs: 2-4.

[0197] FSE-SARS-CoV-2 (SEQ ID NO: 2)

[0198] 5'UTR-SARS-CoV-2 (SEQ ID NO: 3)

[0199] 3'UTR-SARS-CoV-2 (SEQ ID NO:4)

[0200] daPNA-1 was designed based on miRNA-21, wherein the partial sequence of miRNA-21 is shown in SEQ ID NO:5.

[0201] The compositions used in this application are shown in the following table, wherein the first RNA ligand is shown in bold and the second RNA ligand is shown in normal font.

[0202] P5

[0203] H1S2

[0204] daPNA-NC

[0205] daPNA-SFSE-1E-2

[0206] daPNA-1

[0207] 2. Activity Assay of daPNA Targeting the Hairpin Structure of rHP1-5t

[0208] The stability and PRF regulation efficiency of daPNA-H1S2, designed to bind to the rHP1-5t RNA hairpin structure, were determined.

[0209] First, the binding affinity of daPNA-H1S2 to RNA rHP1-5t was analyzed by polyacrylamide gel electrophoresis. D The results showed that daPNA-H1S2 significantly enhanced the PRF efficiency, which was approximately 4±0.2nM.

[0210] FIG2 shows the target of the composition daPNA H1S2 and dbPNA P5 designed based on the RNA hairpin structure of rHP1-5t, and the binding results of the composition to the rHP1-5t hairpin structure and its variants;

[0211] Among them, (a) daPNA H1S2 and dbPNA P5 designed based on rHP1-5t; (b) RNA hairpin structure (5t-Cy3); (c) binding of RNA hairpin and PNA; (d) non-denaturing PAGE data.

[0212] The gels shown are representative gels; the errors given are standard errors.

[0213] FIG3 shows the efficiency of stimulating PRF using the combination of daPNA H1S2 and rHP1-5t complex;

[0214] (a) daPNA H1S2, dbPNA P5, and NC designed based on rHP1-5t and their binding to RNA; (b) PRF efficiency of daPNA-H1S2 bound to rHP1-5t. Extracellular luciferase assays demonstrated that at a PNA concentration of 200 nM, daPNA-H1S2 significantly promoted the frameshift efficiency of rHP1-5m to 14.8±1.6%, while the effect on rHP1-5m-U29C was significantly reduced to 4.5±0.2%. NC had no effect on the frameshift efficiency of rHP1-5m. P5, on the other hand, promoted the frameshift efficiency of rHP1-5m and rHP1-5m-U29C to 3.3±0.9% and 4.5±1.5%, respectively.

[0215] Figure 4 shows the mCherry and eGFP fluorescence intensities of a dual fluorescent protein reporter system using the daPNA H1S2 combination combined with the rHP1-5t complex. The mCherry protein in the dual fluorescent protein reporter system is a -1 coding frame protein produced after ribosomal frameshifting. Addition of 10 μM H1S2 significantly promoted mCherry expression in rHP1-5m. H1S2 expression can bind to the rHP1-5 mRNA structure within cells and promote ribosomal frameshifting efficiency. However, no significant enhancement was observed in the P5 and NC groups. Furthermore, 10 μM H1S2 failed to promote expression of rHP1-5m-U29C mCherry.

[0216] 3. Activity Assay of daPNA Targeting the SARS-CoV-2 Pseudoknot Structure

[0217] The daPNA-SFSE-1E designed to target the pseudoknot structure of the new coronavirus RNA can bind to the corresponding RNA, K D The value was approximately 80±12nM. As verified by extracellular luciferase, daPNA-SFSE-1E strongly inhibited the PRF efficiency of the new coronavirus. The control group had no significant effect. PNA was transfected into ACE2-HEK-293T cells via liposome delivery and was found to significantly inhibit the replication of the new coronavirus mRNA.

[0218] 3.1daPNA regulates ribosome frameshifting efficiency outside the cell

[0219] A series of daPNAs were designed targeting the pseudoknot structure of the new coronavirus to inhibit the efficiency of ribosome frameshifting by intervening in the formation of the pseudoknot structure.

[0220] The results show that daPNA-SFSE-1E-2 has a significant inhibitory effect on the ribosome frameshift of the new coronavirus.

[0221] Among them, Figure 5 shows the result of the composition daPNA-SFSE-1E-2 targeting the pseudoknot structure of the new coronavirus and its regulation of frameshift efficiency, wherein

[0222] (a) Schematic diagram of the designed daPNA binding to the pseudoknot structure of the new coronavirus. (b) Results of daPNA regulating ribosome frameshifting efficiency outside the cell; (c) Antiviral effect of the composition daPNA-SFSE-1E-2 targeting the pseudoknot structure of the new coronavirus;

[0223] The mRNA copy number was calculated based on the CT value using the standard curve formula (Y = -3.7823X + 44.339). Significance testing was performed compared with the liposome control group. ns P > 0.05; *P < 0.1; **P < 0.01; ***P < 0.001. daPNA-SFSE-1E-2 inhibited viral replication in intracellular experiments, with an effective concentration range of 0.1-20 μM.

[0224] 3.2daPNA can inhibit the SARS-CoV-2 PRF efficiency and mRNA replication

[0225] PNA was transfected into ACE2 HEK-293T cells via liposome delivery. Six hours after transfection, the novel coronavirus (COVID-19) was inoculated (MOI = 0.1). Forty-eight hours after infection, cell supernatants were collected, and total RNA was extracted. Real-time quantitative PCR was used to detect the mRNA copy number of the novel coronavirus N gene. The results showed that daPNA-SFSE-1E-2 significantly inhibited the replication of the novel coronavirus, with 2E being the most effective.

[0226] Figure 5(c) shows the SARS-CoV-2 PRF inhibition efficiency and antiviral activity of daPNA-SFSE-1E-2. mRNA copy number was calculated based on the CT value using the standard curve formula (Y = -3.7823X + 44.339). Significance testing was performed compared with the liposome control group. ns P > 0.05; *P < 0.1; **P < 0.01; ***P < 0.001. daPNA-SFSE-1E-2 inhibited viral replication in intracellular experiments.

[0227] 3.3daPNA can inhibit the biological function of microRNA

[0228] A series of daPNAs were designed for targeting microRNA (miRNA), and the targeting effect was successfully verified in vivo and in vitro. Taking miRNA-21 as an example, the binding of daPNA to miRNA-21 precursor was verified in vitro by non-denaturing gel electrophoresis, and its K D value.

[0229] The Cy3-labeled miR-21 precursor (Cy3-pre-miR-21-59nt) was snap-cooled to form a hairpin structure. The structure was then slowly cooled from 65°C to room temperature with a gradient of PNA concentrations and incubated overnight at 4°C. The samples were subjected to native acrylamide gel electrophoresis (PAGE) at 400V for 5 hours to visualize the binding of daPNA to the miRNA precursor. The results are shown in Figure 6.

[0230] Figure 6. Non-denaturing acrylamide gel electrophoresis (PAGE) shows that daPNA-1 can bind to miRNA precursor (Cy3-pre-miR-21-59nt). The results show that daPNA-1 has a high binding affinity for Cy3-pre-miR-21-59nt (A), with a K D The value was 0.52±0.05μM (B).

[0231] Incubation buffer: 200 mM NaCl, 0.5 mM EDTA, 20 mM HEPES, pH 7.5. The concentration of Cy3-pre-miR-21-59nt in all samples was 80 nM.

[0232] HEK293-T cells were seeded at 10,000 cells / well into an opaque 96-well plate. After culturing for 24 hours in an incubator (37°C, 5% CO2), the full-length miRNA-21 precursor (pre-miR-21-59nt) and gradient concentrations of daPNA-1 were co-transfected into HEK-293T cells (anti-miR-21 was used as a positive control). After another 24 hours of culture, the fluorescence intensity of each well was measured using a microplate reader. The ratio of the intensity of firefly luciferase (Firefly) to that of Renilla luciferase (Renilla) represents the expression level of the target protein under miR-21 inhibition. The results are shown in Figure 7.

[0233] Figure 7 Dual luciferase assay results of miRNA-21 and daPNA-1 show that daPNA-1 can inhibit the biological function of miRNA-21 in vivo. After miR-21 inhibited the expression level of the target protein to approximately 50% of the normal level, both anti-miR-21 (SEQ ID NO: 65'-ucaacaucagucugauaagcua-3') and daPNA-1 increased the expression level of the target protein by inhibiting the function of miR-21. The inhibitory effect showed a dose-response effect. Among them, 0.5μM daPNA-1 could restore the protein expression level to approximately 80%. The P value was calculated using the t-test. A P value <0.05 was considered statistically significant.

[0234] Furthermore, the miRNA reporter plasmid and daPNA were transfected into HEK-293T cells via liposome delivery. 24 hours after transfection, the inhibitory effect of daPNA on the biological function of miRNA-21 in cells was verified by measuring the dual luciferase signal.

[0235] The above embodiments are illustrative and are not to be construed as limiting the present invention. Persons skilled in the art may vary, modify, substitute, and modify the above embodiments within the scope of the present invention. Persons skilled in the art may also make improvements and supplements without departing from the present invention, and such improvements and supplements should also be considered within the scope of protection of the present invention.

Claims

1. A composition, characterized in that The composition comprises a first RNA ligand and a second RNA ligand, wherein the first RNA ligand comprises an oligonucleotide capable of specifically targeting a double-stranded segment of a target RNA element, and the second RNA ligand comprises an oligonucleotide capable of specifically targeting a single-stranded segment of a target RNA element, so that the composition can specifically target the double-stranded-single-stranded interface region of the target RNA.

2. The composition according to claim 1, wherein The first RNA ligand comprises a chemically modified oligonucleotide, wherein the chemical modification enables the first RNA ligand to selectively bind to double-stranded RNA, and / or The chemical modification weakens the binding of the first RNA ligand to the single-stranded RNA.

3. The composition according to claim 1 or 2, wherein The first RNA ligand and / or the second RNA ligand comprises one or more of peptide nucleic acid, ribonucleic acid, locked nucleic acid, phosphodiester morpholino oligonucleotide, glycol nucleic acid, threose nucleic acid or bridge nucleic acid; or, The first RNA ligand and / or the second RNA ligand can be linked to functional molecules with different functions; Preferably, the functional molecule is selected from one or more of fluorescent molecules, binding induced luminescence molecules, PET imaging probes, membrane-penetrating small molecules, targeted delivery molecules, nanocarriers, additional RNA ligands, nanoantibodies and / or protein binding ligands.

4. The composition according to any one of claims 1 to 3, wherein The first RNA ligand comprises a chemically modified peptide nucleic acid; and / or The second RNA ligand comprises an antisense peptide nucleic acid.

5. The composition according to any one of claims 1 to 4, wherein The first RNA ligand comprises no less than 3 bases; and / or, The second RNA ligand comprises no less than 3 bases; and / or, The composition comprises no more than 20 bases; and / or A linker may or may not be included between the first RNA ligand and the second RNA ligand; Preferably, the linker is one or more aminoacetic acid or diaminobutyric acid.

6. The composition according to any one of claims 1 to 5, wherein The first RNA ligand comprises one or more peptide nucleic acids selected from the group consisting of: i) A peptide nucleic acid that forms a triplex with GC base pairs as shown below: ii) a peptide nucleic acid that forms a triplex with AU base pairs as shown below; iii) a peptide nucleic acid that forms a triplex with CG base pairs as shown below; and iv) Peptide nucleic acid that forms a triplex with UA base pair as shown below 7. The composition according to any one of claims 1 to 6, characterized in that The target RNA element is selected from one or more of nuclear RNA, cytoplasmic RNA, membrane RNA and extracellular RNA, and the target RNA element comprises a double-stranded segment and a single-stranded segment; Preferably, the nuclear RNA or cytoplasmic RNA is selected from non-coding RNA, mRNA, miRNA, enhancer RNA, nascent RNA, chromosome-enriched RNA, ribosomal RNA, membrane-enriched RNA or mitochondrial RNA.

8. The composition according to claim 7, characterized in that The target RNA element is associated with a disease or disorder.

9. The composition according to any one of claims 1 to 8, wherein the target RNA element comprises an RNA sequence selected from miRNA, a new coronavirus, or a human immunodeficiency syndrome virus HIV; Preferably, the novel coronavirus comprises a pseudoknot structure of the novel coronavirus SARS-CoV-2; Preferably, the miRNA is miRNA-21; Preferably, the composition is a peptide nucleic acid oligomer targeting the double-stranded-single-stranded junction region of the pseudoknot structure of the SARS-CoV-2 virus, and has the following structure: Preferably, the composition is a composition targeting the double-stranded-single-stranded interface region of miRNA-21 and has the following structure: 10 . A nucleic acid drug comprising the composition according to any one of claims 1 to 9 .

11. A pharmaceutical composition, comprising the composition according to any one of claims 1 to 9, or the nucleic acid drug according to claim 10, and a pharmaceutically acceptable carrier.

12. Use of the composition according to any one of claims 1 to 9, the nucleic acid drug according to claim 10 or the pharmaceutical composition according to claim 11 in the preparation of a reagent for regulating the function of RNA elements in an organism, wherein When the composition, the nucleic acid drug or the pharmaceutical composition is contacted with an organism, the structure and / or expression of the RNA element is changed compared to that observed under reference conditions, thereby causing a change in the biological function of the RNA element; The reference condition is selected from the group consisting of: the absence of the composition, the presence of a reference composition, and combinations thereof.

13. Use of the composition according to any one of claims 1 to 9, the nucleic acid drug according to claim 10 or the pharmaceutical composition according to claim 11 in the preparation of a drug for treating a disease, in, The diseases are associated with changes in the function of RNA elements.

14. The use according to claim 13, wherein the disease is selected from cancer; neurodegenerative diseases; metabolic disorders; inflammatory disorders; autoimmune diseases, infectious diseases; or genetic diseases.

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