Targeted RNA modification regulation method

Abstract

Provided in the present application are a method for regulating RNA modification by means of introducing an engineered antisense oligonucleotide targeting an RNA modification site into a host cell to demodify a target RNA, an antisense oligonucleotide for erasing RNA modification used in the method, and a composition comprising same.

Description

Methods for targeting RNA modification regulation

[0001] This application claims priority to the Chinese patent application No. 202410818048.7, filed on June 24, 2024, and entitled “Methods for targeting RNA modification regulation”, the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of molecular biology, and particularly relates to methods for targeting RNA modification regulation. BACKGROUND

[0003] RNA modification refers to chemical modifications that occur in different ways on RNA molecules, which play an important role in regulating processes such as transcription, splicing, degradation, and transport of RNA. In recent years, research has found that RNA modification plays a key role in gene expression regulation, cell fate determination, and disease development, becoming one of the research hotspots in the field of biomedicine. Since the first discovery of RNA chemical modification 60 years ago, more than 170 types of RNA modifications have been identified, and it has been found that about 10 types exist in mRNA in the mammalian transcriptome. These modifications are added to mRNA by different writing proteins, and a part of erasing proteins is responsible for removing the modifications. RNA modifications have important effects on the stability, splicing, localization, and translation efficiency of mRNA metabolism. Functionally, they affect a wide range of physiological and pathological processes. Individual modification sites may play an important role in specific biological processes, such as cell differentiation. The disorder of individual modification sites is often associated with various diseases, including autoimmune diseases, cancer, and neurodegenerative diseases, which indicates that they have significant therapeutic potential in intervention. Studies have shown that individual modification sites have important functions in biological processes such as cell differentiation, and their disorder is associated with various diseases. Recent research has found that most RNA modification enzymes have specific sequence and structure requirements when writing specific RNA modification sites. This means that these enzymes have a certain preference for selectively adding modifications to specific positions of RNA molecules, and this preference may be influenced by the RNA sequence and structure. By understanding the sequence and structure requirements of these modification enzymes, we can better understand their mechanisms of action in RNA modification and help develop intervention strategies targeting specific modification sites.

[0004] Despite recent efforts to explain the role of individual modification sites, challenges remain. Currently, researchers have developed various Cas13 protein-based intervention strategies targeting RNA modifications, however, existing RNA modification erasing tools have some limitations, such as lack of precision and specificity, global non-specific effects, etc., limiting their application in precise regulation of RNA modification sites. For example, it is difficult to accurately control the position and global off-target of erasing proteins. Therefore, developing efficient tools to remove modification sites is crucial for revealing the role of individual modification sites and developing targeted interventions for diseases.

[0005] RCMS (reengineered m5C modification system) developed by Sui et al. is based on CRISPR-Cas13d technology for targeted modification of m5C methylation and demethylation in specific transcripts. The RCMS editor is composed of a dCasRx protein located in the nucleus combined with the catalytic domain of the demethylase Tet2, which can accurately manipulate the methylation modification of specific m5C sites. For m6A modification, Xia et al. developed a bidirectional dCasRx epitranscriptome editing platform composed of a dCasRx protein located in the nucleus combined with methyltransferase METTL3 or demethylase ALKBH5, which is used for editing m6A modification in specific transcripts. Both of these two schemes use CRISPR-Cas technology to achieve specific editing of RNA modification, targeting m5C and m6A modification respectively, providing new tools and platforms for studying RNA epigenetics. The recently developed dCas13-based method for interfering RNA modification has some inherent limitations:

[0006] 1. Lack of precise control over removal of specific modification sites: Although Cas13 can guide the eraser to a specific location, the current method lacks the ability to precisely control the eraser to remove specific modification sites. This may result in inaccurate erasure of target modifications, affecting the precise regulation of modification sites;

[0007] 2. Global non-specific effects: Overexpression of dCas13-erasing proteins can cause global non-specific effects, such as off-target effects. This means that in addition to the target modification site, other unrelated modifications or RNA sequences may also be affected, increasing the complexity and uncertainty of experimental results;

[0008] 3. Lack of universality: dCas-based removal modification tools need to be developed individually for each modification, which increases the complexity and time cost of development. Because different eraser proteins and targeted design and optimization may be needed for each modification type, many important RNA modification types, such as A-to-I RNA editing, pseuU, and m1A, currently lack corresponding eraser proteins, so existing dCas13-based methods are not feasible for these modifications;

[0009] 4. Complex development process: Developing dCas-based removal modification tools involves protein engineering, design optimization, and in vitro and in vivo validation, which may require a large amount of time and resources and has a risk of failure.

[0010] 5. Unable to accurately quantify regulation: Current dCas-based removal modification tools often lack accurate quantitative regulation of the modification removal process, which can lead to over-removal or insufficient removal of modifications, affecting the accuracy and reliability of experimental results.

[0011] 6. Cell toxicity and immunogenicity: The introduction of exogenous proteins (such as Cas13) can cause cell toxicity and immunogenicity, which can have a negative impact on cell survival and homeostasis, and can trigger a response from the host immune system.

[0012] 7. Low delivery efficiency, difficult clinical application: CRISPR-Cas13 system targets RNA specifically to regulate gene expression. However, its delivery efficiency in vivo is affected by many factors, including the choice of vector, the size and structure of the CRISPR component, and the specificity of the target tissue for delivery;

[0013] In summary, although dCas-based removal modification tools have certain potential, their limitations and challenges still exist, and further research and technical improvements are needed to address them. Therefore, developing a more efficient, more accurate, and more targeted RNA modification eraser tool is an urgent need. SUMMARY

[0014] Therefore, the technical problem to be solved by the present application is to provide a method for targeted RNA modification regulation, in order to improve the efficiency and accuracy of RNA modification regulation.

[0015] The method for targeted RNA modification regulation provided by the present application comprises: blocking the recognition and / or action of the RNA modification enzyme on the modification site with an antisense oligonucleotide (ASO) targeting the modification site.

[0016] The antisense oligonucleotide targeting the modification site is reverse complementary to the modification site and fragments upstream and downstream thereof. The method for regulating the RNA modification in the present application is mainly used for removing the RNA modification, and the antisense oligonucleotide used in the method is also referred to as ERASER or ERASER ASO in some embodiments. In the present application, the ASO is a chemically modified antisense oligonucleotide.

[0017] In the method of the present application, the RNA modification refers to a modification occurring on the RNA, for example, at least one base in the sequence of the RNA is modified. The RNA modification includes but is not limited to any one of the following: 5-methylcytosine modification, 5-hydroxymethylcytosine modification, N6-methyladenine modification, 7-methylguanine modification, N6,2'-O-dimethyladenine modification, 2-oxymethylation modification, N1-methyladenine modification, pseudouracil modification, hypoxanthine modification, 5-methoxy cytosine modification, 5-methyluracil modification, N4-acetyl cytosine modification, 5-fluorouracil modification, N7-methylguanine modification, 2'-O-methylation modification.

[0018] In the present application, the type of the RNA is not limited, and the targeted RNA is selected from at least one of the following: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), long non-coding RNA (lncRNA), small nuclear RNA (snRNA), micro-interfering RNA (miRNA) and Piwi-interacting RNA (piRNA).

[0019] In the method of the present application, the ASO acts on the modification site, and thus can hinder the action of various RNA modification enzymes. For example, the RNA modification enzyme can be a methyltransferase, a phosphorylase, an acetylase, a glycosyltransferase, an oxidoreductase, an adenylylase, a hydroxymethylase, an RNA deaminase. For example, the methyltransferase can be NSUN2 / 6 RNA methyltransferase or MELLT3 / 14 RNA methyltransferase.

[0020] In the method of the present application, the length of the antisense oligonucleotide targeting the modification site is not less than 10 bp; preferably, the length of the antisense oligonucleotide targeting the modification site is 10-50 bp; preferably, the length of the antisense oligonucleotide targeting the modification site is 12-40 bp, more preferably, the length of the antisense oligonucleotide targeting the modification site is 14-30 bp, and most preferably, the length of the antisense oligonucleotide targeting the modification site is 17-21 bp. In the embodiments of the present application, the length of the antisense oligonucleotide targeting the modification site is 17 bp, 18 bp, 19 bp, 20 bp or 21 bp.

[0021] In the method of the present application, the modification site is an integer of 1 or more; preferably, the number of the modification site is 1, 2, 3, 4 or 5. In the present application, if the number of the nucleotides corresponding to the modification site on the antisense oligonucleotide ASO targeting the modification site is 1, the ASO only targets one RNA modification site, and if multiple modification sites need to be regulated, multiple ASOs are used. If the number of the modification site is more than 1, one ASO can be used to regulate the occurrence of the modification, or multiple ASOs can be used to regulate the occurrence of the modification, which is not limited in the present application. When the number of the modification site is more than 1, and the length of the region where multiple modification sites exist is not more than the length of the ASO, it is preferred to use the same ASO to regulate it.

[0022] In the embodiments of the present application, the antisense oligonucleotide targeting the modification site comprises nucleotides opposite to the modification site, an upstream homologous fragment and a downstream homologous fragment. The upstream homologous fragment is reverse complementary to the upstream sequence of the RNA where the modification site is located, and the downstream homologous fragment is reverse complementary to the downstream sequence of the RNA where the modification site is located. As preferred, the length of the upstream homologous fragment and the downstream homologous fragment is independently selected from 5-25 bp, preferably, the length of the upstream homologous fragment and the downstream homologous fragment is independently selected from 6-20 bp, more preferably, the length of the upstream homologous fragment and the downstream homologous fragment is independently selected from 7-15 bp. For example, the length of the upstream homologous fragment is 7 bp, 8 bp, 9 bp, 10 bp, 11 bp, 12 bp, 13 bp, 14 bp or 15 bp; the length of the downstream homologous fragment is 7 bp, 8 bp, 9 bp, 10 bp, 11 bp, 12 bp, 13 bp, 14 bp or 15 bp; the length of the upstream homologous fragment and the downstream homologous fragment differs by 0-15 bp; preferably, the length of the upstream homologous fragment and the downstream homologous fragment differs by 0-10 bp; more preferably, the length of the upstream homologous fragment and the downstream homologous fragment differs by 0-5 bp. For example, in the present application, the length of the upstream homologous fragment and the downstream homologous fragment differs by 0 bp, 1 bp, 2 bp, 3 bp, 4 bp or 5 bp.

[0023] In the method of the present application, the nucleotides opposite to the modification site, the upstream homologous fragment and the downstream homologous fragment of the antisense oligonucleotide can also comprise non-paired bases, i.e. not completely complementary. Preferably, the ASO and the target sequence are more than 80% complementary; preferably, the ASO and the target sequence are more than 90% complementary; most preferably, the ASO and the target sequence are completely complementary.

[0024] The antisense oligonucleotide targeting the modification site has at least one base chemically modified, wherein the chemical modification is optionally one or more selected from the group consisting of LNA, UNA, 2'-MOE, 2'-F, 2'-OMe, PNA, Morpholino, BNA, methylation modification, phosphorothioate modification, and DNA base replacement modification.

[0025] Preferably, 1-5 bases at the 3' end and 5' end of the antisense oligonucleotide targeting the modification site are modified.

[0026] Preferably, 1-3 bases at the 3' end and 5' end of the antisense oligonucleotide targeting the modification site are modified.

[0027] Preferably, 3 bases at the 3' end of the antisense oligonucleotide are modified by LNA, 3 bases at the 5' end of the antisense oligonucleotide are modified by LNA, the remaining bases are modified by 2'OME or MOE, and all bases are modified by full phosphorothioate.

[0028] In some embodiments, the antisense oligonucleotide targeting the modification site has a length of 17-21 nt, 3 bases at the 3' end are modified by LNA, 3 bases at the 5' end are modified by LNA, the remaining bases are modified by 2'OMe or MOE, and all bases are modified by full phosphorothioate.

[0029] In some embodiments, the antisense oligonucleotide targeting the modification site has a structure of (N1)x-(N2)y-(N3)z, wherein N is a base selected from any one of A, U, C, and G, N2 is a nucleic acid matching the modification site, (N1)x and (N3)z are respectively a fragment reverse complementary to the upstream and downstream homologous arms, and x, y, and z respectively represent the number of bases of N1, N2, and N3. For example, y=1, x=8, 9, or 10, and z=8, 9, or 10.

[0030] For example, with y=1, x=8, and z=8, the structure of the ASO is LN*LN*LN*mN*mN*mN*mN*mN*mN*mN*mN*mN*mN*mN*LN*LN*LN*.

[0031] For example, with y=1, x=9, and z=9, the structure of the ASO is LN*LN*LN*mN*mN*mN*mN*mN*mN*mN*mN*mN*mN*mN*mN*mN*LN*LN*LN*.

[0032] For example, with y=1, x=10, and z=10, the structure of the ASO is LN*LN*LN*mN*mN*mN*mN*mN*mN*mN*mN*mN*mN*mN*mN*mN*mN*mN*LN*LN*LN*.

[0033] For example, when y = 1, x = 8, and z = 8, the structure of the ASO is LN*LN*LN*eN*eN*eN*eN*eN*eN*eN*eN*eN*eN*eN*LN*LN*LN*.

[0034] For example, when y = 1, x = 9, and z = 9, the structure of the ASO is LN*LN*LN*eN*eN*eN*eN*eN*eN*eN*eN*eN*eN*eN*eN*eN*LN*LN*LN*.

[0035] For example, when y = 1, x = 10, and z = 10, the structure of the ASO is LN*LN*LN*eN*eN*eN*eN*eN*eN*eN*eN*eN*eN*eN*eN*eN*eN*eN*LN*LN*LN*.

[0036] In the ASO structure as described above, L represents LNA modification, m represents 2'OMe modification, e represents 2'MOE modification, and * represents PS modification.

[0037] More specifically, the antisense oligonucleotide targeting the modification site has a nucleic acid sequence as shown in any one of SEQ ID NOs: 1-22 or 65-70. In the sequence listing, the ASO is represented as RNA, but under the ST.26 standard, both U and T are represented as T, and the specific sequences of the ASOs are as follows:

[0038] In one specific embodiment, the antisense oligonucleotide targets the m5C modification at the 156713158 site on chromosome 1, and the antisense oligonucleotide is LG*LG*LT*mU*mC*mC*mC*mA*mG*mU*mU*mU*mG*mC*LA*LG*LG; or LG*LG*LG*mG*mU*mU*mC*mC*mC*mA*mG*mU*mU*mU*mG*mC*mA*mG*LG*LC*LC; or LG*LG*LT*eU*eC*eC*eC*eA*eG*eU*eU*eU*eG*eC*LA*LG*LG; or LG*LG*LG*eG*eU*eU*eC*eC*eC*eA*eG*eU*eU*eU*eG*eC*eA*eG*LG*LC*LC.

[0039] In another specific embodiment, the RNA modification site targeted by the antisense oligonucleotide is a m5C modification at position 135984141 on chromosome 9, and the antisense oligonucleotide is: LT*LG*LA*mG*mC*mC*mU*mG*mG*mC*mA*mU*mG*mU*LT*LG*LA; or is LT*LC*LT*mG*mA*mG*mC*mC*mU*mG*mG*mC*mA*mU*mG*mU*mU*mG*LA*LG*LC; or is LT*LG*LA*eG*eC*eC*eU*eG*eG*eC*eA*eU*eG*eU*LT*LG*LA; or is LT*LC*LT*eG*eA*eG*eC*eC*eU*eG*eG*eC*eA*eU*eG*eU*eU*eG*LA*LG*LC;

[0040] In another specific embodiment, the RNA modification site targeted by the antisense oligonucleotide is a m5C modification at position 48389569 on chromosome 19, and the antisense oligonucleotide is: LT*LG*LT*mA*mG*mC*mC*mA*mC*mC*mG*mC*mU*mG*mG*mA*mG*LG*LC*LT; or is LT*LG*LG*mA*mG*mG*mC*mU*mG*mU*mG*mG*mC*mA*mG*mC*mU*LA*LC*LA; or is LT*LG*LT*eA*eG*eC*eC*eA*eC*eC*eG*eC*eU*eG*eG*eA*eG*LG*LC*LT; or is LT*LG*LG*eA*eG*eG*eC*eU*eG*eU*eG*eG*eC*eA*eG*eC*eU*LA*LC*LA;

[0041] In another specific embodiment, the RNA modification site targeted by the antisense oligonucleotide is a m6A modification at position 40903256 on chromosome 15, and the antisense oligonucleotide is: LC*LG*LT*mC*mG*mU*mA*mG*mU*mC*mC*mA*mU*mG*LT*LA*LC, or is LC*LG*LT*eC*eG*eU*eA*eG*eU*eC*eC*eA*eU*eG*LT*LA*LC;

[0042] In another specific embodiment, the RNA modification site targeted by the antisense oligonucleotide is the m6A modification at position 25677538 of chromosome 9, and the antisense oligonucleotide is LT*LG*LC*mA*mG*mU*mA*mG*mU*mC*mC*mG*mG*mG*LA*LA*LC, or LT*LG*LC*eA*eG*eU*eA*eG*eU*eC*eC*eG*eG*eG*LA*LA*LC.

[0043] In another specific embodiment, the RNA modification site targeted by the antisense oligonucleotide is the m6A modification at position 145806215 of chromosome 6, and the antisense oligonucleotide is LA*LT*LC*mC*mA*mA*mA*mG*mU*mC*mA*mC*mU*mA*LT*LT*LT, or LA*LT*LC*eC*eA*eA*eA*eG*eU*eC*eA*eC*eU*eA*LT*LT*LT.

[0044] In another specific embodiment, the RNA modification site targeted by the antisense oligonucleotide is the m6A modification at position 9264847 of chromosome 1, and the antisense oligonucleotide is LC*LG*LG*mA*mA*mG*mU*mG*mU*mC*mC*mA*mC*mU*LG*LG*LT, or LC*LG*LG*eA*eA*eG*eU*eG*eU*eC*eC*eA*eC*eU*LG*LG*LT.

[0045] In another specific embodiment, the RNA modification site targeted by the antisense oligonucleotide is the m6A modification at position 40903256 of chromosome 6, and the antisense oligonucleotide is LC*LC*LC*mC*mA*mC*mA*mG*mU*mA*mC*mA*mC*mA*LC*LA*LC, or LC*LC*LC*eC*eA*eC*eA*eG*eU*eA*eC*eA*eC*eA*LC*LA*LC.

[0046] In another specific embodiment, the RNA modification site targeted by the antisense oligonucleotide is the m5C modification at the ACTB site, and the antisense oligonucleotide is LG*LT*LC*mA*mU*mA*mG*mU*mC*mC*mG*mC*mC*mU*LA*LG*LA.

[0047] In another specific embodiment, the RNA modification site targeted by the antisense oligonucleotide is the m5C modification at the MALAT1 site, and the antisense oligonucleotide is LA*LC*LT*mC*mA*mA*mA*mG*mU*mC*mC*mA*mA*mU*LG*LC*LA.

[0048] In another embodiment, the antisense oligonucleotide targets the m5C modification at the MYC-1 site, and the antisense oligonucleotide is: LC*LT*LG*mU*mU*mA*mG*mA*mA*mG*mG*mA*mA*mU*LC*LG*LT.

[0049] In another embodiment, the antisense oligonucleotide targets the m5C modification at the MYC-2 site, and the antisense oligonucleotide is: LA*LG*LT*mU*mC*mA*mU*mA*mG*mG*mU*mG*mA*mU*LT*LG*LC.

[0050] In another embodiment, the antisense oligonucleotide targets the m5C modification at the VEGFA site, and the antisense oligonucleotide is: LT*LC*LA*mC*mC*mC*mG*mU*mC*mC*mA*mU*mG*mA*LG*LC*LC.

[0051] In another embodiment, the antisense oligonucleotide targets the m5C modification at the MCM5 site, and the antisense oligonucleotide is: LC*LG*LA*mG*mU*mC*mC*mA*mU*mG*mA*mG*mU*mC*LC*LA*LG.

[0052] Further, the method of the present application comprises: introducing the antisense oligonucleotide into an organism, and inhibiting the occurrence of RNA modification in the cell.

[0053] In the present application, the introduction comprises: contacting a construct containing the antisense oligonucleotide with the organism, and the construct is a GalNAc conjugated or LNP-embedded antisense oligonucleotide.

[0054] In the present application, the introduction can be only one ASO, or multiple ASOs, and the present application does not limit this. One of the introduced ASOs can target only one site, or one ASO can target multiple sites, and the present application does not limit this.

[0055] In the present application, the organism is a eukaryote and / or a prokaryote, which can be from an animal, a plant or a microorganism, and the present application does not limit this. In some embodiments, the organism is a eukaryote, for example, it is a cell, an embryo and / or a tissue from an animal. Or for example, it is a cell, a tissue, a seed or an embryo from a plant. Or for example, it is a cell or a spore from a eukaryotic microorganism. In other embodiments, the organism is a prokaryote, which is a cell of a prokaryotic microorganism.

[0056] In the embodiments of the present application, the cells are human cells and / or animal cells, and the animal body is a mouse.

[0057] The present application also provides an antisense oligonucleotide targeting a modification site as described in the method.

[0058] The present application also provides a complex containing the antisense oligonucleotide and Galnac conjugation or LNP embedding.

[0059] The present application also provides a composition comprising the antisense oligonucleotide as described above and the complex as described above.

[0060] The present application also provides a host cell comprising the antisense oligonucleotide as described above and / or the complex as described above.

[0061] The present application also provides a reagent for targeting RNA modification regulation, comprising the antisense oligonucleotide targeting a modification site as described in the method, or the complex as described above, or the composition as described above, or the host cell as described above.

[0062] The reagent also includes reagents, adjuvants or excipients required in transfection and / or transformation.

[0063] The reagent includes electroporation transfection reagent, ion transfection reagent and / or liposome transfection reagent; for example, the electroporation transfection reagent includes trypsin, PBS buffer, sodium chloride, potassium chloride, lactic acid, acetic acid, Tween, poloxamer, Span, the ion transfection reagent includes calcium phosphate transfection reagent, and the liposome transfection reagent includes 3000, Lipofectamine RNAiMAX, Lipo8000, etc.

[0064] The adjuvant includes at least one of cytokines (such as interferons (e.g. IFN-α, IFN-β and IFN-γ), lymphokines (e.g. IL-2, IL-3, IL-4, IL-5 and IL-10, etc.), monokines (e.g. IL-1, IL-6, IL-8 and IL-12, etc.) and other cytokines such as TNF and GM-CSF, etc.), costimulatory molecules (e.g. B7-1 and B7-2), emulsifying adjuvant (Titer Max Gold (TMG)), Freund's adjuvant, saponin, monophosphoryl acyl lipid.

[0065] The excipient includes polyethylene glycol and / or polylysine.

[0066] The present application also provides the use of the antisense oligonucleotide targeting a modification site as described in the method, or the complex as described above, or the composition as described above, or the host cell as described above, in the preparation of a drug for preventing and treating diseases related to RNA modification.

[0067] In the present application, the diseases related to RNA modification include tumors, nervous system diseases, immune system diseases, metabolic diseases, and / or infectious diseases.

[0068] As preferred, the tumors include malignant tumors and benign tumors, such as breast cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, ovarian cancer, prostate cancer, bladder cancer, skin melanoma, etc.

[0069] The nervous system diseases include Parkinson's disease, Alzheimer's disease, stroke, multiple sclerosis, meningitis, spinal cord injury, brain tumor, etc.

[0070] The immune system diseases include rheumatoid arthritis, systemic lupus erythematosus, autoimmune thyroid disease, ulcerative colitis, Crohn's disease, scleroderma, systemic sclerosis, etc.

[0071] The metabolic diseases include diabetes, obesity, hypertension, hyperlipidemia, hyperthyroidism, hypothyroidism, osteoporosis, etc.

[0072] The infectious diseases include AIDS, hepatitis B, hepatitis C, tuberculosis, malaria, dengue fever, cholera, influenza, pneumonia, etc.

[0073] Further, the present application also provides a drug for preventing and treating diseases related to RNA modification, which comprises an antisense oligonucleotide targeting a modification site as described in the foregoing method, or a complex as described in the foregoing, or a composition as described in the foregoing, or a host cell as described in the foregoing.

[0074] In the drug of the present application, other therapeutic agents, synergistic agents and / or immunomodulatory agents are also included, and the present application does not limit the types of drugs used in combination, the doses of drugs used in combination, and the administration methods of drugs used in combination.

[0075] Further, the present application also provides a method for preventing and treating diseases related to RNA modification, which comprises administering the drug as described in the foregoing to a subject.

[0076] The present application uses antisense oligonucleotides targeting RNA modification sites to regulate RNA modification, which has advantages including but not limited to:

[0077] 1. Precision and specificity: ERASER ASO can achieve precise erasure of RNA modification sites, avoiding the influence on other modification sites, and has higher specificity and accuracy;

[0078] 2. Reduction of global non-specific effects: Compared to methods that overexpress dCas, the ASOs used by ERASER have lower global non-specific effects, reducing interference with cell state and facilitating more accurate assessment of the function and regulatory mechanisms of target modification sites;

[0079] 3. High efficiency: ERASER ASO tools have high modification erasing effects, and use chemical modifications to improve the cell permeability and selectivity of ASOs;

[0080] 4. Universality and versatility: The design concept and method of ERASER ASO can be applied to various RNA modification types, and is not limited to specific modification enzymes or modification types, having wider applicability and versatility.

[0081] 5. Simplified development process: Compared to dCas tools developed for each modification individually, ERASER ASO provides a more simplified development process, reducing tool development time and cost.

[0082] 6. Precise regulation and quantitative adjustment: Since the ASOs used by ERASER can be precisely designed and regulated, precise regulation and quantitative adjustment of modifications can be achieved, providing a more powerful tool for in-depth study of the function and regulatory mechanisms of RNA modifications.

[0083] Therefore, the ERASER ASO provided by the present application has higher precision, specificity, versatility and precise regulation ability compared to dCas-based tools for targeted removal of RNA modifications. Experiments show that the inhibition rate of RNA modification by this method is between 50% and 100%, providing a more effective tool and method for the field of RNA modification research. BRIEF DESCRIPTION OF DRAWINGS

[0084] Figure 1 is a schematic diagram of the design principle of ERASER, wherein:

[0085] (A) Different types of RNA modifications and their modification sites are shown in the schematic diagram. Different types of RNA modifications (such as m5C, m6A) have specific structural characteristics in RNA molecules and are mediated by corresponding modification enzymes to modify specific sites;

[0086] (B) Schematic diagram of the design principle of ERASER. ERASER forms a double-stranded structure by designing specific antisense oligonucleotides (ASO) to bind to target RNA, thereby interfering with the action of modification enzymes and achieving precise erasure of RNA modification sites;

[0087] (C) Schematic diagram of mRNA m5C modification reporter system construction, m5C modification site is selected and constructed into psiCHECK2 reporter plasmid, inserted into the 3'UTR region of hRluc, for evaluating the inhibitory effect of ERASER on m5C modification;

[0088] (D) Evaluation results of the inhibitory effect of ERASER on m5C modification, ERASER ASO centered on m5C site is designed, different modifications (MOE and OMe) and lengths (17nt and 21nt) are used for evaluation, and target BS-PCR is used to detect the m5C level of the substrate, it is found that the m5C modification level is significantly reduced in ERASER transfected cells, which proves the effective inhibitory effect of ERASER on m5C modification;

[0089] Figure 2 is the evaluation of the inhibitory effect of ERASER on endogenous m5C and m6A modification:

[0090] (A) Effective inhibition of ERASER on endogenous m5C site, two m5C sites distributed in different functional regions of RNA are detected in HEK293T cells, and the inhibitory effect on endogenous m5C site is observed, with an inhibition rate of 80% to 95%;

[0091] (B) Design and evaluation of ERASER ASO, comparison of the inhibitory effect of 17nt OMe and MOE modified ASO targeting RALGDS m5C site, it is observed that OMe modified ERASER ASO has stronger inhibitory effect at lower concentration;

[0092] (C) YTH-TadA fusion protein mediated RNA editing detection system, YTH-TadA is a fusion protein composed of YTH binding protein and TadA, used to introduce specific RNA editing modification, in this system, m6A level can be explained as RNA editing level;

[0093] (D) Inhibitory effect of ERASER ASO on YTH-TadA mediated RNA editing, 17nt ERASER ASO centered on m6A is designed, different modifications (OMe and MOE) are used, after transfection of ERASER ASO in HEK293T cells and transfection of YTH-TadA fusion protein after 12 hours, the RNA editing level of the substrate is evaluated by PCR amplification fragment Sanger sequencing, and it is observed that ERASER ASO effectively inhibits YTH-TadA mediated RNA editing, and OMe modified ERASER ASO has more robust inhibitory effect.

[0094] Figure 3 is the effect of ERASER ASO on tumor RNA modification:

[0095] (A) m5C ERASER ASO (HDGF) and m6A ERASER ASO (ACTB, MALAT1, MYC) effects on tumor development related RNA methylation modification sites;

[0096] (B) According to the position (3'UTR or 5'UTR) of the tumor related gene RNA methylation modification site, the front and back 40nt fragments are cloned into the corresponding position of the hRluc of the psiCHECK2 reporter plasmid, wherein the HDGF m5C, MYC m6A and VEGFA m6A sites are located in the 3'UTR, and the MCM5 m6A is located in the 5'UTR;

[0097] (C) HDGF m5C / HDGF m5C mutant / HDGF m5C and ASO, MYC / MYC m5C mutant / MYC m5C and ASO are transfected into bladder cancer cell line T24 respectively, VEGFA / VEGFA m5C mutant / VEGFA m5C and ASO, MCM5 / MCM5 m5C mutant / MCM5 m5C and ASO are transfected into lung cancer cell line A549, and the expression of the reporter plasmid dual luciferase is detected after 48 hours. DETAILED DESCRIPTION

[0098] The present application provides a method for targeting RNA modification regulation, and those skilled in the art can refer to the content herein to appropriately improve the process parameters. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0099] Unless otherwise defined herein, the scientific and technical terms used in connection with the present application shall have the meanings understood by those of ordinary skill in the art.

[0100] In the present application, "and / or" is used to describe the association relationship of the associated objects, which means that there can be three kinds of relationships, for example, A and / or B, which can represent the cases of A alone, A and B together, and B alone. Wherein A and B can be singular or plural.

[0101] In the present application, "at least one" means one or more, and "more" means two or more. "At least one of the following (one / variety)" or similar expressions means any combination of these items, including any combination of single item (one / variety) or multiple items (one / variety).

[0102] In the present application, “including”, “containing” and “having” are used interchangeably and are intended to mean the inclusive, meaning that the described scheme can have other elements in addition to those listed. It should also be understood that the use of “including”, “containing” and “having” to describe the description herein also provides a “consisting of” scheme.

[0103] In the present application, “targeting” refers to targeting a specific modification site on RNA and specifically regulating it, especially removing the modification.

[0104] In the present application, “RNA” is the full name of Ribonucleic Acid, which is a long-chain molecule composed of ribonucleotides condensed by phosphodiester bonds. In the present application, the bases of RNA are mainly A adenine, G guanine, C cytosine and U uracil. Generally, there are three major types of RNA molecules, namely messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA), in addition to some special RNAs such as pre-messenger RNA, long non-coding RNA, small nuclear RNA, microRNA and Piwi-interacting RNA.

[0105] In the present application, “RNA modification” is a post-transcriptional regulation method that exists widely in various types of RNA as described above. The types of RNA modification that have been found so far include but are not limited to methylation, adenylation, phosphorylation and other forms. These modifications play an important regulatory role in transcription, splicing, translation and other processes. In recent years, more and more studies have shown that RNA modification is closely related to the occurrence and development of various diseases. For example, RNA modification plays an important role in cancer. For example, N6-methyladenine (m6A) modification is closely related to the occurrence and prognosis of various cancers. In the pathological process of cancer, abnormal expression of RNA modification enzymes related to m6A such as METTL3, FTO and ALKBH5 can inhibit the normal regulation of m6A modification, leading to instability and abnormal protein translation of RNA, thereby promoting the proliferation and metastasis of cancer cells. In addition, other RNA modification forms such as 5hmC and N4-acetylcytosine (ac4C) also play an important role in tumor growth and metastasis. And RNA modification also plays an important role in nervous system diseases. For example, in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, abnormal RNA modification may lead to neuronal dysfunction and cell death. Changes in specific RNA modification enzymes and modification sites are closely related to the pathogenesis and progression of these diseases. In addition, RNA modification is also related to the occurrence and development of immune system diseases, cardiovascular diseases and other diseases. By regulating RNA modification, we can better understand the pathogenesis of these diseases and provide new ideas and methods for the diagnosis and treatment of diseases.

[0106] In the present application, "antisense oligonucleotide" is a kind of artificially synthesized oligonucleotide fragment, which can combine with target RNA molecule through base complementary principle, interfere and block the recognition and action of RNA modification enzyme to target site.

[0107] In the present application, "LNA modification" (Locked Nucleic Acid) locks the sugar ring into a bicyclic molecular pattern through a methylene bridge between 2' oxygen atom and 4' carbon atom, thereby limiting the flexibility of the sugar ring. It can make the pairing of LNA with DNA or RNA more stable, and improve the thermal stability of duplex.

[0108] In the present application, 2'OMe modification replaces the hydrogen on the 2'-hydroxyl group of ribonucleotide with methoxy (-OCH3). This modification enhances the resistance of oligonucleotide to nuclease, thereby improving its stability.

[0109] In the present application, 2'-MOE modification replaces a hydrogen atom in nucleotide with a methoxyethyl (-OCH2CH2OCH3) at the 2'-position. This modification makes the nucleotide more stable, improves its resistance to nuclease degradation, and thus prolongs its half-life in vivo.

[0110] In the present application, phosphorothioate modification replaces a non-bridging oxygen atom in the phosphate backbone with a sulfur atom. This modification has less disturbance to the structure of nucleic acid than base functionalization, and does not involve base pairing.

[0111] In the present application, "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) or alleviate an undesired physiological change or pathological condition in a subject, such as cancer and tumor. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of the state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Subjects in need of treatment include those already with a condition or disease as well as those in which a condition or disease is to be prevented, or those intending to prevent a condition or disease. When referring to terms such as slowing down, reducing, alleviating, ameliorating, palliating, etc., the meaning also includes elimination, disappearance, non-occurrence, etc.

[0112] In the present application, "drug" refers to a preparation which exists in a form effective for the biological activity of the active ingredient contained therein, and does not contain additional components which have unacceptable toxicity to the subject to which the pharmaceutical composition is administered.

[0113] In the present application, the "drug" can be preferably administered by any of the following modes, such as injection, enteral administration, and topical administration. For example, the injection includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and / or intracranial injection. The enteral administration mainly includes oral and / or rectal administration. The topical administration includes, but is not limited to, spray inhalation, nasal administration, buccal administration, vaginal administration, and the like.

[0114] In the present application, the "subject" refers to an organism receiving treatment for a particular disease or disorder as described in the present application. Exemplarily, the "subject" includes a mammal receiving treatment for a disease or disorder, which mammal includes bovine, equine, ovine, porcine, canine, feline, rodent, primate, and preferably the mammal is human, cat, dog, or pig.

[0115] In the present application, the "effective amount" refers to the amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent, is effective in preventing or alleviating the disease condition or the progression of the disease in a cell, tissue, or other subject. The "effective amount" also refers to the amount of a compound sufficient to alleviate symptoms, such as to treat, cure, prevent, or alleviate the relevant medical condition, or to increase the speed of treating, curing, preventing, or alleviating such conditions. When the active ingredient is administered alone, the therapeutically effective dose refers only to that ingredient. When a combination is used, the therapeutically effective dose refers to the combined amounts of active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously.

[0116] In the present application, the drug can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. Preferably, the dosage form of the drug depends on the intended administration method and therapeutic use. The pharmaceutical composition of the present application should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by incorporating the necessary dosage of ASO of the present application in a suitable solvent, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ion strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterilization by filtration. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) to facilitate storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH-buffered solution (e.g., phosphate-buffered solution), Ringer's solution, and any combination thereof.

[0117] In the present application, "cancer" and "tumor" are not mutually exclusive. "Cancer" refers to or describes a physiological condition in mammals typically characterized by unregulated cell growth. This definition includes both benign and malignant cancers. "Tumor" or "neoplasm" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. More specifically, the tumor or neoplasm refers to a tumor or neoplasm associated with RNA modification.

[0118] The materials used in the present application are all ordinary commercially available products, which can be purchased in the market. The sequence information involved in the examples includes:

[0119] Sequence information:

[0120] The abbreviations and meanings involved in the text include:

[0121] 1. ASO (Antisense Oligonucleotide): an antisense oligonucleotide is a short DNA or RNA sequence that can be complementary to a specific sequence of target RNA, thereby affecting or regulating the expression or function of target RNA.

[0122] 2. m5C: 5-methylcytosine, a common RNA modification, indicates that the cytosine (C) base on the RNA molecule is methylated.

[0123] 3. m6A: N6-methyladenosine, a common RNA modification, indicates that the adenine (A) base on the RNA molecule is methylated.

[0124] 4. pseudU: pseudouridine, a common RNA modification, indicates that the uracil (U) base on the RNA molecule is isomerized to pseudouridine.

[0125] 5. OMe modification (2'-O-methyl ether modification): a chemical modification that replaces the hydrogen atom on the 2' oxygen atom of an RNA or DNA molecule with a methyl oxygen group.

[0126] 6. MOE modification (2'-O-methoxyethyl modification): a chemical modification that replaces the hydrogen atom on the 2' oxygen atom of an RNA or DNA molecule with a methoxyethyl group.

[0127] 7. LNA modification (Locked Nucleic Acid modification): Locked Nucleic Acid is a chemical modification of RNA or DNA that forms a ring structure by connecting the 2' and 4' carbon atoms of the nucleic acid enzyme with oxygen, making the nucleic acid molecule more stable.

[0128] 8. Phosphorothioate (PS) modification: the oxygen atom on the phosphate group is replaced by a sulfur atom, increasing the anti-nuclease degradation ability of DNA or RNA molecules.

[0129] 9. BS treatment of RNA (Bisulfite treatment of RNA): by treating RNA molecules with bisulfite, unmethylated cytosine (C) is converted to uracil (U), which can be used to analyze the methylation status of RNA.

[0130] 10. CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins): CRISPR is a natural immune system, and the CRISPR-Cas system can be used for genome editing, by guiding RNA to complementary pairing with the target DNA sequence, to achieve precise editing of specific sites on the genome.

[0131] The application is further described below in conjunction with examples:

[0132] Examples

[0133] I. ERASER antisense oligonucleotide design

[0134] The design principle of ERASER is based on the need for precise intervention of RNA modification sites. By designing antisense oligonucleotides (ASO) targeting specific RNA modification sites, ASO binds to the target RNA to form a double-stranded structure, thereby interfering with the recognition and action of RNA modification enzymes, and achieving precise erasure of RNA modification sites (Fig. 1 A, B). In Fig. 1 A, the structural characteristics of RNA of different modification types and the modification sites mediated by the corresponding modification enzymes are shown.

[0135] ASO design for the following RNA modification sites:

[0136] Table 1 m5C site

[0137] Table 2 m6A site

[0138] First, the sequence of the modification site and its surrounding 40 bases is obtained. Next, we use Soligo to predict the accessibility of the target and design antisense oligonucleotides. Finally, the candidate antisense oligonucleotides are selected according to the following criteria: 1. Cover the specified site of interest; 2. GC content between 30%≤GC%≤70%; 3. Binding energy ≤-8 kcal / mol; No GGGG in the target sequence. In the case of similar binding energies of multiple antisense oligonucleotides, we select the antisense oligonucleotide centered on the modification site for experiments.

[0139] In addition, chemical modification can make oligonucleotides have drug-like properties, so we use the widely used backbone and 2' modification in ASO to improve the binding affinity to RNA targets, improve selectivity, and enhance their resistance to nuclease degradation. We evaluated ASO of two lengths, 17 nt and 21 nt, and used LNA modification for the three bases at both ends, 2'OMe or MOE modification for the middle part, and complete phosphorothioate (PS) modification for the whole. Since the NSUN6-mediated m5C modification site is located in the loop region of the hairpin structure, we designed an ERASER ASO for NSUN6 m5C, which deviates from the center of the modification site to more effectively unwind the stem structure. Similarly, LNA modification is used for the three bases at both ends of the ASO, and 2'OMe or MOE modification is used for the middle region, respectively.

[0140] The ASO obtained by the design of this example includes:

[0141] Table 3 ERASER ASO for m5C sites in Table 1

[0142] Table 4 ERASER ASO for m6A sites in Table 2 Note: A, U, C, G in Table 3-4 represent the four different bases, L represents LNA modification, m represents 2'OMe modification, e represents MOE modification, and * represents PS modification.

[0143] II. ERASER antisense oligonucleotides for erasing RNA modification

[0144] To establish our system, we chose the mRNA modification m5C as a target, which is functionally important and well-studied. To test the system, we chose 3 m5C methylation sites mediated by NSUN2 or NSUN6 RNA methyltransferases and constructed into psiCHECK2 reporter system, in which the m5C modification site and 40nt sequence before and after were inserted into the 3'UTR region of hRluc (C in Fig. 1). ASO and reporter plasmid were co-transfected into cells, and 48 hours later, the m5C level of the substrate was detected by target BS-PCR. Notably, we found that the m5C level in ERASER ASO transfected cells decreased in all cases (D in Fig. 1). Details as follows:

[0145] 1. Cell culture

[0146] HEK293T and HeLa cell lines were provided by the Chinese Academy of Sciences Cell Bank and verified by STR analysis, and checked by the Chinese Academy of Sciences Cell Bank for whether they were contaminated by mycoplasma. Cell lines were maintained in DMEM medium (Gibco) containing 10% FBS and 1% double antibody, and the cell incubator was set at 37 degrees Celsius, 5% CO2 and 95% humidity environment.

[0147] 2. m5C reporter plasmid construction

[0148] To construct the reporter plasmid, primers (Table 5) were designed to amplify the methylation site and the surrounding 40nt sequence, and the fragment was cloned into the psiCHECK2 hRluc 3'UTR region by homologous recombination.

[0149] Table 5 Primers for constructing m5C site reporter plasmid

[0150] 3. ERASER ASO and plasmid transfection

[0151] Cell transfection experiment was performed when cells reached 70% confluency in 12-well plates. Transfection reagent mix was prepared according to the instruction of transfection reagent Lipo8000. 500 ng m5C reporter plasmid and 40 pmol ERASER ASO targeting m5C were transfected in each well. Transfection solution was added to cells, mixed gently and then put into incubator. Cells were collected 48 hours after transfection for further analysis.

[0152] Cell transfection experiment was performed when cells reached 70% confluency in 12-well plates. Transfection reagent mix was prepared according to the instruction of transfection reagent Lipo8000. 500 ng m5C reporter plasmid and 40 pmol ERASER ASO targeting m5C were transfected in each well. Transfection solution was added to cells, mixed gently and then put into incubator. Cells were collected 48 hours after transfection for further analysis.

[0153] Cell transfection experiment was performed when cells reached 70% confluency in 12-well plates. Transfection reagent mix was prepared according to the instruction of transfection reagent Lipo8000. 500 ng m5C reporter plasmid and 40 pmol ERASER ASO targeting m5C were transfected in each well. Transfection solution was added to cells, mixed gently and then put into incubator. Cells were collected 48 hours after transfection for further analysis.

[0154] 4. BS-PCR

[0155] Total RNA was extracted by TRIzol method, treated with DNase I, BS converted (sulfonation: 3 cycles, 70°C, 10 min, 64°C, 45 min; desulfonation: 25°C, 30 min.) using EZ RNA Methylation Kit (ZYMO research), and reverse transcribed using HiScript II Q RT SuperMix (Vazyme) and random primers. The target sequence was amplified using STARmix Taq DNA Polymerase (GenStar) with the following program: 94°C, 3 min; 94°C, 30 s, 52°C, 30 s, 72°C, 20 s, 30 cycles; 72°C, 1 min. For BS-PCR, Sanger sequencing PCR products were used to measure the methylation level of mRNA substrates.

[0156] Table 6 BS-PCR primers

[0157] 5. Construction of YTH-TadA fusion protein

[0158] TadA was amplified from ABE8e (addgene #138489), YTH fragment was amplified from genome, both fragments were cloned into pcDNA3.1 plasmid vector by homologous recombination.

[0159] Table 7 Primers for constructing YTH-TadA fusion protein

[0160] 6. Cell experiment method for evaluating RNAm6A level

[0161] ERASER ASO was transfected into HEK293T cells with Lipofectamine RNAiMAX, 12 hours later, YTH-TadA fusion protein was further transfected, cells were collected 48 hours later, RNA was extracted using RNA isolation kit (Vazyme), followed by reverse transcription using reverse transcription kit (YESEN), and the RNA editing level of the substrate was evaluated by PCR amplification Sanger sequencing and EditR.

[0162] Table 8 m6A Sanger sequencing primers

[0163] 7. ERASER ASO effectively erased RNAm5C modification

[0164] Firstly, we evaluated the inhibitory effect of ERASER ASO in the m5C reporter plasmid, and the results showed that different modifications and lengths of ERASER ASO all exhibited significant inhibition. Whether it was a class I m5C site located at the 5' end of the hairpin structure (such as HDGF and RALGDS) mediated by NSUN2, or a class II m5C site located in the loop of the hairpin structure (such as SULT2A1) mediated by NSUN6, ERASER ASO could significantly reduce the modification level of these m5C sites (see Figure 1D).

[0165] Subsequently, we detected two m5C sites (HDGF and RALGDS) distributed in different functional regions in endogenous genes in HEK293T cells, and observed the effective inhibition of ERASER ASO on these m5C endogenous sites, with an inhibition rate of 80% to 95% (see Figure 2A). This result indicates that the inhibition of ERASER ASO on m5C modification in cells has high efficiency.

[0166] 8. ERASER ASO effectively erased RNAm6A modification

[0167] To explore whether ERASER ASO can form dsRNA with the target site to destroy the preferred single-stranded environment of METTL3 / 14 and inhibit m6A methylation, we selected five m6A sites identified by the GLORI method in HEK293T cells. We designed 17nt ASO with OMe or MOE modification, because 17nt ERASER showed more robust inhibition effect in m5C (Fig. 1D, Fig. 2A), to introduce dsRNA structure to inhibit m6A methylation.

[0168] YTH-TadA is composed of YTH domain-containing protein and TadA (Tetanus toxin A deaminase), YTH domain-containing protein is usually used to recognize and bind m6A modified sites, while TadA is a deaminase that can catalyze the deamination reaction of adenine (A) base in RNA. In the system of converting m6A modification to adenine (A) base, the m6A level can be explained as the RNA editing level by PCR amplification and Sanger sequencing (Fig. 2C). The results show that ERASER ASO effectively inhibits YTH-TadA8.20-mediated RNA editing, and compared with MOE, OMe-modified ERASER ASO has more robust and significant inhibition effect (see Fig. 2D), which is consistent with the result of our screening of more effective ERASER ASO with lower transfection dose (see Fig. 2B).

[0169] 9. Application potential of ERASER ASO in tumor treatment

[0170] To verify the potential of ERASER ASO in tumor treatment, we designed corresponding ASO targeting the high-level modified sites of RNA methylation related to tumor development and proliferation, and detected the m5C or m6A modification level in RNA. The results show that the inhibition efficiency of ERASER ASO on the RNA modification of these sites reached 50-95% (see Fig. 3A).

[0171] Next, we constructed psiCHECK2 plasmid reporter systems containing these modification sites for high levels of HDGF m5C modification and MYC m6A modification in bladder cancer, and VEGFA and MCM5 m6A modification in lung cancer. The reporter plasmids were constructed with the 40 nt sequence before and after the m5C / m6A modification sites included in the 3'UTR or 5'UTR of hRluc in the psiCHECK2 plasmid (see Fig. 3B), and the mutant plasmids of these RNA modification sites were used as positive controls. Each reporter plasmid (and ERASER ASO) was transfected into bladder cancer cell line T24 or lung cancer cell line A549 cells using Lipo8000, respectively. The results showed that the effect of ERASER ASO was consistent with the mutant reporter plasmid, and both could significantly inhibit the expression of hRluc (see Fig. 3C). In summary, these results strongly demonstrate the inhibitory ability of ERASER ASO at specific RNA modification sites, indicating its broad application prospects in tumor treatment.

[0172] Table 9 Primers for constructing tumor RNA modification related site reporter plasmids

[0173] Table 10 ERASER ASO for m5C or m6A sites in Table 5 Note: A, U, C, G in Table 6 represent the four different bases, L represents LNA modification, m represents 2'OMe modification, e represents MOE modification, and * represents PS modification.

[0174] The above is only the preferred embodiment of the present application, it should be noted that for those skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered within the scope of the present application.

Claims

1. Methods targeting RNA modification regulation, including: Blocking the recognition and / or action of RNA-modifying enzymes on modification sites by antisense oligonucleotides targeting modification sites; The antisense oligonucleotides targeting the modification site are inversely complementary to the modification site and its upstream and downstream fragments.

2. The method according to claim 1, characterized in that, The RNA-modifying enzymes include, but are not limited to, any one of the following enzymes: methyltransferase, phosphorylase, acetyltransferase, glycosyltransferase, oxidoreductase, adenylate acylase, hydroxymethylase, and RNA deaminase.

3. The method according to claim 1, characterized in that, The targeted RNA is selected from at least one of the following: premessenger RNA, messenger RNA, ribosomal RNA, transfer RNA, long noncoding RNA, micro-interfering RNA, and Piwi-interacting RNA.

4. The method according to any one of claims 1 to 3, characterized in that, The RNA modification includes, but is not limited to, any one of the following: 5-methylcytosine modification, 5-hydroxymethylcytosine modification, N6-methyladenine modification, 7-methylguanine modification, N6,2'-O-dimethyladenine modification, 2-oxymethylation modification, N1-methyladenine modification, pseudouracil modification, hypoxanthine modification, 5-methoxycytosine modification, 5-methyluracil modification, N4-acetylcytosine modification, 5-fluorouracil modification, N7-methylguanine modification, and 2'-O-methylation modification.

5. The method according to claim 1 or 4, characterized in that, The modified site is an integer of 1 or greater than 1; preferably, the number of modified sites is 1, 2, 3, 4 or 5.

6. The method according to claim 5, characterized in that, The antisense oligonucleotide targeting the modification site includes a nucleotide opposite to the modification site, an upstream homologous fragment, and a downstream homologous fragment; Preferably, the length difference between the upstream homologous fragment and the downstream homologous fragment is 0–15 bp; Preferably, the length difference between the upstream homologous fragment and the downstream homologous fragment is 0 to 10 bp; Preferably, the length difference between the upstream homologous fragment and the downstream homologous fragment is 0 to 5 bp.

7. The method according to claim 6, characterized in that, The full-length antisense oligonucleotide at the target modification site is not less than 10 bp; Preferably, the antisense oligonucleotide targeting the modification site has a length of 10–50 bp; Preferably, the antisense oligonucleotide at the target modification site has a length of 12–40 bp; Preferably, the antisense oligonucleotide at the target modification site has a length of 14–30 bp; Most preferably, the antisense oligonucleotide at the target modification site has a length of 17–21 bp.

8. The method according to any one of claims 6 to 7, characterized in that, At least one base of the antisense oligonucleotide at the target modification site is chemically modified, wherein the chemical modification is selected from one or more of the following groups: LNA, UNA, 2'-MOE, 2'-F, 2'-OMe, PNA, Morpholino, BNA, methylation modification, thiophosphate modification, and DNA base substitution modification. Preferably, the 3' and 5' ends of the antisense oligonucleotide targeting the modification site are modified by 1 to 5 bases; Preferably, the 3' and 5' ends of the antisense oligonucleotide targeting the modification site are modified by 1 to 3 bases.

9. The method according to claim 8, characterized in that, The antisense oligonucleotide at the target modification site is 17–21 nt in length, with the 3' end 3 bases modified by LNA, the 5' end 3 bases modified by LNA, the remaining bases modified by 2'OMe or MOE, and all bases modified by full-thiophosphate.

10. The method according to any one of claims 1 to 9, characterized in that, The antisense oligonucleotide targeting the modification site has a nucleic acid sequence as shown in any one of SEQ ID NO:1~22 or 65~70.

11. The method according to any one of claims 1 to 10, characterized in that, The method includes: transferring the antisense oligonucleotide into cells to inhibit RNA modification in the cells.

12. The method according to claim 11, characterized in that, The cells are human cells and / or animal cells, and the animal is a human, primate, rodent, feline, and / or canine.

13. The antisense oligonucleotide targeting the modification site as described in any one of claims 1 to 12.

14. A complex comprising Galnac conjugated with the antisense oligonucleotide of claim 13 or encapsulated with LNP.

15. A composition comprising the antisense oligonucleotide of claim 13 and the complex of claim 13.

16. A host cell comprising the antisense oligonucleotide of claim 12 and / or the complex of any one of claims 13.

17. A reagent for targeting RNA modification regulation, comprising an antisense oligonucleotide targeting a modification site as described in any one of claims 1 to 12, or the complex as described in claim 14, or the composition as described in claim 15, or the host cell as described in claim 16.

18. The reagent according to claim 17, characterized in that, It also includes reagents, adjuvants, or excipients required for transfection and / or transformation; The reagents include electroporation transfection reagents, ionotropic transfection reagents, and / or liposome transfection reagents; The adjuvants include at least one of the following: cytokines, co-stimulatory molecules, emulsifying adjuvants, Freund's adjuvants, saponins, monophosphate acyl lipids, TLR agonists, CpG oligonucleotides, oligodeoxynucleotides, and / or immunomodulatory proteins. The excipients include polyethylene glycol and / or polylysine.

19. The use of the antisense oligonucleotide of claim 13, or the complex of claim 14, or the composition of claim 15, the host cell of claim 16, or the reagent of claim 17 or 18, in the preparation of a medicament for the prevention and treatment of diseases related to RNA modification.

20. The application according to claim 19, characterized in that, The diseases associated with RNA modification include: tumors, nervous system diseases, immune system diseases, metabolic diseases, and / or infectious diseases.

21. The application according to claim 20, characterized in that, The tumors include malignant tumors and benign tumors, selected from at least one of breast cancer, lung cancer, colorectal cancer, stomach cancer, liver cancer, pancreatic cancer, ovarian cancer, prostate cancer, bladder cancer, and skin melanoma; The neurological diseases include at least one of Parkinson's disease, Alzheimer's disease, stroke, multiple sclerosis, meningitis, spinal cord injury, and brain tumor; The immune system diseases mentioned include at least one of rheumatoid arthritis, systemic lupus erythematosus, autoimmune thyroid disease, ulcerative colitis, Crohn's disease, scleroderma, and systemic sclerosis; The metabolic diseases mentioned include at least one of diabetes, obesity, hypertension, hyperlipidemia, hyperthyroidism, hypothyroidism, and osteoporosis. The infectious diseases mentioned include at least one of the following: AIDS, hepatitis B, hepatitis C, tuberculosis, malaria, dengue fever, cholera, influenza, and pneumonia.

22. A medicament for the prevention and treatment of diseases related to RNA modification, comprising an antisense oligonucleotide targeting a modification site as described in any one of claims 1 to 12, or the complex as described in claim 14, or the composition as described in claim 15, or the host cell as described in claim 16.

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