Inhibition of target mRNA expression using circular RNA

Abstract

The present invention addresses a novel method for rapidly degrading a target mRNA, and is a technology for artificially binding a circular RNA having EJC to a target mRNA 3' UTR, thereby reducing the expression of a corresponding gene through selective degradation of the target mRNA.

Description

Inhibition of target mRNA expression using circular RNA

[0001] The present invention relates to inhibition of target mRNA expression using circular RNA.

[0002]

[0003] The various biological functions occurring within eukaryotic cells are regulated by the central dogma, which links DNA to mRNA and then to protein. The stability and quality control of mRNA ultimately determine the quantity and quality of the resulting protein, and are tightly regulated within the cell.

[0004] In cells, there are not only linear mRNAs with the well-known 5' cap and 3' poly(A) tail structures, but also circular RNAs called circular RNAs. Circular RNAs are formed when back splicing occurs rather than general splicing, and unlike linear mRNAs, they do not have a 5' cap and 3' poly(A) tail, making them more resistant to exoribonuclease and thus more stable.

[0005] Meanwhile, nonsense-mediated mRNA decay (NMD) is a mechanism in which exon junction complex (EJC) factors and proteins that can induce nonsense-mediated mRNA decay, such as UPF1, UPF2, and PNRC2, form a complex and degrade incorrect mRNA with a premature termination codon (PTC) by endoribonuclease SMG6 and exoribonucleases SMG5 and SMG7.

[0006] Recently, there has been a growing interest in the development of disease treatments through mRNA stability control, with the potential for therapeutic applications of mechanisms that rapidly degrade the mRNA of genes associated with specific diseases, such as microRNA, thereby suppressing their expression.

[0007]

[0008] One object of the present invention is to provide a circular RNA that complementarily binds to the 3' untranslated region of a target mRNA and degrades the target mRNA.

[0009] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating apoptosis-related diseases, which comprises the circular RNA as an active ingredient.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating an autophagy-mediated disease, which comprises the above circular RNA as an active ingredient.

[0011]

[0012] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0013]

[0014] Various embodiments of the present invention are described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques are not described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to an embodiment means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearance of an embodiment in various places throughout this specification does not necessarily indicate the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0015]

[0016] In the present invention, we induced binding of circular RNA and mRNA based on base sequence. Through this, we revealed that when circular RNA with an EJC binds to mRNA, the NMD phenomenon is induced, resulting in mRNA degradation. This mechanism was named circNMD. Furthermore, in addition to this sequence-based circNMD ​​phenomenon, it is possible that circNMD ​​can also occur when binding is induced through various other methods.

[0017] First, circNMD ​​is induced by binding the two RNAs through the interaction between RNA-binding proteins that bind to circular RNA and mRNA, respectively. Specifically, as shown in Fig. 10(i-iii), since the exon junction complex (EJC) is located at the 3' UTR of the target mRNA, which is important for the circNMD ​​phenomenon, even if it is not necessarily a circular RNA that binds complementarily to the target mRNA, the circNMD ​​phenomenon, in which NMD is activated through various interactions between circular RNA and various RNA-binding proteins that bind to the target mRNA, can occur.

[0018] Second, a circular RNA having an exon junction complex (EJC) is synthesized in vitro and artificially positioned to form an exon junction complex (EJC). The circular RNA having the produced exon junction complex (EJC) is reintroduced into cells to induce circNMD. As a method for artificially positioning the EJC, an RNA aptamer capable of binding to an EJC component can be used. Alternatively, an EJC component protein fused to dCas13 and a guide RNA capable of base pairing with the circular RNA can be co-expressed to position the EJC in the circular RNA.

[0019]

[0020] 1. Circular RNA that complementarily binds to the target mRNA and degrades the target mRNA.

[0021] In one embodiment of the present invention, a circular RNA is provided that complementarily binds to a target mRNA and degrades the target mRNA.

[0022] In the present invention, the “circular RNA” refers to a circular RNA, not a linear mRNA having a generally well-known 5’ cap and 3’ poly(A) tail structure. Circular RNA is formed through back splicing rather than general splicing, and unlike linear mRNA, it does not have a 5’ cap and 3’ poly(A) tail, making it more stable by having exoribonuclease resistance.

[0023] In the present invention, the “target mRNA” refers not only to the mRNA of a gene related to a specific disease, but also to RNA encoding a specific gene. The target RNA may be, but is not limited to, tRNA (transfer RNA), mRNA (messenger RNA), rRNA (ribosomal RNA), various small noncoding RNAs, long noncoding RNAs, RNA genomes of pathogens such as viruses / bacteria or RNA produced by them, or ribo-polynucleotides artificially produced by genetic recombination methods. In particular, it may be RNA of a useful candidate substance in biotechnology, medicine, pharmacy, etc., or a substance that causes an action in a living body. Specifically, the target RNA may be a naturally occurring sequence or a newly modified sequence, and may be, but is not limited to, MS2 RNA of bacteriophage, trans-activation response element (TAR) RNA of HIV virus, or human iron-responsive element (IRE) RNA or a variant thereof.

[0024] The “target RNA” of the present invention can be used interchangeably with “target RNA”.

[0025] In some embodiments of the present invention, the circular RNA may have a sequence that complementarily binds to the target mRNA. This complementary binding may optionally include at least one mismatch. For example, the circular RNA may be a sequence that hybridizes or complementarily binds to the target mRNA, wherein the complementary binding may include 0 to 5 mismatches. Alternatively, the circular RNA may be a nucleotide sequence that is at least 70% complementary to the target mRNA.

[0026] In one embodiment, the circular RNA may be a sequence that is at least 70% to 75%, at least 70% to 80%, at least 70% to 85%, at least 70% to 90%, at least 70% to 95%, at least 70% to 100%, at least 75% to 80%, at least 75% to 85%, at least 75% to 90%, at least 75% to 95%, or at least 75% to 100% complementary to the target mRNA. Alternatively, the circular RNA may be a sequence that is at least 80% to 85%, at least 80% to 90%, at least 80% to 95%, at least 80% to 100%, at least 85% to 90%, at least 85% to 95%, or at least 85% to 100% complementary to the target mRNA. Alternatively, the circular RNA may be a sequence that is at least 90% to 95%, at least 90% to 100%, or at least 95% to 100% complementary to the target mRNA. Alternatively, the circular RNA may be a sequence that is at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementary to the target mRNA.

[0027] In another embodiment of the present invention, a circular nucleotide is provided, which comprises a sequence synthesized in vitro or generated through back-splicing within a cell so as to complementarily bind to the untranslated region (UTR) of a target mRNA to remove the target mRNA. The circular nucleotide provided in the present invention can remove the target mRNA by binding to the 3' untranslated region of the target mRNA, even if it is synthesized in vitro and injected into a cell or expressed within a cell through back-splicing.

[0028] In another embodiment of the present invention, the nucleotide is provided as a nucleotide that is RNA or DNA encoding the same.

[0029] In another embodiment of the present invention, the circular RNA or circular nucleotides provide a circular RNA that complementarily binds to the 3' untranslated region (UTR) of the target mRNA.

[0030] In another embodiment of the present invention, a nucleotide is provided that complementarily binds to a target mRNA located at least 100 nucleotides from the stop codon of the target mRNA. In another embodiment of the present invention, a nucleotide is provided that complementarily binds to a target mRNA located at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, or at least 300 nucleotides from the stop codon. If the nucleotide complementarily binds too close to the stop codon, the amount of mRNA cannot be reduced because the exon junction complex present at least 50-55 nucleotides after the stop codon of the mRNA cannot be recognized.

[0031] In the present invention, the “3' untranslated region (UTR)” refers to the portion of messenger RNA (mRNA) following the translation stop codon, and the 3' untranslated region often contains a regulatory region that affects post-transcriptional gene expression. Regulatory regions within the 3'-untranslated region can affect polyadenylation, translation efficiency, localization, and stability of mRNA, and the 3'-untranslated region contains binding sites for both regulatory proteins and microRNAs (miRNAs). miRNAs can reduce gene expression of various mRNAs by inhibiting translation or directly inducing transcript degradation by binding to specific sites within the 3'-untranslated region. The circular RNA according to the present invention can reduce gene expression of various mRNAs by inhibiting translation or directly inducing transcript degradation by binding to the 3'-untranslated region. The 3'-untranslated region also contains a silent region that binds to a repressor protein to inhibit mRNA expression. The 3'-untranslated region may also contain sequences that recruit proteins to associate the mRNA with the cytoskeleton, transport it to or from the nucleus, or perform other types of localization.

[0032] In another embodiment of the present invention, a circular RNA is provided so that an exon junction complex (EJC) can bind to the target mRNA through binding to the circular RNA.

[0033] In another embodiment of the present invention, a nucleotide is provided, wherein the circular nucleotide binds to exon junction complex (EJC) factors.

[0034] In the present invention, the “exon junction complex (EJC)” is a protein complex that binds to the junction of two exons joined together during RNA splicing. The EJC has a major influence on translation, surveillance, localization of spliced ​​mRNA, and m6A methylation. The EJC is first deposited on mRNA during splicing and then transported to the cytoplasm. There, the EJC plays a crucial role in post-transcriptional regulation of mRNA. The EJC is composed of a stable heterotetrameric core that serves as a binding platform for other components required for the mRNA pathway. The core of the EJC includes the protein eukaryotic initiation factor eIF4A3 (DEAD-box RNA helicase) bound to an adenosine triphosphate (ATP) analogue, and additional proteins MAGOH, MLN51, and Y14.

[0035] In another embodiment of the present invention, the exon junction complex provides a nucleotide selected from the group consisting of eIF4A3, MNL51, Y14, and MAGOH.

[0036] In other words, when mRNA is created from DNA through transcription in a eukaryotic cell, its stability is determined by various factors. mRNA stability varies depending on various factors, including mRNA structure, base sequence, mRNA-binding proteins, and internal RNA modifications. This stability leads to the regulation of gene expression.

[0037] The present invention describes a novel method for rapidly degrading target mRNA. When an exon junction complex-bound circular RNA (EJC-loaded circular RNA) or a circular nucleotide binds to the 3' untranslated region of a target mRNA, the mRNA becomes a target of nonsense-mediated mRNA decay (NMD), leading to rapid degradation and, as a result, a decrease in the expression of the corresponding gene.

[0038] In another embodiment of the present invention, the exon junction complex provides a circular RNA that binds to the circular RNA.

[0039] In the present invention, we constructed a circular RNA having a sequence that can complementarily bind to the 3' UTR of a target reporter mRNA whose RNA stability can be experimentally confirmed and an exon junction complex. In other words, we constructed a reporter system that rapidly degrades the mRNA by artificially binding the circular RNA having the exon junction complex to the 3' untranslated region of the target mRNA, and introduced this into a HeLa cell line. Thereafter, the amount of mRNA was quantitatively observed using quantitative real-time polymerase chain reaction (qRT-PCR). As a result, we discovered that the target mRNA was rapidly degraded by the circular RNA binding to the 3' untranslated region of the target mRNA.

[0040] In another embodiment of the present invention, the exon junction complex provides a circular RNA, wherein at least one of the exon junction complexes is selected from the group consisting of eIF4A3, MNL51, RNPS1, Y14, SRm160, Aly / REF, Magoh, and DEK.

[0041] In another embodiment of the present invention, the circular nucleotide provides a nucleotide that induces circNMD.

[0042] In the present invention, the term “circNMD” refers to a mechanism by which the NMD phenomenon is induced when a circular RNA having an EJC binds to mRNA, resulting in mRNA degradation. Specifically, in the present invention, binding of circular RNA and mRNA was induced based on base sequence. Through this, the mechanism by which the NMD phenomenon is induced when circular RNA binds to mRNA, resulting in mRNA degradation, was elucidated, and this mechanism was named circNMD. In addition to the circNMD ​​phenomenon based on base sequence, the circNMD ​​phenomenon may also occur when binding is induced through various other methods.

[0043] First, circNMD ​​is induced by binding the two RNAs through the interaction between RNA-binding proteins that bind to circular RNA and mRNA, respectively. Specifically, as shown in Fig. 10(i-iii), since the exon junction complex (EJC) is located at the 3' UTR of the target mRNA, which is important for the circNMD ​​phenomenon, even if it is not necessarily a circular RNA that binds complementarily to the target mRNA, the circNMD ​​phenomenon, in which NMD is activated through various interactions between circular RNA and various RNA-binding proteins that bind to the target mRNA, can occur.

[0044] Second, a circular RNA having an exon junction complex (EJC) is synthesized in vitro and artificially positioned to form an exon junction complex (EJC). The circular RNA having the produced exon junction complex (EJC) is reintroduced into cells to induce circNMD. As a method for artificially positioning the EJC, an RNA aptamer capable of binding to an EJC component can be used. Alternatively, an EJC component protein fused to dCas13 and a guide RNA capable of base pairing with the circular RNA can be co-expressed to position the EJC in the circular RNA.

[0045] In another embodiment of the present invention, the circNMD ​​provides a nucleotide which is due to nonsense mediated mRNA decay (NMD).

[0046] In another embodiment of the present invention, a circular RNA is provided wherein the target mRNA degradation is by nonsense mediated mRNA decay (NMD).

[0047] In the present invention, the “nonsense mediated mRNA decay (NMD)” is a mechanism in which proteins capable of inducing nonsense mediated mRNA decay, such as exon junction complex (EJC) factors and nonsense mediated mRNA decay factors UPF1, UPF2, and PNRC2, form a complex, and an endoribonuclease SMG6, an exoribonuclease SMG5, SMG7, etc. degrade an incorrect mRNA having a premature termination codon (PTC).

[0048] In another embodiment of the present invention, the circNMD ​​provides a nucleotide that is induced by binding the circular nucleotide to a target mRNA through interaction between the circular nucleotide and an RNA binding protein that binds to the target mRNA, respectively.

[0049] In another embodiment of the present invention, a composition for removing a target nucleotide is provided, comprising the above circular nucleotide.

[0050] In another embodiment of the present invention, a composition for removing a target nucleotide is provided, wherein the nucleotide is RNA or DNA encoding the same.

[0051]

[0052] In one embodiment of the present invention, a method for removing a target nucleotide is provided, comprising the aforementioned circular nucleotide as an active ingredient. When the nucleotide is produced extracellularly and administered to a cell, expressed intracellularly, or administered to a subject, the target nucleotide can be effectively removed.

[0053] In one embodiment of the present invention, a method is provided for removing a target nucleotide, wherein the nucleotide is RNA or DNA encoding the same.

[0054]

[0055] 2. Pharmaceutical composition for preventing or treating apoptosis-related diseases containing the above circular RNA as an active ingredient

[0056] In one embodiment of the present invention, a pharmaceutical composition for preventing or treating a disease related to apoptosis is provided, which comprises the circular RNA as an active ingredient.

[0057] In one embodiment of the present invention, a pharmaceutical composition for preventing or treating apoptosis-related disease is provided, which comprises a circular nucleotide as an active ingredient.

[0058] The circular RNA provided in the present invention complementarily binds to the BCL2L11 gene associated with apoptosis, thereby inhibiting the expression of the protein, thereby reducing apoptosis.

[0059] In the present invention, the protein encoded by the BCL2L11 gene belongs to the BCL-2 protein family. BCL-2 family members form heterodimers or homodimers and act as anti-apoptotic or pro-apoptotic regulators involved in various cellular activities. The protein encoded by this gene contains Bcl-2 homology domain 3 (BH3), which has been shown to interact with other members of the BCL-2 protein family, including BCL2, BCL2L1 / BCL-X(L), and MCL1, and to act as an apoptosis activator. The expression of this gene can be induced by nerve growth factor (NGF) as well as the forkhead transcription factor FKHR-L1 (FoxO3a), suggesting a role for this gene in neuronal and lymphocyte apoptosis. Transgenic studies in mouse counterparts suggest that this gene functions as an essential initiator of apoptosis in thymocyte negative selection.

[0060] In the present invention, the term "apoptosis" refers to a case where a cell naturally dies through a predetermined process. The aforementioned cell suicide, apoptosis, or cell death is a type of programmed cell death observed in multicellular organisms. Apoptosis is defined as the death of a cell due to changes in cell shape and internal biochemical properties. This process includes cell swelling and cracking, changes in the cell membrane, nuclear fragmentation, chromatin condensation and chromosome breakage, and culminates in the cell being eaten by another cell. In contrast to necrosis, which is cell death caused by sudden cell damage, apoptosis is not harmful to the organism and is beneficial to the life cycle. The formation of ten fingers and ten toes during the differentiation process of a human embryo is a representative example of the action of apoptosis. Many proteins in the cytoplasm are involved in the apoptosis pathway (CAD proteins), and these are bound to inhibitors under normal conditions and then activated by apoptosis-inducing substances.

[0061]

[0062] In another embodiment of the present invention, a pharmaceutical composition is provided, wherein the cell death-related disease is an autoimmune disease, graft-versus-host disease, organ transplant rejection, asthma, atopy, or an acute or chronic inflammatory disease.

[0063] To determine whether the same phenomenon occurs in mRNA and circular RNA present in actual cells, we analyzed microarray and RNA in situ conformation sequencing (RIC-seq) data to identify mRNA and circular RNA candidates with a high probability of binding to each other within cells. Among them, we confirmed that BCL2L11 mRNA reduces mRNA stability through binding to circular RNAs provided by the present invention, such as hsa_circ_0002082 or hsa_circ_0008496, and thereby affects intracellular functions such as apoptosis.

[0064] Unlike other cells in the body, approximately half of all neurons die during development. This is known to be due to competition for limited trophic factors, which selectively allows survival of only those cells that form precise synaptic connections with target neurons at synaptic terminals. Furthermore, studies examining cultured neurons and in vivo brains have reported that many neurons die when survival conditions in the adult brain deteriorate (e.g., due to external stress). In many cases, this has been confirmed to be due to apoptotic mechanisms. Therefore, the circular RNA provided by the present invention can exert preventive and therapeutic effects by inhibiting apoptosis for the neurodegenerative disease or neuroinflammatory disease that occurs according to the apoptosis mechanism, and the neurodegenerative disease or neuroinflammatory disease includes stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, nervous system autoimmune disease, spinocerebellar ataxia, inflammatory and neuropathic May be selected from the group consisting of, but not limited to, pain, cerebrovascular disease, spinal cord injury, and tauopathy.

[0065] In the present invention, the "autoimmune disease" refers to a disease manifested by autoimmunity. That is, the immune system mistakenly reacts to normal chemicals and certain body cells. In other words, the immune response mistakenly views healthy cells as harmful and attacks them, causing cell death, etc. The circular RNA provided in the present invention can effectively prevent or treat the autoimmune disease that causes cell death in healthy cells by inhibiting apoptosis. Examples of such autoimmune diseases include not only rheumatoid arthritis, but also type 1 diabetes (insulin-dependent), systemic lupus, Crohn's disease, and psoriasis.

[0066]

[0067] 3. Pharmaceutical composition for preventing or treating autophagy-mediated diseases containing the above circular RNA as an active ingredient

[0068] In one embodiment of the present invention, a pharmaceutical composition for preventing or treating an autophagy-mediated disease is provided, which comprises the circular RNA as an active ingredient.

[0069] In the present invention, the above “autophagy” is a life phenomenon that occurs within a eukaryotic cell, and is one of the life phenomena that maintains cellular homeostasis through destruction and recycling of cellular components. It is a term that refers to the overall process in which intracellular components are assembled into a double membrane called an autophagosome and decomposed by a lysosome.

[0070] In the present invention, a circular RNA containing a sequence that binds to naturally occurring ATG5 or ATG7 mRNA was produced and artificially overexpressed within cells. As a result, it was confirmed that the stability of the corresponding mRNA rapidly decreased. Furthermore, the effect of this process on the intracellular function of autophagy was confirmed.

[0071] Autophagy, as described above, is an intracellular degradation mechanism that maintains cellular homeostasis by degrading unnecessary or dysfunctional proteins, organelles, and nutrients within cells through lysosomes under various external stress environments. Autophagy, which plays a crucial role in maintaining cellular homeostasis, has been shown to be closely related to various diseases, including cancer, degenerative brain diseases, diabetes, and aging, through research in various animal and human disease models. Therefore, the regulation of autophagy is attracting attention as a key target for the development of new drug targets and therapies for various related diseases. In particular, autophagy has been shown to suppress cancer formation in normal cells during the development and growth of cancer. Furthermore, after cancer has formed, it is known to play a role in cancer growth and resistance to anticancer treatment. Therefore, understanding the precise regulatory mechanisms of autophagy according to cancer development and stage and developing modulators are receiving significant attention. In addition, recent studies have revealed that cancer stem cells (CSCs) are significantly dependent on autophagy activity in the process of maintenance, generation, and differentiation, unlike other cells. Therefore, it is expected that the regulation of autophagy will provide a new paradigm for cancer treatment.

[0072] In another embodiment of the present invention, a pharmaceutical composition is provided, wherein the autophagy-mediated disease is at least one selected from the group consisting of cancer, neurological disease, diabetes, myocardial apoptosis disease, obesity, and viral infection.

[0073] In the present invention, "cardiomyocyte apoptosis disease" refers to a cardiovascular disease caused by the death of cardiomyocytes. This is a general term for diseases of the circulatory system, such as the heart and blood vessels, and includes hypertension, ischemic heart disease, angina, myocardial infarction, arteriosclerosis, stroke, and arrhythmia, and is caused by factors such as age, diabetes, obesity, and smoking. While autophagy is an essential cellular mechanism for normal cardiovascular function, excessive autophagy can cause the aforementioned diseases.

[0074] In the present invention, cancer means a malignant tumor and a lump that has grown abnormally due to excessive growth. As examples of the cancer, the cancer may be gastric cancer, breast cancer, lung cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchogenic cancer. One or more selected from the group consisting of, but not limited to, bone marrow tumor.

[0075] In another embodiment of the present invention, a pharmaceutical composition is provided, wherein the neurological disease is a neurodegenerative disease or a neuroinflammatory disease.

[0076] In another embodiment of the present invention, the neurodegenerative disease or neuroinflammatory disease is at least one selected from the group consisting of stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, nervous system autoimmune disease, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and tauopathy. A pharmaceutical composition is provided.

[0077]

[0078] In addition, in the present invention, "treatment" may include, without limitation, any action that improves or benefits the symptoms of a disease by using the pharmaceutical composition of the present invention.

[0079] Meanwhile, but not limited thereto, the method for preventing or treating the disease may be a combination therapy further comprising administering a compound or substance having therapeutic activity against one or more diseases.

[0080] In the present invention, the term "combination" should be understood to refer to simultaneous, separate, or sequential administration. If the administration is sequential or separate, the interval between administrations of the secondary components should be such that the beneficial effects of the combination are not lost.

[0081] In the present invention, the pharmaceutical composition may be characterized as being in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be characterized as being intended for humans.

[0082] In the present invention, the pharmaceutical composition is not limited to these, but may be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, etc., external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, fragrances, etc. for oral administration, and buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. for injections, and bases, excipients, lubricants, preservatives, etc. for topical administration. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in the form of unit dose ampoules or multiple doses. In addition, it can be formulated in the form of solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

[0083] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may be additionally included.

[0084] The routes of administration of the pharmaceutical composition of the present invention include, but are not limited to, direct administration to the eye, orally, intravenously, intramuscularly, intraarterially, intramedullaryly, intrathecally, intracardiacly, transdermally, subcutaneously, intraperitoneally, intranasally, enterally, topically, sublingually, or rectally. Oral or parenteral administration is preferred. Direct administration to the eye is more preferred.

[0085] The term "parenteral" in the present invention includes direct administration to the eye, or subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration. Direct administration to the eye is preferred.

[0086] The "dosage and administration" of the composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug combination, and severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, pharmaceutical form, administration route, and period, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. The administration may be administered once a day or divided into several times. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, a sugar-coated tablet, a capsule, a liquid, a gel, a syrup, a slurry, or a suspension.

[0087]

[0088] The technology for promoting targeted mRNA degradation using circular RNA containing an exon junction complex (EJC) offers the advantages of easily identifying the desired target mRNA by modifying the binding sequence, and utilizing circular RNA that is resistant to intracellular exoribonucleases and thus more stable than linear RNA. Therefore, it is expected to be of great utility in the development of therapeutic technologies based on mRNA stability control.

[0089] Additionally, the exon junction complex (EJC) does not bind to the 3' UTR, and if it binds to a location other than the 3' UTR, NMD does not occur. However, when RNA is generated from DNA through transcription in a cell and circular RNA is generated through back-splicing, the exon junction complex (EJC) naturally binds to the circular RNA. If this circular RNA can bind to the 3' UTR of the target mRNA, NMD is activated, allowing the target mRNA to be specifically degraded.

[0090] Specifically, first, by modifying the linear RNA binding site of a circular RNA containing an exon junction complex (EJC), it is possible to target any desired mRNA without limitation. In other words, any mRNA can be selectively targeted simply by knowing its base sequence.

[0091] Second, it is highly stable because it utilizes circular RNA. Circular RNA is resistant to exoribonuclease, making it more stable than linear RNA. Therefore, using circular RNA offers superior therapeutic stability compared to linear RNA.

[0092] Third, it could contribute to the development of therapeutic technologies based on mRNA stability control. By specifically degrading target mRNA using artificially created circular RNA, it is possible to develop treatments for various diseases.

[0093]

[0094] Figure 1a shows a schematic diagram of the experiment of Example 1-1.

[0095] Figure 1b shows the results of an experiment showing that when a circular effector RNA that binds to a linear reporter mRNA is present together with the linear reporter mRNA, the amount of the linear reporter mRNA decreases.

[0096] Figure 1c shows the results of an experiment conducted in the same manner as Figure 1b, but with the direction of the binding base sequence changed.

[0097] Figure 2a shows a schematic diagram of the experiment of Example 1-2.

[0098] Figure 2b illustrates the relative amounts of circular effector RNA bound to linear reporter mRNA.

[0099] Figure 3a shows a schematic diagram of the experiment of Example 2.

[0100] Figure 3b shows the experimental results showing changes in the amount of each linear reporter mRNA when circular effector RNA is present together with each linear reporter mRNA binding to various sites.

[0101] Figure 4a shows a schematic diagram of the experiment of Example 3.

[0102] Figure 4b shows the results of a Western blotting experiment of various exon junction complex (EJC) factors that bind to purified reporter mRNA.

[0103] Figure 4c shows the results of Western blotting confirming immunoprecipitation with eIF4A3.

[0104] Figure 4d shows the amount of bound circular effector RNA when immunoprecipitated with eIF4A3.

[0105] Figure 4e shows the amount of linear reporter mRNA bound when immunoprecipitated with eIF4A3.

[0106] Figure 5a shows the results of an experiment observing changes in the amount of linear reporter mRNA when nonsense mediated mRNA decay (NMD) factors are reduced.

[0107] Figure 5b shows the results of an experiment observing changes in linear reporter mRNA stability due to a decrease in a key factor of nonsense mediated mRNA decay (NMD).

[0108] Figure 5c shows the results of an experiment in which the direction of the binding base sequence was changed to change the stability of a linear reporter mRNA according to a decrease in the NMD key factor.

[0109] Figure 6a shows a schematic diagram of the microarray experiment conducted in Example 5.

[0110] Figure 6b is a Venn diagram showing the number of linear RNAs and the number of circular RNAs found through the microarray.

[0111] Figure 6c shows the binding pairs between mRNA and circular RNA identified through RIC-seq.

[0112] Figure 7a is an experimental result showing the amount of target BCL2L11 mRNA when the amount of each circular RNA was reduced using siRNA.

[0113] Figure 7b is a graph quantitatively showing the percentage of apoptotic cells after reducing circular RNA.

[0114] Figure 7c shows an image of apoptotic cells stained after reducing circular RNA.

[0115] Figure 8a shows a schematic diagram of the circular effector RNA produced in Example 6-2.

[0116] Figure 8b shows the amount of BCL2L11 mRNA when circular RNA was overexpressed.

[0117] Figure 8c is a graph showing the quantitative results of the proportion of cells that overexpressed circular RNA and experienced apoptosis.

[0118] Figure 8d shows an image of cells overexpressing circular RNA and staining apoptotic cells.

[0119] Figure 9a is a schematic diagram of two circular effector RNAs designed to bind to the ATG5mRNA 3'UTR.

[0120] Figure 9b is an experimental result showing that the amount of ATG5mRNA decreases when two types of circular effector RNAs are expressed separately and together, and that the effect is further enhanced when two types of circular effector RNAs are expressed together.

[0121] Figure 9c is an experimental result showing that as mRNA decreases, the amount of the corresponding protein also decreases, thereby inhibiting autophagy.

[0122] Fig. 10 is a schematic diagram illustrating the present invention. Specifically, Fig. 10(i) illustrates the previously known nonsense mediated mRNA decay (NMD), and Fig. 10(ii) is a diagram illustrating that the circular RNA provided by the present invention binds to the 3'-untranslated region (UTR) of the target RNA together with the exon junction complex (EJC) to activate nonsense mediated mRNA decay (NMD). This phenomenon is named circNMD ​​in the present specification. Fig. 10(iii) is a diagram illustrating that the circular RNA provided by the present invention and an RNA binding protein that commonly binds to the target mRNA bind to the two RNAs to induce circNMD.

[0123] Figure 11 shows the results confirming that when the binding of linear reporter mRNA and circular effector RNA is blocked, the amount of linear mRNA does not decrease. Specifically, Figure 11a is a schematic diagram of the experiment, and Figure 11b is an experimental result showing that when an ASO that binds to linear reporter mRNA or circular effector RNA is treated, the linear reporter mRNA does not bind to circular effector RNA, so the amount of linear reporter mRNA does not decrease.

[0124] Figure 12 shows the results confirming that the phenomenon of linear reporter mRNA reduction does not appear when the formation of normal circular effector RNA is inhibited. Specifically, Figure 12a is a schematic diagram of the experiment, Figure 12b is an experimental result showing that the phenomenon of linear reporter mRNA amount reduction appears only when circular effector RNA is produced, and Figure 12c is the result of the experiment of Figure 12b by changing the direction of the binding sequence.

[0125] Figure 13 shows the results confirming that the linear mRNA reduction phenomenon appears only when the circular effector RNA binds to a position 100 nucleotides or more away from the stop codon. Specifically, Figure 13a is a schematic diagram of the experiment, and Figure 13b is the results of an experiment confirming that when the circular RNA is allowed to bind to various positions of the linear reporter mRNA 3'UTR, the amount of linear reporter mRNA decreases only when the circular effector RNA binds to a position 100 nucleotides or more away from the stop codon of the linear reporter mRNA.

[0126] Figure 14 shows the results confirming that the more circular effector RNA binding sites there are, the greater the decrease in linear reporter mRNA. Specifically, Figure 14a is a schematic diagram of the experiment, and Figure 14b shows the experimental results confirming that the more sites capable of binding circular effector RNA there are, the greater the decrease in the amount of linear reporter mRNA.

[0127] Figure 15 shows the results confirming that the amount of linear reporter mRNA decreases more significantly as the number of circular effector RNAs binding to linear reporter mRNA increases. Specifically, Figure 15a is a schematic diagram of the experiment, and Figure 15b is an experimental result showing that the amount of linear reporter mRNA decreases more significantly as the number of circular effector RNAs binding to linear reporter mRNA increases.

[0128] Figure 16 shows the results confirming that apoptosis increases when the binding of circular RNA and target mRNA is blocked. Specifically, Figure 16a is a schematic diagram of ASO specific to the binding sites of hsa_circ_0008496 and hsa_circ_0002082, Figure 16b is a graph showing the increased amount of BCL2L11mRNA after ASO treatment, Figure 16c is a graph quantitatively showing the percentage of apoptotic cells after ASO treatment, and Figure 16d is a stained image of apoptotic cells after ASO treatment.

[0129]

[0130] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0131]

[0132] [Example]

[0133] [Example 1] Reduction of linear reporter mRNA by circular RNA binding

[0134] [Example 1-1] Confirmation of reduction in linear reporter mRNA amount due to circular RNA binding

[0135] For nonsense-mediated mRNA decay (NMD) to occur, it is important for the exon junction complex (EJC) to bind to the 3' untranslated region (UTR), downstream of the stop codon. Considering this, as shown in Fig. 1a, a linear reporter mRNA and a circular effector RNA were constructed so that a circular RNA containing the exon junction complex can bind to the 3' untranslated region (UTR), downstream of the stop codon of the mRNA, through back-splicing.

[0136] That is, circular effector antisense AS21, which complementarily binds to 21 base pairs in the 3' untranslated region of linear reporter mRNA, and circular effector antisense AS30, which binds to 30 base sequences, were expressed together with the linear reporter in a HeLa cell line, and the amount of each linear reporter mRNA was confirmed.

[0137]

[0138] As a result, as shown in Fig. 1b and Fig. 1c, when a circular effector RNA capable of binding to a linear reporter mRNA was expressed together with the linear reporter mRNA, a decrease in the amount of the reporter mRNA was observed, whereas no change in the amount was observed with a circular effector RNA having a base sequence that cannot bind.

[0139]

[0140] [Example 1-2] Confirmation of binding of linear reporter mRNA and circular RNA

[0141] To confirm whether the circular effector RNA co-expressed directly binds to the linear reporter mRNA, an experiment was conducted. As shown in Figure 2a, a reporter mRNA containing the MS2 binding sequence (MS2bs) to which the MS2-HA-MBP protein can bind, the MS2-HA-MBP protein, and circular effector RNAs with three different binding sequences were co-expressed in cells.

[0142]

[0143] As a result, as shown in Fig. 2b, when an MBP pull-down assay was performed using amylose beads that strongly bind to MBP, it was found that circular effector RNA having a base sequence complementary to the linear reporter mRNA bound most to the linear reporter mRNA.

[0144]

[0145] [Example 1-3] Confirmation of inhibition of binding of linear reporter mRNA and circular effector RNA using antisense oligonucleotides.

[0146] To prevent binding of linear reporter mRNA and circular effector RNA, antisense oligonucleotides (ASOs) that bind to linear reporter mRNA or circular effector RNA, respectively, were prepared, as shown in Fig. 11a.

[0147]

[0148] As a result, as shown in Fig. 11b, it was confirmed that when the binding of linear reporter mRNA and circular effector RNA was prevented using an antisense oligonucleotide (ASO), the effect of reducing linear reporter mRNA did not appear.

[0149]

[0150] [Example 1-4] Confirmation of the effect of linear reporter mRNA reduction according to the form of effector RNA.

[0151] The back-splicing process that generates circular RNA occurs through the repetitive sequences at both ends of the exon. Therefore, as shown in Figure 12a, we compared cases where repetitive sequences exist at both ends of the exon, resulting in normal circular effector RNA production, with cases where one repetitive sequence was removed, preventing the proper production of circular effector RNA.

[0152]

[0153] As a result, as shown in Figures 12b and 12c, when a normally produced circular effector RNA binds to a linear reporter mRNA, the amount of the linear reporter mRNA decreases, whereas when a normal circular effector RNA is not produced due to the removal of the repeat sequence, the amount of the linear mRNA does not decrease.

[0154]

[0155] [Example 2] Reduction of linear reporter mRNA according to the position and number of the above bonds.

[0156] [Example 2-1] Reduction of linear reporter mRNA specifically induced only when the above binding is present in the 3' UTR

[0157] We aimed to determine at which position of a linear reporter mRNA a circular effector RNA binds to reduce the amount of the linear reporter mRNA. As shown in Figure 3a, each linear reporter mRNA was constructed to allow circular effector RNA to bind to the 5' untranslated region, coding sequence (CDS), and 3' untranslated region of the linear reporter mRNA.

[0158]

[0159] As a result, as shown in Fig. 3b, it was observed that the amount of linear reporter mRNA was reduced only when circular effector RNA was bound to the 3' untranslated region of the linear reporter mRNA, and that no reduction was observed when bound to other regions.

[0160]

[0161] [Example 2-2] Confirmation of the linear reporter mRNA reduction effect that is specifically induced only when the above combination is located at a distance of 100 nucleotides or more from the stop codon.

[0162] Nonsense-mediated mRNA decay occurs by recognizing the exon junction complex, which exists 50-55 nucleotides downstream from the mRNA stop codon. Therefore, as shown in Figure 13a, the reduction of the linear reporter mRNA was confirmed by binding a circular effector RNA containing the exon junction complex to various regions of the 3'UTR of the linear mRNA.

[0163]

[0164] As a result, as shown in Fig. 13b, it was observed that the circular effector RNA reduced the amount of linear reporter mRNA only when it was bound at least 100 nucleotides away from the stop codon of the linear reporter mRNA.

[0165]

[0166] [Example 2-3] Confirmation of the enhanced linear reporter mRNA reduction effect when the number of binding sites is increased.

[0167] To determine whether increasing the number of circular effector RNA binding sites in the 3'UTR of the linear reporter mRNA enhances the reduction effect of the linear reporter mRNA, binding base sequences were inserted at positions 100, 150, and 200 nucleotides away from the stop codon, as shown in Fig. 14a.

[0168]

[0169] As a result, as shown in Fig. 14b, it was observed that the decrease in linear reporter mRNA was greater when there were three sites where the circular effector RNA could bind than when there was one site.

[0170]

[0171] [Example 2-4] Confirmation of the phenomenon of a decrease in the amount of enhanced linear reporter mRNA according to the number of circular effector RNAs binding to the linear reporter mRNA.

[0172] To determine whether the effect of reducing the amount of linear reporter mRNA is enhanced when multiple circular effector RNAs that bind to linear reporter mRNA are introduced, three circular effector RNAs that bind to sites 100, 150, and 200 nucleotides away from the stop codon were produced, as shown in Fig. 15a.

[0173]

[0174] As a result, as shown in Fig. 15b, it was observed that the amount of linear reporter mRNA decreased more significantly when all three circular effector RNAs were expressed together than when each circular effector RNA was expressed individually.

[0175]

[0176] [Example 3] Binding of linear reporter mRNA to exon junction complex (EJC) via circular effector RNA

[0177] [Example 3-1] Confirmation of binding of linear reporter mRNA and circular effector RNA using MBP pull-down assay

[0178] We aimed to determine whether the circular effector RNA attached to the linear reporter mRNA contained an exon junction complex (EJC). Based on the experiment conducted in Fig. 2a, we attempted to identify exon junction complex (EJC) factors present in the pulled-down linear reporter mRNA using Western blotting, as shown in Fig. 4a.

[0179]

[0180] As shown in Fig. 4b, the experimental results confirmed that the key factors of the exon junction complex (EJC), eIF4A3, Y14, MAGOH, and MLN51, were present only when a circular effector RNA having a base sequence that binds to a linear reporter mRNA was present. This means that the linear reporter mRNA associates with the exon junction complex (EJC) through binding to the circular effector RNA.

[0181]

[0182] [Example 3-2] Confirmation of binding of linear reporter mRNA and circular effector RNA using exon junction complex (EJC) immunoprecipitation (IP)

[0183] To confirm whether circular RNA having an exon junction complex (EJC) binds to a linear reporter mRNA, as shown in Fig. 4a, immunoprecipitation (IP) was performed with eIF4A3, one of the key factors of the exon junction complex (EJC), to measure the amount of bound linear reporter mRNA.

[0184]

[0185] As a result of the experiment, as shown in Figures 4c to 4e, it was confirmed that eIF4A3, one of the major factors of the exon junction complex (EJC), binds to circular RNA, and that the circular RNA binds to linear reporter mRNA.

[0186]

[0187] [Example 4] Nonsense-mediated mRNA decay (NMD) of linear reporter mRNA induced by circular effector RNA

[0188] We sought to determine whether the decrease in the amount of linear reporter mRNA caused by circular effector RNA binding to the 3' untranslated region (UTR) of linear reporter mRNA was due to nonsense mediated mRNA decay (NMD).

[0189] That is, as shown in Figures 5a, 5b, and 5c, after reducing the protein amount of nonsense mediated mRNA decay (NMD) factors using siRNA, an experiment was conducted to see whether a phenomenon of reducing the amount of linear reporter mRNA by circular effector RNA was observed.

[0190]

[0191] As a result of the experiment, as shown in Fig. 5a, when the nonsense mediated mRNA decay (NMD) factors were reduced, the decrease in the amount of linear reporter mRNA caused by circular effector RNA was not observed, and as shown in Fig. 5b, the stability of the linear reporter mRNA was also observed to increase. This means that the degradation of linear reporter mRNA caused by linear effector RNA is due to nonsense mediated mRNA decay (NMD). And this phenomenon in which circular RNA having an exon junction complex (EJC) binds to the 3' untranslated region (UTR) of linear mRNA and nonsense mediated mRNA decay (NMD) occurs was named nonsense mediated mRNA decay (NMD) intended by circular RNA, i.e., circNMD.

[0192]

[0193] [Example 5] Identification of circNMD ​​target mRNAs and circular RNAs using microarrays and RIC-seq.

[0194] Microarray is an experimental method that uses fluorescent dyes to identify the types and quantities of mRNA present within cells. RIC-seq is an experimental method that identifies RNAs that bind to each other via RNA-binding proteins. The data obtained from these two experiments were comprehensively analyzed to identify mRNAs and circular RNAs that bind to each other within cells.

[0195]

[0196] As a result, as shown in Figures 6a and 6b, when immunoprecipitation was performed with cap binding protein 80 (CBP80), a protein that binds to the structure of the 5' cap of mRNA, and eukaryotic translation initiation factor 4E (eIF4E), the mRNA and circular RNA that were bound were confirmed through microarray.

[0197] Furthermore, to confirm the influence of circNMD, as shown in Figure 6c, only mRNAs that increased when the amount of SMG6 was reduced were analyzed. Furthermore, circular RNAs binding to mRNAs were analyzed using RIC-seq data analysis, which reveals RNA-RNA binding. This allowed us to determine which mRNAs bind to which circular RNAs.

[0198]

[0199] [Example 6] Identification of the intracellular function of the CircNMD ​​mechanism

[0200] [Example 6-1] Effects of circular RNA reduction on cells – Reduction of circular RNA increases cell death.

[0201] The hsa_circ_0002082 and hsa_circ_0008496 circular RNAs found through Fig. 6, confirmed in Example 5 above, commonly bind to the 3' untranslated region (UTR) of BCL2L11 mRNA and induce circNMD ​​of BCL2L11 mRNA. In addition, the BCL2L11 protein plays a role in increasing apoptosis.

[0202] Therefore, as shown in Fig. 7, in order to reduce the amount of hsa_circ_0002082 and hsa_circ_0008496 present in the cell, the amount of each circRNA was reduced using circular RNA-specific siRNA, and then the change in the amount of BCL2L11 mRNA was observed. In order to observe the degree of cell death according to the change, a TUNEL assay (Terminal deoxynucleotidyl transferase dUTP nick end labeling assay; TUNEL assay) experiment was performed to confirm the degree of cell death within the cell.

[0203]

[0204] As a result of the experiment, as shown in Figures 7a to 7c, it was observed that when each circular RNA was reduced, the amount of the target BCL2L11 mRNA increased, and accordingly, it was confirmed that cell death increased.

[0205]

[0206] [Example 6-2] Effect of binding to circular RNA on cells – Interfering with binding of circular RNA to target mRNA increases cell death.

[0207] As shown in Fig. 16a, we observed how cell death changed when ASO that inhibits the binding of BCL2L11mRNA and hsa_circ_0008496 and ASO that inhibits the binding of BCL2L11mRNA and hsa_circ_0002082 were treated separately and together.

[0208]

[0209] As shown in the experimental results in Figures 16b to 16d, when ASO specific to the binding site of each circular RNA was treated, it was observed that the amount of BCL2L11mRNA increased, and accordingly, it was confirmed that cell death increased.

[0210]

[0211] [Example 6-3] Effects of circular RNA overexpression on cells – Overexpression of circular RNA reduces cell death.

[0212] In order to confirm the difference in the degree of cell death when the amount of circular RNA was increased, as opposed to Fig. 7 of the above Example 6-1, two types of circular RNAs capable of binding to BCL2L11 mRNA were introduced into cells, as shown in Fig. 8a. Two types of circular RNAs, circG-BCL2L11-c8496 and circG-BCL2L11-c2082, were prepared to be able to bind to endogenous BCL2L11 mRNA. Next, after treatment with the drug ABT-737, which induces cell death, the effects of the introduced circular RNAs were observed.

[0213]

[0214] As a result of the experiment, as shown in Figures 8b to 8d, it was observed that the amount of BCL2L11 mRNA increased by treatment with ABT-737 and that cell death decreased again when circular RNA capable of binding to BCL2L11 mRNA was introduced.

[0215]

[0216] [Example 6-4] circNMD ​​effect by artificially inserted circular RNA – reduction in mRNA amount of a specific gene

[0217] We aimed to determine whether the amount of target mRNA could be selectively reduced through the circNMD ​​mechanism by artificially introducing circular RNAs that bind to mRNA present in cells, synthesized in vitro. To this end, ATG5 is a protein that functions to increase autophagy. To determine whether the amount of target mRNA could be reduced by artificially introducing circular effector RNAs that bind to mRNA present in cells, two types of circular RNAs capable of binding to the 3'UTR of ATG5 mRNA were produced, as shown in Figure 9a.

[0218] Meanwhile, we experimentally examined whether introducing circular effector RNA into cells reduced the amount of ATG5 mRNA and the resulting decrease in autophagy. Furthermore, we examined whether the effect was further enhanced when various circular effector RNAs were added.

[0219]

[0220] As a result, as shown in FIG. 9b and FIG. 9c, it was revealed that the amount of a specific mRNA can be reduced by artificially expressing a circular effector RNA, and the amount of the corresponding protein can also be reduced, thereby regulating an intracellular function called autophagy.

[0221]

[0222] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0223]

Claims

1. A circular nucleotide synthesized in vitro or created through back-splicing within a cell so that it can complementarily bind to the untranslated region (UTR) of a target mRNA to remove the target mRNA.

2. In paragraph 1, The above nucleotides are nucleotides, which are RNA or DNA encoding it.

3. In paragraph 2, The above nucleotide is a nucleotide that complementarily binds to the 3' untranslated region (UTR) of the target mRNA.

4. In paragraph 3, The above nucleotide is a nucleotide that complementarily binds to a target mRNA located at least 100 nucleotides away from the stop codon of the target mRNA.

5. In paragraph 4, The above circular nucleotide is a nucleotide that binds to exon junction complex (EJC) factors.

6. In paragraph 5, The above exon junction complex comprises at least one nucleotide selected from the group consisting of eIF4A3, MNL51, Y14, and MAGOH.

7. In paragraph 6, The above circular nucleotide is a nucleotide that induces circNMD.

8. In paragraph 7, The above circNMD ​​is a nucleotide that is caused by nonsense mediated mRNA decay (NMD).

9. In paragraph 8, The above circNMD ​​is a nucleotide that is induced by binding the circular nucleotide and the target mRNA through the interaction between the circular nucleotide and the RNA binding protein that binds to the target mRNA, respectively.

10. A composition for removing a target nucleotide, comprising a circular nucleotide of any one of claims 1 to 9.

11. In paragraph 10, A composition for removing a target nucleotide, wherein the nucleotide is RNA or DNA encoding the same.

12. A method for removing a target nucleotide, comprising a circular nucleotide of any one of claims 1 to 9.

13. In paragraph 12, A method for removing a target nucleotide, wherein the target nucleotide is RNA or DNA encoding the same.

14. A pharmaceutical composition for preventing or treating a disease related to apoptosis, comprising the circular nucleotides of claims 1 to 9 as an active ingredient.

15. In paragraph 14, A pharmaceutical composition wherein the above cell death-related disease is an autoimmune disease, a brain and nervous system disease, a graft-versus-host disease, an organ transplant rejection reaction, asthma, atopy, or an acute or chronic inflammatory disease.

16. In paragraph 15, A pharmaceutical composition wherein the above brain and nervous system disease is a neurodegenerative disease or a neuroinflammatory disease.

17. In paragraph 16, A pharmaceutical composition, wherein the neurodegenerative disease or neuroinflammatory disease is at least one selected from the group consisting of stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, nervous system autoimmune diseases, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and tauopathy.

18. A pharmaceutical composition for preventing or treating an autophagy-mediated disease, comprising the circular nucleotides of claims 1 to 9 as an active ingredient.

19. In Article 18, A pharmaceutical composition wherein the autophagy-mediated disease is at least one selected from the group consisting of cancer, neurological disease, diabetes, myocardial apoptosis disease, obesity, and viral infection.

20. In paragraph 19, A pharmaceutical composition wherein the cancer is gastric cancer, liver cancer, glioma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma or lung cancer.

21. A method for preventing or treating a disease related to apoptosis, comprising a step of administering to a subject the nucleotides in the circular form of claims 1 to 9.

22. In paragraph 21, The above cell death-related disease is an autoimmune disease, a brain and nervous system disease, graft-versus-host disease, organ transplant rejection, asthma, atopy, or an acute or chronic inflammatory disease.

23. In paragraph 22, A method wherein the above brain and nervous system disease is a neurodegenerative disease or a neuroinflammatory disease.

24. In paragraph 23, A method according to claim 1, wherein the neurodegenerative disease or neuroinflammatory disease is at least one selected from the group consisting of stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, autoimmune nervous system diseases, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and tauopathy.

25. A method for preventing or treating an autophagy-mediated disease, comprising a step of administering to a subject the circular nucleotides of claims 1 to 9.

26. In paragraph 25, A method wherein the above autophagy-mediated disease is at least one selected from the group consisting of cancer, neurological disease, diabetes, myocardial apoptosis disease, obesity, and viral infection.

27. In paragraph 26, The method wherein the cancer is gastric cancer, liver cancer, glioma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma or lung cancer.

Images