AgRNAs and methods for treating hepatolenticular degenerative diseases

Abstract

The application belongs to the field of biological medicine, and relates to ADAR guide RNA and a method for treating hepatolenticular degeneration. The ADAR guide RNA provided by the application corrects a pathogenic mutation of an ATP7B gene through RNA editing technology, and restores protein function.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an ADAR guide RNA and method for treating Wilson's disease. Background Technology

[0002] Wilson's disease (WD), also known as hepatolenticular degeneration, is a copper metabolism disorder caused by mutations in the ATPase copper transporting beta (ATP7B) gene. The ATP7B gene, located on chromosome 13q14.3, encodes a protein containing 1465 amino acids, 20 introns, and 21 exons, including a phosphatase domain (PD), a phosphorylation domain, and eight transmembrane ion channels. ATP7B is primarily expressed on the Golgi apparatus membrane of hepatocytes and is responsible for copper transport and excretion. Normally, copper is absorbed from the intestine and transported to hepatocytes via the portal vein. It enters the hepatocyte via copper transporter 1 (CTR1), binds to a molecular chaperone, and is transported to the corresponding protein. Copper ions are then transported to ATP7B via antioxidant protein 1 (Atox1), and then transported to the Golgi apparatus, where they bind to ceruloplasmin precursors to form functional ceruloplasmin, which is then secreted into the bloodstream. When ATP7B function is impaired, the binding and transport capacity of ceruloplasmin decreases, leading to copper accumulation in hepatocytes and causing hepatocyte damage, fibrosis, and cirrhosis. Copper can also deposit in the brain, kidneys, cornea, and joints, causing corresponding clinical manifestations. Unbound ceruloplasmin has a shortened half-life in the blood, resulting in decreased serum ceruloplasmin levels in WD patients.

[0003] Despite the availability of treatments, managing Wilson's disease remains a significant challenge. Primary treatments include copper chelators (such as penicillamine and triethanolamine) and zinc salts, which help the body excrete excess copper. However, these medications typically require lifelong use and can have serious side effects, such as skin and kidney problems, worsening neurological symptoms, and bone marrow suppression. For patients with severe liver damage, liver transplantation may be the only option, but this is an invasive procedure, and donor liver availability is limited. Furthermore, existing treatments present challenges in managing neuropsychiatric symptoms, as these symptoms can overlap with common mental illnesses, leading to diagnostic delays. In summary, while current treatments can control copper metabolism, they cannot completely correct mutations in the ATP7B gene, thus necessitating more effective treatment strategies.

[0004] RNA editing technology can precisely repair disease-causing mutation sites, restore the function of the ATP7B protein, thereby improving copper transport and excretion, reducing copper accumulation in hepatocytes and other tissues, and alleviating Wilson's disease symptoms. RNA editing technology is highly efficient, precise, and reversible, and can target specific mutation sites for repair, offering new possibilities for the treatment of Wilson's disease. Summary of the Invention

[0005] In some embodiments, the present invention provides an ADAR guide RNA comprising any one of the sequences shown in SEQ ID NO: 4-33, wherein the ADAR guide RNA is modified.

[0006] In some implementations, the modification includes skeletal modification, sugar modification, or base modification.

[0007] In some implementations, the modifications include 2'F modification, 2'OMe modification, thiophosphate bond modification, locked nucleic acid modification, or deoxyribose modification.

[0008] In some implementations, the ADAR guide RNA has a sequence length of 10-200 bases.

[0009] In some implementations, the ADAR guide RNA has a sequence length of 15-150 bases.

[0010] In some implementations, the ADAR guide RNA has a sequence length of 20-100 bases.

[0011] In some implementations, the ADAR guide RNA has a sequence length of 25-50 bases.

[0012] In some implementations, the ADAR guide RNA can bind to the target RNA to form a structure capable of recruiting ADAR within the cell, thereby editing the target adenosine in the target RNA through the recruited ADAR.

[0013] In some implementations, the distance between the 3' end of the ADAR guide RNA and the target adenosine is 1 to 40 nt.

[0014] In some implementations, the distance between the 3' end of the ADAR guide RNA and the target adenosine is 2-35 nt.

[0015] In some implementations, the distance between the 3' end of the ADAR guide RNA and the target adenosine is 5-35 nt.

[0016] In some implementations, the target RNA is the ATP7B transcript.

[0017] In some implementations, the target RNA is the human ATP7B transcript.

[0018] In some implementations, the target RNA contains the 1531C>T mutation.

[0019] In some implementations, the target RNA encodes a copper-transporting ATPase β.

[0020] In some implementations, the target adenosine is at base 1532A of the human ATP7B transcript.

[0021] In some implementations, the editing is ADAR-mediated A-to-I editing.

[0022] In some implementations, the ADAR is selected from ADAR1 or ADAR2.

[0023] In some implementations, the editing is capable of mutating 1532A to I in the human ATP7B transcript.

[0024] In some implementations, the ADAR guide RNA is either fully complementary or partially complementary to the target RNA.

[0025] In some implementations, the imperfect complementary pairing is a complementary pairing that has one or more mismatches, wobbles, omissions, and / or protrusions with respect to the target region.

[0026] In some implementations, the ADAR guide RNA has at least one mismatch with the target RNA.

[0027] In some embodiments, the complementary strands are base-complementary paired at non-mismatched, non-deleted, non-protruding, non-inner-loop, or non-wobbly base-pairing sites.

[0028] In some implementations, the ratio of complementary base pairs in the double-stranded RNA formed by the ADAR guide RNA and the target RNA is greater than 60%.

[0029] In some implementations, the ratio of complementary base pairs in the double-stranded RNA formed by the ADAR guide RNA and the target RNA is greater than 75%.

[0030] In some embodiments, the ADAR guide RNA has the same base sequence as shown in SEQ ID NO: 4~33.

[0031] In some implementations, the sequence of the ADAR guide RNA is shown in SEQ ID NO: 4~33.

[0032] In some embodiments, the present invention provides a vector comprising a polynucleotide encoding or containing the ADAR guide RNA.

[0033] In some implementations, the vector is a recombinant expression vector.

[0034] In some implementations, the vector includes a plasmid or a viral vector.

[0035] In some implementations, the carrier is a delivery carrier.

[0036] In some embodiments, the vector is a plasmid or viral vector for expression in higher eukaryotic or prokaryotic cells.

[0037] In some embodiments, the present invention provides compositions comprising plasmids or viral vectors encoding or containing the ADAR guide RNA.

[0038] In some embodiments, the present invention provides a host cell containing the ADAR guide RNA, the vector, or the composition.

[0039] In some embodiments, the present invention provides a pharmaceutical composition comprising the ADAR guide RNA and pharmaceutically acceptable excipients.

[0040] In some embodiments, the present invention provides the use of the ADAR guide RNA, the vector, the composition of claim 5, the host cell, or the pharmaceutical composition in the preparation of a medicament for altering the expression of a target protein in the cells of a subject, the cells having a target RNA encoding the target protein, the target RNA being a human ATP7B transcript containing the 1531C>T mutation.

[0041] In some implementations, the target protein is copper-transporting ATPase β.

[0042] In some implementations, the drug is a drug used to treat Wilson's disease.

[0043] In some implementations, the ADAR guide RNA can bind to the target RNA to form a structure capable of recruiting ADAR within the cell, thereby editing the target adenosine in the target RNA through the recruited ADAR.

[0044] In some implementations, the target adenosine is at base 1532A of the human ATP7B transcript.

[0045] In some implementations, the editing is ADAR-mediated A-to-I editing.

[0046] In some implementations, the ADAR is selected from ADAR1 or ADAR2.

[0047] In some implementations, the editing is capable of mutating 1532A to I in the human ATP7B transcript.

[0048] In some implementations, the agRNA provided by this invention restores the normal function of the ATP7B protein by correcting the stop codon in the ATP7B mutant gene, thereby improving the patient's symptoms and prognosis.

[0049] In some implementations, the agRNA design strategy provided by this invention demonstrates the innovation and flexibility of gene editing technology in the treatment of hereditary diseases. By restoring protein function through alteration of adjacent sites rather than directly repairing mutated sites, it offers new insights for future gene therapy. Attached Figure Description

[0050] Figure 1 This illustrates the editing level of agRNAs (SEQ ID NO: 4~SEQ ID NO: 8) with different nucleotide base sequences and various chemical modifications provided in Example 1 of this application in HeLa cells carrying a mutant ATP7B gene fragment.

[0051] Figure 2 This illustrates the editing level of agRNAs (SEQ ID NO: 9~SEQ ID NO: 14) with different nucleotide base sequences and various chemical modifications provided in Example 2 of this application in HeLa cells carrying a mutant ATP7B gene fragment.

[0052] Figure 3 This demonstrates the editing level of agRNAs (SEQ ID NO:15~SEQ ID NO:33) with different lengths, different nucleotide sequences, and various chemical modifications provided in Example 3 of this application in HeLa cells carrying a mutant ATP7B gene fragment.

[0053] Figure 4 The illustration shows the editing level of the agRNA (SEQ ID NO: 4) provided in Example 4 of this application compared with the agRNA (SEQ ID NO: 34~SEQ ID NO: 35) designed using the prior art RESTORE strategy in different cell lines carrying the mutated ATP7B gene fragment. Detailed Implementation

[0054] The following specific embodiments further illustrate the technical solution of the present invention. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0055] The technical terms used in this field are explained below: The term "ADAR guide RNA," abbreviated as "agRNA," typically refers to artificially synthesized single-stranded or double-stranded oligonucleotides. AgRNAs can be modified to alter their structure, binding sites, and other characteristics. In this disclosure, agRNA can be single-stranded, and its function can be to pair complementaryly with a specific sequence of the target RNA for editing.

[0056] The term "modification" generally refers to the alteration of natural or synthetic components. Modification can include modifications to bases, nucleosides, sugars, and internucleotide bonds; it can include both chemical and non-chemical modifications.

[0057] The term "link" refers to the phosphate backbone between two oligonucleotides, including but not limited to phosphate esters, thiophosphate esters, etc.

[0058] The terms "complementary pairing" and "complementary" are used interchangeably and generally refer to Watson-Crick or Hoogsteen base pairing between nucleotide units of a nucleic acid molecule. In this disclosure, base pairing can refer to AT, CG. In this disclosure, complementary pairing can be perfect complementary pairing, or it can involve one or more protrusions, wobbles, deletions, and / or mismatches between nucleic acid molecules. In this disclosure, agRNA can form a double-stranded complex with target RNA through complementary pairing.

[0059] The term "protrusion" usually refers to a region where the base upstream and downstream of the protrusion is complementary to the target RNA strand, and the corresponding two bases of the target RNA strand are consecutive.

[0060] The term "swing" usually refers to GU pairing.

[0061] The term "deletion" usually refers to a region where the upstream and downstream bases are continuous, and the target RNA strand in the deleted region has a corresponding number of bases.

[0062] The term "mismatch" generally refers to a double-stranded RNA complex in which the opposing nucleotides do not form a perfect base pair according to the Watson-Crick base pairing rule. Mismatches can be one of the following types: AA, AG, AC, UU, UC, GG, GA, CA, CC, or CU.

[0063] The term "perfectly complementary pairing" generally refers to a nucleic acid molecule where there is only strict Watson-Crick or Hoogsteen base pairing between nucleotide units. Perfectly complementary pairing does not involve bulges, wobbles, deletions, and / or mismatches.

[0064] The term “treatment” generally refers to: (1) preventing the development of a disease, condition and / or symptom in a patient who may be susceptible to the disease, condition and / or symptom but has not yet been diagnosed with the disease; (2) suppressing the disease, condition or symptom, i.e., curbing its development; and (3) alleviating the disease, condition or symptom, i.e., achieving the resolution of the disease, condition and / or symptom and / or symptoms associated with the disease, condition and / or symptom.

[0065] The term "delivery vector" generally refers to a vector that delivers one or more nucleotides into cells. The vector can include viral vectors and non-viral vectors. Viral vectors can include lentivirus (LV) vectors, adenovirus (AdV) vectors, and adeno-associated virus (AAV) vectors, etc. Non-viral vectors can include liposomes, molecularly coupled receptors, polymers, composite vectors, and nanoparticle vectors, etc. In this disclosure, the delivery vector can deliver the agRNA or isolated nucleic acid molecules described herein into cells.

[0066] The term “subject” generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0067] The term "RNA editing" generally refers to a co-transcriptional or post-transcriptional modification process that introduces changes into the genome-encoded RNA sequence, resulting in RNA mutations. Adenosine editing in double-stranded RNA (dsRNA) to inosine (A-to-I), catalyzed by adenosine deaminases (ADARs) acting on the RNase family, is a common type of RNA editing in mammals. In vertebrates, the family of three ADAR proteins—ADAR1, ADAR2, and ADAR3—has been previously characterized. ADAR1 and ADAR2 (ADARs) catalyze all currently known A-to-I editing sites. ADAR3 has no known deaminase activity. Inosine (I) mimics guanosine (G), thus ADAR proteins introduce a dummy A-to-G substitution in the transcript. This change can lead to specific amino acid substitutions, alternative splicing, miRNA-mediated gene silencing, or alterations in transcript localization and stability.

[0068] The term "nucleotide" refers to its respective ribosyl phosphate or ribosyl deoxyribosyl phosphate. In this disclosure, the terms "adenosine" and "adenine" (abbreviated "A"), "guanosine" and "guanine" (abbreviated "G"), "cytidine" and "cytosine" (abbreviated "C"), "uridine" and "uracil" (abbreviated "U"), and "thymidine" and "thymidine" (abbreviated "T") are used interchangeably.

[0069] The term "cell" generally includes both prokaryotic and eukaryotic cells. Nucleic acids can be transfected into cells, plasmids can proliferate in prokaryotic cells, and nucleic acids encoding polypeptides can be expressed in eukaryotic cells. For example, cells may include agRNA and / or delivery vectors. Cells can originate from any of the following organs: for example, skin, lungs, heart, kidneys, liver, pancreas, intestines, muscles, glands, eyes, brain, blood, etc. For example, cells can be human cells or mouse cells. For example, cells can be immune cells. For example, immune cells can be T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoprogenitor cells, and / or pluripotent stem cells. For example, immune cells can be T cells.

[0070] As used herein and in the appended claims, the singular forms “a / an,” “a / an,” and “the” include plural references unless the context clearly specifies otherwise. Thus, for example, reference to “a method” includes multiple such methods, and reference to “the segment” includes reference to one or more segments and their equivalents known to those skilled in the art, and so on.

[0071] As used herein, the term "and / or" refers to and covers any and all possible combinations of one or more of the associated listed items. When used in a list of two or more items, the term "and / or" means that any one of the listed items may be used alone, or any combination of two or more listed items may be used. For example, if a composition, combination, construction, etc., is described as comprising (or containing) components A, B, C, and / or D, then the composition may contain A alone; contain B alone; contain C alone; contain D alone; contain a combination of A and B; contain a combination of A and C; contain a combination of A and D; contain a combination of B and C; contain a combination of B and D; contain a combination of C and D; contain a combination of A, B, and C; contain a combination of A, B, and D; contain a combination of A, C, and D; contain a combination of B, C, and D; or use a combination of A, B, C, and D.

[0072] It should be further understood that, when the term “comprising” is used to describe various embodiments, those skilled in the art will understand that, in certain specific circumstances, the language “substantially consisting of” or “consisting of” may be used instead to describe the embodiments.

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While many methods and reagents are similar to or equivalent to those described herein, exemplary methods and materials are disclosed herein. Where scientific terms involve multiple different meanings, the one generally used or recognized in the art shall prevail.

[0074] It should be understood that this disclosure is not limited to the specific methods, schemes, and reagents described herein, and these methods are subject to change. The terminology used herein is for the purpose of describing specific embodiments or aspects only and is not intended to limit the scope of this disclosure.

[0075] As used herein, the term “pharmaceutically acceptable” means compounds, materials, compositions, and / or dosage forms that (within the scope of reasonable medical judgment) are suitable for contact with a patient’s tissues without causing additional toxicity, irritation, allergic reactions, or other problems or complications (i.e., with a reasonable risk / benefit ratio).

[0076] As used herein, “pharmaceutical composition” refers to a therapeutically effective amount of a drug and pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, excipients, and / or carriers. “Therapeutically effective amount” as used herein refers to the amount that provides a therapeutic effect for a given condition and dosing regimen. In some embodiments, these compositions are liquid or lyophilized formulations, or otherwise dry formulations, containing various buffered contents (e.g., Tris-HCl, acetate, phosphate), diluents of pH and ionic strength, additives such as albumin or gelatin to prevent adsorption to surfaces, and denaturing agents (e.g., Tween 20, Tween 80, Pluronic F68, bile salts). Solubilizers (e.g., glycerol, polyethylene glycol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), thickeners or tension modifiers (e.g., lactose, mannitol), covalent linkages of polymers such as polyethylene glycol to proteins, complexations with metal ions, or incorporation of materials into or onto specific preparations of polymeric compounds (e.g., polylactic acid, polyglycolic acid, hydrogels, etc.), or onto liposomes, microemulsions, micelles, monolayer or multilayer vesicles, erythrocyte shadows, or protoplasmic spheres. Such compositions will affect physical state, solubility, stability, in vivo release rate, and in vivo clearance rate. Controlled-release or sustained-release compositions include formulations in lipophilic reservoirs (e.g., fatty acids, waxes, oils).

[0077] The terms “oligonucleotide,” “nucleotide sequence,” and “nucleic acid” are used interchangeably. They refer to nucleotides, deoxyribonucleotides, or ribonucleotides of any length in polymeric form, or their analogues.

[0078] In some embodiments, ADAR is naturally present in host cells, such as eukaryotic cells (preferably mammalian cells, more preferably human cells). In some embodiments, the ADAR is introduced into the host cells.

[0079] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0080] In this article, for modified sequences, the modifications are as described in this specification.

[0081] In the following examples, A, U, C, and G in sequences SEQ ID NO: 4 to SEQ ID NO: 35 (containing modified sequences) are RNA bases, dN represents DNA bases, and T represents thymine DNA bases; "fN" represents 2' F-modified oligonucleotides; "mN" represents 2' OMe-modified oligonucleotides; "*" represents phosphate thioester bonds; "L" is LNA, and "LN" represents oligonucleotides modified with locked nucleic acids (LNA).

[0082] In the embodiments of this article, the structural diagrams of phosphate thioester bond modification (Formula (I)), 2' LNA modification (Formula (II)), DNA modification (Formula (III)), 2' F (Formula (IV)), and 2' OMe modification (Formula (V)) are shown below ("Base" in the structural formula refers to a base): (I); (II); (III); (IV); (V).

[0083] Unless otherwise specified, the sequence from left to right in this article is 5'→3'.

[0084] In the embodiments described herein, considering that the ATP7B gene is highly expressed in hepatocytes and that all cell lines derived from hepatocytes express the wild-type ATP7B gene, which may affect the evaluation of the binding and editing efficiency of the drug to the mutant ATP7B, the embodiments described herein all utilize HeLa cells or other cells that do not express the endogenous ATP7B gene.

[0085] Unless otherwise specified, the final concentration of agRNA used in this embodiment is 40 nM.

[0086] In this embodiment, the distance between the 3' end of the agRNA and the editing site is the distance between the bases on the ATP7B mutant gene fragment that are complementary to the 3' end of the agRNA and the editing site.

[0087] In this embodiment, the A base at position 1532 on the human ATP7B transcript (NM_000053.4) (as shown in Table 1, the underlined A base in the SEQ ID NO:1 sequence) was used as the editing site. The C base adjacent to this A site in the mutant ATP7B gene used in this embodiment was mutated to a T base.

[0088] Materials and methods Carrier construction This application uses a vector to express the ATP7B gene editing site and upstream and downstream sequences (SEQ ID NO:1, see Table 1). The specific construction method is the same as that in the patent application with application number PCT / CN2023 / 085802. The mutation of the ATP7B gene is the ATP7Bc.1531C>T mutation, that is, the codon encoding glutamine is mutated to a stop codon, resulting in premature termination (the source of this mutation site is: Dong Y, Ni W, Chen WJ, et al. Spectrum and Classification of ATP7BVariants in a Large Cohort of Chinese Patients with Wilson's Disease Guides Genetic Diagnosis. Theranostics. 2016;6(5):638-649. Published 2016 Mar 3. doi:10.7150 / thno.14596).

[0089] Cell culture The various cell lines used in this application (293T, A549, HeLa, SH-SY5Y, and U2OS cells) were all purchased from the Cell Bank of the Chinese Academy of Sciences. All cells were thawed, cultured, passaged, and cryopreserved according to their instructions. Thawing followed the principle of slow freezing and rapid thawing. Cells were cultured in a 37°C, 5% CO2 incubator, with the medium changed every 1-2 days. When cell confluence reached approximately 90%, cells were passaged and digested with 0.25% trypsin. Cells could then be passaged into specific-sized wells for transfection experiments or cryopreserved. For cryopreservation, the cell suspension was first placed in cryovials and stored at -80°C for 24 hours, then transferred to liquid nitrogen for long-term preservation.

[0090] Methods for constructing cells carrying the mutated ATP7B gene fragment Transfect the vector containing the mutant ATP7B gene fragment into the corresponding cells using Lipofectamine 3000 (see product instructions for specific experimental steps). This completes the construction of cells carrying the mutant ATP7B gene. Change the medium after 6 hours and proceed with subsequent experimental procedures.

[0091] Cell transfection After passage, cells were cultured in an incubator for 24 hours. Transfection was then performed according to the instructions in the Lipofectamine RNAiMAX transfection reagent manual. The specific steps for 40 nM agRNA transfection were as follows: 40 pmol of agRNA was mixed with 4 μL of Lipofectamine RNAiMAX transfection reagent in 100 μL of Opti-MEM medium. After mixing, the mixture was thoroughly inverted and mixed with the liposome complex to ensure a stable complex formation. This mixture was incubated at room temperature for 20 minutes to promote liposome complex formation. After incubation, it was added to a 1 mL cell culture volume and gently shaken to allow the transfection complex to bind fully to the cells, completing the transfection process. For 4 nM agRNA transfection, 4 pmol of agRNA was mixed with 0.4 μL of Lipofectamine RNAiMAX transfection reagent in 100 μL of Opti-MEM medium, and subsequent procedures were the same as for the 40 nM concentration. The cell culture medium should be changed promptly within 24 hours after transfection to remove unadsorbed transfection reagents and other impurities in order to optimize the transfection effect.

[0092] agRNA editing level verification RNA was extracted from cells using an RNA isolation kit (Novizan, catalog number: RC102-01) following the supplier's instructions. DNase I (Thermo) digestion was followed by reverse transcription using a random primer (Promega). Simultaneously, PCR amplification of the target site was performed using primers ATP7B-F and ATP7B-R (SEQ ID NO:2 and SEQ ID NO:3, see Table 2) and DNA Taq enzyme (GeneStar). After agarose gel electrophoresis analysis, the target fragment was sequenced using Sanger sequencing. Sequencing results were analyzed using EditR, and the A-to-I editing efficiency was quantified according to the G / (G+A) peak height. If reverse primers were used for sequencing, the C and T peak heights were adjusted accordingly.

[0093] Table 1. Transcript sequence of the mutant ATP7B gene fragment Table 2 PCR primer sequences for target sites Example 1: Editing levels of agRNA at different distances from the 3' end of the agRNA to the editing site The distance between the 3' end of the agRNA and the editing site affects the editing level of the agRNA. In this embodiment, multiple chemically modified agRNAs (SEQ ID NO:4~SEQ ID NO:8) with different distances between the 3' end and the editing site were designed, as shown in Table 3. These were tested in HeLa cells carrying the ATP7B mutant gene fragment. The results showed that agRNAs with different distances between the 3' end and the editing site could all achieve a high (>40%) editing level. Figure 1 ).

[0094] Table 3. agRNA sequences with different distances from the 3' end to the editing site Example 2: Editing levels of different agRNA sequences with the same 3' end distance from the edit site. In this embodiment, different nucleotide base sequences with the same 3' end distance from the editing site, different types of sugar modifications (2' LNA, 2' OMe, 2' F, DNA), and agRNAs with phosphate-thiolated linkages were designed and synthesized, as shown in Table 4 (SEQ ID NO: 9~SEQ ID NO: 14). The distance from the 3' end of the agRNA provided in this embodiment to the editing site is 9 nt. Figure 2 The results showed that in HeLa cells carrying the ATP7B mutant gene fragment, agRNAs that form different complementary pairing structures with the target gene transcripts can all edit the editing site, and different secondary structures and chemical modifications will result in different editing levels.

[0095] Table 4. Sequences of different base sequences and modified agRNAs Example 3: Editing levels of agRNAs with different distances from the 3' end to the editing site and of different lengths at the editing site. This embodiment designed agRNAs with different distances between the 3' end and the editing site, as shown in Table 5 (SEQ ID NO:15~SEQ ID NO:33). The agRNAs shown had different lengths and chemical modifications, and their editing levels at the editing site were tested in HeLa cells carrying the ATP7B mutant gene fragment at two final concentrations of 4 nM and 40 nM. The results showed that agRNAs of different lengths could edit the editing site. Under the premise that the distance between the 3' end of the agRNA and the editing site was the same, different agRNA sequences exhibited different editing levels, and the closer the editing site was to the 3' end of the agRNA, the better the editing level. Figure 3 ).

[0096] Table 5. Sequences of different base sequences and modified agRNAs Example 4: Comparison of editing levels of agRNA designed with existing technology at editing sites in different cell lines In this embodiment, RC-39 (SEQ ID NO: 4) from Example 1 was selected as the comparison sequence and its editing level was compared with that of agRNAs designed using the existing RESTORE design strategy (SEQ ID NO: 34~SEQ ID NO: 35, as shown in Table 6). The RESTORE design has two high-editing strategies, v9.5 and v25 (RESTORE design strategy reference: Merkle, T., Merz, S., Reautschnig, P. et al. Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides. Nat Biotechnol 37, 133–138 (2019). https: / / doi.org / 10.1038 / s41587-019-0013-6). The results showed that in different cell lines carrying the ATP7B mutant gene fragment (293T, A549, HeLa, SH-SY5Y, and U2OS cells), the editing levels of agRNA designed by RESTORE v25 were higher than those designed by RESTORE v9.5, but all were lower than those of RC-39 provided in this application (e.g., Figure 4 (As shown).

[0097] Table 6. agRNA sequences designed using the RESTORE strategy The above embodiments are only for further elaboration and explanation of the technical solutions of the present invention, so that those skilled in the art can more accurately understand the inventive concept and operation scheme of the present invention, and are not intended to further limit the present invention. Any non-prominent substantive features and non-significant improvements made by those skilled in the art on this basis shall fall within the protection scope of the present invention.

Claims

1. An ADAR guide RNA, characterized in that, The ADAR guide RNA is modified and includes any one of the sequences shown in SEQ ID NO: 4-33.

2. The ADAR guide RNA as described in claim 1, characterized in that, The modifications include skeletal modifications, sugar modifications, or base modifications; Preferably, the modification includes 2' F modification, 2' OMe modification, phosphate thioester bond modification, locked nucleic acid modification, or deoxyribose modification; Preferably, the ADAR guide RNA has a sequence length of 10-200 bases; Preferably, the ADAR guide RNA has a sequence length of 15-150 bases; Preferably, the ADAR guide RNA has a sequence length of 20-100 bases; Preferably, the ADAR guide RNA has a sequence length of 25-50 bases.

3. The ADAR guide RNA as described in claim 1, characterized in that, The ADAR guide RNA can bind to the target RNA to form a structure that can recruit ADARs within the cell, and edit the target adenosine in the target RNA through the recruited ADARs; Preferably, the distance between the 3' end of the ADAR guide RNA and the target adenosine is 1~40 nt; Preferably, the distance between the 3' end of the ADAR guide RNA and the target adenosine is 2-35 nt; Preferably, the distance between the 3' end of the ADAR guide RNA and the target adenosine is 5-35 nt; Preferably, the target RNA is an ATP7B transcript; Preferably, the target RNA is a human ATP7B transcript; Preferably, the target RNA contains the 1531C>T mutation; Preferably, the protein encoded by the target RNA is copper-transporting ATPase β; Preferably, the target adenosine is at base 1532A in the human ATP7B transcript; Preferably, the editing is ADAR-mediated A-to-I editing; Preferably, the ADAR is selected from ADAR1 or ADAR2; Preferably, the editing is capable of mutating 1532A to I in the human ATP7B transcript; Preferably, the ADAR guide RNA is either completely complementary or partially complementary to the target RNA; Preferably, the incomplete complementary pairing is a complementary pairing that has one or more mismatches, wobbles, omissions, and / or protrusions with the target region; Preferably, the ADAR guide RNA has at least one mismatch with the target RNA; Preferably, the complementary chains are base-complementary pairs at non-mismatched, non-deleted, non-protruding, non-inner-loop, or non-wobbly base-pairing sites. Preferably, the proportion of complementary base pairs in the double-stranded RNA formed by the ADAR guide RNA and the target RNA is greater than 60%. Preferably, the proportion of complementary base pairs in the double-stranded RNA formed by the ADAR guide RNA and the target RNA is greater than 75%. Preferably, the ADAR guide RNA has the same base sequence as shown in SEQ ID NO: 4~33; Preferably, the sequence of the ADAR guide RNA is shown in SEQ ID NO: 4~33.

4. A carrier, characterized in that, The vector contains a polynucleotide encoding or containing any of the ADAR guide RNAs according to claims 1-3; Preferably, the vector is a recombinant expression vector; Preferably, the vector comprises a plasmid or a viral vector; Preferably, the carrier is a delivery carrier; Preferably, the vector is a plasmid or viral vector for expression in higher eukaryotic or prokaryotic cells.

5. A composition comprising a plasmid or viral vector encoding or comprising the ADAR guide RNA of any one of claims 1-3.

6. A host cell, characterized in that, It comprises the ADAR guide RNA according to any one of claims 1-3, the vector according to claim 4, or the composition according to claim 5.

7. A pharmaceutical composition, characterized in that, It contains the ADAR guide RNA as described in any one of claims 1-3, and pharmaceutically acceptable excipients.

8. The use of the ADAR guide RNA of any one of claims 1-3, the vector of claim 4, the composition of claim 5, the host cell of claim 6, or the pharmaceutical composition of claim 7 in the preparation of a medicament for altering the expression of a target protein in the cells of a subject, characterized in that, The cell has a target RNA encoding a target protein, the target RNA being a human ATP7B transcript containing a 1531C>T mutation; Preferably, the target protein is copper-transporting ATPase β; Preferably, the drug is a drug used to treat Wilson's disease; Preferably, the ADAR guide RNA can bind to the target RNA to form a structure that can recruit ADAR within the cell, and edit the target adenosine in the target RNA through the recruited ADAR; Preferably, the target adenosine is at base 1532A in the human ATP7B transcript; Preferably, the editing is ADAR-mediated A-to-I editing; Preferably, the ADAR is selected from ADAR1 or ADAR2; Preferably, the editing is capable of mutating 1532A to I in the human ATP7B transcript.

Images