Modified guide agrna

Abstract

The present application relates to a modified guide agRNA (ADAR guide RNA, agRNA), which has a phosphorothioate modification at an agRNA site where off-target editing is present, and further has other modifications. The present application further provides a composition containing the modified agRNA composition, a method for editing RNA, and the use.

Description

A modified guide agRNA TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a modified guide agRNA (ADAR guide RNA, agRNA), a method for editing RNA, and uses. BACKGROUND

[0002] RNA editing mainly refers to the processing and modification of RNA after transcription of genetic information DNA in organisms, and common examples include A-to-I based on ADAR protein and C-to-U RNA editing based on APOBEC protein. Compared with the heritable and irreversible characteristics of DNA editing, RNA editing has controllability and reversibility, does not affect the stability of the genome, only needs to introduce an engineered RNA to exert the editing function and does not induce an immune response.

[0003] ADAR protein mainly binds to double-stranded RNA (dsRNA) in cells, and generates I base by deamination of A base at a specific site, while I base is recognized as G base during translation. In the human transcriptome, there are thousands of A-to-I editing sites in the coding and non-coding regions of mRNA, and ADAR targeting codons can have a profound impact on the proteome. How ADAR targets specific A-to-I RNA editing sites is a long-standing problem, and the primary sequence and secondary structure around the editing site can affect the preference and selectivity of ADAR. ADAR has a preference for the adjacent motif of the targeted adenosine, while the length of the dsRNA and whether the base pairing is interrupted due to mismatch, bulge or loop will affect the number of adenosine editing in the dsRNA, and through the engineering of guide RNA, endogenous ADAR protein can be recruited in cells to perform A-to-G single base editing at specific sites.

[0004] Existing methods for reducing the off-target effect of RNA editing mainly include: 1) A-G mismatch at the off-target site (LEAPER 1.0); 2) deletion of U base at the off-target site (LEAPER 2.0); 3) chemical modification of the sequence (AIMer).

[0005] LEAPER (Leveraging Endogenous ADAR for Programmable Editing on RNA) RNA editing technology, mainly by introducing a length of 111 nt to 151 nt of arRNA (ADAR recruiting RNA) complementary to the sequence of the target editing site into the cell, recruiting endogenous ADAR protein to edit the target editing site of RNA A-to-I. Due to the formation of relatively long double-stranded RNA, the system shows a large number of off-target editing in the target RNA region covered by arRNA. In order to reduce off-target effects, A-G mismatches are made at sites that may produce editing. For single-base mismatches that cannot reduce off-target editing motifs (UAG and AAG), double mismatches are used. This editing method achieves higher editing specificity while not increasing the loss of editing rate on target sites.

[0006] LEAPER2.0 uses covalently closed circular RNA (cir-arRNA) instead of linear arRNA in LEAPER1.0, with 3.1-fold higher editing efficiency and higher stability. In LEAPER1.0, A-G mismatches can significantly reduce off-target editing, because adenosine deamination requires ADAR to flip the reaction base out of the RNA double helix to access its active site. Based on the catalytic characteristics of ADAR1 / 2, deleting the nucleotides at the non-targeted adenosine position significantly reduces site-specific off-target editing in the circ-arRNA coverage area.

[0007] The RESTORE editing system is mainly composed of two parts, namely the specificity domain and the ADAR recruiting domain, both of which are chemically modified. In 2022, the RESTORE editing system was further optimized by adding three recruitment sequences (Recruitment Sequence, RS) with a length of 7-20 nt at the 3' end of the agRNA, and named CLUSTER guide RNA. The RS binding site selected by the CLUSTER algorithm does not contain adenosine, which can avoid off-target editing and reduce unnecessary folding. Even if the RS is distributed over 1,000 nucleotides, the CLUSTER guide RNA can still produce high editing efficiency with very few off-target editing.

[0008] AIMer structures recruit endogenous ADAR enzymes to efficiently perform A-to-I RNA editing. AIMer is an oligonucleotide, short in length (30-mers), backbone fully modified with stereo pure phosphorothioate (PS) and phosphor guanydyl based nitrogen (PN) linkages, and 2'-ribose modifications within the targeted editing region. AIMer can induce high specific editing on the targeted sequence with very low off-target editing. In vitro, chimeric modification backbone enhances potency and A-to-I editing efficiency compared to PS-modified AIMer, while in vivo, n-acetylgalactosamine (GalNAc)-modified AIMer achieves up to 50% editing in non-human primate (NHP) liver with no off-target editing and a duration of 1 month.

[0009] However, the above methods for reducing the off-target effect of RNA editing all have certain defects. The sequence length of LEAPER is relatively long, so that base mismatches at off-target sites have no significant effect on the editing efficiency of LEAPER, while the shorter agRNA will be greatly reduced in editing efficiency due to base mismatches affecting the secondary structure formed by the agRNA and the target RNA. In AIMer, the DNA bases are used to replace the original RNA bases within the targeted editing region, but the length is relatively short, and the editing efficiency will also be reduced. Therefore, there is an urgent need to improve the prior art and invent a new method for reducing the off-target effect of RNA editing, which greatly reduces off-target editing without affecting the editing efficiency. SUMMARY

[0010] The present application provides a modified guide agRNA (ADAR guide RNA, agRNA) having phosphorothioate modification at the agRNA site where off-target editing exists, and also having other modifications. The other modifications can be two or more than two different types of modifications, and the modifications are selected from the group consisting of LNA, UNA, 2'-MOE, 2'-F, 2'-OMe, and DNA base replacement modification. Using the modified agRNA provided by the present application for RNA editing, there is basically no off-target editing, and the chemical modification at the off-target position basically has no effect or little effect on the editing level of the target site, and the method for reducing the off-target effect of RNA editing provided by the present application can be used for different target sites.

[0011] In one aspect, the present application provides a modified guide agRNA (ADAR guide RNA, agRNA) having phosphorothioate modification at the agRNA site where off-target editing exists, and also having other modifications.

[0012] In some embodiments, in the modified agRNA, the modification can reduce off-target editing of the target RNA.

[0013] In certain embodiments, in the modified agRNA, the Watson-Crick base pairs of the target site do not comprise 2’-MOE or 2’-OMe modifications.

[0014] In certain embodiments, in the modified agRNA, the modification comprises at least one structure of any one of Formula 1 to Formula 6:

[0015] wherein Formula 1 is including but not limited to constrained ethyl nucleotides, UNA-nucleotides, LNA nucleotides;

[0016] R1 is hydrogen, hydroxyl, halogen, amino, O-C1-C6 alkoxy, or O-methoxyethyl;

[0017] R2 is hydroxyl, halogen, or C1-C6 alkoxy;

[0018] R3 is hydrogen, hydroxyl, halogen, or C1-C6 alkoxy;

[0019] R4 is hydrogen, hydroxyl, halogen, or C1-C6 alkoxy; and

[0020] R5 is hydrogen, hydroxyl, halogen, or C1-C6 alkoxy.

[0021] In certain embodiments, in the modified agRNA, all nucleotides that pair to sites other than the target site and off-target site of the target RNA do not have the structure of any one of Formula 1 to Formula 6.

[0022] In certain embodiments, in the modified agRNA, the chemical modification comprises at least one structure of any one of Formula 1 to Formula 6.

[0023] In certain embodiments, in the modified agRNA, the agRNA site where off-target editing exists comprises two and more than two different kinds of modifications, which are optionally selected from the structures of Formula 1 to Formula 6.

[0024] In certain embodiments, in the modified agRNA, the Watson-Crick base pairs of the target site and off-target site do not have the modification selected from the group consisting of LNA, UNA, 2’-MOE, 2’-F, 2’-OMe, 2’-F, and DNA base replacement modification.

[0025] In certain embodiments, in the modified agRNA, all agRNA sites further comprise phosphorothioate modification.

[0026] In certain embodiments, in the modified agRNA, the modification comprises sugar modification and / or base modification.

[0027] In certain embodiments, in the modified agRNA, the modification comprises a chemical modification.

[0028] In certain embodiments, in the modified agRNA, the chemical modification comprises: LNA, UNA, 2’-MOE, 2’-F, 2’-OMe.

[0029] In certain embodiments, in the modified agRNA, the modification comprises a DNA base substitution modification.

[0030] In certain embodiments, in the modified agRNA, the agRNA site with off-target editing comprises two or more different kinds of modifications selected from the group consisting of: LNA, UNA, 2’-MOE, 2’-F, 2’-OMe, and DNA base substitution modification.

[0031] In certain embodiments, in the modified agRNA, it further comprises a phosphorothioate modification.

[0032] In certain embodiments, in the modified agRNA, wherein the off-target site is located at the -1 or +1 position of the target site, the modification of the agRNA site with off-target editing is selected from the group consisting of DNA base substitution modification and 2’-F.

[0033] In certain embodiments, in the modified agRNA, wherein the off-target site is within 10 bases of the target site, the agRNA site with off-target editing is each independently selected from the group consisting of modifications of: 2’-F, 2’-OMe, DNA base substitution modification, 2’-MOE, LNA and UNA.

[0034] In certain embodiments, in the modified agRNA, wherein the off-target site is more than 10 bases away from the target site, the agRNA site with off-target editing is each independently selected from the group consisting of modifications of: 2’-MOE, LNA, UNA, 2’-F and 2’-OMe.

[0035] In certain embodiments, in the modified agRNA, the agRNA is 25-70 nt in length.

[0036] In certain embodiments, in the modified agRNA, the agRNA is 41 nt or 51 nt in length.

[0037] In certain embodiments, in the modified agRNA, the off-target site is adenosine.

[0038] In some embodiments, in the modified agRNA, the target site is adenosine.

[0039] In some embodiments, in the modified agRNA, the target RNA is selected from one or more of the group consisting of pre-mRNA, mRNA, rRNA, tRNA, and Inc-RNA.

[0040] In some embodiments, in the modified agRNA, the agRNA is fully complementary to the target RNA.

[0041] In some embodiments, in the modified agRNA, the agRNA is complementary to the target RNA with one or more mismatches, wobbles, deletions, and / or bulges.

[0042] In some embodiments, in the modified agRNA, the agRNA can recruit endogenous deaminases to deaminate specific nucleotide sites.

[0043] In another aspect, the present application provides a method of editing RNA, comprising using the modified agRNA.

[0044] In some embodiments, the method can reduce off-target editing of the target RNA.

[0045] In some embodiments, the method comprises the following steps:

[0046] (1) providing the modified agRNA of any one of claims 1-20;

[0047] (2) introducing the modified agRNA into a cell;

[0048] (3) allowing the agRNA to bind to the target RNA.

[0049] In another aspect, the present application provides a delivery vehicle comprising the modified agRNA.

[0050] In another aspect, the present application provides a cell comprising the modified agRNA, and / or the delivery vehicle.

[0051] In some embodiments, the cell is a eukaryotic cell.

[0052] In some embodiments, the cell is a human cell or a mouse cell.

[0053] In some embodiments, the cell is a liver cell.

[0054] In some embodiments, the cell is a neural cell.

[0055] In another aspect, the present application provides a pharmaceutical composition comprising the modified agRNA, the delivery vehicle, the cell, and / or a pharmaceutically acceptable carrier.

[0056] In another aspect, the present application provides the modified agRNA, the delivery vehicle, the cell, the pharmaceutical composition for use in preventing and / or treating a disease and / or a disorder.

[0057] In another aspect, the present application provides use of the modified agRNA, the delivery vehicle, the cell, the pharmaceutical composition in the manufacture of a medicament for preventing and / or treating a disease and / or a disorder.

[0058] In another aspect, the present application provides a method of preventing and / or treating a disease and / or a disorder, comprising administering to a subject in need thereof an effective amount of the modified agRNA, the delivery vehicle, the cell, the pharmaceutical composition.

[0059] Other aspects and advantages of the present application will be readily appreciated from the detailed description that follows, when considered in connection with the accompanying drawings. The exemplary embodiments of the present application shown and described in the detailed description are only exemplary and are not intended to be limiting of the application. The content of the specification is merely exemplary and is not intended to be limiting of the application. Accordingly, the drawings and description are illustrative only and are not intended to be limiting. BRIEF DESCRIPTION OF DRAWINGS

[0060] The features and advantages of the present application involved in the application will be better understood by referring to the detailed description in conjunction with the accompanying drawings. The drawings are briefly described as follows:

[0061] FIG. 1 shows the raw peak plot of editing level obtained by next-generation sequencing of agRNA transfection into mouse primary hepatocytes targeting mouse GAPDH gene with GAPDH-41nt-V1 and GAPDH-51nt-V1. FIG. 1A is the raw peak plot of editing level obtained by next-generation sequencing of agRNA transfection into mouse primary hepatocytes targeting mouse GAPDH gene with GAPDH-41nt-V1. FIG. 1B is the raw peak plot of editing level obtained by next-generation sequencing of agRNA transfection into mouse primary hepatocytes targeting mouse GAPDH gene with GAPDH-51nt-V1.

[0062] Figure 2 shows raw peak plots of editing levels obtained by next generation sequencing for GAPDH-41nt-V2, GAPDH-41nt-V3 and GAPDH-41nt-V4 transfected into mouse primary hepatocytes as described herein.

[0063] Figure 3 shows raw peak plots of editing levels obtained by next generation sequencing for GAPDH-41nt-V5, GAPDH-41nt-V6 and GAPDH-41nt-V7 transfected into mouse primary hepatocytes as described herein.

[0064] Figure 4 shows raw peak plots of editing levels obtained by next generation sequencing for GAPDH-51nt-V2, GAPDH-51nt-V3 and GAPDH-51nt-V4 transfected into mouse primary hepatocytes as described herein.

[0065] Figure 5 shows raw peak plots of editing levels obtained by next generation sequencing for GAPDH-51nt-V5, GAPDH-51nt-V6 and GAPDH-51nt-V7 transfected into mouse primary hepatocytes as described herein.

[0066] Figure 6 shows raw peak plots of editing levels obtained by next generation sequencing for GAPDH-51nt-V3, GAPDH-51nt-V8 and GAPDH-51nt-V9 transfected into mouse primary hepatocytes as described herein.

[0067] Figure 7 shows raw peak plots of editing levels obtained by next generation sequencing for GAPDH-51nt-V4, GAPDH-51nt-V10 and GAPDH-51nt-V11 transfected into mouse primary hepatocytes as described herein.

[0068] Figure 8 shows raw peak plots of editing levels obtained by next generation sequencing for GAPDH-51nt-V4, GAPDH-51nt-V12 and GAPDH-51nt-V13 transfected into mouse primary hepatocytes as described herein.

[0069] Figure 9 shows raw peak plots of editing levels obtained by next generation sequencing for GAPDH-51nt-V2, GAPDH-51nt-V14, GAPDH-51nt-V15 and GAPDH-51nt-V16 transfected into mouse primary hepatocytes as described herein.

[0070] Figure 10 shows raw peak plots of editing levels obtained by next generation sequencing for GAPDH-51nt-V17, GAPDH-51nt-V18, GAPDH-51nt-V19, GAPDH-51nt-V20, GAPDH-51nt-V21, and GAPDH-51nt-V22 transfection into mouse primary hepatocytes.

[0071] Figure 11 shows raw peak plots of editing levels obtained by next generation sequencing for RAB7A-V1, RAB7A-V2, and RAB7A-V3 transfection into Hela cells.

[0072] Figure 12 shows raw peak plots of editing levels obtained by next generation sequencing for PPIA-S1-V1, PPIA-S1-V2, and PPIA-S1-V3 transfection into Hela cells.

[0073] Figure 13 shows raw peak plots of editing levels obtained by next generation sequencing for PPIA-S2-V1, PPIA-S2-V2, and PPIA-S2-V3 transfection into Hela cells. DETAILED DESCRIPTION

[0074] The following specific examples illustrate the embodiments of the application, and other advantages and effects of the application can be easily understood by those skilled in the art from the disclosure of the specification.

[0075] Definitions of terms

[0076] In the present application, the term "guide agRNA (ADRA guide RNA)", also known as "Antisense Oligonucleotide guide RNA", is abbreviated as "agRNA", which generally refers to an artificially synthesized single-stranded or double-stranded oligonucleotide. The agRNA can be modified to change its structure, binding site, etc. In the present application, the agRNA can be in single-stranded form, and its function can be to complementarily pair with a specific sequence of target RNA and edit it.

[0077] In the present application, the term "modification" generally refers to the modification of natural or artificially synthesized components. The modification can include modification of bases, modification of nucleosides, modification of sugars, modification of internucleotide linkage; can include chemical modification and non-chemical modification. In the present application, the modification can include LNA, UNA, 2'-MOE, 2'-OMe, 2'-F, phosphorothioate modification, DNA base substitution modification, etc. LNA refers to a modification in which the ribose ring is "locked" by a methylene bridge connecting the 2'-O atom and the 4'-C atom. UNA refers to a modification in which the ribose ring lacks a C2' to C3' chemical bond. 2'-MOE refers to the replacement of the hydrogen on the 2'-hydroxyl group of a ribonucleotide with a methoxyethyl group. 2'-OMe refers to the replacement of the hydrogen on the 2'-hydroxyl group of a ribonucleotide with a methoxy group. 2'-F replaces the hydrogen on the 2'-hydroxyl group of a ribonucleotide with fluorine. Phosphorothioate modification replaces one of the non-bridging oxygen atoms in the original phosphate bond with a sulfur atom. DNA base substitution modification replaces the RNA base in the agRNA with a DNA base.

[0078] In the present application, the term "target site", also referred to as "editing site", can be used interchangeably in the present application, and generally refers to the site in the RNA editing to which the agRNA is directed. In the present application, the target site can be adenosine.

[0079] In the present application, the term "off-target site", also referred to as "non-editing site", can be used interchangeably in the present application, and generally refers to the site on the target RNA that initiates off-target editing in RNA editing. In the present application, the off-target site can be adenosine.

[0080] In the present application, the term "agRNA site with off-target editing", also referred to as "counter-nucleotide of off-target site", can be used interchangeably in the present application, and generally refers to the nucleotide in the agRNA that pairs with the off-target site.

[0081] In the present application, the term "target RNA", also referred to as "target RNA" or "Target RNA", can be used interchangeably in the present application, and generally refers to the RNA containing the target site. The target RNA can be pre-mRNA, mRNA, rRNA, tRNA, IncRNA, sRNA, etc. Mutation of certain nucleotide sites can cause different types of functional differences in the target RNA, such as abnormal splicing, alternative splicing of RNA, truncation, extension, misfolding of protein, etc.

[0082] In the present application, the term "complementary pairing" is used interchangeably with "complementarity" and generally refers to Watson-Crick or Hoogsteen base pairing between nucleotide units of nucleic acid molecules. In the present application, base pairing can refer to A-T, C-G, T*A / T, C*G / C. In the present application, complementary pairing can be perfect complementary pairing, or there can be one or more bulges, wobbles, deletions, and / or mismatches between nucleic acid molecules. In the present application, an agRNA can form a duplex complex with a target RNA through complementary pairing.

[0083] In the present application, the term "bulge" generally refers to a region where the upstream and downstream bases of the bulge region are both complementary paired to the target RNA strand and the corresponding two bases of the target RNA strand are consecutive.

[0084] In the present application, the term "wobble" generally refers to a G-U pairing.

[0085] In the present application, the term "deletion" generally refers to a region where the upstream and downstream bases of the deletion region are consecutive and there are corresponding number of bases of the target RNA strand in the deletion region.

[0086] In the present application, the term "mismatch" generally refers to a situation where the opposite nucleotides in a duplex RNA complex are not perfect base pairs according to Watson-Crick base pairing rules. The type of mismatch can be one of A-A, A-G, A-C, U-U, U-C, G-G, G-A, C-A, C-C, C-U.

[0087] In the present application, the term "perfect complementary pairing" generally refers to a situation where there are only strict Watson-Crick or Hoogsteen base pairing between nucleotide units of nucleic acid molecules. There are no bulges, wobbles, deletions, and / or mismatches in perfect complementary pairing.

[0088] In the present application, the term "secondary structure" generally refers to a structure formed by nucleic acid molecules that are not perfectly complementary paired. The imperfect complementary pairing can lead to bulges, wobbles, deletions, and / or mismatches. The secondary structure of a DNA molecule can refer to the helical conformation of the DNA. The secondary structure of an RNA molecule can refer to the helical conformation, loops, stems, and / or arms. In the present application, the secondary structure can refer to the secondary structure of an RNA formed by intramolecular base interactions, or the secondary structure of an RNA formed by intermolecular base interactions. A loop can refer to a secondary structure formed by unpaired bases sticking out, a stem can refer to a local A-form double helical secondary structure formed by complementary base pairing, and an arm can refer to a secondary structure of unpaired nucleotides next to a stem but not belonging to a loop.

[0089] In the present application, the term "delivery vehicle" generally refers to a vehicle that delivers one or more nucleotides to a cell. The vehicle can include a viral vehicle, a non-viral vehicle. The viral vehicle can include a lentivirus (LV) vehicle, an adenovirus (AdV) vehicle, and an adeno-associated virus (AAV) vehicle, etc. The non-viral vehicle can include a liposome, a molecular conjugate, a polymer, a complex vehicle, and a nanoparticle vehicle, etc. In the present application, the delivery vehicle can deliver the agRNA or the isolated nucleic acid molecule described in the present application into a cell.

[0090] In the present application, the term "cell" generally includes prokaryotic cells and eukaryotic cells. The nucleic acid can be transfected in the cell, the plasmid can be propagated in the prokaryotic cell, and the nucleic acid, the polypeptide can be expressed in the eukaryotic cell. For example, the cell can include the agRNA and / or the delivery vehicle. The cell can be from any organ, such as skin, lung, heart, kidney, liver, pancreas, intestine, muscle, gland, eye, brain, blood, etc. For example, the cell can be a human cell or a mouse cell. For example, the cell can be an immune cell. For example, the immune cell can be a T cell, a B cell, a natural killer cell (NK cell), a macrophage, a NKT cell, a monocyte, a dendritic cell, a granulocyte, a lymphocyte, a leukocyte, a peripheral blood mononuclear cell, an embryonic stem cell, a lymphoid progenitor cell, and / or a pluripotent stem cell. For example, the immune cell can be a T cell.

[0091] In the present application, the term "pharmaceutical composition" generally refers to a chemical or biological composition suitable for administration to an individual. For example, the pharmaceutical composition can include the agRNA, the delivery vehicle and / or the cell, and optionally a pharmaceutically acceptable carrier. For example, the individual can be a mammal.

[0092] In the present application, the term "pharmaceutically acceptable carrier" generally refers to a material and / or ingredient that is acceptable to administer to a subject in a pharmaceutical composition without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition. For example, a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethylene glycol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations. Pharmaceutically acceptable carriers include pharmaceutically acceptable salts, wherein the term "pharmaceutically acceptable salt" includes salts of the active compounds which are produced by using a relatively nontoxic acid or base, depending on the particular substituents found on the compounds described herein.

[0093] In the present application, the term "treatment" generally refers to: (1) preventing a disease, disorder, and / or condition from occurring in a subject that can be predisposed to the disease, disorder, and / or condition, but has not yet been diagnosed as having it; (2) inhibiting the disease, disorder, or condition, i.e., arresting its development; and (3) relieving the disease, disorder, or condition, i.e., causing the disease, disorder, and / or condition and / or symptoms associated with the disease, disorder, and / or condition to regress.

[0094] In the present application, the term "subject" generally refers to a human or non-human animal, including, but not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0095] In the present application, the term "RNA editing" generally refers to a cotranscriptional or posttranscriptional modification process that introduces changes in the genomic-encoded RNA sequence, thereby resulting in RNA mutations. Adenosine editing in double-stranded RNA (dsRNA) from adenosine to inosine (A-to-I) is catalyzed by adenosine deaminases of the RNA-acting (ADAR) enzyme family and is a common type of RNA editing in mammals. In vertebrates, three ADAR proteins, ADAR1, ADAR2, and ADAR3, have been previously characterized in the ADAR family. ADAR1 and ADAR2 (ADARs) catalyze all currently known A-to-I editing sites. ADAR3 has no known deaminase activity. Inosine (I) mimics guanosine (G), so ADAR proteins introduce a virtual A-to-G substitution in the transcript. This change can lead to specific amino acid substitutions, alternative splicing, miRNA-mediated gene silencing, or changes in transcript localization and stability.

[0096] In the present application, the terms "adenine", "guanine", "cytosine", "thymine", "uracil", and "hypoxanthine" refer to the nucleobases themselves. The terms "adenosine", "guanosine", "cytidine", "thymidine", "uridine", and "inosine" refer to the nucleobases linked to a ribose or deoxyribose sugar moiety. The term "nucleoside" refers to a nucleobase linked to a ribose or deoxyribose.

[0097] In the present application, the term "nucleotide" refers to the respective nucleobase- ribosyl-phosphate or nucleobase-deoxyribosyl-phosphate. In the present application, the terms "adenosine" and "adenine" (abbreviated "A"), "guanosine" and "guanine" (abbreviated "G"), "cytidine" and "cytosine" (abbreviated "C"), "uridine" and "uracil" (abbreviated "U"), "thymidine" and "thymine" (abbreviated "T"), "inosine" and "hypoxanthine" (abbreviated "I") are used interchangeably.

[0098] In the present application, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in a cell culture, etc., and not within an organism (e.g., an animal, a plant, and / or a microorganism).

[0099] In the present application, the term "in vivo" refers to events that occur within an organism (e.g., an animal, a plant, and / or a microorganism).

[0100] In the present application, the term "and / or" should be understood to mean either one of the items or any combination of the items.

[0101] In the present application, the term "comprising" generally means including, but not limited to, the specifically recited components. In certain circumstances, "comprising" can also encompass only including the specifically recited components. For example, "comprising" also means "consisting of."

[0102] In the present application, the term "about" generally means a range of variation above or below the specified value of 0.5-10%, for example, a range of variation above or below the specified value of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

[0103] In the present application, the term "comprising" generally means the meaning of including, encompassing, containing or comprising. In some cases, it also means the meaning of "consisting of" or "consisting essentially of".

[0104] DETAILED DESCRIPTION

[0105] Modified guide agRNA

[0106] In one aspect, the present application provides a modified guide agRNA, which has a phosphorothioate modification at the agRNA site where off-target editing occurs, and also has other modifications that can reduce off-target editing of target RNA. RNA editing using the modified agRNA provided by the present application has substantially no off-target editing, and the chemical modification of the off-target position has substantially no effect or little effect on the editing level of the target site, and the method provided by the present application for reducing the off-target effect of RNA editing can be used for different target sites.

[0107] In the present application, the paring nucleotides of the target site in the modified agRNA can not contain 2'-MOE or 2'-OMe modification.

[0108] In the present application, the paring nucleotides of all sites other than the target site and the off-target site in the modified agRNA can not have a modification selected from the group consisting of LNA, UNA, 2'-MOE, 2'-F, 2'-OMe, and DNA base replacement modification.

[0109] For example, all sites other than the target site and the off-target site do not have the same modification, which can be the same modification or different modifications. For example, all sites other than the target site and the off-target site do not have a 2’-MOE modification. For example, all sites other than the target site and the off-target site do not have a 2’-F modification. For example, all sites other than the target site and the off-target site do not have a 2’-OMe modification. For example, all sites other than the target site and the off-target site do not have a DNA base replacement modification. For example, all sites other than the target site and the off-target site do not have a combination of modifications selected from LNA, UNA, 2’-MOE, 2’-F, 2’-OMe, and DNA base replacement. For example, all sites other than the target site and the off-target site can have 1-10 sites (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) or more sites that do not contain a modification.

[0110] For example, in the modified agRNA described, the agRNA can comprise an isotopic modification. For example, the agRNA can comprise an isotopic modification of one or more elements (e.g., hydrogen, carbon, nitrogen, etc.). For example, the agRNA can comprise one or more isotopic modifications.

[0111] For example, in the modified agRNA, the modified can be a nucleobase. The modified nucleobase can have at least one function of a nucleobase. For example, capable of base pairing. For example, the modified nucleobase can be A, T, C, G, or U. For example, the base can be modified by substitution, which can be a DNA base substitution. For example, the agRNA can comprise one or more modified nucleobases. For example, in the agRNA, the modified nucleobases can account for about 5%-100%, about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.

[0112] For example, in the modified agRNA, the modified can be a ribose. The modified sugar can have the spatial arrangement, electronic properties, or some other physical-chemical properties of unmodified ribose. For example, the modified sugar is a substituted ribose or deoxyribose. For example, the modified sugar comprises a 2’-modification, which can be 2’-MOE, 2’-, 2’-OMe, 2’-OR, wherein R can be an optionally substituted C1-10 aliphatic group. For example, the modified sugar can comprise a LNA modification. For example, the modified sugar can comprise a UNA modification. For example, the agRNA can comprise one or more modified sugars. For example, in the agRNA, the modified sugars can be about 5%-100%, about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.

[0113] In the present application, in the modified agRNA, the modification comprises at least one structure of any one of Formula 1 to Formula 6:

[0114] wherein Formula 1 is including but not limited to constrained ethyl nucleotides, UNA-nucleotides, LNA nucleotides;

[0115] R1 is hydrogen, hydroxyl, halogen, amino, O-C1-C6 alkoxy, or O methoxyethyl;

[0116] R2 is hydroxyl, halogen, or C1-C6 alkoxy;

[0117] R3 is hydrogen, hydroxyl, halogen, or C1-C6 alkoxy;

[0118] R4 is hydrogen, hydroxyl, halogen, or C1-C6 alkoxy; and

[0119] R5 is hydrogen, hydroxyl, halogen, or Ci-C6alkoxy.

[0120] In the present application, the modified agRNA has no other modification than the modification selected from any one of Formula 1 to Formula 6 at the agRNA site of RNA target editing and at all other agRNA sites except the agRNA site where off-target editing exists.

[0121] For example, the modified agRNA has no modification than the modification at the agRNA site of RNA target editing and at all other agRNA sites except the agRNA site where off-target editing exists. For example, the modified agRNA has no modification than the structure of Formula 1 at the agRNA site of RNA target editing and at all other agRNA sites except the agRNA site where off-target editing exists. For example, the modified agRNA has no modification than the structure of Formula 2 at the agRNA site of RNA target editing and at all other agRNA sites except the agRNA site where off-target editing exists. For example, the modified agRNA has no modification than the structure of Formula 3 at the agRNA site of RNA target editing and at all other agRNA sites except the agRNA site where off-target editing exists. For example, the modified agRNA has no modification than the structure of Formula 4 at the agRNA site of RNA target editing and at all other agRNA sites except the agRNA site where off-target editing exists. For example, the modified agRNA has no modification than the structure of Formula 5 at the agRNA site of RNA target editing and at all other agRNA sites except the agRNA site where off-target editing exists. For example, the modified agRNA has no modification than the structure of Formula 6 at the agRNA site of RNA target editing and at all other agRNA sites except the agRNA site where off-target editing exists. For example, the modified agRNA has no combination of the structure of Formula 1 to Formula 6 at the agRNA site of RNA target editing and at all other agRNA sites except the agRNA site where off-target editing exists. For example, the modified agRNA has 1-10 sites (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) or more sites that do not contain the structure selected from any one of Formula 1 to Formula 6 at the agRNA site of RNA target editing and at all other agRNA sites except the agRNA site where off-target editing exists.

[0122] In the present application, the agRNA site with off-target editing can comprise two and more than two different kinds of modifications selected from the group consisting of LNA, UNA, 2’-MOE, 2’-F, 2’-OMe, and DNA base replacement modification. In the present application, the agRNA site with off-target editing can also comprise phosphorothioation modification.

[0123] In the present application, the agRNA site with off-target editing can comprise two and more than two different kinds of modifications selected from the group consisting of LNA, UNA, 2’-MOE, 2’-F, 2’-OMe, and DNA base replacement modification. In the present application, the agRNA site with off-target editing can also comprise phosphorothioation modification.

[0124] For example, the combination of the modifications can be: DNA base replacement modification, 2’-F. For example, the modification can be optionally selected from the group consisting of LNA, UNA, 2’-MOE, 2’-F, 2’-OMe, and DNA base replacement modification. For example, the combination of the modifications can be optionally selected from the group consisting of 2’-MOE, LNA, UNA, 2’-F, 2’-OMe. For example, the modification can be optionally selected from the group consisting of phosphorothioation modification, DNA base replacement modification, 2’-F. For example, the modification can be optionally selected from the group consisting of phosphorothioation modification, LNA, UNA, 2’-MOE, 2’-F, 2’-OMe, and DNA base replacement modification. For example, the combination of the modifications can be optionally selected from the group consisting of phosphorothioation modification, 2’-MOE, LNA, UNA, 2’-F and 2’-OMe.

[0125] For example, the modification can be selected and optimized according to the distance between the off-target site and the target site.

[0126] For example, when the off-target site is proximal to the target site, the combination of the modification of the agRNA site with off-target editing can be selected from the group consisting of DNA base replacement modification and 2’-F. For example, when the off-target site is proximal to the target site, the modification can be DNA base replacement modification, which further comprises phosphorothioation modification. For example, when the off-target site is proximal to the target site, the modification can be 2’-F, which further comprises phosphorothioation modification.

[0127] For example, the proximal end can refer to the off-target site being located at position -1 or +1 from the target site. For example, when the off-target site is located at position -1 or +1 from the target site, the modification of the agRNA site in the presence of off-target editing can be a modification selected from the group consisting of: a DNA base replacement modification and 2’-F. For example, when the off-target site is located at position -1 or +1 from the target site, the modification can be a DNA base replacement modification, the modification further comprising a phosphorothioated modification. For example, when the off-target site is located at position -1 or +1 from the target site, the modification can be 2’-F, the modification further comprising a phosphorothioated modification.

[0128] For example, when the off-target site is within 10 bases from the target site, the agRNA site in the presence of off-target editing is independently selected from the group consisting of: LNA, UNA, 2’-MOE, 2’-F, 2’-OMe, and a DNA base replacement modification. For example, when the off-target site is within 10 bases from the target site, the agRNA site in the presence of off-target editing is independently selected from the group consisting of: a 2’-F modification and a 2’-OMe modification, the modification further comprising a phosphorothioated modification. For example, when the off-target site is within 10 bases from the target site, the agRNA site in the presence of off-target editing is independently selected from the group consisting of: a DNA base replacement modification, a 2’-MOE modification, a LNA modification, and a UNA modification, the modification further comprising a phosphorothioated modification.

[0129] For example, when the off-target site is distal to the target site, the agRNA site in the presence of off-target editing is independently selected from the group consisting of: 2’-MOE, LNA, UNA, 2’-F, and 2’-OMe. For example, when the off-target site is distal to the target site, the agRNA site in the presence of off-target editing is independently selected from the group consisting of: a 2’-MOE modification, a LNA modification, and a UNA modification, the modification further comprising a phosphorothioated modification. For example, when the off-target site is distal to the target site, the agRNA site in the presence of off-target editing is independently selected from the group consisting of: a 2’-F modification and a 2’-OMe, the modification further comprising a phosphorothioated modification.

[0130] For example, the off-target site located distal to the target site can mean that the distance between the off-target site and the target site is greater than 10 bases. For example, when the distance between the off-target site and the target site is greater than 10 bases, the agRNA site with off-target editing is each independently selected from the following modifications: 2'-MOE, LNA, UNA, 2'-F, and 2'-OMe. For example, when the distance between the off-target site and the target site is greater than 10 bases, the agRNA site with off-target editing is each independently selected from the following modifications: 2'-MOE modification, LNA modification, and UNA modification, which further comprises phosphorothioate modification. For example, when the distance between the off-target site and the target site is greater than 10 bases, the agRNA site with off-target editing is each independently selected from the following modifications: 2'-F modification and 2'-OMe, which further comprises phosphorothioate modification.

[0131] For example, the agRNA can comprise any one of the modification combinations as shown in the sequence XX-XX.

[0132] In the present application, the length of the agRNA can be 25-70 nt. For example, the length of the agRNA can be about 25-30 nt, about 25-35 nt, about 25-40 nt, about 25-45 nt, about 25-50 nt, about 25-55 nt, about 25-60 nt, about 25-60 nt, about 30-35 nt, about 30-40 nt, about 30-45 nt, about 30-50 nt, about 30-55 nt, about 30-60 nt, about 30-60 nt, about 30-70 nt, about 40-45 nt, about 40-50 nt, about 40-55 nt, about 40-60 nt, about 40-60 nt, about 40-70 nt, about 50-55 nt, about 50-60 nt, about 50-60 nt, about 50-70 nt, about 60-70 nt. For example, the length of the agRNA can be 41 nt. For example, the length of the agRNA can be 51 nt.

[0133] In the present application, the off-target site of the agRNA can be adenosine. In the present application, the target site of the agRNA can be adenosine. The selection of the target site and the off-target site can be selected by conventional means known in the art, or can be selected by writing a script by oneself.

[0134] In the present application, the agRNA can bind to the target RNA to form a double-stranded complex.

[0135] For example, the target RNA can be selected from one or more of the following group: pre-mRNA, mRNA, rRNA, tRNA, lnc-RNA.

[0136] For example, the agRNA pairs completely complementary to the target RNA. For example, the complementary pairing principle is Watson-Crick or Hoogsteen base pairing. For example, the complementary pairing can refer to A-T, C-G, T*A / T, C*G / C.

[0137] For example, the agRNA is complementary paired to the target RNA with one or more mismatches, wobbles, deletions, and / or bulges. For example, there can be 1-10 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) base mismatches. For example, there can be 1-10 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) wobbles. For example, there can be 1-10 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) deletions. For example, there can be 1-10 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) bulges. For example, the complementarity between the agRNA and the target RNA is about 50-100% (e.g., about 50-80%, 50-85%, 50-90%, 50-95%, 60-80%, 60-85%, 60-90%, 60-95%, 60-100%, 65-80%, 65-85%, 65-90%, 65-95%, 65-100%, 70-80%, 70-85%, 70-90%, 70-95%, 70-100%, 75-80%, 75-85%, 75-90%, 75-95%, 75-100%, 80-85%, 80-90%, 80-95%, 80-100%, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.). For example, the complementarity is at least about 60%.In some embodiments, the complementarity is at least about 65%. For example, the complementarity is at least about 70%. For example, the complementarity is at least about 75%. For example, the complementarity is at least about 80%. For example, the complementarity is at least about 85%. For example, the complementarity is at least about 90%. For example, the complementarity is at least about 95%.

[0138] In the present application, the agRNA can recruit endogenous deaminases to perform deamination reactions on specific nucleotide sites.

[0139] In the present application, the agRNA can comprise any of the sequences as shown in XX-XX.

[0140] Method for editing RNA

[0141] In another aspect, the present application provides a method for editing RNA, comprising using the modified agRNA described in the present application.

[0142] In the present application, the method can reduce off-target editing of the target RNA.

[0143] For example, the off-target editing is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%. For example, the method can reduce the editing level of all off-target sites to the background state. For example, the chemical modification of the off-target site has little or little effect on the editing level of the target site.

[0144] In the present application, the method can comprise the following steps:

[0145] (1) providing the modified agRNA described in the present application;

[0146] (2) introducing the modified agRNA into the cell;

[0147] (3) allowing the agRNA to bind to the target RNA.

[0148] For example, the RNA editing method can not only be used for editing RNA of animal cells, such as mammalian cells, but also be used for editing RNA of plants or fungi, for example, genetically engineered plants and fungi with improved properties can be generated using the RNA editing method provided in the present application.

[0149] Delivery vector

[0150] In another aspect, the present application provides a delivery vector, which can comprise the modified agRNA described in the present application, and can deliver the agRNA in the present application to the cell.

[0151] For example, the delivery vector can include a viral vector, a non-viral vector. The viral vector can include a lentivirus (LV) vector, an adenovirus (AdV) vector, and an adeno-associated virus (AAV) vector, etc. For example, the non-viral vector can include a liposome, a molecular conjugate receptor, a polymer, a complex vector, and a nanoparticle vector, etc. The agRNA can be delivered to the cell for RNA editing using the delivery vector.

[0152] Cell

[0153] In another aspect, the present application provides a cell, which can comprise the agRNA, the delivery vector described in the present application.

[0154] For example, the agRNA can be transfected into the cell, the agRNA can be delivered to the cell using the delivery vector, and RNA editing can be performed.

[0155] For example, the operation on the cell can be performed in vivo. For example, the cell can be isolated from the body, or the cell line can be cultured and operated in vitro.

[0156] Pharmaceutical composition

[0157] In another aspect, the present application provides a pharmaceutical composition, which can comprise the agRNA, one or more isolated nucleic acid molecules, the expression vector, the delivery vector, the cell, and / or the pharmaceutically acceptable carrier described in the present application.

[0158] For example, the pharmaceutical composition can be administered by intravenous infusion over a period of time. For example, the pharmaceutical composition can be administered by subcutaneous delivery. For example, the pharmaceutical composition can be administered periodically, such as once a week, once every two weeks, once a month, two months, three months, four months. For example, after administration every week or every two weeks for three months, the administration can be once a month for six months or one year or more.

[0159] Application

[0160] In another aspect, the present application provides the agRNA, the delivery vector, the cell, and the pharmaceutical composition described for preventing and / or treating diseases and / or conditions.

[0161] In another aspect, the present application provides the use of the agRNA, the delivery vector, the cell, and the pharmaceutical composition described in the preparation of a medicament for preventing and / or treating diseases and / or conditions.

[0162] In another aspect, the present application provides a method for preventing and / or treating a disease and / or a disorder, comprising administering to a subject in need thereof an effective amount of the agRNA, the delivery vector, the cell, or the pharmaceutical composition.

[0163] For example, the disease and / or disorder can be a monogenic or polygenic mutation related disease. For example, the gene can be GAPDH, ATP7B, FGFR, TP53, APC, SERPINA1, MECP2, SCN1A.

[0164] Without wishing to be bound by any theory, the examples below are merely to illustrate the agRNA, the method of preparation and use thereof, etc. of the present application, and are not intended to limit the scope of the present application.

[0165] Examples

[0166] Example 1 Off-target editing of agRNA in mouse primary hepatocytes

[0167] The preparation process is based on the following steps:

[0168] 1. Isolation and culture of mouse primary hepatocytes:

[0169] (1) Preparation of 1x HBSS solution: Take 100 mL of 10x HBSS into a beaker, add 850 mL of double distilled water, add sodium bicarbonate powder to adjust the pH of the solution to 7.5, and finally make up to 1000 mL.

[0170] (2) Preparation of pre-perfusion solution: Add 500 μL of 0.5 M EGTA solution (final concentration: 0.5 mM) to 500 mL of 1x HBSS solution, filter sterilize, and prepare immediately for use.

[0171] (3) Preparation of perfusion solution: Add calcium chloride (final concentration: 3 mM) and collagenase IV (final concentration: 100 U / mL) to 500 mL of 1x HBSS solution, dissolve thoroughly, filter sterilize, and prepare immediately for use.

[0172] (4) Isotonic Percoll separation solution: Take 1 mL of 10x HBSS solution and add it to 10 mL of Percoll solution to prepare an isotonic Percoll separation solution with a concentration of 90%.

[0173] (5) Turn on the electric heating water bath and preheat it to 42°C. The reagents that need to be preheated are the pre-perfusion solution and the perfusion solution.

[0174] (6) Surface disinfection: Disinfect the surgical instruments and operating table with 75% alcohol.

[0175] (7) Precool DMEM on ice.

[0176] (8) Anesthetize the mouse by intraperitoneal injection.

[0177] (9) After the mouse is completely anesthetized, fix the mouse to the foam board, and sterilize the whole mouse with 75% alcohol.

[0178] (10) Cut the skin and peritoneum of the mouse abdomen along the midline and fix with a needle.

[0179] (11) Push the small and large intestines of the mouse to the right side with a cotton swab, and expose the portal vein (the vein that carries blood into the liver, between the liver and the stomach) and inferior vena cava (the vein that carries blood out of the liver).

[0180] (12) Gently push the mouse liver down with a cotton swab, find the superior vena cava at the junction of the liver and diaphragm, and clamp the superior vena cava with a vein clamp to block the return of blood to the heart.

[0181] (13) Insert a retention needle into the hepatic portal vein, and when the needle core is withdrawn, see blood flowing back in the hose, which indicates successful insertion of the blood vessel. Connect the hose of the peristaltic pump to the retention needle, and cut off the inferior vena cava.

[0182] (14) Start the peristaltic pump, and perfuse the mouse liver with the pre-perfusion solution, which is always placed in a 42°C water bath. The perfusion flow rate is 5 mL / min, and the perfusion time is 5 min. During the perfusion process, press the inferior vena cava with a cotton swab every 30 s for 7 s, so that the pre-perfusion solution fills the mouse liver (the liver swells due to water filling). The perfused liver should be white, and there should be no red liquid left in the inferior vena cava.

[0183] (15) Stop the peristaltic pump, replace the pre-perfusion solution with perfusion solution (which is always placed in a 42°C water bath), and continue to perfuse the liver. The flow rate is adjusted to 3 mL / min, and the perfusion time is 8 min. During the perfusion process, press the inferior vena cava with a cotton swab every 30 s for 7 s, so that the perfusion solution fills the mouse liver. The perfused liver should be soft and inelastic, and there should be visible cracking on the surface of the liver.

[0184] (16) Gently separate the liver with scissors and tweezers (at this time the liver becomes very fragile, avoid breaking the capsule to cause liver cells to flow out) and place it in a culture dish containing DMEM. Gently rinse the surface of the liver, and remove the gallbladder (the whole process is operated on ice).

[0185] (17) Transfer the liver to another clean culture dish containing DMEM, gently tear the liver capsule with a curved forceps, and shake the liver. Liver cells will flow out, and the whole solution will become turbid (the whole process is operated on ice).

[0186] (18) Shake the liver until all the hepatocytes flow out, at this time the whole liver is only left with fibrous tissue. Use a Pasteur pipette to suck the rinse solution through a 70 μm cell strainer into a 50 mL centrifuge tube (the whole process is operated on ice).

[0187] (19) 50g, 4°C centrifugation for 2 min, a large amount of hepatocytes can be seen in the bottom precipitate.

[0188] (20) Remove the supernatant, resuspend the hepatocytes with 7.5 mL of DMEM, then add 5 mL of isotonic Percoll separation solution, mix well, 200g, 4°C centrifugation for 15 min.

[0189] (21) After density separation, the hepatocytes in the bottom precipitate are good active cells, the cells suspended in the solution are dead cells and poor active cells, and the supernatant is vacuumed.

[0190] (22) Use 15 mL of DMEM to rinse the hepatocytes twice, resuspend the precipitate with hepatocyte plating culture solution, count the cells, adjust the cell density to 2x10^5 cells / mL, and inoculate the cells into a 12-well cell culture plate.

[0191] (23) After 4 hours, when the hepatocytes are completely adherent, replace them with hepatocyte maintenance medium for the next step experiment.

[0192] 2. Cell transfection experiment steps:

[0193] (1) Seed mouse primary hepatocytes in a 24-well cell culture plate (1*10 5 cells / well), and place in a cell culture incubator at 37°C, 5% CO2.

[0194] (2) After 4 hours, when the cells are completely adherent, replace them with fresh maintenance medium.

[0195] (3) According to the instructions provided by the transfection reagent Lipofectamine TM RNAiMAX, prepare the transfection reagent mixture, and stand at room temperature for 5 min.

[0196] (4) Take out the cells and carefully add the transfection solution to the cells, mix gently and evenly, and then place in the incubator.

[0197] 3. Sanger sample preparation for first-generation sequencing

[0198] The total RNA of the mouse primary hepatocytes was extracted 48 hours after transfection of the agRNA using a FastPure Cell / Tissue Total RNA Isolation Kit (manufacturer: Novogene, item number: RC101-01) according to the instructions. Then, the RNA was reverse transcribed to obtain cDNA using a HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (manufacturer: Novogene, item number: R312-02) according to the instructions. Then, the cDNA was used as a template for PCR amplification using 2x Taq PCR StarMix (manufacturer: GenStar, item number: A012) according to the instructions, and the PCR products were subjected to Sanger sequencing using the primers mGAPDH-F and mGAPDH-R in the following table.

[0199] 4. Sanger data RNA editing level measurement

[0200] The raw sequencing files obtained by Sanger sequencing need to measure the editing level of the editing site. The present application uses the EditR software (https: / / moriaritylab.shinyapps.io / editr_v10 / ) for calculation and analysis. The software analyzes the peak value of each base of A, T, G, and C in the upstream and downstream regions of the editing site. The calculation method of the editing level is 100*(1-A peak value / A+T+C+G peak value sum).

[0201] The experimental results are shown in the following figures:

[0202] Figure 1A shows that the A base editing level of the target site of the GAPDH gene in the mouse primary hepatocytes transfected with the agRNA GAPDH-41nt-V1 is 87%, the editing level of the A base at the off-target site-8 is 21%, the editing level of the A base at the off-target site-11 is 21%, the editing level of the A base at the off-target site-13 is 45%, and the editing level of the A base at the off-target site-14 is 61% (the A base at the editing site is defined as 0, the base connected to the A base at the editing site and located at the 5' end of the target RNA is-1, and the base connected to the A base at the editing site and located at the 3' end of the target RNA is +1).

[0203] Figure 1B shows that the A base editing level of the target site of the GAPDH gene in mouse primary hepatocytes transfected with agRNAGAPDH-51nt-V1 is 88%, the editing level of the -8th A base of the off-target site is 19%, the editing level of the -11th A base is 15%, the editing level of the -13th A base is 16%, the editing level of the -14th A base is 25%, the editing level of the -16th A base is 47%, and the editing level of the -17th A base is 82%.

[0204] Table 1 Editing levels of the target site and off-target sites of the mouse GAPDH gene after transfection

[0205] The above experimental results show that:

[0206] The two agRNAs targeting the mouse GAPDH gene with different lengths have high off-target editing at the off-target sites. According to the distance between the off-target site and the editing site, the off-target editing shows different editing levels.

[0207] Note: When the agRNA binds to the target RNA to form double-stranded RNA, the A base of the target site is 0.

[0208] Example 2 Chemical modification reduces the editing efficiency of off-target sites

[0209] From Example 1, we found that agRNAs with 5'-end 3 LNA modifications and 3'-end 3 LNA modifications, and full-thio-modified backbone, can cause severe off-target editing of the target RNA in in vitro cell experiments (Table 1). According to the original Sanger sequencing graph (Figure 1), we can determine which A base positions have off-target editing. In order to reduce the off-target editing of each off-target site, we first modified the bases paired with the off-target sites of the target RNA on the basis of the original agRNAs (GAPDH-41nt-V1 and GAPDH-51nt-V1) to include but not limited to the following chemical modifications: 2'-F modification, 2'-OMe modification, 2'-MOE modification, LNA modification, UNA modification, and DNA base replacement modification, and synthesized the corresponding agRNAs. Mouse primary hepatocytes were seeded in a 24-well cell culture plate, and after 4 hours, the culture medium was replaced with fresh maintenance medium, and 20 pmol of agRNA was transfected into the cells using Lipofectamine TM RNAiMAX, and 48 hours after transfection, Sanger sequencing samples were prepared according to the experimental method in Example 1 and subjected to Sanger sequencing to analyze the editing levels of the target editing site and the off-target site of the target RNA.

[0210] Table 2 agRNA sequences with different chemical modifications Note: N = RNA base, dN = DNA base, LN = LNA base, fN = 2'-F RNA base, mN = 2'-OMe RNA base, eN = 2'-MOE RNA base, unaN = UNA RNA base, * = phosphorothioate bond

[0211] The experimental results are shown in the figures:

[0212] Figures 2 and 3 show the Sanger sequencing raw images of the editing levels of mouse primary hepatocyte GAPDH gene mediated by agRNAs with different chemical modifications designed on the basis of GAPDH-41nt-V1, and the editing levels of the editing sites and off-target sites are shown in Table 3.

[0213] Table 3: Editing levels of mouse GAPDH gene targeting region editing sites and off-target sites after transfection

[0214] The experimental results show that:

[0215] Compared with GAPDH-41nt-V1, the agRNAs with 2'-F modification, 2'-OMe modification, 2'-MOE modification, LNA modification, UNA modification and DNA base substitution modification on the base paired with the target RNA off-target site can greatly reduce the editing level of the off-target site, among which 2'-OMe modification, 2'-MOE modification, LNA modification and UNA modification can reduce the editing level of all off-target sites to the background state, showing that there is basically no off-target editing, and the chemical modification of the off-target position basically does not affect or slightly affects the editing level of the target site.

[0216] The experimental results are shown in the figures:

[0217] Figures 4 and 5 show the Sanger sequencing raw images of the editing levels of mouse primary hepatocyte GAPDH gene mediated by agRNAs with different chemical modifications designed on the basis of GAPDH-51nt-V1, and the editing levels of the editing sites and off-target sites are shown in Table 4.

[0218] Table 4: Editing levels of mouse GAPDH gene targeting region editing sites and off-target sites after transfection

[0219] The experimental results show that:

[0220] Compared with GAPDH-51nt-V1, 2'-F modification, 2'-OMe modification, 2'-MOE modification, LNA modification, UNA modification and DNA base substitution modification on the base paired with the target RNA off-target site can greatly reduce the editing level of the off-target site, among which 2'-MOE modification, LNA modification and UNA modification can reduce the editing level of all off-target sites to the background state, showing that there is basically no off-target editing, and the chemical modification of the off-target position basically does not affect or slightly affects the editing level of the target site.

[0221] Example 3 Combination of various chemical modifications reduces off-target editing of off-target sites

[0222] This example studies the selection of two or more chemical modifications from the following chemical modifications, including but not limited to: 2'-F modification, 2'-OMe modification, 2'-MOE modification, LNA modification, UNA modification and DNA base substitution modification, to combine the modification of the base paired with the target RNA off-target site in the agRNA, so as to better reduce the editing level of the off-target site.

[0223] From Example 2, we found that when a single chemical modification was made to the base paired with the target RNA off-target site based on GAPDH-51nt-V1, 2'-MOE modification, LNA modification and UNA modification could reduce the editing level of all off-target sites to no editing, and 2'-F modification, 2'-OMe modification and DNA base substitution modification could effectively reduce off-target editing, but could not reduce the off-target editing of all off-target sites to no editing. We further introduced, based on the agRNA with single modification of 2'-F modification, 2'-OMe modification and DNA base substitution modification, including but not limited to 2'-F modification, 2'-OMe modification, 2'-MOE modification, LNA modification, UNA modification and DNA base substitution modification to the off-target sites still having off-target editing, so that the agRNA has two or more chemical modifications.

[0224] Mouse primary hepatocytes were seeded in a 24-well cell culture plate, and after 4 hours, the culture medium was replaced with fresh maintenance medium, and 20 pmol of agRNA was transfected into the cells with Lipofectamine TM RNAiMAX, and 48 hours after transfection, Sanger sequencing samples were prepared according to the experimental method in Example 1 and subjected to Sanger sequencing, and the editing levels of the target RNA target editing site and the off-target site were analyzed.

[0225] Table 5.1 agRNA sequence with multiple modifications Note: N = RNA base, dN = DNA base, LN = LNA RNA base, fN = 2’-F RNA base, mN = 2’-OMe RNA base, eN = 2’-MOE RNA base, unaN = UNA RNA base, * = phosphorothioate bond

[0226] The experimental results are shown in the figures:

[0227] Figure 6 shows the Sanger sequencing raw images of the editing levels of mouse primary hepatocyte GAPDH gene mediated by agRNA using various combinations of chemical modifications designed on the basis of GAPDH-51nt-V3. The editing levels of the editing site and off-target site are shown in Table 5.2.

[0228] Table 5.2: Editing levels of the editing site and off-target site in the targeted region of mouse GAPDH gene after transfection

[0229] Figures 7 and 8 show the Sanger sequencing raw images of the editing levels of mouse primary hepatocyte GAPDH gene mediated by agRNA using various combinations of chemical modifications designed on the basis of GAPDH-51nt-V4. The editing levels of the editing site and off-target site are shown in Table 6.

[0230] Table 6: Editing levels of the editing site and off-target site in the targeted region of mouse GAPDH gene after transfection

[0231] Figures 9 and 10 show the Sanger sequencing raw images of the editing levels of mouse primary hepatocyte GAPDH gene mediated by agRNA using various combinations of chemical modifications designed on the basis of GAPDH-51nt-V2. The editing levels of the editing site and off-target site are shown in Table 7.

[0232] Table 7: Editing levels of the editing site and off-target site in the targeted region of mouse GAPDH gene after transfection

[0233] The experimental results show that:

[0234] Further, on the basis of the single modification of the agRNA with 2'-F modification, 2'-OMe modification, and DNA base substitution modification, introduce modifications including but not limited to 2'-F modification, 2'-OMe modification, 2'-MOE modification, LNA modification, UNA modification, and DNA base substitution modification on the off-target sites still having off-target editing, so that the agRNA has two or more chemical modifications. When the agRNA with two or more chemical modifications is transfected into mouse primary hepatocytes to mediate RNA editing of the GAPDH gene, the editing level of all off-target sites can be reduced to the background level, showing that there is essentially no off-target editing, and the chemical modification of the off-target site has little or no effect on the editing level of the target site.

[0235] Example 4. Reducing off-target editing of human RAB7A gene by using multiple chemical modification combinations

[0236] This example studies the combination modification of the bases in the agRNA that pair with the target RNA off-target sites by selecting two or more chemical modifications from the following chemical modifications including but not limited to 2'-F modification, 2'-OMe modification, 2'-MOE modification, LNA modification, UNA modification, and DNA base substitution modification, so as to better reduce the editing level of the off-target sites.

[0237] Hela cells were seeded in a 24-well cell culture plate, and 24 hours later, the culture medium was replaced with fresh complete culture medium, and 20 pmol of agRNA was transfected into the cells using Lipofectamine TM RNAiMAX, and 48 hours after transfection, the Sanger sample for next-generation sequencing was prepared according to the experimental method in Example 1 and subjected to Sanger sequencing, and the editing levels of the target RNA target editing site and the off-target site were analyzed.

[0238] Table 8. Sequences of agRNAs with multiple modifications targeting human RAB7A gene

[0239] Figure 11 shows the Sanger sequencing raw graph of the editing level of the RAB7A gene mediated by the agRNA with multiple chemical modification combinations targeting the human RAB7A gene transfected into Hela cells, and the editing levels of the editing site and the off-target site are shown in Table 9.

[0240] Table 9: Editing levels of the target region editing site and the off-target site of the human RAB7A gene after transfection

[0241] The experimental results show that:

[0242] When the agRNAs with two or more than two chemical modifications were transfected into Hela cells to mediate the RNA editing of human RAB7A gene, the editing levels of all off-target sites were reduced to the background level, showing that there was almost no off-target editing, and the chemical modifications of off-target sites had little or no effect on the editing level of target sites.

[0243] Example 5 Combination of multiple chemical modifications reduces off-target editing of human PPIA gene site 1

[0244] This example studies the combination of two or more chemical modifications selected from the following chemical modifications, but not limited to: 2'-F modification, 2'-OMe modification, 2'-MOE modification, LNA modification, UNA modification and DNA base substitution modification, to modify the bases in the agRNA that pair with the off-target sites of the target RNA, so as to better reduce the editing level of the off-target sites.

[0245] Hela cells were seeded in 24-well cell culture plates, 24 hours later the culture medium was replaced with fresh complete culture medium, and 20 pmol of agRNA was transfected into the cells with Lipofectamine TM RNAiMAX, 48 hours after transfection, Sanger sequencing samples were prepared according to the experimental method in Example 1 and Sanger sequencing was performed, and the editing levels of the target RNA target editing site and off-target site were analyzed.

[0246] Table 10: agRNA sequences with multiple modifications targeting human RAB7A gene site 1

[0247] Figure 12 shows the Sanger sequencing raw image of the editing level of the agRNA with multiple chemical modification combinations targeting human PPIA gene site 1 mediated by transfection into Hela cells, and the editing levels of the editing site and off-target site are shown in Table 9.

[0248] Table 11: Editing levels of the target region of human RAB7A gene and off-target sites after transfection

[0249] The experimental results show that:

[0250] When the agRNAs with two or more than two chemical modifications were transfected into Hela cells to mediate the RNA editing of human PPIA gene site 1, the editing levels of all off-target sites were reduced to the background level, showing that there was almost no off-target editing, and the chemical modifications of off-target sites had little or no effect on the editing level of target sites.

[0251] Example 6 Combination of multiple chemical modifications reduces off-target editing of human RAB7A gene site 2

[0252] This example studies the combination modification of the bases in the agRNA that pair with the off-target sites of the target RNA by selecting two or more chemical modifications from the following, but not limited to: 2'-F modification, 2'-OMe modification, 2'-MOE modification, LNA modification, UNA modification and DNA base substitution modification, so as to achieve better reduction of the editing level of off-target sites.

[0253] Hela cells were seeded in 24-well cell culture plates, 24 hours later the culture medium was replaced with fresh complete culture medium, and 20 pmol of agRNA was transfected into the cells with Lipofectamine TM RNAiMAX, 48 hours after transfection, the Sanger sample for next generation sequencing was prepared according to the experimental method in Example 1 and Sanger sequencing was performed, and the editing level of the target RNA target editing site and off-target site was analyzed.

[0254] Table 12: agRNA sequences of various modifications targeting human RAB7A gene site 2

[0255] Figure 13 shows the Sanger sequencing raw graph of the editing level of the RAB7A gene mediated by the agRNA of various chemical modification combinations targeting human PPIA gene site 2 transfected into Hela cells, and the editing level of the editing site and off-target site is shown in Table 9.

[0256] Table 13: Editing level of the target site and off-target site of the human RAB7A gene after transfection

[0257] The experimental results show that:

[0258] When the agRNA with two or more chemical modifications is transfected into Hela cells to mediate RNA editing of the human PPIA gene site 2, the editing level of all off-target sites can be reduced to the background state, showing that there is essentially no off-target editing, and the chemical modification of the off-target position has little or no effect on the editing level of the target site.

Claims

1. A modified guide agRNA (ADAR guide RNA, agRNA) having phosphorothioated modification at agRNA sites where off-target editing exists, and further having other modifications.

2. The modified agRNA of claim 1, the modifications capable of reducing off-target editing to a target RNA.

3. The modified agRNA of any one of claims 1-2, wherein the paring nucleotides of the target site do not comprise 2’-MOE or 2’-OMe modification.

4. The modified agRNA of any one of claims 1-3, wherein the other modification comprises at least one having the structure of any one of Formula 1 to Formula 6: wherein Formula 1 is including but not limited to constrained ethyl-nucleotides, UNA- nucleotides, LNA-nucleotides; R1 is hydrogen, hydroxyl, halogen, amino, O-C1-C6 alkoxy, or O-methoxyethyl; R2 is hydroxyl, halogen, or C1-C6 alkoxy; R3 is hydrogen, hydroxyl, halogen, or C1-C6 alkoxy; R4 is hydrogen, hydroxyl, halogen, or C1-C6 alkoxy; and R5 is hydrogen, hydroxyl, halogen, or C1-C6 alkoxy.

5. The modified agRNA of any one of claims 1-4, having other modifications at the agRNA sites of RNA target editing and at all other agRNA sites except the agRNA sites where off-target editing exists, the other modifications selected from the structures of any one of Formula 1 to Formula 6.

6. The modified agRNA of any one of claims 1-5, wherein the agRNA sites where off-target editing exists comprise two and more than two different kinds of modifications, the modifications optionally selected from the structures of Formula 1 to Formula 6.

7. The modified agRNA of any one of claims 1-6, wherein the agRNA sites where off-target editing exists comprise two and more than two different kinds of modifications, the modifications selected from the group consisting of LNA, UNA, 2’-MOE, 2’-F, 2’-OMe, and DNA base replacement modification.

8. The modified agRNA of any one of claims 1-7, all agRNA sites further comprising phosphorothioated modification.

9. The modified agRNA of any one of claims 1-8, wherein the modifications of the agRNA sites where off-target editing exists are selected from DNA base replacement modification and 2’-F when the off-target site is located at the -1 or +1 position of the target site.

10. The modified agRNA of any one of claims 1-8, wherein the agRNA sites where off-target editing exists are each independently selected from the modifications of the group consisting of 2’-F, 2’-OMe, DNA base replacement modification, 2’-MOE, LNA, and UNA when the off-target site is within 10 bases of the target site.

11. The modified agRNA of any one of claims 1-8, wherein the agRNA sites where off-target editing exists are each independently selected from the modifications of the group consisting of 2’-MOE, LNA, UNA, 2’-F, and 2’-OMe when the off-target site is more than 10 bases from the target site.

12. The modified agRNA of any one of claims 1-11, wherein the agRNA is 25-70 nt in length.

13. The modified agRNA of any one of claims 1-12, wherein the agRNA is 41 nt or 51 nt in length.

14. The modified agRNA of any one of claims 1-13, wherein the off-target site is adenosine.

15. The modified agRNA of any one of claims 1-14, wherein the target site is adenosine.

16. The agRNA of any one of claims 1-15, wherein the target RNA is selected from one or more of the group consisting of pre-mRNA, mRNA, rRNA, tRNA, and Inc-RNA.

17. The modified agRNA of any one of claims 1-16, wherein the agRNA pairs completely complementary to the target RNA.

18. The modified agRNA of any one of claims 1-16, wherein the agRNA pairs complementary to the target RNA with one or more mismatches, wobbles, deletions, and / or bulges.

19. The modified agRNA of any one of claims 1-18, wherein the agRNA can recruit endogenous deaminases to deaminate a specific nucleotide site.

20. A method of editing RNA, comprising using the modified agRNA of any one of claims 1-19.

21. The method of claim 20, which is capable of reducing off-target editing of a target RNA.

22. The method of any one of claims 20-21, comprising the steps of: (1) providing the modified agRNA of any one of claims 1-19; (2) introducing the modified agRNA into a cell; and (3) allowing the agRNA to bind to a target RNA.

23. A delivery vehicle comprising the modified agRNA of any one of claims 1-19.

24. A cell comprising the modified agRNA of any one of claims 1-19, and / or the delivery vehicle of claim 23.

25. The cell of claim 24, wherein the cell is a eukaryotic cell.

26. The cell of any one of claims 24-25, wherein the cell is a human cell or a mouse cell.

27. The cell of any one of claims 24-26, wherein the cell is a liver cell.

28. The cell of any one of claims 24-27, wherein the cell is a neural cell.

29. A pharmaceutical composition comprising the modified agRNA of any one of claims 1-19, the delivery vehicle of claim 23, the cell of any one of claims 24-28.

30. The modified agRNA of any one of claims 1-19, the delivery vehicle of claim 23, the cell of any one of claims 24-28, and / or the pharmaceutical composition of claim 29 for use in the prevention and / or treatment of a disease and / or disorder.

31. Use of the modified agRNA of any one of claims 1-19, the delivery vehicle of claim 23, the cell of any one of claims 24-28, and / or the pharmaceutical composition of claim 29 in the manufacture of a medicament for the prevention and / or treatment of a disease and / or disorder.

32. A method of preventing and / or treating a disease and / or disorder comprising administering to a subject in need thereof an effective amount of the modified agRNA of any one of claims 1-19, the delivery vehicle of claim 23, the cell of any one of claims 24-28, and / or the pharmaceutical composition of claim 29.

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