RNA editing oligonucleotides and uses thereof

Abstract

The present disclosure features compositions and methods useful for treating a condition for which deamination of adenosine in RNA results in a therapeutic outcome in a subject in need thereof.

Description

[0001] By incorporating materials submitted electronically. As a separate part of this disclosure, this application contains a sequence list in computer-readable form (filename: 50004_Seqlisting.txt; size: 1,229,802 bytes; created: July 11, 2024), which is incorporated herein by reference in its entirety. Background Technology

[0002] RNA-acting adenosine deaminases (ADARs) are enzymes that bind to double-stranded RNA (dsRNA) and convert adenosine to inosine via deamination. In RNA, inosine functions similarly to guanosine for translation and replication. Therefore, the conversion of adenosine to inosine in mRNA can cause codon changes, which may alter the encoded protein and its function. Three ADAR proteins are known to be expressed in humans: ADAR1, ADAR2, and ADAR3. ADAR1 and ADAR2 are expressed systemically, while ADAR3 is expressed only in the brain. ADAR1 and ADAR2 are catalytically active, while ADAR3 is considered inactive.

[0003] Synthetic single-stranded oligonucleotides have been shown to utilize ADAR proteins to edit target RNA by deaminating specific adenosines in the target RNA. The oligonucleotides are complementary to the target RNA, except for at least one mismatch with the adenosine to be deainated. However, previously disclosed methods have not yet demonstrated the selectivity and / or stability required for their therapeutic use. Therefore, there is a need for novel oligonucleotides capable of selectively editing target RNA using ADAR proteins in a therapeutically effective manner. Summary of the Invention

[0004] This disclosure provides oligonucleotides, compositions, and methods for, for example, deaminating adenosine (e.g., adenosine that can be deaminated to produce a therapeutic effect) in target RNA in subjects in need. In some embodiments, the target RNA is mRNA.

[0005] Adenosine deaminase (ADAR) acting on RNA is an editing enzyme that recognizes certain structural motifs of double-stranded RNA (dsRNA) and edits adenosine into inosine, thereby recoding the amino acid codons. This recoding can lead to changes in the encoded protein and its function. Nucleobases surrounding the editing site, particularly the 5' and 3' nucleotides adjacent to the editing site (forming a triad with the editing site), play a crucial role in adenosine deamination. The preference of U at the 5' position and G at the 3' position relative to the editing site was revealed through analysis of yeast RNA efficiently edited by overexpression of human ADAR2 and ADAR1. See Wang et al., (2018) *Biochemistry*, 57: 1640-1651; Eifler et al., (2013) *Biochemistry*, 52: 7857-7869; and Eggington et al., (2011) *Nature Communications*, 319: 1-9. Regardless of adjacent bases, recruiting ADAR to specific sites on selected transcripts and deaminating adenosine offers great promise for disease treatment. Based on structural and modeling studies of editing sites in dsRNA / ADAR complexes, several structural features that can be incorporated into guide oligonucleotides have been identified, which can enhance ADAR recruitment and improve the editing efficiency of target RNA. Novel chemically modified oligonucleotides, such as α-homologous DNA, are shown that can recruit ADAR proteins and deamate adenosine in target RNA with different peripheral base compositions. In addition, structure-activity relationship (SAR) studies revealed that, in addition to triplet modification, 2'-O-methyl (2'-OMe) modification of the ribose of some, but not all, nucleosides in the guide oligonucleotides is compatible with efficient ADAR recruitment and editing.

[0006] In one aspect, this paper describes an oligonucleotide comprising the following structure: [A m ]-X 1 -X 2 -X 3 -[B n ] in m + n is 24 to 50, n is at least 4, and m is at least 20; -X 1 -X 2 -X 3 - is the central triplet of the oligonucleotide; X 1 For the position -1, X of the oligonucleotide2 The position of the oligonucleotide is 0, and X 3 +1 to the position of the oligonucleotide; [A] m This is the first domain located at positions -(m+1) to -2 of the oligonucleotide; [B] n This is a second domain located at positions +2 to +(n+1) of the oligonucleotide; Each A and B is a nucleotide containing a nucleobase, a sugar (“A / B sugar”), and an internucleotide bond; Each X 1 X 2 and X 3 It contains nucleobases, sugars ("X sugars"), and bonds between nucleotides; The A / B sugar and the X 3 The sugars are selected from 2'-methoxy-ribose, 2'-MOE-ribose, 2'-deoxy-2'-fluororibose, 2'-fluoro-arabinose, 2-methoxy-arabinose, 2'-deoxyribose and locked nucleic acids (LNA). The X 1 The sugar is 2'-deoxy-2'-fluororibose or 2'-deoxyribose; The X 2 The sugars are selected from 2'-methoxy-ribose, 2'-MOE-ribose, 2'-deoxy-2'-fluororibose, 2'-fluoro-arabinose, 2-methoxy-arabinose, 2'-deoxyribose, locked nucleic acid (LNA), and β-homo-DNA sugars; The A / B sugar and the X sugar together constitute 10-70% 2'-deoxy-2'-fluoro-ribose; The oligonucleotide has 30%-100% thiophosphate and aminophosphate bonds between its nucleotides, and 3 to 20 nucleotide bonds are aminophosphate bonds. (i) the bond between the nucleotide at position -(m+1) and the nucleotide at position -(m) (5' end), (ii) the bond between the nucleotide at position +(n) and the nucleotide at position +(n+1) (3' end), or (iii) the bond between the nucleotides at each of the 5' and 3' ends of the oligonucleotide is an aminophosphate bond; and The nucleotide bonds between the nucleotide at position -(m) and the nucleotide at position -(m-1), and the nucleotide bonds between the nucleotide at position +(n-1) and the nucleotide at position +(n), ​​are independently thiophosphate bonds or aminophosphate bonds.

[0007] 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. This document describes methods and materials used in this disclosure; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of any conflict, this specification (including definitions) shall prevail.

[0008] definition For convenience, the following provides the meanings of some terms and phrases used in this specification, examples, and appended claims. Unless otherwise stated or implied from the context, the following terms and phrases include the meanings provided below. These meanings are provided to aid in describing particular embodiments, and these definitions are not intended to limit the claimed technology, as the scope of the technology is limited only by the claims. 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 they pertain. If there is a significant deviation in the use of terminology in the art from the definitions provided herein, the definitions provided in this specification shall prevail.

[0009] In this application, unless the context clearly indicates otherwise, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; and (iii) the terms “comprising” and “including” may be understood to encompass sub-components or steps, whether such sub-components or steps are presented individually or together with one or more other components or steps.

[0010] As used herein, the term “about” refers to a value that is within 10% of or higher than the described value. For example, the term “about 5 nM” indicates a range of 4.5 nM to 5.5 nM.

[0011] The term "at least" preceding a number or series of numbers is understood to include the number adjacent to the term "at least," as well as all subsequent numbers or integers that can logically be included, as is clearly apparent from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For instance, "at least 18 nucleotides in a nucleic acid molecule of 21 nucleotides" means that 18, 19, 20, or 21 nucleotides have the indicated property. When "at least" appears before a series of numbers or a range, it should be understood that "at least" can modify each number in the series or range.

[0012] As used herein, “no more than” or “less than” is understood to be the value and logical lower bound or integer adjacent to the phrase, such that, in context, the logical value is zero. For example, an oligonucleotide having “no more than 5 unmodified nucleotides” has 5, 4, 3, 2, 1, or 0 unmodified nucleotides. When “no more than” appears before a series of numbers or ranges, it should be understood that “no more than” can modify each number in the series or range.

[0013] As used herein, the term "administration" means administering a composition (e.g., a compound as described herein or a formulation comprising said compound) to a subject or system. Administration to animal subjects (e.g., to humans) may be performed via any suitable route as described herein.

[0014] As used herein, the term "oligonucleotide" is a molecule comprising two or more nucleotides. The term "nucleotide" refers to the nucleobase, sugar moiety, and internucleotide bond. Oligonucleotides are typically prepared in the laboratory by solid-phase chemical synthesis followed by purification. When referring to the sequence of an oligonucleotide, it means the sequence or order of the nucleobase moiety or its modifications of a covalently linked nucleotide or nucleoside. The oligonucleotides described herein may be artificial and chemically synthesized, and are typically purified or isolated. Oligonucleotides are also intended to include (i) compounds having one or more furanose moieties, said furanose moieties being replaced by furanose derivatives or any structure (cyclic or acyclic) that can serve as covalent linkage sites for the base moieties; (ii) compounds having one or more phosphodiester bonds, said phosphodiester bonds being modified, such as in the case of aminophosphate or thiophosphate bonds, or replaced by suitable linking moieties, such as in the case of methyl acetal or riboacetal bonds; and / or (iii) compounds having one or more linked sugar-phosphodiester bond moieties, said one or more linked sugar-phosphodiester bond moieties being replaced by any structure (cyclic or acyclic) that can serve as covalent linkage sites for the nucleobase moieties. Oligonucleotides described herein may include one or more alternative nucleotides (e.g., including the alternative nucleotides described herein). It should also be understood that oligonucleotides include compositions lacking a sugar moiety or nucleobase but still capable of pairing or hybridizing with a target sequence. As used herein, oligonucleotides contain 100 or fewer nucleotides.

[0015] The terms “nucleobase” and “base” include the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) portions present in nucleosides and nucleotides that form hydrogen bonds during nucleic acid hybridization. The term nucleobase also encompasses alternative nucleobases that may differ from naturally occurring nucleobases but are functional during nucleic acid hybridization. In this context, “nucleobase” refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, and alternative nucleobases. Such variants are described, for example, in Hirao et al. (2012), *Accounts of Chemical Research*, Vol. 45, p. 2055, and Bergstrom (2009), *Current Protocols in Nucleic Acid Chemistry*, Supplement 371.4.1.

[0016] The letters “G”, “C”, “A”, “T”, and “U” typically represent naturally occurring nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as nucleotide bases, respectively. However, G, C, A, T, and U can also refer to guanine, cytosine, adenine, thymidine, and uracil nucleotide bases with a sugar moiety other than ribose (or deoxyribose). This article discusses such alternative sugar moieties.

[0017] In some embodiments, the nucleobase portion is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or substituted pyrimidine, such as a “substitute nucleobase” selected from the following: isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazo-uracil, 2-thiouracil, pseudouracil, 1-methylpseudouracil, 5-methoxyuracil, 2'-thio-thymidine, hypoxanthine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0018] The nucleobase portion can be represented by a letter code for each corresponding nucleobase, such as A, T, G, C, or U, where each letter may optionally include an alternative nucleobase with equivalent function.

[0019] "Sugar" or "sugar moiety" includes sugars having a furanose ring (e.g., ribose, deoxyribose, arabinose). Sugars also include "alternative sugars" defined as structures capable of replacing the furanose ring in a nucleoside. In some embodiments, the alternative sugar is a non-furanose (or 4'-substituted furanose) ring or ring system or open system. Such structures include six-membered rings (e.g., pyranose rings) or acyclic moieties, such as those used in peptide nucleic acids. Alternative sugars may also include morpholine rings, pyranose rings, or hexitol ring systems. The sugar moiety that can be used to prepare oligonucleotides with motifs includes, but is not limited to, β-D-ribose, β-D-2'-deoxyribose, methoxy-substituted sugars (e.g., β-D-2'-methoxyribose), MOE-substituted sugars (e.g., β-D-2'-methoxyethylribose), fluorinated sugars (e.g., 2'-deoxy-2-fluororibose and β-D-2'-deoxy-2'-fluoroarabinofuranose, which is also referred to herein as 2'-fluoroarabinose), and substituted sugars. Sugars (such as 2', 5', and disubstituted sugars), 4'-S-sugars (such as 4'-S-ribose, 4'-S-2'-deoxyribose, and 4'-S-2'-substituted ribose), bicyclic substituted sugars (such as locked nucleic acids (LNAs) with ribose-derived bicyclic sugars bridged by 2'-O—CH2-4' or 2'-O—(CH2)2-4'), and sugar substitutes (such as when the ribose ring is replaced by a morpholine ring, a pyran ring, or a hexitol ring system, such as β-D-homogeneous DNA). The β-D-homogeneous DNA sugar moiety is the substituted pyran ring shown in the structure (where N is a nucleobase): .

[0020] The internucleotide bonds in a nucleotide can be phosphate ester bonds. Other internucleotide bonds are known in the art, including but not limited to thiophosphates or borophosphates. Other internucleotide bonds include phosphate triesters, thiophosphates, aminophosphates, and other variants of the phosphate ester backbone.

[0021] The term "nucleoside" refers to a monomeric unit of an oligonucleotide or polynucleotide that has a nucleobase and a sugar moiety.

[0022] Oligonucleotides can have any length that allows for base modification (e.g., deamination of target adenosine) of a desired target RNA via ADAR-mediated pathways, and the length can be in the range of about 27-50 base pairs, for example, about 30-45 base pairs or about 35-45 base pairs, for example, about 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 base pairs. The ranges in between are also considered part of the oligonucleotides described herein.

[0023] As used herein, and unless otherwise stated, when used to describe a first nucleotide or nucleoside sequence relative to a second nucleotide or nucleoside sequence, the term "complementarity" refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide or nucleoside sequence to hybridize with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions and form a double-stranded structure, as will be understood by those skilled in the art. Such conditions, for example, can be stringent conditions, which may include: 400 mM NaCl, 40 mM PIPES at pH 6.4, 1 mM EDTA, 50°C or 70°C for 12–16 hours, followed by washing (see, for example, *Molecular Cloning: A Laboratory Manual*, Sambrook et al. (1989), Cold Spring Harbor Laboratory Press). Other conditions may be applied, such as physiologically relevant conditions that may be encountered within an organism. Those skilled in the art will be able to determine the set of conditions best suited for the complementarity test of the two sequences based on the final application of the hybridized nucleotide or nucleoside.

[0024] As used herein, a “complementary” sequence may also include non-Watson-Crickbase pairs and / or base pairs formed from non-natural and alternative nucleotides, or may be formed entirely from said base pairs, provided that the above requirements regarding their hybridization ability are met. Such non-Watson-Crickbase pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing. The complementary sequence between the oligonucleotide and the target sequence described herein comprises an oligonucleotide or polynucleotide including the first nucleotide sequence and an oligonucleotide or polynucleotide including the second nucleotide sequence with base pairings along the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as “perfectly complementary” to each other. However, when the first sequence is referred to herein as “substantially complementary” to the second sequence, the two sequences may be perfectly complementary, or they may form, after hybridization, one or more, but generally no more than 5, 4, 3, or 2 mismatched base pairs of a duplex of up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to its final application (e.g., deamination of adenosine). "Substantially complementary" can also refer to an oligonucleotide that is substantially complementary to a continuous portion of the mRNA of interest (e.g., an mRNA containing a target adenosine). For example, if the sequence is substantially complementary to an uninterrupted portion of the mRNA of interest, then the oligonucleotide is complementary to at least a portion of the mRNA of interest.

[0025] As used herein, the term "complementary region" refers to a region on an oligonucleotide that is substantially complementary to all or part of a gene, primary transcript, sequence (e.g., target sequence; e.g., target sequence containing target nucleobases (e.g., adenosine), or treated mRNA, thereby interfering with the expression of an endogenous gene. In cases where the complementary region is not perfectly complementary to the target sequence, mismatches can be located within the molecule or in terminal regions. Typically, the most tolerable mismatches are located in terminal regions, for example, within five, four, three, or two nucleotides at the 5' and / or 3' ends of the oligonucleotide.

[0026] The phrase “contacting cells with oligonucleotides (as described herein)” includes contacting cells by any possible means. Contacting cells with oligonucleotides includes contacting cells with oligonucleotides in vitro or in vivo. Contact can be direct or indirect. Thus, for example, the individual performing the method may physically contact the oligonucleotides with the cells, or alternatively, the oligonucleotide agent may be placed in a position that will allow or will subsequently contact the cells.

[0027] In vitro cell contact can be achieved, for example, by incubating cells with oligonucleotides. In vivo cell contact can be achieved, for example, by injecting oligonucleotides into or near the tissue where the cells are located, or by injecting an oligonucleotide agent into another area (e.g., the bloodstream or subcutaneous space) so that the agent subsequently reaches the tissue where the cells to be contacted are located. For example, the oligonucleotide may contain a ligand (e.g., GalNAc3) and / or may be conjugated to said ligand, which guides the oligonucleotide to the site of interest, such as the liver. Combinations of in vitro and in vivo contact methods are also possible. For example, cells can also be contacted in vitro with oligonucleotides and subsequently transplanted into a subject.

[0028] In one embodiment, contacting cells with oligonucleotides includes “introducing” or “delivering” the oligonucleotide into the cells by promoting or influencing uptake or absorption within the cells. The uptake or absorption of the oligonucleotide can occur through unassisted diffusion or active cellular processes or via an adjuvant or auxiliary device. Introducing the oligonucleotide into cells can be performed in vitro and / or in vivo. For example, for in vivo introduction, the oligonucleotide can be injected into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipid transfection. Other methods are described below and / or are known in the art.

[0029] As used herein, "lipid nanoparticles" or "LNPs" are vesicles comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, for example, an oligonucleotide. LNPs refer to stable nucleic acid-lipid particles. LNPs typically contain cationic lipids, ionizable lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). LNPs are described, for example, in U.S. Patent Nos. 6,858,225; 6,815,432; 8,158,601; and 8,058,069, the entire contents of which are hereby incorporated by reference.

[0030] As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer (e.g., one or more bilayers). Liposomes include monolayer and multilayer vesicles having a membrane formed of a lipophilic material and an aqueous interior. The aqueous portion contains an oligonucleotide composition. The lipophilic material separates the aqueous interior from the aqueous exterior, which typically does not contain the oligonucleotide composition, but in some instances may include it. Liposomes also include "sterically stable" liposomes; as used herein, this term refers to a liposome comprising one or more specific lipids that, upon incorporation, increase cycle life compared to liposomes lacking such specific lipids.

[0031] "Micelles" are defined in this paper as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all hydrophobic portions of the molecules face inward, thereby allowing the hydrophilic portions to come into contact with the surrounding water. If the environment is hydrophobic, the opposite arrangement exists.

[0032] As used herein, the terms “effective amount,” “therapeutic effective amount,” and “adequate amount” for pharmaceutical agents that produce a therapeutic effect (e.g., in cells or subjects) refer to an amount sufficient to produce a beneficial or desired outcome (including clinical outcomes) when administered to a subject (including a person), and therefore, “effective amount” or its synonyms depend on the context in which they are applied. For example, in the case of treating a condition, the amount is an amount of pharmaceutical agent sufficient to achieve a therapeutic response relative to a response obtained without administration. The amount of a given pharmaceutical agent will vary depending on various factors such as the given pharmaceutical agent, the pharmaceutical formulation, the route of administration, the type of disease or condition, the identity of the subject being treated (e.g., age, sex, and / or weight), or the host, but can still be routinely determined by those skilled in the art. Furthermore, as used herein, a “therapeutic effective amount” of a pharmaceutical agent is an amount that produces a beneficial or desired outcome in a subject relative to a control. As defined herein, a therapeutically effective amount of a pharmaceutical agent can be readily determined by those skilled in the art using methods known in the art. Dosing regimens can be adjusted to provide an optimal therapeutic response.

[0033] "Therapeutic effective amount" refers to the amount of oligonucleotide that produces a desired local or systemic effect (whether administered in a single dose or multiple doses) in a reasonable benefit / risk ratio applicable to any treatment. The oligonucleotides used in the methods disclosed herein can be administered in amounts sufficient to produce a reasonable benefit / risk ratio applicable to such treatments.

[0034] "Determining protein levels" means directly or indirectly detecting a protein or the mRNA encoding the protein by methods known in the art. "Direct determination" means performing a process (e.g., performing an assay or test on a sample or "analyzing a sample" as defined herein) to obtain a physical entity or value. "Indirect determination" means receiving a physical entity or value from another party or source (e.g., a third-party laboratory that directly obtains the physical entity or value). Methods for measuring protein levels generally include, but are not limited to, Western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surface plasmon resonance, chemiluminescence, fluorescence polarization, phosphorescence, immunohistochemistry, matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry, liquid chromatography (LC)-mass spectrometry, microcell counting, microscopy, fluorescence-activated cell sorting (FACS), and flow cytometry, as well as assays based on protein properties, including but not limited to enzyme activity or interactions with other protein chaperones. Methods for measuring mRNA levels are known in the art.

[0035] The "sequence identity percentage (%)" relative to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to nucleotides or amino acids in the reference polynucleotide or polypeptide sequence after alignment and the introduction of gaps (if necessary) to achieve the maximum sequence identity percentage. Alignments used to determine the sequence identity percentage can be performed in various ways within the capabilities of those skilled in the art, such as using publicly available computer software like BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. For example, the sequence identity percentage value can be generated using the sequence comparison computer program BLAST. As an illustration, the sequence identity percentage of a given sequence A with respect to, and or relative to, a given sequence B (which can be alternatively expressed as a given sequence A having a certain sequence identity percentage with, and or relative to, a given sequence B) is calculated as follows: 100 multiplied by (fraction X / Y) Where X is the number of nucleotides or amino acids that are scored as identical matches by a sequence alignment program (e.g., BLAST) in the alignment of A and B, and Y is the total number of nucleotides or amino acids in B. It should be understood that when the length of sequence A is not equal to the length of sequence B, the percentage of sequence identity between A and B will not be equal to the percentage of sequence identity between B and A.

[0036] “Level” refers to the level or activity of a protein or its encoding mRNA relative to a reference. A reference can be any available reference as defined herein. A “reduced level” or “increased level” of a protein means a decrease or increase in protein level relative to a reference (e.g., a decrease or increase of approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%, or more; a decrease or increase of more than...). Approximately 10%, approximately 15%, approximately 20%, approximately 50%, approximately 75%, approximately 100%, or approximately 200%; a decrease or increase of less than approximately 0.01, approximately 0.02, approximately 0.1, approximately 0.3, approximately 0.5, approximately 0.8, or less; or an increase of approximately more than approximately 1.2, approximately 1.4, approximately 1.5, approximately 1.8, approximately 2.0, approximately 3.0, approximately 3.5, approximately 4.5, approximately 5.0, approximately 10, approximately 15, approximately 20, approximately 30, approximately 40, approximately 50, approximately 100, approximately 1000, or more. Protein levels can be expressed as mass / vol (e.g., g / dL, mg / mL, μg / mL, ng / mL) or as a percentage of total protein or mRNA in the sample.

[0037] As used herein, the term "pharmaceutical composition" means a composition containing the compounds described herein formulated with pharmaceutically acceptable excipients and preferably manufactured or sold as part of a treatment regimen for treating diseases in mammals with the approval of a government regulatory agency. Pharmaceutical compositions may be formulated for, for example, oral administration in unit dosage forms (e.g., tablets, capsules, pouches, soft capsules, or syrups); for topical administration (e.g., in the form of creams, gels, lotions, or ointments); for intravenous administration (e.g., in the form of a sterile solution without microparticle plugs and in a solvent system suitable for intravenous use); for intrathecal injection; for intraventricular injection; for intraparenchymal injection; or formulated in any other pharmaceutically acceptable formulation.

[0038] As used herein, “pharmaceuticalally acceptable excipient” means any ingredient other than the compounds described herein (e.g., mediators capable of suspending or dissolving active compounds) and that has properties that are substantially non-toxic and non-inflammatory in patients. Excipients may include, for example: anti-adhesives, antioxidants, adhesives, coatings, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film-forming agents or coatings, flavorings, fragrances, flow enhancers (flow improvers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydrated water. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (binary), calcium stearate, croscarmellose, croscarmellose, citric acid, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silica, sodium carboxymethyl cellulose, sodium citrate, sodium starch carbonyl acetate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0039] As used herein, the term "pharmaceuticalally acceptable salt" means any pharmaceutically acceptable salt of the oligonucleotides described herein. For example, pharmaceutically acceptable salts include salts suitable for contact with tissues in humans and animals without excessive toxicity, irritation, anaphylactic reactions, etc., within the bounds of reasonable medical judgment and in proportion to a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in the following literature: Berge et al., *Journal of Pharmaceutical Sciences* 66:1-19, 1977, and *Pharmaceutical Salts: Properties, Selection and Use*, (edited by PH Stahl and CG Wermuth), Wiley-VCH, 2008. Salts may be prepared in situ during the final isolation and purification of the compounds described herein, or may be prepared separately by reacting a free base group with a suitable organic acid.

[0040] Pharmaceutically acceptable salts can be acid addition salts involving inorganic or organic acids, or salts can be prepared from inorganic or organic bases. Typically, pharmaceutically acceptable salts are prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases, and methods for preparing appropriate salts, are well known in the art. Salts can be prepared from pharmaceutically acceptable, non-toxic acids and bases, including inorganic and organic acids and bases. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentylpropionate, diglucuronate, dodecyl sulfate, ethanesulfonate, fumarate, glucohepanoate, glyceryl phosphate, hemisulfate, heptaate, hexanoate, hydroiodide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, dodecyl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pyrate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0041] “Reference” means any useful reference used for comparing protein or mRNA levels or activities. A reference can be any sample, standard, standard curve, or level used for comparative purposes. A reference can be a normal reference sample or a reference standard or level. A “reference sample” can be, for example, a control, such as a predetermined negative control value, like a “normal control”, or a previous sample taken from the same subject; a sample from a normal healthy subject, such as normal cells or normal tissue; a sample (e.g., cells or tissue) from a subject without disease; a sample from a subject diagnosed with a disease but not yet treated with the compounds described herein; a sample from a subject treated with the compounds described herein; or a sample of purified protein (e.g., any protein described herein) at a known normal concentration. “Reference standard or level” means a value or number derived from a reference sample. A “normal control value” is a predetermined value representing a non-disease state, such as a value expected in healthy control subjects. Typically, normal control values ​​are expressed as a range (“between X and Y”), a high threshold (“not higher than X”), or a low threshold (“not lower than X”). Subjects whose measured values ​​for a specific biomarker fall within the normal control range are generally referred to as being "within the normal limit" of the biomarker. The normal reference standard or level may be derived from a healthy subject without disease or condition; or from a value or number of a subject who has been treated with the compounds described herein. In a preferred embodiment, the reference sample, standard, or level is matched to the sample subject sample by at least one of the following criteria: age, weight, sex, disease stage, and overall health. A standard curve of a purified protein (e.g., any protein described herein) within the normal reference range may also be used as a reference.

[0042] As used herein, the term "subject" means any organism to which the compositions according to this disclosure may be administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). Subjects may be people or animals seeking or potentially needing treatment, requiring treatment, currently receiving treatment, or in the future receiving treatment, or being cared for by a trained professional for a specific disease or condition.

[0043] As used herein, the terms “treatment,” “treated,” or “under treatment” mean both therapeutic treatment and preventative or preventative measures aimed at preventing or slowing (alleviating) undesirable physical symptoms, conditions, or diseases, or at achieving beneficial or desired clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief; reduction in the severity of symptoms, conditions, or diseases; stabilization of symptoms, conditions, or disease status (i.e., no worsening); delayed onset or slowed progression of symptoms, conditions, or diseases; improvement or relief of symptoms, conditions, or disease status (whether partial or complete) (whether detectable or undetectable); improvement in at least one measurable physical parameter (which the patient may not necessarily perceive); or enhancement or improvement of symptoms, conditions, or diseases. Treatment includes evoking a clinically significant response without producing excessive levels of side effects. Treatment also includes prolonged survival compared to expected survival without treatment.

[0044] As used herein, the terms “variant” and “derivative” are used interchangeably and refer to naturally occurring, synthetic, and semi-synthetic analogs of the compounds, peptides, proteins, or other substances described herein. The variants or derivatives of the compounds, peptides, proteins, or other substances described herein may retain or enhance the bioactivity of the original material.

[0045] Details of one or more embodiments described herein are set forth in the following description. Other features, objectives, and advantages described herein will become apparent from the description and claims. Attached Figure Description

[0046] Figure 1 (A) Schematic diagram of 42-mer oligonucleotides designed to test the effect of PA-1 bonds (oligonucleotides #1, #2, and #3). Circles between nucleobases represent PA-1 bonds, while triangles represent PS bonds; all other bonds between nucleobases not represented by triangles or circles are phosphate ester bonds. (B) Chemical structure of PA-1 bonds within the oligonucleotide backbone. (C) Percentage of in vivo editing of E342K SERPINA1 RNA isolated from PiZ mouse livers on days 1, 4, and 7 (n = 3 / group).

[0047] Figure 2 (A) Schematic diagram of 30-mer oligonucleotides designed to test the effects of altered 2' modification patterns. (B) Percentage of E342K SERPINA1 RNA edited in vivo from PiZ mouse livers on day 1 and day 4 (n = 3 / group).

[0048] Figure 3(A) Schematic diagram of oligonucleotides targeting the 3' UTR region of cynomolgus cowrie ACTB. 5' or 3' tri-GalNAc is represented by a hexagon at either end of the oligonucleotide. Squares between nucleobases represent PA-1 bonds, while triangles represent phosphate thioester linkers. (B) Percentage of in vitro editing of ACTB RNA in cynomolgus cowrie hepatocytes at 10 nM and 100 nM in the presence of RNAiMAX (“LIPO”) or at 100 nM and 1000 nM in the absence of transfection reagents (free uptake or “FU”) (n = 4 / group).

[0049] Figure 4 (A) Schematic diagram of parental oligonucleotides containing five PA-1 bonds, indicated by circles between nucleobases. (B) Schematic diagram of each oligonucleotide containing an additional PA-1 at different positions within the oligonucleotide (left panel), and the corresponding in vitro editing percentage of E342KSERPINA1 RNA in PiZ mouse hepatocytes at two concentrations in the absence of lipofectamine (right panel) (n = 3 / group). (C) Analysis of editing fold changes (calculated by normalizing the editing level of the oligonucleotide relative to the parental oligonucleotides represented in (A). The X-axis represents the position where the PA-1 bond was subsequently added. Editing fold changes were calculated and normalized relative to parental 30-mer editing (free uptake at 10 nM or 100 nM).

[0050] Figure 5 (A) Schematic diagram of oligonucleotides containing four or five PA-1 bonds, indicated by circles between nucleobases. (B) Fold change in editing of E342K SERPINA1 in PiZ mouse hepatocytes in the absence of lipofectamine (“free access”) and in the presence of RNAiMAX (“transfection”) (n = 3 / group). The fold change in editing was calculated by dividing the editing level of the oligonucleotide indicated on the X-axis by the editing level of oligonucleotide 15.

[0051] Figure 6 A schematic diagram of oligonucleotides containing the PA-1 corridor on oligonucleotide #3 (left) and the corresponding edits of these oligonucleotides to E342K SERPINA1 in ZZ HLC (right). Each oligonucleotide contains an additional PA-1 bond at a different position on the oligonucleotide. The edits performed in ZZ HLC were measured at 1 nM, 10 nM, and 100 nM (n = 4 / group).

[0052] Figure 7(A) Schematic diagram of 42-mer and 30-mer oligonucleotides targeting the 3' UTR region of mouse ACTB. Oligonucleotides #120, #121, and #123 contain PA-1 bonds, while oligonucleotides #119 and #122 do not. (B) Quantification of editing of mouse ACTB RNA in primary mouse hepatocytes (“PMH”) at 10 nM and 100 nM in the absence of lipofectamine.

[0053] Figure 8 (A) Schematic diagram of oligonucleotides containing a single substitution of 2'OMe for 2'F or 2'MOE at different positions. (B) Percentage of E342K SERPINA1 RNA edited in PiZ mouse hepatocytes in the presence ("transfection") and absence ("free access") of the transfection reagent RNAiMAX.

[0054] Figure 9 Top: Schematic diagram of the parental oligonucleotides. Circled positions indicate where the substitution of 2'OMe for 2'F leads to a decrease in editing. Bottom: Fold changes in editing at different positions with 2'OMe substitution in PiZ mouse hepatocytes indicate which 2'F residues are crucial for editing activity. The fold change in editing is calculated by dividing the editing activity of the oligonucleotide with the 2'OMe substitution by the editing activity of the parental oligonucleotide lacking the substitution. At +3, -5, -16, and -20, editing is halved when 2'F is replaced by O-Me.

[0055] Figure 10 - Quantification of mouse ACTB RNA edited at 10 nM in PMH after transfection with oligonucleotides using lipofectamine (RNAiMax).

[0056] Figure 11 - Percentage of in vivo edited ACTB RNA isolated from mouse liver seven days after final administration (n = 3 / group).

[0057] Figure 12 - Percentage of in vitro editing of UGP2 RNA in mouse hepatocytes at 10 nM and 100 nM oligonucleotides in the presence of RNAiMAX (“TFX”) (A) or at 500 nM (n = 4 / group) in the absence of transfection reagent (free uptake or “FU”) (B).

[0058] Figure 13 - Percentage of NRF2 RNA edited in vitro in mouse hepatocytes at 1 nM, 5 nM, and 25 nM oligonucleotides in the presence of RNAiMAX (“TFX”).

[0059] Figure 14 - In vitro editing of E342K SERPINA1 in PiZ mouse hepatocytes in the absence of lipofectamine (“free access”) (A) and in the presence of RNAiMAX (“transfection”) (B) (n = 3 / group).

[0060] Figure 15 - In vitro editing of E342K SERPINA1 in PiZ mouse hepatocytes in the absence of lipofectamine (“free access”) (A) and in the presence of RNAiMAX (“transfection”) (B) (n = 3 / group). Detailed Implementation

[0061] This document provides oligonucleotides that can be used to modify nucleobases on target RNA. Therefore, this disclosure provides oligonucleotides, compositions containing said oligonucleotides, and methods for modifying target nucleobases on RNA (e.g., deaminating target adenosine), wherein said modification produces a therapeutic outcome, such as in a subject in need. In some embodiments, said target RNA is mRNA.

[0062] I. Symptoms This disclosure also includes oligonucleotides as described herein for methods of altering target RNA sequences in mammalian cells (preferably human cells). Similarly, this disclosure provides the use of these oligonucleotides as described herein in the manufacture of medicaments for altering target RNA sequences in mammalian cells (preferably human cells). In some embodiments, the target RNA is mRNA.

[0063] This disclosure also relates to a method for deaminating at least one specific target adenosine present in a target RNA sequence in a cell, the method comprising the steps of: providing the cell with an oligonucleotide as described herein; allowing the cell to take up the oligonucleotide; allowing the oligonucleotide to anneal to the target RNA sequence; allowing a mammalian ADAR enzyme to deaminate the target adenosine in the target RNA sequence to inosine; and optionally identifying the presence of inosine in the RNA sequence.

[0064] In some embodiments, this document provides oligonucleotides, compositions, and methods in which two adjacent adenosines are co-deaminated by an RNA editing enzyme such as ADAR. In this particular case, the UAA stop codon is converted to the UII Trp encoding codon. Other examples of modifications resulting from the deamination of the target adenosine within the target codon are provided in Tables 1 and 2 below.

[0065] Table 1 Table 2. Base composition of the triplet and the resulting edited triplets Because the deamination of adenosine to inosine may result in the absence of the mutated A protein at the target site, the identification of the deamination to inosine can be a functional readout, such as an assessment of the presence of a functional protein or even an assessment of whether the disease caused by the presence of adenosine is (partially) reversed. Functional assessments for each disease mentioned herein will generally be performed according to methods known to those skilled in the art. When the presence of target adenosine leads to aberrant splicing, the readout can be an assessment of whether aberrant splicing still occurs, does not occur, or occurs less frequently. On the other hand, when it is desired that the deamination of target adenosine introduces a splicing site, a similar approach can be used to check whether the desired type of splicing is indeed occurring. Suitable methods for identifying the presence of inosine after the deamination of target adenosine are RT-PCR and sequencing performed using methods well known to those skilled in the art.

[0066] Typically, a G-to-A mutation in any target RNA that can be reversed using the oligonucleotide constructs disclosed herein is a G-to-A mutation, and the oligonucleotide constructs can be designed accordingly. Mutations that can be targeted using the oligonucleotide constructs also include C-to-A and U-to-A (T-to-A at the DNA level) mutations that recruit adenosine deaminase. Although in the latter case, RNA editing may not necessarily restore the mutation to the wild type, the edited nucleotides may produce improvements superior to the original mutation. For example, a mutation that produces an in-frame stop codon resulting in a truncated protein during translation can be changed to a codon encoding an amino acid at said position, which may not be the original amino acid, but produces a (full-length) protein with at least some function (at least more than the function of the truncated protein).

[0067] The oligonucleotides described herein are particularly suitable for treating genetic diseases such as cystic fibrosis, albinism, alpha-1-antitrypsin (A1AT) deficiency, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), asthma, 11-thalassemia, Cadasil syndrome, Charcot-Marie-Tooth disease, chronic obstructive pulmonary disease (COPD), distal spinal muscular atrophy (DSMA), Duchenne / Becker muscular dystrophy, dystrophic epidermolysis bullosa, epidermolysis bullosa, Fabry disease, Factor V Leiden associated disorder, familial adenoma, polyposis, galactosemia, Gaucher's disease, glucose-6-phosphate dehydrogenase deficiency, hemophilia, hereditary hemochromatosis, and Hunter syndrome. syndromes including Huntington's disease, Hurler syndrome, inflammatory bowel disease (IBD), hereditary polyagglutination syndrome, Leber congenital amaurosis, Lesch-Nyhan syndrome, Lynch syndrome, Marfan syndrome, mucopolysaccharidosis, muscular dystrophy, myotonic dystrophy type I and II, neurofibromatosis, Niemann-Pick disease type A, B and C, NY-NY-ESO-1 related cancers, Parkinson's disease, Peutz-Jeghers syndrome, phenylketonuria, Pompe's disease, primary ciliary disease, and prothrombin mutation-related disorders (e.g.,Prothrombin G20210A mutation), pulmonary hypertension, retinitis pigmentosa, Sandhoff disease, severe combined immunodeficiency syndrome (SCID), sickle cell anemia, spinal muscular atrophy, Stargardt's disease, Tay-Sachs disease, Usher syndrome, X-linked immunodeficiency, Sturge-Weber syndrome, Rett syndrome, and various forms of cancer (e.g., BRCA1 and 2-linked breast and ovarian cancer).

[0068] The oligonucleotides described in this article can mutate and deaminate adenosine, thereby increasing protein activity.

[0069] In some embodiments, the treatment is administered to subjects who have been diagnosed with a mutation in a gene but do not yet have disease symptoms (e.g., infants aged 1 to 12 months or subjects under 2 years of age). In other embodiments, the treatment is administered to individuals with at least one symptom.

[0070] Treatment can be administered to subjects of any age, from infancy to adulthood. Subjects may begin treatment, for example, at birth, six months, or at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, or 18 years of age.

[0071] In some embodiments, the oligonucleotide increases (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more, or by more than 1.2, 1.4, 1.5, 1.8, 2.0, 3.0, 3.5, 4.5, 5.0, 10, 15, 20, 30, 40, 50, 100, 1000 or more times) the protein activity in vitro and / or in vivo.

[0072] In some embodiments, oligonucleotides increase (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more, or by more than 1.2, 1.4, 1.5, 1.8, 2.0, 3.0, 3.5, 4.5, 5.0, 10, 15, 20, 30, 40, 50, 100, 1000 or more times) protein activity in the brain.

[0073] II. Oligonucleotides The oligonucleotides described herein are complementary to the target RNA and are capable of recruiting an ADAR enzyme for editing target nucleobases on the target RNA, for example, for deaminating adenosine on the target RNA. In some embodiments, only one nucleobase (e.g., one adenosine) is edited (e.g., deamination). In some embodiments, one, two, or three nucleobases are edited. In some embodiments, the oligonucleotide includes at least one mismatch, wobble, insertion, or deletion. In some cases, the oligonucleotide includes a mismatch opposite to the target nucleobase, for example, located at X. 2 The mismatches at the sites (see structures below). The oligonucleotides described herein may further include modifications (e.g., alternative nucleotides) for increasing stability and / or improving deamination efficiency. In some embodiments, the oligonucleotides described herein contain one, two, three, four, or five mismatches, wobbles, insertions, or deletions (or any combination thereof).

[0074] In some embodiments, one or more nucleobases of the oligonucleotides described herein are chemically modified to enhance stability or other beneficial properties. Without being bound by theory, it is believed that certain modifications can increase nuclease resistance and / or serum stability or reduce immunogenicity. For example, the oligonucleotides described herein may contain nucleotides found naturally in DNA or RNA (e.g., adenine, thymidine, guanosine, cytidine, uridine, or inosine), or may contain nucleotides with one or more chemical modifications to one or more components of the nucleotide (e.g., nucleobases, sugars, or nucleotide bonds).

[0075] The oligonucleotides described in this article contain the following structures: [A m ]-X 1 -X 2 -X 3 -[B n ] in m + n is 24 to 50, n is at least 4, and m is at least 20; -X 1 -X 2 -X 3 - is the central triplet of the oligonucleotide; X 1 For the position -1, X of the oligonucleotide 2 The position of the oligonucleotide is 0, and X 3 +1 to the position of the oligonucleotide; [A] m This is the first domain located at positions -(m+1) to -2 of the oligonucleotide; [B] n This is a second domain located at positions +2 to +(n+1) of the oligonucleotide; Each A and B is a nucleotide containing a nucleobase, a sugar (“A / B sugar”), and an internucleotide bond; Each X 1 X 2 and X 3 It contains nucleobases, sugars ("X sugars"), and bonds between nucleotides; The A / B sugar and the X 3 The sugars are selected from 2'-methoxy-ribose, 2'-MOE-ribose, 2'-deoxy-2'-fluororibose, 2'-fluoro-arabinose, 2-methoxy-arabinose, 2'-deoxyribose and locked nucleic acids (LNA). The X 1 The sugar is 2'-deoxy-2'-fluororibose or 2'-deoxyribose; The X 2 The sugars are selected from 2'-methoxy-ribose, 2'-MOE-ribose, 2'-deoxy-2'-fluororibose, 2'-fluoro-arabinose, 2-methoxy-arabinose, 2'-deoxyribose, locked nucleic acid (LNA), and β-homo-DNA sugars; The A / B sugar and the X sugar together constitute 10-70% 2'-deoxy-2'-fluoro-ribose; The oligonucleotide has 30%-100% thiophosphate and aminophosphate bonds between its nucleotides, and 3 to 20 nucleotide bonds are aminophosphate bonds. (i) the bond between the nucleotide at position -(m+1) and the nucleotide at position -(m) (5' end), (ii) the bond between the nucleotide at position +(n) and the nucleotide at position +(n+1) (3' end), or (iii) the bond between the nucleotides at each of the 5' and 3' ends of the oligonucleotide is an aminophosphate bond; and The nucleotide bonds between the nucleotide at position -(m) and the nucleotide at position -(m-1), and the nucleotide bonds between the nucleotide at position +(n-1) and the nucleotide at position +(n), ​​are independently thiophosphate bonds or aminophosphate bonds.

[0076] In some embodiments, the X 2 The nucleobase is cytosine or isodU. In some embodiments, the X 3 The nucleobases are guanosine, hypoxanthine, or 7-denitroguanine.

[0077] The oligonucleotides disclosed in this article have X 1 The sugar is 2'-deoxy-2'-fluororibose (sometimes referred to as 2'-fluororibose). or 2'-deoxyribose .

[0078] The oligonucleotides disclosed in this article have A / B sugars and X sugars. 3 Each sugar is individually 2'-methoxyribose. 2'-Deoxy-2'-fluororibose (sometimes referred to as 2'-deoxy-2'-fluororibose) 2'-Methoxyethyl ribose (sometimes referred to as 2'-MOE-ribose) 2'-Fluoroarabinose 2'-deoxyribose Locked nucleic acid (LNA).

[0079] The oligonucleotides disclosed in this article have X 2 Each sugar is individually 2'-methoxyribose. 2'-deoxy-2'-fluororibose 2'-Methoxyethylribose 2'-Fluoroarabinose 2'-deoxyribose β-D-homogeneous DNA sugars Locked nucleic acid (LNA).

[0080] In some embodiments, 10-70% (e.g., 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%) of A / B sugar and X sugar together constitute 2'-deoxy-2'-fluororibose. In some embodiments, 20-50% (e.g., 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%) of A / B sugar and X sugar together constitute 2'-deoxy-2'-fluororibose.

[0081] In some embodiments, no more than four consecutive A / B sugars are 2'-deoxy-2'-fluororibose. In some embodiments, the A / B sugars are selected from 2'-methoxy-ribose, 2'-MOE-ribose, 2'-deoxy-2'-fluororibose, and 2'-deoxyribose.

[0082] In some embodiments, the A / B sugar at position +3 is 2'-deoxy-2'-fluororibose. In some embodiments, the A / B sugar at position -5 is 2'-deoxy-2'-fluororibose. In some embodiments, the A / B sugar at position -16 is 2'-deoxy-2'-fluororibose. In some embodiments, the A / B sugar at position -20 is 2'-deoxy-2'-fluororibose. In some embodiments, the A / B sugar at each of positions -5, -16, and -20 is 2'-deoxy-2'-fluororibose. In some embodiments, the A / B sugar at each of positions +3, -5, -16, and -20 is 2'-deoxy-2'-fluororibose.

[0083] In some embodiments, the X 1 The sugar is 2'-fluororibose or 2-deoxyribose. In some embodiments, the X 1 The sugar is 2'-fluororibose. In some embodiments, the X 1 The sugar is 2-deoxyribose.

[0084] In some embodiments, the X 2 The nucleobase is cytosine. In some embodiments, the X 2 The sugar is a β-homo-DNA sugar or a 2'-deoxyribose. In some embodiments, the X 2 The sugar is a β-homo-DNA sugar. In some embodiments, the X 2 The sugar is 2'-deoxyribose.

[0085] In some embodiments, the X 3 The nucleobase is hypoxanthine.

[0086] In some embodiments, the internucleotide bond is an aminophosphate, a thiophosphate, a dithiophosphate, a methylphosphate, a thiophosphate, a 3'-thiophosphate, or a 5'-thiophosphate.

[0087] aminophosphate bonds (e.g., (where R is a suitable substituent on nitrogen, such as alkyl, sulfoxide, etc.) including methanesulfonylaminophosphate. In some embodiments, the phosphoramidite bond is methanesulfonylphosphamide. In some embodiments, each aminophosphate bond in the oligonucleotide is methanesulfonylphosphamide.

[0088] In some embodiments, the internucleotide bonds of the oligonucleotides described herein comprise at least 30% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) aminophosphate and / or thiophosphate bonds. In some embodiments, the oligonucleotides described herein have 30-70% (e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%) thiophosphate and aminophosphate bonds. In some embodiments, the oligonucleotides described herein have 40-60% (e.g., 40%, 45%, 50%, 55%, 60%) thiophosphate and aminophosphate bonds.

[0089] In some embodiments, the thiophosphate bond is an Sp thiophosphate bond. In other embodiments, the thiophosphate bond is an Rp thiophosphate bond. In some cases, the thiophosphate bond is a mixture of Sp and Rp.

[0090] In some embodiments, at least three of the internucleotide bonds in the oligonucleotide described herein (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) are phosphoramidite bonds.

[0091] In some embodiments, X 1 With X 2 The internucleotide bonds between them are phosphate thioester bonds or phosphodiester bonds. In some embodiments, X 1 With X 2 The nucleotide bonds between them are thiophosphate bonds.

[0092] In some embodiments, X 2 With X 3 The internucleotide bonds between them are phosphate thioester bonds or phosphodiester bonds. In some embodiments, X 2 With X 3 The nucleotide bonds between them are thiophosphate bonds.

[0093] In some embodiments, the nucleoside located at position -2 is related to X. 1 The internucleotide bonds between them are phosphate thioester bonds or phosphodiester bonds. In some embodiments, the nucleotide at position -2 is bonded to X. 1 The nucleotide bonds between them are thiophosphate bonds.

[0094] In some embodiments, the internucleotide bond between the nucleotide at position -9 and the nucleotide at position -8 is a phosphoramidite bond.

[0095] In some embodiments, the internucleotide bond between the nucleotide at position -11 and the nucleotide at position -10 is a phosphoramidite bond.

[0096] In some embodiments, the internucleotide bond between the nucleotide at position +1 and the nucleotide at position +2 is a phosphoramide bond.

[0097] In some embodiments, the nucleotide bond between the nucleotide at position +4 and the nucleotide at position +5 is an aminophosphoester bond. In some embodiments, the nucleotide bond between the nucleotide at position +5 and the nucleotide at position +6 is an aminophosphoester bond. In some embodiments, the nucleotide bond between the nucleotide at position +9 and the nucleotide at position +10 is an aminophosphoester bond. In some embodiments, the nucleotide bonds between the nucleotide at position +1 and the nucleotide at position +2, and between the nucleotide at position +9 and the nucleotide at position +10 are aminophosphoester bonds. In some embodiments, the nucleotide bonds between the nucleotide at position +1 and the nucleotide at position +2, the nucleotide bonds between the nucleotide at position +5 and the nucleotide at position +6, and the nucleotide bonds between the nucleotide at position +9 and the nucleotide at position +10 are aminophosphoester bonds.

[0098] In some embodiments, n and m (together) are integers ranging from 24 to 40 (e.g., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40). In some embodiments, n and m (together) are 27. In some embodiments, n and m (together) are 39.

[0099] In some embodiments, m is at least 20 (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35). In some embodiments, n is at least 4 (e.g., 4, 5, 6, 7, 8, or 9).

[0100] In some embodiments, the oligonucleotide described herein has a GalNAc moiety at the 5' end. In some embodiments, the oligonucleotide described herein has a GalNAc moiety at the 3' end.

[0101] In some embodiments, n is an integer ranging from 8 to 10. In some embodiments, n is 8, 9, or 10. In some embodiments, n is 9.

[0102] Table 3 below shows the exemplary oligonucleotides described herein (Hierarchical Editing Language for Macromolecules (HELM) - syntax) (Zhang et al., *Journal of Chemical Information and Modeling*, 2012, 52, 10, 2796-2806), where each nucleotide is represented by a term between dots (.); the first term represents the sugar moiety, the next term is the nucleobase, and the last term is the internucleotide bond. For clarity, the terms may be separated by punctuation marks, such as square brackets and parentheses. Thus, the nucleotide name “.f(A)P.” signifies the 2'-deoxy-2'-fluororibose moiety and the adenosine nucleobase linked by a phosphodiester bond. The sugar components are named as follows: "f" represents 2'-deoxy-2'-fluororibose, "m" represents 2'-methoxyribose, "fana" represents 2'-fluoroarabinose, "d" represents deoxyribose, "dH" represents β-homogeneous DNA, and "moe" represents 2'-MOE ribose. For bonds, "msPA" represents a methanesulfonylaminophosphate nucleotide bond, "sP" represents a thiophosphate bond, and "P" represents a phosphate bond. Additional chemical modifications are represented by CHEM1{tri-GalNAc2}|CHEM2{P} (representing tri-GalNAc conjugated to the 5' end of an oligonucleotide) and CHEM3{P}|CHEM4{tri-GalNAc1} (representing tri-GalNAc conjugated to the 3' end).

[0103] Table 3. Exemplary Oligonucleotides In some embodiments, the oligonucleotides disclosed herein do not include stem-loop structures.

[0104] In some embodiments, the oligonucleotides disclosed herein include stem-loop structures. Stem-loop structures can serve as recruitment domains of ADAR enzymes (e.g., ADAR recruitment domains), but oligonucleotides disclosed herein may also affect ADAR recruitment and activity for target adenosine in target RNA in the absence of such stem-loop structures.

[0105] In some embodiments, the oligonucleotides described herein may further include a 5' cap structure. In some embodiments, the 5' cap structure is a 2,2,7-trimethylguanosine cap.

[0106] In some embodiments, the oligonucleotide described herein includes a GalNAc portion at the 5' end of the oligonucleotide. In some embodiments, the oligonucleotide described herein includes a GalNAc portion at the 3' end of the oligonucleotide.

[0107] The oligonucleotides described herein can be synthesized using standard methods known in the art, as further discussed below, for example, by using an automated DNA synthesizer, such as a DNA synthesizer commercially available from, for example, Biosearch, Applied Biosystems, Inc.

[0108] Oligonucleotides can be prepared using solution-phase synthesis, solid-phase organic synthesis, or both. Organic synthesis offers the advantage of readily preparing oligonucleotides that include non-natural or alternative nucleotides. The single-stranded oligonucleotides described herein can be prepared using solution-phase synthesis, solid-phase organic synthesis, or both.

[0109] It is envisioned that for any sequence identified herein, further optimization could be achieved by systematically adding or removing linking nucleotides to produce longer or shorter sequences. Such optimized sequences could be modified, for example, by introducing alternative nucleosides, alternative sugar moieties, and / or alternative internucleotide bonds as described herein or known in the art, including alternative nucleosides, alternative sugar moieties, and / or alternative internucleotide bonds as known in the art and / or discussed herein, to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting specific locations or cell types, and / or increasing interaction with RNA editing enzymes (e.g., ADAR).

[0110] The oligonucleotides described herein were synthesized and / or modified using methods recognized in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA, which are hereby incorporated herein by reference. Representative U.S. patents teaching the preparation of oligonucleotides include, but are not limited to, U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, and 5,489,677. Nos. 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439, the entire contents of each of these documents are hereby incorporated herein by reference.

[0111] Some embodiments include oligonucleotides having a thiophosphate backbone and / or a heteroatom backbone, and particularly the oligonucleotides of -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [referred to as methylene (methylimino) or MMI backbone], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- [wherein the native phosphodiester backbone is represented as -OPO-CH2-] and the oligonucleotides of the amide backbone of U.S. Patent No. 5,602,240 cited above. In some embodiments, the oligonucleotides characterized herein have the morpholine backbone structure of U.S. Patent No. 5,034,506 cited above. In some embodiments, the oligonucleotides described herein include phosphoryldiamine morpholine oligomers (PMOs), wherein the deoxyribose moiety is replaced by a morpholine ring and the charged phosphodiester subunit interunit bonds are replaced by uncharged phosphoryldiamine bonds, as described in Summerton et al., Antisense Nucleic Acid Drug Dev. 1997, 7:63-70.

[0112] Various modifications can be introduced into sugars and / or nucleobases according to this disclosure. For example, in some embodiments, the modifications are those described in US 9006198. In some embodiments, the modifications are US 9394333, US 9744183, US9605019, US 9982257, US 20170037399, US 20180216108, US 20180216107, US 9598458, WO2017 / 062862, WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679, WO 2017 / 210647, WO2018 / 098264, WO 2018 / 022473, WO 2018 / 223056, WO 2018 / 223073, WO The modifications described in WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 032612, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, WO 2019 / 032612, WO 2020 / 191252 and / or WO 2021 / 071858, wherein the sugar, base and nucleotide bonds of each of the modifications are independently incorporated herein by reference.

[0113] Alternative nucleotides and nucleosides include nucleotides and nucleosides having modifications including: for example, end modifications, such as 5' end modifications (phosphorylation, conjugation, antibonding), or 3' end modifications (conjugation, DNA nucleotide, antibonding, etc.); base modifications, such as substitution with a stable base, an unstable base, or a base paired with an extended chaperone library base, removal of a base (a baseless nucleotide), or conjugated base; sugar modifications (e.g., at the 2' or 4' position) or sugar substitution; and / or backbone modifications, including modifications or substitutions of phosphodiester bonds. The nucleobase may also be an isonucleotide in which the nucleobase has moved from the C1 position of the sugar moiety to a different position (e.g., C2, C3, C4, or C5). Specific examples of oligonucleotide compounds that can be used in the embodiments described herein include, but are not limited to, alternative nucleosides containing a modified backbone or lacking natural nucleotide inter-bonding. Among other things, nucleotides and nucleosides having a modified backbone include sequences that do not have phosphorus atoms in the backbone. For the purposes of this specification, and as sometimes mentioned in the art, alternative RNAs that do not have phosphorus atoms in their internucleotide backbone may also be considered oligonucleotides. In some embodiments, the oligonucleotide will have phosphorus atoms in its internucleotide backbone.

[0114] Alternative nucleotide internucleotide bonds include, for example, thiophosphates, chiral thiophosphates, thiophosphate diesters, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkylphosphonates (including 3'-alkylene phosphonates and chiral phosphonates) with normal 3'-5' bonds, hypophosphonates, aminophosphates (including 3'-aminoaminophosphates and aminoalkylaminophosphates), thiocarbonylaminophosphates, thiocarbonylalkylaminophosphates, thiocarbonylalkyl phosphate triesters and borophosphates, analogs of these esters with 2'-5' linkages, and esters with reverse polarity, wherein adjacent pairs of nucleoside units are linked by 3'-5' to 5'-3' or 2'-5' to 5'-2' linkages. Various salts, mixed salts, and free acid forms are also included.

[0115] Representative U.S. patents teaching the preparation of the aforementioned phosphorus-containing bonds include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, and 5,321. No. 131; No. 5,399,676; No. 5,405,939; No. 5,453,496; No. 5,455,233; No. 5,466,677; No. 5,476,925; No. 5,519,126; No. 5,536,821; No. 5,541,316; No. 5,550,111; No. 5,563,253; No. 5,571,799; No. 5,587 No. 361; No. 5,625,050; No. 6,028,188; No. 6,124,445; No. 6,160,109; No. 6,169,170; No. 6,172,209; No. 6,239,265; No. 6,277,603; No. 6,326,199; No. 6,346,614; No. 6,444,423; No. 6,531,590; No. 6,534 The entire contents of each of the following documents are hereby incorporated herein by reference: No. 639; No. 6,608,035; No. 6,683,167; No. 6,858,715; No. 6,867,294; No. 6,878,805; No. 7,015,315; No. 7,041,816; No. 7,273,933; No. 7,321,029; and U.S. Patent No. RE39464.

[0116] Alternative nucleotide inter-bonds that do not include phosphorus atoms have backbones formed by: short-chain alkyl or cycloalkyl nucleotide inter-bonds, mixed heteroatom and alkyl or cycloalkyl nucleotide inter-bonds, or one or more short-chain heteroatom or heterocyclic nucleotide inter-bonds. These nucleotide inter-bonds include those having: morpholine bonds (partially formed from the sugar moiety of a nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formylacetyl and thioformylacetyl backbones; methyleneformylacetyl and thioformylacetyl backbones; olefin-containing backbones; aminosulfonate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones having mixed N, O, S, and CH2 component moieties.

[0117] Representative U.S. patents teaching the preparation of the aforementioned oligonucleotides include, but are not limited to, U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, and 5,489. No. 677; No. 5,541,307; No. 5,561,225; No. 5,596,086; No. 5,602,240; No. 5,608,046; No. 5,610,289; No. 5,618,704; No. 5,623,070; No. 5,663,312; No. 5,633,360; No. 5,677,437; and No. 5,677,439, the entire contents of each of these documents are hereby incorporated herein by reference.

[0118] In other embodiments, suitable oligonucleotides include those in which both the sugar of the nucleotide and the internucleotide bonds (i.e., the backbone) are replaced. The base units are maintained to hybridize with suitable nucleic acid target compounds. One such oligomeric compound, a mimic that has shown excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar of the nucleoside is replaced by an amide-containing backbone, particularly aminoethylglycine. The nucleobases are retained and are directly or indirectly bound to the aza-nitrogen atom of the amide moiety of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Further PNA compounds suitable for use with the oligonucleotides described herein are disclosed, for example, in Nielsen et al., *Science*, 1991, 254, 1497-1500.

[0119] Alternative nucleosides and nucleotides may also contain one or more substituted sugar moieties. Oligonucleotides, for example, the oligonucleotides characterized herein, may include one of the following oligonucleotides located at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-ynyl; or O-alkyl-O-alkyl; wherein the alkyl, alkenyl, and ynyl groups may be substituted or unsubstituted C1 to C2 groups. 10 Alkyl or C2 to C 10 Alkenyl and ynyl groups. Exemplary suitable modifications include -O[(CH2)] n O] m CH3, -O(CH2) n OCH3, -O(CH2) n -NH2、-O(CH2) n CH3, -O(CH2) n -ONH2 and -O(CH2) n -ON[(CH2) n CH3]2, where n and m are 1 to about 10. In other embodiments, the oligonucleotide includes one of the following oligonucleotides located at the 2' position: C1 to C2. 10 Lower alkyl groups, substituted lower alkyl groups, alkylaryl groups, aryl groups, O-alkylaryl or O-aryl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkylaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-O-MOE) (Martin et al., Helv. Chin. Acta, 1995, 78:486-504), i.e., alkoxy-alkoxy groups. 2'-O-MOE nucleoside endows oligonucleotides with several beneficial properties, including but not limited to increased nuclease resistance, improved pharmacokinetic properties, reduced nonspecific protein binding, reduced toxicity, reduced immunostimulatory properties, and enhanced target affinity compared to unmodified oligonucleotides.

[0120] Another exemplary alternative contains a 2'-dimethylaminoethoxy group, i.e., -O(CH2)2ON(CH3)2, as described herein in the examples below, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-(CH2)2-O-(CH2)2-N(CH3)2. Further exemplary alternatives include: 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both the R and S isomers of these three families); 2'-alkoxyalkyl groups; and 2'-NMA (N-methylacetamide).

[0121] Other alternative forms include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the nucleoside and nucleotide of the oligonucleotide, specifically at the 3' position of the sugar on the 3' terminal nucleotide or the 5' position of the 5' terminal nucleotide. The oligonucleotide can also have sugar mimicry, such as replacing the cyclobutyl moiety of the pentofuranosyl sugar. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, and 5,567. Nos. 811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, some of which are commonly owned with this application. The entire contents of each of the foregoing items are hereby incorporated herein by reference.

[0122] The oligonucleotides described herein may also include nucleobase (generally referred to in the art simply as "base") substitutions (e.g., modifications or substitutions). Unmodified or native nucleobases include purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Substitute nucleobases include other synthetic and native nucleobases such as 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-carboxycytosine, pyrrolocytosine, dideoxycytosine, uracil, 5-methoxyuracil, 5-hydroxydeoxyuracil, dihydrouracil, 4-thiouracil, pseudouracil, 1-methyl-pseudouracil, deoxyuracil, and 5-hydroxybutyl-2'-deoxyuracil. Pyrimidine, xanthine, hypoxanthine, 7-denitroxanthine, thienoguanine, 8-aza-7-denitroguanine, 7-methylguanine, 7-denitroguanine, 6-aminomethyl-7-denitroguanine, 8-aminoguanine, 2,2,7-trimethylguanine, 8-methyladenine, 8-azaadenine, 7-methyladenine, 7-denitroadenine, 3-denitroadenine, 2,6-diaminoguanine 2-Aminopurine, 7-deazo-8-aza-adenine, 8-aminoadenine, thymine, dideoxythymine, 5-nitroindole, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine Pyridine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 4-thionuracil, 8-halogenated, 8-amino, 8-thio, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogenated, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 8-azaguanine and 8-azaadenine and 3-deazoguanine.Other nucleobases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in modified nucleosides in *Biochemistry, Biotechnology and Medicine*, Herdewijn, P. (ed.), John Wiley-VCH, 2008; those disclosed in *The Concise Encyclopedia of Polymer Science and Engineering*, pp. 858-859, Kroschwitz, JL (ed.), John Wiley & Sons, 1990; those disclosed in *Angewandte Chemie, International Edition*, Englisch et al. (1991), 30:613; and those disclosed in Sanghvi, Y.S., Chapter 15, "Antisense Research and Applications". Nucleotides disclosed in *Applications*, pp. 289-302, edited by Crooke, ST. and Lebleu, B., CRC Press, 1993. Certain nucleotides in these nucleotides can be used specifically to enhance the binding affinity of the oligomers described herein. These nucleotides include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitution has been shown to increase the stability of nucleic acid duplexes by 0.6–1.2 °C (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., *Antisense Research and Applications*, CRC Press, Boca Raton, 1993, pp. 276–278), and is an exemplary base substitution, even more specifically in combination with 2'-O-methoxyethyl sugar modifications.

[0123] Representative U.S. patents teaching the preparation of the aforementioned alternative nucleobases and other alternative nucleobases include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,30, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, and 5,594,1... No. 21; No. 5,596,091; No. 5,614,617; No. 5,681,941; No. 5,750,692; No. 6,015,886; No. 6,147,200; No. 6,166,197; No. 6,222,025; No. 6,235,887; No. 6,380,368; No. 6,528,640; No. 6,639,062; No. 6,617,438; No. 7,045,610; No. 7,427,672; and No. 7,495,088, the entire contents of each of these documents are hereby incorporated herein by reference.

[0124] In other embodiments, the sugar portion of the nucleotide may be a ribose molecule, optionally having a ribose molecule modified with 2'-O-methyl, 2'-O-MOE, 2'-F, 2'-amino, 2'-O-propyl, 2'-aminopropyl, or 2'-OH.

[0125] In some embodiments, the oligonucleotides described herein comprise one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanyl ring modified by a two-atom bridging. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting the two carbon atoms of the sugar ring, thereby forming a bicyclic system. In some embodiments, the bridge connects the 4' and 2' carbons of the sugar ring. Therefore, in some embodiments, the oligonucleotides described herein may comprise one or more locked nucleosides. A locked nucleoside is a nucleoside having a modified ribose moiety comprising an additional bridge connecting the 2' and 4' carbons. In other words, a locked nucleoside is a nucleoside comprising a bicyclic sugar moiety comprising 4'-CH2-O-2'. This structure effectively “locks” the ribose in a 3'-inner conformation. Adding locked nucleosides to oligonucleotides has been shown to increase oligonucleotide stability in serum and reduce off-target effects (Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides include, but are not limited to, nucleosides comprising a bridge between the 4' and 2' ribosyl ring atoms. In some embodiments, the oligonucleotide comprises one or more bicyclic nucleosides comprising a 4' to 2' bridge. Examples of such 4' to 2'-bridged bicyclic nucleotides include, but are not limited to, 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also referred to as "bound ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and their analogues; see, for example, U.S. Patent No. 7,399,845); 4'- C(CH3)(CH3)-O-2' (and its analogues; see, for example, U.S. Patent No. 8,278,283); 4'-CH2-N(OCH3)-2' (and its analogues; see, for example, U.S. Patent No. 8,278,425); 4'-CH2-ON(CH3)2-2' (see, for example, U.S. Patent Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2', wherein R is H, Cl-C 12 Alkyl groups or protecting groups (see, for example, U.S. Patent No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, for example, Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and their analogues; see, for example, U.S. Patent No. 8,278,426). The entire contents of each of the foregoing items are hereby incorporated herein by reference.

[0126] Other representative U.S. patents and U.S. patent publications teaching the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; and others. The entire contents of each of the aforementioned U.S. patents, namely U.S. Patent No. 2008 / 0039618 and U.S. Patent No. 2009 / 0012281, are hereby incorporated herein by reference.

[0127] Any of the aforementioned bicyclic nucleotides having one or more stereochemical sugar configurations can be prepared, including, for example, α-L-ribofuranosyl and β-D-ribofuranosyl (see WO 99 / 14226).

[0128] The oligonucleotides described herein can also be modified to include one or more restricted ethyl nucleotides. As used herein, "restricted ethyl nucleotide" or "cEt" refers to a locked nucleic acid comprising a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge. In one embodiment, the restricted ethyl nucleotide is in the S conformation referred to herein as "S-cEt".

[0129] The oligonucleotides described herein may also include one or more “conformation-restricted nucleotides” (“CRNs”). A CRN is a nucleotide analog with a linker connecting the C2' and C4' carbons of the ribose or the C3 and -C5' carbons of the ribose. CRNs lock the ribose ring into a stable conformation and increase hybridization affinity to mRNA. The linker is long enough to place oxygen in the optimal position for achieving stability and affinity, resulting in fewer ribose ring wrinkles.

[0130] Representative publications teaching the preparation of the CRN mentioned above include, but are not limited to, U.S. Patent Publication No. 2013 / 0190383; and PCT Publication WO 2013 / 036868, the entire contents of each of which are hereby incorporated herein by reference.

[0131] In some embodiments, the oligonucleotides described herein comprise one or more monomers that are UNA (unlocked nucleic acid) nucleotides. An UNA is an unlocked acyclic nucleic acid in which any bonds of the sugar have been removed, resulting in unlocked “sugar” residues. In one instance, UNA also encompasses monomers in which the bond between C1' and C4' has been removed (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another instance, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Molecular Biology Systems, 2009, 10, 1039, which are hereby incorporated by reference).

[0132] Representative U.S. publications teaching the preparation of UNA include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Publication Nos. 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are hereby incorporated herein by reference.

[0133] Ribose molecules can also be modified with a cyclopropane ring to produce tricyclic deoxyribonucleic acid (tricyclic DNA). The ribose moiety can be replaced by another sugar, such as 1,5'-anhydrohexitol, threose used to produce threononucleotides (TNAs), or arabinose used to produce arabinonucleotides. Ribose molecules can also be replaced by non-carbohydrates, such as cyclohexene used to produce cyclohexene nucleotides or ethylene glycol used to produce ethylene glycol nucleotides.

[0134] Ribose molecules can also be replaced by non-carbohydrates, such as cyclohexene, to produce cyclohexene nucleic acids (CeNA), or by ethylene glycol, to produce ethylene glycol nucleic acids (GNA). Potential stabilizing modifications to the ends of nucleotide molecules can include N-(acetamidohexanoyl)-4-hydroxyproline (Hyp-C6-NHAc), N-(hexanoyl-4-hydroxyproline) (Hyp-C6), N-(acetyl-4-hydroxyproline) (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminohexanoyl)-4-hydroxyproline (Hyp-C6-amino), 2-docosyl-uridine-3''-phosphate, and inverse base dT (idT), etc. Publications on such modifications can be found in PCT Publication WO 2011 / 005861.

[0135] Other alternative chemical components of the oligonucleotides described herein include 5' phosphate esters or 5' phosphate ester mimics, such as the 5'-terminal phosphate ester or phosphate ester mimic of the oligonucleotide. Suitable phosphate ester mimics are disclosed, for example, in U.S. Patent Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0136] The exemplary oligonucleotides described herein include sugar-modified nucleosides and may also include DNA or RNA nucleosides. In some embodiments, the oligonucleotides include both sugar-modified nucleosides and DNA nucleosides. Incorporating alternative nucleosides into the oligonucleotides described herein can enhance the affinity of the oligonucleotides for target nucleic acids. In such cases, the alternative nucleosides may be referred to as affinity-enhancing alternative nucleotides.

[0137] In some embodiments, the oligonucleotides described herein comprise at least one alternative nucleoside, such as at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, or at least sixteen alternative nucleosides. In other embodiments, the oligonucleotide comprises one to ten alternative nucleosides, such as two to nine, three to eight, four to seven, six, or seven alternative nucleosides. In one embodiment, the oligonucleotides described herein may comprise alternative forms independently selected from these three types of alternative forms (alternative sugar moieties, alternative nucleobases, and alternative nucleotide internucleotide bonds) or combinations thereof. Preferably, the oligonucleotide comprises one or more nucleosides comprising an alternative sugar moieties, such as 2'-sugar alternative nucleosides. In some embodiments, the oligonucleotides described herein comprise one or more 2'-sugar substitute nucleosides, said one or more 2'-sugar substitute nucleosides being independently selected from the group consisting of: 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, ANA, 2'-fluoro-ANA, and BNA (e.g., LNA) nucleosides. In some embodiments, one or more substitute nucleosides are BNA.

[0138] Oligonucleotides conjugated with ligands The oligonucleotides described herein may be chemically linked to one or more ligands, portions, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotides.Such fractions include, but are not limited to, lipid fractions, such as cholesterol fractions (Letsinger et al., (1989) *Proceedings of the National Academy of Sciences of the United States of America*, 86:6553-6556); bile acids (Manoharan et al., (1994) *Biorg. Med. Chem. Let.*, 4:1053-1060); thioethers, for example, beryl-S-triphenylmethanethiol (Manoharan et al., (1992) *Annals of the New York Academy of Sciences*, 660:306-309; Manoharan et al., (1993) *Biorg. Med. Chem. Let.*, 3:2765-2770); mercaptocholesterol (Oberhauser et al., (1992) *Nucleic Acid Research*, ...). 20:533-538); aliphatic chains, for example, dodecanediol or undecyl residues (Saison-Behmoaras et al., (1991) *European Organization for Molecular Biology Journal (EMBO J)*, 10:1111-1118; Kabanov et al., (1990) *FEBS Letters*, 259:327-330; Svinarchuk et al., (1993) *Biochimie*, 75:49-54); phospholipids, for example, di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycerol-3-phosphonate triethyl-ammonium (Manoharan et al., (1995) *Tetrahedron Letters*, 36:3651-3654; Shea et al., (1990) *Nucleic Acid Research*, 18:3777-3783); polyamine or polyethylene glycol chain (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973) or adamantane acetic acid (Manoharan et al., (1995) Tetrahedral Letters, 36:3651-3654); palmityl moiety (Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229-237); or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

[0139] In some embodiments, ligands alter the distribution, targeting, or lifetime of the oligonucleotide agent to which they are incorporated. In some embodiments, for example, ligands provide enhanced affinity for selected targets (e.g., molecules, cells, or cell types) and compartments (e.g., cellular or organ compartments, tissues, organs, or body regions) compared to species in which such ligands are absent.

[0140] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, for example, synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolic acid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphonazine. Examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, pseudopeptide-polyamines, dendritic polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0141] Ligands may also include targeting groups, such as cell or tissue targets, like lectins, glycoproteins, lipids, or proteins, such as antibodies that bind to specific cell types (e.g., kidney cells). Targeting groups can be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polylactose, polygalactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polymannose, polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamate, polyaspartate, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or RGD peptides or RGD peptide mimics.

[0142] Other examples of ligands include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), and lipophilic molecules (e.g., cholesterol, bile acids, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O-hexadecylglycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl) chalcedony). Acids, O3-(oleoyl)cholenic acid, dimethoxytriphenylmethyl or phenoxazine), peptide conjugates (e.g., tentacles, Tat peptides), alkylating agents, phosphates, amino groups, thiol groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, diimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraaza macrocyclic compounds), dinitrophenyl, HRP or AP.

[0143] Ligands can be proteins (e.g., glycoproteins), peptides (e.g., molecules with specific affinity for the coligand), or antibodies (e.g., antibodies that bind to specific cell types, such as hepatocytes). Ligands can also include hormones and hormone receptors. Ligands can also include non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, or polyvalent fucose.

[0144] Ligands can be substances that can increase the uptake of oligonucleotide agents into cells, for example, by disrupting the cell's cytoskeleton, such as by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments. Drugs can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoseverin.

[0145] In some embodiments, ligands linked to oligonucleotides as described herein act as pharmacokinetic modulators (PK modulators). PK modulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEGs, vitamins, etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, bile acids, lithocholic acids, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. Oligonucleotides comprising numerous thiophosphate bonds are also known to bind to serum proteins; therefore, short oligonucleotides, such as those of about 5, 10, 15, or 20 bases, including thiophosphate bonds in their backbone as ligands (e.g., as PK-regulating ligands), are also suitable as the oligonucleotides disclosed herein. Additionally, aptamers that bind to serum components (e.g., serum proteins) are also suitable as PK-regulating ligands in the embodiments described herein.

[0146] The ligand-conjugated oligonucleotides described herein can be synthesized using oligonucleotides with side-group reactive capabilities, such as oligonucleotides derived from linkers attached to oligonucleotides (as described below). These reactive oligonucleotides can react directly with commercially available ligands, synthetic ligands with any of a variety of protecting groups, or ligands having a linker motif attached to them.

[0147] The oligonucleotides used in the conjugates described herein can be conveniently and routinely prepared using well-known solid-phase synthesis techniques. Equipment for such synthesis is sold by several suppliers, including, for example, Applied Biosystems (Foster City, California). Any other methods known in the art for such synthesis can be used alternatively or as an alternative. The use of similar techniques to prepare other oligonucleotides, such as phosphate thioides and alkylated derivatives, is also known.

[0148] In the ligand-conjugated oligonucleotides described herein, such as ligand molecules containing sequence-specific links to nucleosides of the oligonucleotides described herein, the oligonucleotides and oligonucleotides can be assembled onto a suitable DNA synthesizer using standard nucleotides or nucleoside precursors, or nucleotides or nucleoside conjugate precursors already containing linking portions, ligand-nucleotide or nucleoside conjugate precursors already containing ligand molecules, or building blocks containing non-nucleoside ligands.

[0149] When using a nucleotide conjugate precursor that already has a linker moiety, the synthesis of a sequence-specific linked nucleoside is typically performed, and then the ligand molecule reacts with the linker moiety to form a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides described herein are synthesized via an automated synthesizer using phosphorusamide derived from the ligand-nucleoside conjugate, along with commercially available standard and non-standard phosphorusamides commonly used in oligonucleotide synthesis.

[0150] lipid conjugates In some embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such lipid or lipid-based molecules preferably bind to a serum protein, such as human serum albumin (HSA). HSA-binding ligands allow the conjugate to distribute to target tissues, such as non-renal target tissues of the body. For example, the target tissue could be the liver, including hepatic parenchymal cells. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipid or lipid-based ligands can (a) enhance resistance to conjugate degradation; (b) increase targeting or transport to target cells or cell membranes; and / or (c) can be used to modulate binding to serum proteins, such as HSA.

[0151] Lipid-based ligands can be used to inhibit (e.g., control) the binding of conjugates to target tissues. For example, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidney and therefore less likely to be cleared from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target conjugates to the kidney.

[0152] In some embodiments, the ligand is a portion taken up by target cells (e.g., proliferating cells), such as a vitamin. Exemplary vitamins include vitamins A, E, and K.

[0153] Cell permeabilizer In some embodiments, the ligand is a cell permeabilizer, preferably a helical cell permeabilizer. Preferably, the agent is amphiphilic. Exemplary agents are peptides, such as tat or tentacles. If the agent is a peptide, it can be modified, including by peptide analogs, inversions, non-peptide or pseudopeptide bonds, and the use of D-amino acids. The helical agent is preferably an α-helical agent having both a lipophilic and a lipophobic phase.

[0154] The ligand can be a peptide or a peptide mimic. A peptide mimic (also referred to herein as an oligopeptide mimic) is a molecule that, like a natural peptide, can fold into a defined three-dimensional structure. The conjugation of peptides and peptide mimics to oligonucleotide drugs can affect the pharmacokinetic distribution of oligonucleotides, such as by enhancing cellular recognition and uptake. The length of the peptide or peptide mimic moiety can be approximately 5–50 amino acids, for example, approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.

[0155] The peptide or peptide mimic can be, for example, a cell-permeable peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., primarily composed of Tyr, Trp, or Phe). The peptide moiety can be a dendritic peptide, a bound peptide, or a cross-linked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary peptide containing a hydrophobic MTS is an RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 152). RFGF analogs containing hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 153)) can also be targeting moieties. The peptide moiety can be a "delivery" peptide that can carry highly polar molecules, including peptides, oligonucleotides, and proteins, across the cell membrane. For example, sequences from HIVTat protein (GRKKRRQRRRPPQ (SEQ ID NO: 154) and Drosophila tentacles peptide protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 155)) have been found to act as delivery peptides. Peptides or peptide mimics can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-to-one-compound (OBOC) combinatorial libraries (Lam et al., Nature 354:82-84, 1991). Examples of peptides or peptide mimics tethered to oligonucleotide agents by incorporating monomeric units for cell-targeting purposes are arginine-glycine-aspartic acid (RGD)-peptides or RGD mimics. The length of the peptide moiety can range from about 5 amino acids to about 40 amino acids. The peptide moiety can have structural modifications, such as structural modifications to increase stability or direct conformational properties. Any structural modifications described below can be used.

[0156] RGD peptides used in the compositions and methods described herein can be linear or cyclic and can be modified, for example, by glycosylation or methylation, to facilitate targeting to specific tissues. RGD-containing peptides and peptide mimics may include D-amino acids as well as synthetic RGD mimics. In addition to RGD, other parts of the targeting integrin ligand may be used. Some conjugates of this ligand target PECAM-1 or VEGF.

[0157] "Cell-permeable peptides" are capable of penetrating cells, such as microbial cells (e.g., bacterial or fungal cells) or mammalian cells (e.g., human cells). Microbial cell-permeable peptides can be, for example, α-helical linear peptides (e.g., LL-37 or CeropinP1), disulfide-containing peptides (e.g., α-defensins, β-defensins, or bacteriocins), or peptides containing only one or two major amino acids (e.g., PR-39 or indolicidin). Cell-permeable peptides can also include nuclear localization signals (NLS). For example, cell-permeable peptides can be amphiphilic peptides derived from the NLS of the fusion peptide domain of HIV-1 gp41 and the SV40 large T antigen, such as MPG (Simeoni et al., Nucleic Acid Research 31:2717-2724, 2003).

[0158] carbohydrate conjugates In some embodiments, the oligonucleotides described herein further include carbohydrates. Carbohydrate-conjugated oligonucleotides are advantageous for in vivo delivery of nucleic acids and for compositions suitable for in vivo therapeutic use as described herein. As used herein, "carbohydrate" means a compound that is itself composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) wherein oxygen, nitrogen, or sulfur atoms are bonded to each carbon atom; or a compound having as part of a carbohydrate portion composed of one or more monosaccharide units each having at least six carbon atoms (which may be linear, branched, or cyclic) wherein oxygen, nitrogen, or sulfur atoms are bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Certain monosaccharides include sugars with 5 or more saccharides (e.g., C5, C6, C7, or C8); disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0159] In some embodiments, the carbohydrate conjugate is a monosaccharide.

[0160] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, PK regulators and / or cell-penetrating peptides.

[0161] Additional carbohydrate conjugates (and linkers) applicable to the oligonucleotides described herein include the carbohydrates described in PCT Publications WO 2014 / 179620 and WO 2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0162] connector In some embodiments, the conjugates or ligands described herein can be linked to oligonucleotides via various linkers, which can be cleavable or non-cleavable.

[0163] Linkages typically include direct bonds or atoms (such as oxygen or sulfur), units (such as NR8, C(O), C(O)NH, SO, SO2, SO2NH), or atomic chains, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, aralkyl, areneyl, arynyl, heteroaralkyl, heteroareneyl, heteroarynyl, heterocyclic alkyl, heterocyclic alkenyl, heterocyclic alynyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, alkylaralkyl, alkylareneyl, alkylarynyl, alkenylaralkyl, alkenylareneyl, alkenylarynyl, alynylaralkyl, alynylareneyl, alynylarynyl, alkylheteroalkyl, alkylheteroalkenyl, alkylheteroalynyl, alkenylheteroalkyl, alkylheteroalynyl, alkenylheteroalkyl, alkylheteroalkyl, alkylheteroalynyl, alkenylheteroalkylane Alkyl, alkenyl heteroaryl, alkenyl heteroarylynyl, alkyneyl heteroarylalkyl, alkyneyl heteroaryl, alkyneyl heteroarylynyl, alkyl heterocyclic alkyl, alkyl heterocyclic alkenyl, alkyl heterocyclic alkyne, alkenyl heterocyclic alkenyl, alkenyl heterocyclic alkyne, alkyneyl heterocyclic alkyl, alkyneyl heterocyclic alkenyl, alkyneyl heterocyclic alkyne, alkyl aryl, alkenyl aryl, alkyneyl aryl, alkyl heteroaryl, alkenyl heteroaryl, alkyneyl heteroaryl, alkyl aryl, alkenyl aryl, alkyneyl aryl, alkyl heteroaryl, alkenyl heteroaryl, alkyneyl heteroaryl, alkyl aryl, alkenyl heteroaryl, alkyneyl heteroaryl, alkyl aryl, alkenyl heteroaryl, alkynyl heteroaryl, wherein one or more methylene groups may be interrupted or capped by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle; wherein R8 is hydrogen, acyl, aliphatic compound or substituted aliphatic compound. In some embodiments, the connector is about 1-24 atoms, 2-24 atoms, 3-24 atoms, 4-24 atoms, 5-24 atoms, 6-24 atoms, 6-18 atoms, 7-18 atoms, 8-18 atoms, 7-17 atoms, 8-17 atoms, 6-16 atoms, 7-17 atoms, or 8-16 atoms.

[0164] A cleavable linker is a linker that is sufficiently stable outside the cell but is cleaved upon entry into a target cell to release the two parts of the linker that remain together. In a preferred embodiment, the cleavage rate of the cleavable linker in the target cell or under a first reference condition (which may be selected, for example, to simulate or represent intracellular conditions) is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or more times, or at least about 100 times, the cleavage rate in the subject's blood or under a second reference condition (which may be selected, for example, to simulate or represent conditions present in blood or serum).

[0165] Cleavable linker groups are susceptible to the effects of cleaving agents (e.g., pH, redox potential, or the presence of degrading molecules). Generally, cleaving agents are more prevalent or present at higher levels or with higher activity inside cells than in serum or blood. Examples of such degrading agents include: redox agents that are selective or non-substrate-specific to a particular substrate, including, for example, oxidases or reductases (e.g., thiols) present in cells that can degrade redox-cleavable linker groups by reduction; esterases; endosomes or agents that can produce an acidic environment, for example, endosomes or agents that produce a pH of five or lower; and enzymes that can hydrolyze or degrade acid-cleavable linker groups by acting as universal acids, peptidases (which may be substrate-specific), and phosphatases.

[0166] Cleavable linker groups (such as disulfide bonds) may be susceptible to pH influence. Human serum has a pH of 7.4, while the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH, in the range of 5.5 to 6.0, and lysosomes have a pH of approximately 5.0 or even more acidic. Some linkers will have cleavable linker groups that are cleaved at a preferred pH, thereby releasing cationic lipids from intracellular ligands or into desired compartments of the cell.

[0167] Linkers can include cleavable linker groups that can be cleaved by specific enzymes. The type of cleavable linker group incorporated into the linker can depend on the cell type to be targeted. For example, liver-targeting ligands can be linked to cationic lipids via linkers that include ester groups. Hepatocytes are rich in esterases, and therefore the linker will cleave more efficiently in hepatocytes compared to non-esterase-rich cell types. Other esterase-rich cell types include lung, renal cortex, and testicular cells.

[0168] When targeting cell types rich in peptidase, such as hepatocytes and synovial cells, a linker containing peptide bonds can be used.

[0169] Generally, the suitability of a candidate cleavable linker can be evaluated by testing its ability to cleave the candidate linker group by a degrading agent (or condition). It will also be necessary to test the ability of the candidate cleavable linker group to resist cleavage in blood or upon contact with other non-target tissues. Thus, a relative sensitivity to cleavage can be determined between a first condition and a second condition, wherein the first condition is selected to indicate cleavage in target cells, and the second condition is selected to indicate cleavage in other tissues or biological fluids (e.g., blood or serum). Evaluation can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or in whole animals. Initial evaluation under cell-free or culture conditions, followed by confirmation through further evaluation in whole animals, may be useful. In some embodiments, the cleavage rate of a useful candidate compound in cells (or under in vitro conditions selected to simulate intracellular conditions) is at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times that in blood or serum (or under in vitro conditions selected to simulate extracellular conditions).

[0170] Oxidative-reduction cleavage linker In some embodiments, the cleavable linker is an oxidoreductile linker that is cleaved upon reduction or oxidation. An example of an oxidoreductile linker is a disulfide linker (--S--S--). To determine whether a candidate cleavable linker is a suitable “oxidoreductile linker,” or, for example, whether it is suitable for use with a specific oligonucleotide moiety and a specific target, reference can be made to the methods described herein. For example, candidates can be evaluated using reagents known in the art by incubation with dithiothreitol (DTT) or other reducing agents that simulate the cleavage rate to be observed in cells (e.g., target cells). Candidates can also be evaluated under conditions selected to simulate blood or serum conditions. In one embodiment, the candidate compound is cleaved by up to about 10% in blood. In other embodiments, the degradation rate of the useful candidate compound in cells (or under in vitro conditions selected to simulate intracellular conditions) is at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times that in blood (or under in vitro conditions selected to simulate extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to simulate intracellular media and compared to conditions selected to simulate extracellular media.

[0171] Phosphate-based cleavable linker groups In another embodiment, the cleavable connector comprises a phosphate ester-based cleavable linker group. The phosphate ester-based cleavable linker group is cleaved by an agent that degrades or hydrolyzes the phosphate ester group. Examples of agents in cells that cleave phosphate ester groups are cellular enzymes, such as phosphatases. Examples of phosphate ester-based linker groups are -OP(O)(ORk)-O-, OP(S)(ORk) O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, OP(S)(ORk) S, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, SP(O)(Rk)-S-, -OP(S)(Rk)-S-. These candidates can be evaluated using methods similar to those described above.

[0172] Acid-cutting linker In another embodiment, the cleavable connector includes an acid-cleavable linker group. An acid-cleavable linker group is a linker group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linker group is cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0 or lower) or by an agent (such as an enzyme) that can act as a generalized acid. In cells, specific low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linker group. Examples of acid-cleavable linker groups include, but are not limited to, hydrazones, esters, and esters of amino acids. The acid-cleavable group can have the general formula -C=NN--, C(O)O, or --OC(O). A preferred embodiment is when the carbon (alkoxy group) attached to the oxygen of the ester is aryl, substituted alkyl, or tertiary alkyl (such as dimethylpentyl or tert-butyl). These candidates can be evaluated using methods similar to those described above.

[0173] Based on ester linking groups In some embodiments, the cleavable linker comprises an ester-based cleavable linker group. Ester-based cleavable linker groups are cleaved by cellular enzymes such as esterases and amidases. Examples of ester-based cleavable linker groups include, but are not limited to, alkylene, alkenene, and ynylene esters. Ester-cleavable linker groups have the general formula -C(O)O-- or --OC(O)--. These candidates can be evaluated using methods similar to those described above.

[0174] Based on peptide cleavage groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linker group. The peptide-based cleavable linker group is cleaved by enzymes in the cell, such as peptidases and proteases. The peptide-based cleavable linker group is a peptide bond formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable linker group does not include an amide group (--C(O)NH--). The amide group can be formed between any alkylene, alkenyl, or alkyne groups. A peptide bond is a specific type of amide bond formed between amino acids to produce peptides and proteins. The peptide-based cleavable linker group is generally limited to peptide bonds (i.e., amide bonds) formed between amino acids that produce peptides and proteins, and does not include the entire amide functional group. The peptide-based cleavable linker group has the general formula --NHCHRAC(O)NHCHRBC(O)--, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0175] In one embodiment, the oligonucleotide described herein is conjugated to a carbohydrate via a linker. The linker comprises divalent and trivalent branched linker groups. Exemplary oligonucleotide-carbohydrate conjugates having linkers include, but are not limited to, the conjugates described in Formulas 24-35 of PCT Publication No. WO 2018 / 195165.

[0176] Representative U.S. patents teaching the preparation of oligonucleotide conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,045, 5,414,077, and 5,486,603. No. 5,512,439; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,762,779; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4,958,013; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,082,830; No. 5,1 No. 12,963; No. 5,214,136; No. 5,245,022; No. 5,254,469; No. 5,258,506; No. 5,262,536; No. 5,272,250; No. 5,292,873; No. 5,317,098; No. 5,371,241; No. 5,391,723; No. 5,416,203; No. 5,451,463; No. 5,510,475; No. 5,512,667; No. 5,514,785; No. 5,565,552; No. 5,567,81 No. 0; No. 5,574,142; No. 5,585,481; No. 5,587,371; No. 5,595,726; No. 5,597,696; No. 5,599,923; No. 5,599,928; and No. 5,688,941; No. 6,294,664; No. 6,320,017; No. 6,576,752; No. 6,783,931; No. 6,900,297; No. 7,037,646; No. 8,106,022, the entire contents of each of these documents are hereby incorporated herein by reference.

[0177] In some cases, the oligonucleotides described herein can be modified with non-ligand groups. Many non-ligand molecules have been conjugated to oligonucleotides to enhance their activity, cellular distribution, or cellular uptake, and procedures for such conjugation are available in the scientific literature. Such non-ligand moieties already include lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proceedings of the National Academy of Sciences, 1989, 86:6553); bile acids (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053); thioethers, for example, hexyl-S-triphenylmethylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765); mercaptocholesterol (Oberhauser et al., Nucleic Acid Research, 1992, 20:533); aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., Journal of the European Society for Molecular Biology, 1991, 10:111; Kabanov et al., FEBS Letters, 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49); phospholipids, such as di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycerol-3-H-phosphonate triethylammonium (Manoharan et al., Tetrahedral Letters, 1995, 36:3651; Shea et al., Nucleic Acid Research, 1990, 18:3777); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides and Nucleotides, 1995, 14:969); or adamantaneacetic acid (Manoharan et al., Tetrahedral Letters, 1995, 36:3651); palmityl moiety (Mishra et al., Chinese Journal of Biochemistry and Biophysics, 1995, 1264229); or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., Journal of Pharmacology and Experimental Therapeutics, 1996, 277:923). The above lists representative U.S. patents teaching the preparation of such oligonucleotide conjugates. Typical conjugation schemes involve the synthesis of oligonucleotides with amino linkers at one or more positions in the sequence.The amino group is then reacted with the conjugated molecule using a suitable coupling or activating reagent. The conjugation reaction can be carried out in solution while the oligonucleotide is still bound to the solid support or after oligonucleotide cleavage. Purification of the oligonucleotide conjugate by HPLC typically yields a pure conjugate.

[0178] III. Drug Use The oligonucleotides described herein can be used to treat any condition that can be treated by deamination of adenosine. For example, any condition caused by mutations that convert guanosine to adenosine, the introduction of a premature stop codon, or the expression of an unwanted protein. In some embodiments, the oligonucleotides described herein, when administered to a subject, may cause correction of mutations that convert guanosine to adenosine. In some embodiments, the oligonucleotides described herein may cause premature stop codons to be turned off, allowing the desired protein to be expressed. In some embodiments, the oligonucleotides described herein may cause inhibition of the expression of an unwanted protein.

[0179] The target adenosine of particular concern for editing with the oligonucleotides described herein is a portion of the codon defined for amino acid residues that is part of the following key functions or properties for co-translation or post-translational modifications (such as glycosylation, hydroxylation, myristylation) and protein cleavage by proteases (to mature the protein and / or as part of intracellular routing): catalytic site, binding site for other proteins, substrate binding, and localization domain.

[0180] Many genetic diseases are caused by G-to-A mutations, and these diseases are potential candidates for treatment with the oligonucleotides described herein because adenosine deamination at the mutated target adenosine reverses the mutation to the wild type. However, reversal to the wild type is not always necessary to achieve beneficial effects. A-to-G modifications in the target RNA can also be beneficial if the wild-type nucleotide is not G. In some cases, this may be the case, but in others, some testing may be required. In some cases, A-to-G modifications in the target RNA may be silent (not translated into different amino acids) when the wild-type is not G, or alternatively, insignificant (e.g., amino acid substitutions that are conserved and do not disrupt protein structure and function), or the amino acid is a part of a functional domain that is robust to the change. If the A-to-G transition caused by editing occurs in non-coding RNA or a non-coding portion of RNA, the result may also be insignificant or less severe than the original mutation. Those skilled in the art will understand that the methods described herein are applicable in a wide range of ways and are not limited to the prevention or treatment of diseases. The described methods can also be used to modify transcripts to study their effects, even in cases where such modification induces disease states, such as in cellular or non-human animal models.

[0181] Examples of genetic diseases that can be prevented and / or treated with oligonucleotides described herein include any disease in which modification of one or more adenosines in the target RNA will result in (potentially) beneficial changes.

[0182] This disclosure is not limited to correcting mutations, as it can instead be used to alter wild-type sequences to mutated sequences by applying the oligonucleotides described herein. One example where it may be advantageous to modify wild-type adenosine is to achieve exon skipping, for example, by modifying adenosine at a branching site that happens to be required for splicing of said exon. Another example is where the adenosine defines a recognition sequence for protein binding or is part of said sequence, or relates to secondary structures defining RNA stability. As mentioned above, therefore, the oligonucleotides and methods described herein can be used to provide research tools for diseases to introduce novel mutations that are less harmful than existing mutations. Deamination of adenosine using the oligonucleotides disclosed herein includes any level of adenosine deamination, for example, having at least one deamination of adenosine within the target sequence (e.g., having at least one, two, three, or more deaminations of adenosine in the target sequence).

[0183] Adenosine deamination can be assessed by a reduction in the absolute or relative level of adenosine within the target sequence relative to a control level. The control level can be any type of control level used in the art, such as baseline levels before administration or levels determined from untreated or similar subjects, cells, or samples treated with a control (e.g., a buffer-only control or an inactive agent control).

[0184] Because the enzymatic activity of ADAR converts adenosine to inosine, adenosine deamination can also be assessed alternatively by an increase in the absolute or relative level of inosine within the target sequence relative to a control level. Similarly, a control level can be any type of control level used in the art, such as baseline levels before administration, or levels determined from similar subjects, cells, or samples that are untreated or treated with a control (e.g., a buffer-only control or an inactive agent control).

[0185] The levels of adenosine and / or inosine within the target sequence can be assessed using any method known in the art for determining the nucleotide composition of a polynucleotide sequence. For example, the relative or absolute levels of adenosine or inosine within the target sequence can be assessed using nucleic acid sequencing techniques, including but not limited to Sanger sequencing, next-generation sequencing (NGS; e.g., pyrosequencing, sequencing by reversible termination chemistry, ligation sequencing, and real-time sequencing), provided on commercially available platforms (e.g., Qiagen, Pacific Biosciences, Thermo Fisher, Roche, and Oxford Nanopore Technologies). Cloning and amplification of the target sequence against NGS can be performed using real-time polymerase chain reaction (also known as qPCR) on platforms commercially available from Applied Biosystems, Roche, Stratagene, Cepheid, Eppendorf, or Bio-Rad Laboratories. Alternatively, emulsion PCR can be used to amplify the target sequence using a commercially available platform, such as Bio-Rad Laboratories' droplet digital PCR.

[0186] In some embodiments, alternative biomarkers may be used to detect adenosine deamination within the target sequence. For example, effective treatment of a subject with a genetic condition involving a mutation that changes from G to A, using oligonucleotides of this disclosure, as demonstrated by acceptable diagnostic and monitoring criteria, can be understood as exhibiting clinically relevant adenosine deamination. In some embodiments, the method includes clinically relevant adenosine deamination, for example, demonstrated by clinically relevant results achieved after treating a subject with oligonucleotides of this disclosure.

[0187] Adenosine deamination of the gene of interest can be indicated by an increase or decrease in the level of mRNA expressed in a first cell or cell group (e.g., such cells may be present in a sample, for example, derived from a subject), where the gene of interest in the first cell or cell group has been transcribed and the first cell or cell group has been treated (e.g., by contacting one or more cells with an oligonucleotide of this disclosure, or by administering the oligonucleotide described herein to a subject in which said cells are present or were previously present), such that the expression of the gene of interest is increased or decreased compared to a second cell or cell group that is substantially the same as the first cell or cell group but has not been so treated (control cells not treated with oligonucleotides or not treated with oligonucleotides targeting the gene of interest). The extent to which the mRNA level of the gene of interest (e.g., SERPINA1) is increased or decreased can be represented by the following: In other embodiments, changes in gene levels can be assessed based on a decrease in parameters related to the expression of the gene of interest, such as protein expression of the gene of interest or downstream signaling functions of the protein. Changes in the levels of the gene of interest can be determined in any cell expressing the gene of interest (whether endogenous or heterologous to the expression construct) and by any assay known in the art.

[0188] Changes in the expression level of the gene of interest may manifest as an increase or decrease in the level of the protein produced by the gene of interest expressed in cells or cell groups (e.g., the level of the protein expressed in a sample derived from a subject). As explained above, to assess mRNA repression, changes in protein expression levels in treated cells or cell groups can be similarly expressed as a percentage of protein levels in control cells or cell groups.

[0189] Control cells or cell groups that can be used to assess changes in the expression of the three genes of interest include cells or cell groups that have not yet been exposed to the oligonucleotides disclosed herein. For example, control cells or cell groups may be derived from a single subject (e.g., a human or animal subject) prior to treatment with the oligonucleotide.

[0190] The level of mRNA of the gene of interest expressed by cells or cell groups can be determined using any method known in the art for assessing mRNA expression. In one embodiment, the expression level of the gene of interest in a sample is determined by detecting transcribed polynucleotides or portions thereof, such as the mRNA of the gene of interest. RNA can be extracted from cells using RNA extraction techniques, including, for example, extraction using acid phenol / guanidine isothiocyanate (RNAzol B; Biogenesis), RNEASY. TM RNA preparation kits (Qiagen) or PAXgene (PreAnalytix, Switzerland). Typical assays using ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, northern blotting, in situ hybridization, and microarray analysis. Circulating mRNA of the gene of interest can be detected using the methods described in PCT publication WO2012 / 177906, the entire contents of which are hereby incorporated by reference. In some embodiments, the expression level of the gene of interest is determined using nucleic acid probes. As used herein, the term "probe" refers to any molecule capable of selectively binding to a specific sequence, such as mRNA or polypeptide. Probes can be synthesized by those skilled in the art or derived from suitable biological agents. Probes can be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0191] Isolated mRNA can be used for hybridization or amplification assays, including but not limited to DNA blotting (Southern) analysis or RNA blotting analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method for determining mRNA levels involves contacting isolated mRNA with a nucleic acid molecule (probe) capable of hybridizing with the mRNA of a gene of interest. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe is immobilized on a solid surface and the mRNA is contacted with the probe, for example, in an AFFYMETRIX gene chip array. Those skilled in the art can readily use known mRNA detection methods to determine the mRNA levels of a gene of interest.

[0192] Alternative methods for determining the expression level of a gene of interest in a sample involve procedures such as nucleic acid amplification and / or reverse transcriptase (to prepare cDNA) of mRNA in the sample, for example, by: RT-PCR (experimental examples described in Mullis, 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany (1991), Proceedings of the National Academy of Sciences 88:189-193), autonomous sequence replication (Guatelli et al. (1990), Proceedings of the National Academy of Sciences 87:1874-1878), transcription amplification systems (Kwoh et al. (1989), Proceedings of the National Academy of Sciences 86:1173-1177), Q-β replicase (Lizardi et al. (1988), Bio / Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Patent No. 5,854,033 or any other nucleic acid amplification method, followed by detection of the amplified molecule using techniques well known to those skilled in the art. These detection protocols are particularly useful for detecting such molecules if they are present in very low quantities. In some embodiments, the expression level of the gene of interest is measured by quantitative fluorescent reverse RT-PCR (i.e., TAQMAN). TM The assay result is determined by either the system or the DUAL-GLO® luciferase assay.

[0193] The expression level of the mRNA of the gene of interest can be monitored using membrane blotting (e.g., for hybridization analysis, such as RNA blotting, DNA blotting, dot blotting, etc.) or microwells, sample tubes, gels, beads, or fibers (or any solid carrier including bound nucleic acids). See U.S. Patents 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which are incorporated herein by reference. Determining gene expression levels can also involve using nucleic acid probes in solution.

[0194] In some embodiments, the level of mRNA expression is assessed using branched-strand DNA (bDNA) assays or real-time PCR (qPCR). The use of this PCR method is described and illustrated in the examples presented herein. Such methods can also be used to detect the nucleic acids of the gene of interest.

[0195] The level of protein produced by the expression of the gene of interest can be determined using any method known in the art for measuring protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), ultradiffusion chromatography, fluid or gel precipitation reactions, absorption spectroscopy, colorimetric assays, spectrophotometry, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assays, electrochemiluminescence assays, etc. These assays can also be used to detect proteins that indicate the presence or replication of proteins produced by the gene of interest. Furthermore, the above assays can be used to report changes in the mRNA sequence of interest that restore or alter protein function, thereby providing therapeutic effects and benefits to subjects, treating their conditions and / or reducing their symptoms.

[0196] In some embodiments, the oligonucleotides described herein are administered to a subject such that the oligonucleotides are delivered to a specific site within the subject's body. Changes in the expression of the gene of interest can be assessed using measurements or changes in the levels of mRNA or protein of the gene of interest derived from a sample from a specific site within the subject's body.

[0197] In other embodiments, the oligonucleotide is administered in an amount and at a time sufficient to cause one or more of the following (e.g., two or more, three or more, four or more): (a) a reduction in the amount of adenosine within the target sequence of the gene of interest, (b) a delay in the onset of symptoms, (c) an increase in the survival of the subject, (d) an increase in the progression-free survival of the subject, (e) restoration or alteration of protein function, and (f) a reduction in symptoms.

[0198] Compared to the untreated population, treatment of the condition associated with the G-to-A mutation may also reduce mortality in the treated population. For example, mortality may be reduced by more than 2% (e.g., 5%, 10%, or 25%). This reduction in mortality in the treated population can be achieved in any reproducible manner, such as by calculating the average number of disease-related deaths per unit time following the initiation of treatment with the compound described herein or a pharmaceutically acceptable salt of the compound. A reduction in mortality may also be achieved, for example, by calculating the average number of disease-related deaths per unit time following completion of the first round of treatment with the compound described herein or a pharmaceutically acceptable salt of the compound.

[0199] A. Delivery of oligonucleotides Delivery of the oligonucleotides described herein to cells, such as those of a subject, including human subjects (e.g., subjects in need, such as those with a disease), can be achieved in a variety of different ways. For example, delivery can be performed by contacting cells with the oligonucleotides described herein, either ex vivo, in vitro, or in vivo. In vivo delivery can also be performed directly by administering a composition comprising the oligonucleotide to the subject. Alternatively, in vivo delivery can also be performed indirectly by administering one or more carriers encoding and directing the expression of the oligonucleotide. Combinations of in vitro and in vivo methods of cell contact are also possible. As discussed above, cell contact can be direct or indirect. Furthermore, cell contact can be achieved using targeted ligands, including any ligands described herein or known in the art. In some embodiments, the targeted ligand is a carbohydrate moiety, such as the GalNAc3 ligand, or any other ligand that directs the oligonucleotide to the site of interest. Cells can include central nervous system cells or muscle cells. These alternative forms will be discussed further below.

[0200] Cellular contact with oligonucleotides can be accomplished in vitro or in vivo. For in vivo delivery, factors to consider include, for example, the biostability of the delivered molecule, prevention of nonspecific effects, and accumulation of the delivered molecule in the target tissue. Nonspecific effects of oligonucleotides can be minimized through local application, such as by direct injection or implantation into tissues, or by topical administration of the formulation. Local application to the treatment site maximizes the local concentration of the agent, limits the exposure of the agent to systemic tissues, and allows for the administration of a lower total dose of oligonucleotide molecules that may be additionally damaged or degraded by the agent.

[0201] For systemic administration of oligonucleotides to treat diseases, oligonucleotides may include alternative nucleobases, alternative sugar moieties, and / or alternative nucleotide internucleotide bonds, or may be delivered alternatively using a drug delivery system; both methods aim to prevent rapid degradation of oligonucleotides in vivo due to endonucleases and exonucleases. Modification of oligonucleotides or drug carriers can also allow for targeting of oligonucleotide compositions to target tissues and avoid undesirable off-target effects. Oligonucleotide molecules can also be modified by chemical conjugation with lipophilic groups (such as cholesterol) to enhance cellular uptake and prevent degradation. In an alternative embodiment, oligonucleotides can be delivered using drug delivery systems such as nanoparticles, lipid nanoparticles, polymer complex nanoparticles, dendritic macromolecules, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems promote the binding of negatively charged oligonucleotide molecules and also enhance interactions at negatively charged cell membranes to allow for efficient cellular uptake of oligonucleotides. Cationic lipids, dendritic macromolecules, or polymers can either bind to oligonucleotides or be induced to form vesicles or micelles encapsulating oligonucleotides. When administered systemically, the formation of vesicles or micelles further prevents the degradation of oligonucleotides. In general, any method known in the art for delivering nucleic acids is applicable to the delivery of oligonucleotides described herein. Methods for preparing and administering cationic oligonucleotide complexes are entirely within the capabilities of those skilled in the art (see, for example, Sorensen, DR. et al. (2003) *J. Mol. Biol* 327:761-766; Verma, UN. et al. (2003) *Clin. Cancer Res.* 9:1291-1300; Arnold, AS et al. (2007) *J. Hypertens.* 25:197-205, all of which are incorporated herein by reference in their entirety).Some non-limiting examples of drug delivery systems that can be used for systemic delivery of oligonucleotides include DOTAP (Sorensen, D R. et al. (2003), ibid.; Verma, U N. et al. (2003), ibid.); Oligofectamine, i.e., “solid nucleic acid lipid particles” (Zimmermann, T S. et al. (2006) Nature 441:111-114); cardiolipin (Chien, P Y. et al. (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al. (2005) International Journal of Oncology 26:1087-1091); polyethyleneimine (Bonnet M E. et al. (2008) Pharmaceutical Research, August 16 electronic preprint; Aigner, A. (2006) *Journal of Biomedical and Biotechnology* 71659; Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) *Molecular Pharmacology* 3:472-487); and polyamide amines (Tomalia, D A. et al. (2007) *Biochem. Soc. Trans.* 35:61-67; Yoo, H. et al. (1999) *Pharmaceutical Research* 16:1799-1804). In some embodiments, the oligonucleotide and cyclodextrin form a complex for systemic administration. Methods for administering oligonucleotides and cyclodextrins, and pharmaceutical compositions of oligonucleotides and cyclodextrins, can be found in U.S. Patent No. 7,427,605, which is incorporated herein by reference in its entirety. In some embodiments, the oligonucleotides described herein are delivered via polymer complex nanoparticles or lipid complex nanoparticles.Methods for administering oligonucleotide and polymer complex nanoparticles and lipid complex nanoparticles, as well as pharmaceutical compositions thereof, can be found in the following U.S. Patent Applications Nos. 2017 / 0121454, 2016 / 0369269, 2016 / 0279256, 2016 / 0251478, 2016 / 0230189, 2015 / 0335764, 2015 / 0307554, 2015 / 0174549, 2014 / 0342003, 2014 / 0135376, and 2013 / 0317086, all of which are incorporated herein by reference in their entirety.

[0202] i. Membrane-based molecular assembly and delivery methods The oligonucleotides described herein can also be delivered using a variety of membrane-based molecular assembly delivery methods, including polymers, biodegradable microparticles, or microcapsule delivery devices known in the art. For example, colloidal dispersion systems can be used to target and deliver the oligonucleotide agents described herein. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Liposomes are artificial membrane vesicles that can be used as delivery mediators in vitro and in vivo. It has been shown that large monolayer vesicles (LUVs) ranging in size from 0.2 to 4.0 µm can encapsulate a considerable percentage of aqueous buffers containing large macromolecules. Liposomes can be used to transfer and deliver active ingredients to the site of action. Because the liposome membrane is structurally similar to a biological membrane, when liposomes are applied to tissues, the liposome bilayer fuses with the cell membrane bilayer. As the fusion of the liposome and the cell progresses, the internal aqueous contents, including the oligonucleotides, are delivered into the cell, where the oligonucleotides can specifically bind to target RNA and mediate RNase H-mediated gene silencing. In some cases, liposomes are also specifically targeted, for example, to deliver oligonucleotides to specific cell types. Liposome compositions are typically combinations of phospholipids, often in combination with steroids, particularly cholesterol. Other phospholipids or other lipids may also be used. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0203] Liposomes containing oligonucleotides can be prepared by a variety of methods. In one example, the lipid component of the liposome is dissolved in a detergent, such that micelles are formed together with the lipid component. For example, the lipid component can be an amphiphilic cationic lipid or a lipid conjugate. The detergent can have a high critical micelle concentration and can be nonionic. Exemplary detergents include bile salts, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. An oligonucleotide formulation is then added to the micelles comprising the lipid component. The cationic groups on the lipids interact with the oligonucleotide and condense around the oligonucleotide to form liposomes. After condensation, the detergent is removed, for example by dialysis, to produce a liposome formulation of the oligonucleotide.

[0204] If desired, a carrier compound that facilitates condensation can be added during the condensation reaction, for example, through controlled addition. For instance, the carrier compound could be a polymer other than nucleic acids (e.g., spermine or spermidine). The pH can also be adjusted to favor condensation.

[0205] Methods for generating stable oligonucleotide delivery mediators incorporating structural components of oligonucleotide / cationic lipid complexes as delivery mediators are further described, for example, in WO 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation may also include one or more aspects of the exemplary methods described in the following: Feigner, PL et al., (1987) Proceedings of the National Academy of Sciences 8:7413-7417; U.S. Patent No. 4,897,355; U.S. Patent No. 5,171,678; Bangham et al., (1965) M. Molecular Biology 23:238; Olson et al., (1979) Acta Biochimica et Biophysica Sinica 557:9; Szoka et al., (1978) Proceedings of the National Academy of Sciences 75:4194; Mayhew et al., (1984) Acta Biochimica et Biophysica Sinica 775:169; Kim et al., (1983) Acta Biochimica et Biophysica Sinica 728:339; and Fukunaga et al., (1984) Endocrinol. 115:757. Common techniques for preparing lipid aggregates of appropriate size for use as delivery media include sonication and freeze-thaw extrusion (see, for example, Mayer et al., (1986) *Chinese Journal of Biochemistry and Biophysics* 858:161). When consistently small (50 nm to 200 nm) and relatively homogeneous aggregates are desired, microfluidics can be used (Mayhew et al., (1984) *Chinese Journal of Biochemistry and Biophysics* 775:169). These methods are readily applicable to packaging oligonucleotide formulations into liposomes.

[0206] Liposomes fall into two broad categories. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid / liposome complexes bind to the negatively charged cell surface and are internalized in endosomes. Due to the acidic pH in the endosomes, the liposomes rupture, thereby releasing their contents into the cytoplasm (Wang et al. (1987), Biochem. Biophys. Res. Commun., 147:980-985).

[0207] Liposomes trap nucleic acids rather than complexing with them. Since both nucleic acids and lipids carry similar charges, repulsion rather than complex formation occurs. However, some nucleic acids are trapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene into cultured cell layers. Expression of the exogenous gene was detected in the target cells (Zhou et al. (1992), *Journal of Controlled Release*, 19:269-274).

[0208] One major type of liposome composition comprises phospholipids other than naturally derived phosphatidylcholine. Neutral liposome compositions may be formed, for example, from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are typically formed from dimyristoyl phosphatidylglycerol, while anionic fused liposomes are primarily formed from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Yet another type is formed from phospholipids and / or mixtures of phosphatidylcholine and / or cholesterol.

[0209] Examples of other methods for introducing liposomes into cells in vitro and in vivo include U.S. Patent Nos. 5,283,185; 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Felgner, (1994) Journal of Biol. Chem. 269:2550; Nabel, (1993) Proceedings of the National Academy of Sciences 90:11307; Nabel, (1992) Human Gene Therapy 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) Journal of the European Society for Molecular Biology 11:417.

[0210] Nonionic liposome systems were also examined to determine their efficacy in drug delivery to cells, particularly systems involving nonionic surfactants and cholesterol. NOVASOME was used. TM I (Dilaurate / Cholesterol / Polyoxy-10-Stearyl Ether) and NOVASOME TMA nonionic liposome formulation of glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether was used to deliver cyclosporine A into the dermis of mouse skin. Results showed that this type of nonionic liposome system was effective in promoting the deposition of cyclosporine A into different layers of the skin (Hu et al., (1994) STP Pharma. Sci., 4(6):466).

[0211] Liposomes can also be stereostable liposomes comprising one or more specific lipids, which, compared to liposomes lacking such specific lipids, result in enhanced cycle life. An example of a stereostable liposome is one in which a portion of the vesicle-forming lipid portion of liposome (A) comprises one or more glycolipids, such as monosialotetrahexosylganglioside G. M1 (A) or (B) liposomes partially derived from one or more hydrophilic polymers, such as polyethylene glycol (PEG). While not wishing to be bound by any particular theory, it is believed in the art that, at least for spatially stable liposomes containing gangliosides, sphingomyelins, or PEG-derived lipids, the enhanced circulating half-life of these spatially stable liposomes is due to reduced uptake into cells entering the reticuloendothelial system (RES) (Allen et al., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).

[0212] Various liposomes, including one or more glycolipids, are known in the art. Papahadjopoulos et al. (Annals of the New York Academy of Sciences, (1987), 507:64) reported monosialotetrahexosylganglioside G M1 Galactocerebroside sulfate and phosphatidylinositol can improve the blood half-life of liposomes. These findings are described in Gabizon et al., Proceedings of the National Academy of Sciences, (1988), 85:6949. Both U.S. Patent Nos. 4,837,028 and WO 88 / 04924 to Allen et al. disclose liposomes comprising: (1) sphingomyelin and (2) ganglioside G. M1 Or galactocerebroside sulfate. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. WO 97 / 13499 (Lim et al.) discloses liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine.

[0213] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with cell membranes. Although non-cationic liposomes cannot fuse efficiently with the plasma membrane, they are absorbed by macrophages in vivo and can be used to deliver oligonucleotides to macrophages.

[0214] Other advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can be incorporated into a variety of water-soluble and lipid-soluble drugs; and liposomes can protect oligonucleotides encapsulated in their internal compartments from metabolism and degradation (Rosoff, “Pharmaceutical Dosage Forms”, Lieberman, Rieger, and Banker (eds.), 1988, Vol. 1, p. 245). Important considerations in the preparation of liposome formulations are lipid surface charge, vesicle size, and the aqueous volume of the liposome.

[0215] Small liposomes can be formed using the positively charged synthetic cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), which spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that are capable of fusing with negatively charged lipids in the cell membranes of tissue cultured cells to deliver oligonucleotides (see, for example, Felgner, PL et al., (1987) Proceedings of the National Academy of Sciences 8:7413-7417; and U.S. Patent No. 4,897,355, which describes DOTMA and its use with DNA).

[0216] The DOTMA analog, 1,2-bis(oleoyloxy)-3-(trimethylamino)propane (DOTAP), can be used in combination with phospholipids to form DNA complex vesicles. LIPOFECTIN TM(Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids into living tissue culture cells comprising positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form a complex. When sufficiently positively charged liposomes are used, the resulting complex also has a positive net charge. The positively charged complex prepared in this way spontaneously attaches to the negatively charged cell surface, fuses with the plasma membrane, and efficiently delivers functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylamino)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Indiana) differs from DOTMA in that the oleoyl moiety is linked by an ester bond rather than an ether bond.

[0217] Other reported cationic lipid compounds include those conjugated with multiple portions, including, for example, those conjugated with one of two types of lipids and including, for example, 5-carboxy-arginine-glycine-octadecylamide (“DOGS”). TM The compounds such as Promega, Madison, Wisconsin (Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxy-arginyl-amide (“DPPES”) are carboxy-arginine compounds (see, for example, U.S. Patent No. 5,171,678).

[0218] Another type of cationic lipid conjugate includes lipid-cholesterol derivatives (“DC-Chol”), which have been formulated into liposomes in combination with DOPE (see, Gao, X. and Huang, L., (1991) Biochemical and Biophysical Research Communications 179:280). Lipopolylysine, prepared by conjugating polylysine with DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Acta Biochimica and Biophysica Sinica 1065:8). For certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection compared to compositions containing DOTMA. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (LifeTechnology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for delivering oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0219] Liposome formulations are particularly suitable for topical application, exhibiting several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to deliver oligonucleotides to the skin. In some embodiments, liposomes are used to deliver oligonucleotides to epidermal cells and also to enhance the penetration of oligonucleotides into dermal tissues, such as the skin. For example, liposomes can be applied topically. Local delivery of drugs formulated as liposomes to the skin has been documented (see, for example, Weiner et al., (1992) *Journal of Drug Targeting*, Vol. 2, 405-410; du Plessis et al., (1992) *Antiviral Research*, 18:259-265; Mannino, RJ and Fould-Fogerite, S., (1998) *Biotechniques*, 6:682-690; Itani, T. et al., (1987) *Gene*, 56:267-276; Nicolau, C. et al. (1987) *Meth. Enzymol.*, 149:157-176; Straubinger, RM and Papahadjopoulos, D. (1983) *Enzymological Methods*). 101:512-527; Wang, CY and Huang, L., (1987) Proceedings of the National Academy of Sciences 84:7851-7855.

[0220] Nonionic liposome systems were also examined to determine their efficacy in drug delivery to cells, particularly systems comprising nonionic surfactants and cholesterol. Nonionic liposome formulations comprising Novasome I (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver drugs to the dermis of mouse skin. Such formulations containing oligonucleotides may be used to treat dermatological conditions.

[0221] The targeting of liposomes can also be achieved based on factors such as organ specificity, cell specificity, and organelle specificity, and is known in the art. In the case of a liposome-targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome to maintain stable association between the targeting ligand and the liposome bilayer. Various linker groups can be used to bind the lipid chain to the targeting ligand. Other methods are known in the art and described, for example, in U.S. Application Publication No. 20060058255, whose linker groups are incorporated herein by reference.

[0222] Liposomes containing oligonucleotides can be made highly deformable. This deformability allows the liposomes to penetrate through pores smaller than the average radius of the liposome. For example, delivery bodies are another type of liposome and are highly deformable lipid aggregates, which are attractive candidates for drug delivery mediators. Delivery bodies can be described as lipid droplets that are highly deformable, allowing them to easily penetrate through pores smaller than the droplets. Delivery bodies can be prepared by adding a surface-edge surfactant (typically a surfactant) to a standard liposome composition. Delivery bodies containing oligonucleotides can be delivered, for example, via subcutaneous infection to deliver the oligonucleotides to keratinocytes in the skin. To penetrate intact mammalian skin, lipid vesicles must pass through a series of pores less than 50 nm in diameter under the influence of a suitable transdermal gradient. Furthermore, due to their lipid properties, these delivery bodies can self-optimize (adapt to the shape of the pores, such as those in the skin), self-repair, and often reach their targets without fragmentation, and often self-load. Delivery bodies have been used to deliver serum albumin into the skin. Serum albumin delivery mediated by a serum albumin translocator has been shown to be as effective as subcutaneous injection of a serum albumin-containing solution.

[0223] Other formulations suitable for the disclosed oligonucleotides and methods are described in WO 2009 / 086558 and WO2009 / 088891. WO 2008 / 042973 also describes formulations suitable for the oligonucleotides and methods disclosed herein.

[0224] Surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the properties of many different types of surfactants (both natural and synthetic) is by using the hydrophilic / lipophilic balance (HLB). The properties of the hydrophilic group (also known as the “head”) provide the most useful way to classify the different surfactants used in formulations (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0225] If a surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants are widely used in pharmaceutical and cosmetic products and are available in a wide range of pH values. Typically, their HLB values ​​range from 2 to approximately 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglycerol esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this category. Polyoxyethylene surfactants are the most popular members of the nonionic surfactant category.

[0226] Surfactants are classified as anionic if their molecules carry a negative charge when dissolved or dispersed in water. Anionic surfactants include carboxylic acid esters, such as soaps, lactic acid esters, acylamides of amino acids, esters of sulfuric acid (such as alkyl sulfates and ethoxylated alkyl sulfates), sulfonates (such as alkyl benzenesulfonates, isothioctanoic acid acyl esters, taurine acyl esters, and succinate sulfonates), and phosphate esters. The most important members of the anionic surfactant category are alkyl sulfates and soaps.

[0227] Surfactants are classified as cationic if their molecules carry a positive charge when dissolved or dispersed in water. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this category of cationic surfactants.

[0228] Surfactants are classified as amphoteric if their molecules can carry either a positive or negative charge. Amphoteric surfactants include acrylic acid derivatives, substituted alkyl amides, N-alkyl betaines, and phospholipids.

[0229] The use of surfactants in pharmaceutical products, formulations and emulsions is reviewed (Rieger, Pharmaceutical Dosage Forms, Marcel Decker, NY, 1988, p. 285).

[0230] The oligonucleotides used in the methods described herein can also be provided in the form of micelle formulations. Micelles are a specific type of molecular assembly in which amphiphilic molecules are arranged in a globular structure such that all hydrophobic portions of the molecules face inward, thereby allowing the hydrophilic portions to come into contact with the surrounding water. If the environment is hydrophobic, the opposite arrangement exists.

[0231] ii. Lipid nanoparticle-based delivery methods The oligonucleotides described herein can be completely encapsulated in lipid formulations, such as lipid nanoparticles (LNPs) or other nucleic acid-lipid particles. LNPs are particularly useful for systemic application because they exhibit prolonged circulation life after intravenous (iv) injection and accumulate at distant sites (e.g., sites physically separate from the application site). LNPs include “pSPLP”, which comprises an encapsulated condenser-nucleic acid complex as shown in PCT Publication WO 00 / 03683. The particles of this disclosure typically have an average diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially non-toxic. Furthermore, the nucleic acids, when present in the nucleic acid-lipid particles, are resistant to degradation by nucleases in aqueous solutions. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Patent Nos. 5,976,567, 5,981,501, 6,534,484, 6,586,410, and 6,815,432; U.S. Publication No. 2010 / 0324120; and PCT Publication No. WO 96 / 40964.

[0232] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to oligonucleotide ratio) will be in the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges between these ranges are also contemplated as part of this description.

[0233] Non-limiting examples of cationic lipids include N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N,N-distearate-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-(dioleoyloxy)propylamine (DODMA), and 1,2-dilinoleoyloxy-N,N-dimethylamine. 1,2-Dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinoleoylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleoylcarbamoyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dialkylcarbamoyloxy-3-morpholinopropane (DLin-MA), 1,2-dihydrooleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleoylthio-3 -Dimethylaminopropane (DLin-S-DMA), 1-linoleyl-2-linoleyl-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dihydrooleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazine)propane (DLin-MPZ) or 3-(N,N-dioleylamino)-1,2-propanediol (DLinAP), 3-(N,N- Dioleoylamino)-1,2-propanediol (DOAP), 1,2-dioleo-oxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleo-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-DMA) or analogues thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-bis((9Z,12Z)-octadec-9,12-dienyl)tetrahydro- 3aH-cyclopenteno[d][1,3]-dioxacyclopenten-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazadiyl)docosahexadecane-2-ol (Tech G1), or mixtures thereof.Cationic lipids may comprise, for example, about 20 mol% to about 50 mol% or about 40 mol% of the total lipids present in the particles.

[0234] Ionizable / non-cationic lipids can be anionic or neutral lipids, including but not limited to distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), and palmitoylphosphatidylethanolamine (P... OPE, dioleoyl-phosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearate-phosphatidyl-ethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. Non-cationic lipids may constitute, for example, about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipids present in the particles (if cholesterol is included).

[0235] The conjugated lipids that inhibit particle aggregation can be, for example, polyethylene glycol (PEG) lipids, including but not limited to PEG-diacylglycerol (DAG), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof. PEG-DAA conjugates can be, for example, PEG-1-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dispalmityloxypropyl (Ci6), or PEG-distearateloxypropyl (Ci8). The conjugated lipids that prevent particle aggregation can comprise, for example, from 0 mol% to about 20 mol% or about 2 mol% of the total lipids present in the particles.

[0236] In some embodiments, the nucleic acid-lipid particles further include cholesterol, for example, cholesterol comprising about 10 mol% to about 60 mol% or about 50 mol% of the total lipids present in the particles.

[0237] IV. Pharmaceutical Compositions The oligonucleotides described herein are preferably formulated into pharmaceutical compositions for administration to human subjects in a biocompatible form suitable for in vivo administration.

[0238] The oligonucleotides described herein can be administered, for example, orally, parenterally, intrathecally, intraventricularly, intra-organ parenchymally, buccally, sublingually, nasally, rectally, via patch, pump, intratumorally, or percutaneously, as well as in corresponding formulated pharmaceutical compositions. Parenterical administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, intraventricular, intra-organ parenchymal, rectal, and local administration. Parenterical administration can be achieved through continuous infusion over a selected time period.

[0239] The oligonucleotides described herein can be administered orally, for example, with an inert diluent or with an assimilated edible carrier, or the oligonucleotides can be encapsulated in hard or soft-shell capsules, or the oligonucleotides can be compressed into tablets, or the oligonucleotides can be directly incorporated into dietary foods. For oral therapeutic use, the oligonucleotides described herein can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, and flakes. The oligonucleotides described herein can also be administered parenterally. Solutions of the oligonucleotides described herein can be prepared in water with a suitable surfactant, such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, DMSO, and mixtures thereof with or without alcohol, as well as in oils. Under normal storage and use conditions, these formulations may contain preservatives to prevent microbial growth. The routine procedures and ingredients for selecting and preparing suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2012, 22nd edition) and The United States Pharmacopeia: The National Formulary (USP 41 NF 36), published in mid-2018. Suitable drug forms for injectable use include sterile aqueous solutions or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid in a manner readily applicable by syringe. Compositions for nasal administration can routinely be formulated as aerosols, drops, gels, and powders. Aerosol formulations typically comprise solutions or fine suspensions of the active substance in physiologically acceptable aqueous or non-aqueous solvents and are typically presented sterilely in single- or multiple-dose amounts in sealed containers, which may be in the form of boxes or refillable for use with nebulizers. Alternatively, the sealed container can be a single dispensing device, such as a single-dose nasal inhaler or an aerosol dispenser equipped with a metering valve intended for post-use disposal. In dosage forms that include aerosol dispensers, the dispenser will contain a propellant, which can be a compressed gas, such as compressed air, or an organic propellant, such as chlorofluorocarbons. Aerosol dosage forms can also be in the form of a pump-nebulizer. Compositions suitable for buccal or sublingual administration include tablets, lozenges, and soft lozenges in which the active ingredient is formulated with a carrier (such as sugar, guar gum, gum, gelatin, and glycerin). Compositions for rectal administration are conveniently presented as suppositories containing a conventional suppository base, such as cocoa butter. The oligonucleotides described herein can also be administered intratumorally, for example, via intratumoral injection. Intratumoral injection involves direct injection into the tumor's blood vessels and is specifically envisioned for discrete, solid, and easily accessible tumors.Local, regional, or systemic application may also be appropriate.

[0240] The oligonucleotides described herein may be administered to animals, such as humans, alone or in combination with pharmaceutically acceptable carriers as mentioned herein, in proportions determined by the solubility and chemical properties of the oligonucleotides, the chosen route of administration, and standard pharmaceutical practice.

[0241] V. Dosage The dosage of the compositions described herein (e.g., compositions comprising oligonucleotides) can vary depending on many factors, such as the pharmacokinetic properties of the compound; the administration method; the recipient's age, health, and weight; the nature and severity of symptoms; the frequency of treatment and the type of concurrent treatment (if any); and the clearance of the compound in the animal to be treated. Those skilled in the art can determine an appropriate dosage based on these factors. The compositions described herein can be initially administered at a suitable dosage, which can be adjusted as needed based on clinical response. In some embodiments, the dosage of the composition (e.g., a composition comprising oligonucleotides) is a preventative or therapeutically effective amount.

[0242] VI. Reagent Kit This document provides a kit comprising (a) a pharmaceutical composition comprising an oligonucleotide that deamination adenosine in mRNA in cells or a subject described herein, and (b) a packaging insert having instructions for performing any of the methods described herein. In some embodiments, the kit comprises (a) a pharmaceutical composition comprising an oligonucleotide that deamination adenosine in mRNA in cells or a subject described herein, (b) an additional therapeutic agent, and (c) a packaging insert having instructions for performing any of the methods described herein.

[0243] Example: 1. An oligonucleotide comprising the following structure: [A m ]-X 1 -X 2 -X 3 -[B n ] in m + n is 24 to 50, n is at least 4, and m is at least 20; -X 1 -X 2 -X 3 - is the central triplet of the oligonucleotide; X 1 For the position -1, X of the oligonucleotide 2 The position of the oligonucleotide is 0, and X3 +1 to the position of the oligonucleotide; [A] m This is the first domain located at positions -(m+1) to -2 of the oligonucleotide; [B] n This is a second domain located at positions +2 to +(n+1) of the oligonucleotide; Each A and B is a nucleotide containing a nucleobase, a sugar (“A / B sugar”), and an internucleotide bond; Each X 1 X 2 and X 3 It contains nucleobases, sugars ("X sugars"), and bonds between nucleotides; The A / B sugar and the X 3 The sugars are selected from 2'-methoxy-ribose, 2'-MOE-ribose, 2'-deoxy-2'-fluororibose, 2'-fluoro-arabinose, 2-methoxy-arabinose, 2'-deoxyribose and locked nucleic acids (LNA). The X 1 The sugar is 2'-deoxy-2'-fluororibose or 2'-deoxyribose; The X 2 The sugars are selected from 2'-methoxy-ribose, 2'-MOE-ribose, 2'-deoxy-2'-fluororibose, 2'-fluoro-arabinose, 2-methoxy-arabinose, 2'-deoxyribose, locked nucleic acid (LNA), and β-homo-DNA sugars; The A / B sugar and the X sugar together constitute 10-70% 2'-deoxy-2'-fluoro-ribose; The oligonucleotide has 30%-100% thiophosphate and aminophosphate bonds between its nucleotides, and 3 to 20 nucleotide bonds are aminophosphate bonds. (i) the bond between the nucleotide at position -(m+1) and the nucleotide at position -(m) (5' end), (ii) the bond between the nucleotide at position +(n) and the nucleotide at position +(n+1) (3' end), or (iii) the bond between the nucleotides at each of the 5' and 3' ends of the oligonucleotide is an aminophosphate bond; and The nucleotide bonds between the nucleotide at position -(m) and the nucleotide at position -(m-1), and the nucleotide bonds between the nucleotide at position +(n-1) and the nucleotide at position +(n), ​​are independently thiophosphate bonds or aminophosphate bonds.

[0244] 2. The oligonucleotide according to Example 1, wherein X 2 The nucleobase is cytosine or isodU.

[0245] 3. The oligonucleotide according to Example 1 or Example 2, wherein X 3 The nucleobases are guanosine, hypoxanthine, or 7-denitroguanine.

[0246] 4. The oligonucleotide according to any one of Examples 1 to 3, wherein no more than four consecutive A / B sugars are 2'-deoxy-2'-fluororibose.

[0247] 5. The oligonucleotide according to any one of Examples 1 to 4, wherein the A / B sugar and the X sugar together are 20-50% 2'-deoxy-2'-fluororibose.

[0248] 6. The oligonucleotide according to any one of Examples 1 to 5, wherein the A / B sugar is selected from 2'-methoxy-ribose, 2'-MOE-ribose, 2'-deoxy-2'-fluororibose and 2'-deoxyribose.

[0249] 7. The oligonucleotide according to any one of Examples 1 to 6, wherein the X 2 The nucleobase is cytosine.

[0250] 8. The oligonucleotide according to any one of Examples 1 to 7, wherein the X 2 The sugar is a β-homo-DNA sugar.

[0251] 9. The oligonucleotide according to any one of Examples 1 to 7, wherein the X 2 The sugar is 2'-deoxyribose.

[0252] 10. The oligonucleotide according to any one of Examples 1 to 9, wherein the X 1 The sugar is 2'-deoxy-2'-fluororibose.

[0253] 11. The oligonucleotide according to any one of Examples 1 to 9, wherein the X 1 The sugar is 2'-deoxyribose.

[0254] 12. The oligonucleotide according to any one of Examples 1 to 11, wherein the X 3 The nucleobase is hypoxanthine.

[0255] 13. The oligonucleotide according to any one of Examples 1 to 12, wherein X 1 With X 2 The internucleotide bonds between them are thiophosphate bonds or phosphodiester bonds.

[0256] 14. The oligonucleotide according to Example 13, wherein X 1 With X 2 The nucleotide bonds between them are thiophosphates.

[0257] 15. The oligonucleotide according to any one of Examples 1 to 14, wherein X 2 With X 3 The internucleotide bonds between them are thiophosphate bonds or phosphodiester bonds.

[0258] 16. The oligonucleotide according to Example 15, wherein X 2 With X 3 The nucleotide bonds between them are thiophosphates.

[0259] 17. The oligonucleotide according to any one of Examples 1 to 16, wherein the nucleotide at position -2 is associated with X. 1 The internucleotide bonds between them are thiophosphate bonds or phosphodiester bonds.

[0260] 18. The oligonucleotide according to Example 17, wherein the nucleotide at position -2 is associated with X. 1 The nucleotide bonds between them are thiophosphates.

[0261] 19. The oligonucleotide according to any one of Examples 1 to 18, wherein the internucleotide bond between the nucleotide at position -9 and the nucleotide at position -8 is an aminophosphate.

[0262] 20. The oligonucleotide according to any one of Examples 1 to 19, wherein the internucleotide bond between the nucleotide at position -11 and the nucleotide at position -10 is an aminophosphate.

[0263] 21. The oligonucleotide according to any one of Examples 1 to 20, wherein the internucleotide bond between the nucleotide at position +1 and the nucleotide at position +2 is an aminophosphate ester.

[0264] 22. The oligonucleotide according to any one of Examples 1 to 21, wherein the internucleotide bond between the nucleotide at position +4 and the nucleotide at position +5 is an aminophosphate.

[0265] 23. The oligonucleotide according to any one of Examples 1 to 22, wherein the internucleotide bond between the nucleotide at position +5 and the nucleotide at position +6 is an aminophosphate ester.

[0266] 24. The oligonucleotide according to any one of Examples 1 to 23, wherein the internucleotide bond between the nucleotide at position +9 and the nucleotide at position +10 is an aminophosphate.

[0267] 25. The oligonucleotide according to Example 24, wherein the internucleotide bond between the nucleotide at position +1 and the nucleotide at position +2, and the internucleotide bond between the nucleotide at position +9 and the nucleotide at position +10, are aminophosphates.

[0268] 26. The oligonucleotide according to Example 24 or 25, wherein the internucleotide bond between the nucleotide at position +1 and the nucleotide at position +2, the internucleotide bond between the nucleotide at position +5 and the nucleotide at position +6, and the internucleotide bond between the nucleotide at position +9 and the nucleotide at position +10 are aminophosphates.

[0269] 27. The oligonucleotide according to any one of Examples 1 to 26, having 30-70% thiophosphate and aminophosphate bonds.

[0270] 28. The oligonucleotide according to Example 27 has 40-60% thiophosphate and aminophosphate bonds.

[0271] 29. The oligonucleotide according to any one of Examples 1 to 28, wherein n + m is 27.

[0272] 30. The oligonucleotide according to any one of Examples 1 to 28, wherein n + m is 39.

[0273] 31. The oligonucleotide according to any one of Examples 1 to 30, wherein n is 4, 5, 6, 7, 8 or 9.

[0274] 32. The oligonucleotide according to Example 31, wherein n is 4.

[0275] 33. The oligonucleotide according to Example 31, wherein n is 5.

[0276] 34. The oligonucleotide according to Example 31, wherein n is 9.

[0277] 35. The oligonucleotide according to any one of Examples 1 to 34, wherein the A / B sugar at position +3 is 2'-deoxy-2'-fluororibose.

[0278] 36. The oligonucleotide according to any one of Examples 1 to 35, wherein the A / B sugar at position -5 is 2'-deoxy-2'-fluororibose.

[0279] 37. The oligonucleotide according to any one of Examples 1 to 36, wherein the A / B sugar at position -16 is 2'-deoxy-2'-fluororibose.

[0280] 38. The oligonucleotide according to any one of Examples 1 to 37, wherein the A / B sugar at position -20 is 2'-deoxy-2'-fluororibose.

[0281] 39. The oligonucleotide according to any one of Examples 1 to 34, wherein the A / B sugar at positions -5, -16 and -20 is 2'-deoxy-2'-fluororibose.

[0282] 40. The oligonucleotide according to any one of Examples 1 to 34, wherein the A / B sugar at positions +3, -5, -16 and -20 is 2'-deoxy-2'-fluororibose.

[0283] 41. The oligonucleotide according to any one of Examples 1 to 40, having a GalNAc moiety at the 5' end.

[0284] 42. The oligonucleotide according to any one of Examples 1 to 41, having a GalNAc moiety at the 3' end.

[0285] 43. The oligonucleotide according to any one of Examples 1 to 42, wherein at least one aminophosphate is a methanesulfonyl aminophosphate.

[0286] 44. The oligonucleotide according to Example 43, wherein each aminophosphate is a methanesulfonyl aminophosphate.

[0287] 45. The oligonucleotide according to any one of Examples 1 to 44, which is sufficiently complementary to the target RNA having a target adenosine moiety and is capable of forming a complex with the target RNA.

[0288] 46. ​​The oligonucleotide according to Example 45, wherein, when forming the complex with the target RNA, the nucleotide of the oligonucleotide opposite to the target adenosine is X. 2 .

[0289] 47. The oligonucleotide according to Example 45 or 46, which is capable of binding to and recruiting the ADAR enzyme to perform editing on the target adenosine of the target RNA.

[0290] 48. The oligonucleotide according to any one of Examples 1 to 47, wherein the oligonucleotide does not contain a portion capable of forming an intramolecular stem-loop structure.

[0291] 49. The oligonucleotide according to any one of Examples 1 to 47, comprising a portion capable of forming an intramolecular stem-loop structure.

[0292] 50. A complex comprising an oligonucleotide according to any one of Examples 1 to 49 and a target RNA, the complex being formed by hybridization between the oligonucleotide and the target RNA.

[0293] 51. The complex according to Example 50, comprising one, two, three, four or five mismatches, wobbles, insertions or deletions.

[0294] 52. A method for editing a target adenosine in a target RNA in a cell, the method comprising contacting the cell with an oligonucleotide according to any one of Examples 1 to 49, to (i) form a complex between the oligonucleotide and the target RNA, such that the X of the oligonucleotide 2 In contrast to the target adenosine, and (ii) recruiting ADAR from the cells to the complex such that the ADAR edits the target adenosine.

[0295] 53. A pharmaceutical composition comprising an oligonucleotide according to any one of Examples 1 to 49 and a pharmaceutically acceptable excipient.

[0296] 54. The pharmaceutical composition according to Example 53, wherein the oligonucleotide is encapsulated in lipid nanoparticles (LNP).

[0297] Example General methods All guide oligonucleotides were chemically synthesized using standard β-cyanoethyl phosphoramide chemistry and universal solid supports, such as controlled-porosity glass (CPG), on an automated RNA / DNA synthesizer. N-protected β-homo-DNA phosphoramide was synthesized using a reported procedure. See Matheus Froeyen et al., (2001) *Chem. Eur. J.*, 7: 5183-5794; Herdewijn, (2010) *Chem. Biodivers.*, 7: 1-59; Jabgunde et al., (2019) *Tetrahedron*, 75: 1107-1114. Other 5'-O-DMT-3'-phosphoramide RNA, 2'-O-methyl RNA, and DNA monomers, namely A, C, G, U, and T, were purchased from commercial sources. All oligonucleotides were synthesized at a 200 nmol scale by BioSpring GmbH (Frankfurt, Germany). Following synthesis, the oligonucleotides were cleaved from the solid support, deprotected, and purified using standard protocols via HPLC. The oligonucleotides were desalted, dialyzed, and lyophilized. The purity of each lyophilized oligonucleotide was ≥ 95%, as determined by analytical reversed-phase HPLC. The sequence integrity of the oligonucleotides was determined by ESI-MS. (Sequences of the various oligonucleotides are provided in Tables 3 and 4).

[0298] The human ADAR2 sequence (NM_001112.4) was cloned into the pcDNA3.1 plasmid using BamHI and XbaI restriction sites (Quintara Bio, Berkeley, CA) under the control of the CMV promoter, and the correct insert was validated. This plasmid will therefore be designated ADAR2 / pcDNA3.1. For editing experiments, 25 μL of culture medium was used per 10 cm² culture dish. L of Lipofectamine 3000 and 24 L's P3000 (Life Technologies) will 2 g of ADAR2 / pcDNA3.1 plasmid was transfected into 5 x 10⁻⁶ cells. 6HEK293T cells (American Type Culture Collection (ATCC)). Four hours later, the medium was replenished with freshly heated DMEM high-glucose medium (Lifetechnologies, Inc.). 12–16 hours post-transfection, the transfected HEK293T cells were transfected with guide oligonucleotides to achieve a final concentration of 100 nM in each well. All transfections were performed using Lipofectamine 3000 (0.4 g / L). Cells were transfected in 96-well batches (L / well) according to the manufacturer's instructions. 12–16 hours after the second transfection, cells were washed once with ice-cold PBS, and total mRNA isolation was performed using the DynaBeads mRNA Direct Kit for KingFisher Flex Purification (Lifetech Corporation) according to the manufacturer's instructions. Samples were treated with TURBO DNase (Lifetech Corporation) prior to elution. The resulting isolated mRNA was used for cDNA synthesis using SuperScript IV Vilo according to the manufacturer's instructions (Lifetech Corporation). 1 µl of cDNA was used as a template for PCR using gene-specific primers (Platinum II Hot-Start PCR Master Mixture; Lifetech Corporation) to generate amplicons for Sanger sequencing. Sanger sequencing was performed by Quintiles Biosciences (Berkeley, CA). The editing yield of adenosine to guanosine was quantified by measuring the peak heights of adenosine and guanosine and dividing the guanosine peak height by the combined peak height of adenosine and guanosine.

[0299] Example 1 - Editing efficiency of 42-meric oligonucleotides with PA1 bonds relative to those without PA1 bonds Single-stranded antisense oligonucleotides (“ASOs”) were designed to measure the effect of PA1 linkers on SERPINA1 editing in mice over time. Guided oligonucleotides were chemically synthesized using standard β-cyanoethylphosphoramide chemistry and universal solid supports, such as controlled-pore glass (CPG), on an automated RNA / DNA synthesizer. 5'-O-DMT-3'-phosphoramide RNA, 2'-O-methyl-RNA, 2'-fluoro-arabinose-RNA (FANA), and DNA monomers, namely A, C, G, U, and T, were purchased from commercial sources. The PA-1 bond-containing oligonucleotides were prepared using methods known in the art, such as those described by Anderson et al. Following synthesis, the oligonucleotides were cleaved from the solid support, deprotected, and purified using standard protocols via an HPLC system. The oligonucleotides were desalted, dialyzed, and lyophilized. The purity of each lyophilized oligonucleotide was ≥90%, as determined by analytical reversed-phase HPLC. Sequence integrity of the oligonucleotides was determined by ESI-MS. Then, using MC3 as an ionizable lipid, oligonucleotides were formulated into lipid nanoparticles (“LNPs”) using microfluidic mixing.

[0300] Oligonucleotides in LNPs were tested in PiZ mice to measure SERPINA1 editing in the liver at different time points. PiZ transgenic mice with the E342K mutation were intravenously injected with LNPs containing oligonucleotides #1, #2, or #3 at a dose of 3 mg / kg. Liver was isolated from the mice on day 1, day 4, or day 7 post-injection, and total RNA was isolated. The isolated RNA was used for cDNA synthesis using SuperScript IV VILO™ according to the manufacturer's instructions (Life Technologies). 10 µl of cDNA was used for next-generation sequencing (NGS), i.e., amplicon sequencing, by Quintiles Biotechnology. The percentage of editing at the site of interest was quantified as a percentage of the number of edited nucleotides based on NGS counts. Each oligonucleotide was determined in at least three replicates. Primer sequences used for NGS are shown in Table 4 below.

[0301] Table 4. PCR and sequencing primers Guided oligonucleotides showed higher levels of editing in the presence of PA-1 bonds, especially at later time points (days 4 and 7). Figure 1 (C) These data indicate that the PA-1 bond on oligonucleotide #3 improves the durability of the in vivo response compared to the PS bond on oligonucleotide #1. On days 4 and 7, non-PA1 oligonucleotides and single-PA1-bonded oligonucleotides showed less durability. After day 4 or 7, five-PA1-bonded oligonucleotides showed approximately 2.7 times the activity of non-PA1 oligonucleotides.

[0302] Example 2 - Editing efficiency of 30-meric oligonucleotides with different 2' sugar modification patterns Guided oligonucleotides were designed to measure the effect of different 2' sugar modification patterns on in vivo SERPINA1 editing. The 2' modification patterns and PS content varied among three oligonucleotides (i.e., oligonucleotides #4, #5, and #6), while the length and number of PA1 bonds remained constant. ASOs of 30 nucleotides in length containing five PA1 bonds were synthesized and formulated into LNPs as described in Example 1. The tests and subsequent analyses performed in mice were substantially the same as those in Example 1 above. Editing was measured on days 1 and 4.

[0303] When a specific 2' modification pattern is used on oligonucleotide #6, the guide oligonucleotide with a PA1 bond shows an increased level of editing. Figure 2 B). Oligonucleotides with the same length and number of PA1 bonds (oligonucleotides #4 and #5) showed lower editing, demonstrating the importance of 2'F modification. 2'F modification plays a crucial role compared to PS bonds. The pattern of 2'F modification increased in vivo potency by approximately 2x (5 vs. 6). The percentage of 2'F or PS content had no effect on increasing activity (4 vs. 5).

[0304] Example 3 - Editing efficiency of 30-meric oligonucleotides with PA1 bonds relative to PS bonds The guide oligonucleotide was designed to measure the effect of PA-1 bond addition on editing. The guide oligonucleotide was synthesized in the same manner as in Example 1. The editing efficiency of the oligonucleotide in primary cynomolgus (“cyno”) hepatocytes was tested in the absence of interferon-α. These were tested using RNAiMAX at 10 nM and 100 nM, and at 100 nM and 1000 nM without any lipofectamine.

[0305] Hepatocytes from cynomolgus monkeys were thawed in 50 ml tubes containing Universal Cryopreservation Restore Medium (UCRM - Discovery Life Sciences) at 37°C. After centrifugation at 100 xg for 5 min, the supernatant was aspirated, and the cell pellet was resuspended in Universal Primary Cell Plating Medium (Discovery Life Sciences). Cells were plated at 40,000 cells / well or 10,000 cells / well onto 96-well or 384-well collagen-coated tissue culture plates, respectively. Cells were transferred to an incubator (37°C) and, after 4 to 6 hours, the medium was replaced with hepatocyte induction medium (Discovery Life Sciences), and the cells were transfected with ASO at the desired concentration, with or without RNAiMax (Life Technologies, California), according to the manufacturer's protocol, and then returned to the incubator.

[0306] Forty-eight hours after the addition of oligonucleotides, mRNA was isolated from cynomolgus hepatocytes using Oligo(dT)25 magnetic beads and relevant buffers from New England BioLabs. Following elution, the samples were processed with EZ DNase (Lifetech Corporation). The resulting isolated mRNA was used for cDNA synthesis using SuperScript IV VILO™ according to the manufacturer's instructions (Lifetech Corporation). 10 µl of cDNA was used for next-generation sequencing (NGS), i.e., amplicon sequencing, by Quintiles Biotechnology.

[0307] DNA amplicon was used directly for amplicon next-generation sequencing (NGS). The percentage of editing at the site of interest was quantified as a percentage of the number of edited nucleotides based on the NGS count. Each oligonucleotide was determined in at least three replicates. Primer sequences used for NGS are shown in Table 5 below.

[0308] Table 5. PCR and sequencing primers Guide oligonucleotides containing PA-1 bonds exhibited high editing efficiency through both transfection (“Lipo”) and free uptake in the absence of transfection reagent (“FU”). Figure 3 B). When the PA-1 bond was incorporated into the oligonucleotide, a 16-fold increase in editing was observed via free uptake. The activity of PA1 in the 30-mer ACTB oligonucleotide (oligonucleotide #7) was approximately 16x greater than that in the fully modified PS oligonucleotide (oligonucleotide #10).

[0309] Example 4 - Additions and subtractions to PA-1 demonstrate the positional importance of the PA-1 bond. Oligonucleotides are engineered to contain the addition or removal of a single PA-1 bond at each position of the guide oligonucleotide. Figure 4 A, Figure 5 A). Then, the editing changes of oligonucleotides at different positions of PA-1 are measured to determine where the editing increases or decreases. Oligonucleotide synthesis is performed as described in Example 1.

[0310] The efficiency of oligonucleotide editing in Piz mouse hepatocytes was tested in the absence of interferon-alpha. These were tested using RNAiMAX and at desired concentrations without lipofectamine. Piz mouse hepatocytes were thawed in 50 ml tubes containing cryopreserved hepatocyte recovery medium (CHRM-Life Technologies) at 37°C. After centrifugation at 80 xg for 6 min, the supernatant was aspirated, and the cell pellet was resuspended in hepatocyte plating medium (MB Bioscience). Cells were plated at 20,000 cells / well onto 96-well collagen-coated tissue culture plates. Cells were transferred to an incubator (37°C) and, after 4 to 6 hours, the medium was replaced with hepatocyte maintenance medium (MB Bioscience), and cells were transfected with ASO at the desired concentration with or without RNAiMAX (Life Technologies, California), according to the manufacturer's protocol, and then returned to the incubator.

[0311] Forty-eight hours after the addition of oligonucleotides, mRNA was isolated from PiZ hepatocytes using Oligo(dT)25 magnetic beads and relevant buffer from New England Biolabs. Following elution, the samples were processed with EZ DNase (Lifetech Corporation). The resulting isolated mRNA was used for cDNA synthesis using SuperScript IV VILO™ according to the manufacturer's instructions (Lifetech Corporation). 10 µl of cDNA was used for next-generation sequencing (NGS), i.e., amplicon sequencing, by Quintiles Biotechnology.

[0312] DNA amplicon was used directly for amplicon next-generation sequencing (NGS). The percentage of editing at the site of interest was quantified based on NGS counts, as a percentage of the number of edited nucleotides. Each oligonucleotide was measured in at least three replicates. The primers used for sequencing were the same as described in Example 1.

[0313] Guide oligonucleotides containing PA-1 bonds at positions -23 to -5 showed similar editing, suggesting that PA-1 bonds far from the triplet are well-tolerated. Figure 4BC). However, compared to the same oligonucleotides without PA-1 at these positions, PA-1 bonds closer to the triplet (positions -4 to +4) showed reduced editing (BC). Figure 4 BC). When the PA-1 bond is removed from oligonucleotide #15 and replaced with a PS bond, editing is reduced when the bond is located at either end of the molecule. Figure 5 (AB). These results demonstrate the importance of having PA-1 bonds at both ends of the molecule.

[0314] Example 5 - The role of the PA-1 key in editing efficiency Oligonucleotides are engineered to contain the addition or removal of a single PA-1 bond at each position of the guide oligonucleotide. Figure 6 Similar to Example 4, but using 42-mer oligonucleotides instead of 30-mer oligonucleotides, with the PA-1 bond substituted at each position of the oligonucleotide. The oligonucleotide synthesis was performed as described in Example 1.

[0315] The editing efficiency of ASO was measured in α1-antitrypsin disease ZZ hepatocyte-like cells (ZZ HLC; DefiniGEN) at doses of 1 U / µL interferon α at 1 nM, 10 nM, and 100 nM. ZZ HLCs were thawed in 50 mL tubes containing complete Def-Hep thawing medium (DTM; DefiniGEN) prepared according to the manufacturer's instructions at 37°C. After centrifugation at 100 xg for 5 min, the supernatant was aspirated, and the cell pellet was resuspended in Def-Hep recovery and maintenance medium (DefiniGEN) containing 1 U / mL Rock inhibitor (Selleckchem) according to the manufacturer's instructions. ZZ HLCs were seeded at 15,000 cells / well onto 384-well tissue culture-treated plates. Cells were transferred to a hypoxic incubator (37°C, 5% CO2, 6% O2) and fed again every 48-72 hours at 40 µL DTM per well for 12-14 days.

[0316] On day 12, day 13, or day 14, ZZ HLCs were transfected with ASO at the desired concentration using RNAiMax (Life Technologies, California) according to the manufacturer's protocol and then returned to the hypoxic incubator. Further treatment was performed as described in Example 4.

[0317] The editing of all oligonucleotides was observed to be largely consistent, suggesting that the PA-1 bond is well tolerated in this model. Figure 6 When in X 2 Position and X 3Increased editing was observed when PA-1 bonds were used at the +7 and +8 positions. At 10 nM, the editing of oligonucleotide #111 was significantly greater than that of oligonucleotide #3. At both 100 nM and M10 nM doses, the editing of oligonucleotide #111 was significantly greater than that of oligonucleotide #3.

[0318] Next, targeting another specific target, mouse ACTB, the effect of PA-1 addition on two oligonucleotides of different lengths was tested. Oligonucleotides with or without five PA-1 bonds, ranging in length from 42 to 30 nucleotides, were synthesized. Figure 7 A). Perform oligonucleotide synthesis as described in Example 1.

[0319] Editing was measured in primary mouse hepatocytes. Mouse hepatocytes were thawed in 50 ml tubes containing Lonza rodent cryopreserved hepatocyte thawing medium (MCRT50 - Lonza) at 37°C. After centrifugation at 100 xg for 5 min, the supernatant was aspirated, and the cell pellet was resuspended in Lonza plating medium (Lonza). Cells were plated at 20,000 cells / well on 96-well collagen-coated tissue culture plates. Cells were transferred to an incubator (37°C) and, after 4 to 6 hours, the medium was replaced with Lonza maintenance medium (CC-3198, LonzA), and the cells were transfected with ASO at the desired concentration with or without RNAiMax (Life Technologies, California) according to the manufacturer's protocol, and the cells were returned to the incubator. Subsequent treatment was the same as described in Example 3. Primers used for NGS are shown in Table 6 below.

[0320] Table 6. PCR and sequencing primers Similar to other targets, increased editing of oligonucleotides containing PA-1 was observed. Figure 7 B). When PA-1 was included only near the 3' and 5' ends of the 42-mer oligonucleotide molecule, a decrease in editing was observed relative to oligonucleotides with five PA-1 bonds, but still higher than editing of oligonucleotides without PA-1. Transfection editing in Piz mouse hepatocytes (PMH) was also increased for oligonucleotides containing PA-1 bonds. Figure 10 ).

[0321] Example 6 - Oligonucleotides with one or more 2' modifications and their related effects on editing efficiency The effect of 2' modification of the 30-meric oligonucleotide was determined by replacing each 2'F with 2'OMe. Figure 8A). The PA-1 bond and length remain constant in all oligonucleotides. Oligonucleotide synthesis was performed as described in Example 1. Editing was measured in PiZ mouse hepatocytes by free uptake and transfection as described in Example 4.

[0322] Several residues demonstrate the importance of 2' modification. Reduced editing was observed when 2'F-ribose was replaced with 2'OMe-ribose at positions -20, -16, -5, and +3. Some of the top editors (oligonucleotides #63, #64, #67, #74, #75, #81, and #82) replaced 2'MOE with 2'Ome. Other editors (oligonucleotides #66 and #71) broke the 2'F cluster.

[0323] Example 7 - Editing of GalNAc-modified oligonucleotides Oligonucleotides containing trianthopteric GalNAc were tested in C57BL / 6 wild-type mice to measure ACTB editing in the liver seven days after the last administration. Mice were subcutaneously injected with the oligonucleotides at a dose of 10 mg / kg daily for five consecutive days (10 mg / kg once daily x 5). Seven days after the final injection, the liver was isolated from the mice, and total RNA was isolated. The isolated RNA was used for cDNA synthesis using SuperScript IV VILO™ according to the manufacturer's instructions (Life Technologies). 10 µl of cDNA was used for next-generation sequencing (NGS), i.e., amplicon sequencing, by Quintiles Biotechnology. The percentage of editing at the site of interest was quantified as a percentage of the number of edited nucleotides based on the NGS count. Each oligonucleotide was determined in at least three replicates. Primer sequences used for NGS are shown in Table 6 below.

[0324] For PA-1 oligonucleotides containing trianthraquinone GalNAc modification ( Figure 11 In both 30-mer oligonucleotides and 42-mer oligonucleotides, editing of more than 50% was observed.

[0325] Example 8 - In vitro editing of UGP2 Editing was measured in primary mouse hepatocytes. Mouse hepatocytes were thawed in 50 ml tubes containing Lonza rodent cryopreserved hepatocyte thawing medium (MCRT50 - Lonza) at 37°C. After centrifugation at 100 xg for 5 min, the supernatant was aspirated, and the cell pellet was resuspended in Lonza plating medium (Lonza). Cells were plated at 20,000 cells / well on 96-well collagen-coated tissue culture plates. Cells were transferred to an incubator (37°C) and, after 4 to 6 hours, the medium was replaced with Lonza maintenance medium (CC-3198, LonzA), and the cells were transfected with ASO at the desired concentration, with or without RNAiMax (Thermo Fisher Scientific, California), according to the manufacturer's protocol, and the cells were returned to the incubator. Subsequent treatment was the same as described in Example 3. Primers used for NGS are shown in Table 7 below.

[0326] Table 7. PCR and sequencing primers like Figure 12 As shown, under all conditions, the PA-1 / PS / PO variant of the 42-mer oligonucleotide #198 exhibited the highest experimental editing. Specifically, the presence of PA-1 at the +5 position appears to be particularly beneficial in this chemotype (see, for example, oligonucleotides 203 and 204). Optimal editing was observed for oligonucleotides with at least five PA-1 bonds, and additional PA-1 bonds did not appear to reduce editing. Furthermore, editing was generally higher in oligonucleotides containing 75% PS than in other oligonucleotides.

[0327] Example 9 - In vitro editing of NRF2 Editing was measured in the human hepatocellular carcinoma line Hep3B. Hep3B cells were grown in complete MEM medium (minimum necessary medium with 10% fetal bovine serum (Thermo Fisher Scientific, California)). On the day of transfection, Hep3B cells were trypsinized and washed once in complete MEM medium. Cells were then reverse stained with oligonucleotides by adding 12,000 cells / well of oligonucleotides to a solution of RNAiMax (Thermo Fisher Scientific, California) on 384-well collagen-coated tissue culture plates. Cells were then transfected at 37°C for 48 hours. Subsequent treatment was the same as described in Example 3. Primers used for NGS are shown in Table 8 below.

[0328] Table 8. PCR and sequencing primers For variants of KB021602, oligonucleotides with two or more PA-1s (oligonucleotide #218, oligonucleotide #219, and oligonucleotide #220) showed a significant reduction in editing. At all tested doses, the reduction in editing for oligonucleotides with 5x PA-1 (oligonucleotide #218), 3x PA-1 (oligonucleotide #219), and 2x PA-1 (oligonucleotide #220) was similar to the reduction in editing for oligonucleotide #217. For a single modification change compared to oligonucleotide #217, a single addition of PA-1 at the 5' or 3' end also reduced editing at a 5 nM dose, with the single addition of PA-1 at the +1 position having the least effect on editing. See also Figure 13 .

[0329] Example 10 - In Vitro Editing of A1AT As described in Example 4, oligonucleotides were tested in vitro in PiZ hepatocytes.

[0330] like Figure 14 As shown, oligonucleotides containing PA-1 bonds exhibited a significant increase in editing via free uptake, and editing via transfection was similar to that of oligonucleotides containing only PS.

[0331] Perform filtering to determine the optimal PA-1 placement for A1AT editing ( Figure 15 PA-1 was found to be located as a flexible 5' triplet, with the best-performing oligonucleotides containing internal PA-1 at positions -21, -16, -9, and +1. The combination of the 2' F sugar at -20 and -19 with PA-1 at -21 produced the highest editing (…). Figure 14 ).

[0332] A time-dependent study was conducted in mice on the intravenous administration of PA-1 oligonucleotides in the form of LNP formulations. At all time points, the PA-1-containing oligonucleotides showed improved editing compared to oligonucleotides lacking the PA-1 bond.

Claims

1. An oligonucleotide comprising the following structure: [A m ]-X 1 -X 2 -X 3 -[B n ] in m + n is 24 to 50, n is at least 4, and m is at least 20; -X 1 -X 2 -X 3 - is the central triplet of the oligonucleotide; X 1 For the position -1, X of the oligonucleotide 2 The position of the oligonucleotide is 0, and X 3 +1 to the position of the oligonucleotide; [A] m This is the first domain located at positions -(m+1) to -2 of the oligonucleotide; [B] n This is a second domain located at positions +2 to +(n+1) of the oligonucleotide; Each A and B is a nucleotide containing a nucleobase, a sugar ("A / B sugar"), and an internucleotide bond; Each X 1 X 2 and X 3 It contains nucleobases, sugars ("X sugars"), and bonds between nucleotides; The A / B sugar and the X 3 The sugars are selected from 2'-methoxy-ribose, 2'-MOE-ribose, 2'-deoxy-2'-fluororibose, 2'-fluoro-arabinose, 2-methoxy-arabinose, 2'-deoxyribose and locked nucleic acids (LNA). The X 1 The sugar is 2'-deoxy-2'-fluororibose or 2'-deoxyribose; The X 2 The sugars are selected from 2'-methoxy-ribose, 2'-MOE-ribose, 2'-deoxy-2'-fluororibose, 2'-fluoro-arabinose, 2-methoxy-arabinose, 2'-deoxyribose, locked nucleic acid (LNA), and β-homo-DNA sugars; The A / B sugar and the X sugar together constitute 10-70% 2'-deoxy-2'-fluororibose; The oligonucleotide has 30%-100% thiophosphate and aminophosphate bonds between its nucleotides, and 3 to 20 nucleotide bonds are aminophosphate bonds. (i) the bond between the nucleotide at position -(m+1) and the nucleotide at position -(m) (5' end), (ii) the bond between the nucleotide at position +(n) and the nucleotide at position +(n+1) (3' end), or (iii) the bond between the nucleotides at each of the 5' and 3' ends of the oligonucleotide is an aminophosphate bond; and The nucleotide bonds between the nucleotide at position -(m) and the nucleotide at position -(m-1), and the nucleotide bonds between the nucleotide at position +(n-1) and the nucleotide at position +(n), ​​are independently thiophosphate bonds or aminophosphate bonds.

2. The oligonucleotide of claim 1, wherein the X 2 The nucleobase is cytosine or isodU.

3. The oligonucleotide according to claim 1 or claim 2, wherein the X 3 The nucleobases are guanosine, hypoxanthine, or 7-denitroguanine.

4. The oligonucleotide according to any one of claims 1 to 3, wherein the not more than four consecutive A / B sugars are 2'-deoxy-2'-fluororibose.

5. The oligonucleotide according to any one of claims 1 to 4, wherein the A / B sugar and the X sugar together constitute 20-50% 2'-deoxy-2'-fluororibose.

6. The oligonucleotide according to any one of claims 1 to 5, wherein the A / B sugar is selected from 2'-methoxy-ribose, 2'-MOE-ribose, 2'-deoxy-2'-fluororibose and 2'-deoxyribose.

7. The oligonucleotide according to any one of claims 1 to 6, wherein the X 2 The nucleobase is cytosine.

8. The oligonucleotide according to any one of claims 1 to 7, wherein the X 2 The sugar is a β-homo-DNA sugar.

9. The oligonucleotide according to any one of claims 1 to 7, wherein the X 2 The sugar is 2'-deoxyribose.

10. The oligonucleotide according to any one of claims 1 to 9, wherein the X 1 The sugar is 2'-deoxy-2'-fluororibose.

11. The oligonucleotide according to any one of claims 1 to 9, wherein the X 1 The sugar is 2'-deoxyribose.

12. The oligonucleotide according to any one of claims 1 to 11, wherein the X 3 The nucleobase is hypoxanthine.

13. The oligonucleotide according to any one of claims 1 to 12, wherein X 1 With X 2 The internucleotide bonds between them are thiophosphate bonds or phosphodiester bonds.

14. The oligonucleotide of claim 13, wherein X 1 With X 2 The nucleotide bonds between them are thiophosphates.

15. The oligonucleotide according to any one of claims 1 to 14, wherein X 2 With X 3 The internucleotide bonds between them are thiophosphate bonds or phosphodiester bonds.

16. The oligonucleotide of claim 15, wherein X 2 With X 3 The nucleotide bonds between them are thiophosphates.

17. The oligonucleotide according to any one of claims 1 to 16, wherein the nucleotide at position -2 is associated with X. 1 The internucleotide bonds between them are thiophosphate bonds or phosphodiester bonds.

18. The oligonucleotide of claim 17, wherein the nucleotide at position -2 is associated with X. 1 The nucleotide bonds between them are thiophosphates.

19. The oligonucleotide according to any one of claims 1 to 18, wherein the internucleotide bond between the nucleotide at position -9 and the nucleotide at position -8 is an aminophosphate.

20. The oligonucleotide according to any one of claims 1 to 19, wherein the internucleotide bond between the nucleotide at position -11 and the nucleotide at position -10 is an aminophosphate.

21. The oligonucleotide according to any one of claims 1 to 20, wherein the internucleotide bond between the nucleotide at position +1 and the nucleotide at position +2 is an aminophosphate.

22. The oligonucleotide according to any one of claims 1 to 21, wherein the internucleotide bond between the nucleotide at position +4 and the nucleotide at position +5 is an aminophosphate.

23. The oligonucleotide according to any one of claims 1 to 22, wherein the internucleotide bond between the nucleotide at position +5 and the nucleotide at position +6 is an aminophosphate.

24. The oligonucleotide according to any one of claims 1 to 23, wherein the internucleotide bond between the nucleotide at position +9 and the nucleotide at position +10 is an aminophosphate.

25. The oligonucleotide of claim 24, wherein the internucleotide bond between the nucleotide at position +1 and the nucleotide at position +2, and the internucleotide bond between the nucleotide at position +9 and the nucleotide at position +10, are aminophosphates.

26. The oligonucleotide according to claim 24 or 25, wherein the internucleotide bond between the nucleotide at position +1 and the nucleotide at position +2, the internucleotide bond between the nucleotide at position +5 and the nucleotide at position +6, and the internucleotide bond between the nucleotide at position +9 and the nucleotide at position +10 are aminophosphates.

27. The oligonucleotide according to any one of claims 1 to 26, having 30-70% thiophosphate and aminophosphate bonds.

28. The oligonucleotide according to claim 27, having 40-60% thiophosphate and aminophosphate bonds.

29. The oligonucleotide according to any one of claims 1 to 28, wherein n + m is 27.

30. The oligonucleotide according to any one of claims 1 to 28, wherein n + m is 39.

31. The oligonucleotide according to any one of claims 1 to 30, wherein n is 4, 5, 6, 7, 8 or 9.

32. The oligonucleotide according to claim 31, wherein n is 4.

33. The oligonucleotide according to claim 31, wherein n is 5.

34. The oligonucleotide of claim 31, wherein n is 9.

35. The oligonucleotide according to any one of claims 1 to 34, wherein the A / B sugar at position +3 is 2'-deoxy-2'-fluororibose.

36. The oligonucleotide according to any one of claims 1 to 35, wherein the A / B sugar at position -5 is 2'-deoxy-2'-fluororibose.

37. The oligonucleotide according to any one of claims 1 to 36, wherein the A / B sugar at position -16 is 2'-deoxy-2'-fluororibose.

38. The oligonucleotide according to any one of claims 1 to 37, wherein the A / B sugar at position -20 is 2'-deoxy-2'-fluororibose.

39. The oligonucleotide according to any one of claims 1 to 34, wherein the A / B sugar at each of positions -5, -16 and -20 is 2'-deoxy-2'-fluororibose.

40. The oligonucleotide according to any one of claims 1 to 34, wherein the A / B sugar at each of positions +3, -5, -16 and -20 is 2'-deoxy-2'-fluororibose.

41. The oligonucleotide according to any one of claims 1 to 40, wherein it has a GalNAc moiety at the 5' end.

42. The oligonucleotide according to any one of claims 1 to 41, wherein it has a GalNAc moiety at the 3' end.

43. The oligonucleotide according to any one of claims 1 to 42, wherein at least one aminophosphate is a methanesulfonyl aminophosphate.

44. The oligonucleotide of claim 43, wherein each aminophosphate is a methanesulfonyl aminophosphate.

45. The oligonucleotide according to any one of claims 1 to 44, which is sufficiently complementary to the target RNA having a target adenosine moiety and is capable of forming a complex with said target RNA.

46. ​​The oligonucleotide of claim 45, wherein, when forming the complex with the target RNA, the nucleotide of the oligonucleotide opposite to the target adenosine is X. 2 .

47. The oligonucleotide of claim 45 or 46, which is capable of binding to and recruiting the ADAR enzyme to perform editing on the target adenosine of the target RNA.

48. The oligonucleotide according to any one of claims 1 to 47, wherein the oligonucleotide does not contain a portion capable of forming an intramolecular stem-loop structure.

49. The oligonucleotide according to any one of claims 1 to 47, comprising a portion capable of forming an intramolecular stem-loop structure.

50. A complex comprising an oligonucleotide according to any one of claims 1 to 49 and a target RNA, the complex being formed by hybridization between the oligonucleotide and the target RNA.

51. The complex of claim 50, comprising one, two, three, four, or five mismatches, wobbles, insertions, or deletions.

52. A method for editing a target adenosine in a target RNA in a cell, the method comprising contacting the cell with an oligonucleotide according to any one of claims 1 to 49, to (i) form a complex between the oligonucleotide and the target RNA, such that the X of the oligonucleotide 2 In contrast to the target adenosine, and (ii) recruiting ADAR from the cells to the complex such that the ADAR edits the target adenosine.

53. A pharmaceutical composition comprising an oligonucleotide according to any one of claims 1 to 49 and a pharmaceutically acceptable excipient.

54. The pharmaceutical composition of claim 53, wherein the oligonucleotide is encapsulated in lipid nanoparticles (LNP).

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