Oligonucleotide editing

Abstract

The present invention relates to oligonucleotides for editing target nucleic acids, as well as conjugates, salts and pharmaceutical compositions thereof. The present invention also relates to the use of such oligonucleotides, conjugates, salts and pharmaceutical compositions in methods for editing target nucleic acids and in medical uses and methods for treating diseases.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to oligonucleotides for editing target nucleic acids, as well as conjugates, salts and pharmaceutical compositions thereof. The present invention also relates to methods for editing target nucleic acids and the use of such oligonucleotides, conjugates, salts and pharmaceutical compositions in medical uses and methods for treating diseases. [Background technology]

[0002] background Nucleic acid editing has the potential to treat or prevent disease by altering or removing gene sequences that produce dysfunctional gene products that result in disease phenotypes.

[0003] RNA sequences, particularly mRNA sequences that encode proteins, can be edited using adenosine deaminase acting on RNA (ADAR) enzymes. ADAR binds to double-stranded RNA and deaminates adenine nucleobases to form hypoxanthine nucleobases, thereby converting adenosine (A) nucleosides to inosine (I) nucleosides. Inosine nucleosides are structurally similar to guanosine (G) and form complementary base pairs with cytidine (C). Therefore, inosine nucleosides are "read" as guanosine (G) nucleosides by the cellular translation machinery. Therefore, editing adenosine to inosine codons in an mRNA sequence can change the amino acid sequence and, ultimately, the function of the protein encoded by the mRNA. ADARs can also be used to edit non-coding sequences. Three ADAR genes (ADAR1, ADAR2, and ADAR3) have been identified in mammals (Nishikura 2016 Nat Rev Mol Cell Bio 17 83-96 (Non-Patent Document 1)).

[0004] ADAR can be recruited to single-stranded nucleic acids such as mRNA by a guide oligonucleotide that forms complementary base pairs with the target nucleic acid, thereby forming a double-stranded molecule that ADAR can bind to.Therefore, the sequence of the guide oligonucleotide determines the site(s) at which ADAR acts, i.e., the adenosine that ADAR can convert to inosine.Therefore, endogenous ADAR can be induced to convert specific adenosine to inosine by introducing a guide oligonucleotide of a specific sequence into cells.In therapeutic situations, the use of endogenous ADAR advantageously avoids the ectopic expression of exogenous proteins (Merkle et al.2019 Nat Biotech 37 133-138; Qu et al.Nat Biotech 37 1059-1069 (Non-Patent Document 2)).

[0005] Alpha-1 antitrypsin (A1AT) deficiency (A1AD) is a disease that may be treatable using RNA editing technology. A1AD is associated with mutations in the SERPINA1 gene. SERPINA1 encodes A1AT, a serine protease inhibitor synthesized in the liver and released into other tissues to protect against endogenous inflammatory serine proteases such as neutrophil elastase. Subjects with A1AD express reduced levels of A1AT, which can lead to excessive breakdown of elastin in the lungs, thereby reducing lung elasticity and related health problems such as emphysema. Accumulation of misfolded A1AT in the liver can also lead to liver-related problems such as cirrhosis and jaundice. The most severe form of A1AD is associated with a single base pair substitution resulting in an E342 to K mutation (E342K mutation) in A1AT. Less severe A1AD is associated with a single base pair substitution resulting in an E264 to V mutation in A1AT (E264V mutation). SERPINA1 mRNA in cultured cells has been targeted using RNA editing techniques to attempt to correct the E342K mutation (see WO 2021 / 071858 and WO 2021 / 243023). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2021 / 071858 [Patent Document 2] International Publication No. 2021 / 243023 [Non-patent literature]

[0007] [Non-Patent Document 1] Nishikura 2016 Nat Rev Mol Cell Bio 17 83-96 [Non-patent document 2] Merkle et al.2019 Nat Biotech 37 133-138;Qu et al.Nat Biotech 37 1059-1069 Summary of the Invention

[0008] The present invention relates to oligonucleotides that provide ADAR-mediated editing of target nucleic acids, such as RNA. The oligonucleotides of the present invention comprise a mixmer structure.

[0009] The present invention provides an oligonucleotide comprising an editing region comprising an editing nucleoside, a 5' mixmer region located 5' to the editing region, and a 3' mixmer region located 3' to the editing region.

[0010] The present invention also provides an oligonucleotide conjugate comprising an oligonucleotide of the invention covalently attached to at least one conjugate moiety.

[0011] The present invention also provides a pharmaceutical composition comprising an oligonucleotide or oligonucleotide conjugate of the invention and a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0012] The present invention also provides in vitro and in vivo methods for editing a target nucleic acid in a target cell, comprising administering to the target cell an effective amount of an oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the present invention.

[0013] The present invention also provides a method for treating or preventing a disease, comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the present invention to a subject suffering from or susceptible to the disease. The present invention also provides an oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the present invention for use in treating or preventing a disease in a subject. The present invention also provides use of an oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the present invention for the preparation of a medicament for treating or preventing a disease in a subject. In a preferred embodiment, the disease is A1AD. [Brief explanation of the drawings]

[0014] [Figure 1] Degradation over time of oligonucleotides containing mixmer regions of alternating 2'-O-methyl-RNA and 2'-fluoro-RNA nucleosides (CMP nos. 3_3 and 91_1) and an oligonucleotide containing a region of RNA nucleosides (CMP no. 91_2) is shown, where the oligonucleotides are incubated in fetal bovine serum (FBS), cerebrospinal fluid (CSF), or lysosomes isolated from rat liver (rat liver Tritosomes - Trit). [Figure 2] The editing efficiency of oligonucleotides containing mixmer or non-mixmer regions on GAPDH mRNA (A) and SERPINA1 mRNA (B) is shown. [Figure 3] The editing efficiency of oligonucleotides containing mixmer or non-mixmer region SERPINA1 mRNA (A and B) is shown. [Figure 4]The editing efficiency of an oligonucleotide containing a guanosine (G) nucleoside at position -1 (CMP number 2_1) and an oligonucleotide containing an inosine (I) nucleoside at position -1 (CMP number 3_1) on SERPINA1 mRNA is compared. [Figure 5] The editing efficiency of an oligonucleotide containing a DNA cytidine (C) nucleoside at position 0 (CMP number 91_3) and an oligonucleotide containing a DNA nucleoside with a Benner base at position 0 (CMP number 93_1) on GAPDH mRNA is compared. [Figure 6-1] Figure 6 shows the editing efficiency of SERPINA1 mRNA using oligonucleotides with different symmetries, specifically oligonucleotides with different numbers of nucleosides 3' to the editing nucleoside (A and B) and 5' to the editing nucleoside (C). The effect of inverting the symmetry of the oligonucleotide was also tested (D). [Figure 6-2] See description of Figure 6-1. [Figure 7] 1 shows the editing efficiency of oligonucleotides with different symmetries on SERPINA1 mRNA. [Figure 8] The editing efficiency of oligonucleotides with different symmetries on GAPDH mRNA is shown (A and B). [Figure 9] 1 shows the editing efficiency of oligonucleotides with different symmetries on GAPDH mRNA. [Figure 10-1] FIG. 10 shows the editing efficiency of oligonucleotides with different symmetries on mHprt mRNA, specifically oligonucleotides with different numbers of nucleosides 3′ (A) to the editing nucleoside and 5′ (B) to the editing nucleoside. [Figure 10-2] See description of Figure 10-1. [Figure 11] 1 shows the editing efficiency of oligonucleotides with different modifications to the editing nucleoside on SERPINA1 mRNA. [Figure 12]1 shows the editing efficiency of oligonucleotides with different modifications to the editing nucleoside on SERPINA1 mRNA. [Figure 13] 1 shows the editing efficiency of oligonucleotides with different nucleic acid base and sugar moiety modifications for the editing nucleoside on GAPDH mRNA. [Figure 14] 1 shows the editing efficiency of oligonucleotides with different nucleobase and sugar moiety modifications for editing nucleosides expressing exogenous human ADAR1 p110 on GAPDH mRNA. [Figure 15] 1 shows the editing efficiency of oligonucleotides with different nucleobase and sugar moiety modifications on the editing nucleoside for GAPDH mRNA in HEK293 cells expressing exogenous human ADAR1 p150. [Figure 16] 1 shows the editing efficiency of oligonucleotides with different nucleobase and sugar moiety modifications on the editing nucleoside for GAPDH mRNA in HEK293 cells expressing exogenous human ADAR2. [Figure 17] 1 shows the editing efficiency of oligonucleotides with different nucleobase and sugar moiety modifications in the editing triplet on GAPDH mRNA in HEK293 cells expressing exogenous human ADAR2 (A) and exogenous human ADAR1 p110 (B). [Figure 18] 1 shows the editing efficiency of oligonucleotides with different nucleobase modifications in the editing triplet on mHprt mRNA. [Figure 19] 1 shows the editing efficiency of oligonucleotides containing different numbers of phosphorothioate (PS) internucleoside linkages on GAPDH mRNA. [Figure 20] Figure 1 shows the editing efficiency of oligonucleotides in which all internucleoside linkages are phosphorothioate (PS) internucleoside linkages except for phosphodiester (PO) linkages at the indicated positions on GAPDH mRNA in cells expressing human ADAR2 (A) and human ADAR1 p110 (B). [Figure 21]Figure 1 shows the editing efficiency of oligonucleotides in which all internucleoside linkages are phosphorothioate (PS) internucleoside linkages except for phosphodiester (PO) linkages at the indicated positions on GAPDH mRNA in cells expressing human ADAR2 (A) and human ADAR1 p110 (B). [Figure 22] 1 shows the editing efficiency of oligonucleotides in which all internucleoside linkages are phosphorothioate (PS) internucleoside linkages except for phosphodiester (PO) linkages at the indicated positions on GAPDH mRNA in Huh7 cells. [Figure 23] 1 shows the editing efficiency of oligonucleotides in which all internucleoside linkages are phosphorothioate (PS) internucleoside linkages except for phosphodiester (PO) linkages at the indicated positions on mHprt mRNA. [Figure 24] 1 shows the editing efficiency of oligonucleotides containing a mixer region of 2'-fluoro RNA nucleosides and 2'-O-methyl RNA nucleosides in a first (A) or second (B) alternating pattern, with additional 2'-O-methyl RNA nucleosides at specific positions, on SERPINA1 mRNA. [Figure 25] 1 shows the editing efficiency of oligonucleotides containing mixmer regions of 2'-fluoro and 2'-O-methyl RNA nucleosides in a first (A) or second (B) alternating pattern, with additional 2'-fluoro nucleosides at specific positions, on SERPINA1 mRNA. [Figure 26-1] Figure 26 shows the editing efficiency of oligonucleotides with or without MOE flanking regions on GAPDH mRNA in cells expressing human ADAR2 (A) or human ADAR1 p110 (B) and HuH-7 cells (C). [Figure 26-2] See description of Figure 26-1. [Figure 27] The editing efficiency of the oligonucleotide and the corresponding oligonucleotide conjugate (CNJ number 126_2_1) on mHprt mRNA is shown. [Figure 28] 1 shows the editing efficiency of oligonucleotides delivered to cells by reverse gymnosis on mHprt mRNA. [Figure 29-1] Figure 29 shows the chemical structure of CMP No. 32_1. Due to the length of oligonucleotide CMP No. 32_1, Figure 29 is split across three pages to ensure that all atoms in the structure are legible. The structure on page 1 of Figure 29 (page 32 of the figure) is connected to the structure on page 2 of Figure 29 (page 33 of the figure) by a carbon-carbon bond that is split between the two pages. The end of this split carbon-carbon bond on page 1 of Figure 29 is indicated by "*1," and the end of this split carbon-carbon bond on page 2 of Figure 29 is indicated by "*2." Similarly, the structure on page 2 of Figure 29 is connected to the structure on page 3 of Figure 29 (page 34 of the figure) by a carbon-carbon bond that is split between the two pages. The end of this split carbon-carbon bond on page 2 of Figure 29 is indicated by "*3," and the end of this split carbon-carbon bond on page 3 of Figure 29 is indicated by "*4." [Figure 29-2] See description of Figure 29-1. [Figure 29-3] See description of Figure 29-1. DETAILED DESCRIPTION OF THE INVENTION

[0015] The chemical diagrams of oligonucleotides herein show the protonated form of the oligonucleotide, and it is understood that each hydrogen atom on the sulfur atom in the phosphorothioate internucleoside bond can be independently present or absent. It is understood that the presence of protons depends on the acidity of the molecule's environment. In salt form, one or more hydrogen atoms can be replaced with, for example, a cation, such as a metal cation (e.g., sodium or potassium cation). Protonated phosphorothioates exist in tautomeric forms.

[0016] Detailed Description of the Invention Unless otherwise stated, all ranges are inclusive of the starting and ending values.

[0017] The present invention provides an oligonucleotide comprising an editing region comprising an editing nucleoside, a 5' mixmer region located 5' to the editing region, and a 3' mixmer region located 3' to the editing region.

[0018] The present invention also provides an oligonucleotide comprising an edited region comprising an edited nucleoside.

[0019] Oligonucleotides As used herein, the term "oligonucleotide" is defined as commonly understood by those skilled in the art as a molecule containing two or more covalently linked nucleosides. Such covalently linked nucleosides may also be referred to as a nucleic acid molecule or oligomer.

[0020] Oligonucleotide is usually produced in laboratory by solid phase chemical synthesis and then purified and isolated.When referring to the sequence of oligonucleotide, it refers to the sequence or order of the nucleic acid base moiety of covalently linked nucleotide or nucleoside, or its modification.The oligonucleotide of the present invention is artificial, is chemically synthesized, and is usually purified or isolated.

[0021] Nucleotides and Nucleosides Nucleotide and nucleoside are the building blocks of oligonucleotide and polynucleotide, and for the purpose of the present invention, include both naturally occurring nucleotide and nucleoside and non-naturally occurring nucleotide and nucleoside.Naturally, nucleotide, such as DNA and RNA nucleotide, comprises a ribose sugar moiety, a nucleic acid base moiety and one or more phosphate groups.One or more phosphate groups are not present in nucleoside.Nucleoside and nucleotide can also be referred to interchangeably as "unit" or "monomer".The terms "nucleoside" and "nucleotide" can be used interchangeably herein when referring to these units in the context of the oligonucleotide of the present invention.

[0022] The nucleosides of the oligonucleotide of the present invention can be referred to by their position in the oligonucleotide relative to the editing nucleoside.Therefore, in some embodiments of the oligonucleotide of the present invention, the editing nucleoside is designated as position 0, and each nucleoside that is 5' relative to the editing nucleoside is designated as position +x, where x is the number of nucleosides that are 5' relative to the editing nucleoside at that position, including the nucleoside at that position, and each nucleoside that is 3' relative to the editing nucleoside at that position is designated as position -y, where y is the number of nucleosides that are 3' relative to the editing nucleoside at that position, including the nucleoside at that position.For example, the nucleoside that is 2 nucleosides 5' relative to the editing nucleoside is position +2, and the nucleoside that is 3 nucleosides 3' relative to the editing nucleoside is position -3. As a further example, SEQ ID NO: 32 is shown below with the edited region underlined and the edited nucleosides in bold: TIFF2025525564000001.tif4139.

[0023] The editing nucleoside C is at position 0. The T in the editing region is at position +1, the C immediately 5' to it is at position +2, etc. The I in the editing region is at position -1, the U immediately 3' to it is at position -2, etc.

[0024] In some embodiments, oligonucleotides of the invention comprise inosine nucleosides (i.e., nucleosides containing the nucleobase hypoxanthine). In some embodiments, oligonucleotides of the invention comprise one or more abasic nucleosides (i.e., nucleosides without a nucleobase). In some embodiments, oligonucleotides of the invention comprise one or more TNA nucleosides. In some embodiments, oligonucleotides of the invention comprise one or more SNA nucleosides. In some embodiments, oligonucleotides of the invention comprise one or more iDNA nucleosides. In some embodiments, oligonucleotides of the invention comprise one or more ScEt nucleosides. Examples of such nucleosides are shown below: TIFF2025525564000002.tif94156.

[0025] Nucleic acid bases The term "nucleobase" includes the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds during nucleic acid hybridization.In the context of the present invention, the term "nucleobase" also encompasses modified 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 non-naturally occurring variants.Such variants are described, for example, in Hirao et al., 2012, Accounts of Chemical Research, 45, 2055-2065 and Bergstrom, 2009, Curr.Protoc.Nucleic Acid Chem., 37, 1.4.1-1.4.32.

[0026] In some embodiments, the nucleobase moiety is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, e.g., a substituted purine or substituted pyrimidine, e.g., a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 7-deaza-8-azaguanine, 5-propynyl-uracil, 5-bromouracil 5-thiazolo-uracil, 2-thio-uracil, 2'thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0027] The structures of modified nucleobases that may be included in the oligonucleotides of the invention are shown below, along with hypoxanthine: TIFF2025525564000003.tif132167.

[0028] Nucleobase moieties may be represented by the letter code for each corresponding nucleobase, e.g., A, T, G, C, or U, and each letter may optionally include functionally equivalent modified nucleobases. 5-methylcytosine may be represented as "E." 7-deaza-8-azaguanine may be represented as "F." Hypoxanthine (such as inosine nucleoside) may be represented as "I."

[0029] Editing of target nucleic acid The oligonucleotide of the present invention is suitable for editing a target nucleic acid. The oligonucleotide of the present invention can edit a target nucleic acid. The term "editing" refers to changing the nucleic acid base sequence of a target nucleic acid. The oligonucleotide of the present invention can be called an "editing oligonucleotide." The target nucleic acid is the nucleic acid that is intended to be edited.

[0030] The target nucleic acid comprises target adenosine.The term "target adenosine" refers to the adenosine nucleoside of the target nucleic acid, which is converted into inosine nucleoside by deamination of adenine nucleobase to form hypoxanthine nucleobase.Therefore, the oligonucleotide of the present invention can cause the conversion of target adenosine (A) into inosine (I).

[0031] In some embodiments, the target nucleic acid is RNA. In some embodiments, the target nucleic acid encodes a protein. In some embodiments, the target nucleic acid is mRNA. In some embodiments, the target nucleic acid is non-coding RNA.

[0032] ADAR The oligonucleotide of the present invention is a guide oligonucleotide for adenosine deaminase acting on RNA (ADAR). Therefore, the oligonucleotide of the present invention can be referred to as a "guide oligonucleotide" or an "editing guide oligonucleotide."

[0033] ADAR is an enzyme that binds to double-stranded RNA and deaminates adenine nucleobases to form hypoxanthine nucleobases, thereby converting adenosine (A) nucleosides to inosine (I) nucleosides. Three ADAR genes (ADAR1, ADAR2, and ADAR3) have been identified in mammals. ADAR1 and ADAR2 are expressed in many tissues, while ADAR3 is specifically expressed in the brain and may be catalytically inactive. In some embodiments, the ADAR for which the oligonucleotide of the present invention is a guide oligonucleotide is ADAR1 or ADAR2. In some embodiments, the ADAR is endogenous human ADAR1 or ADAR2. The reference sequences for human ADAR1 and ADAR2 in the UniProtKB and NCBI databases are shown in Table 1 below.

[0034] [Table 1]

[0035] The term "guide oligonucleotide" refers to the oligonucleotide of the present invention that can direct ADAR to a target nucleic acid and a specific target adenosine on the target nucleic acid, so that ADAR deaminates the adenine of the target adenosine.The oligonucleotide achieves this by hybridizing with the target nucleic acid in the region of the target adenosine to form a double-stranded molecule.Therefore, the oligonucleotide of the present invention can bind to the target nucleic acid through complementary base pairing.ADAR can associate with the stretch of double-stranded nucleic acid.Therefore, the oligonucleotide of the present invention can recruit ADAR to the target nucleic acid.Therefore, the sequence of the oligonucleotide determines which target nucleic acid the oligonucleotide can hybridize with, and therefore the specific sequence of the oligonucleotide determines which adenosine the deamination activity of ADAR is directed to.

[0036] Editing SERPINA1 In some embodiments of the present invention, the target nucleic acid encodes alpha-1 antitrypsin (A1AT). In other words, in some embodiments, the oligonucleotide of the present invention is for editing a target nucleic acid encoding A1AT.

[0037] A1AT is a serine protease inhibitor synthesized in the liver and released into other tissues to protect against endogenous inflammatory serine proteases such as neutrophil elastase. A1AT is encoded by the SERPINA1 (serpin family A member 1) gene. Thus, in some embodiments, the target nucleic acid is SERPINA1 mRNA. In other words, in some embodiments, the oligonucleotide of the present invention is for editing SERPINA1 mRNA. The term "SERPINA1 mRNA" refers to any mRNA transcribed from the SERPINA1 gene. The UnitProtKB entry for A1AT is P01009, and the reference sequence number for the SERPINA1 gene in the NCBI database is NG_008290.1. The reference sequence numbers of 11 known mRNA transcript variants derived from the SERPINA1 gene are listed in Table 2 below.

[0038] [Table 2]

[0039] In some embodiments, the SERPINA1 mRNA comprises or consists of the sequence of any one of the mRNA transcripts listed in Table 2.

[0040] Mutations in the SERPINA1 gene are associated with a disorder called A1AT deficiency (A1AD). The most severe form of A1AD is associated with a single base pair substitution resulting in a glutamic acid (E)-342 to lysine (K) mutation in A1AT (E342K mutation).

[0041] As described herein, oligonucleotides of the present invention can be used to treat A1AD by editing mutant SERPINA1 mRNA. In particular, A1AD can be treated by editing the mutant codon encoding the E342K mutation. Glutamic acid (E) is encoded by the codons GAA and GAG. Lysine (K) is encoded by the codons AAA and AAG. In some embodiments, oligonucleotides of the present invention can convert a lysine-encoding AAA codon to a glutamic acid-encoding IAA codon on a target nucleic acid. In such embodiments, oligonucleotides of the present invention recruit ADARs that convert the first adenosine (target adenosine) of the AAA codon (encoding lysine) to inosine, generating an IAA codon that is read as GAA (encoding glutamic acid), thereby correcting the glutamic acid to lysine mutation. In some embodiments, oligonucleotides of the present invention can convert a lysine-encoding AAG codon to a glutamic acid-encoding IAG codon on a target nucleic acid. In such an embodiment, the oligonucleotide of the invention recruits an ADAR that converts the first adenosine (target adenosine) of the AAG codon (encoding lysine) to inosine, generating an IAG codon that is read as GAG (encoding glutamic acid), thereby correcting the glutamic acid to lysine mutation.

[0042] In some embodiments, the SERPINA1 mRNA comprises or consists of the sequence of any one of the mRNA transcripts listed in Table 2, wherein the GAG codon is mutated to AAG. In some embodiments, the SERPINA1 mRNA comprises or consists of the sequence of any one of the mRNA transcripts listed in Table 2, wherein the GAG codon encoding E342 in the A1AT protein is mutated to AAG.

[0043] The coding sequence from SERPINA1 mRNA transcript variant 11 (NM_001127707.2) containing the E342K mutation is shown below as SEQ ID NO: 186. The codon for K342 is AAG (shown underlined in the sequence below), and the first adenosine in the AAG codon is A1024 (shown in bold below).

[0044] SERPINA1 mRNA E342K mutant coding sequence - SEQ ID NO: 186: TIFF2025525564000006.tif79159

[0045] In some embodiments, the target adenosine corresponds to A1024 of SEQ ID NO: 186. The term "corresponding to" means that the target adenosine does not have to be the 1024th nucleoside of the target nucleic acid, and the sequence of the target nucleic acid is not particularly limited to SEQ ID NO: 186, but must be the first nucleoside within the AAG codon encoding K342 in the coding sequence of A1AT, such as A1024. Thus, in some embodiments, the target adenosine is the first nucleoside within the AAG codon encoding K342 in the coding sequence of A1AT.

[0046] In some embodiments, the target nucleic acid comprises a sequence having at least 80% identity to SEQ ID NO: 186, e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 186, wherein the target nucleic acid comprises A1024 of SEQ ID NO: 186. In some embodiments, the target nucleic acid comprises a sequence according to SEQ ID NO: 186. In some embodiments, the target nucleic acid consists of a sequence having at least 80% identity to SEQ ID NO: 186, e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 186, wherein the target nucleic acid comprises A1024 of SEQ ID NO: 186. In some embodiments, the target nucleic acid consists of the sequence according to SEQ ID NO:186.

[0047] Edited nucleosides and regions The term "edited nucleoside" refers to a nucleoside in an oligonucleotide of the present invention that is opposite to the target adenosine when the oligonucleotide of the present invention hybridizes to a target nucleic acid. In other words, the oligonucleotide of the present invention forms a double-stranded molecule with the target nucleic acid through complementary base pairing between the nucleoside of the oligonucleotide and the target nucleic acid, and the sequences of the oligonucleotide and the target nucleic acid are aligned so that the edited nucleoside aligns with the target adenosine. The edited nucleoside is not complementary to the target adenosine. In other words, the edited nucleoside forms a mismatch with the target adenosine. In some embodiments, the nucleobase of the edited nucleoside is selected from the group consisting of cytosine, 5-methylcytosine, guanine, and hypoxanthine. In some embodiments, the nucleoside is cytidine (C). In some embodiments, the nucleobase of the edited nucleoside is cytosine (C) or 5-methylcytosine (m5C). In some embodiments, the nucleobase of the edited nucleoside is 5-methylcytosine (m5C). In some embodiments, the edited nucleoside is guanosine (G). In some embodiments, the edited nucleoside is inosine (I).

[0048] The term "edited region" refers to one or more consecutive nucleosides (i.e., nucleosides linked by internucleoside linkages) comprising an edited nucleoside. The edited region can be defined to distinguish the nucleosides in the edited region from the nucleosides in other regions of the oligonucleotide of the present invention, such as the mixmer region and the flanking region. The nucleosides in the edited region can have common characteristics as described herein.

[0049] In some embodiments, the edited region consists of the edited nucleoside. In other words, there are no nucleosides in the edited region other than the edited nucleoside. In such embodiments, the edited nucleoside and the edited region are synonymous.

[0050] In some embodiments, the editing region comprises nucleosides at positions +1, 0, and -1. In some embodiments, the editing region consists of nucleosides at positions +1, 0, and -1. In some embodiments, the editing region comprises an editing triplet consisting of three nucleosides, and the editing nucleoside is the middle nucleoside of the editing triplet. In some embodiments, the editing region consists of an editing triplet consisting of three nucleosides, and the editing nucleoside is the middle nucleoside of the editing triplet. In some embodiments, the editing triplet is 5'-thymidine-cytidine-inosine-3' (TCI). Thus, in such embodiments, the middle C is the editing nucleoside, the T nucleoside is immediately 5', and the inosine nucleoside is immediately 3'.

[0051] In some embodiments of the oligonucleotides of the present invention, each nucleoside in the edited region contains the same sugar moiety. In some embodiments, each nucleoside in the edited region is independently selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, 2'-fluoro-RNA, MOE-RNA, LNA, ANA, and FANA nucleosides. Different types of sugar moieties that can be included in the oligonucleotides of the present invention are described elsewhere herein.

[0052] In some embodiments, the edited region comprises one or more DNA nucleosides. In some embodiments, the edited nucleosides are DNA nucleosides. In some embodiments, all nucleosides in the edited region are DNA nucleosides. In some embodiments, the nucleosides at one or more of positions +1, 0, and -1, for example, two or more of positions +1, 0, and -1, are DNA nucleosides. In some embodiments, the nucleoside at position +1 is a DNA nucleoside. In some embodiments, the nucleoside at position 0 is a DNA nucleoside. In some embodiments, the nucleoside at position -1 is a DNA nucleoside. In some embodiments, the nucleoside at each of positions +1, 0, and -1 is a DNA nucleoside. In some embodiments, the editing region consists of nucleosides at positions +1, 0 and -1 of the oligonucleotide, wherein the nucleoside at position +1 is a thymidine DNA nucleoside, the nucleoside at position 0 is a cytidine DNA nucleoside, and the nucleoside at position -1 is an inosine DNA nucleoside.

[0053] In some embodiments, the editing nucleoside is a FANA nucleoside. In some embodiments, the nucleoside at position 0 is a FANA nucleoside. In some embodiments, the editing nucleoside is a FANA nucleoside, and all other nucleosides in the editing region are DNA nucleosides. In some embodiments, the nucleoside at position 0 is a FANA nucleoside, and (a) the nucleoside at position +1 is a DNA nucleoside, or (b) the nucleoside at position −1 is a DNA nucleoside, or (c) the nucleoside at each of positions +1 and −1 is a DNA nucleoside. In some embodiments, the editing region consists of nucleosides at positions +1, 0 and -1 of the oligonucleotide, wherein the nucleoside at position +1 is a thymidine DNA nucleoside, the nucleoside at position 0 is a cytidine FANA nucleoside, and the nucleoside at position -1 is an inosine DNA nucleoside.

[0054] In some embodiments, the editing region comprises one or more nucleosides comprising a sugar moiety that differs from that of a first type of 5' mixmar nucleoside. In some embodiments, the editing region comprises one or more nucleosides comprising a sugar moiety that differs from that of a second type of 5' mixmar nucleoside. In some embodiments, the editing region comprises one or more nucleosides comprising a sugar moiety that differs from that of a first type of 5' mixmar nucleoside and a second type of 5' mixmar nucleoside. In some embodiments, the editing region comprises one or more nucleosides comprising a sugar moiety that differs from that of a first type of 3' mixmar nucleoside. In some embodiments, the editing region comprises one or more nucleosides comprising a sugar moiety that differs from that of a second type of 3' mixmar nucleoside. In some embodiments, the editing region comprises one or more nucleosides comprising a sugar moiety that differs from that of a first type of 3' mixmar nucleoside and a second type of 3' mixmar nucleoside.

[0055] In some embodiments, the editing region includes one or more nucleosides that include a sugar moiety that is different from the sugar moiety of a first type of 5' mixmar nucleoside, a second type of 5' mixmar nucleoside, a first type of 3' mixmar nucleoside, and a second type of 3' mixmar nucleoside.

[0056] In some embodiments, each nucleoside in the editing region comprises a sugar moiety that is different from that of a first type of 5' mixmar nucleoside. In some embodiments, each nucleoside in the editing region comprises a sugar moiety that is different from that of a second type of 5' mixmar nucleoside. In some embodiments, each nucleoside in the editing region comprises a sugar moiety that is different from that of a first type of 3' mixmar nucleoside. In some embodiments, each nucleoside in the editing region comprises a sugar moiety that is different from that of a second type of 3' mixmar nucleoside. In some embodiments, each nucleoside in the editing region comprises a sugar moiety that is different from that of a first type of 5' mixmar nucleoside, a second type of 5' mixmar nucleoside, a first type of 3' mixmar nucleoside, and a second type of 3' mixmar nucleoside.

[0057] Mixmar area The oligonucleotide of the present invention comprises one or more mixmer regions. The oligonucleotide of the present invention comprises a 5' mixmer region located 5' to the edited region and a 3' mixmer region located 3' to the edited region.

[0058] The term "mixmer region" refers to two or more consecutive nucleosides (i.e., nucleosides linked by internucleoside linkages) that contain more than one type of sugar moiety. In other words, a mixmer region contains nucleosides with different types of sugar modifications. Nucleosides with different types of sugar moieties or sugar modifications are sometimes referred to herein as "different types of nucleosides" or "different types of sugar-modified nucleosides."

[0059] In some embodiments, the oligonucleotide comprises a mixer region. In some embodiments, the oligonucleotide is a mixer. In some embodiments, the oligonucleotide comprises a mixer region divided into two parts by an editing region. In other words, the mixer region comprises an editing region. In some embodiments, the editing region divides the mixer region into a 5' mixer region and a 3' mixer region.

[0060] In some embodiments, the 5' mixmer region is located immediately 5' to the editing region. In other words, there is no additional nucleoside between the 3'-most nucleoside of the 5' mixmer region and the 5'-most nucleoside of the editing region; the 3'-most nucleoside of the 5' mixmer region and the 5'-most nucleoside of the editing region are connected by an internucleoside bond. In some embodiments, the 3' mixmer region is located immediately 3' to the editing region. In other words, there is no additional nucleoside between the 5'-most nucleoside of the 3' mixmer region and the 3'-most nucleoside of the editing region; the 5'-most nucleoside of the 3' mixmer region and the 3'-most nucleoside of the editing region are connected by an internucleoside bond. In some embodiments, the 5' mixmer region is located immediately 5' to the editing region, and the 3' mixmer region is located immediately 3' to the editing region.

[0061] In some embodiments, the oligonucleotide comprises the following structure: X +1 -X 0 -X -1 During the ceremony, X +1 is the 5' mixmer region, X 0 is the editing region, X -1 is the 3' mixmer region.

[0062] Therefore, according to the above formula, the 5' mixmer region, the editing region, and the 3' mixmer region are contiguous (i.e., connected by an internucleoside bond). The hyphen in the above formula represents an internucleoside bond.

[0063] The mixer region contains more than one type of sugar-modified nucleoside. The 5' mixer region contains more than one type of sugar-modified nucleoside. The 3' mixer region contains more than one type of sugar-modified nucleoside. In some embodiments, the mixer region contains more than one type of sugar-modified nucleoside. In some embodiments, the 5' mixer region contains exactly two types of sugar-modified nucleosides. In some embodiments, the 3' mixer region contains exactly two types of sugar-modified nucleosides.

[0064] In some embodiments, the mixmer region comprises a first type of mixmer nucleoside and a second type of mixmer nucleoside, and the sugar moiety of the first type of mixmer nucleoside is different from the sugar moiety of the second type of mixmer nucleoside. In other words, the first type of mixmer nucleoside and the second type of mixmer nucleoside are distinguished by the type of sugar moiety they contain.

[0065] In some embodiments, the 5' mixmer region comprises a first type of 5' mixmer nucleoside and a second type of 5' mixmer nucleoside, and the sugar moiety of the first type of 5' mixmer nucleoside is different from the sugar moiety of the second type of 5' mixmer nucleoside. In other words, the first type of 5' mixmer nucleoside and the second type of 5' mixmer nucleoside are distinguished by the type of sugar moiety they contain.

[0066] In some embodiments, the 3' mixmer region comprises a first type of 3' mixmer nucleoside and a second type of 3' mixmer nucleoside, and the sugar moiety of the first type of 3' mixmer nucleoside is different from the sugar moiety of the second type of 3' mixmer nucleoside. In other words, the first type of 3' mixmer nucleoside and the second type of 3' mixmer nucleoside are distinguished by the type of sugar moiety they contain.

[0067] Suitable sugar moieties (ie, different types of sugar-modified nucleosides or sugar modifications) are described elsewhere herein.

[0068] In some embodiments, the mixmir region comprises a first type of mixmir nucleoside and a second type of mixmir nucleoside. In some embodiments, the first type of mixmir nucleoside is a 2'-O-methyl-RNA nucleoside and the second type of mixmir nucleoside is a 2'-fluoro-RNA nucleoside. In some embodiments, the first type of mixmir nucleoside is a 2'-fluoro-RNA nucleoside and the second type of mixmir nucleoside is a 2'-O-methyl-RNA nucleoside.

[0069] In particular, other characteristics (e.g., nucleobase) of a given nucleoside of the first type of mixomer nucleoside may be different from those of a given nucleoside of the second type, but it is the different sugar moieties that determine which type of mixomer nucleoside the nucleoside belongs to.For example, two nucleosides may have different nucleobases, but have the same type of sugar moiety, so they can be the same type of mixomer nucleoside.Conversely, two nucleosides may have the same nucleobase, but can be different types of mixomer nucleosides due to their different sugar moieties.

[0070] In some embodiments, the first type of 5'-mixmar nucleoside and the first type of 3'-mixmar nucleoside are the same type of sugar-modified nucleoside (i.e., they have the same sugar moiety). In some embodiments, the first type of 5'-mixmar nucleoside and the first type of 3'-mixmar nucleoside are the same type of sugar-modified nucleoside and are collectively referred to as "first type of mixmar nucleoside." In some embodiments, the second type of 5'-mixmar nucleoside and the second type of 3'-mixmar nucleoside are the same type of sugar-modified nucleoside (i.e., they have the same sugar moiety). In some embodiments, the second type of 5'-mixmar nucleoside and the second type of 3'-mixmar nucleoside are the same type of sugar-modified nucleoside and are collectively referred to as "second type of mixmar nucleoside."

[0071] In some embodiments, the first type of 5' mixmer nucleosides and the second type of 5' mixmer nucleosides are arranged in an alternating pattern within the 5' mixmer region. In other words, a stretch of nucleosides of the first type of 5' mixmer nucleosides is followed by a stretch of nucleosides of the second type of 5' mixmer nucleosides, which is then followed by a stretch of nucleosides of the first type of 5' mixmer nucleosides, etc. In other words, in some embodiments, the 5' mixmer region comprises an alternating pattern of the first type of 5' mixmer nucleosides and the second type of 5' mixmer nucleosides.

[0072] Similarly, in some embodiments, the first type of 3' mixmer nucleosides and the second type of 3' mixmer nucleosides are arranged in an alternating pattern within the 3' mixmer region. In other words, a stretch of nucleosides of the first type of 3' mixmer nucleosides is followed by a stretch of nucleosides of the second type of 3' mixmer nucleosides, which is then followed by a stretch of nucleosides of the first type of 3' mixmer nucleosides, etc. In other words, in some embodiments, the 3' mixmer region comprises an alternating pattern of the first type of 3' mixmer nucleosides and the second type of 3' mixmer nucleosides.

[0073] There can be from 1 to about 4 nucleosides in each stretch of nucleosides of a first type or a second type of mixed nucleosides. In some embodiments, the number of nucleosides in each stretch of a given type of nucleoside varies independently. Thus, in some embodiments, the number of nucleosides in each stretch varies between nucleoside types (e.g., a specific number of nucleosides of the first type can be present in each stretch of the first type, and a different number of nucleosides of the second type can be present in each stretch of the second type). In some embodiments, the number of nucleosides varies between stretches of nucleosides of the same type (e.g., a specific number of nucleosides can be present in a given stretch of nucleosides of a first type, but a different number of nucleosides can be present in different stretches of nucleosides of the first type).

[0074] In some embodiments, each stretch of nucleosides of the same type comprises the same number of nucleosides. For example, all stretches of nucleosides of the first type can be two nucleosides long, and all stretches of nucleosides of the second type can be one nucleoside long. In some embodiments, each stretch of nucleosides comprises the same number of nucleosides. For example, all stretches of nucleosides of the first and second types can be two nucleosides long.

[0075] In some embodiments, each stretch of nucleosides of the first or second type is a single nucleoside. In other words, in some embodiments, the 5' mixmer region comprises a single nucleoside (i.e., one nucleoside) of a first type of 5' mixmer oligonucleoside, followed by a single nucleoside of a second type of 5' mixmer oligonucleoside, followed by a single nucleoside of a first type of 5' mixmer oligonucleoside, etc. In some embodiments, the first type of 5' mixmer nucleosides and the second type of 5' mixmer nucleosides are arranged in a single nucleoside alternating pattern. In other words, in some embodiments, the 5' mixmer region comprises an alternating pattern of a single nucleoside of a first type of 5' mixmer nucleoside and a single nucleoside of a second type of 5' mixmer nucleoside.

[0076] Similarly, in some embodiments, the 3' mixmer region comprises a single nucleoside (i.e., one nucleoside) of a first type of 3' mixmer oligonucleoside, followed by a single nucleoside of a second type of 3' mixmer oligonucleoside, followed by a single nucleoside of the first type of 3' mixmer oligonucleoside, etc. In some embodiments, the first type of 3' mixmer nucleosides and the second type of 3' mixmer nucleosides are arranged in a single nucleoside alternating pattern. In other words, in some embodiments, the 3' mixmer region comprises an alternating pattern of a single nucleoside of a first type of 3' mixmer nucleoside and a single nucleoside of a second type of 3' mixmer nucleoside.

[0077] In some embodiments, the 5' mixmer region comprises: (a) one or more first 5' mixmer subregions of one or more nucleosides of a first type of 5' mixmer nucleoside; and (b) one or more second 5' mixmer subregions of one or more nucleosides of a second type of 5' mixmer nucleoside; The first and second 5' mixmer subregions are arranged in an alternating pattern, i.e., a first 5' mixmer subregion is followed by a second 5' mixmer subregion, which is followed by another first 5' mixmer subregion, which is followed by a second 5' mixmer subregion, etc.

[0078] In some embodiments, the 5' mixmer region comprises: (a) one or more first 5' mixmer subregions of 1, 2, 3, or 4 nucleosides of a first type of 5' mixmer nucleoside; and (b) one or more second 5' mixmer subregions of 1, 2, 3, or 4 nucleosides of a second type of 5' mixmer nucleoside; The first 5' mixmer subregion and the second 5' mixmer subregion are arranged in an alternating pattern.

[0079] In some embodiments, the 5' mixmer region comprises: (a) one or more first 5' mixmer subregions of one nucleoside of a first type of 5' mixmer nucleoside; and (b) one or more second 5' mixmer subregions of one nucleoside of a second type of 5' mixmer nucleoside; The first 5' mixmer subregion and the second 5' mixmer subregion are arranged in an alternating pattern.

[0080] Similarly, in some embodiments, the 3' mixmer region comprises: (a) one or more first 3' mixmer subregions of one or more nucleosides of a first type of 3' mixmer nucleoside; and (b) one or more second 3' mixmer subregions of one or more nucleosides of a second type of 3' mixmer nucleoside; The first and second 3' mixmer subregions are arranged in an alternating pattern, i.e., a first 3' mixmer subregion is followed by a second 3' mixmer subregion, which is followed by another first 3' mixmer subregion, which is followed by a second 3' mixmer subregion, etc.

[0081] In some embodiments, the 3' mixmer region is (a) one or more first 3' mixmer subregions of 1, 2, 3, or 4 nucleosides of a first type of 3' mixmer nucleoside; and (b) one or more second 3' mixmer subregions of 1, 2, 3, or 4 nucleosides of a second type of 3' mixmer nucleoside; The first 3' mixmer subregion and the second 3' mixmer subregion are arranged in an alternating pattern.

[0082] In some embodiments, the 3' mixmer region is (a) one or more first 3' mixmer subregions of one nucleoside of a first type of 3' mixmer nucleoside; and (b) one or more second 3' mixmer subregions of one nucleoside of a second type of 3' mixmer nucleoside; The first 3' mixmer subregion and the second 3' mixmer subregion are arranged in an alternating pattern.

[0083] In some embodiments, the 5' mixmer region comprises one or more subregions of one or more nucleosides of a first type of 5' mixmer nucleoside, each subregion being separated by one or more nucleosides of a second type of 5' mixmer nucleoside. In some embodiments, the 3' mixmer region comprises one or more subregions of one or more nucleosides of a first type of 3' mixmer nucleoside, each subregion being separated by one or more nucleosides of a second type of 3' mixmer nucleoside.

[0084] In some embodiments, the 5' mixmer region comprises one or more subregions of 1, 2, 3, or 4 nucleosides of a first type of 5' mixmer nucleoside, each subregion separated by 1, 2, 3, or 4 nucleosides of a second type of 5' mixmer nucleoside. In some embodiments, the 3' mixmer region comprises one or more subregions of 1, 2, 3, or 4 nucleosides of a first type of 3' mixmer nucleoside, each subregion separated by 1, 2, 3, or 4 nucleosides of a second type of 3' mixmer nucleoside.

[0085] In some embodiments, the 5' mixmer region comprises one or more subregions of one nucleoside of a first type of 5' mixmer nucleoside, each subregion being separated by one nucleoside of a second type of 5' mixmer nucleoside. In some embodiments, the 3' mixmer region comprises one or more subregions of one nucleoside of a first type of 3' mixmer nucleoside, each subregion being separated by one nucleoside of a second type of 3' mixmer nucleoside.

[0086] The structure of the mixmer region can be represented by the formula: Thus, in some embodiments, the 5' mixmer region comprises the following structure: (Y 5 -Z 5 -) m During the ceremony, Y 5 is one or more nucleosides of a first type of 5'-mixmer nucleoside, Z 5 is one or more nucleosides of a second type of 5'-mixmer nucleoside, m is a number between 1 and 20.

[0087] In some embodiments, the 3' mixmer region comprises the following structure: (Y 3 -Z 3 -) n During the ceremony, Y 3 is one or more nucleosides of a first type of 3'-mixmer nucleoside, Z 3 is one or more nucleosides of a second type of 3'-mixmer nucleoside, n is a number between 1 and 20.

[0088] The nucleosides in each mixer region are contiguous (i.e., linked by internucleoside linkages). The hyphen in the above formula represents an internucleoside linkage. In some embodiments, each Y 5 is independently 1, 2, 3, or 4 nucleosides. 5 is independently one nucleoside. In some embodiments, each Z 5 are independently 1, 2, 3, or 4 nucleosides. 5 is independently one nucleoside. In some embodiments, each Y 3 is independently 1, 2, 3, or 4 nucleosides. 3 is independently one nucleoside. In some embodiments, each Z 3 are independently 1, 2, 3, or 4 nucleosides. 3 is independently one nucleoside.

[0089] In some embodiments, each Y 5 is independently one nucleoside, and each Z 5 is independently one nucleoside, and each Y 3 is independently one nucleoside, and each Z 3 is independently one nucleoside.

[0090] In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, m is 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, m is 9.

[0091] In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, n is 3, 4, 5, 6, 7, 8, or 9. In some embodiments, n is 5.

[0092] In other words, the 5' mixmer region can be represented as follows: Y 5 -Z 5 -Y 5 -Z 5 -Y 5 -Z 5 -Y 5 -Z 5 -Y 5 -Z 5 -Y 5 -Z 5 … In the formula, each Y 5 is a stretch of nucleosides of the first type 5'mixmer nucleosides, and each Z 5 is a stretch of nucleosides of the second type of 5' mixmer nucleosides. The stretch of nucleosides is linked by internucleoside bonds, thus forming a continuous 5' mixmer region. Each Y of nucleosides 5 The stretches are independently 1, 2, 3, or 4 nucleosides. 5 The stretches may be independently 1, 2, 3, or 4 nucleosides. 5 and Z 5 exists.

[0093] Similarly, the 3' mixmer region can be represented as follows: Y 3 -Z 3 -Y 3 -Z 3 -Y 3 -Z 3 -Y 3 -Z 3 -Y 3 -Z 3 -Y 3 -Z 3 … In the formula, each Y 3 is a stretch of nucleosides of the first type 3'mixmer nucleosides, and each Z 3 is a stretch of nucleosides of the second type of 3' mixmer nucleosides. The stretch of nucleosides is linked by internucleoside bonds, thus forming a continuous 3' mixmer region. Each Y of nucleosides 3 The stretches are independently 1, 2, 3, or 4 nucleosides. 3 The stretches may be independently 1, 2, 3, or 4 nucleosides. 3 and Z 3 exists.

[0094] Mixmer region length In some embodiments of the oligonucleotide, the 5' mixmer region is 15 to 25 nucleosides in length. In some embodiments, the 5' mixmer region is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides in length. In some embodiments, the 5' mixmer region is 18 nucleosides in length.

[0095] In some embodiments of the oligonucleotide, the 3' mixmer region is 4 to 12 nucleosides in length. In some embodiments, the 3' mixmer region is 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleosides in length. In some embodiments, the 3' mixmer region is 9 nucleosides in length.

[0096] In some embodiments, the 5' mixmer region is 24 nucleosides long and the 3' mixmer region is 9 nucleosides long. In some embodiments, the 5' mixmer region is 24 nucleosides long and the 3' mixmer region is 8 nucleosides long. In some embodiments, the 5' mixmer region is 24 nucleosides long and the 3' mixmer region is 7 nucleosides long. In some embodiments, the 5' mixmer region is 24 nucleosides long and the 3' mixmer region is 6 nucleosides long. In some embodiments, the 5' mixmer region is 23 nucleosides long and the 3' mixmer region is 9 nucleosides long. In some embodiments, the 5' mixmer region is 23 nucleosides long and the 3' mixmer region is 4 nucleosides long. In some embodiments, the 5' mixmer region is 22 nucleosides long and the 3' mixmer region is 9 nucleosides long. In some embodiments, the 5' mixmer region is 22 nucleosides long and the 3' mixmer region is 5 nucleosides long. In some embodiments, the 5' mixmer region is 21 nucleosides long and the 3' mixmer region is 9 nucleosides long. In some embodiments, the 5' mixmer region is 21 nucleosides long and the 3' mixmer region is 6 nucleosides long. In some embodiments, the 5' mixmer region is 20 nucleosides long and the 3' mixmer region is 9 nucleosides long. In some embodiments, the 5' mixmer region is 20 nucleosides long and the 3' mixmer region is 7 nucleosides long. In some embodiments, the 5' mixmer region is 19 nucleosides in length and the 3' mixmer region is 9 nucleosides in length. In some embodiments, the 5' mixmer region is 19 nucleosides in length and the 3' mixmer region is 8 nucleosides in length. In some embodiments, the 5' mixmer region is 19 nucleosides in length and the 3' mixmer region is 7 nucleosides in length. In some embodiments, the 5' mixmer region is 19 nucleosides in length and the 3' mixmer region is 6 nucleosides in length.In some embodiments, the 5' mixmer region is 18 nucleosides in length and the 3' mixmer region is 9 nucleosides in length. In some embodiments, the 5' mixmer region is 17 nucleosides in length and the 3' mixmer region is 9 nucleosides in length.

[0097] In some embodiments, the oligonucleotide is 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleosides in length, the 5' mixmer region is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides in length, and the 3' mixmer region is 5, 6, 7, 8, 9, 10, 11, or 12 nucleosides in length. In some embodiments, the oligonucleotide is 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleosides in length, the 5' mixmer region is 16, 17, 18, 19, 20, 21, 22, or 23 nucleosides in length, and the 3' mixmer region is 8 or 9 nucleosides in length. In some embodiments, the oligonucleotide is 40 nucleosides in length, the 5' mixmer region is 18 nucleosides in length, and the 3' mixmer region is 9 nucleosides in length.

[0098] In some embodiments of the oligonucleotides of the present invention, the 5' mixmer region comprises nucleosides at positions +1 to +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, or +25. In other words, the 5' mixmer region comprises nucleosides at positions +1 to +10, or at positions +1 to +11, or at positions +1 to +12, etc. In some embodiments, the 5' mixmer region comprises nucleosides at positions +2 to +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, or +25. In some embodiments, the 5' mixmer region comprises a nucleoside at position +2 to position +15, +16, +17, +18, +19, +20, +21, +22, +23, or +24. In some embodiments, the 5' mixmer region comprises a nucleoside at position +2 to position +19, +20, +21, +22, +23, or +24.

[0099] In some embodiments, the 5' mixmer region consists of nucleosides from position +1 to position +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, or +25. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, or +25. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +15, +16, +17, +18, +19, +20, +21, +22, +23, or +24. In some embodiments, the 5' mixmer region consists of nucleosides at position +2 through position +19, +20, +21, +22, +23, or +24.

[0100] In some embodiments, the 5' mixmer region comprises nucleosides at positions +2 to +19. In some embodiments, the 5' mixmer region consists of nucleosides at positions +2 to +19.

[0101] In some embodiments of the oligonucleotides of the present invention, the 3' mixmer region comprises nucleosides at positions -1 to -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, or -17. In other words, the 3' mixmer region comprises nucleosides at positions -1 to -3, or -1 to -4, or -1 to -5, etc.

[0102] In some embodiments, the 3' mixmer region comprises a nucleoside at position -2 through position -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, or -17. In some embodiments, the 3' mixmer region comprises a nucleoside at position -2 through position -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, or -15. In some embodiments, the 3' mixmer region comprises a nucleoside at position -2 through position -5, -6, -7, -8, -9, or -10.

[0103] In some embodiments, the 3' mixmer region comprises nucleosides at positions -2 through -10. In some embodiments, the 3' mixmer region consists of nucleosides at positions -2 through -10.

[0104] In some embodiments of the oligonucleotides of the present invention, the 5' mixmer region consists of nucleosides from position +2 to position +15, +16, +17, +18, +19, +20, +21, +22, +23, or +24; the 3' mixmer region consists of nucleosides from position -2 to position -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, or -15. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +17, +18, +19, +20, +21, +22, +23, or +24; and the 3' mixmer region consists of nucleosides from position -2 to position -7, -8, -9, or -10.

[0105] In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +17, +18, +19, +20, +21, +22, +23, or +24; the 3' mixmer region consists of nucleosides from position -2 to position -10. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +17; the 3' mixmer region consists of nucleosides from position -2 to position -10. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +18; the 3' mixmer region consists of nucleosides from position -2 to position -10. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +19; the 3' mixmer region consists of nucleosides from position -2 to position -10. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +20; the 3' mixmer region consists of nucleosides from position -2 to position -10. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +21; the 3' mixmer region consists of nucleosides from position -2 to position -10. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +22; the 3' mixmer region consists of nucleosides from position -2 to position -10. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +23; the 3' mixmer region consists of nucleosides from position -2 to position -10. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +24; the 3' mixmer region consists of nucleosides from position -2 to position -10. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +25; and the 3' mixmer region consists of nucleosides from position -2 to position -10.

[0106] In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +25; the 3' mixmer region consists of nucleosides from position -2 to position -9. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +25; the 3' mixmer region consists of nucleosides from position -2 to position -8. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +25; and the 3' mixmer region consists of nucleosides from position -2 to position -7.

[0107] In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +20; the 3' mixmer region consists of nucleosides from position -2 to position -8. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +20; and the 3' mixmer region consists of nucleosides from position -2 to position -7.

[0108] In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +20; the 3' mixmer region consists of nucleosides from position -2 to position -9. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +21; the 3' mixmer region consists of nucleosides from position -2 to position -8. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +22; the 3' mixmer region consists of nucleosides from position -2 to position -7. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +23; the 3' mixmer region consists of nucleosides from position -2 to position -6. In some embodiments, the 5' mixmer region consists of nucleosides from position +2 to position +24; the 3' mixmer region consists of nucleosides from position -2 to position -5.

[0109] In some embodiments, the oligonucleotide is 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleosides in length, wherein the 5' mixmer region consists of nucleosides from position +2 to position +15, +16, +17, +18, +19, +20, +21, +22, +23, or +24; and the 3' mixmer region consists of nucleosides from position -2 to position -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, or -15. In some embodiments, the oligonucleotide is 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleosides in length, and the 5' mixmer region consists of nucleosides from position +2 to position +17, +18, +19, +20, +21, +22, +23, or +24; and the 3' mixmer region consists of nucleosides from position -2 to position -7, -8, -9, or -10. In some embodiments, the oligonucleotide is 40 nucleosides in length, and the 5' mixmer region consists of nucleosides from position +2 to position +17, +18, +19, +20, +21, +22, +23, or +24; and the 3' mixmer region consists of nucleosides from position -2 to position -7, -8, -9, or -10. In some embodiments, the oligonucleotide is 40 nucleosides in length, with the 5' mixmer region consisting of nucleosides from position +2 to position +19; and the 3' mixmer region consisting of nucleosides from position -2 to position -10.

[0110] Mixer region patterns In some embodiments, the nucleosides at one or more of positions +3, +5, +7, +9, +11, +13, +15, +17, and +19 are first type of mixumer nucleosides. In some embodiments, the nucleosides at one or more of positions +3, +5, +7, +9, +11, +13, +15, +17, and +19 are first type of 5' mixumer nucleosides. In some embodiments, the nucleoside at position +3 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +5 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +7 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +9 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +11 is a 5' mixmer nucleoside of a first type. is a first type of 5' mixmer nucleoside, and / or the nucleoside at position +13 is a first type of 5' mixmer nucleoside, and / or the nucleoside at position +15 is a first type of 5' mixmer nucleoside, and / or the nucleoside at position +17 is a first type of 5' mixmer nucleoside, and / or the nucleoside at position +19 is a first type of 5' mixmer nucleoside. In some embodiments, the nucleoside at each of positions +3, +5, +7, +9, +11, +13, +15, +17 and +19 is a first type of 5' mixmer nucleoside.

[0111] In some embodiments, the nucleoside at one or more of positions -3, -5, -7, and -9 is a first type of mixer nucleoside. In some embodiments, the nucleoside at one or more of positions -3, -5, -7, and -9 is a first type of 3' mixer nucleoside. In some embodiments, the nucleoside at position -3 is a first type of 3' mixer nucleoside, and / or the nucleoside at position -5 is a first type of 3' mixer nucleoside, and / or the nucleoside at position -7 is a first type of 3' mixer nucleoside, and / or the nucleoside at position -9 is a first type of 3' mixer nucleoside. In some embodiments, the nucleoside at each of positions -3, -5, -7, and -9 is a first type of 3' mixer nucleoside.

[0112] In some embodiments, the nucleoside at one or more of positions +2, +4, +6, +8, +10, +12, +14 and +18 is a second type of mixumer nucleoside. In some embodiments, the nucleoside at one or more of positions +2, +4, +6, +8, +10, +12, +14 and +18 is a second type of 5' mixumer nucleoside. In some embodiments, the nucleoside at position +2 is a second type of 5' mixmer nucleoside, and / or the nucleoside at position +4 is a second type of 5' mixmer nucleoside, and / or the nucleoside at position +6 is a second type of 5' mixmer nucleoside, and / or the nucleoside at position +8 is a second type of 5' mixmer nucleoside, and / or the nucleoside at position +10 is a second type of 5' mixmer nucleoside, and / or the nucleoside at position +12 is a second type of 5' mixmer nucleoside, and / or the nucleoside at position +14 is a second type of 5' mixmer nucleoside, and / or the nucleoside at position +18 is a second type of 5' mixmer nucleoside. In some embodiments, the nucleoside at each of positions +2, +4, +6, +8, +10, +12, +14 and +18 is a 5' mixer nucleoside of the second type.

[0113] In some embodiments, the nucleoside at one or more of positions -2, -4, and -6 is a second type of mixer nucleoside. In some embodiments, the nucleoside at one or more of positions -2, -4, and -6 is a second type of 3' mixer nucleoside. In some embodiments, the nucleoside at position -2 is a second type of 3' mixer nucleoside, and / or the nucleoside at position -4 is a second type of 3' mixer nucleoside, and / or the nucleoside at position -6 is a second type of 3' mixer nucleoside. In some embodiments, the nucleoside at each of positions -2, -4, and -6 is a second type of 3' mixer nucleoside.

[0114] In some embodiments, the nucleosides at one or more of positions +3, +5, +7, +9, +11, +13, +15, +17, +19, +21 and +23 are first type of mixumer nucleosides. In some embodiments, the nucleosides at one or more of positions +3, +5, +7, +9, +11, +13, +15, +17, +19, +21 and +23 are first type of 5' mixumer nucleosides. In some embodiments, the nucleoside at position +3 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +5 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +7 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +9 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +11 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +13 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +5 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +6 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +7 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +9 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +11 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +13 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +14 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +15 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +16 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +17 is a 5' mixmer nucleoside of a first type, and / or the nucleoside at position +19 is a 5' mixmer is a first type of 5' mixmer nucleoside, and / or the nucleoside at position +15 is a first type of 5' mixmer nucleoside, and / or the nucleoside at position +17 is a first type of 5' mixmer nucleoside, and / or the nucleoside at position +19 is a first type of 5' mixmer nucleoside, and / or the nucleoside at position +21 is a first type of 5' mixmer nucleoside, and / or the nucleoside at position +23 is a first type of 5' mixmer nucleoside. In some embodiments, the nucleoside at each of positions +3, +5, +7, +9, +11, +13, +15, +17, +19, +21 and +23 is a first type of 5' mixmer nucleoside.

[0115] In some embodiments, the nucleoside at one or more of positions -3, -5, -7, -9, -11, -13, and -15 is a first type of mixumer nucleoside. In some embodiments, the nucleoside at one or more of positions -3, -5, -7, -9, -11, -13, and -15 is a first type of 3' mixumer nucleoside. In some embodiments, the nucleoside at position-3 is a first type of 3' mixmer nucleoside, and / or the nucleoside at position-5 is a first type of 3' mixmer nucleoside, and / or the nucleoside at position-7 is a first type of 3' mixmer nucleoside, and / or the nucleoside at position-9 is a first type of 3' mixmer nucleoside, and / or the nucleoside at position-11 is a first type of 3' mixmer nucleoside, and / or the nucleoside at position-13 is a first type of 3' mixmer nucleoside, and / or the nucleoside at position-15 is a first type of 3' mixmer nucleoside. In some embodiments, the nucleoside at each of positions -3, -5, -7, -9, -11, -13 and -15 is a 3' mixer nucleoside of the first type.

[0116] In some embodiments, the nucleosides at one or more of positions +2, +4, +6, +8, +10, +12, +14, +18, +20, +22, and +24 are mixumer nucleosides of the second type. In some embodiments, the nucleosides at one or more of positions +2, +4, +6, +8, +10, +12, +14, +18, +20, +22, and +24 are 5' mixumer nucleosides of the second type. In some embodiments, the nucleoside at position +2 is a 5' mixmer nucleoside of a second type, and / or the nucleoside at position +4 is a 5' mixmer nucleoside of a second type, and / or the nucleoside at position +6 is a 5' mixmer nucleoside of a second type, and / or the nucleoside at position +8 is a 5' mixmer nucleoside of a second type, and / or the nucleoside at position +10 is a 5' mixmer nucleoside of a second type, and / or the nucleoside at position +12 is a 5' mixmer nucleoside of a second type, and / or the nucleoside at position +3 is a 5' mixmer nucleoside of a second type, and / or the nucleoside at position +4 is a 5' mixmer nucleoside of a second type, and / or the nucleoside at position +5 is a 5' mixmer nucleoside of a second type, and / or the nucleoside at position +6 is a 5' mixmer nucleoside of a second type, and / or the nucleoside at position +7 is a 5' mixmer nucleoside of a second type, and / or the nucleoside at position +8 is a 5' mixmer nucleoside of a second type, and / or the nucleoside at position +9 is a 5' mixmer nucleoside of a second type, and / or the nucleoside at position +10 is a 5' mixmer nucleoside of a second type, and / or the nucleoside at position +12 is a 5' mixmer nucleoside of a second type, and / or the nucleoside at position +13 is a 5' mixmer nucleoside of a second type, and / or the nucleoside at position +14 is a 5' mixmer nucleo is a 5' mixmer nucleoside of the second type, and / or the nucleoside at position +14 is a 5' mixmer nucleoside of the second type, and / or the nucleoside at position +18 is a 5' mixmer nucleoside of the second type, and / or the nucleoside at position +20 is a 5' mixmer nucleoside of the second type, and / or the nucleoside at position +22 is a 5' mixmer nucleoside of the second type, and / or the nucleoside at position +24 is a 5' mixmer nucleoside of the second type. In some embodiments, the nucleoside at each of positions +2, +4, +6, +8, +10, +12, +14, +18, +20, +22 and +24 is a 5' mixmer nucleoside of the second type.

[0117] In some embodiments, the nucleoside at one or more of positions -2, -4, -6, -10, -12, and -14 is a second type of mixumer nucleoside. In some embodiments, the nucleoside at one or more of positions -2, -4, -6, -10, -12, and -14 is a second type of 3' mixumer nucleoside. In some embodiments, the nucleoside at position -2 is a 3'-mixmer nucleoside of the second type, and / or the nucleoside at position -4 is a 3'-mixmer nucleoside of the second type, and / or the nucleoside at position -6 is a 3'-mixmer nucleoside of the second type, and / or the nucleoside at position -10 is a 3'-mixmer nucleoside of the second type, and / or the nucleoside at position -12 is a 3'-mixmer nucleoside of the second type, and / or the nucleoside at position -14 is a 3'-mixmer nucleoside of the second type. In some embodiments, the nucleoside at each of positions -2, -4, -6, -10, -12, and -14 is a 3'-mixmer nucleoside of the second type.

[0118] In some embodiments of the oligonucleotides of the present invention, the nucleosides at positions +2 through +19 are a 5' mixmer region, the nucleosides at each of positions +3, +5, +7, +9, +11, +13, +15, +17, and +19 are 5' mixmer nucleosides of a first type, and the nucleosides at each of positions +2, +4, +6, +8, +10, +12, +14, +16, and +18 are 5' mixmer nucleosides of a second type.

[0119] In some embodiments, the nucleoside at position +16 is a 3' mixmer nucleoside of the second type. In some embodiments, the nucleoside at position -8 is a 3' mixmer nucleoside of the second type.

[0120] In some embodiments of the oligonucleotides of the invention, the nucleosides at positions -2 through -10 are a 3' mixmer region, the nucleosides at each of positions -3, -5, -7, and -9 are 3' mixmer nucleosides of a first type, and the nucleosides at each of positions -2, -4, -6, -8, and -10 are 3' mixmer nucleosides of a second type.

[0121] In some embodiments of the oligonucleotides of the present invention, the nucleosides at positions +2 through +24 are a 5' mixmer region, the nucleosides at each of positions +3, +5, +7, +9, +11, +13, +15, +17, +19, +21, and +23 are 5' mixmer nucleosides of a first type, and the nucleosides at each of positions +2, +4, +6, +8, +10, +12, +14, +16, +18, +20, +22, and +24 are 5' mixmer nucleosides of a second type.

[0122] In some embodiments of the oligonucleotides of the invention, the nucleosides at positions -2 through -15 are a 3' mixmer region, the nucleosides at each of positions -3, -5, -7, -9, -11, -13, and -15 are 3' mixmer nucleosides of a first type, and the nucleosides at each of positions -2, -4, -6, -8, -10, -12, and -14 are 3' mixmer nucleosides of a second type.

[0123] Mixumare nucleoside type In some embodiments of the oligonucleotides of the present invention, the mixmer region comprises a first type of mixmer nucleoside and a second type of mixmer nucleoside, and the sugar moiety of the first type of mixmer nucleoside is different from the sugar moiety of the second type of mixmer nucleoside.

[0124] In some embodiments, the first type of Mixmir nucleoside is selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, 2'-O-methoxyethyl-RNA (MOE-RNA), 2'-fluoro-RNA, linked nucleic acid (LNA), arabinonucleic acid (ANA), and 2'-fluoroarabinonucleic acid (FANA) nucleosides. In some embodiments, the first type of Mixmir nucleoside is selected from the group consisting of RNA, 2'-O-methyl-RNA, and 2'-fluoro-RNA nucleosides. In some embodiments, the first type of Mixmir nucleoside is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. In some embodiments, the first type of Mixmir nucleoside is a 2'-fluoro-RNA nucleoside.

[0125] In some embodiments, the second type of Mixmir nucleoside is selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, MOE-RNA, 2'-fluoro-RNA, LNA, ANA, and FANA nucleosides. In some embodiments, the second type of Mixmir nucleoside is selected from the group consisting of RNA, 2'-O-methyl-RNA, and 2'-fluoro-RNA nucleosides. In some embodiments, the second type of Mixmir nucleoside is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. In some embodiments, the second type of Mixmir nucleoside is a 2'-O-methyl-RNA nucleoside.

[0126] In some embodiments, the first type of 5'-mixmir nucleoside is selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, MOE-RNA, 2'-fluoro-RNA, LNA, ANA, and FANA nucleosides. In some embodiments, the first type of 5'-mixmir nucleoside is selected from the group consisting of RNA, 2'-O-methyl-RNA, and 2'-fluoro-RNA nucleosides. In some embodiments, the first type of 5'-mixmir nucleoside is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. In some embodiments, the first type of 5'-mixmir nucleoside is a 2'-fluoro-RNA nucleoside.

[0127] In some embodiments, the second type of 5'-mixmir nucleoside is selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, MOE-RNA, 2'-fluoro-RNA, LNA, ANA, and FANA nucleosides. In some embodiments, the second type of 5'-mixmir nucleoside is selected from the group consisting of RNA, 2'-O-methyl-RNA, and 2'-fluoro-RNA nucleosides. In some embodiments, the second type of 5'-mixmir nucleoside is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. In some embodiments, the second type of 5'-mixmir nucleoside is a 2'-O-methyl-RNA nucleoside.

[0128] In some embodiments, the first type of 3'-mixmer nucleoside is selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, MOE-RNA, 2'-fluoro-RNA, LNA, ANA, and FANA nucleosides. In some embodiments, the first type of 3'-mixmer nucleoside is selected from the group consisting of RNA, 2'-O-methyl-RNA, and 2'-fluoro-RNA nucleosides. In some embodiments, the first type of 3'-mixmer nucleoside is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. In some embodiments, the first type of 3'-mixmer nucleoside is a 2'-fluoro-RNA nucleoside.

[0129] In some embodiments, the second type of 3'-mixmer nucleoside is selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, MOE-RNA, 2'-fluoro-RNA, LNA, ANA, and FANA nucleosides. In some embodiments, the second type of 3'-mixmer nucleoside is selected from the group consisting of RNA, 2'-O-methyl-RNA, and 2'-fluoro-RNA nucleosides. In some embodiments, the second type of 3'-mixmer nucleoside is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. In some embodiments, the second type of 3'-mixmer nucleoside is a 2'-O-methyl-RNA nucleoside.

[0130] In some embodiments, (a) the first type of 5'-mixmer nucleoside is selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, MOE-RNA, 2'-fluoro-RNA, LNA, ANA, and FANA nucleosides; and / or (b) the second type of 5'-mixmer nucleoside is selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, MOE-RNA, 2'-fluoro-RNA, LNA, ANA, and FANA nucleosides; and / or (c) the first type of 3'-mixmer nucleoside is selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, MOE-RNA, 2'-fluoro-RNA, LNA, ANA, and FANA nucleosides; and / or (d) The second type of 3'mixmer nucleoside is selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, MOE-RNA, 2'-fluoro-RNA, LNA, ANA, and FANA nucleosides.

[0131] In some embodiments, the first type of 5'-mixmer nucleoside is a 2'-fluoro-RNA nucleoside, and / or the second type of 5'-mixmer nucleoside is a 2'-O-methyl-RNA nucleoside, and / or the first type of 3'-mixmer nucleoside is a 2'-fluoro-RNA nucleoside, and / or the second type of 3'-mixmer nucleoside is a 2'-O-methyl-RNA nucleoside.

[0132] In some embodiments, the first type of mixmir nucleoside is a 2'-fluoro-RNA nucleoside and the second type of mixmir nucleoside is a 2'-O-methyl-RNA nucleoside. In some embodiments, the first type of mixmir nucleoside is a 2'-O-methyl-RNA nucleoside and the second type of mixmir nucleoside is a 2'-fluoro-RNA nucleoside. In some embodiments, the first type of 5' mixmir nucleoside is a 2'-fluoro-RNA nucleoside and the second type of 5' mixmir nucleoside is a 2'-O-methyl-RNA nucleoside, the first type of 3' mixmir nucleoside is a 2'-fluoro-RNA nucleoside and the second type of 3' mixmir nucleoside is a 2'-O-methyl-RNA nucleoside.

[0133] The different types of sugar-modified nucleosides listed above are described in detail elsewhere herein.

[0134] 2'-Fluoro-RNA and 2'-O-methyl-RNA mixmer regions In some embodiments, the mixmer region comprises 2'-fluoro-RNA nucleosides and 2'-O-methyl-RNA nucleosides. In some embodiments, the mixmer region consists of 2'-fluoro-RNA nucleosides and 2'-O-methyl-RNA nucleosides. In some embodiments, each nucleoside in the mixmer region is either a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. In some embodiments, the mixmer region comprises alternating 2'-fluoro-RNA nucleosides and 2'-O-methyl-RNA nucleosides. In some embodiments, the mixmer region consists of alternating 2'-fluoro-RNA nucleosides and 2'-O-methyl-RNA nucleosides.

[0135] In some embodiments, the 5' mixmer region comprises 2'-fluoro-RNA nucleosides and 2'-O-methyl-RNA nucleosides. In some embodiments, the 5' mixmer region consists of 2'-fluoro-RNA nucleosides and 2'-O-methyl-RNA nucleosides. In some embodiments, each nucleoside in the 5' mixmer region is either a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. In some embodiments, the 5' mixmer region comprises alternating 2'-fluoro-RNA nucleosides and 2'-O-methyl-RNA nucleosides. In some embodiments, the 5' mixmer region consists of alternating 2'-fluoro-RNA nucleosides and 2'-O-methyl-RNA nucleosides.

[0136] In some embodiments, the 3' mixmer region comprises 2'-fluoro-RNA nucleosides and 2'-O-methyl-RNA nucleosides. In some embodiments, each nucleoside in the 3' mixmer region is either a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. In some embodiments, the 3' mixmer region comprises alternating 2'-fluoro-RNA nucleosides and 2'-O-methyl-RNA nucleosides. In some embodiments, the 3' mixmer region consists of alternating 2'-fluoro-RNA nucleosides and 2'-O-methyl-RNA nucleosides.

[0137] In some embodiments, each nucleoside at positions +2 through +19 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. In some embodiments, the nucleoside at position +2 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +3 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +4 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +5 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. and / or the nucleoside at position +6 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +7 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +8 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. and / or the nucleoside at position +9 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +10 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +11 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +12 is a 2'-fluoro- an RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +13 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +14 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +15 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside;and / or the nucleoside at position +16 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +17 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +18 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +19 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside.

[0138] In some embodiments, each nucleoside at positions -2 through -9 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. In some embodiments, the nucleoside at position -2 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position -3 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position -4 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position -5 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. and / or the nucleoside at position-6 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position-7 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position-8 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position-9 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside.

[0139] In some embodiments, each nucleoside at positions +2 through +24 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. In some embodiments, the nucleoside at position +2 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +3 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +4 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +5 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. and / or the nucleoside at position +6 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +7 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +8 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. and / or the nucleoside at position +9 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +10 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +11 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +12 is a 2'-fluoro-R and / or the nucleoside at position +13 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +14 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +15 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside;and / or the nucleoside at position +16 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +17 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +18 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +19 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +20 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. A nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +21 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +22 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +23 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +24 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside.

[0140] In some embodiments, each nucleoside at positions -2 through -15 is a 2'-O-methyl-RNA nucleoside or a 2'-fluoro-RNA nucleoside. In some embodiments, the nucleoside at position -2 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position -3 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position -4 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position -5 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. and / or the nucleoside at position-6 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position-7 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position-8 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. and / or the nucleoside at position-9 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position-10 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position-11 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position-12 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside. A nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position -13 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position -14 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position -15 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside.

[0141] A 2'-fluoro-RNA nucleoside or 2'-O-methyl-RNA nucleoside at a specific position increases editing efficiency. Thus, in some embodiments, the nucleoside at position -2 is a 2'-O-methyl-RNA nucleoside. In some embodiments, the nucleoside at position -3 is a 2'-fluoro-RNA nucleoside. In some embodiments, the nucleoside at position -8 is a 2'-fluoro-RNA nucleoside. In some embodiments, the nucleoside at position +16 is a 2'-fluoro-RNA nucleoside. In some embodiments, the nucleoside at position +17 is a 2'-fluoro-RNA nucleoside.

[0142] In some embodiments, the nucleoside at one or more of positions +3, +5, +7, +9, +11, +13, +15, +17 and +19 is a 2'-fluoro-RNA nucleoside. In some embodiments, the nucleoside at position +3 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position +5 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position +7 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position +9 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position +11 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position +13 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position +15 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position +17 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position +19 is a 2'-fluoro-RNA nucleoside. In some embodiments, the nucleoside at each of positions +3, +5, +7, +9, +11, +13, +15, +17 and +19 is a 2'-fluoro-RNA nucleoside.

[0143] In some embodiments, the nucleoside at one or more of positions -3, -5, -7, and -9 is a 2'-fluoro-RNA nucleoside. In some embodiments, the nucleoside at position -3 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position -5 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position -7 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position -9 is a 2'-fluoro-RNA nucleoside. In some embodiments, the nucleoside at each of positions -3, -5, -7, and -9 is a 2'-fluoro-RNA nucleoside.

[0144] In some embodiments, the nucleoside at one or more of positions +2, +4, +6, +8, +10, +12, +14 and +18 is a 2'-O-methyl-RNA nucleoside. In some embodiments, the nucleoside at position +2 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +4 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +6 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +8 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +10 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +12 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +14 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +18 is a 2'-O-methyl-RNA nucleoside. In some embodiments, the nucleoside at each of positions +2, +4, +6, +8, +10, +12, +14 and +18 is a 2'-O-methyl-RNA nucleoside.

[0145] In some embodiments, the nucleoside at one or more of positions -2, -4, and -6 is a 2'-O-methyl-RNA nucleoside. In some embodiments, the nucleoside at position -2 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position -4 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position -6 is a 2'-O-methyl-RNA nucleoside. In some embodiments, the nucleoside at each of positions -2, -4, and -6 is a 2'-O-methyl-RNA nucleoside.

[0146] In some embodiments, the nucleoside at one or more of positions +3, +5, +7, +9, +11, +13, +15, +17, +19, +21, and +23 is a 2'-fluoro-RNA nucleoside. In some embodiments, the nucleoside at position +3 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position +5 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position +7 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position +9 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position +11 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position +13 is a 2'-fluoro-RNA nucleoside. and / or the nucleoside at position +15 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position +17 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position +19 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position +21 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position +23 is a 2'-fluoro-RNA nucleoside. In some embodiments, the nucleoside at each of positions +3, +5, +7, +9, +11, +13, +15, +17, +19, +21, and +23 is a 2'-fluoro-RNA nucleoside.

[0147] In some embodiments, the nucleoside at one or more of positions -3, -5, -7, -9, -11, -13, and -15 is a 2'-fluoro-RNA nucleoside. In some embodiments, the nucleoside at position -3 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position -5 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position -7 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position -9 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position -11 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position -13 is a 2'-fluoro-RNA nucleoside, and / or the nucleoside at position -15 is a 2'-fluoro-RNA nucleoside. In some embodiments, the nucleoside at each of positions -3, -5, -7, -9, -11, -13, and -15 is a 2'-fluoro-RNA nucleoside.

[0148] In some embodiments, the nucleoside at one or more of positions +2, +4, +6, +8, +10, +12, +14, +18, +20, +22, and +24 is a 2'-O-methyl-RNA nucleoside. In some embodiments, the nucleoside at position +2 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +4 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +6 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +8 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +10 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +12 is a 2'-O-methyl-RNA nucleoside. and / or the nucleoside at position +14 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +18 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +20 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +22 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position +24 is a 2'-O-methyl-RNA nucleoside. In some embodiments, the nucleoside at each of positions +2, +4, +6, +8, +10, +12, +14, +18, +20, +22, and +24 is a 2'-O-methyl-RNA nucleoside.

[0149] In some embodiments, the nucleoside at one or more of positions -2, -4, -6, -10, -12, and -14 is a 2'-O-methyl-RNA nucleoside. In some embodiments, the nucleoside at position -2 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position -4 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position -6 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position -10 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position -12 is a 2'-O-methyl-RNA nucleoside, and / or the nucleoside at position -14 is a 2'-O-methyl-RNA nucleoside. In some embodiments, the nucleoside at each of positions -2, -4, -6, -10, -12 and -14 is a 2'-O-methyl-RNA nucleoside.

[0150] In some embodiments, the nucleoside at position -8 is a 2'-O-methyl-RNA nucleoside. In some embodiments, the nucleoside at position +16 is a 2'-O-methyl-RNA nucleoside.

[0151] Sugar-modified nucleosides In some embodiments, the oligonucleotide of the present invention comprises one or more sugar-modified nucleosides. In other words, the oligonucleotide of the present invention can comprise one or more nucleosides with modified sugar moieties, i.e., sugar moieties modified compared to the ribose sugar moiety found in DNA and RNA. In particular, as described herein, the mixer region of the oligonucleotide of the present invention comprises sugar-modified nucleosides.

[0152] Numerous nucleosides with modifications in the ribose sugar moiety have been developed primarily with the goal of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance.

[0153] Such modifications include those in which the ribose ring structure has been modified, for example, by replacing it with a hexose ring (HNA), or a bicyclic ring (LNA) typically having a biradical bridge between the C2 and C4 carbons on the ribose ring, or an unlinked ribose ring (e.g., UNA) typically lacking a bond between the C2 and C3 carbons. Other sugar-modified nucleosides include, for example, bicyclohexose nucleic acids (WO 2011 / 017521) or tricyclic nucleic acids (WO 2013 / 154798). Modified nucleosides also include nucleosides in which the sugar moiety has been replaced with a non-sugar moiety, for example, in the case of peptide nucleic acids (PNAs) or morpholino nucleic acids.

[0154] Sugar modifications also include modifications made by changing the substituent on the ribose ring to a group other than hydrogen or to the 2'-OH group naturally occurring in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4', or 5' position.

[0155] 2' sugar-modified nucleosides In some embodiments, the oligonucleotide of the present invention comprises one or more 2' sugar-modified nucleosides, which are nucleosides having a substituent other than -H or -OH at the 2' position (2'-substituted nucleosides), or nucleosides containing a 2'-linked biradical that can form a bridge between the 2' carbon and a second carbon of the ribose ring, such as LNA (2'-4' biradical bridge) nucleosides.

[0156] In fact, much attention has been paid to the development of 2' sugar-substituted nucleosides, and many 2'-substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides. For example, 2'-modified sugars can confer improved binding affinity and / or increased nuclease resistance to oligonucleotides. Examples of 2'-substituted modified nucleosides include 2'-O-alkyl-RNA, 2'-O-methyl-RNA (2'OMe), 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleosides. For further examples, see, e.g., Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development 2000, 3(2), 203-213, and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are examples of some 2'-substituted modified nucleosides. TIFF2025525564000007.tif76129

[0157] Locked nucleic acid nucleosides (LNA nucleosides) In some embodiments, the oligonucleotide comprises one or more LNA nucleosides.

[0158] An "LNA nucleoside" is a 2'-modified nucleoside containing a biradical (also referred to as a "2'-4' bridge") linking C2' and C4' of the ribose sugar ring of the nucleoside, restricting or locking the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNAs). Locking the conformation of the ribose is associated with improved hybridization affinity (duplex stabilization) when LNAs are incorporated into oligonucleotides of complementary RNA or DNA molecules. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complementary duplex.

[0159] Non-limiting exemplary LNA nucleosides include those described in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, WO 2004 / 046160, WO 00 / 047599, WO 2007 / 134181, WO 2010 / 077578, WO 2010 / 036698, WO 2007 / 090071, WO 2009 / 006478, WO 2011 / 156202, WO 2008 / 154401, WO 2009 / 067647, WO 2008 / 150729, Morita et al. al., Bioorganic & Med. Chem. Lett., 12, 73-76, Seth et al., J. Org. Chem., 2010, Vol 75(5) pp. 1569-81, Mitsuoka et al., Nucleic Acids Research, 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry, 2016, 59, 9645-9667.

[0160] Further non-limiting exemplary LNA nucleosides are disclosed in Scheme 1. Scheme 1: TIFF2025525564000008.tif164152

[0161] Particular LNA nucleosides are β-D-oxy-LNA, 6′-methyl-β-D-oxy LNA, such as (S)-6′-methyl-β-D-oxy-LNA (ScET) and ENA.

[0162] In some embodiments, the oligonucleotide comprises one or more LNA β-D-oxy-LNA nucleosides.

[0163] Oligonucleotide length In some embodiments, the oligonucleotides of the invention are 30 to 61 nucleosides in length, 30 to 50 nucleosides in length, 35 to 45 nucleosides in length.

[0164] In some embodiments, the oligonucleotide is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleosides in length. In some embodiments, the oligonucleotide is 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleosides in length. In some embodiments, the oligonucleotide is 40 nucleosides in length.

[0165] Oligonucleotide Symmetry The term "symmetry" refers to the number of nucleosides on both sides of the editing nucleoside in an oligonucleotide.In other words, the symmetry of an oligonucleotide represents the number of nucleosides 5' to the editing nucleoside and the number of nucleosides 3' to the editing nucleoside.The symmetry of the oligonucleotide of the present invention can be described herein using the formula "X-1-Y", where "X" is the number of nucleosides 5' to the editing nucleoside, "1" represents the editing nucleoside, and "Y" is the number of nucleosides 3' to the editing nucleoside.For example, an oligonucleotide with 24-1-15 symmetry is 40 nucleosides long, and has exactly 24 nucleosides 5' to the editing nucleoside and exactly 15 nucleosides 3' to the editing nucleoside.

[0166] In some embodiments, the oligonucleotide comprises 20-30 nucleosides 5' to the edited nucleoside. In some embodiments, the oligonucleotide comprises 22-29 nucleosides 5' to the edited nucleoside. In some embodiments, the oligonucleotide comprises 24-27 nucleosides 5' to the edited nucleoside. In some embodiments, the oligonucleotide comprises 24 nucleosides 5' to the edited nucleoside.

[0167] In some embodiments, the oligonucleotide comprises exactly 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleosides 5' to the edited nucleoside. In some embodiments, the oligonucleotide comprises exactly 22, 23, 24, 25, 26, 27, 28, or 29 nucleosides 5' to the edited nucleoside. In some embodiments, the oligonucleotide comprises exactly 24, 25, 26, or 27 nucleosides 5' to the edited nucleoside. In some embodiments, the oligonucleotide comprises exactly 24 nucleosides 5' to the edited nucleoside.

[0168] In some embodiments, the oligonucleotide comprises 4 to 20 nucleosides 3' to the edited nucleoside. In some embodiments, the oligonucleotide comprises 12 to 15 nucleosides 3' to the edited nucleoside. In some embodiments, the oligonucleotide comprises 15 nucleosides 3' to the edited nucleoside.

[0169] In some embodiments, the oligonucleotide comprises exactly 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides 3' to the edited nucleoside. In some embodiments, the oligonucleotide comprises exactly 12, 13, 14, or 15 nucleosides 3' to the edited nucleoside. In some embodiments, the oligonucleotide comprises exactly 15 nucleosides 3' to the edited nucleoside.

[0170] In some embodiments, the oligonucleotide contains exactly 22-29 nucleosides 5' to the edited nucleoside and 12-15 nucleosides 3' to the edited nucleoside, hi some embodiments, the oligonucleotide contains 24-27 nucleosides 5' to the edited nucleoside and 14 or 15 nucleosides 3' to the edited nucleoside.

[0171] In some embodiments, the oligonucleotide comprises exactly 22, 23, 24, 25, 26, 27, 28, or 29 nucleosides 5' to the edited nucleoside and exactly 12, 13, 14, or 15 nucleosides 3' to the edited nucleoside, hi some embodiments, the oligonucleotide comprises 24, 25, 26, or 27 nucleosides 5' to the edited nucleoside and exactly 14 or 15 nucleosides 3' to the edited nucleoside.

[0172] In some embodiments, the oligonucleotide comprises 21-30 nucleosides 5' to the edited nucleoside and 15 nucleosides 3' to the edited nucleoside. In some embodiments, the oligonucleotide comprises exactly 22-27 nucleosides 5' to the edited nucleoside and 15 nucleosides 3' to the edited nucleoside. In some embodiments, the oligonucleotide comprises 24 nucleosides 5' to the edited nucleoside and 15 nucleosides 3' to the edited nucleoside.

[0173] In some embodiments, the oligonucleotide comprises exactly 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleosides 5' to the edited nucleoside and exactly 15 nucleosides 3' to the edited nucleoside. In some embodiments, the oligonucleotide comprises exactly 22, 23, 24, 25, 26, or 27 nucleosides 5' to the edited nucleoside and exactly 15 nucleosides 3' to the edited nucleoside. In some embodiments, the oligonucleotide comprises exactly 24 nucleosides 5' to the edited nucleoside and exactly 15 nucleosides 3' to the edited nucleoside.

[0174] In some embodiments, the oligonucleotide comprises exactly 25 nucleosides 5' to the edited nucleoside and exactly 14 nucleosides 3' to the edited nucleoside. In some embodiments, the oligonucleotide comprises exactly 26 nucleosides 5' to the edited nucleoside and exactly 13 nucleosides 3' to the edited nucleoside. In some embodiments, the oligonucleotide comprises exactly 27 nucleosides 5' to the edited nucleoside and exactly 12 nucleosides 3' to the edited nucleoside. In some embodiments, the oligonucleotide comprises exactly 28 nucleosides 5' to the edited nucleoside and exactly 11 nucleosides 3' to the edited nucleoside. In some embodiments, the oligonucleotide comprises exactly 29 nucleosides 5' to the edited nucleoside and exactly 10 nucleosides 3' to the edited nucleoside.

[0175] Frank region In some embodiments, the oligonucleotide of the present invention comprises one or more flanking regions. The term "flanking region" refers to a stretch of nucleosides in an oligonucleotide that is flanking the editing nucleoside and the mixer region. In other words, the flanking region is outside both the editing region and the mixer region in the oligonucleotide. In other words, the flanking region is either 5' or 3' to both the editing region and the mixer region.

[0176] In some embodiments, the oligonucleotide comprises a 5' flanking region located 5' of the 5' mixmer region. In some embodiments, the oligonucleotide comprises a 3' flanking region located 3' of the 3' mixmer region. In some embodiments, the oligonucleotide comprises a 5' flanking region located 5' of the 5' mixmer region and a 3' flanking region located 3' of the 3' mixmer region.

[0177] In some embodiments, the 5'-flank region is located immediately 5' to the 5'-mixmer region. In other words, there is no additional nucleoside between the 3'-most nucleoside of the 5'-flank region and the 5'-most nucleoside of the 5'-mixmer region; the 3'-most nucleoside of the 5'-flank region and the 5'-most nucleoside of the 5'-mixmer region are connected by an internucleoside bond. In some embodiments, the 3'-flank region is located immediately 3' to the 3'-mixmer region. In other words, there is no additional nucleoside between the 5'-most nucleoside of the 3'-flank region and the 3'-most nucleoside of the 3'-mixmer region; the 5'-most nucleoside of the 3'-flank region and the 3'-most nucleoside of the 3'-mixmer region are connected by an internucleoside bond. In some embodiments, the 5' flanking region is located immediately 5' to the 5' mixmer region and the 3' flanking region is located immediately 3' to the 3' mixmer region.

[0178] In some embodiments, the oligonucleotide comprises the following structure: X +2 -X +1 -X 0 -X -1 -X -2 During the ceremony, X +2 is the 5' flanking region, X +1 is the 5' mixmer region, X 0 is the editing region, X -1 is the 3' mixmer region, X -2 is the 3' flanking region.

[0179] In other words, in some embodiments, the 5' flanking region is immediately 5' to the 5' mixmer region, which is immediately 5' to the editing nucleoside, which is immediately 5' to the 3' mixmer region, which is immediately 5' to the 3' flanking region. Thus, the 5' flanking region, 5' mixmer region, editing region, 3' mixmer region and 3' flanking region are contiguous (i.e., linked by internucleoside linkages).

[0180] In some embodiments, the 5' flanking region is 1, 2, 3, 4, or 5 nucleosides in length. In some embodiments, the 5' flanking region is 5 nucleosides in length. In some embodiments, the 3' flanking region is 1, 2, 3, 4, or 5 nucleosides in length. In some embodiments, the 3' flanking region is 5 nucleosides in length. In some embodiments, the 5' flanking region and the 3' flanking region are each 5 nucleosides in length.

[0181] In some embodiments, the flanking region is at the end of the oligonucleotide. In some embodiments, the 5'-flank region is at the 5'-end of the oligonucleotide. In other words, in some embodiments, the 5'-most nucleoside of the oligonucleotide is the 5'-flank region. In some embodiments, the 3'-flank region is at the 3'-end of the oligonucleotide. In other words, in some embodiments, the 3'-most nucleoside of the oligonucleotide is the 3'-flank region. In some embodiments, the 5'-flank region consists of 1, 2, 3, 4, or 5 nucleosides at the 5'-end of the oligonucleotide. In some embodiments, the 5'-flank region consists of 5 nucleosides at the 5'-end of the oligonucleotide. In some embodiments, the 3'-flank region consists of 1, 2, 3, 4, or 5 nucleosides at the 3'-end of the oligonucleotide. In some embodiments, the 3'-flank region consists of 5 nucleosides at the 3'-end of the oligonucleotide.

[0182] In some embodiments, the 5'-flank region comprises one or more sugar-modified nucleosides. In some embodiments, the 3'-flank region comprises one or more sugar-modified nucleosides. In some embodiments, the 5'-flank region and the 3'-flank region each comprise one or more sugar-modified nucleosides. In some embodiments, the one or more sugar-modified nucleosides in the 5'-flank region or the 3'-flank region are independently selected from the group consisting of 2'-O-methyl-RNA, 2'-fluoro-RNA, 2'-O-methoxyethyl-RNA (MOE-RNA), and LNA nucleosides.

[0183] In some embodiments, all nucleosides in the 5' flanking region are sugar-modified nucleosides. In some embodiments, all nucleosides in the 3' flanking region are sugar-modified nucleosides. In some embodiments, all nucleosides in both the 5' flanking region and the 3' flanking region are sugar-modified nucleosides. In some embodiments, all nucleosides in the 5' flanking region are 2'-O-methyl-RNA nucleosides. In some embodiments, all nucleosides in the 3' flanking region are 2'-O-methyl-RNA nucleosides. In some embodiments, all nucleosides in both the 5' flanking region and the 3' flanking region are 2'-O-methyl-RNA nucleosides.

[0184] In some embodiments, the 5' flanking region consists of five 2'-O-methyl-RNA nucleosides. In some embodiments, the 3' flanking region consists of five 2'-O-methyl-RNA nucleosides. In some embodiments, the 5' flanking region consists of five 2'-O-methyl-RNA nucleosides and the 3' flanking region consists of five 2'-O-methyl-RNA nucleosides.

[0185] In some embodiments, the 5'-flank region consists of five 2'-O-methyl-RNA nucleosides at the 5'-end of the oligonucleotide. In some embodiments, the 3'-flank region consists of five 2'-O-methyl-RNA nucleosides at the 3'-end of the oligonucleotide. In some embodiments, the 5'-flank region consists of five 2'-O-methyl-RNA nucleosides at the 5'-end of the oligonucleotide, and the 3'-flank region consists of five 2'-O-methyl-RNA nucleosides at the 3'-end of the oligonucleotide.

[0186] In some embodiments, the 5'-flank region comprises nucleosides at positions +20 to +24 (i.e., positions +20, +21, +22, +23, and +24). In some embodiments, the nucleosides at positions +20 to +24 are 2'-O-methyl-RNA nucleosides. In some embodiments, the 3'-flank region comprises nucleosides at positions -11 to -15 (i.e., positions -11, -12, -13, -14, and -15). In some embodiments, the nucleosides at positions -11 to -15 are 2'-O-methyl-RNA nucleosides.

[0187] Modified internucleoside linkages In some embodiments, oligonucleotides of the invention may contain one or more modified internucleoside linkages.

[0188] The term "modified internucleoside linkage" is defined as a linkage other than a phosphodiester (PO) bond that covalently joins two nucleosides to one another as commonly understood by those skilled in the art.

[0189] In some embodiments, all internucleoside linkages in the oligonucleotide are modified internucleoside linkages.

[0190] In some embodiments, each modified internucleoside bond is independently selected from the group consisting of phosphorothioate internucleoside bond and phosphorodithioate internucleoside bond.In some embodiments, the oligonucleotide comprises one or more phosphorothioate internucleoside bond.In some embodiments, all internucleoside bond of the oligonucleotide is phosphorothioate internucleoside bond.

[0191] Phosphorothioate internucleoside linkages and phosphorodithioate internucleoside linkages are useful because they can make oligonucleotides more resistant to degradation by nucleases.In phosphorothioate internucleoside linkages, compared to naturally occurring phosphodiester internucleoside linkages, one of the oxygen atoms of the phosphate group that is not bonded to the carbon of the nucleoside sugar moiety is replaced with a sulfur atom.In phosphorodithioate internucleoside linkages, each of the two oxygen atoms of the phosphate group that is not bonded to the carbon of the nucleoside sugar moiety is replaced with a sulfur atom.Therefore, when a phosphodiester linkage can be represented by the formula -OP(O)2-O-, a phosphorothioate internucleoside linkage can be represented by the formula -OP(O,S)-O-, and a phosphorodithioate internucleoside linkage can be represented by the formula -OP(S)2-O-.

[0192] Phosphorothioate internucleoside linkages are chiral (see, for example, Jahns et al. 2022 Nucleic Acids Research Vol. 50, No. 3, 1221-1240) and have right-handed (Rp) and left-handed (Sp) isomers. The Rp diastereomer may be referred to as an R-PS internucleoside linkage or an srP internucleoside linkage. The Sp diastereomer may be referred to as an S-PS internucleoside linkage or an ssP internucleoside linkage. In some embodiments, an oligonucleotide contains one or more srP internucleoside linkages. In some embodiments, an oligonucleotide contains one or more ssP internucleoside linkages. When the chirality of a phosphorothioate internucleoside linkage is not specified, the phosphorothioate internucleoside linkage may be either an srP linkage or an ssP linkage. The structures of srP and ssP linkages are shown below: TIFF2025525564000009.tif52128.

[0193] Particular types of internucleoside linkages between particular nucleosides may improve the editing efficiency of oligonucleotides.

[0194] In some embodiments, the internucleoside linkages between nucleosides at positions +1 and +0 are phosphodiester internucleoside linkages. In some embodiments, the internucleoside linkages between nucleosides at positions +1 and +0 are phosphodiester internucleoside linkages, and all other internucleoside linkages are phosphorothioate internucleoside linkages.

[0195] In some embodiments, the internucleoside linkages between nucleosides at positions 0 and -1 are phosphodiester internucleoside linkages. In some embodiments, the internucleoside linkages between nucleosides at positions 0 and -1 are phosphodiester internucleoside linkages, and all other internucleoside linkages are phosphorothioate internucleoside linkages.

[0196] In some embodiments, the internucleoside linkage between the nucleosides at positions -1 and -2 is a phosphorothioate internucleoside linkage.

[0197] In some embodiments, the internucleoside linkages between nucleosides at positions -2 and -3 are phosphodiester internucleoside linkages, hi some embodiments, the internucleoside linkages between nucleosides at positions -2 and -3 are phosphodiester internucleoside linkages, and all other internucleoside linkages are phosphorothioate internucleoside linkages.

[0198] In some embodiments, all internucleoside linkages in the oligonucleotide are phosphorothioate internucleoside linkages, except for: the internucleoside linkages between the nucleosides at positions +1 and +0 are phosphodiester internucleoside linkages, and / or the internucleoside linkages between the nucleosides at positions 0 and -1 are phosphodiester internucleoside linkages, and / or The internucleoside linkages between the nucleosides at positions -2 and -3 are phosphodiester internucleoside linkages.

[0199] Complementarity In some embodiments, the oligonucleotide of the present invention is complementary to a target nucleic acid. In some embodiments, the oligonucleotide of the present invention comprises or consists of a sequence complementary to the target nucleic acid. In some embodiments, the target nucleic acid is SERPINA1 mRNA. Thus, in some embodiments, the oligonucleotide of the present invention is complementary to SERPINA1 mRNA. In some embodiments, the oligonucleotide of the present invention comprises or consists of a sequence complementary to SERPINA1 mRNA.

[0200] The term "complementarity" describes the Watson-Crick base pairing ability of nucleosides / nucleotides. Watson-Crick base pairs are cytosine-guanine (CG) and adenine-thymine / uracil (AT / U). It is understood that oligonucleotides may contain nucleosides with modified nucleobases. For example, 5-methylcytosine (E) may be used in place of cytosine, and 7-deaza-8-azaguanine (F) may be used in place of guanine. The term "complementarity" encompasses Watson-Crick base pairing between unmodified and modified nucleobases (see, e.g., Hirao et al., 2012, Accounts of Chemical Research, 45, 2055 and Bergstrom, 2009, Curr. Protoc. Nucleic Acid Chem., 37, 1.4.1). In particular, Watson-Crick base pairing encompasses EG, CF, and EF base pairs. Thus, an oligonucleotide complementary to a target nucleic acid will base pair with and thereby bind to the target nucleic acid, and an oligonucleotide complementary to SERPINA1 mRNA will base pair with and thereby bind to SERPINA1 mRNA.

[0201] "Complementarity" does not require that the oligonucleotide be complementary to the target nucleic acid over the entire length of the target nucleic acid. The oligonucleotide is typically shorter than the target nucleic acid. Rather, "complementarity" refers to the proportion of nucleobases / nucleotides in the oligonucleotide that base pair with the target nucleic acid. In some embodiments, the oligonucleotide is complementary to a portion of the target nucleic acid. In other words, the oligonucleotide is complementary to a sequence in the target nucleic acid. In some embodiments, the oligonucleotide is complementary to a portion of the target nucleic acid surrounding the target adenosine. In some embodiments, the oligonucleotide is complementary to a portion of the SERPINA1 mRNA. In other words, the oligonucleotide is complementary to a sequence in the SERPINA1 mRNA. In some embodiments, the oligonucleotide is complementary to a portion of the SERPINA1 mRNA surrounding the target adenosine.

[0202] As described herein, A1AD is associated with a single base pair substitution resulting in a mutation of glutamic acid (E)342 to lysine (K) in A1AT (E342K mutation). Thus, in some embodiments, the oligonucleotide is complementary to a portion of the SERPINA1 mRNA surrounding the mutant AAG codon that encodes lysine. In other words, in some embodiments, the oligonucleotide is complementary to a portion of the SERPINA1 mRNA surrounding the target adenosine, which is the first nucleoside of the mutant AAG codon that encodes lysine.

[0203] "Complementarity" does not require that the oligonucleotide is complementary to the continuous sequence in the target nucleic acid. There may be mismatches between the oligonucleotide and the target nucleic acid. Therefore, the complementarity between the oligonucleotide of the present invention and the target nucleic acid may be expressed as a percentage. Furthermore, it should be understood that the target adenosine on the target nucleic acid and the editing nucleoside on the oligonucleotide do not form a base pair. In other words, the target adenosine and the editing nucleoside form a mismatch.

[0204] Thus, in some embodiments, the sequence of the oligonucleotide of the present invention comprises or consists of a sequence having at least 80% complementarity to a sequence in the target nucleic acid, and any matches or mismatches between the editing nucleoside and the target adenosine are not included when determining the percentage of complementarity. In some embodiments, the sequence of the oligonucleotide of the present invention comprises or consists of a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementarity to a sequence in the target nucleic acid, and any matches or mismatches between the editing nucleoside and the target adenosine are not included when determining the percentage of complementarity.

[0205] In some embodiments, the sequence of the oligonucleotide of the present invention comprises or consists of a sequence having at least 80% complementarity to a sequence in SERPINA1 mRNA, and any matches or mismatches between the editing nucleoside and the target adenosine are not included when determining the percentage of complementarity. In some embodiments, the sequence of the oligonucleotide of the present invention comprises or consists of a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementarity to a sequence in SERPINA1 mRNA, and any matches or mismatches between the editing nucleoside and the target adenosine are not included when determining the percentage of complementarity.

[0206] As used herein, the term "% complementary" refers to the percentage of nucleotides in an oligonucleotide of the present invention that are complementary to a sequence in a target nucleic acid, such as SERPINA1 mRNA, excluding base pairing (or lack thereof) between an editing nucleoside and a target adenosine. In other words, any matches or mismatches between an editing nucleoside and a target adenosine are not included when determining the percentage complementary. Therefore, the percentage complementary is calculated by counting the number of aligned nucleobases that are complementary (i.e., form Watson-Crick base pairs) between two sequences, excluding the editing nucleoside and the target adenosine, dividing that number by the total number of nucleotides in the oligonucleotide (excluding the editing nucleoside), and multiplying by 100. In such a comparison, nucleobases / nucleotides that do not align (form base pairs) are referred to as mismatches. Insertions and deletions are not allowed in calculating the percent complementary of a contiguous nucleotide sequence. It will be understood that in determining complementarity, chemical modifications of a nucleobase are disregarded so long as the nucleobase retains the functional ability to form Watson-Crick base pairs (e.g., 5'-methylcytosine is considered to be identical to cytosine, and 7-deaza-8-azaguanine is considered to be identical to guanine for purposes of calculating % complementarity).

[0207] The term "complementary" (such as in phrases like "the oligonucleotide is complementary to a sequence in SERPINA1 mRNA") does not require 100% complementarity. Rather, in the present invention, the term "complementary" requires that the oligonucleotide is at least 75% complementary to the target nucleic acid. In some embodiments, the term "complementary" requires that the oligonucleotide is at least 75% complementary to the SERPINA1 mRNA. In some embodiments, the oligonucleotide is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary, or 100% complementary to the target nucleic acid. In some embodiments, the oligonucleotide is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to SERPINA1 mRNA.

[0208] The term "fully complementary" refers to 100% complementarity. In some embodiments, the oligonucleotide is fully complementary to a sequence in a target nucleic acid. In other words, all nucleobases in the oligonucleotide, except for the nucleobases of the editing nucleosides, form base pairs with the nucleobases of the target nucleic acid. In some embodiments, the oligonucleotide is fully complementary to a sequence in SERPINA1 mRNA. In other words, all nucleobases in the oligonucleotide, except for the nucleobases of the editing nucleosides, form base pairs with the nucleobases of the SERPINA1 mRNA.

[0209] In some embodiments, an oligonucleotide of the invention is complementary to any one of the SERPINA1 mRNA transcripts listed in Table 2 herein. In some embodiments, an oligonucleotide of the invention is complementary to SEQ ID NO: 186 (SERPINA1 mRNA transcript variant 11 (NM_001127707.2) containing the E342K mutation).

[0210] The exemplified edited oligonucleotide compound binds to SEQ ID NO: 186 at positions 986-1062. Thus, in some embodiments, the sequence of an oligonucleotide of the invention comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementarity to the sequence within positions 986-1062 of SEQ ID NO: 186, and any matches or mismatches between the edited nucleoside and the target adenosine are not included when determining the percentage of complementarity. Positions 986-1062 of SEQ ID NO: 186 are represented below as SEQ ID NO: 187. The AAG codon encoding K342 is underlined, and the target adenosine is in bold. The target adenosine is A39 of SEQ ID NO: 187. TIFF2025525564000010.tif11159

[0211] In some embodiments, the sequence of an oligonucleotide of the invention comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementarity to SEQ ID NO: 187, and any matches or mismatches between the editing nucleosides and the target adenosines are not included when determining the percentage of complementarity. The oligonucleotide may form complementary base pairs with any stretch of nucleosides within the sequence of SEQ ID NO: 187.

[0212] array As used herein, the term "sequence" refers to the order of nucleic acid bases in a nucleic acid, such as an oligonucleotide. When used to refer to the oligonucleotide or oligonucleotide conjugate of the present invention, the term "sequence" does not limit the type of sugar moiety of the nucleic acid, nor does it limit the type of internucleoside linkage of the nucleic acid. Thus, a given sequence of an oligonucleotide or oligonucleotide conjugate may contain any type of nucleoside sugar moiety (e.g., RNA, DNA, LNA, 2'-O-methyl-RNA, MOE-RNA as described herein) in any combination, and may contain any type of internucleoside linkage (e.g., phosphodiester, phosphorothioate, phosphorodithioate as described herein). Each reference to a SEQ ID NO (SEQ ID NO.) herein refers to the sequence represented by that SEQ ID NO. Unless otherwise specified, all sequences herein are presented in the 5' to 3' direction, as is conventional in the art.

[0213] In some embodiments of the oligonucleotides of the present invention, the sequence of the oligonucleotide has the following sequence: The oligonucleotide may comprise or consist of 30 to 61 consecutive nucleosides from SEQ ID NO: 87, or a variant of SEQ ID NO: 87 containing exactly one, exactly two, or exactly three single nucleoside substitutions, where I is inosine, and the oligonucleotide comprises positions 38, 39, and 40 of SEQ ID NO: 87. Positions 38, 39, and 40 (underlined above) of SEQ ID NO: 87 are edited triplets. Position 39 of SEQ ID NO: 87 is an edited triplet. The statement that an oligonucleotide sequence "comprises positions 38, 39, and 40" means that the sequence must contain nucleosides corresponding to these positions. For example, it is not permissible for the sequence to contain only nucleosides 5' to position 38 (e.g., positions 1-37) or 3' to position 40 (e.g., positions 41-61). However, it is not necessary that the nucleosides at positions 38, 39, and 40 have the sequence TCI. The nucleosides at these positions can be substituted with different types of nucleosides (within the limit of exactly three nucleoside substitutions throughout the sequence); however, there must be corresponding nucleosides at these positions in the sequence.

[0214] In some embodiments, the sequence of the oligonucleotide comprises or consists of 30 to 50 consecutive nucleosides from SEQ ID NO: 87 or a variant thereof. In some embodiments, the sequence comprises or consists of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive nucleosides from SEQ ID NO: 87 or a variant thereof. In some embodiments, the sequence comprises or consists of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 consecutive nucleosides from SEQ ID NO: 87 or a variant thereof. In some embodiments, the sequence comprises or consists of 40 consecutive nucleosides from SEQ ID NO: 87 or a variant thereof.

[0215] In some embodiments, a variant of SEQ ID NO: 87 comprises exactly one single nucleoside substitution. In some embodiments, a variant of SEQ ID NO: 87 comprises a nucleoside substitution at positions 38, 39 and / or 40 of SEQ ID NO: 87. In some embodiments, a variant of SEQ ID NO: 87 comprises a nucleoside substitution at position 39 of SEQ ID NO: 87.

[0216] Sequence of example compounds In some embodiments, the sequence of an oligonucleotide of the present invention comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 1-86 (shown in Table 3 of Example 1 herein). In some embodiments, the sequence of an oligonucleotide comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to any one of SEQ ID NOs: 1-86. In some embodiments, the sequence of an oligonucleotide comprises the sequence of any one of SEQ ID NOs: 1-86.

[0217] In some embodiments, the sequence of the oligonucleotide of the present invention consists of a sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 86. In some embodiments, the sequence of the oligonucleotide consists of a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to any one of SEQ ID NOs: 1 to 86. In some embodiments, the sequence of the oligonucleotide consists of the sequence of any one of SEQ ID NOs: 1 to 86.

[0218] In some embodiments, the sequence of an oligonucleotide of the invention is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 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, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 and 86. In some embodiments, the sequence of the oligonucleotide is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 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, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 and 86.In some embodiments, the sequence of the oligonucleotide is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 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, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 and 86.

[0219] In some embodiments, the sequence of an oligonucleotide of the invention is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 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, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 and 86. In some embodiments, the sequence of the oligonucleotide is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 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, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 and 86, or a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity to any one ofIn some embodiments, the sequence of the oligonucleotide is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 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, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 and 86.

[0220] In some embodiments, the sequence of an oligonucleotide of the invention comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 3, 7, 8, 9, 10, 19, 20, 21, 22, 23, 26, 29, 30, 31, 32, 33, 34, 35, 36, 41, 42, 43, 45, 60, 61, 62 and 63. In some embodiments, the sequence of the oligonucleotide comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity to any one of SEQ ID NOs: 3, 7, 8, 9, 10, 19, 20, 21, 22, 23, 26, 29, 30, 31, 32, 33, 34, 35, 36, 41, 42, 43, 45, 60, 61, 62 and 63. In some embodiments, the sequence of the oligonucleotide comprises any one of SEQ ID NOs: 2, 3, 7, 8, 9, 10, 19, 20, 21, 22, 23, 26, 29, 30, 31, 32, 33, 34, 35, 36, 41, 42, 43, 45, 60, 61, 62 and 63.

[0221] In some embodiments, the sequence of the oligonucleotide of the present invention consists of a sequence having at least 80% identity to any one of SEQ ID NOs: 3, 7, 8, 9, 10, 19, 20, 21, 22, 23, 26, 29, 30, 31, 32, 33, 34, 35, 36, 41, 42, 43, 45, 60, 61, 62 and 63. In some embodiments, the sequence of the oligonucleotide consists of a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity to any one of SEQ ID NOs: 3, 7, 8, 9, 10, 19, 20, 21, 22, 23, 26, 29, 30, 31, 32, 33, 34, 35, 36, 41, 42, 43, 45, 60, 61, 62 and 63. In some embodiments, the sequence of the oligonucleotide consists of any one of SEQ ID NOs: 3, 7, 8, 9, 10, 19, 20, 21, 22, 23, 26, 29, 30, 31, 32, 33, 34, 35, 36, 41, 42, 43, 45, 60, 61, 62 and 63.

[0222] In some embodiments, the sequence of the oligonucleotide of the invention comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 3, 7, 8, 9, 19, 20, 21, 22, 29, 30, 31, 32, 33, 34, 42, 43, 62, and 63. In some embodiments, the sequence of the oligonucleotide comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to any one of SEQ ID NOs: 3, 7, 8, 9, 19, 20, 21, 22, 29, 30, 31, 32, 33, 34, 42, 43, 62, and 63. In some embodiments, the sequence of the oligonucleotide comprises the sequence of any one of SEQ ID NOs: 3, 7, 8, 9, 19, 20, 21, 22, 29, 30, 31, 32, 33, 34, 42, 43, 62 and 63.

[0223] In some embodiments, the sequence of the oligonucleotide of the invention consists of a sequence having at least 80% identity to any one of SEQ ID NOs: 3, 7, 8, 9, 19, 20, 21, 22, 29, 30, 31, 32, 33, 34, 42, 43, 62, and 63. In some embodiments, the sequence of the oligonucleotide consists of a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to any one of SEQ ID NOs: 3, 7, 8, 9, 19, 20, 21, 22, 29, 30, 31, 32, 33, 34, 42, 43, 62, and 63. In some embodiments, the sequence of the oligonucleotide consists of any one of SEQ ID NOs: 3, 7, 8, 9, 19, 20, 21, 22, 29, 30, 31, 32, 33, 34, 42, 43, 62 and 63.

[0224] In some embodiments, the sequence of an oligonucleotide of the present invention comprises a sequence having at least 80% identity to SEQ ID NO: 32. In some embodiments, the sequence of an oligonucleotide comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 32. In some embodiments, the sequence of an oligonucleotide comprises the sequence of SEQ ID NO: 32. In some embodiments, the sequence of an oligonucleotide consists of a sequence having at least 80% identity to SEQ ID NO: 32. In some embodiments, the sequence of an oligonucleotide consists of a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 32. In some embodiments, the sequence of an oligonucleotide consists of the sequence of SEQ ID NO: 32.

[0225] identity The term "identity" as used herein refers to the percentage (expressed as a percentage) of nucleotides in a nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide, e.g., an oligonucleotide of the present invention) that are identical across the nucleotide sequence compared to a reference sequence. The nucleotide sequence that is compared to a reference sequence can be referred to herein as a "query sequence" in the context of determining sequence identity.

[0226] For determining sequence identity, sequence comparison can be carried out by any method known in the art.Suitably, sequence comparison can be carried out by eye, or more commonly, with the aid of readily available sequence comparison programs.These publicly available computer programs can calculate the sequence identity between two or more sequences.

[0227] Sequence identity can be calculated over a continuous sequence, that is, one sequence is aligned with another sequence, and each nucleotide in one sequence is directly compared with the corresponding nucleotide in the other sequence one nucleotide at a time.This is called " ungapped " alignment.Usually, this ungapped alignment is only carried out over a relatively short number of nucleotides (for example, less than 50 consecutive nucleotides).

[0228] Although this is a very simple and consistent method, it fails to take into account that, for example, in an otherwise identical sequence pair, a single insertion or deletion can cause the next nucleotide to be out of alignment, thus potentially significantly reducing the percentage identity when a global alignment is performed. As a result, most sequence comparison methods are designed to produce an optimal alignment that takes into account possible insertions and deletions without excessively compromising overall identity. This is achieved by inserting "gaps" into the sequence alignment to attempt to maximize local identity.

[0229] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, so that for the same number of identical nucleotides, a sequence alignment with as few gaps as possible (reflecting a higher relatedness between the two compared sequences) achieves a higher score than a sequence alignment with many gaps. An "affine gap cost" is typically used, which imposes a relatively high cost for the existence of a gap and a smaller penalty for each subsequent nucleotide in the gap. This is the most commonly used gap scoring system. A high gap penalty naturally produces an optimized alignment with fewer gaps. Most alignment programs allow for the gap penalty to be modified. However, when using such software for sequence comparison, it is preferable to use the default value.

[0230] Therefore, the calculation of maximum percentage sequence identity requires first generating an optimal alignment, taking into account gap penalties. A suitable computer program for performing such alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. 1984 Nucleic Acids Research 12:387). Examples of other software that can perform sequence comparison include, but are not limited to, the BLAST package (see Ausubel et al. 1999 ibid-Chapter 18), FASTA (Atschul et al. 1990, J. Mol. Biol., 403-410) and the GENEWORKS comparison tool suite. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al. 1999 ibid, pages 7-58 to 7-60). However, it is preferred to use the GCG Bestfit program.

[0231] Suitably, sequence identity may be determined over the entire sequence. Suitably, sequence identity may be determined over the entire query sequence compared to a reference sequence.

[0232] Although the final sequence identity can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is generally used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs generally use either public default values or custom symbol comparison tables, if provided (see user manuals for further details). Preferably, the public default values of the GCG package or, in the case of other software, a default matrix such as BLOSUM62 is used.

[0233] Once the software has produced an optimal alignment, it is possible to calculate percentage sequence identity, which the software typically does as part of the sequence comparison and generates a numerical result.

[0234] In some embodiments, the percentage of identity is calculated by counting the number of identical (matching) aligned nucleobases between the query and reference sequences, dividing that number by the total number of nucleotides in the reference sequence, and multiplying by 100. Thus, the percentage of identity = (match x 100) / length of the reference sequence).

[0235] Thus, in embodiments where an oligonucleotide of the invention or a sequence of an oligonucleotide of the invention is stated to have at least a particular identity to another sequence, the oligonucleotide of the invention or the sequence of an oligonucleotide of the invention is the query sequence, and the other sequence is the reference sequence. In embodiments where an oligonucleotide of the invention or a sequence of an oligonucleotide of the invention is stated to have at least a particular identity to a SEQ ID NO (i.e., the sequence represented by a SEQ ID NO), the oligonucleotide of the invention or the sequence of an oligonucleotide of the invention is the query sequence, and the SEQ ID NO is the reference sequence.

[0236] For example, for an oligonucleotide of the present invention having at least 80% identity with SEQ ID NO: 32, the oligonucleotide of the present invention is the query sequence and SEQ ID NO: 32 (i.e., the sequence represented by the SEQ ID NO:) is the reference sequence. For an oligonucleotide of the present invention having at least 80% identity with SEQ ID NO: 32, the sequence of the oligonucleotide of the present invention is the query sequence and SEQ ID NO: 32 is the reference sequence.

[0237] Suitably, insertions and deletions are not allowed in calculating percentage identity of nucleotide sequences. It will be understood that in determining identity, chemical modifications of nucleobases are disregarded so long as the functional ability of the nucleobase to form Watson-Crick base pairs is retained (e.g., 5-methylcytosine is considered identical to cytosine, and 7-deaza-8-azaguanine is considered identical to guanine for purposes of calculating percent identity).

[0238] compound The term "compound" is used herein to refer to the combination of sequence (i.e., the order of nucleobases), sugar moieties, and internucleoside linkages in the oligonucleotide of the present invention.For a given compound, the nucleobases, sugar moiety types, and internucleoside linkages of each nucleotide are specified.Therefore, the order of sugar moieties and internucleoside linkages is also specified in a given compound.However, unless otherwise indicated, a compound may contain other elements in addition to a specific sequence, sugar moiety, and internucleoside linkage.

[0239] In some embodiments, the term "oligonucleotide" is interchangeable with the term "compound." In other words, in some embodiments, an oligonucleotide of the invention is a compound of the invention, and vice versa.

[0240] Particular compounds are referred to herein using compound identification numbers (CMP numbers) of the form X_Y, where X and Y are each numbers. For each CMP number, X is the number of the SEQ ID NO: corresponding to the compound's sequence. For example, a compound designated CMP number 2_1 has the same nucleobase sequence as SEQ ID NO:2.

[0241] The structures of the compounds referred to herein are shown in Table 4 of Example 1 herein using HELM strings. Another section of this specification explains how to read HELM strings. HELM strings will not be written out again here for brevity, but it will be understood that each reference to a CMP number herein refers to the compound represented by the HELM string for that CMP number, as shown in Table 4. CMP numbers and the HELM string in Table 4 that corresponds to that CMP number are interchangeable.

[0242] In some embodiments, the oligonucleotides of the invention are selected from the group consisting of CMP numbers 1_1, 2_1, 3_1, 3_2, 3_3, 4_1, 5_1, 6_1, 7_1, 8_1, 9_1, 10_1, 11_1, 12_1, 13_1, 14_1, 15_1, 16_1, 17_1, 18_1, 19_1, 20_1, 21_1, 22_1, 23_1, 24_1, 25_1, 26_1, 26_2, 26_3, 26_4, 26_5, 26_6, 26_7, 26_8, 26_9, 27_1, 28_1, 29_1, 30_1, 31_1, 32_1, 32_2, 32_3, 32_4, 32_5, 32_6, 32_7, 32_8, 32_9, 33_1, 33_2, 33_3, 33_4, 33_5, 33_6, 33_7, 33_8, 33_9, 34_1, 34_2, 34_3, 34_4, 34_5, 34_6, 34_7, 34_8, 34_9, 35_1, 35_2, 35_3, 35_4, 35_5, 35_6, 35_7, 35_8, 35_9, 36_1, 36_2, 36_3, 36_4, 36_5, 36_6, 36 2_5, 32_6, 32_7, 32_8, 32_9, 32_10, 32_11, 32_12, 32_13, 32_14, 32_15, 32_16, 32_17, 32_18, 32_19, 32_20, 32_21, 32_22, 32_23, 32_24, 32_25, 32_26 , 32_27, 32_28, 32_29, 32_30, 32_31, 32_32, 32_33, 32_34, 32_35, 32_36, 32_37, 32_38, 32_39, 32_40, 32_41, 32_42, 32_43, 32_44, 32_45, 32_46, 32_4 7, 32_48, 32_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_59, 32_60, 32_61, 32_62, 32_63, 32_64, 32_65, 32_66, 32_67, 32_ 68, 32_69, 32_70, 32_71, 32_72, 32_73, 32_74, 32_75, 32_76, 32_77, 32_78, 32_79, 32_80, 32_81, 32_82, 32_83, 33_1, 34_1, 35_1, 36_1, 37_1, 38_1, 39 _1, 40_1, 40_2, 40_3, 40_4, 40_5, 40_6, 40_7, 40_8, 40_9, 40_10, 41_1, 41_2, 41_3, 41_4, 41_5, 41_6, 41_7, 41_8, 41_9, 41_10, 42_1, 42_2, 42_3, 42_4, 42_5, 42_6, 43_1, 43_2, 43_3, 44_1, 45_1, 46_1, 47_1, 47_2, 48_1, 49_1, 50_1, 51_1, 52_1, 53_1, 54_1, 55_1, 56_1, 57_1, 58_1, 59_1, 60_1, 61_1, 62_1,Includes any one of 63_1, 64_1, 65_1, 66_1, 67_1, 68_1, 69_1, 70_1, 71_1, 72_1, 73_1, 74_1, 75_1, 76_1, 77_1, 78_1, 79_1, 80_1, 81_1, 82_1, 83_1, 84_1, 85_1 and 86_1. In some embodiments, the antisense oligonucleotide is selected from the group consisting of CMP numbers 1_1, 2_1, 3_1, 3_2, 3_3, 4_1, 5_1, 6_1, 7_1, 8_1, 9_1, 10_1, 11_1, 12_1, 13_1, 14_1, 15_1, 16_1, 17_1, 18_1, 19_1, 20_1, 21_1, 22_1, 23_1, 24_1, 25_1, 26_1, 26_2, 26_3, 26_4, 26_5, 26_6, 26_7, 26_8, 26_9, 27_1, 28_1, 29_2, 30_3, 31_4, 32_5, 33_6, 34_7, 34_8, 35_9, 36_1, 37_2, 38_3, 39_4, 40_5, 41_6, 42_7, 43_8, 44_9, 45_1, 46_1, 47_2, 48_3, 49_4, 50_1, 51_1, 52_1, 53_1, 54_1, 55_1, 56_1, 57_2, 58_3, 59_4, 60_1, 61_1, 62_1, 63_1, 64_1, 65_1, 66_1, 67_1, 68_2, 69_3, 70_1, 71_1, 72_1, 73_1 9_1, 30_1, 31_1, 32_1, 32_2, 32_3, 32_4, 32_5, 32_6, 32_7, 32_8, 32_9, 32_10, 32_11, 32_12, 32_13, 32_14, 32_15, 32_16, 32_17, 32_18, 32_19, 32_20, 32_21, 32_22, 32_23, 32_24, 32_25, 32_26, 32_27, 32_28, 32_29, 32_30, 32_31, 32_32, 32_33, 32_34, 32_35, 32 _36, 32_37, 32_38, 32_39, 32_40, 32_41, 32_42, 32_43, 32_44, 32_45, 32_46, 32_47, 32_48, 32_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_59, 32_60, 32_61, 32_62, 32_63, 32_64, 32_65, 32_66, 32_67, 32_68, 32_69, 32_70, 32_71, 32 _72, 32_73, 32_74, 32_75, 32_76, 32_77, 32_78, 32_79, 32_80, 32_81, 32_82, 32_83, 33_1, 34_1, 35_1, 36_1, 37_1, 38_1, 39_1, 40_1, 40_2, 40_3, 40_4, 40_5, 40_6, 40_7, 40_8, 40_9, 40_10, 41_1, 41_2, 41_3, 41_4, 41_5, 41_6, 41_7, 41_8, 41_9, 41_10, 42_1, 42_2,42_3, 42_4, 42_5, 42_6, 43_1, 43_2, 43_3, 44_1, 45_1, 46_1, 47_1, 47_2, 48_1, 49_1, 50_1, 51_1, 52_1, 53_1, 54_1, 55_1, 56_1, 57_1, 58_1, 59_1, 60_1, 61_1, 62_1 , 63_1, 64_1, 65_1, 66_1, 67_1, 68_1, 69_1, 70_1, 71_1, 72_1, 73_1, 74_1, 75_1, 76_1, 77_1, 78_1, 79_1, 80_1, 81_1, 82_1, 83_1, 84_1, 85_1 and 86_1.

[0243] In some embodiments, the oligonucleotides of the invention are selected from the group consisting of CMP numbers 3_3, 7_1, 8_1, 9_1, 10_1, 19_1, 20_1, 21_1, 22_1, 23_1, 26_8, 29_1, 30_1, 31_1, 32_1, 32_2, 32_4, 32_5, 32_6, 32_7, 32_8, 32_9, 32_10, 32_11, 32_12, 32_13, 32_14, 32_16, 32_17, 32_18, 32_19, 32_20, 32_21, 32_22, 32_23, 32_24, 32_25, 32_26, 32_27, 32_28, 32_29, 32_30, 32_31, 32_32, 32_33, 32_34, 32_35, 32_36, 32_37, 32_38, 32_39, 32_40, 32_41, 32_42, 32_43, 32_44, 32_45, 32_46, 32_47, 32_48, 32_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_59, 32_60, 32_61, 32_6 Includes any one of 2_23, 32_29, 32_33, 32_34, 32_47, 32_48, 32_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_59, 32_60, 32_61, 32_62, 32_63, 32_64, 32_65, 33_1, 34_1, 35_1, 36_1, 41_8, 42_6, 43_1, 43_3, 45_1, 60_1, 61_1, 62_1 and 63_1. In some embodiments, the oligonucleotides of the invention are selected from the group consisting of CMP numbers 3_3, 7_1, 8_1, 9_1, 10_1, 19_1, 20_1, 21_1, 22_1, 23_1, 26_8, 29_1, 30_1, 31_1, 32_1, 32_2, 32_4, 32_5, 32_6, 32_7, 32_8, 32_9, 32_10, 32_11, 32_12, 32_13, 32_14, 32_16, 32_17, 32_18, 32_19, 32_20, 32_21, 32_22 , 32_23, 32_29, 32_33, 32_34, 32_47, 32_48, 32_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_60, 32_61, 32_62, 32_63, 32_64, 32_65, 33_1, 34_1, 35_1, 36_1, 41_8, 42_6, 43_1, 43_3, 45_1, 60_1, 61_1, 62_1 and 63_1.

[0244] In some embodiments, the oligonucleotides of the invention are selected from the group consisting of CMP numbers 7_1, 8_1, 9_1, 19_1, 20_1, 21_1, 22_1, 29_1, 30_1, 31_1, 19_1, 32_1, 32_4, 32_5, 32_6, 32_7, 32_8, 32_9, 32_10, 32_11, 32_12, 32_13, 32_14, 32_16, 32_17, 32_18, 32_19, 32_20, 32_21, 32_22, 32_23, 32_24, 32_25, 32_26, 32_27, 32_28, 32_29, 32_30, 32_31, 32_32, 32_33, 32_34, 32_35, 32_36, 32_37, 32_38, 32_39, 32_40, 32_41, 32_42, 32_43, 32_44, 32_45, 32_46, 32_47, 32_48, 32_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_59, 32_60, 32_61, 32_62, 32_63, 32_64, 32_65, Includes any one of 20, 32_21, 32_22, 32_23, 32_33, 32_47, 32_48, 32_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_60, 32_61, 32_62, 32_63, 32_64, 32_65, 33_1, 34_1, 42_6, 43_1, 62_1 and 63_1. In some embodiments, the oligonucleotides of the invention are selected from the group consisting of CMP numbers 7_1, 8_1, 9_1, 19_1, 20_1, 21_1, 22_1, 29_1, 30_1, 31_1, 19_1, 32_1, 32_4, 32_5, 32_6, 32_7, 32_8, 32_9, 32_10, 32_11, 32_12, 32_13, 32_14, 32_16, 32_17, 32_18, 32_19, 32_20, 32_21, 32_22, 32_23, 32_24, 32_25, 32_26, 32_27, 32_28, 32_29, 32_30, 32_31, 32_32, 32_33, 32_34, 32_35, 32_36, 32_37, 32_38, 32_39, 32_40, 32_41, 32_42, 32_43, 32_44, 32_45, 32_46, 32_47, 32_48, 32_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_59, 32_60, 32_61, 32_62, 32_63, 32_64, 32_65, 20, 32_21, 32_22, 32_23, 32_33, 32_47, 32_48, 32_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_60, 32_61, 32_62, 32_63, 32_64, 32_65, 33_1, 34_1, 42_6, 43_1, 62_1 and 63_1.

[0245] In some embodiments, the oligonucleotides of the invention are selected from the group consisting of CMP numbers 32_1, 32_2, 32_3, 32_4, 32_5, 32_6, 32_7, 32_8, 32_9, 32_10, 32_11, 32_12, 32_13, 32_14, 32_15, 32_16, 32_17, 32_18, 32_19, 32_20, 32_21, 32_22, 32_23, 32_24, 32_25, 32_26, 32_27, 32_28, 32_29, 32_30, 32_31, 32_32, 32_33, 32_34, 32_35, 32_36, 32_37, 32_38, 32_39, 32_40, 32_41, 32_42, 32_43, 32_44, 32_45, 32_46, 32_47, 32_48, 32_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_59, 32_60, 32_61, 32_62, 32_63, 32_64, 32_65, 32_66, 32_67, 32_68, 32_69, 32_70, 32_71, 3 41, 32_42, 32_43, 32_44, 32_45, 32_46, 32_47, 32_48, 32_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_59, 32_60, 32_61, 32_62, 32_6 Includes any one of 3, 32_64, 32_65, 32_66, 32_67, 32_68, 32_69, 32_70, 32_71, 32_72, 32_73, 32_74, 32_75, 32_76, 32_77, 32_78, 32_79, 32_80, 32_81, 32_82 and 32_83.In some embodiments, the oligonucleotides of the invention are selected from the group consisting of CMP numbers 32_1, 32_2, 32_3, 32_4, 32_5, 32_6, 32_7, 32_8, 32_9, 32_10, 32_11, 32_12, 32_13, 32_14, 32_15, 32_16, 32_17, 32_18, 32_19, 32_20, 32_21, 32_22, 32_23, 32_24, 32_25, 32_26, 32_27, 32_28, 32_29, 32_30, 32_31, 32_32, 32_33, 32_34, 32_35, 32_36, 32_37, 32_38, 32_39, 32_40, 32_41, 32_42, 32_43, 32_44, 32_45, 32_46, 32_47, 32_48, 32_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_59, 32_60, 32_61, 32_62, 32_63, 32_64, 32_65, 32_66, 32_67, 32_68, 32_69, 32_70, 32_71, 3 41, 32_42, 32_43, 32_44, 32_45, 32_46, 32_47, 32_48, 32_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_59, 32_60, 32_61, 32_62, 32_63 , 32_64, 32_65, 32_66, 32_67, 32_68, 32_69, 32_70, 32_71, 32_72, 32_73, 32_74, 32_75, 32_76, 32_77, 32_78, 32_79, 32_80, 32_81, 32_82 and 32_83.

[0246] In some embodiments, an oligonucleotide of the invention comprises any one of CMP numbers 32_1, 32_4, 32_5, 32_6, 32_7, 32_8, 32_9, 32_10, 32_11, 32_12, 32_13, 32_14, 32_16, 32_17, 32_18, 32_19, 32_20, 32_21, 32_22, 32_23, 32_33, 32_47, 32_48, 32_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_60, 32_61, 32_62, 32_63, 32_64, and 32_65. In some embodiments, an oligonucleotide of the invention consists of any one of CMP numbers 32_1, 32_4, 32_5, 32_6, 32_7, 32_8, 32_9, 32_10, 32_11, 32_12, 32_13, 32_14, 32_16, 32_17, 32_18, 32_19, 32_20, 32_21, 32_22, 32_23, 32_33, 32_47, 32_48, 32_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_60, 32_61, 32_62, 32_63, 32_64, and 32_65.

[0247] In some embodiments, the oligonucleotide of the present invention comprises CMP No. 32_1. In some embodiments, the oligonucleotide of the present invention consists of CMP No. 32_1. In some embodiments, the oligonucleotide of the present invention is CMP No. 32_1. In some embodiments, the oligonucleotide of the present invention is the compound shown in Figure 29.

[0248] Conjugates The present invention provides an oligonucleotide conjugate comprising an oligonucleotide of the present invention covalently linked to at least one conjugate moiety, in other words, an oligonucleotide covalently linked to at least one conjugate moiety.

[0249] The term "oligonucleotide conjugate" is used herein interchangeably with the terms "conjugate" and "conjugate of the present invention." The term "conjugate moiety" refers to a non-nucleotide moiety that can be covalently bound to an oligonucleotide of the present invention. Thus, as used herein, the term "conjugate" refers to an oligonucleotide of the present invention that is covalently bound to a non-nucleotide moiety (conjugate moiety).

[0250] Oligonucleotide conjugates and their synthesis are also reported in comprehensive reviews by Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, S.T. Crooke, ed., Ch. 16, Marcel Dekker, Inc., 2001 and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103.

[0251] The term "conjugate" can be used herein to refer to the combination of sequence (i.e., the order of nucleobases), sugar moieties, internucleoside linkages and conjugate moieties in a given oligonucleotide conjugate of the present invention.For a given conjugate, the nucleobases, sugar moiety type and internucleoside linkage of each nucleotide in the oligonucleotide of the conjugate are specified.Therefore, the order of sugar moieties and internucleoside linkages is also specified in a given conjugate.However, unless otherwise indicated, a conjugate can include other elements in addition to a specific sequence, sugar moiety, internucleoside linkage and conjugate moiety.

[0252] Specific conjugates are referred to herein using conjugate identification numbers (CNJ numbers) of the form X_Y_Z, where X, Y, and Z are each numbers. For each CNJ number, X_Y is the CMP number of the oligonucleotide compound of the conjugate (recalling that X is the SEQ ID NO of the nucleobase sequence of that compound). For example, a conjugate designated CNJ number 126_2_1 contains oligonucleotide compound CMP number 126_2 having the nucleobase sequence of SEQ ID NO: 126.

[0253] In some embodiments, at least one conjugate moiety is covalently attached to the 5' end of the oligonucleotide. In some embodiments, at least one conjugate moiety is covalently attached to the 3' end of the oligonucleotide.

[0254] Conjugate moiety In some embodiments, the conjugate moiety (i.e., the non-nucleotide moiety) is selected from the group consisting of a carbohydrate (e.g., GalNAc), a cell surface receptor ligand, a drug substance, a hormone, a lipophile, a polymer, a protein, a peptide, a toxin (e.g., a bacterial toxin), a vitamin, a viral protein (e.g., a capsid), or a combination thereof.

[0255] In some embodiments, the conjugate moiety can bind to an asialoglycoprotein receptor, such as the human asialoglycoprotein receptor (ASGPR). For example, the conjugate moiety may comprise at least one asialoglycoprotein receptor targeting moiety selected from the group consisting of galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, Nn-butanoyl-galactosamine, and N-isobutanoylgalactosamine.

[0256] In some embodiments, the asialoglycoprotein receptor targeting moiety is N-acetylgalactosamine (GalNAc). In some embodiments, the conjugate moiety is an N-acetylgalactosamine (GalNAc) conjugate moiety. Thus, the oligonucleotide of the present invention can be conjugated to at least one conjugate moiety comprising at least one N-acetylgalactosamine (GalNAc) moiety, for example, at least one conjugate moiety comprising at least one N-acetylgalactosamine (GalNAc) moiety described below.

[0257] In some embodiments, the conjugate moiety is at least divalent, for example, divalent, trivalent or tetravalent GalNAc.In a preferred embodiment, the conjugate moiety is trivalent GalNAc.Trivalent N-acetylgalactosamine conjugate moiety is suitable for binding to ASGPR, for example, see WO2014 / 076196, WO2014 / 207232 and WO2014 / 179620.Such conjugate moiety is useful for enhancing the uptake of oligonucleotide into the liver.As used herein, the term "trivalent GalNAc" refers to a residue that comprises three N-acetylgalactosamine moieties, that is, preferably three moieties of the following formula: TIFF2025525564000012.tif35128

[0258] In some embodiments, the GalNAc conjugate moiety is an aminohexyl-conjugated tri(N-acetyl-galactosamine), as shown below. TIFF2025525564000013.tif85138

[0259] The trivalent N-acetylgalactosamine (GalNAc) shown above is also referred to herein as "5gn2c6."

[0260] "Aminohexyl-conjugated tri(N-acetyl-galactosamine)" is sometimes referred to as "-hexylene-NH-tri(N-acetyl-galactosamine)."

[0261] In some embodiments, the conjugate moiety is covalently attached to the oligonucleotide via a phosphodiester bond. In some embodiments, the conjugate moiety is covalently attached to the linker via a phosphodiester bond.

[0262] Linker In some embodiments of the oligonucleotide conjugates of the present invention, the conjugate moiety is covalently attached to the oligonucleotide via a linker. Thus, in some embodiments, the conjugate comprises a linker. In some embodiments, the conjugate comprises a linker located between the oligonucleotide and the conjugate moiety.

[0263] In some embodiments of the oligonucleotide conjugates of the invention, the conjugate moiety is covalently attached to the oligonucleotide via a linker nucleoside sequence.

[0264] In some embodiments, the linker nucleoside sequence comprises or consists of 1 to 10 linked nucleosides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linked nucleosides, e.g., 2 to 6 linked nucleosides, e.g., 2 to 5 linked nucleosides, e.g., 2 to 4 linked nucleosides. In some embodiments, the linker nucleoside sequence is 2 nucleosides in length. In some embodiments, the linker comprises two linked nucleosides. In some embodiments, the linker consists of two linked nucleosides.

[0265] In some embodiments, the linker nucleoside sequence comprises DNA nucleosides. In some embodiments, the linker nucleoside sequence consists of DNA nucleosides. In other words, in some embodiments, all nucleosides in the linker nucleoside sequence are DNA nucleosides.

[0266] In some embodiments, the nucleosides of the linker nucleoside sequence are linked via phosphodiester internucleoside linkages. In some embodiments, the linker is linked to the oligonucleotide via a phosphodiester internucleoside linkage. In some embodiments, the linker is linked to the conjugate moiety via a phosphodiester bond.

[0267] As used herein, the terms "phosphodiester internucleoside bond" and "phosphodiester bond" refer to the same chemical structure known in the art, in which a first chemical entity is linked to a second chemical entity via an intermediate phosphate group. The term "phosphodiester internucleoside bond" is particularly used when the linked first and second chemical entities are nucleosides, such as the nucleosides of the oligonucleotide of the present invention. As known in the art, phosphodiester internucleoside bond is the naturally occurring internucleoside bond in naturally occurring nucleic acids, such as genomic DNA. The term "phosphodiester bond" is used particularly in the context of the present invention to refer to the covalent attachment of a conjugate moiety to a linker, such as the oligonucleotide or nucleoside linker sequence of the present invention, because the conjugate moiety is not a nucleoside, and therefore the term "phosphodiester internucleoside bond" is not appropriate.

[0268] In some embodiments, the linker comprises or consists of a DNA dinucleotide having a sequence selected from the group consisting of AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, or GG, wherein there is a phosphodiester bond between the two DNA nucleosides, and at least one additional phosphodiester is present at the 5' or 3' end of the dinucleotide that links the oligonucleotide to the dinucleotide or the conjugate moiety to the dinucleotide. For example, the linker can be a CA dinucleotide. In some embodiments, the linker comprises or consists of the sequence AAA, AAT, AAC, AAG, ATA, ATT, ATC, ATG, ACA, ACT, ACC, ACG, AGA, AGT, AGC, AGG, TAA, TAT, TAC, TAG, TTA, TTT, TTC, TAG, TCA, TCT, TCC, TCG, TGA, TGT, TGC, TGG, CAA, CAT, CAC, CAG, CTA, CTG, CTC, CTT, CCA, CCT, CCC, CCG, CGA, CGT, CGC, CGG, GAA, GAT, GAC, CAG, GTA, GTT, GTC, GTG, GCA, GCT, GCC, GCG, GGA, GGT, GGC, or GGG, and wherein there are phosphodiester bonds between the DNA nucleosides and, potentially, additional phosphodiester bonds at the 5' or 3' end of the trinucleotide.

[0269] In some embodiments, the linker nucleoside sequence is CA. In other words, in some embodiments, the sequence of the linker nucleoside sequence is CA. In some embodiments, the linker nucleotide sequence is 5'-CA-3'. In some embodiments, the linker nucleoside sequence is the dinucleotide CA, where the C nucleoside is linked to the conjugate moiety by a phosphodiester bond, the C nucleoside is linked to the A nucleoside by a phosphodiester internucleoside bond, and the A nucleoside is linked to the 5' nucleoside of the antisense oligonucleotide of the present invention by a phosphodiester internucleoside bond.

[0270] In some embodiments, the linker is a biocleavable linker. A biocleavable linker comprises or consists of a physiologically labile bond that is cleavable under conditions typically encountered or similar to those encountered in a mammalian body. Conditions under which a physiologically labile linker undergoes chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidizing or reducing conditions or oxidizing or reducing agents, and salt concentrations similar to those found or encountered in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activities typically present in mammalian cells, such as proteolytic or hydrolytic enzymes or nucleases. In one embodiment, the biocleavable linker is susceptible to S1 nuclease cleavage. In some embodiments, the nuclease-sensitive linker comprises one to five nucleosides, such as DNA nucleosides containing at least two consecutive phosphodiester bonds. Phosphodiester-containing biocleavable linkers are described in more detail in WO2014 / 076195 (incorporated herein by reference). For conjugates having biocleavable linkers, when compared to a standard, at least about 50% of the conjugated moieties are cleaved from the oligonucleotide, such as at least about 60% are cleaved, for example, at least about 70% are cleaved, for example, at least about 80% are cleaved, for example, at least about 85% are cleaved, for example, at least about 90% are cleaved, for example, at least about 95% of the conjugated moieties are cleaved from the oligonucleotide.

[0271] Pharmaceutically acceptable salts In some embodiments, the oligonucleotide or oligonucleotide conjugate of the present invention is in the form of a pharmaceutically acceptable salt. In some embodiments, the oligonucleotide is in the form of a pharmaceutically acceptable salt. In some embodiments, the oligonucleotide conjugate is in the form of a pharmaceutically acceptable salt.

[0272] As used herein, the term "salt" conforms to its commonly known meaning, i.e., an ionic assembly of anions and cations.

[0273] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the free base or free acid, without being biologically or otherwise undesirable. Salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid, especially hydrochloric acid, as well as organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and N-acetylcysteine. In addition, these salts can be prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, and polyamine resin salts.The compounds of the present invention can also exist in zwitterionic form.The particularly preferred pharmaceutically acceptable salts of the oligonucleotide or oligonucleotide conjugate of the present invention are salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and methanesulfonic acid.

[0274] In some embodiments, the pharmaceutically acceptable salt is a sodium salt or a potassium salt. In some embodiments, the oligonucleotide or oligonucleotide conjugate of the present invention is in the form of a sodium salt. In some embodiments, the oligonucleotide of the present invention is in the form of a sodium salt. In some embodiments, the oligonucleotide conjugate of the present invention is in the form of a sodium salt. In some embodiments, the oligonucleotide or oligonucleotide conjugate of the present invention is in the form of a potassium salt. In some embodiments, the oligonucleotide of the present invention is in the form of a potassium salt. In some embodiments, the oligonucleotide conjugate of the present invention is in the form of a potassium salt.

[0275] Delivery of antisense oligonucleotides In some embodiments, the oligonucleotide or oligonucleotide conjugate of the present invention is encapsulated in a lipid-based delivery vehicle, covalently linked to a dendrimer, or encapsulated in a dendrimer, or conjugated to an aptamer.This may be for the purpose of delivering the oligonucleotide of the present invention to target cells and / or to improve the pharmacokinetics of the oligonucleotide.Examples of lipid-based delivery vehicles include oil-in-water emulsions, micelles, liposomes, and lipid nanoparticles.

[0276] In some embodiments, the oligonucleotide is encapsulated in a lipid-based delivery vehicle. In some embodiments, the oligonucleotide is covalently linked to a dendrimer. In some embodiments, the oligonucleotide is encapsulated in a dendrimer. In some embodiments, the oligonucleotide is conjugated to an aptamer.

[0277] In some embodiments, the oligonucleotide conjugate is encapsulated in a lipid-based delivery vehicle. In some embodiments, the oligonucleotide conjugate is covalently linked to a dendrimer. In some embodiments, the oligonucleotide conjugate is encapsulated in a dendrimer. In some embodiments, the oligonucleotide conjugate is conjugated to an aptamer.

[0278] Pharmaceutical Composition The present invention provides pharmaceutical compositions comprising an oligonucleotide or oligonucleotide conjugate of the present invention and a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant. In some embodiments, the pharmaceutical composition comprises an aqueous diluent or solvent. In some embodiments, the aqueous diluent or solvent is phosphate buffered saline. In some embodiments, the aqueous diluent or solvent is sterile.

[0279] The present invention provides a pharmaceutical composition comprising an oligonucleotide of the present invention and a pharmaceutically acceptable salt. In some embodiments, the salt comprises a metal cation. In some embodiments, the pharmaceutically acceptable salt is selected from the group consisting of sodium salt, potassium salt, and ammonium salt.

[0280] The present invention also provides a pharmaceutical solution of the oligonucleotide of the present invention or a conjugate thereof, comprising the oligonucleotide of the present invention or a conjugate thereof and a pharmaceutically acceptable solvent such as physiological saline.

[0281] The present invention also provides an oligonucleotide or conjugate thereof of the present invention in solid powder form, such as in the form of a lyophilized powder.

[0282] Methods for editing target nucleic acids The present invention provides an in vitro or in vivo method for editing a target nucleic acid in a target cell, comprising administering to the target cell an effective amount of an oligonucleotide or oligonucleotide conjugate of the present invention or a pharmaceutical composition of the present invention.

[0283] The present invention also provides an in vivo method for editing a target nucleic acid in a target cell, the method comprising administering to the target cell an effective amount of an oligonucleotide or oligonucleotide conjugate of the present invention or a pharmaceutical composition of the present invention.

[0284] The target nucleic acid comprises target adenosine.The oligonucleotide or oligonucleotide conjugate binds to the target nucleic acid by complementary base pairing.The oligonucleotide or oligonucleotide conjugate recruits ADAR to the target nucleic acid.In some embodiments, the ADAR is ADAR1 or ADAR2.In some embodiments of the method of the present invention, the target adenosine (A) is converted to inosine (I).

[0285] In some embodiments of the methods of the present invention, the target nucleic acid is RNA. In some embodiments, the target nucleic acid is RNA. In some embodiments, the target nucleic acid encodes a protein. In some embodiments, the target nucleic acid is mRNA. In some embodiments, the target nucleic acid encodes alpha-1 antitrypsin (A1AT). In some embodiments, the target nucleic acid is SERPINA1 mRNA.

[0286] In some embodiments of the methods of the present invention, the oligonucleotide or oligonucleotide conjugate can effect conversion of a AAA codon encoding lysine on a target nucleic acid to an IAA codon encoding glutamic acid. In some embodiments of the methods of the present invention, the oligonucleotide or oligonucleotide conjugate can effect conversion of an AAG codon encoding lysine on a target nucleic acid to an IAG codon encoding glutamic acid.

[0287] In some embodiments of the methods of the invention, the target adenosine corresponds to A1024 of SEQ ID NO: 186. In some embodiments, the target nucleic acid comprises or consists of a sequence according to SEQ ID NO:186.

[0288] In some embodiments of the methods of the present invention, the target cell is a mammalian cell. In some embodiments, the target cell is a human cell. In some embodiments of the methods, the target cell is a lung cell or a liver cell.

[0289] In some embodiments of the methods of the present invention, the amount of edited target nucleic acid is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to a control. In some embodiments, the control is a cell that has not been exposed to the oligonucleotide or oligonucleotide conjugate. The amount of edited target nucleic acid can be determined by techniques known in the art. For example, the target nucleic acid can be amplified using PCR, and then the percentage of edited target nucleic acid can be determined by sequencing. Exemplary methods for determining the amount of edited target nucleic acid are described in Example 3 herein, Merkle et al. 2019 Nature Biotechnology, Vol. 37, 133-138, and Qu et al. 2019 Nature Biotechnology, Vol. 37, 1059-1069.

[0290] Treatment, prevention and medical uses The present invention provides a method for treating or preventing a disease, comprising administering to a subject suffering from or susceptible to the disease a therapeutically or prophylactically effective amount of an oligonucleotide or oligonucleotide conjugate of the invention, or a pharmaceutical composition of the invention.

[0291] The present invention provides an oligonucleotide or oligonucleotide conjugate of the present invention, or a pharmaceutical composition of the present invention for use in treating or preventing a disease in a subject.The present invention provides an oligonucleotide of the present invention, an oligonucleotide conjugate of the present invention, or a pharmaceutical composition of the present invention for use as a medicament.The present invention provides an oligonucleotide of the present invention, an oligonucleotide conjugate of the present invention, or a pharmaceutical composition of the present invention for use in therapy.

[0292] The present invention provides use of the oligonucleotide or oligonucleotide conjugate of the present invention, or the pharmaceutical composition of the present invention, for the preparation of a medicament for the treatment or prevention of a disease in a subject. The present invention provides the oligonucleotide of the present invention, the oligonucleotide conjugate of the present invention, or the pharmaceutical composition of the present invention for preparing a medicament.

[0293] As used herein, the terms "treating," "treatment," and "treatment" refer to both the treatment of an existing disease (e.g., a disease or disorder referred to herein) or the prevention of disease, i.e., prophylaxis. Accordingly, it will be recognized that the treatment referred to herein may, in some embodiments, be prophylactic. Prophylactic may be understood to prevent A1AD.

[0294] As used herein, the terms "preventing," "prevention," or "prevent" refer to prophylactic treatment, i.e., measures or measures the purpose of which is to prevent rather than cure a disease. Prevention means that the desired pharmacological and / or physiological effect is obtained prophylactically, in terms of completely or partially preventing a disease or its symptoms.

[0295] The term "disease" as used herein refers to a state of dysfunction in the body. The term disease is used herein synonymously with similar terms such as "condition" or "disorder."

[0296] Many diseases are associated with mutations. Those skilled in the art will understand that the oligonucleotides provided herein (which facilitate the correction of G to A mutations) can be used to treat many such diseases associated with mutations, particularly G to A mutations. In some embodiments, the disease is associated with a mutation of a G nucleotide (i.e., guanosine) to an A nucleotide (i.e., adenosine). In some embodiments, the disease is caused by a mutation of a G nucleotide to an A nucleotide. In some embodiments, the disease is caused by a mutation of a G nucleotide to an A nucleotide in the genome of a subject.

[0297] Alpha-1 antitrypsin deficiency (A1AD) In some embodiments of the method, the oligonucleotide for use, the oligonucleotide conjugate for use, or the pharmaceutical composition for use of the invention, or the use of the invention for treating or preventing a disease, the disease is a disease associated with a mutation in the SERPINA1 gene.

[0298] Alpha-1 antitrypsin (A1AT) deficiency (A1AD) is a disease associated with mutations in the SERPINA1 gene.SERPINA1 encodes A1AT, a serine protease inhibitor, which is synthesized in the liver and released into other tissues to protect against endogenous inflammatory serine proteases such as neutrophil elastase.Subjects suffering from A1AD express reduced levels of A1AT, which can lead to excessive destruction of elastin in the lung, thereby reducing lung elasticity and related health problems such as emphysema.The accumulation of misfolded A1AT in the liver can also lead to liver-related problems such as cirrhosis and jaundice.

[0299] In some embodiments of the method, the oligonucleotide for use, the oligonucleotide conjugate for use, or the pharmaceutical composition for use of the invention, or the use of the invention for treating or preventing a disease, the disease is alpha 1 antitrypsin deficiency (A1AD).

[0300] Thus, the present invention provides a method for treating or preventing A1AD, comprising administering to a subject suffering from or susceptible to A1AD a therapeutically or prophylactically effective amount of an oligonucleotide or oligonucleotide conjugate of the present invention, or a pharmaceutical composition of the present invention.

[0301] The present invention provides an oligonucleotide or oligonucleotide conjugate of the present invention, or a pharmaceutical composition of the present invention for use in treating or preventing A1AD in a subject.

[0302] The present invention provides use of an oligonucleotide or oligonucleotide conjugate of the present invention, or a pharmaceutical composition of the present invention, for the preparation of a medicament for the treatment or prevention of A1AD in a subject.

[0303] Here, "preventing A1AD" includes preventing the occurrence of A1AD in a subject, and preventing the occurrence of symptoms of A1AD in a subject.

[0304] In some embodiments, the treatment or prevention includes treating or preventing one or more symptoms of A1AD selected from liver damage, liver failure, cirrhosis, jaundice, elastin breakdown in the lungs, emphysema, and chronic obstructive pulmonary disease (COPD).

[0305] subject For the purposes of the present invention, a "subject" or "patient" may be a vertebrate. In the context of the present invention, the term "subject" includes both humans and other animals, particularly mammals, and other organisms. Thus, the means and methods provided herein are applicable to both human therapy and veterinary use. Thus, herein, a subject may be an animal such as a mouse, rat, hamster, rabbit, guinea pig, ferret, cat, dog, chicken, sheep, bovine species, horse, camel, or primate. Preferably, the subject is a mammal. More preferably, the subject is a human. In some embodiments, the subject is suffering from a disease referred to herein, such as A1AD. In some embodiments, the subject is susceptible to the disease.

[0306] Sequence Listing The sequence listing submitted with this application is incorporated herein by reference. In the event of any discrepancy between the sequence listing and the specification or drawings, the information disclosed in the specification (including the drawings) shall be presumed to be correct.

[0307] Each reference herein to a SEQ ID NO (Sequence Identifier Number) refers to the sequence represented by that SEQ ID NO. Each reference herein to a CMP Number (Compound Identification Number) refers to the compound represented by that CMP Number. Each reference herein to a CNJ Number (Conjugate Identifier Number) refers to the conjugate represented by that CNJ Number.

[0308] HELM notation The oligonucleotides (compounds) of the invention and the oligonucleotide conjugates (conjugates) of the invention are depicted herein using the Hierarchical Editing Language for Macromolecules (HELM) notation.

[0309] HELM is a notation format designed to depict the structure of macromolecules. Full details of the HELM notation can be found at www.pistoiaalliance.org / helm-tools / , in Zhang et al. J. Chem. Inf. Model. 2012, 52, 2796-2806 (which first described the HELM notation) and Milton et al. J. Chem Inf. Model. 2017, 57, 1233-1239 (which describes HELM version 2.0).

[0310] Briefly, a macromolecule is depicted as a "HELM string" and is divided into sections. The first section of a HELM string lists the molecules contained in the macromolecule. The second section lists the connections between molecules within the macromolecule. The third, fourth, and fifth sections (which may be used in HELM strings for more complex macromolecules) are not used in HELM strings herein. One or more dollar signs $ mark the end of a section of a HELM string.

[0311] The compounds of the present invention are represented by HELM strings consisting of a single first section that defines an oligonucleotide.

[0312] The conjugates of the present invention are represented by a HELM string consisting of two sections: a first section defining the oligonucleotide (which may include a nucleotide linker) and the conjugate moiety, and a second section defining the linkage between the oligonucleotide and the conjugate moiety.

[0313] Each molecule listed in the first section of a HELM string is given an identifier (e.g., "RNA1" for a nucleic acid, "PEPTIDE1" for an amino acid sequence, or "CHEM1" for a chemical structure), and the structure of the molecule is defined by the notation in brackets {} immediately following the identifier. "RNA1" is an identifier for the oligonucleotide portion of the compound or conjugate; the oligonucleotide is defined in brackets {} after the first instance of "RNA1"; the second instance of "RNA1" in each conjugate string defines the position of the linkage between the oligonucleotide and the conjugate moiety. "CHEM1" is an identifier for the conjugate moiety of the conjugate; the conjugate moiety is defined in brackets {} after the first instance of "CHEM1"; the second instance of "CHEM1" in each conjugate string defines the position of the linkage between the oligonucleotide and the conjugate moiety. "V2.0" indicates use of HELM version 2.0.

[0314] The HELM notation used to define the structure of each molecule in the braces {} in the first section of the HELM string for compounds and conjugates of the invention is as follows: defines a nucleoside, R(A) is an RNA adenine nucleoside; R(C) is an RNA cytosine nucleoside; R(G) is an RNA guanine nucleoside; R([In]) is an RNA hypoxanthine nucleoside; R(U) is an RNA uracil nucleoside; [dR] is a DNA nucleoside lacking a nucleobase (abasic), [dR](A) is a DNA adenine nucleoside; [dR](C) is a DNA cytosine nucleoside; [dR](G) is a DNA guanine nucleoside; [dR]([In]) is a DNA hypoxanthine nucleoside; [dR](T) is a DNA thymine nucleoside; [dR]([2AP]) is a DNA 2-aminopurine nucleoside; [dR]([apC]) is a DNA aminoethylphenoxazine nucleoside; [dR]([5BrC]) is the DNA 5-bromocytosine nucleoside, [dR]([prpC]) is a DNA 5-propynylcytosine nucleoside; [dR] ([PyrC]) is a DNA pyrrolocytosine nucleoside; [dR]([PPG]) is a DNA 7-deaza-8-azaguanine nucleoside; [dR](Z) is a DNA nucleoside containing a Benner base, [mR](A) is 2'-O-methyl RNA adenine nucleoside; [mR](C) is a 2'-O-methyl RNA cytosine nucleoside; [mR](G) is a 2'-O-methyl RNA guanine nucleoside; [mR]([In]) is 2'-O-methyl RNA hypoxanthine nucleoside; [mR](U) is 2'-O-methyl RNA uracil nucleoside; [fR](A) is a 2'-fluoro RNA adenine nucleoside; [fR](C) is a 2'-fluoro RNA cytosine nucleoside; [fR](G) is a 2'-fluoro RNA guanine nucleoside; [fR]([In]) is 2'-fluoro RNA hypoxanthine nucleoside; [fR](U) is a 2'-fluoroRNA uracil nucleoside; [FANA](A) is FANA adenine nucleoside; [FANA](G) is FANA guanine nucleoside; [FANA](C) is a FANA cytosine nucleoside; [LR](A) is a β-D-oxy-LNA adenine nucleoside; [LR](G) is a β-D-oxy-LNA guanine nucleoside; [LR] ([5meC]) is β-D-oxy-LNA 5-methylcytosine nucleoside, [MOE] is a 2'-MOE RNA nucleoside lacking a nucleobase (abasic), [MOE](A) is a 2'-MOE RNA adenine nucleoside; [MOE](G) is a 2'-MOE RNA guanine nucleoside; [MOE](T) is 2'-MOE RNA thymine nucleoside; [MOE] ([5meC]) is 2'-MOE RNA 5-methylcytosine nucleoside; [idR](A) is an iDNA adenine nucleoside; [idR](C) is an iDNA cytosine nucleoside; [idR](G) is an iDNA guanine nucleoside; [ScEt](G) is ScET guanine nucleoside; [SNA](A) is an SNA adenine nucleoside; [SNA](G) is SNA guanine nucleoside; [SNA] ([5meC]) is SNA 5-methylcytosine nucleoside; [TNA](A) is TNA adenine nucleoside; [TNA](G) is TNA guanine nucleoside; [TNA] ([5meC]) is TNA 5-methylcytosine nucleoside; [P] is a phosphodiester internucleoside linkage, [sP] is a phosphorothioate internucleoside linkage; [ssP] is the left-handed stereodefined isomer (Sp) of the phosphorothioate internucleoside linkage; [srP] is the right-handed stereodefined isomer (Rp) of the phosphorothioate internucleoside linkage, [PS2] is a phosphorodithioate internucleoside linkage.

[0315] As mentioned above, in the context of the present invention, the second section is only used in the HELM string representing the conjugate of the present invention. This second section lists the connections between the molecules listed in the first section. Each pair of molecules to be linked is defined by listing their identifiers, and then the attachment points between them (i.e., the points at which the covalent bond exists between the molecules) are defined.

[0316] In the HELM strings representing the conjugates of the present invention, there is a single linkage (between the conjugate moiety and the oligonucleotide). This single connection is represented in all HELM strings herein as follows: CHEM1, RNA1, 1:R2-1:R1.

[0317] This shows that the conjugate moiety (CHEM1) is attached to the oligonucleotide (RNA1) by a covalent bond between the R2 attachment point (which is the entire conjugate moiety) of the first monomer of CHEM1 (denoted "1:R2") and the R1 attachment point of the first monomer of RNA1 (denoted "1:R1").

[0318] As noted above, "RNA1" indicates that the molecule is a nucleic acid, the brackets {} define the portion of the HELM string that refers to a nucleic acid molecule (oligonucleotide), "$$$$" indicate the end of the molecule, and "V2.0" indicates that HELM version 2.0 is being used. Thus, in HELM strings that refer to oligonucleotides (i.e., compounds) of the present invention, these characters are unnecessary because they do not provide any additional information that is not evident from the molecule being an oligonucleotide. Thus, a simplified form of any HELM string herein can be formed by removing RNA1, {}, $$$$, and V2.0 from the HELM string. Such a simplified HELM string is referred to herein as a "simplified HELM string." A simplified HELM string refers to exactly the same molecule as the corresponding full HELM string. A simplified HELM string may be a more readable way to present a HELM string.

[0319] Thus, any HELM string herein that refers to an oligonucleotide (i.e., a compound) of the invention can alternatively be depicted using a simplified HELM string. In other words, the simplified HELM string for a given compound is interchangeable with the full HELM string for that compound.

[0320] HELM notation example For example, CMP number 32_1 is represented by the following HELM string (shown in Table 4): RNA1{[mR](C)[sP].[mR](C)[sP].[mR](C)[sP].[mR](C)[sP].[mR](A)[ sP].[fR](G)[sP].[mR](C)[sP].[fR](A)[sP].[mR](G)[sP].[fR](C)[sP ].[mR](U)[sP].[fR](U)[sP].[mR](C)[sP].[fR](A)[sP].[mR](G)[sP] .[fR](U)[sP].[mR](C)[sP].[fR](C)[sP].[mR](C)[sP].[fR](U)[sP].[ mR](U)[sP].[fR](U)[sP].[mR](C)[sP].[dR](T)[sP].[dR](C)[sP].[d R]([In])[sP].[mR](U)[sP].[fR](C)[sP].[mR](G)[sP].[fR](A)[sP].[ mR](U)[sP].[fR](G)[sP].[mR](G)[sP].[fR](U)[sP].[mR](C)[sP].[m R](A)[sP].[mR](G)[sP].[mR](C)[sP].[mR](A)[sP].[mR](C)}$$$$V2.0

[0321] This HELM string consists of a single section listing the oligonucleotide with CMP number 32_1. The initial "RNA1" indicates that the molecule is a nucleic acid (oligonucleotide). The structure of the oligonucleotide is shown using HELM notation in brackets {} following RNA1. "$$$$" indicates the end of the section and the entire HELM string. "V2.0" indicates the use of HELM version 2.0. A simplified HELM string pointing to the same molecule with RNA1, {}, $$$$, and V2.0 removed is as follows: [mR](C)[sP].[mR](C)[sP].[mR](C)[sP].[mR](C)[sP].[mR](A)[sP] .[fR](G)[sP].[mR](C)[sP].[fR](A)[sP].[mR](G)[sP].[fR](C)[sP] .[mR](U)[sP].[fR](U)[sP].[mR](C)[sP].[fR](A)[sP].[mR](G)[sP] .[fR](U)[sP].[mR](C)[sP].[fR](C)[sP].[mR](C)[sP].[fR](U)[sP] .[mR](U)[sP].[fR](U)[sP].[mR](C)[sP].[dR](T)[sP].[dR](C)[sP ].[dR]([In])[sP].[mR](U)[sP].[fR](C)[sP].[mR](G)[sP].[fR](A) [sP].[mR](U)[sP].[fR](G)[sP].[mR](G)[sP].[fR](U)[sP].[mR](C) [sP].[mR](A)[sP].[mR](G)[sP].[mR](C)[sP].[mR](A)[sP].[mR](C)

[0322] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology, biochemistry, cell biology, virology, or related fields are intended to be within the scope of the following claims.

[0323] Numbered paragraphs Particular embodiments of the present invention are as defined in the following numbered paragraphs. 1. An editing region comprising an editing nucleoside; a 5' mixmer region located 5' to the editing region; a 3' mixmer region located 3' to the editing region; An oligonucleotide comprising:

[0324] 2. An oligonucleotide containing an editing region that includes an editing nucleoside.

[0325] 3. The editing nucleoside is designated as position 0, each nucleoside 5' to an editing nucleoside is designated position +x, where x is the number of nucleosides 5' to the editing nucleoside at that position, including the nucleoside at that position; Each nucleoside 3' to an editing nucleoside is designated position -y, where y is the number of nucleosides 3' to the editing nucleoside at that position, including the nucleoside at that position. 3. The oligonucleotide of paragraph 1 or 2.

[0326] 4. The following structure: X +1 -X 0 -X -1 During the ceremony, X +1 is the 5' mixmer region, X 0 is the editing region, X -1 is the 3' mixmer region, The oligonucleotide of any one of the preceding paragraphs, comprising:

[0327] 5. The oligonucleotide of any one of the preceding paragraphs, wherein the 5' mixmer region comprises a first type of 5' mixmer nucleoside and a second type of 5' mixmer nucleoside, and wherein the sugar moieties of the first type of 5' mixmer nucleoside are different from the sugar moieties of the second type of 5' mixmer nucleoside.

[0328] 6. The oligonucleotide of any one of the preceding paragraphs, wherein the 3' mixmer region comprises a first type of 3' mixmer nucleoside and a second type of 3' mixmer nucleoside, and wherein the sugar moiety of the first type of 3' mixmer nucleoside is different from the sugar moiety of the second type of 3' mixmer nucleoside.

[0329] 7. The oligonucleotide of any one of the preceding paragraphs, wherein the 5' mixmer region comprises an alternating pattern of a first type of 5' mixmer nucleoside and a second type of 5' mixmer nucleoside.

[0330] 8. The oligonucleotide of paragraph 7, wherein the 5' mixmer region comprises an alternating pattern of a single nucleoside of a first type of 5' mixmer nucleoside and a single nucleoside of a second type of 5' mixmer nucleoside.

[0331] 9. The oligonucleotide of any one of the preceding paragraphs, wherein the 3' mixmer region comprises an alternating pattern of a first type of 3' mixmer nucleoside and a second type of 3' mixmer nucleoside.

[0332] 10. The oligonucleotide of paragraph 9, wherein the 3' mixmer region comprises an alternating pattern of a single nucleoside of a first type of 3' mixmer nucleoside and a single nucleoside of a second type of 3' mixmer nucleoside.

[0333] 11. The 5' mixmer region has the following structure: (Y 5 -Z 5 -) m During the ceremony, Y 5 is one or more nucleosides of a first type of 5'-mixmer nucleoside, Z 5 is one or more nucleosides of a second type of 5'-mixmer nucleoside, m is a number between 1 and 20 The oligonucleotide of any one of the preceding paragraphs, comprising:

[0334] 12. The 3' mixmer region has the following structure: (Y 3 -Z 3 -) n During the ceremony, Y 3 is one or more nucleosides of a first type of 3'-mixmer nucleoside, Z 3 is one or more nucleosides of a second type of 3'-mixmer nucleoside, n is a number between 1 and 20 The oligonucleotide of any one of the preceding paragraphs, comprising:

[0335] 13. Each Y 5 are independently 1, 2, 3 or 4 nucleosides, preferably 1 nucleoside; each Z 5 are independently 1, 2, 3 or 4 nucleosides, preferably 1 nucleoside; Each Y 3 are independently 1, 2, 3 or 4 nucleosides, preferably 1 nucleoside; and / or each Z 3 are independently 1, 2, 3 or 4 nucleosides, preferably 1 nucleoside; 13. The oligonucleotide of paragraph 12.

[0336] 14. m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, more preferably 9; and / or n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 3, 4, 5, 6, 7, 8 or 9, more preferably 4; 14. The oligonucleotide of paragraph 12 or 13.

[0337] 15. The oligonucleotide of any one of the preceding paragraphs, wherein the 5' mixmer region is 15 to 25 nucleosides in length.

[0338] 16. The oligonucleotide of any one of the preceding paragraphs, wherein the 5' mixmer region is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleosides in length, preferably 18 nucleosides in length.

[0339] 17. The oligonucleotide of any one of the preceding paragraphs, wherein the 3' mixmer region is 4 to 12 nucleosides in length.

[0340] 18. The oligonucleotide of any one of the preceding paragraphs, wherein the 3' mixmer region is 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleosides in length, preferably 9 nucleosides in length.

[0341] 19. The 5' mixmer region is from position +1 to position +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24 or +25, preferably from position +2 to position +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, 19. The oligonucleotide of any one of paragraphs 3 to 18, comprising or consisting of a nucleoside at +24 or +25, more preferably at position +2 to position +15, +16, +17, +18, +19, +20, +21, +22, +23 or +24, more preferably at position +2 to position +19, +20, +21, +22, +23 or +24.

[0342] 20. The oligonucleotide of paragraph 19, wherein the 5' mixmer region comprises or consists of nucleosides at positions +2 to +19.

[0343] 21. The oligonucleotide of any one of paragraphs 3 to 20, wherein the 3' mixmer region comprises or consists of nucleosides at position -1 to position -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16 or -17, preferably at position -2 to position -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16 or -17, more preferably at position -2 to position -5, -6, -7, -8, -9, -10, -11, -12, -13, -14 or -15, more preferably at position -2 to position -5, -6, -7, -8, -9 or -10.

[0344] 22. The oligonucleotide of paragraph 21, wherein the 3' mixmer region comprises or consists of nucleosides at positions -2 to -10.

[0345] 23. The 5' mixmer region consists of nucleosides from position +2 to position +15, +16, +17, +18, +19, +20, +21, +22, +23 or +24, preferably from position +2 to position +17, +18, +19, +20, +21, +22, +23 or +24; and 23. The oligonucleotide of any of paragraphs 3 to 22, wherein the 3' mixmer region consists of nucleosides from position -2 to position -5, -6, -7, -8, -9, -10, -11, -12, -13, -14 or -15, more preferably from position -2 to position -5, -6, -7, -8, -9 or -10.

[0346] 24. The 5' mixmer region consists of nucleosides at positions +2 to +19; and The 3' mixmer region consists of nucleosides at positions -2 to -10. 24. The oligonucleotide of paragraph 23.

[0347] 25. (a) the first type of 5'-mixmer nucleoside is selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, 2'-O-methoxyethyl-RNA (MOE-RNA), 2'-fluoro-RNA, linked nucleic acid (LNA), arabinonucleic acid (ANA) and 2'-fluoroarabinonucleic acid (FANA) nucleosides, preferably the first type of 5'-mixmer nucleoside is a 2'-O-methyl-RNA nucleoside or a 2'-fluoro-RNA nucleoside, more preferably a 2'-fluoro-RNA nucleoside; and / or (b) the second type of 5'-mixmer nucleoside is selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, MOE-RNA, 2'-fluoro-RNA, LNA, ANA, and FANA nucleosides, preferably the second type of 5'-mixmer nucleoside is a 2'-O-methyl-RNA nucleoside or a 2'-fluoro-RNA nucleoside, more preferably a 2'-O-methyl-RNA nucleoside; and / or (c) the first type of 3'-mixmer nucleoside is selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, MOE-RNA, 2'-fluoro-RNA, LNA, ANA, and FANA nucleosides, preferably the first type of 3'-mixmer nucleoside is a 2'-O-methyl-RNA nucleoside or a 2'-fluoro-RNA nucleoside, more preferably a 2'-O-methyl-RNA nucleoside; and / or (d) The second type of 3'-mixmer nucleoside is selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, MOE-RNA, 2'-fluoro-RNA, LNA, ANA and FANA nucleosides, preferably the second type of 3'-mixmer nucleoside is a 2'-O-methyl-RNA nucleoside or a 2'-fluoro-RNA nucleoside, more preferably a 2'-fluoro-RNA nucleoside; 25. The oligonucleotide according to any one of paragraphs 5 to 24.

[0348] 26. The first type of 5'-mixmer nucleoside is a 2'-fluoro-RNA nucleoside; and / or The second type of 5'-mixmer nucleoside is a 2'-O-methyl-RNA nucleoside; and / or The first type of 3'mixmer nucleoside is a 2'-fluoro-RNA nucleoside; and / or The second type of 3'-mixmer nucleoside is a 2'-O-methyl-RNA nucleoside. 26. The oligonucleotide of paragraph 25.

[0349] 27. The first type of 5'-mixmer nucleoside is a 2'-fluoro-RNA nucleoside, The second type of 5'-mixmer nucleoside is a 2'-O-methyl-RNA nucleoside, The first type of 3'mixmer nucleoside is a 2'-fluoro-RNA nucleoside, The second type of 3'-mixmer nucleoside is a 2'-O-methyl-RNA nucleoside. 27. The oligonucleotide of paragraph 26.

[0350] 28. The oligonucleotide of any one of the preceding paragraphs, wherein the 5' mixmer region comprises or consists of 2'-fluoro-RNA nucleosides and 2'-O-methyl-RNA nucleosides.

[0351] 29. The oligonucleotide of any one of the preceding paragraphs, wherein the 5' mixmer region comprises or consists of alternating 2'-fluoro-RNA nucleosides and 2'-O-methyl-RNA nucleosides, preferably alternating a single 2'-fluoro-RNA nucleoside and a single 2'-O-methyl-RNA nucleoside.

[0352] 30. The oligonucleotide of any one of the preceding paragraphs, wherein the 3' mixmer region comprises or consists of 2'-fluoro-RNA nucleosides and 2'-O-methyl-RNA nucleosides.

[0353] 31. The oligonucleotide of any one of the preceding paragraphs, wherein the 3' mixmer region comprises or consists of alternating 2'-fluoro-RNA nucleosides and 2'-O-methyl-RNA nucleosides, preferably alternating a single 2'-fluoro-RNA nucleoside and a single 2'-O-methyl-RNA nucleoside.

[0354] 32. The oligonucleotide of any one of paragraphs 3 to 31, wherein each nucleoside at positions +2 to +19 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside.

[0355] 33. The oligonucleotide of any one of paragraphs 3 to 32, wherein each nucleoside at positions -2 to -10 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside.

[0356] 34. The oligonucleotide of any one of paragraphs 3 to 33, wherein each nucleoside at positions +2 to +24 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside.

[0357] 35. The oligonucleotide of any one of paragraphs 3 to 34, wherein each nucleoside at positions -2 to -15 is a 2'-fluoro-RNA nucleoside or a 2'-O-methyl-RNA nucleoside.

[0358] 36. The oligonucleotide according to any one of paragraphs 3 to 35, wherein the nucleoside at one or more of positions +3, +5, +7, +9, +11, +13, +15, +17 and +19 is a 2'-fluoro-RNA nucleoside, preferably wherein the nucleoside at each of positions +3, +5, +7, +9, +11, +13, +15, +17 and +19 is a 2'-fluoro-RNA nucleoside.

[0359] 37. The oligonucleotide according to any one of paragraphs 3 to 36, wherein the nucleoside at one or more of positions -3, -5, -7 and -9 is a 2'-fluoro-RNA nucleoside, preferably wherein the nucleoside at each of positions -3, -5, -7 and -9 is a 2'-fluoro-RNA nucleoside.

[0360] 38. The oligonucleotide according to any one of paragraphs 3 to 37, wherein the nucleoside at one or more of positions +2, +4, +6, +8, +10, +12, +14 and +18 is a 2'-O-methyl-RNA nucleoside, preferably wherein the nucleoside at each of positions +2, +4, +6, +8, +10, +12, +14 and +18 is a 2'-O-methyl-RNA nucleoside.

[0361] 39. The oligonucleotide according to any one of paragraphs 3 to 38, wherein the nucleoside at one or more of positions -2, -4 and -6 is a 2'-O-methyl-RNA nucleoside, preferably wherein the nucleoside at each of positions -2, -4 and -6 is a 2'-O-methyl-RNA nucleoside.

[0362] 40. The oligonucleotide of any one of paragraphs 3 to 39, wherein the nucleoside at one or more of positions +3, +5, +7, +9, +11, +13, +15, +17, +19, +21 and +23 is a 2'-fluoro-RNA nucleoside, preferably wherein the nucleoside at each of positions +3, +5, +7, +9, +11, +13, +15, +17, +19, +21 and +23 is a 2'-fluoro-RNA nucleoside.

[0363] 41. The oligonucleotide of any one of paragraphs 3 to 40, wherein the nucleoside at one or more of positions -3, -5, -7, -9, -11, -13, and -15 is a 2'-fluoro-RNA nucleoside, preferably, the nucleoside at each of positions -3, -5, -7, -9, -11, -13, and -15 is a 2'-fluoro-RNA nucleoside.

[0364] 42. The oligonucleotide according to any one of paragraphs 3 to 41, wherein the nucleoside at one or more of positions +2, +4, +6, +8, +10, +12, +14, +18, +20, +22 and +24 is a 2'-O-methyl-RNA nucleoside, preferably wherein the nucleoside at each of positions +2, +4, +6, +8, +10, +12, +14, +18, +20, +22 and +24 is a 2'-O-methyl-RNA nucleoside.

[0365] 43. The oligonucleotide according to any one of paragraphs 3 to 42, wherein the nucleoside at one or more of positions -2, -4, -6, -10, -12 and -14 is a 2'-O-methyl-RNA nucleoside, preferably, the nucleoside at each of positions -2, -4, -6, -10, -12 and -14 is a 2'-O-methyl-RNA nucleoside.

[0366] 44. The oligonucleotide of any one of paragraphs 3 to 43, wherein the nucleoside at position -2 is a 2'-O-methyl-RNA nucleoside.

[0367] 45. The oligonucleotide of any one of paragraphs 3 to 44, wherein the nucleoside at position -3 is a 2'-fluoro-RNA nucleoside.

[0368] 46. The oligonucleotide of any one of paragraphs 3 to 45, wherein the nucleoside at position -8 is a 2'-fluoro-RNA nucleoside.

[0369] 47. The oligonucleotide of any one of paragraphs 3 to 46, wherein the nucleoside at position +16 is a 2'-fluoro-RNA nucleoside.

[0370] 48. The oligonucleotide of any one of paragraphs 3 to 47, wherein the nucleoside at position +17 is a 2'-fluoro-RNA nucleoside.

[0371] 49. The oligonucleotide of any one of paragraphs 3 to 45, 47, or 48, wherein the nucleoside at position -8 is a 2'-O-methyl-RNA nucleoside.

[0372] 50. The oligonucleotide of any one of paragraphs 3 to 46, 48, or 49, wherein the nucleoside at position +16 is a 2'-O-methyl-RNA nucleoside.

[0373] 51. The oligonucleotide of any one of the preceding paragraphs, wherein the oligonucleotide is for editing a target nucleic acid.

[0374] 52. The oligonucleotide according to paragraph 51, wherein the target nucleic acid comprises a target adenosine.

[0375] 53. The oligonucleotide according to paragraph 51 or paragraph 52, which is capable of binding to a target nucleic acid by complementary base pairing.

[0376] 54. The oligonucleotide of any one of the preceding paragraphs, wherein the oligonucleotide is a guide oligonucleotide for adenosine deaminase acting on RNA (ADAR).

[0377] 55. The oligonucleotide of any one of the preceding paragraphs, wherein the oligonucleotide is capable of recruiting ADAR to a target nucleic acid.

[0378] 56. The oligonucleotide according to paragraph 54 or 55, wherein the ADAR is ADAR1 or ADAR2.

[0379] 57. The oligonucleotide according to any one of paragraphs 52 to 56, wherein the oligonucleotide is capable of effecting the conversion of a target adenosine (A) to inosine (I).

[0380] 58. The oligonucleotide of any one of paragraphs 51 to 57, wherein the target nucleic acid is RNA.

[0381] 59. The oligonucleotide according to any one of paragraphs 51 to 58, wherein the target nucleic acid encodes a protein.

[0382] 60. The oligonucleotide of any one of paragraphs 51 to 59, wherein the target nucleic acid is mRNA.

[0383] 61. The oligonucleotide according to any one of paragraphs 51 to 60, wherein the target nucleic acid encodes alpha-1 antitrypsin (A1AT).

[0384] 62. The oligonucleotide according to any one of paragraphs 51 to 61, wherein the target nucleic acid is SERPINA1 mRNA.

[0385] 63. The oligonucleotide according to any one of paragraphs 51 to 62, wherein the oligonucleotide is capable of effecting conversion of an AAA codon encoding lysine to an IAA codon encoding glutamic acid on the target nucleic acid.

[0386] 64. The oligonucleotide according to any one of paragraphs 51 to 63, wherein the oligonucleotide is capable of effecting conversion of an AAG codon encoding lysine to an IAG codon encoding glutamic acid on the target nucleic acid.

[0387] 65. The oligonucleotide according to any one of paragraphs 52 to 64, wherein the target adenosine corresponds to A1024 of SEQ ID NO: 186.

[0388] 66. The oligonucleotide according to any one of paragraphs 51 to 65, wherein the target nucleic acid comprises or consists of a sequence according to SEQ ID NO: 186.

[0389] 67. The oligonucleotide of any one of paragraphs 51 to 66, wherein the editing nucleoside is not complementary to the target adenosine.

[0390] 68. The oligonucleotide of any one of the preceding paragraphs, wherein the nucleobase of the editing nucleoside is selected from the group consisting of cytosine, 5-methylcytosine, guanine, and hypoxanthine.

[0391] 69. The oligonucleotide of any one of the preceding paragraphs, wherein the editing nucleoside is cytidine (C).

[0392] 70. The oligonucleotide of any one of the preceding paragraphs, wherein the edited region consists of edited nucleosides.

[0393] 71. The oligonucleotide of any one of paragraphs 3 to 70, wherein the editing region comprises or consists of nucleosides at positions +1, 0 and -1.

[0394] 72. The oligonucleotide of any one of the preceding paragraphs, wherein the edited region comprises or consists of an edited triplet consisting of three nucleosides, and the edited nucleoside is the middle nucleoside of the edited triplet.

[0395] 73. The oligonucleotide according to paragraph 72, wherein the editing triplet is 5'-thymidine-cytidine-inosine-3' (TCI).

[0396] 74. The oligonucleotide of any one of paragraphs 5 to 73, wherein the editing region comprises one or more nucleosides comprising a sugar moiety that is different from the sugar moieties of a first type of 5' mixmar nucleoside, a second type of 5' mixmar nucleoside, a first type of 3' mixmar nucleoside, and / or a second type of 3' mixmar nucleoside.

[0397] 75. The oligonucleotide of paragraph 74, wherein each nucleoside of the editing region comprises a sugar moiety that differs from the sugar moieties of a first type of 5' mixmar nucleoside, a second type of 5' mixmar nucleoside, a first type of 3' mixmar nucleoside, and a second type of 3' mixmar nucleoside.

[0398] 76. The oligonucleotide of any one of the preceding paragraphs, wherein each nucleoside of the editing region contains the same sugar moiety.

[0399] 77. The oligonucleotide of any one of the preceding paragraphs, wherein each nucleoside of the editing region is independently selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, 2'-fluoro-RNA, MOE-RNA, LNA, ANA, and FANA nucleosides.

[0400] 78. The oligonucleotide of any one of the preceding paragraphs, wherein the editing region comprises one or more DNA nucleosides.

[0401] 79. The oligonucleotide of any one of the preceding paragraphs, wherein the editing nucleoside is a DNA nucleoside.

[0402] 80. The oligonucleotide of any one of the preceding paragraphs, wherein all nucleosides in the editing region are DNA nucleosides.

[0403] 81. The oligonucleotide of any one of paragraphs 3 to 80, wherein the nucleoside at one or more of positions +1, 0 and −1 is a DNA nucleoside, preferably the nucleoside at each of positions +1, 0 and −1 is a DNA nucleoside.

[0404] 82. The oligonucleotide of paragraph 81, wherein the editing region consists of nucleosides at positions +1, 0 and -1 of the oligonucleotide, wherein the nucleoside at position +1 is a thymidine DNA nucleoside, the nucleoside at position 0 is a cytidine DNA nucleoside, and the nucleoside at position -1 is an inosine DNA nucleoside.

[0405] 83. The oligonucleotide of any one of paragraphs 1 to 78, wherein the editing nucleoside is a FANA nucleoside.

[0406] 84. The oligonucleotide of paragraph 83, wherein the editing nucleoside is a FANA nucleoside and all other nucleosides in the editing region are DNA nucleosides.

[0407] 85. The oligonucleotide of any one of paragraphs 3 to 78, wherein the nucleoside at position 0 is a FANA nucleoside.

[0408] 86.(a) The nucleoside at position +1 is a DNA nucleoside, or (b) the nucleoside at position -1 is a DNA nucleoside, or (c) The oligonucleotide of paragraph 85, wherein the nucleosides at positions +1 and −1 are DNA nucleosides.

[0409] 87. The oligonucleotide according to paragraph 86, wherein the editing region consists of nucleosides at positions +1, 0 and -1 of the oligonucleotide, wherein the nucleoside at position +1 is a thymidine DNA nucleoside, the nucleoside at position 0 is a cytidine FANA nucleoside, and the nucleoside at position -1 is an inosine DNA nucleoside.

[0410] 88. The oligonucleotide of any one of the preceding paragraphs, wherein the oligonucleotide is 30 to 61 nucleosides in length, preferably 30 to 50 nucleosides in length, more preferably 35 to 45 nucleosides in length.

[0411] 89. The oligonucleotide of any one of the preceding paragraphs, wherein the oligonucleotide is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleosides in length, preferably 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 nucleosides in length, more preferably 40 nucleosides in length.

[0412] 90. The oligonucleotide of any one of the preceding paragraphs, wherein the oligonucleotide comprises exactly 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleosides 5' to the edited nucleoside, preferably 24, 25, 26 or 27 nucleosides 5' to the edited nucleoside, more preferably exactly 24 nucleosides 5' to the edited nucleoside.

[0413] 91. The oligonucleotide of any one of the preceding paragraphs, wherein the oligonucleotide comprises exactly 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides 3' to the edited nucleoside, preferably exactly 12, 13, 14 or 15 nucleotides 3' to the edited nucleoside, more preferably exactly 15 nucleotides 3' to the edited nucleoside.

[0414] 92. The oligonucleotide of any one of the preceding paragraphs, wherein the oligonucleotide comprises exactly 24 nucleotides 5' to the edited nucleoside and exactly 15 nucleotides 3' to the edited nucleoside.

[0415] 93. The oligonucleotide of any one of the preceding paragraphs, wherein the oligonucleotide comprises one or more phosphorothioate internucleoside linkages.

[0416] 94. The oligonucleotide according to any one of paragraphs 3 to 93, wherein the internucleoside linkages between the nucleosides at positions +1 and +0 are phosphodiester internucleoside linkages.

[0417] 95. The oligonucleotide according to any one of paragraphs 3 to 94, wherein the internucleoside linkages between the nucleosides at positions 0 and -1 are phosphodiester internucleoside linkages.

[0418] 96. The oligonucleotide according to any one of paragraphs 3 to 95, wherein the internucleoside linkages between the nucleosides at positions -1 and -2 are phosphorothioate internucleoside linkages.

[0419] 97. The oligonucleotide according to any one of paragraphs 3 to 96, wherein the internucleoside linkages between the nucleosides at positions -2 and -3 are phosphodiester internucleoside linkages.

[0420] 98. The oligonucleotide according to any one of paragraphs 94 to 97, wherein all other internucleoside linkages are phosphorothioate internucleoside linkages.

[0421] 99. The oligonucleotide according to any one of paragraphs 1 to 93, wherein all internucleoside linkages of the oligonucleotide are phosphorothioate internucleoside linkages.

[0422] 100. The oligonucleotide of any one of the preceding paragraphs, wherein the oligonucleotide comprises a 5' flank region located 5' to the 5' mixmer region.

[0423] 101. The oligonucleotide of any one of the preceding paragraphs, wherein the oligonucleotide comprises a 3' flank region located 3' to the 3' mixmer region.

[0424] 102. The following structure: X +2 -X +1 -X 0 -X -1 -X -2 During the ceremony, X +2 is the 5' flanking region, X +1 is the 5' mixmer region, X 0 is the editing region, X -1 is the 3' mixmer region, X -2 is the 3' flanking region, 103. The oligonucleotide of Paragraph 102, comprising:

[0425] 103. The oligonucleotide according to any one of paragraphs 100 to 102, wherein the 5' flanking region is 1, 2, 3, 4 or 5 nucleosides in length, preferably 5 nucleosides in length.

[0426] 104. The oligonucleotide according to any one of paragraphs 101 to 103, wherein the 3' flanking region is 1, 2, 3, 4 or 5 nucleosides in length, preferably 5 nucleosides in length.

[0427] 105. The oligonucleotide according to any one of paragraphs 100 to 104, wherein the 5' flanking region consists of 1, 2, 3, 4 or 5 nucleosides at the 5' end of the oligonucleotide, preferably 5 nucleosides at the 5' end of the oligonucleotide.

[0428] 106. The oligonucleotide according to any one of paragraphs 101 to 105, wherein the 3' flanking region consists of 1, 2, 3, 4 or 5 nucleosides at the 3' end of the oligonucleotide, preferably 5 nucleosides at the 3' end of the oligonucleotide.

[0429] 107. The oligonucleotide of any one of paragraphs 100 to 106, wherein the 5' flanking region comprises one or more sugar-modified nucleosides.

[0430] 108. The oligonucleotide of any one of paragraphs 101 to 107, wherein the 3' flanking region comprises one or more sugar-modified nucleosides.

[0431] 109. The oligonucleotide according to paragraph 107 or paragraph 108, wherein one or more sugar-modified nucleosides are independently selected from the group consisting of 2'-O-methyl-RNA, 2'-fluoro-RNA, 2'-O-methoxyethyl-RNA (MOE-RNA) and LNA nucleosides.

[0432] 110. The oligonucleotide of any one of paragraphs 100 to 109, wherein all nucleosides in the 5' flanking region are sugar-modified nucleosides.

[0433] 111. The oligonucleotide of any one of paragraphs 101 to 110, wherein all nucleosides in the 3' flanking region are sugar-modified nucleosides.

[0434] 112. The oligonucleotide of any one of paragraphs 100 to 111, wherein all nucleosides of the 5' flanking region are 2'-O-methyl-RNA.

[0435] 113. The oligonucleotide of any one of paragraphs 101 to 112, wherein all nucleosides of the 3' flanking region are 2'-O-methyl-RNA.

[0436] 114. The oligonucleotide of any one of the preceding paragraphs, wherein the oligonucleotide comprises or consists of a sequence complementary to SERPINA1 mRNA.

[0437] 115. The oligonucleotide of any one of the preceding paragraphs, wherein the sequence of the oligonucleotide comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementarity to positions 986 to 1062 of SEQ ID NO: 186, and wherein any matches or mismatches between the editing nucleoside and the target adenosine are not included when determining the percentage of complementarity.

[0438] 116. The following sequence: TIFF2025525564000014.tif11157, or a variant of SEQ ID NO: 87 containing exactly one, exactly two, or exactly three single nucleoside substitutions; In the sequence, I is inosine, The oligonucleotide of any one of the preceding paragraphs, wherein the oligonucleotide comprises positions 38, 39 and 40 of SEQ ID NO:87.

[0439] 117. The oligonucleotide according to paragraph 116, wherein positions 38, 39 and 40 of SEQ ID NO: 87 are edited triplets.

[0440] 118. The oligonucleotide according to paragraph 116 or paragraph 117, wherein the sequence of the oligonucleotide comprises or consists of 30 to 50 consecutive nucleosides from SEQ ID NO: 87 or a variant thereof.

[0441] 119. The oligonucleotide according to paragraph 118, wherein the sequence of the oligonucleotide comprises or consists of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 consecutive nucleosides, preferably 40 consecutive nucleosides, from SEQ ID NO: 87 or a variant thereof.

[0442] 120. The oligonucleotide according to any one of paragraphs 116 to 119, wherein the variant of SEQ ID NO: 87 comprises exactly one single nucleoside substitution.

[0443] 121. The oligonucleotide of any one of paragraphs 116 to 120, wherein the variant of SEQ ID NO: 87 comprises a nucleoside substitution at position 38, 39 and / or 40 of SEQ ID NO: 87.

[0444] 122. The oligonucleotide according to any one of the preceding paragraphs, wherein the sequence of the oligonucleotide comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any one of SEQ ID NOs: 1-86.

[0445] 123. The sequence of the oligonucleotide is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 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, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 and 86, comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any one of 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 and 86.

[0446] 124. The oligonucleotide of any one of the preceding paragraphs, wherein the sequence of the oligonucleotide comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 2, 3, 7, 8, 9, 10, 13, 19, 20, 21, 22, 23, 26, 29, 30, 31, 32, 33, 34, 35, 36, 41, 42, 43, 45, 59, 60, 61, 62, 63, 64, and 65.

[0447] 125. The oligonucleotide of any one of the preceding paragraphs, wherein the sequence of the oligonucleotide comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 3, 7, 8, 9, 19, 20, 21, 22, 29, 30, 31, 32, 33, 34, 42, 43, 62, and 63.

[0448] 126. The oligonucleotide of any one of the preceding paragraphs, wherein the sequence of the oligonucleotide comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 32.

[0449] 127. Oligonucleotides are CMP numbers 1_1, 2_1, 3_1, 3_2, 3_3, 4_1, 5_1, 6_1, 7_1, 8_1, 9_1, 10_1, 11_1, 12_1, 13_1, 14_1, 15_1, 16_1, 17_1, 18_1, 19_1, 20_1, 21_1, 22_1, 23_1, 24_1, 25_1, 26_1, 26_2, 26_3, 26_4, 26_5, 26_6, 26_7, 26_8, 26_9, 27_1, 28_1, 29_1, 30_1, 31_1, 32_1, 32_2, 32_3, 32_4, 32_5, 32_6, 32_7, 32_8, 32_9, 33_1, 34_1, 34_2, 34_3, 34_4, 34_5, 34_6, 34_7, 34_8, 34_9, 35_1, 35_2, 35_3, 35_4, 35_5, 35_6, 35_7, 35_8, 35_9, 36_1, 36_2, 36_3, 36_4, 36_5, 36_6, 36_7, 36_8, 36_9, 37_1, 37_2, 37_3, 37_4, 37_5, 37_6, 37_7 2_8, 32_9, 32_10, 32_11, 32_12, 32_13, 32_14, 32_15, 32_16, 32_17, 32_18, 32_19, 32_20, 32_21, 32_22, 32_23, 32_24, 32_25, 32_26, 32_27, 32_28, 32 _29, 32_30, 32_31, 32_32, 32_33, 32_34, 32_35, 32_36, 32_37, 32_38, 32_39, 32_40, 32_41, 32_42, 32_43, 32_44, 32_45, 32_46, 32_47, 32_48, 32_49, 3 2_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_59, 32_60, 32_61, 32_62, 32_63, 32_64, 32_65, 32_66, 32_67, 32_68, 32_69, 32_70, 32_71, 32_72, 32_73, 32_74, 32_75, 32_76, 32_77, 32_78, 32_79, 32_80, 32_81, 32_82, 32_83, 33_1, 34_1, 35_1, 36_1, 37_1, 38_1, 39_1, 40_1, 40_2, 40 _3, 40_4, 40_5, 40_6, 40_7, 40_8, 40_9, 40_10, 41_1, 41_2, 41_3, 41_4, 41_5, 41_6, 41_7, 41_8, 41_9, 41_10, 42_1, 42_2, 42_3, 42_4, 42_5, 42_6, 43_1, 43_2, 43_3, 44_1, 45_1, 46_1, 47_1, 47_2, 48_1, 49_1, 50_1, 51_1, 52_1, 53_1, 54_1, 55_1, 56_1, 57_1, 58_1, 59_1, 60_1, 61_1, 62_1, 63_1, 64_1, 65_1,The oligonucleotide of any one of the preceding paragraphs, comprising or consisting of any one of 66_1, 67_1, 68_1, 69_1, 70_1, 71_1, 72_1, 73_1, 74_1, 75_1, 76_1, 77_1, 78_1, 79_1, 80_1, 81_1, 82_1, 83_1, 84_1, 85_1 and 86_1.

[0450] 128. Oligonucleotides are CMP numbers 2_1, 3_1, 3_3, 7_1, 8_1, 9_1, 10_1, 13_1, 19_1, 20_1, 21_1, 22_1, 23_1, 26_8, 29_1, 30_1, 31_1, 32_1, 32_2, 32_4, 32_5, 32_6, 32_7, 32_8, 32_9, 32_10, 32_11, 32_12, 32_13, 32_14, 32_16, 32_17, 32_18, 32_19, 32_20, 32_21, 32_22, 32_23, 32_29, 32_33, 32_34, 32_4 7, 32_48, 32_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_59, 32_60, 32_61, 32_62, 32_63, 32_64, 32_65, 33_1, 34_1, 35_1, 36_1, 41_8, 42_6, 43_1, 43_3, 45_1, 59_1, 60_1, 61_1, 62_1, 63_1, 64_1 and 65_1.

[0451] 129. Oligonucleotides are CMP numbers 3_1, 7_1, 8_1, 9_1, 19_1, 20_1, 21_1, 22_1, 29_1, 30_1, 31_1, 19_1, 32_1, 32_4, 32_5, 32_6, 32_7, 32_8, 32_9, 32_10, 32_11, 32_12, 32_13, 32_14, 32_16, 32_17, 32_18, 32_19, 32_20, 32_21, 32_22, 32_23, 32_24, 32_25, 32_26, 32_27, 32_28, 32_29, 32_30, 32_31, 32_32, 32_33, 32_34, 32_35, 32_36, 32_37, 32_38, 32_39, 32_40, 32_41, 32_42, 32_43, 32_44, 32_45, 32_46, 32_47, 32_48, 32_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_59, 32_60, 32_61, 32_62, 32_63, 32_64, 32_65, 32 32_6, 32_6, 32_6_1, 32_6_2, 32_6_3, 32_6_4, 32_6_5, 32_6_6, 32_6_7, 32_6_8, 32_6_9, 32_7_10, 32_7_21, 32_7_32, 32_7_4, 32_7_5, 32_7_6, 32_7_7, 32_7_8, 32_7_9, 32_8_11, 32_8_12, 32_8_13, 32_8_14, 32_8_15, 32_8_16, 32_8_17, 32_8_18, 32_8_19, 32_9_20, 32_9_21, 32_9_22, 32_9_23, 32_9_24, 32_9_25, 32_9_26, 32_9_27, 32_9_28, 32_9_30, 32_9_31, 32_9_32, 32_9_33, 32_9_47, 32_9_48, 32_9_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_60, 32_61, 32_62, 32_63, 32_64, 32_65, 33_1, 34_1, 42_6, 43_

[0452] 130. Oligonucleotides are CMP numbers 32_1, 32_2, 32_3, 32_4, 32_5, 32_6, 32_7, 32_8, 32_9, 32_10, 32_11, 32_12, 32_13, 32_14, 32_15, 32_16, 32_17, 32_18, 32_19, 32_20, 32_21, 32 _22, 32_23, 32_24, 32_25, 32_26, 32_27, 32_28, 32_29, 32_30, 32_31, 32_32, 32_33, 32_34, 32_35, 32_36, 32_37, 32_38, 32_39, 32_40, 32_41, 32_42, 32_43, 32_44, 32_4 5, 32_46, 32_47, 32_48, 32_49, 32_50, 32_51, 32_52, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_59, 32_60, 32_61, 32_62, 32_63, 32_64, 32_65, 32_66, 32_67, 32_68, The oligonucleotide of any one of the preceding paragraphs, comprising or consisting of any one of 32_69, 32_70, 32_71, 32_72, 32_73, 32_74, 32_75, 32_76, 32_77, 32_78, 32_79, 32_80, 32_81, 32_82 and 32_83.

[0453] 131. The oligonucleotide according to any one of the preceding paragraphs, wherein the oligonucleotide comprises or consists of CMP No. 32_1.

[0454] 132. The oligonucleotide shown in Figure 29.

[0455] 133. An oligonucleotide conjugate comprising an oligonucleotide according to any one of the preceding paragraphs covalently attached to at least one conjugate moiety.

[0456] 134. The oligonucleotide conjugate according to paragraph 133, wherein the conjugate moiety is covalently attached to the oligonucleotide via a linker.

[0457] 135. The oligonucleotide conjugate of paragraph 134 or paragraph 135, wherein the conjugate moiety is selected from the group consisting of a carbohydrate, a cell surface receptor ligand, a drug substance, a hormone, a lipophilic substance, a polymer, a protein, a peptide, a toxin (e.g., a bacterial toxin), a vitamin, a viral protein (e.g., a capsid), or a combination thereof.

[0458] 136. The oligonucleotide conjugate according to paragraph 135, wherein the conjugate moiety is an N-acetylgalactosamine (GalNAc) conjugate moiety.

[0459] 137. The oligonucleotide or oligonucleotide conjugate according to any one of the preceding paragraphs, wherein the oligonucleotide or oligonucleotide conjugate is in the form of a pharmaceutically acceptable salt.

[0460] 138. The oligonucleotide or oligonucleotide conjugate according to paragraph 137, wherein the salt is a sodium salt or a potassium salt.

[0461] 139. The oligonucleotide or oligonucleotide conjugate of any one of the preceding paragraphs, wherein the oligonucleotide or oligonucleotide conjugate is encapsulated in a lipid-based delivery vehicle, covalently bound or encapsulated in a dendrimer, or conjugated to an aptamer.

[0462] 140. A pharmaceutical composition comprising an oligonucleotide or oligonucleotide conjugate according to any one of paragraphs 1 to 139 and a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0463] 141. A pharmaceutical composition according to paragraph 140, comprising an aqueous diluent or solvent, for example phosphate buffered saline.

[0464] 142. An in vitro method for editing a target nucleic acid in a target cell, comprising administering to the target cell an effective amount of an oligonucleotide or oligonucleotide conjugate described in any one of paragraphs 1 to 139 or a pharmaceutical composition described in paragraph 140 or paragraph 141.

[0465] 143. An in vivo method for editing a target nucleic acid in a target cell, comprising administering to the target cell an effective amount of an oligonucleotide or oligonucleotide conjugate according to any one of paragraphs 1 to 139 or a pharmaceutical composition according to paragraph 140 or paragraph 141.

[0466] 144. The method of paragraph 142 or 143, wherein the target nucleic acid comprises a target adenosine.

[0467] 145. The method of any one of paragraphs 142 to 144, wherein the target cell is a mammalian cell.

[0468] 146. The method of paragraph 145, wherein the target cells are human cells.

[0469] 147. The method of any one of paragraphs 142 to 146, wherein the target cells are lung cells or liver cells.

[0470] 148. The method of any one of paragraphs 142 to 147, wherein the amount of edited target nucleic acid is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the control.

[0471] 149. The method of paragraph 148, wherein the control is a cell that has not been exposed to the oligonucleotide or oligonucleotide conjugate.

[0472] 150. The method of any one of paragraphs 142-149, wherein the oligonucleotide binds to the target nucleic acid by complementary base pairing.

[0473] 151. The method of any one of paragraphs 142-150, wherein the oligonucleotide recruits ADAR to the target nucleic acid.

[0474] 152. The method of paragraph 151, wherein the ADAR is ADAR1 or ADAR2.

[0475] 153. The method of any one of paragraphs 142-152, wherein the target adenosine (A) is converted to inosine (I).

[0476] 154. The method of any one of paragraphs 142-153, wherein the target nucleic acid encodes a protein.

[0477] 155. The method of any one of paragraphs 142 to 154, wherein the target nucleic acid is mRNA.

[0478] 156. The method of any one of paragraphs 142 to 155, wherein the target nucleic acid encodes alpha-1 antitrypsin (A1AT).

[0479] 157. The method of any one of paragraphs 142 to 156, wherein the target nucleic acid is SERPINA1 mRNA.

[0480] 158. The method of any one of paragraphs 142 to 157, wherein on the target nucleic acid, an AAA codon encoding lysine is converted to an IAA codon encoding glutamic acid.

[0481] 159. The method of any one of paragraphs 142 to 158, wherein on the target nucleic acid, an AAG codon encoding lysine is converted to an IAG codon encoding glutamic acid.

[0482] 160. The method of any one of paragraphs 142-159, wherein the target adenosine is A1024 on the target nucleic acid defined by SEQ ID NO: 186.

[0483] 161. The method according to any one of paragraphs 142 to 160, wherein the target nucleic acid comprises or consists of a sequence according to SEQ ID NO: 186.

[0484] 162. A method for treating or preventing a disease, comprising administering to a subject suffering from or susceptible to the disease a therapeutically or prophylactically effective amount of an oligonucleotide or oligonucleotide conjugate according to any one of paragraphs 1 to 139 or a pharmaceutical composition according to paragraph 140 or paragraph 141.

[0485] 163. An oligonucleotide or oligonucleotide conjugate according to any one of paragraphs 1 to 139, or a pharmaceutical composition according to paragraph 140 or paragraph 141, for use in the treatment or prevention of a disease in a subject.

[0486] 164. Use of an oligonucleotide or oligonucleotide conjugate according to any one of paragraphs 1 to 139, or a pharmaceutical composition according to paragraph 140 or paragraph 141, for the preparation of a medicament for the treatment or prevention of a disease in a subject.

[0487] 165. The method according to paragraph 162, the oligonucleotide for use, the oligonucleotide conjugate for use or the pharmaceutical composition for use according to paragraph 163, or the use according to paragraph 164, wherein the disease is alpha 1 antitrypsin deficiency (A1AD).

[0488] 166. The method, oligonucleotide for use, oligonucleotide conjugate for use, pharmaceutical composition for use, or use of paragraph 165, wherein the treatment or prevention comprises treating or preventing one or more symptoms of A1AD selected from liver damage, liver failure, cirrhosis, jaundice, elastin breakdown in the lungs, emphysema, and chronic obstructive pulmonary disease (COPD). [Example]

[0489] Example 1 - Oligonucleotide Sequences and Compounds Oligonucleotides (compounds) and conjugates for editing SERPINA1, GAPDH, or mHprt mRNA were synthesized.

[0490] Oligonucleotide synthesis Oligonucleotide synthesis is generally known in the art.The oligonucleotide and conjugate of the present invention can be synthesized by any such method known in the art.The following is the protocol that can be applied.The oligonucleotide of the present invention may be produced by a method that is slightly different in terms of the device, support and concentration used.

[0491] Oligonucleotides are synthesized on a uridine universal support using the phosphoramidite approach of Mermade 192 on a 1 μmol scale. At the end of the synthesis, the oligonucleotides are cleaved from the solid support using aqueous ammonia at 60 °C for 5–16 h. The oligonucleotides are purified by reverse-phase HPLC (RP-HPLC), ion-exchange chromatography, or solid-phase extraction, characterized by UPLC, and their molecular weights are further confirmed by ESI-MS.

[0492] Oligonucleotide extension: Coupling of β-cyanoethyl phosphoramidites (DNA-A(Bz), DNA-G(ibu), DNA-C(Bz), DNA-T, LNA-5-methyl-C(Bz), LNA-A(Bz), LNA-G(dmf), or LNA-T) is carried out using 0.1 M of the 5'-O-DMT-protected amidite in acetonitrile and a solution of DCI (4,5-dicyanoimidazole) in acetonitrile (0.25 M) as the activator. In the final cycle, a phosphoramidite bearing the desired modification, such as a C6 linker for attaching a conjugate group, or such a conjugate group, can be used. Thiolation to introduce a phosphorothioate bond is carried out using hydrogenated xanthan gum (0.01 M in acetonitrile / pyridine 9:1). Phosphodiester bonds can be introduced using 0.02 M iodine in THF / pyridine / water 7:2:1. The remaining reagents are those commonly used in oligonucleotide synthesis.

[0493] For post-solid-phase synthesis conjugation, commercially available C6 amino linker phosphoramidites can be used in the final cycle of solid-phase synthesis, and after deprotection and cleavage from the solid support, the amino-linked, deprotected oligonucleotide is isolated. The conjugate is introduced by activation of functional groups using standard synthetic methods.

[0494] Purification by RP-HPLC: The crude compound is purified by preparative RP-HPLC on a Phenomenex Jupiter® C18 10 μm 150 × 10 mm column. 0.1 M ammonium acetate pH 8 and acetonitrile are used as buffers at a flow rate of 5 mL / min. The collected fractions are lyophilized to give the purified compound, typically as a white solid.

[0495] Abbreviation: DCI: 4,5-dicyanoimidazole DCM: dichloromethane DMF: dimethylformamide DMT: 4,4'-dimethoxytrityl THF: tetrahydrofuran Bz: benzoyl Ibu: Isobutyryl RP-HPLC: reversed-phase high-performance liquid chromatography

[0496] Table of Sequences, Compounds and Conjugates Tables 3, 5, and 7 below show the sequences of oligonucleotides for editing SERPINA1, GAPDH, and mHprt, respectively. The codes used in Tables 3, 5, and 7 are as follows:

[0497] Nucleobase code: A = adenine, ab = abasic (i.e., no nucleobase), C = cytosine, E = 5-methylcytosine, F = 7-deaza-8-azaguanine, G = guanine, I = hypoxanthine (i.e., inosine nucleotide), prpC = 5-propynylcytosine, pyrC = pyrrolocytosine, T = thymine, U = uracil, Z = Benner base, 2AP = 2-aminopurine, apC = aminoethylphenoxazine, 5brC = 5-bromocytosine

[0498] Tables 4, 6, and 8 below show the oligonucleotide sequences, sugar modification patterns, and internucleoside linkage patterns for editing SERPINA1, GAPDH, and mHprt, respectively, using HELM strings. Table 9 also describes conjugates using HELM strings.

[0499] An explanation of the HELM notation used in Tables 4, 6, 8 and 9 is provided in the detailed description herein.

[0500] [Table 3] TIFF2025525564000016.tif242155TIFF2025525564000017.tif242153TIFF2025525564000018.tif24229

[0501]

Table 4

[0502]

Table 5

[0503]

Table 6

[0504]

Table 7

[0505]

Table 8

[0506]

Table 9

[0507] Example 2 - Effect of Mixmar on stability We investigated the effect of changing an editing oligonucleotide containing primarily RNA nucleosides to one containing a mixmer of alternating 2'-O-methyl-RNA and 2'-fluoro-RNA nucleosides on the stability of the editing oligonucleotide (Figure 1).

[0508] Oligonucleotides were diluted to a concentration of 5 μM in PBS, FBS (Sigma-Aldrich), human cerebrospinal fluid (CSF, Zen-Bio), or a 10% solution of rat liver tritosomes in catabolic buffer (SEKISUI XenoTech) and incubated at 37°C. After different time intervals as indicated, samples were diluted to 0.5 μM in sample loading buffer and treated with proteinase K (Thermo Fisher Scientific) for 30 minutes according to the manufacturer's protocol. After inactivation of proteinase K, samples were loaded and run on a 15% TBE-urea gel (Thermo Fisher Scientific), and bands were visualized by SYBR Gold (Thermo Fisher Scientific) staining.

[0509] These data indicate that oligonucleotides containing mixmer structures of alternating 2'-O-methyl-RNA and 2'-fluoro-RNA nucleosides (CMP numbers 3_3 and 91_1 in Figure 1) are much more resistant to degradation than oligonucleotides composed primarily of RNA nucleosides (CMP number 91_2 in Figure 1).

[0510] Example 3 - Method for determining editing efficiency The efficiency of editing a specific target mRNA (SERPINA1, GAPDH, or mHprt) when using different guide oligonucleotides was determined using the following methodology.

[0511] Generally, cells were transfected with editing oligonucleotides, RNA was extracted, the target sequence was amplified by PCR, and the percentage of edited RNA was determined by sequencing. Specific cell types and editing targets are indicated in each example or figure.

[0512] HuH-7 / 3T3 cell experiments Horizon Discovery custom CRISPR-engineered HuH-7-SERPINA1-PiZ cells to introduce the SERPINA1-PiZ E342K mutation into the wild-type HuH-7 cell line.

[0513] HuH-7 wt (JCRB), HuH-7-SERPINA1-PiZ, or 3T3-L1 (ATCC) cells were seeded at 7500 cells / well in 96-well plates in the respective medium recommended by the vendor. 24 hours after seeding, the medium was changed and the cells were transfected with 20 μL of a mixture of ASO (50 nM final concentration in the well unless otherwise specified) in OptiMEM (Thermo Fisher Scientific) and 0.3 μL of Lipofectamine RNAiMAX (Thermo Fisher Scientific) per well.

[0514] Flp-In-T-REx 293-hADAR cells Stable expression of Flp-In-T-REx 293-hADAR1 p110 / hADAR1 p150 / hADAR2 was generated by cotransfecting the hADAR1 p110 / hADAR1 p150 / hADAR2 cDNAs into the pcDNA5 vector, derived from commercially available cDNA vectors (ADAR1 (NM_001111) and RED1 (ADARB1) (NM_001112) Origene), using Flp recombinase according to the manufacturer's protocol (Thermo Fisher Scientific). This was followed by selection with hygromycin (100 μg / mL) and blasticidin S (15 μg / mL) for 3 weeks. Cells were maintained in selective medium but seeded (15,000 cells / well in a 96-well plate) 24 h prior to transfection in medium containing 1 μg / mL tetracycline but without the selection antibiotic.

[0515] primary mouse hepatocytes Freshly isolated primary mouse hepatocytes were reverse transfected by adding 25,000 cells in Williams E medium supplemented with 10% FBS and 2 mM glutamine (Sigma-Aldrich) to collagen-coated plates (Corning) containing a transfection mix of ASO (50 nM final) in OptiMEM (Thermo Fisher Scientific) and 0.3 μL Lipofectamine RNAiMAX (Thermo Fisher Scientific). For reverse gymnosis, 25,000 cells in medium were added to collagen-coated plates containing ASO diluted in PBS.

[0516] RNA extraction, PCR and sequencing After 24 hours (unless otherwise stated), cells were harvested with RLT buffer, and RNA was isolated using the RNeasy 96 kit (Qiagen) with on-column DNase digestion according to the manufacturer's protocol. Before performing one-step RT-PCR (QuantaBio qScript XLT One Step Mix), an oligonucleotide complementary to the editing oligonucleotide ("sense") and transcript-specific forward and reverse primers (Table 10) were added to the isolated RNA and incubated at 90°C for 2 minutes. The PCR products were then diluted 1:10,000 in water, and a second PCR (Phusion High Fidelity PCR Master Mix with GC Buffer, Thermo Fisher Scientific) was performed using primers with individual barcodes and Illumina adapters for NGS sequencing (Table 11). After the second PCR, PCR products from individual wells were pooled and purified using the Monarch PCR & DNA Cleanup Kit (NEB).

[0517] Indexed NGS libraries were sequenced using an Illumina Mini-Seq system according to the manufacturer's instructions. The generated fastq files were analyzed using CLC Genomics Workbench Version 20.0.4 software (Qiagen). Primer-derived reads were trimmed, sequences were mapped to target transcripts, and variant calling was performed on the mapped reads from each sample. A minimum cutoff of 1% was used as a filter for variant detection, and the %G at the editing target site was plotted as editing efficiency.

[0518] [Table 10]

[0519] " / 3InvdT / " stands for "3' inverted dT" and refers to a DNA thymine nucleotide that has been "inverted" to form a 3'-3' bond according to the following structure: TIFF2025525564000070.tif50128.

[0520] [Table 11] TIFF2025525564000072.tif242161TIFF2025525564000073.tif242162TIFF2025525564000074.tif24216 2TIFF2025525564000075.tif242162TIFF2025525564000076.tif242162TIFF2025525564000077.tif86167

[0521] Example 4 - Effect of Mixmar on editing efficiency We examined the effect of including a mixmer sequence in the editing oligonucleotide for mRNA on editing of GAPDH in HEK-293 ADAR1 p150 cells (Fig. 2A) and SERPINA1 in HuH7 cells (Fig. 2B and Fig. 3).

[0522] Although the highest editing efficiency was achieved with non-mixmer RNA sequences, such oligonucleotides have low stability, as demonstrated by Example 2 herein. Good editing efficiency was also achieved with 2'-O-methyl-RNA / 2'-fluoro-RNA mixmers and RNA / DNA mixmers.

[0523] Example 5 - Effect of inosine in the edited region on editing efficiency Substitution of inosine for guanosine at position −1 of the editing oligonucleotide significantly enhanced editing of SERPINA1 mRNA (Fig. 4 ).

[0524] Example 6 - Effect of Benner's base in editing nucleosides on editing efficiency Substitution of cytosine with Benner's base (Z) at position 0 (editing nucleoside) of the editing oligonucleotide significantly enhanced editing of GAPDH mRNA (Figure 5).

[0525] Example 7 - Effect of length and symmetry on editing efficiency The effect of varying the length and symmetry of the editing oligonucleotides on editing of SERPINA1 (Figures 6 and 7), GAPDH (Figures 8 and 9), and mHprt (Figure 10) mRNAs was examined.

[0526] Figures 6, 8, and 9 show that increasing the length of the editing nucleoside 3' to the oligonucleotide from 4 to 15 nucleosides improves editing efficiency. Figure 7 shows that good editing efficiency is achieved with a symmetry range of about 30-19 to about 22-17. Figure 10A shows that good editing efficiency can be achieved using about 8 to 25 nucleosides 3' to the editing nucleoside. Figure 10B shows that good editing efficiency can be achieved using about 22 to 30 nucleosides 5' to the editing nucleoside.

[0527] Example 8 - Effect of editing region modification on editing efficiency We investigated the effect of nucleoside modifications in the editing region of editing oligonucleotides on editing of SERPINA1 (Figures 11 and 12), GAPDH (Figures 13-17), and mHprt (Figure 18) mRNA. Editing nucleosides that are DNA nucleosides or FANA nucleosides particularly enhance editing efficiency (see Figures 11, 12, 13, 14, and 16).

[0528] Example 9 - Effect of internucleoside linkages on editing efficiency The effect of modifying the internucleoside linkage of editing oligonucleotides on the editing of GAPDH (Figures 19-22) and mHprt (Figure 23) mRNA was investigated. Figure 19 shows that a high phosphorothioate (PS) content in editing oligonucleotides is tolerated and even preferred.

[0529] Example 10 - Effect of modified sugar moieties on editing efficiency The effects of 2'-O-methyl and 2'-fluoro sugar modifications at specific positions in the editing oligonucleotide on editing of SERPINA1 mRNA were examined (Figures 24 and 25).

[0530] 2'-O-methyl-RNA at position-2 enhances editing (see Figure 24B, CMP number 32_29). 2'-fluoro-RNA at position-3 enhances editing (see Figure 25B, CMP number 32_77).

[0531] Example 11 - Effect of flanking regions on editing efficiency The effect of the presence of flanking regions containing 2'-O-methyl or 2'-MOE sugar modifications in the editing oligonucleotide on the editing of GAPDH mRNA was examined (Figure 26).

[0532] Example 12 - Effect of conjugates on editing efficiency Mouse primary hepatocytes were exposed to editing oligonucleotides targeting mHprt, including oligonucleotides conjugated to a GalNAc conjugate moiety. The conjugate (CNJ No. 126_2_1) was as effective at editing mHprt mRNA as a non-conjugated compound (CMP No. 126_2) with the same oligonucleotide sequence and modification pattern (Figures 27 and 28).

Claims

1. below: an edited region comprising edited nucleosides; a 5' mixmer region located 5' to the editing region; a 3' mixmer region located 3' to the editing region; Including, the editing nucleoside is designated as position 0; each nucleoside 5' to said editing nucleoside is designated position +x, where x is the number of nucleosides 5' to said editing nucleoside at that position, including said nucleoside at that position; each nucleoside 3' to the editing nucleoside is designated position -y, where y is the number of nucleosides 3' to the editing nucleoside at that position, including the nucleoside at that position; Oligonucleotides.

2. (a) the 5' mixmer region comprises a first type of 5' mixmer nucleoside and a second type of 5' mixmer nucleoside, and the sugar moiety of the first type of 5' mixmer nucleoside is different from the sugar moiety of the second type of 5' mixmer nucleoside; and / or (b) the 3' mixmer region comprises a first type of 3' mixmer nucleoside and a second type of 3' mixmer nucleoside, and the sugar moiety of the first type of 3' mixmer nucleoside is different from the sugar moiety of the second type of 3' mixmer nucleoside; The oligonucleotide of claim 1.

3. (a) the 5' mixmer region comprises an alternating pattern of a single nucleoside of the first type of 5' mixmer nucleoside and a single nucleoside of the second type of 5' mixmer nucleoside; and / or (b) the 3' mixmer region comprises an alternating pattern of a single nucleoside of the first type of 3' mixmer nucleoside and a single nucleoside of the second type of 3' mixmer nucleoside; The oligonucleotide according to claim 1 or claim 2.

4. (a) the 5' mixmer region comprises or consists of nucleosides from position +1 to position +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24 or +25, preferably from position +2 to position +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24 or +25, more preferably from position +2 to position +15, +16, +17, +18, +19, +20, +21, +22, +23 or +24, more preferably from position +2 to position +19, +20, +21, +22, +23 or +24; and / or (b) the 3' mixmer region comprises or consists of nucleosides at positions -1 to -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16 or -17, preferably at positions -2 to -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16 or -17, more preferably at positions -2 to -5, -6, -7, -8, -9, -10, -11, -12, -13, -14 or -15, more preferably at positions -2 to -5, -6, -7, -8, -9 or -10; The oligonucleotide according to any one of claims 1 to 3.

5. (a) the first type of 5' mixumer nucleoside is selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, 2'-O-methoxyethyl-RNA (MOE-RNA), 2'-fluoro-RNA, linked nucleic acid (LNA), arabinonucleic acid (ANA) and 2'-fluoroarabinonucleic acid (FANA) nucleosides, preferably the first type of 5' mixumer nucleoside is a 2'-O-methyl-RNA nucleoside or a 2'-fluoro-RNA nucleoside, more preferably a 2'-fluoro-RNA nucleoside; and / or (b) the second type of 5'-mixmer nucleoside is selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, MOE-RNA, 2'-fluoro-RNA, LNA, ANA and FANA nucleosides, preferably the second type of 5'-mixmer nucleoside is a 2'-O-methyl-RNA nucleoside or a 2'-fluoro-RNA nucleoside, more preferably a 2'-O-methyl-RNA nucleoside; and / or (c) the first type of 3'-mixmer nucleoside is selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, MOE-RNA, 2'-fluoro-RNA, LNA, ANA and FANA nucleosides, preferably the first type of 3'-mixmer nucleoside is a 2'-O-methyl-RNA nucleoside or a 2'-fluoro-RNA nucleoside, more preferably a 2'-O-methyl-RNA nucleoside; and / or (d) the second type of 3'-mixmer nucleoside is selected from the group consisting of DNA, RNA, 2'-O-methyl-RNA, MOE-RNA, 2'-fluoro-RNA, LNA, ANA and FANA nucleosides, preferably the second type of 3'-mixmer nucleoside is a 2'-O-methyl-RNA nucleoside or a 2'-fluoro-RNA nucleoside, more preferably a 2'-fluoro-RNA nucleoside; The oligonucleotide according to any one of claims 2 to 4.

6. the first type of 5'mixmer nucleoside is a 2'-fluoro-RNA nucleoside; the second type of 5'-mixmer nucleoside is a 2'-O-methyl-RNA nucleoside; the first type of 3'mixmer nucleoside is a 2'-fluoro-RNA nucleoside; The second type of 3'-mixmer nucleoside is a 2'-O-methyl-RNA nucleoside; The oligonucleotide of claim 5.

7. (a) the nucleoside at one or more of positions +3, +5, +7, +9, +11, +13, +15, +17, and +19 is a 2'-fluoro-RNA nucleoside, preferably the nucleoside at each of positions +3, +5, +7, +9, +11, +13, +15, +17, and +19 is a 2'-fluoro-RNA nucleoside; and / or (b) the nucleoside at one or more of positions -3, -5, -7, and -9 is a 2'-fluoro-RNA nucleoside, preferably the nucleoside at each of positions -3, -5, -7, and -9 is a 2'-fluoro-RNA nucleoside; and / or (c) the nucleoside at one or more of positions +2, +4, +6, +8, +10, +12, +14, and +18 is a 2'-O-methyl-RNA nucleoside, preferably the nucleoside at each of positions +2, +4, +6, +8, +10, +12, +14, and +18 is a 2'-O-methyl-RNA nucleoside; and / or (d) the nucleoside at one or more of positions -2, -4, and -6 is a 2'-O-methyl-RNA nucleoside, preferably the nucleoside at each of positions -2, -4, and -6 is a 2'-O-methyl-RNA nucleoside; The oligonucleotide according to any one of claims 1 to 6.

8. (a) the nucleoside at position -2 is a 2'-O-methyl-RNA nucleoside; and / or (b) the nucleoside at position -3 is a 2'-fluoro-RNA nucleoside; and / or (c) the nucleoside at position -8 is a 2'-fluoro-RNA nucleoside; and / or (d) the nucleoside at position +16 is a 2'-fluoro-RNA nucleoside; and / or (e) the nucleoside at position +17 is a 2'-fluoro-RNA nucleoside; The oligonucleotide according to any one of claims 1 to 7.

9. (a) the nucleoside at position -8 is a 2'-O-methyl-RNA nucleoside; and / or (b) the nucleoside at position +16 is a 2'-O-methyl-RNA nucleoside; The oligonucleotide according to any one of claims 1 to 7.

10. 10. The oligonucleotide of any one of the preceding claims, for editing a target nucleic acid containing a target adenosine, said oligonucleotide being capable of effecting conversion of said target adenosine (A) to inosine (I).

11. The oligonucleotide of claim 10 , wherein the target nucleic acid is SERPINA1 mRNA.

12. The oligonucleotide according to any one of claims 1 to 11, wherein all nucleosides in the edited region are DNA nucleosides.

13. The oligonucleotide of any one of claims 1 to 11, wherein the edited nucleoside is a FANA nucleoside and all other nucleosides in the edited region are DNA nucleosides.

14. 2. The oligonucleotide according to any one of the preceding claims, which is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleosides in length, preferably 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 nucleosides in length, more preferably 40 nucleosides in length.

15. (a) comprising exactly 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleosides 5' to said edited nucleoside, preferably 24, 25, 26, or 27 nucleosides 5' to said edited nucleoside, more preferably exactly 24 nucleosides 5' to said edited nucleoside; and / or (b) comprising exactly 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleosides 3' to the edited nucleoside, preferably exactly 12, 13, 14 or 15 nucleosides 3' to the edited nucleoside, more preferably exactly 15 nucleosides 3' to the edited nucleoside; An oligonucleotide according to any one of the preceding claims.

16. The sequence of the oligonucleotide is the following sequence: or a variant of SEQ ID NO: 87 comprising exactly 1, exactly 2 or exactly 3 single nucleoside substitutions; In the sequence, I is inosine, the oligonucleotide comprises positions 38, 39 and 40 of SEQ ID NO: 87; An oligonucleotide according to any one of the preceding claims.

17. The sequences of the oligonucleotides are SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 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, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 and 86, comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any one of

18. 10. The oligonucleotide of any one of the preceding claims, wherein the sequence of the oligonucleotide comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:

32.

19. CMP numbers 1_1, 2_1, 3_1, 3_2, 3_3, 4_1, 5_1, 6_1, 7_1, 8_1, 9_1, 10_1, 11_1, 12_1, 13_1, 14_1, 15_1, 16_1, 17_1, 18_1, 19_1, 20_1, 21_1, 22_1, 23_1, 24_1, 2 5_1, 26_1, 26_2, 26_3, 26_4, 26_5, 26_6, 26_7, 26_8, 26_9, 27_1, 28_1, 29_1, 30_1, 31_1, 32_1, 32_2, 32_3, 32_4, 32_5, 32_6, 32_7, 32_8, 32_9, 32_10, 32_11, 32_12, 32_13, 32_14, 32_15, 32_16, 32_17, 32_18, 32_19, 32_20, 32_21, 32_22, 32_23, 32_24, 32_25, 32_26, 32_27, 32_28, 32_29, 32_30, 32_31 , 32_32, 32_33, 32_34, 32_35, 32_36, 32_37, 32_38, 32_39, 32_40, 32_41, 32_42, 32_43, 32_44, 32_45, 32_46, 32_47, 32_48, 32_49, 32_50, 32_51, 32_5 2, 32_53, 32_54, 32_55, 32_56, 32_57, 32_58, 32_59, 32_60, 32_61, 32_62, 32_63, 32_64, 32_65, 32_66, 32_67, 32_68, 32_69, 32_70, 32_71, 32_72, 32_ 73, 32_74, 32_75, 32_76, 32_77, 32_78, 32_79, 32_80, 32_81, 32_82, 32_83, 33_1, 34_1, 35_1, 36_1, 37_1, 38_1, 39_1, 40_1, 40_2, 40_3, 40_4, 40_5, 40 _6, 40_7, 40_8, 40_9, 40_10, 41_1, 41_2, 41_3, 41_4, 41_5, 41_6, 41_7, 41_8, 41_9, 41_10, 42_1, 42_2, 42_3, 42_4, 42_5, 42_6, 43_1, 43_2, 43_3, 44_1, 45_1, 46_1, 47_1, 47_2, 48_1, 49_1, 50_1, 51_1, 52_1, 53_1, 54_1, 55_1, 56_1, 57_1, 58_1, 59_1, 60_1, 61_1, 62_1, 63_1, 64_1, 65_1, 66_1, 67_1, 68_1,The oligonucleotide according to any one of the preceding claims, comprising or consisting of any one of 69_1, 70_1, 71_1, 72_1, 73_1, 74_1, 75_1, 76_1, 77_1, 78_1, 79_1, 80_1, 81_1, 82_1, 83_1, 84_1, 85_1 and 86_1.

20. 10. The oligonucleotide of claim 1, comprising or consisting of CMP number 32_1.

21. The oligonucleotides shown in FIG.

22. 10. An oligonucleotide conjugate comprising an oligonucleotide according to any one of the preceding claims covalently attached to at least one conjugate moiety.

23. 10. The oligonucleotide or oligonucleotide conjugate of claim 1, wherein the oligonucleotide or oligonucleotide conjugate is in the form of a pharmaceutically acceptable salt.

24. A pharmaceutical composition comprising an oligonucleotide or oligonucleotide conjugate according to any one of claims 1 to 23 and a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

25. 1. An in vitro or in vivo method for editing a target nucleic acid in a target cell, comprising: administering to said target cells an effective amount of the oligonucleotide or oligonucleotide conjugate according to any one of claims 1 to 23 or the pharmaceutical composition according to claim 24. An in vitro or in vivo method comprising:

26. An oligonucleotide or oligonucleotide conjugate according to any one of claims 1 to 23, or a pharmaceutical composition according to claim 24, for use in the treatment or prevention of a disease in a subject.

27. 27. The oligonucleotide for use, the oligonucleotide conjugate for use, or the pharmaceutical composition for use of claim 26, wherein the disease is alpha 1 antitrypsin deficiency (A1AD).

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