Chemically modified antisense oligonucleotides (ASOs) and compositions containing same for RNA editing

Abstract

The present invention provides a method for the preparation of a nucleic acid molecule comprising a sequence of 23 to 80 nucleotides in length capable of binding to a target sequence in a target RNA, the nucleic acid molecule comprising a central base triplet (CBT) (I) of three nucleotides having a central nucleotide (N0) directly opposite a target adenosine in said target RNA. [Formula 1] JPEG2025541721000056.jpg5169 wherein the core oligonucleotide has the following sequence (II): [chemical 2] JPEG2025541721000057.jpg5169 The present disclosure also provides an oligonucleotide and its composition for use in treating or preventing genetic disorders, conditions, or diseases. Also provided is a method for editing a target adenosine or deaminating at least one specific adenosine in a target nucleic acid.

Description

Detailed Description of the Invention

[0001] FIELD OF THE INVENTION The present invention relates to the field of medicine, in particular to the field of site-specific RNA editing, whereby RNA sequences are targeted by single-stranded antisense oligonucleotides (ASOs) for RNA editing of specific genetic mutations ("compensatory editing") or for editing RNA derived from wild-type alleles ("beneficial editing").

[0002] BACKGROUND OF THE INVENTION RNA editing is a natural process that allows some cells to individually modify specific nucleotide sequences within RNA molecules in a site-specific manner. Unlike DNA editing, RNA editing offers the advantage of a more efficient method for modifying genetic information. This is because RNA is generally rapidly degraded, meaning that errors introduced by off-target modifications are washed away rather than remaining permanently in the target DNA. Furthermore, because RNA editing is a naturally occurring editing mechanism in humans, it may be less likely to trigger an immune response. Furthermore, RNA editing may result in a more natural response than introducing an externally engineered gene.

[0003] For many years, oligonucleotide therapeutics have been developed to specifically silence, restore, or correct the expression of disease-causing or disease-associated genes, for example, in cancer and other genetic disorders. These therapeutics include, for example, antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and microRNAs, which interfere with coding and noncoding RNAs. Because ASO sequences are relatively easy to customize and precise, they can target virtually any mutated gene. As a result, ASOs have become the most clinically advanced, with several already approved by the U.S. Food and Drug Administration (FDA) and in clinical trials (Cideciyan et al., 2019; Gagliardi and Ashizawa, 2021).

[0004] In general, a vast group of proteins is involved in mediating the RNA editing process within cells (Quinones-Valdez et al., 2019). Specifically, site-directed RNA editing (SDRE) refers to altering RNA sequences by introducing or removing nucleotides from RNA or by changing the nature of nucleobases through deamination. RNA editing enzymes are known in the art. The first RNA editing process discovered in mammals was the deamination of cytidine (C) by APOBEC proteins to form uridine (U) (Zinshteyn and Nishikura, 2009). To date, the two most useful and most studied types of RNA editing are cytidine (C) to uridine (U) conversion ("C-to-U") and adenosine (A) to inosine (I) conversion ("A-to-I"). Especially for therapeutic purposes, "A-to-I" conversion is the most common type of RNA editing in higher eukaryotes. This conversion is catalyzed by the adenosine deaminase (ADAR) family, which acts on RNA. Over the years, three vertebrate ADAR genes have been identified, generating several ADAR proteins derived from alternative promoters or exhibiting splice variants (Wulff and Nishikura, 2010). These proteins are expressed across various types of human tissues and can alter the splicing and translation machinery, double-stranded RNA (dsRNA) structure, and binding affinity between RNA and RNA-binding proteins (Tomaselli et al., 2014; Zinshteyn and Nishikura, 2009). Of the three known ADAR genes, hADAR1 and hADAR2 are expressed in most tissues and encode active deaminases. Human ADAR3 (hADAR3) is expressed exclusively in the central nervous system and has been reported to lack deaminase activity in vitro.All ADARs are multi-domain proteins that comprise targeting domain or dsRNA binding domain (dsRBD) and catalytic domain, but ADAR1 protein also comprises one or more Z-binding domains, and splice variants ADAR2R and ADAR3 comprise R domains (Zinshteyn and Nishikura, 2009; Wulff and Nishikura, 2010).Therefore, in some embodiments, ADAR is hADAR1, hADAR2 or hADAR3.

[0005] "A-to-I" editing was initially identified in Xenopus eggs (Bass and Weintraub, 1987; Rebagliati and Melton, 1987). A human cDNA encoding "double-stranded RNA adenosine deaminase" was first cloned by Kim et al. (1994), and recombinant expression in insect cells confirmed the adenosine-to-inosine ("A-to-I") conversion activity of this protein. "A-to-I" editing alters the information content of RNA molecules because inosine preferentially base pairs with cytidine and is therefore interpreted as guanosine (G) by the translation and splicing machinery. During this enzymatically catalyzed reaction, adenosine is converted to inosine via a hydrated intermediate. While guanosine can form three hydrogen bonds with its complementary base, cytidine, inosine can only form two hydrogen bonds with cytidine. The translation machinery reads inosine as guanosine. Thus, ADARs have the effect of introducing functional adenosine-to-guanosine mutations at the RNA level. The ability of ADARs to alter RNA sequences has also been used to artificially target RNA in vitro within cells for RNA editing. Potentially, this approach could be used to repair genetic defects and alter genetic information at the RNA level.

[0006] ASOs are generally short, single-stranded synthetic RNA or DNA molecules ranging in length from 18 to 25 nucleobases that bind specifically to target RNA sequences using Watson-Crick base pairing. ASOs are broadly classified as first-, second-, and third-generation ASOs. The first ASO was employed to inhibit the translation of Rous sarcoma virus ribosomal RNA (Stephenson and Zamecnik, 1978). First-generation ASOs are characterized by modified backbones in which the nucleotide linkages are modified with sulfur, methyl, or amine groups to generate phosphorothioates (PS), methylphosphonates, and phosphoramidates, respectively, while second-generation ASOs additionally possess alkyl modifications at the 2' position of the ribose. These second-generation ASOs tend to be less toxic than PS-modified ASOs and have slightly higher affinity for their targets. In comparison, third-generation ASOs tend to be more heterogeneous, containing numerous chemical modifications aimed at improving binding affinity, stability, and pharmacokinetics (Quemener et al., 2019). The diversity of chemical modifications, along with the ASO sequence, provides considerable flexibility regarding therapeutic approaches. Depending on their mechanism of action, ASOs can be used to degrade target mRNAs to reduce protein levels, alter or correct splicing events, regulate RNA translation, or target pathological coding and noncoding RNAs (Quemener et al., 2019).

[0007] ASOs can act through many mechanisms, depending in part on the region of the targeted RNA sequence and the design / chemical characteristics of the ASO. To ensure specificity, ASOs ideally have sequences that are complementary or at least partially complementary to the target RNA. However, in the case of site-directed mutagenesis, or "A-to-I" RNA editing, the ASO targeting domain contains a mismatch opposite the targeted adenosine. It should be noted that some endogenous substrates of ADAR contain mismatches and / or bulges (Thomas and Beal, 2017), and therefore, mimicking these features in the ASO / generated dsRNA may alter or improve substrate recognition.

[0008] Furthermore, ASOs can be chemically modified to improve their properties. For example, ASOs can be modified to protect them from nucleases and enhance their efficacy. While phosphorothioate (PS) modifications appear to have a positive effect on ASO stability and pharmacokinetics, differences in the chirality of the PS bond can significantly affect the overall properties of ASOs. PS bonds can be found in two stereoisomers, Rp and Sp, and it is known in the art that Rp and Sp bonds affect properties such as thermal stability, binding affinity, and pharmacological properties of ASOs. However, the advantages of the Rp and Sp stereoisomers have been debated (Iwamoto et al., 2017; Crooke et al., 2020).

[0009] While the use of antisense oligonucleotides in RNA editing is generally known in the art (Vogel et al., 2014; Merkle et al., 2019), ASO-based therapies have gained increasing support over the past few years for the treatment of various conditions and diseases, particularly genetic disorders. RNA editing systems using endogenous adenosine deaminase enzymes have been widely studied. That is, exogenous oligonucleotides are used to specifically recruit endogenous adenosine deaminase to specific target sites in target RNA, resulting in improved systems for targeted RNA editing. Oligonucleotide constructs for site-specific RNA editing are described in patent applications WO2016 / 097212 and WO2017 / 010556, which utilize an endogenous cellular pathway, i.e., endogenous ADAR, to edit endogenous RNA. Using available structural information on ADAR-RNA complexes, new designs of nucleoside analogs are constantly being investigated (Doherty et al., 2021). The use of stereopure, chemically modified oligonucleotides to induce endogenous ADAR-mediated RNA editing has previously been reported in non-human primates (Monian et al., 2022). These oligonucleotides typically contain a high concentration of 2'-F modifications within the 5' half, with this modification occurring within either 3' end of the CBT as a block of 2'-F modifications and a block of uniform 2'-O-methyl modifications. Furthermore, these oligonucleotides contain a highly stereopure PS-modified backbone, a highly stereopure PS linkage, and an additional charge-neutral PN linkage (also stereopure), although the latter has not yet been applied clinically. Merkle et al. (2019) previously demonstrated that precise, site-specific RNA editing can be achieved by targeting endogenous ADARs with antisense oligonucleotides. Merkle et al. (2019) demonstrated that they could target endogenous human ADARs using chemically optimized ASOs, enabling them to demonstrate the ability to edit endogenous transcripts in a simple and programmable manner with minimal off-target editing.

[0010] In WO2020 / 001793, the inventors of the present application provide artificial nucleic acids for site-specific editing of RNA ("A-to-I" editing), wherein the nucleic acid comprises a targeting sequence and a recruiting moiety. Similarly, WO2018 / 041973 relates to ASOs capable of specifically editing adenosines in target RNA sequences, but the ASOs do not form intramolecular hairpin or stem-loop structures. Specifically, WO2018 / 041973 relates to chemically modified single-stranded RNA editing oligonucleotides for deamination of target adenosines by ADAR enzymes, whereby the central base triplet (CBT) of three consecutive nucleotides contains a sugar modification and / or a base modification. It has been found that deoxyribose is well tolerated at all three positions of the CBT, resulting in substantial stabilization against nuclease digestion.

[0011] Other prior art, such as WO2021 / 071858, relates to oligonucleotides comprising a first and a second domain, where the first domain comprises one or more 2'-F modifications and the second domain comprises one or more sugars without 2'-F modifications. WO2022 / 099159 relates to oligonucleotides having a first and a second domain, where the domains comprise a specific proportion of 2'-F modifications and aliphatic substitutions.

[0012] Research in the field of ASO optimization has led to the identification and more thorough investigation of the CBT and the immediately surrounding 5' and 3' regions. In addition to specifically examining CBT modifications (e.g., 2'-F and 2'-FANA), WO2021 / 243023 explores the modification of nucleobases immediately outside the CBT ([Am]-X). 1 -X 2 -X 3 -X 4 -position +2 of an oligonucleotide containing the structure [Bn], where X 4They also mention 3' guide or targeting domain modifications (where 2'-F corresponds to the +2 position). The choice of the nucleotide at the +2 position of the guide oligonucleotide triplet was found to affect the rate of editing of the target. Enhanced editing was observed when the 2'-F modification was at the +2 position.

[0013] However, despite its promising nature, few ASOs have been marketed. This is due to difficulties with stability, intracellular delivery and uptake, and clinical efficacy, as well as off-target effects and / or preclinical toxicological challenges. Overcoming these various challenges is therefore crucial for ultimately achieving widespread clinical success with ASO-based therapies. Thus, there is currently an unmet need for improved ASOs and effective therapies for the treatment of genetic disorders, including these improved ASOs.

[0014] The present inventors have discovered that the artificial, chemically modified oligonucleotides of the present invention are suitable for editing a wide variety of endogenous RNA transcripts, such as endogenous mRNAs of housekeeping genes, as well as endogenous transcripts of disease-related genes, such as STAT1, SERPINA 1, LRRK2, CRB1, NLRP3, CTNNB1, PEX1, and PDE6A. Surprisingly, the present inventors have discovered that the present invention provides improvements to ASOs. For example, the ASOs of the present application have improved editing efficacy and effectiveness. Furthermore, the ASOs of the present application have the advantages of increased lysosomal half-life, i.e., improved lysosomal stability, and facilitating ease of production (e.g., cost, purity, quality control). The ASOs of the present invention also have the advantage of reducing off-target RNA editing. Summary of the Invention

[0015] Therefore, the problem solved by the present application is to provide improved synthetic, chemically modified ASOs that can mediate the functional conversion of adenosine (A) to guanosine (G) and correct otherwise deleterious point mutations. The present invention solves this problem by providing synthetic ASOs that contain specific nucleoside modifications, particularly modifications at the 2' position of the sugar, backbone linkage modifications, and combinations thereof. Overall, the ASOs of the present application exhibit several differences and associated advantages over those disclosed in the prior art. Among other things, the present invention is characterized by the specific combinations of nucleoside and linkage modifications provided herein. To date, no prior art has been identified that teaches or suggests the oligonucleotides, compositions, and methods disclosed in the present application, all of which are based on the inventors' discovery that specific combinations of nucleobase, nucleoside, and linkage modifications are particularly effective in providing stable and effective ASOs and compositions containing them.

[0016] The solution to the problem addressed by this application is achieved by the embodiments described herein and defined by the appended claims.

[0017] The present invention generally provides oligonucleotides, compositions comprising said oligonucleotides, and their use in treating or preventing genetic disorders, conditions or diseases.Also provided herein is an in vitro method for editing target adenosine in target nucleic acid, and an in vitro method for deaminating at least one specific adenosine present in target RNA sequence in cells.Also provided herein is a method for treating or preventing genetic disorders, conditions or diseases, and the method comprises administering an effective amount of the oligonucleotide of the present invention.

[0018] In a first aspect, the present invention provides a nucleic acid molecule comprising a sequence of 23 to 80 nucleotides in length capable of binding to a target sequence in a target RNA, the nucleic acid molecule comprising a central base triplet (CBT) of three nucleotides having a central nucleotide (N0) directly opposite a target adenosine in said target RNA. [ka] wherein the core oligonucleotide comprises the following sequence: [ka] Includes; where (a) at least two of the three nucleotides of said CBT are chemically modified at the 2' position of the sugar moiety, are deoxyribonucleosides, or are a combination thereof, and d and e are internucleoside linkage modifications; (b) N +2 The nucleotide is 2'-O-alkyl-modified; +3 The nucleotides are 2'-fluoro (2'-F)-modified; (c) at least 10% of the nucleotides are 2'-F-modified and at least 10% of the nucleotides are 2'-O-alkyl-modified, where no more than six consecutive nucleotides have the same 2'-modification; (d) the internucleoside linkage modification content is at least 15%; and (e) Bonds h and i are not phosphorothioate (PS) bonds.

[0019] In a second aspect of the present specification, a central base triplet (CBT) of three nucleotides is provided, which comprises a sequence of 23 to 50 nucleotides in length capable of binding to a target sequence in a target RNA, and has a central nucleotide (N0) directly opposite a target adenosine in the target RNA. [ka] wherein the core oligonucleotide comprises the following sequence: [ka] Includes; where (a) at least two of the three nucleotides of said CBT are chemically modified at the 2' position of the sugar moiety, are deoxyribonucleosides, or are a combination thereof, and d and e are internucleoside linkage modifications; (b) N +2 The nucleotide is 2'-O-alkyl-modified; +3 The nucleotides are 2'-fluoro (2'-F)-modified; (c) at least 10% of the nucleotides are 2'-F-modified and at least 10% of the nucleotides are 2'-O-alkyl-modified, where no more than six consecutive nucleotides have the same 2'-modification; (d) the regions located 3' and 5' of said CBT do not contain a total of more than six deoxyribonucleosides; (e) an internucleoside linkage modification content of at least 30%;

[0020] In a third aspect of the present specification, a central base triplet (CBT) of three nucleotides is provided, which comprises a sequence of 40 to 80 nucleotides in length capable of binding to a target sequence in a target RNA, and has a central nucleotide (N0) directly opposite a target adenosine in the target RNA. [ka] wherein the core oligonucleotide comprises the following sequence: [ka] Includes; where (a) at least two of the three nucleotides of said CBT are chemically modified at the 2' position of the sugar moiety, are deoxyribonucleosides, or are a combination thereof, and d and e are internucleoside linkage modifications; (b) N +2 The nucleotide is 2'-O-alkyl-modified; +3 The nucleotides are 2'-fluoro (2'-F)-modified; (c) at least 10% of the nucleotides are 2'-F-modified and at least 10% of the nucleotides are 2'-O-alkyl-modified, where no more than six consecutive nucleotides have the same 2'-modification; (d) the regions located 3' and 5' of the CBT have a total deoxyribonucleoside content of 5 to 50%;

[0021] In a fourth aspect herein, there is provided a pharmaceutical composition comprising the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof.

[0022] In a fifth aspect herein, there is provided a chemically modified oligonucleotide of the present invention or a pharmaceutical composition of the present invention for use in the treatment or prevention of a genetic disorder, condition or disease.

[0023] In a sixth aspect herein there is provided an in vitro method for editing a target adenosine in a target nucleic acid, the method comprising contacting the target nucleic acid with an oligonucleotide of the present invention.

[0024] In a seventh aspect herein there is provided an in vitro method for deaminating at least one specific adenosine present in a target RNA sequence in a cell, the method comprising the steps of: (a) contacting the target nucleic acid with a chemically modified oligonucleotide according to any one of claims 1 to 36; (b) allowing the chemically modified oligonucleotide to be taken up by the cell; (c) allowing the chemically modified oligonucleotide to anneal to the target RNA sequence; and (d) enabling a mammalian ADAR enzyme containing a naturally occurring dsRNA-binding domain found in a wild-type enzyme to deaminate a target adenosine in said target RNA sequence to inosine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The figures shown below are merely examples and further illustrate the invention and should not be construed as limiting the invention thereto.

[0026] FIG. 1 is a graph showing the editing efficacy and lysosomal stability of oligonucleotides targeting SERPINA.

[0027] FIG. 2 is a graph showing the SERPINA-editing effect of 2′-F-modified oligonucleotides.

[0028] FIG. 3 is a graph showing the editing efficacy and lysosomal stability of oligonucleotides targeting STAT1 Y701.

[0029] Figure 4 is a graph showing the editing efficacy and lysosomal stability of oligonucleotides targeting CRB1 C948Y.

[0030] FIG. 5 is a graph showing the editing efficacy and lysosomal stability of oligonucleotides targeting LRRK2 G2019S.

[0031] FIG. 6 is a graph showing the editing effect of oligonucleotides targeting mouse PDE6A (mPDE6A) V685M.

[0032] Figure 7 is a graph showing the editing effect of oligonucleotides targeting NLRP3 Y166 expressed from a plasmid (A) and expressed after integration into the genome.

[0033] FIG. 8 is a graph showing data regarding the editing effect of oligonucleotides targeting the GAPDH 3′UTR.

[0034] Figure 9 is a graph showing data on the editing effects of truncated variants (31 nt, 40 nt, 45 nt, 50 nt, 59 nt) of oligonucleotides targeting SERPINA.

[0035] FIG. 10 shows data on the editing effects of 5′ and / or 3′ truncated variants of SERPINA-targeting oligonucleotides.

[0036] FIG. 11 is a graph showing the editing effect of 32-nt and 33-nt long SERPINA-targeting oligonucleotides.

[0037] FIG. 12 is a graph showing the editing effect of oligonucleotides targeting 3′-truncated STAT1 Y701.

[0038] Figure 13 is a graph showing the editing effect of oligonucleotides targeting 3'-truncated CTNNB1 T41.

[0039] FIG. 14 is a graph showing the editing effect of oligonucleotides targeting 5′- and / or 3′-truncated CRB1 C948Y.

[0040] Figure 15 is a graph showing the editing effect of oligonucleotides targeting STAT1 Y701.

[0041] FIG. 16 is a graph showing the editing effect of oligonucleotides targeting SERPINA with modifications in the extended hotspot region 3′ to the CBT (optimal versions: +2 (2′-OMe) and +3 (2′-F)).

[0042] Figure 17 is a graph showing the editing effect of oligonucleotides targeting CTNNB1 T41 with modifications in the extended hotspot region 3' to the CBT (optimal versions: +2 (2'-OMe) and +3 (2'-F)).

[0043] FIG. 18 is a graph showing the editing efficacy, lysosomal stability, and relative toxicity of SERPINA-targeting oligonucleotides containing 2′-MOE end blocks.

[0044] Figure 19 is a graph showing the editing efficacy and lysosomal stability of a long SERPINA-targeting oligonucleotide (59 nt) with reduced PS binding using a genomic system (A) and a plasmid system (B).

[0045] FIG. 20 is a graph showing data on the editing effect of short SERPINA-targeting oligonucleotides (40 nt) with reduced PS-binding modifications.

[0046] FIG. 21 is a graph showing the editing effect of oligonucleotides targeting SERPINA containing a continuous region of PS binding.

[0047] Figure 22 is a graph showing the effect of LNA modifications on the editing effect and editing efficacy of SERPINA-targeting oligonucleotides.

[0048] FIG. 23 is a graph showing the effect of LNA modifications at the 5′ end of SERPINA-targeting oligonucleotides.

[0049] FIG. 24 is a graph showing 5′-terminal LNA modification of short SERPINA-targeted oligonucleotides.

[0050] FIG. 25 is a graph showing 5′ and 3′ end block disruption of SERPINA-targeting oligonucleotides.

[0051] FIG. 26 is a graph showing block disruption of short STAT1-targeted oligonucleotides.

[0052] FIG. 27 is a graph showing 2′-FANA modification of CBT of oligonucleotides targeting CRB1 C948Y. Detailed Description Glossary

[0053] In order that the present invention may be more readily understood, certain terms are first defined.

[0054] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an" means one element or more than one element, e.g., a plurality of elements.

[0055] The terms "about" and "approximately" may be understood to allow for standard variations as appreciated by those of ordinary skill in the art.

[0056] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including, but not limited to." Similarly, the term "comprising" is used herein to mean, and is used interchangeably with, the phrase "comprising, but not limited to."

[0057] As used herein, the term "nucleic acid" is intended to include any DNA molecule (e.g., cDNA or genomic DNA) and any RNA molecule (e.g., mRNA), as well as analogs of DNA or RNA produced using nucleotide analogs. For example, in one embodiment, oligonucleotides include, for example, UNA (unlocked nucleic acid), PMO (phosphorodiamidate-linked morpholino), or PNA (peptide nucleic acid). Nucleic acids can be single-stranded or double-stranded. Oligonucleotides can be single-stranded (ss) or double-stranded (ds). A single-stranded oligonucleotide can have a double-stranded region (formed by two portions of a single-stranded oligonucleotide), and a double-stranded oligonucleotide containing two oligonucleotide strands can have a single-stranded region, for example, in a region where the two oligonucleotide strands are not complementary to each other. Each component of the DNA or RNA structure can be modified and can be classified by modifications of (1) the internucleoside linkage, (2) the deoxyribose / ribose, and / or (3) the nucleobase.

[0058] As used herein, the term "oligonucleotide" or "oligonucleotides" is defined as a molecule comprising two or more covalently linked nucleosides (e.g., short nucleic acid polymers), as generally understood by those skilled in the art. These may comprise DNA and / or RNA. The oligonucleotides provided herein have a backbone comprising deoxyribonucleotides and / or ribonucleotides.

[0059] The term "nucleobase" refers to the nitrogen-containing biological building blocks that form nucleosides, or in other words, the building blocks of nucleotides. Naturally occurring bases [guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)] are derivatives of purines or pyrimidines, but it should be understood that naturally occurring and non-naturally occurring base analogs are also included, and the term "nucleobase" also includes "modified nucleobases."

[0060] In the context of the present invention, the terms "modified nucleobase" and "modified base" can be used interchangeably with the term "nucleobase." A nucleobase may be modified or unmodified. Thus, in some embodiments, a modified nucleobase is a nucleobase that includes a modification. In some embodiments, a modified nucleobase can form a moiety in a polymer that can base pair with a nucleic acid that contains at least one nucleobase function, e.g., at least a complementary sequence of bases. In one embodiment, a modified nucleobase can increase hydrogen bonding, base pair stacking interactions, and / or stabilize a nucleic acid complex. In another embodiment, a modified nucleobase (e.g., a Bener base) can mimic an N3 protonated cytosine base. In some embodiments, a modified nucleobase is a substituted A, T, C, G, or U, or a substituted tautomer of A, T, C, G, or U. In some embodiments, a modified nucleobase in the context of an oligonucleotide refers to a nucleobase that is not A, T, C, G, or U. Modifications include the non-standard nucleobase 5-methyl-2'-deoxycytidine (m-methyl-2'-deoxycytidine). 5Modifications include, but are not limited to, (N) heterocycles (e.g., nebularine) or aromatic rings that stack well in RNA duplexes, such as Benar base Z (and / or analogs) or 8-oxo-adenosine (8-oxo-A). As used herein, the term "Benar base Z" refers to the pyrimidine analog 6-amino-5-nitro-3-(1'-β-D-2'-deoxyribofuranosyl)-2(1H)-pyridone (dZ). In one embodiment, modifications include the introduction of a nucleobase analog or a simple heterocycle that enhances editing. As used herein, and as generally understood by those skilled in the art, the phrase "derivative thereof" refers to a derivative of a (modified) nucleobase, nucleoside, or nucleotide. For example, a derivative can be a corresponding nucleic acid base, nucleoside, or nucleotide chemically derived from the nucleic acid base, nucleoside, or nucleotide. For example, derivatives of deoxycytidine include fluoro-modified deoxycytidine, 5-methyl-2'-deoxycytidine (m-methyl-2'-deoxycytidine), and 5-methyl-2'-deoxycytidine (m-methyl-2'-deoxycytidine). 5 C), or ribocytidine.

[0061] The term "nucleoside" refers to a moiety in which a nucleobase or modified nucleobase is covalently linked to a sugar or modified sugar. In some embodiments, "nucleoside" refers to a nucleoside unit in an oligonucleotide or nucleic acid. The term "nucleoside" encompasses all modified versions and derivatives, including "modified nucleobases."

[0062] As used herein, the term "nucleotide" refers to a monomeric unit of a polynucleotide consisting of a nucleobase, a sugar, and one or more linkages (e.g., phosphate linkages in natural DNA and RNA). In some cases, the linkage may be a non-naturally occurring and / or modified linkage. In some embodiments, the linkage may be an internucleoside linkage as described herein. In certain embodiments, the modified linkage is a PS linkage. In some embodiments, "nucleotide" refers to a nucleotide unit in an oligonucleotide or nucleic acid. The term "nucleotide" encompasses all modified versions and derivatives of "nucleosides" and "modified nucleobases."

[0063] As used herein, the term "internucleoside linkage" refers to the bond between adjacent nucleosides. "Internucleoside linkage" and "linkage" can be used interchangeably. The linkage can be continuous or consecutive. The linkage can be discontinuous or intermittent. As used herein, the term "discontinuous" or "intermittent" means that there are, for example, more than four, more than five, more than six, more than seven, or more consecutive internucleoside linkage modifications of the same modification. In some embodiments, a naturally occurring P-O linkage is replaced with a modified internucleoside linkage. Thus, in some embodiments, the linkage is a non-naturally occurring internucleoside linkage. In some embodiments, the internucleoside linkages include, but are not limited to, phosphorothioate (PS), 3'-methylene phosphonate, 5'-methylene phosphonate, 3'-phosphoramidate, 2'-5'-phosphodiester, and phosphorylguanidine (PN) linkages. In another embodiment, the internucleoside linkage modification is a 3'-3' or 5'-5' phosphate linkage (3'-P-3' and 5'-P-5'). The internucleoside linkages can be stereopure or stereorandom. Thus, within a particular oligonucleotide, the internucleoside linkages can include stereopure and stereorandom linkages. In one embodiment, the natural 3'-5' phosphodiester linkage is replaced with a modified internucleoside linkage. In some embodiments, one or more naturally occurring PO linkages are replaced with modified internucleoside linkages to introduce one or more PS linkages or non-phosphorus derived internucleoside linkages.

[0064] As used herein, the terms "sterically pure" or "sterically random" refer to chemically modified oligonucleotides. Specifically, the term "sterically pure" refers to chirally pure (i.e., "sterechemically pure") oligonucleotides. The term "sterically random" refers to racemic (i.e., "sterically random," "chirally controlled") oligonucleotides. Thus, the oligonucleotides of the present invention contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more stereorandom internucleoside linkages (mixtures of Rp-type and Sp-type phosphorus internucleoside linkages, e.g., derived from conventional non-chirally controlled oligonucleotide synthesis). In one embodiment, the internucleoside linkages are phosphorothioate (PS) linkages. In one embodiment, the internucleoside linkages are stereorandom PS linkages. In one embodiment, the internucleoside linkages are chirally controlled PS linkages. In one embodiment, the internucleoside linkage is a phosphorylguanidine (PN) linkage.

[0065] The term "hydroxy" as used herein refers to an --OH group.

[0066] As used herein, the term "antisense oligonucleotide" or "ASO" refers to a short chain of nucleotide analogues that hybridizes with complementary mRNA in a sequence-specific manner through Watson-Crick base pairing.ASO includes DNA and RNA.ASO may be chemically modified.As used herein, the terms "antisense oligonucleotide" (ASO) and "oligonucleotide" can be used interchangeably.

[0067] The term "modified sugar" refers to a moiety that can replace a naturally occurring sugar. Modified sugars mimic the spatial arrangement, electronic properties, or other physicochemical properties of the sugar. Naturally occurring sugars are pentoses (sugars with five carbon atoms), deoxyribose (which form DNA), or ribose (which form RNA), but it should be understood that naturally occurring and non-naturally occurring sugar analogs are also included. For example, other sugars can include, e.g., C4, C5, and / or C6 sugars. In some embodiments, the modified sugar is a substituted ribose or substituted deoxyribose. In some embodiments, the modified sugar comprises a 2'-modification. Examples of useful 2'-sugar modifications, such as 2'-ribose, 2'-deoxyribose, and 2'-arabinose, are widely used in the art and are described herein. One of skill in the art, after reading this disclosure, will understand that various types of 2'-sugar modifications are known and can be utilized in accordance with the present disclosure. In some embodiments, the modified sugar is a bicyclic sugar (e.g., a sugar used in LNA, BNA, etc.). In some embodiments, the modified sugar is an LNA sugar. The term "locked nucleic acid" (LNA) or "locked nucleic acids" (LNAs), also known as bridged nucleic acids (BNA), refers to a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon. In some embodiments, the sugar modification is 2'-OMe, 2'-O-methoxy-ethyl (2'-MOE), 2'-F, 5'-vinyl, or S-constrained ethyl (S-cEt). In one embodiment, the 2'-modification is the C2-stereoisomer of 2'-F-ribose. In one embodiment, the 2'-modification is 2'-F. In one embodiment, the 2'-modification is 2'-FANA. In one embodiment, the modified sugar is a morpholino sugar. In one embodiment, the oligonucleotide comprises, for example, a UNA (unlocked nucleic acid), a PMO (phosphorodiamidate-linked morpholino), or a PNA (peptide nucleic acid).Thus, in one embodiment, the nucleic acid analog is a PNA (peptide nucleic acid). In one embodiment, the nucleic acid analog is a PMO (phosphorodiamidate-linked morpholino). In one embodiment, the 2'-modification is a 2'-O-alkyl-modification. In one embodiment, the 2'-O-alkyl-modification is a 2'-O-methyl-, 2'-O-ethyl-, 2'-O-propyl-, or 2'-MOE-modification. In a preferred embodiment, the 2'-modification is 2'-OMe. In some embodiments, the 2'-modification is 2'-MOE. In some embodiments, the 2'-modification is 2'-OR, where R is a substituted C1-10 aliphatic. In some embodiments, in the context of an oligonucleotide, the modified sugar is a sugar that is not a ribose or deoxyribose as typically found in natural RNA or DNA (e.g., arabinose). In some embodiments, the 2'-O-alkyl-modification is not a 2'-MOE.

[0068] The term "FANA" or "modified FANA" refers to 2'-fluoroarabinoside-modified nucleobases and / or oligonucleotides containing such nucleobases. For example, the term "FANA-cytidine" refers to cytidine containing a 2'-fluoro-β-D-arabino nucleoside sugar modification. In the context of the present invention, the term "a derivative thereof" refers to a nucleotide or oligonucleotide chemically derived from the corresponding nucleotide or oligonucleotide.

[0069] As used herein, the terms "complementary," "partially complementary," or "substantially complementary" refer to nucleic acid sequences capable of specific intermolecular base pairing via complementary nucleotides. For example, an oligonucleotide may contain a nucleic acid sequence complementary to a target sequence, such as SERPINA1 or another target sequence. As those skilled in the art will appreciate, perfect complementarity is often not required, and one or more wobbles (wobble base pairs), bulges, mismatches, etc. may be adequately accommodated. One or more wobbles, bulges, or mismatches may be located inside or outside the CBT. For example, an ASO of the present invention includes a mismatch opposite the target adenosine. Thus, the complementarity of an ASO of the present invention may be 100%, excluding the nucleoside opposite the target nucleoside to be edited. In one embodiment, the complementarity is at least 80%, 85%, 90%, or 95%. In another embodiment, the complementarity is 85% to 99%. In another embodiment, the ASO contains 1, 2, 3, 4, or 5 mismatches when aligned with the target nucleic acid. In one embodiment, the ASO contains wobble bases outside the CBT. In one embodiment, one or more mismatches are independently wobble base pairs. In one embodiment, the ASO contains up to four mismatches or wobble bases outside the CBT. In one embodiment, the ASO contains up to three mismatches or wobble bases outside the CBT.

[0070] As used herein, the term "mutation" refers to the substitution of a residue in a sequence, for example, a nucleic acid sequence or an amino acid sequence, with another residue, or the deletion or insertion of one or more residues in a sequence. Here, a mutation is typically described by identifying the original residue, then the position of the residue in the sequence, and the identity of the newly substituted residue. Notably, the present invention is not limited to correcting mutations; instead, it may be useful to change a wild-type sequence into a mutant sequence by applying an ASO according to the present invention. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided, for example, by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)).

[0071] As used herein, the term " beneficial editing " refers to the editing of the RNA derived from wild-type allele (not mutant allele), for example, to regulate the function of wild-type protein in a manner useful for preventing or treating disease.For example, beneficial editing includes sites that are not the cause of genetic disease, but rather represent the site of wild-type protein, such as STAT1 Y701, NLRP3 Y166, and CTNNB1 T41.These sites are mutated (there is no underlying G-to-A mutation) to change the function of wild-type protein.

[0072] The term "compensatory editing" refers to the modification of RNA nucleotides to alter or correct one or more deleterious or undesirable changes in the RNA sequence compared to the wild type. For example, compensatory A-to-I conversion can serve to functionally compensate for otherwise uneditable mutations to improve disease phenotypes.

[0073] As used herein, the term "off-target" refers to non-specific and unintended genetic modification of a target. Specifically, off-target editing includes unintended point mutations, deletions, insertions, inversions, and translocations.

[0074] The term "adenosine deaminase" or "adenosine deaminase acting on RNA" (ADAR), as used herein, refers to any (poly)peptide, protein, or protein domain or fragment thereof that can catalyze the hydrolytic deamination of adenosine to inosine. Thus, this term refers not only to full-length and wild-type ADARs, but also to functional fragments or functional variants of ADARs. In some embodiments, ADARs are (endogenous) adenosine deaminases that catalyze the deamination of adenosine to inosine or deoxyadenosine to deoxyinosine. In some embodiments, ADARs catalyze the deamination of adenine or adenosine in deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). ADARs may be human ADARs. ADARs may be endogenous ADARs. Thus, in some embodiments, the ADAR is endogenous human ADAR1, ADAR2, or ADAR3 (hADAR1, hADAR2, or hADAR3), or any fragment or isoform thereof (eg, hADAR1 p110 and p150).

[0075] As used herein, the term "guide RNA" (gRNA) or "guide oligonucleotide" refers to an RNA fragment or oligonucleotide (including RNA and / or DNA) that functions as a guide for an enzyme that forms a complex. The guide RNA or guide oligonucleotide may contain endogenous and / or exogenous sequences. The guide may be used in vitro and in vivo. For example, the guide RNA or guide oligonucleotide guides a base-modifying activity / editing function (e.g., ADAR) to a target to be edited in trans.

[0076] As used herein, the term "target RNA" refers to the RNA that is "targeted" by each ASO of the present invention, typically subjected to an editing reaction.

[0077] As used herein, the terms "disease" or "disorder" are used interchangeably to refer to a condition in a subject. In certain embodiments, the condition is a disease in a subject, the severity of which is reduced by inducing an immune response in the subject via administration of a pharmaceutical composition.

[0078] As used herein, the term "effective amount" in the context of administration of a therapy to a subject refers to the amount of the therapy that has a prophylactic and / or therapeutic effect.

[0079] As used herein, the term "combination" in the context of administering two or more therapies to a subject refers to the use of two or more therapies (e.g., two or more prophylactic and / or therapeutic agents). The use of the term "combination" does not restrict the order in which the therapies are administered to a subject.

[0080] As used herein, the terms "prevent," "preventing," and "prevention," in the context of the present invention, and the administration of a therapy to a subject, refer to the inhibition of the development or onset of a disease or its symptoms. In one embodiment, this relates to the administration of a compound to a patient known to be at increased risk of developing a particular condition, disorder, or disease.

[0081] As used herein, the terms "treat," "treatment," and "treating" refer, in the context of the present invention, to the administration of a compound to a patient who already exhibits signs and / or symptoms of a particular condition, disorder, or disease. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; a reduction in the severity of the condition, disorder, or disease; a stabilized (i.e., not worsening) condition, disorder, or disease; a delay in the onset of the condition, disorder, or disease; or a delay in the progression of the condition, disorder, or disease; an improvement or remission (whether partial or total) of the condition, disorder, or disease (whether detectable or undetectable); an improvement in at least one measurable physical parameter (not necessarily discernible by the patient); or an enhancement or amelioration of the condition, disorder, or disease. Treatment includes eliciting a clinically significant response without undue side effects. Treatment also includes prolonging survival compared to the expected survival if not receiving treatment.

[0082] The terms "subject" or "patient" are used interchangeably and refer to a human or animal (e.g., a mammal) that may require administration of a compound of the present invention in the field of veterinary medicine. In a specific embodiment, the subject is a human. The subject may be administered the oligonucleotide of the present invention for beneficial editing. The subject may be administered the oligonucleotide of the present invention for compensatory editing.

[0083] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle into which a pharmaceutical composition is administered. Physiological saline, aqueous solutions of dextrose and glycerol can also be used as liquid carriers, especially for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. The dosage form should suit the mode of administration.

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in this disclosure; other, suitable methods and materials known in the art can also be used. (Antisense) oligonucleotides

[0085] In particular, chemically modified (antisense) oligonucleotides (ASOs) are provided herein. Without intending to be bound by any particular theory of operation, it is believed that modifications of the nucleobase and backbone bonds of the ASOs are useful for stabilizing and improving the editing effect and lysosomal stability of the oligonucleotides of the present invention. Furthermore, these modifications may also reduce off-target editing of different ASOs. Because one or more modifications can be synthetically transferred to various oligonucleotide sequences, such modifications may improve the editing effect of oligonucleotides with different specificities. The ASOs of the present invention can be used for several purposes. Advantageously, the oligonucleotides provided herein may be useful for editing one or more G-to-A mutations. It is noteworthy that the ASOs of the present invention are not limited to correcting G-to-A mutations, but are also useful for changing wild-type sequences to mutant sequences ("beneficial editing") to regulate protein expression and / or function, or for compensating for mutations other than G-to-A mutations. Thus, the oligonucleotides and compositions comprising them may be useful as active agents in medicines for treating genetic disorders, conditions or diseases associated with one or more G-to-A mutations.

[0086] The present inventors have recognized that in providing oligonucleotides for RNA editing, achieving a beneficial balance between high editing efficiency and lysosomal stability requires incorporating certain features into the oligonucleotide. Specifically, the inventors found that oligonucleotides should have a mixture of different modifications at the 2'-position of the sugar residue and avoid stretches of more than six nucleotides with the same 2'-modification. Avoiding uniform blocks of more than six nucleotides with the same 2'-modification prevented a significant loss of editing activity by native ADARs. In addition to this design feature, modified oligonucleotides of the present invention require that at least two of the three nucleotides in the CBT are modified at the 2'-position of the sugar base or are deoxyribonucleosides, which further stabilizes the oligonucleotide against nuclease digestion. Furthermore, it was found that phosphorothioate (PS) linkages should be avoided at positions h and i of the core sequence. PS linkages at these positions were found to strongly impair editing. However, the present inventors have found that the oligonucleotide of the present invention is beneficial to have at least 15% modifications in order to achieve good RNA editing, and it is therefore beneficial to have base-level internucleoside bond modifications at other positions.It has been found that it is beneficial for oligonucleotides to incorporate modifications at the 2'-position of nucleotides, and that such modifications should be composed of different groups.Therefore, it has been found that a mixture of 2'-F- and 2'-O-alkyl-modifications is beneficial, with a minimum of 10% each being desirable.The combination of these features allows oligonucleotides to achieve high levels of lysosomal stability and RNA editing effect.Therefore, the oligonucleotide of the present invention is preferably modified and designed accordingly.

[0087] According to the present invention, the core oligonucleotide has the sequence: [ka] The core sequence comprises a specific pattern of 2'-modification and internucleoside bond, which contributes to the advantageous properties of the oligonucleotide.The core sequence can comprise PS bond, for example, at positions d, e, and optionally a.Surprisingly, the region that is particularly sensitive to the discovered bond pattern is CBT and adjacent hotspot ("extended hotspot"), where highly specific nucleotide modification is required to be ideally introduced into the enzyme active site of ADAR for editing effect.Generally, the mixture of 2'-F-, 2'-OMe, and 2'-H modification, at least 15% internucleoside bond modification, not more than 6 consecutive nucleotides with the same 2'-modification, and N +2 2'-O-alkyl-modified and N +3 Oligonucleotides containing 2'-F-modifications of the formula (I) confer optimal editing and lysosomal stability (for "extended hotspots") (e.g., Example 15 for "extended hotspots").

[0088] Thus, as used herein, a central base triplet (CBT) of three nucleotides comprises a sequence of 23 to 80 nucleotides in length capable of binding to a target sequence in a target RNA, with a central nucleotide (N0) directly opposite a target adenosine in said target RNA. [ka] 1. A chemically modified oligonucleotide comprising the following core oligonucleotide: [ka] Includes; where: (a) at least two of the three nucleotides of said CBT are chemically modified at the 2' position of the sugar moiety, are deoxyribonucleosides, or are a combination thereof, and d and e are internucleoside linkage modifications; (b) N +2 The nucleotide is 2'-O-alkyl-modified;+3 The nucleotides are 2'-fluoro (2'-F)-modified; (c) at least 10% of the nucleotides are 2'-F-modified and at least 10% of the nucleotides are 2'-O-alkyl-modified, where no more than six consecutive nucleotides have the same 2'-modification; (d) the internucleoside linkage modification content is at least 15%; and (e) Chemically modified oligonucleotides are provided, wherein linkages h and i are not phosphorothioate (PS) linkages.

[0089] Evidence for the beneficial effects of having a 2'-O-alkyl-modification at the +2 position and a 2'-fluoro (2'-F)-modification at the +3 position has been provided by the inventors in this application ("modification hotspots"). Specifically, N +2 2'-OMe at N +3 It has been shown that constructs with a 2'-F at the N +2 In one embodiment, the nucleotide of N +3 In one embodiment, the nucleotide of N +2 The nucleotides are 2'-O-alkyl-modified and N +3 The nucleotides are 2'-F-modified.

[0090] The present inventors have also shown that uniform blocks or continuous stretches of nucleotides with the same chemical modification can interfere with ASO activity. Therefore, the oligonucleotides of the present invention may be modified so as not to contain uniform blocks or continuous stretches of the same 2'-sugar modification. In one embodiment, no more than six consecutive nucleotides have the same 2'-modification. In one embodiment, no more than five consecutive nucleotides have the same 2'-modification. In one embodiment, no more than four consecutive nucleotides have the same 2'-modification. In one embodiment, no more than three consecutive nucleotides have the same 2'-modification. In one embodiment, no more than two consecutive nucleotides have the same 2'-modification.

[0091] In general, metabolically unstable ASOs may be desirable for specific, very transient therapeutic effects, such as wound healing. Restoring point mutations that cause classic diseases requires metabolically stable ASOs that allow for less frequent administration. While embodiments with a low content of 2'-modifications are also possible, the inventors aim to maximize stability by replacing each RNA nucleoside with either 2'-modified RNA or 2'-modified DNA. As shown in this application, the inventors have recognized that oligonucleotides can tolerate a high percentage of 2'-modifications without adversely affecting activity, as long as no block of more than six consecutive nucleotides has the same 2'-modification. Thus, in one embodiment, 20 to 100%, 30 to 100%, 40 to 100%, 50 to 100%, 60 to 100%, 70 to 100%, 80 to 100%, or 90 to 100% of the nucleotides are deoxyribonucleosides (DNA) or 2'-modified. In one embodiment, 20 to 100% of the nucleotides are DNA or 2'-modified. In one embodiment, 50 to 100% of the nucleotides are DNA or 2'-modified nucleotides. In one embodiment, 100% of the nucleotides are DNA or 2'-modified nucleotides. In one embodiment, 30 to 95%, 40 to 95%, 40 to 90%, 50 to 95%, 50 to 90%, 60 to 95%, or 60 to 90% of the nucleotides are DNA or 2'-modified nucleotides. In one embodiment, the above percentages are filled only with 2'-modified nucleotides and do not include DNA.

[0092] According to the present invention, the oligonucleotides of the present invention may contain modifications at the 2'-position on the nucleotides, in which different modifying groups are used. In one embodiment, 20 to 70% of the nucleotides are 2'-F-modified. In one embodiment, 35 to 65% of the nucleotides are 2'-F-modified. In one embodiment, 20 to 60% of the nucleotides are 2'-O-methyl (2'-OMe)-modified. In one embodiment, 25 to 55% of the nucleotides are 2'-OMe-modified.

[0093] The inventors have also recognized that modified oligonucleotides of the present invention do not require all internucleoside linkages to be modified to withstand lysosomal degradation, provided that a minimum level of internucleoside modification is incorporated and that linkages d and e of the core oligonucleotide sequence (as described above) are modified. In one embodiment, the content of internucleoside linkage modifications is at least 15%. In one embodiment, the content of internucleoside linkage modifications is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, or 90%. In one embodiment, 95% or less, 90% or less, 85% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, or 30% or less of the linkages are internucleoside linkage modifications.

[0094] Internucleoside bond modifications, such as PS linkages, tend to have a particularly positive effect on the pharmacokinetics of ASOs, as well as their stability, protein binding, and intracellular localization. However, it is also desirable to simultaneously reduce the overall PS content, for example, to reduce toxicity and nonspecific protein binding. In one embodiment, (a) internucleoside bond modifications do not account for more than 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% of the outer bonds of the CBT; or (b) 15-90% of the bonds are internucleoside bond modifications, preferably 40-80%, and most preferably 45-60% of the bonds are internucleoside bond modifications. In one embodiment, internucleoside bond modifications do not account for more than 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% of the outer bonds of the CBT. In one embodiment, 15-90% of the linkages are internucleoside linkage modifications, preferably 40-80%, and most preferably 45-60% of the linkages are internucleoside linkage modifications. In one embodiment, the internucleoside linkage modification content is at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In one embodiment, the internucleoside linkage modification content is 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, or 20% or less. In one embodiment, the internucleoside bond modification content is 10 to 90%, 15 to 90%, 15 to 80%, 15 to 70%, 15 to 60%, 20 to 90%, 10 to 80%, 20 to 80%, 25 to 80%, 30 to 80%, 30 to 90%, 40 to 90%, 40 to 80%, 40 to 70%, 45 to 90%, 45 to 85%, 45 to 75%, 45 to 70%, 45 to 60%, or 45 to 55%. In one embodiment, 15 to 90% of the bonds are internucleoside bond modifications. In one embodiment, 40 to 80% of the bonds are internucleoside bond modifications. In one embodiment, 45 to 60% of the bonds are internucleoside bond modifications.In one embodiment, the internucleoside linkage modification content is 20%, 30%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%. In one embodiment, the internucleoside linkage modification content is 30%. In one embodiment, the internucleoside linkage modification content is 15%.

[0095] To achieve optimal RNA editing, oligonucleotides of different lengths may require different mixtures of specific 2'-modifications and internucleoside linkage modifications. The shorter the oligonucleotide, the better the endosomal escape. Furthermore, the toxicity of a particular oligonucleotide depends on its length. Shorter oligonucleotides may also exhibit higher specificity. Longer oligonucleotides may bind more strongly or quickly to their respective RNA targets, but editing-facilitating bulges, mismatches, and wobbles may also be more effective in longer oligonucleotides. Consequently, both long and short oligonucleotides of the present invention have advantages and / or tradeoffs. Thus, the oligonucleotides of the present invention may be of various lengths. The oligonucleotides may range in length from about 23 to 80 nucleotides, for example, from about 23 to 50 nucleotides or from about 40 to 80 nucleotides. In one embodiment, the oligonucleotide is 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, or 80 nucleotides. In a preferred embodiment, the oligonucleotide is 59 nucleotides in length. In some embodiments, the oligonucleotide is 23 to 80 nucleotides in length. In some embodiments, the oligonucleotides have a length of 23 to 80, 23 to 70, 23 to 60, 23 to 50, 23 to 40, 23 to 33, or 23 to 38 nucleotides. In some embodiments, the oligonucleotides have a length of 25 to 80, 25 to 70, 25 to 60, 25 to 50, or 25 to 40 nucleotides. In some embodiments, the oligonucleotides have a length of 30 to 80, 30 to 70, 30 to 60, 30 to 50, or 30 to 40 nucleotides.In some embodiments, the oligonucleotide has a length of 40 to 80, 50 to 80, 60 to 80, or 70 to 80 nucleotides. In some embodiments, the oligonucleotide is at least 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, or 80 nucleotides. In one embodiment, the oligonucleotide has a length of 28 to 60, 28 to 55, 28 to 50, 28 to 45, 28 to 40, 28 to 35, or 28 to 30 nucleotides. In one embodiment, the oligonucleotide has a length of 25 to 60, 25 to 55, 25 to 50, 25 to 45, 25 to 40, 25 to 35, or 25 to 30 nucleotides. In one embodiment, the oligonucleotide has a length of 28 to 70 nucleotides. In one embodiment, the oligonucleotide has the following lengths: (i) 28 to 60, 28 to 55, or 28 to 45 nucleotides; (ii) 59 nucleotides; or (iii) 45 or fewer nucleotides. Ranges and lengths intermediate to the above ranges and lengths are also contemplated as part of the present invention. In one embodiment, the oligonucleotide has a length of 40 nucleotides. In one embodiment, the oligonucleotide has a length of 45 nucleotides or less, with no more than four nucleotides outside the CBT being deoxyribonucleotides. In one embodiment, the oligonucleotide has a length of 40 nucleotides. In one embodiment, the oligonucleotide has a length of 45 nucleotides. In one embodiment, the oligonucleotide has a length of 35 nucleotides. In one embodiment, the oligonucleotide has a length of 33 nucleotides. In one embodiment, the oligonucleotide has a length of 32 nucleotides. In one embodiment, the oligonucleotide has a length of 30 nucleotides.In one embodiment, the oligonucleotide has a length of 25 nucleotides.

[0096] The inventors have found that longer oligonucleotides (approximately 40-80 nucleotides in length) containing the above-described mixture of modifications and designs can tolerate a total deoxyribonucleoside content of 5-50%. Furthermore, the inventors have surprisingly found that oligonucleotides containing such modifications and designs can be shortened to even shorter sequences (≤45 nt) while still providing good RNA editing (e.g., Examples 8-14). Furthermore, shorter oligonucleotides containing a total of six or fewer deoxyribonucleosides outside the CBT and with at least 30% internucleoside linkage modification content resulted in good RNA editing (e.g., Example 17). Contiguous regions and high levels of internucleoside linkage modification content typically resulted in increased editing.

[0097] Therefore, as used herein, a central base triplet (CBT) of three nucleotides is also defined as a nucleic acid molecule that comprises a sequence of 23 to 50 nucleotides in length capable of binding to a target sequence in a target RNA, and has a central nucleotide (N0) directly opposite a target adenosine in the target RNA. [ka] 1. A chemically modified oligonucleotide comprising the following core oligonucleotide: [ka] Includes; where: (a) at least two of the three nucleotides of said CBT are chemically modified at the 2' position of the sugar moiety, are deoxyribonucleosides, or are a combination thereof, and d and e are internucleoside linkage modifications; (b) N +2 The nucleotide is 2'-O-alkyl-modified;+3 The nucleotides are 2'-fluoro (2'-F)-modified; (c) at least 10% of the nucleotides are 2'-F-modified and at least 10% of the nucleotides are 2'-O-alkyl-modified, where no more than six consecutive nucleotides have the same 2'-modification; (d) the regions located 3' and 5' of said CBT do not contain a total of more than six deoxyribonucleosides; (e) A chemically modified oligonucleotide having an internucleoside bond modification content of at least 30%. Although oligonucleotides of the present invention tolerate DNA outside the CBT, placement of very high levels of DNA (or high DNA:RNA ratios) tends to hinder editing efficiency. Nevertheless, potentially useful embodiments containing a significant number of deoxyribonucleotides can be created to strike a reasonable balance between stability and editing efficiency. Thus, oligonucleotides of the present invention may contain different amounts of DNA. Specifically, oligonucleotides may contain different amounts of DNA (2'-H modified) outside the CBT. Thus, in one embodiment, the regions located 3' and 5' of the CBT do not contain a total of more than six deoxyribonucleosides. In one embodiment, the regions located 3' and 5' of the CBT do not contain a total of more than five, four, or three deoxyribonucleosides.

[0098] Outside the CBT, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleobases may be deoxyribonucleotides. In one embodiment, outside the CBT, the oligonucleotide does not contain any deoxyribonucleosides. In one embodiment, outside the CBT, no more than 1, 2, 3, or 4 nucleobases are deoxyribonucleotides. In one embodiment, outside the CBT, no more than 3 nucleobases are deoxyribonucleotides. Compared to shorter ASOs, longer ASOs tend to tolerate more DNA outside the CBT. Thus, as used herein, a central base triplet (CBT) of three nucleotides, comprising a sequence of 40 to 80 nucleotides in length capable of binding to a target sequence in a target RNA, the central nucleotide (N0) being directly opposite a target adenosine in said target RNA; [ka] 1. A chemically modified oligonucleotide comprising the following core oligonucleotide: [ka] Includes; where: (a) at least two of the three nucleotides of said CBT are chemically modified at the 2' position of the sugar moiety, are deoxyribonucleosides, or are a combination thereof, and d and e are internucleoside linkage modifications; (b) N +2 The nucleotide is 2'-O-alkyl-modified; +3 The nucleotides are 2'-fluoro (2'-F)-modified; (c) at least 10% of the nucleotides are 2'-F-modified and at least 10% of the nucleotides are 2'-O-alkyl-modified, where no more than six consecutive nucleotides have the same 2'-modification; (d) A chemically modified oligonucleotide, wherein the regions located 3' and 5' of the CBT have a total deoxyribonucleoside content of 5 to 50%.

[0099] In one embodiment, the regions located 3' and 5' of the CBT have a total deoxyribonucleoside content of 10 to 50%. In one embodiment, the deoxyribonucleoside content outside the CBT is 10 to 40%, more preferably 11 to 30%, and even more preferably 13 to 25%.

[0100] According to the present invention, the chemically modified oligonucleotide comprises an internucleoside linkage modification. In one embodiment, the oligonucleotide comprises at least one internucleoside linkage modification selected from the group consisting of phosphorothioate (PS), 3'-methylene phosphonate, 5'-methylene phosphonate, 3'-phosphoramidate, 2'-5' phosphodiester, and phosphorylguanidine (PN). In a preferred embodiment, the internucleoside linkage modification is a PS linkage. In one embodiment, the internucleoside linkage modification is a 3'-methylene phosphonate linkage. In one embodiment, the internucleoside linkage modification is a 5'-methylene phosphonate linkage. In one embodiment, the internucleoside linkage modification is a 3'-phosphoramidate linkage. In one embodiment, the internucleoside linkage modification is a 2'-5'-phosphodiester linkage. In one embodiment, the internucleoside linkage modification is a phosphorylguanidine (PN) linkage. In one embodiment, the nucleic acid analog is a PNA (peptide nucleic acid). In one embodiment, the nucleic acid analog is a PMO (phosphorodiamidate-linked morpholino). In one embodiment, the oligonucleotide contains a PS linkage, a phosphate (PO) linkage, and / or a phosphorodiamidate linkage. In one embodiment, at least one internucleoside linkage modification is PS. In one embodiment, the oligonucleotide contains a continuous region of PS linkages. In one embodiment, the continuous region of PS linkages is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more linkages in length.

[0101] The positioning of internucleoside linkages within an oligonucleotide plays an important role in determining the balance between high editing yield and long half-life. At the same time, careful consideration of the introduction of backbone modifications is necessary, as it affects the susceptibility of the CBT and bases surrounding the CBT to subtle chemical modifications to the ribose, nucleobase, or bond. For example, positions h and i may be chemically modified or unmodified. However, the inventors of the present application have found that PS linkages should not be placed at positions h and i, which are clearly detrimental to the editing efficacy of the construct. Thus, in one embodiment, bonds h and i are not chemically modified. In one embodiment, bond h is not chemically modified. In one embodiment, bond i is not chemically modified. In some embodiments, bonds h and i are phosphate (PO) linkages. In some embodiments, bonds h and i are not phosphorothioate (PS) linkages. In one embodiment, up to three linkages selected from the group consisting of bonds b, c, f, g, and j are also PS linkages. However, it is excluded that all of bonds a through j are PS linkages. In a particularly preferred embodiment, bond f is a PS linkage. In a particularly preferred embodiment, bonds a, d and e are PS bonds, while bonds h and i are PO bonds.

[0102] The stability and editing effect of various oligonucleotides can be affected by the amount and sequential placement of specific 2'-modifications. That is, repeated modifications of the same type of 2'-modification have been found to be detrimental to the RNA editing effect of oligonucleotides. Therefore, the present inventors have begun to investigate the effect of separating consecutive regions or blocks of the same 2'-modification. As shown in the present application, oligonucleotides containing small blocks of no more than six consecutive nucleotides with the same 2'-modification have produced the best editing effect. Therefore, the oligonucleotides of the present invention do not contain uniform blocks of more than about six nucleotides with the same 2'-modification. For example, in one embodiment, the oligonucleotide does not contain more than six consecutive nucleotides that are 2'-F-modified and / or 2'-O-alkyl-modified. In one embodiment, the number of consecutive nucleotides that are 2'-F-modified does not exceed four, five, or six; and / or the number of consecutive nucleotides that are 2'-O-alkyl-modified does not exceed four, five, or six. In one embodiment, the number of consecutive nucleotides that are 2'-F-modified does not exceed 4, 5, or 6. In one embodiment, the number of consecutive nucleotides that are 2'-O-alkyl-modified does not exceed 4, 5, or 6. In one embodiment, the number of consecutive nucleotides that are 2'-F- and / or 2'-O-alkyl-modified does not exceed 4, 5, or 6. In one embodiment, the oligonucleotide contains 4 consecutive nucleotides that are 2'-F- and / or 2'-O-alkyl-modified. In one embodiment, the oligonucleotide contains 5 consecutive nucleotides that are 2'-F- and / or 2'-O-alkyl-modified. In one embodiment, the oligonucleotide contains 6 consecutive nucleotides that are 2'-F- and / or 2'-O-alkyl-modified. The oligonucleotide may contain fewer than 4 consecutive nucleotides with the same 2'-modification. In a preferred embodiment, the 2'-O-alkyl-modification is a 2'-OMe-modification.

[0103] Furthermore, it has been found that uniform, block-wise 2'-modification of oligonucleotides, as used in the prior art, leads to a significant loss of editing activity. Without being bound by any theory, the present inventors believe that this is due to the negative effect of bulky 2'-modifications on the binding of dsRNA-binding domains to dsRNA substrates. dsRNA recognition (e.g., by ADAR deaminase and / or dsRBD) and dsRNA binding typically occur through interaction between the protein and the minor groove of the RNA helix. 2'-ribose modifications protrude into the minor groove, creating steric demands and altering the hydration state of the RNA helix. 2'-F-modifications are sterically most similar to the natural 2'-OH in ribose, but are highly hydrophobic and may disrupt hydration. 2'-O-methyl-modifications are more sterically demanding, and 2'-MOE-modifications are even more so. Therefore, bulky 2'-modifications are poorly tolerated, especially in large blocks, and can reject dsRBD binding. This is particularly true for 2'-MOE, but also for large blocks of 2'-O-methyl modifications. However, contiguous stretches of 2'-F are also more readily tolerated than 2'-O-methyl, though perhaps not ideal due to their strong hydrophobicity. Thus, mixing 2'-F and 2'-O-methyl modifications provides a means of generating duplexes that provide easy access for ADAR binding. However, for certain reasons, 2'-F is more readily tolerated.

[0104] Therefore, the present inventors have found that avoiding a uniform block of more than 6 nucleotides with the same 2'-modification can prevent the strong loss of editing activity by natural ADAR.Therefore, in one embodiment, less than 6, less than 5, less than 4, or less than 3 consecutive nucleotides have the same 2'-modification.In one embodiment, more than 6 consecutive nucleotides are not 2'-F-modified.In one embodiment, more than 5 consecutive nucleotides are not 2'-F-modified.In one embodiment, more than 4 consecutive nucleotides are not 2'-F-modified.In one embodiment, more than 6 consecutive nucleotides are not 2'-O-alkyl-modified.In one embodiment, more than 5 consecutive nucleotides are not 2'-O-alkyl-modified.In one embodiment, more than 4 consecutive nucleotides are not 2'-OMe-modified, and optionally more than 4 consecutive nucleotides are not 2'-OMe-modified. In one embodiment, the oligonucleotide comprises 2, 3, 4, 5, or 6 consecutive nucleotides with the same 2'-modification, for example, 5 consecutive nucleotides are 2'-F-modified.

[0105] Various types of PS-modifications can affect the efficacy of oligonucleotides. Therefore, the present application also provides chemically modified ASOs with reduced sterically pure bond chemistry. That is, the ASOs of the present invention relate to sterically random PS-modified ASOs that are generally easy and inexpensive to manufacture. Without being bound by any theory, the inventors believe that, contrary to prior art ASOs, a high level of sterically pure bond formation may not be useful or necessary to achieve efficient RNA editing. Considering that the active site of a deaminase must bind to the target RNA / oligonucleotide drug duplex around the N0 position, certain bonds, such as those where proteins interact with the phosphate backbone, may benefit from the insertion of sterically pure bond modifications, such as sterically pure PS (or PN) modifications. However, the inventors believe that, if present at all, these beneficial effects of sterically pure bond modifications apply only to a small number of sites; likely no more than 10 bonds per ASO, more likely no more than 5 bonds per ASO. Therefore, the inventors believe that the very high degree of sterically pure binding used in the prior art is not necessary to design ASOs with efficient editing.

[0106] However, when applying an optimal 2'-sterically random bond modification pattern in an optimized length and asymmetric oligonucleotide, the introduction of (some) stereo-pure bonds can be used. Generally, an oligonucleotide contains stereo-random internucleoside linkages. In one embodiment, an oligonucleotide contains one or more stereo-random internucleoside bond modifications. In one embodiment, an oligonucleotide does not contain stereo-pure PS bond modifications. In one embodiment, an oligonucleotide contains 10 or less, preferably 5 or less, stereo-pure internucleoside linkages. In one embodiment, an oligonucleotide contains 5 or less stereo-pure internucleoside linkages. In one embodiment, an oligonucleotide contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 stereo-pure internucleoside linkages.

[0107] Although the oligonucleotides of the present invention contain different internucleoside bond modifications, the inventors have shown that an optimized PS bond modification combining balanced 2'-modification and reduced block size is beneficial for producing oligonucleotides with good RNA editing. Notably, without being bound by any theory, the inventors believe that different bond modifications (or a particular type, for example, many phosphorylguanidine (PN) bonds) may result in oligonucleotides that do not exhibit sufficient RNA editing effects. Therefore, in one embodiment, the stereochemically pure bond is a PS bond. In one embodiment, the stereochemically pure bond is a PS bond and / or a PN bond. In one embodiment, the stereochemically pure bond is a PN bond. In one embodiment, the oligonucleotide does not contain a stereochemically pure PS bond and / or a stereochemically pure PN bond. In one embodiment, the oligonucleotide does not contain a stereochemically pure PS bond. In one embodiment, the oligonucleotide does not contain a stereochemically pure PN bond. In one embodiment, the chemically modified oligonucleotide does not contain a stereochemically pure PS bond modification.

[0108] As mentioned above, incorporating a high level of RNA into an oligonucleotide tends to make the oligonucleotide metabolically unstable, but this can be an attractive feature.To achieve the necessary metabolic stability of an oligonucleotide, the final oligonucleotide should ideally not contain any unmodified RNA nucleobases.In one embodiment, an oligonucleotide contains unmodified RNA nucleobases.In one embodiment, an oligonucleotide contains more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% modified nucleotides.In one embodiment, an oligonucleotide contains less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% unmodified RNA nucleotides.In this context, modification includes having deoxyribonucleotides.

[0109] 2'-MOE residues are used in splice-switching oligonucleotides and typically exhibit very low toxicity. However, due to their bulkiness, 2'-MOE residues are not well tolerated in large quantities. The present inventors observed that 2'-MOE modification at the terminus of an oligonucleotide does not affect the overall editing yield, while surprisingly reducing the cytotoxicity of the test construct. Specifically, the present inventors found that the amount of 2'-MOE modification can be limited to about six, seven, or eight nucleotides to achieve good RNA editing. Similarly, the present inventors found that oligonucleotides containing two to six LNAs resulted in good RNA editing.

[0110] Thus, an oligonucleotide may contain no more than six, seven, or eight 2'-MOE modifications within the oligonucleotide. In one embodiment, the oligonucleotide comprises: (a) 2'-O-(2-methoxyethyl)-oligoribonucleotide (2'-MOE) end blocks at the 3'- and 5'-ends, where each end has no more than four nucleotides with 2'-MOE, preferably no more than three nucleotides with 2'-MOE; or (b) terminal-locked nucleic acids (LNAs), where the oligonucleotide contains two to six LNAs at each end or the 5'-end; preferably, the oligonucleotide contains two LNAs at each end or the 5'-end. In one embodiment, each end has no more than four nucleotides with 2'-MOE, preferably no more than three nucleotides with 2'-MOE. In one embodiment, the oligonucleotide comprises 2'-MOE end blocks at the 3'- and 5'-ends, wherein there are no more than four nucleotides with 2'-MOE at each end. In one embodiment, the oligonucleotide comprises 2'-MOE end blocks at the 3'- and 5'-ends, wherein there are no more than three nucleotides with 2'-MOE at each end. In one embodiment, the oligonucleotide comprises a terminal-locked nucleic acid (LNA), wherein the oligonucleotide comprises 2 to 6 LNAs at each end. In one embodiment, the oligonucleotide comprises a terminal-locked nucleic acid (LNA), wherein the oligonucleotide comprises 2 to 6 LNAs at the 5'-end. In one embodiment, the oligonucleotide comprises two LNAs at each end. In one embodiment, the oligonucleotide comprises two LNAs at the 5'-end. In this context, the term "end" refers to the last or terminal nucleotide at either end of the oligonucleotide; for example, "no more than four nucleotides at each end" refers to the last four nucleotides at each end of the oligonucleotide. In one embodiment, there are no 2'-MOE modifications within the CBT.In one embodiment, there are no 2'-MOE modifications in the +2 and / or +3 positions.

[0111] In one embodiment, bond g is not a PS bond. In one embodiment, bond g is a phosphate (PO) bond.

[0112] In one embodiment, the 2'-O-alkyl-modification is a 2'-OMe-modification. Notably, in some embodiments, the 2'-O-alkyl-modification is not a 2'-MOE-modification.

[0113] CBT is highly sensitive to position-specific bond modification, because it prevents ADAR active site binding.Therefore, in order to achieve efficient editing and stabilization of oligonucleotide, the present inventors have shown that specific bond modification (mixture) must be placed at specific position in oligonucleotide.In one embodiment, d and e are PS bond modification, and optionally f is internucleoside bond modification.In one embodiment, d and e are PS bond modification.In one embodiment, f is PS bond.

[0114] In one embodiment, the modification at the 2'-position of the sugar moiety is (i) a 2'-O-alkyl-modification, (ii) a 2'-F-modification, or (iii) a 2'-fluoroarabinoside (FANA) modification.

[0115] CBT(5'-N -1 -N0-N +1- 3') can be subjected to different modifications and various permutations of modifications. In one embodiment, the CBT is chemically modified. -1 Rank, N0 rank and / or N +1 The 2' position may be modified. In one embodiment, only one position in CBT is chemically modified. In one embodiment, two positions in CBT are chemically modified. In one embodiment, all positions in CBT are chemically modified. According to one embodiment, each of the three nucleosides of CBT is either alone or in combination of the following: (a) deoxyribonucleotides; and / or (b) 2'-FANA-modified; and / or (c) 2'-O-methyl-modified; and / or (d) 2'-F-modification. In one embodiment, at least one of the three oligonucleotides of the CBT is a deoxyribonucleotide. In one embodiment, at least one of the three oligonucleotides is 2'-FANA-modified. In one embodiment, at least one of the three oligonucleotides is -O-methyl-modified. In one embodiment, at least one of the three oligonucleotides is 2'-F-modified. In one embodiment, (i) N -1 is 2'-F, 2'-FANA, DNA, or 2'-O-methyl; and / or (ii) N0 is 2'-FANA or DNA; and / or (iii) N +1 is 2'-FANA, DNA, or 2'-O-methyl. In some embodiments, N -1 In some embodiments, position N is 2'-fluoro-RNA, 2'-FANA, or DNA. In some embodiments, position N is 2'-FANA or DNA. +1 The CBT modification may include any permutation of the above modifications.

[0116] In one embodiment, N0 is deoxycytidine or FANA-cytidine. In one embodiment, N0 is deoxycytidine. In one embodiment, N0 is FANA-cytidine. Other modifications include replacement of the nucleobase with an (N) heterocyclic or aromatic ring that stacks well in an RNA duplex, such as, for example, Benner base Z (dZ) (and / or analogs) or 8-oxo-adenosine (8-oxo-A). Thus, in one embodiment, N0 is a Benner base. In one embodiment, N0 is 8-oxo-adenosine.

[0117] The region outside the CBT and one or more nucleotides may be modified. For example, the nucleotides located 5' and / or 3' of the CBT may be chemically modified to have a 2'-modification. The oligonucleotides of the present invention may be modified within the region defining the "hot spot site" or "hot spot region." In one embodiment, the oligonucleotide is modified at the first nucleotide located directly 3' of the CBT (i.e., +2 position). [ka] In one embodiment, the oligonucleotide has a second nucleotide located directly 3' of the CBT (i.e., position +3). [ka] In a preferred embodiment, the oligonucleotide is modified at the first two nucleotides located directly 3' of the CBT (i.e., positions +2 and +3). [ka] .

[0118] The nucleotides located at 5' and / or 3' of the CBT may be chemically modified to be 2'-modified. In one embodiment, the -5, -4, and -3 positions are 2'-O-alkyl-modified; and / or the -2 position is 2'-F-modified. In one embodiment, the -5, -4, and -3 positions are 2'-O-alkyl-modified. In one embodiment, the -2 position is 2'-F-modified. In one embodiment, the -5, -4, and -3 positions are 2'-O-alkyl-modified and the -2 position is 2'-F-modified. In one embodiment, the -5, -4, and -3 positions are 2'-O-alkyl-modified or the -2 position is 2'-F-modified. In one embodiment, the 2'-OMe-modification is at the +2 position. In one embodiment, the 2'-F-modification is at the +2 position. In one embodiment, the 2'-F-modification is at the -2 position. In one embodiment, the 2'-OMe-modifications are at the -5, -4, and -3 positions. In one embodiment, the 2'-OMe-modifications are at the -5, -4, or -3 positions. Other modifications include nucleobase substitution with (N) heterocyclic or aromatic rings that stack well in RNA duplexes, such as, for example, Benner base Z (dZ) (and / or analogs) or 8-oxo-adenosine (8-oxo-A). In one embodiment, N is a Benner base. In one embodiment, N is 8-oxo-adenosine (8-oxo-A). Specifically, in a preferred embodiment, N +2 The nucleotides are 2'-O-alkyl-modified. +3 The nucleotides are 2'-fluoro (2'-F)-modified. +2 The nucleotides are 2'-O-alkyl-modified and N +3 The nucleotides are 2'-fluoro (2'-F)-modified.

[0119] In one embodiment, 2'-OMe-modifications are preferred over DNA close to the CBT. In one embodiment, 2'-F-modifications are preferred over DNA close to the CBT. In one embodiment, there is no DNA at the +2 or +3 positions.

[0120] As mentioned above, internucleoside bond modifications such as PS bonds tend to have a positive effect on, for example, the stability of ASOs, so the introduction of these bonds within ASOs appears to play an important role. In particular, the present inventors have found that, in terms of the stability of the tritosome, a PS bond 3' to the DNA appears to be more important than a PS bond 5' to the DNA. Therefore, in some embodiments, the PS bond is located 3' to the DNA. In one embodiment, the PS bond is located directly 3' to the DNA. In some embodiments, the PS bond is located 3' and 5' to the DNA. In one embodiment, only 2'-modified nucleotides are present in the ASO, and no DNA is present. In one embodiment, the stability of the ASO is improved by placing the PS bond 3' to the DNA.

[0121] Uniform blocks or stretches of large 2'-sugar modifications within an ASO tend to interfere with the binding of ADAR dsRNA-binding proteins (dsRBDs). Therefore, the oligonucleotides of the present invention can be modified to avoid such interference. For example, the oligonucleotides are modified so as not to contain consecutive stretches or uniform blocks of nucleotides with the same chemical modification (i.e., avoiding block-like modification structures). Thus, in one embodiment, the oligonucleotides are not uniformly modified. In one embodiment, the oligonucleotides contain non-uniform blocks and / or non-block-like modification structures. In one embodiment, the oligonucleotides do not contain consecutive stretches or uniform blocks of nucleotides with the same chemical modification at the 2'-position of the sugar moiety. In a preferred embodiment, the oligonucleotides are modified to avoid uniform blocks of 2'-F-modifications and / or 2'-OMe-modifications. In some embodiments, the oligonucleotides do not contain any blocks of 2'-H(DNA). In a preferred embodiment, the oligonucleotides are modified to avoid uniform blocks of 2'-F-modifications, 2'-OMe-modifications, and / or 2'-H groups. In particular, the maximum block size of 2'-F-modification and 2'-OMe-modification may be different. Thus, in one embodiment, an oligonucleotide comprises a larger block of 2'F-modified nucleotides. In one embodiment, an oligonucleotide comprises a larger block of 2'-OMe-modified nucleotides. In one embodiment, 2'-OMe-modifications are accepted in smaller blocks than 2'-F-modifications. In one embodiment, 2'-F-modifications are accepted in larger blocks than 2'-OMe-modifications.

[0122] The oligonucleotides of the present invention may contain a "continuous stretch" or "uniform block" of a particular length. In one embodiment, the size or length of the "continuous stretch" or "uniform block" is 2, 3, 4, 5, or 6 nucleotides in length. In one embodiment, the size or length of the "continuous stretch" or "uniform block" is not more than 2, 3, 4, 5, or 6 nucleotides in length. In one embodiment, the oligonucleotide contains not more than 2, 3, 4, 5, or 6 consecutive nucleotides containing a 2'-F-modification. In one embodiment, the oligonucleotide contains not more than 2, 3, 4, 5, or 6 consecutive nucleotides containing a 2'-OMe-modification. In one embodiment, one or more uniform blocks are interrupted. The interruption can be caused by any other chemical modification (e.g., DNA, RNA, 2'-F, 2'-OMe, 2'-MOE, LNA, etc.). In one embodiment, one or more uniform blocks of 2'-F-modified nucleotides are preferably interrupted by 2'-OMe-modified nucleotides. In one embodiment, one or more uniform blocks of 2'-OMe-modified nucleotides are preferably interrupted by 2'-F-modified nucleotides. In some embodiments, the blocks are interrupted by DNA.

[0123] According to the present invention, the oligonucleotide does not contain a block of more than six consecutive 2'-OMe-modified nucleotides. According to the present invention, the oligonucleotide does not contain a block of more than six consecutive 2'-F-modified nucleotides. In one embodiment, the oligonucleotide does not contain a block of more than five, more than four, or more than three consecutive 2'-OMe-modified nucleotides. In one embodiment, the oligonucleotide does not contain a block of more than four consecutive 2'-OM-modified nucleotides.

[0124] Linkage g can be unmodified or modified. In one embodiment, linkage g is a phosphate (PO) linkage. In one embodiment, linkage g is a 3',5'-phosphodiester linkage. In one embodiment, linkage g is a PS linkage. In one embodiment, the oligonucleotide comprises (a) at least 10 consecutive internucleoside linkage modifications; and / or (b) three consecutive internucleoside linkage modifications at each end. In one embodiment, the oligonucleotide comprises at least 10 consecutive internucleoside linkage modifications. In one embodiment, the oligonucleotide comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more consecutive PS linkages. In some embodiments, each end comprises three consecutive internucleoside linkage modifications. In some embodiments, each end contains 4, 5, or 6 consecutive internucleoside linkage modifications. In some embodiments, each end does not contain more than 8, 7, 6, 5, 4, or 3 consecutive internucleoside linkage modifications. In preferred embodiments, each end contains 3 consecutive internucleoside linkage modifications. In some embodiments, the modifications are 3'-methylene phosphonate, 5'-methylene phosphonate, 3'-phosphoramidate, 2'-5' phosphodiester, or phosphorylguanidine (PN) modifications. In one embodiment, the internucleoside linkage modification is a PS linkage modification. In another embodiment, the internucleoside linkage modification is a 3'-3' or 5'-5' phosphate linkage (3'-P-3' and 5'-P-5').

[0125] Without being bound by any theory, we believe that the ideal asymmetry for each target may depend on the length and specific underlying sequence of the particular oligonucleotide. ADARs are known to function as asymmetric dimers with a footprint of up to 50 bp. Some substrates are edited more efficiently by the deaminase domain alone than by the full-length protein, while the opposite is true for other substrates. This suggests that ADARs bind and register differently (monomers or dimers) depending on the size of the target / drug RNA helix (without dsRBD, one, two, or up to six dsRBDs). This creates a situation where specific symmetries on the target adenosine and specific modification patterns (e.g., ribose and linkage) are preferred depending on the length of the ASO. A short 3' end appears sufficient for optimal deaminase binding (at least 4 nt flanking the CBT). Longer 3' ends can even lead to loss of target editing. Meanwhile, the 5' end provides binding space for the dsRBD and therefore typically requires more nucleotides (at least 16 nt). Some well-functioning embodiments of symmetry provided herein and identified by the inventors of this application are shown in Table A.

[0126] Thus, in some embodiments, the ASO may be asymmetric, i.e., there may be different numbers of nucleotides (nt) at the 3' and 5' ends of the oligonucleotide. For example, there may be 20-40 nt at the 5' end and 5-15 nt at the 3' end. In one embodiment, there are: a) at least 4 nucleotides 3' of the CBT; or b) at least 16 nucleotides 5' of the CBT. In some embodiments, the CBT is present 4 nt to 30 nt 3' of the CBT. In some embodiments, the CBT is present 10 nt or less 3' of the CBT. In some embodiments, the 3' end is truncated to a length of 5 nt 3' of the CBT. In some embodiments, the 3' end is truncated to a length of 4 nt 3' of the CBT. In some embodiments, the region 3' to the CBT comprises 4 nt, 5 nt, or 6 nt. In some embodiments, the CBT is present 4 nt to 30 nt 5' of the CBT. In some embodiments, the CBT is present 35 nt or less 5' of the CBT. In some embodiments, the 5' end is truncated to a length of 25 nt or 26 nt 5' of the CBT. In one embodiment, the region 5' of the CBT comprises 24 nt, 25 nt, or 26 nt.

[0127] According to the present invention, oligonucleotides may have the symmetries and lengths as disclosed in Table A below. The oligonucleotides have the following scheme: (5' end length)-(CBT length)-(3' end length). For example, an ASO of the present invention having a length of 32 nt has a 5' end that is 24 nt long, a CBT that is 3 nt long, and a 3' end that is 5 nt long (scheme "24-3-5"). Thus, in one embodiment, the oligonucleotide has any one of the symmetries listed in Table A. [Table 1]

[0128] Depending on the length and / or minimal degree of linkage modification of a particular oligonucleotide, it may be differently modified as described herein. DNA has a different sugar puckering preference than RNA, and 2'-modified RNA is known to favor a B-form helix. Furthermore, DNA is fairly hydrophobic, altering the hydration of the double helix. Therefore, DNA is only accepted at certain positions and less accepted in large blocks. Therefore, the oligonucleotides provided herein may contain different ratios and amounts of DNA and / or RNA. In some embodiments, the oligonucleotides contain a combination of RNA and DNA. In some embodiments, the oligonucleotides contain a combination of RNA and DNA outside the CBT.

[0129] Furthermore, oligonucleotides of the present invention may have limited DNA content outside the CBT. In one embodiment, the DNA is located outside the CBT. In one embodiment, the DNA is located 3' and / or 5' of the CBT. In one embodiment, the DNA is located 3' of the CBT. In one embodiment, the DNA is located 5' of the CBT. Shorter oligonucleotides (≦45 nt) may have lower DNA content than longer oligonucleotides (≧50 nt). In one embodiment, the oligonucleotide has a length of 45 nt. In one embodiment, the oligonucleotide has a length of 45 nt or less nucleotides, with no more than three nucleotides that are deoxyribonucleotides outside the CBT. In one embodiment, no more than one, two, three, or four nucleotides that are deoxyribonucleotides outside the CBT. In one embodiment, the nucleotides have a length of 28 to 60, 28 to 55, or 28 to 45 nucleotides. In one embodiment, the nucleotides have a length of 28 to 60 nucleotides and a deoxyribonucleoside content outside the CBT of 10 to 40%, more preferably 11 to 30%, and even more preferably 13 to 25%. In one embodiment, the oligonucleotide does not contain any unmodified RNA nucleotides.

[0130] In one embodiment, at least one of the three nucleotides of the CBT is chemically modified at the 2' position of the sugar moiety, wherein said modification is a 2'-F-modification.

[0131] Oligonucleotides with loop hairpin structure have been previously described (WO2020 / 001793), and chemically modified oligonucleotides have been successfully used to utilize ADAR. However, they are relatively large, and without being bound by any theory, the inventors believe that more intelligent design of ASOs can form substrate duplexes that are very well and quickly recognized by endogenous ADARs, so that large recruitment motifs can be omitted. This is a clear advantage for delivery and manufacturing, as shorter ASOs can be designed. Therefore, oligonucleotides may or may not contain recruitment motifs for deaminase. Instead, the chemically modified nucleic acids of the present invention form RNA duplexes to which ADAR enzymes attach, thereby improving editing efficiency. In one embodiment, the oligonucleotide does not contain the ADAR recruitment motif of loop hairpin structure.

[0132] In certain cases, the ASO targeting domain, or the nucleobase opposite the target nucleobase to be edited, contains one or more wobble bases to compensate for variability in the target sequence. That is, the non-stringent base pairing requirement of wobble bases (e.g., GU, IA, GA, IU, IC, etc.) allows the ASO to pair with more than one target nucleic acid. Thus, in some embodiments, mismatches and / or wobbles allow for targeting of different target nucleic acids. In one embodiment, the oligonucleotide contains one or more additional mismatches, wobble bases, and / or bulges. In some embodiments, the oligonucleotide of the present invention can contain bulges of one, two, three, or more nucleotides. In one embodiment, the oligonucleotide contains one or more mismatches, wobble bases, and / or bulges relative to its target and / or a mismatch at N0. In one embodiment, the oligonucleotide contains one or more mismatches, wobble bases, and / or bulges relative to its target. In one embodiment, the oligonucleotide contains a mismatch at N0.

[0133] The targeting sequence of the artificial nucleic acid typically comprises a nucleic acid sequence that is complementary or at least partially complementary to the nucleic acid sequence in the target RNA. In some embodiments, the targeting sequence comprises a nucleic acid sequence that is complementary or at least 60%, 70%, 80%, 90%, 95% or 99% complementary to the nucleic acid sequence in the target RNA.

[0134] The oligonucleotide may contain DNA and / or RNA, but may also contain further modifications. LNA improves the binding strength of ASOs by maintaining the nucleoside in a preferred sugar confirmation (entropically favorable state). However, this sugar preorganization by additional bridges also reduces flexibility. The double-stranded RNA (dsRNA) structure is strongly perturbed at the active site of ADAR (flip-out mechanism). LNA may interfere with this process, so it is desirable to position LNA not within or too close to the CBT. In one embodiment, the oligonucleotide contains LNA. In one embodiment, the oligonucleotide contains DNA and / or RNA and / or LNA. In one embodiment, the oligonucleotide contains DNA, RNA, and LNA. In one embodiment, the oligonucleotide contains DNA. In one embodiment, the oligonucleotide contains RNA. In one embodiment, the oligonucleotide contains LNA.

[0135] In addition to specific backbone bond modification patterns and modifications at the 2'-position of the sugar moiety, purines and / or pyrimidines of oligonucleotides can be specifically targeted. The purines and / or pyrimidines can be modified or unmodified. In one embodiment, the purines and / or pyrimidines are modified. In some embodiments, the nucleobases are substituted purine base residues. In some embodiments, the nucleobases are substituted pyrimidine base residues. In one embodiment, the purines are modified with 2'-OMe, 2'-F, or 2'-deoxy. In one embodiment, the pyrimidines are modified with 2'-OMe, 2'-F, or 2'-deoxy. In some embodiments, the nucleobases are substituted heterocyclic base analogs. In some embodiments, the heterocyclic base analogs are nitrogen (N), oxygen (O), sulfur (S), or boron (B) heterocyclic base analogs. In certain embodiments, the modifications include the Benner base Z and / or analogs thereof.

[0136] Oligonucleotides of the present invention may be modified at their 5' and / or 3' ends. Oligonucleotides of the present invention may contain one or more different linkers, tags, or coupling agents at either or both ends. For example, oligonucleotides may contain an amino linker, preferably a C6-amino linker. Thus, in some embodiments, oligonucleotides of the present invention contain a C6-amino linker at the 5' end. In some embodiments, oligonucleotides contain a C6-amino linker at the 3' end. Oligonucleotides may contain a moiety that enhances cellular uptake of the oligonucleotide, such as N-acetylgalactosamine (GalNAc). Thus, in some embodiments, chemically modified oligonucleotides contain or are conjugated to a moiety that enhances cellular uptake of the oligonucleotide. Preferably, the moiety that enhances cellular uptake is triantennaryl N-acetylgalactosamine (GalNAc3), which is preferably conjugated to the 3' or 5' end of the oligonucleotide. composition

[0137] The nucleic acids or oligonucleotides (or ASOs) provided herein may be incorporated into compositions. For example, targeted delivery of oligonucleotides to liver hepatocytes using bivalent or trivalent N-acetylgalactosamine (GalNAc) conjugates has been previously described for the treatment of liver diseases, including hepatitis B virus (HBV), non-alcoholic fatty liver disease, and genetic diseases (Debacker et al., 2020).

[0138] Therefore, compositions comprising the oligonucleotides of the present invention are provided herein. In some embodiments, the present disclosure provides oligonucleotide compositions of the oligonucleotides described herein. In some embodiments, the compositions are pharmaceutical compositions. As used herein, pharmaceutical compositions refer to a mixture of substances suitable for administration to an individual. For example, pharmaceutical compositions may include one or more active pharmaceutical agents (such as oligonucleotides) and a sterile aqueous solution. In one embodiment, the compositions include one or more oligonucleotides of the present invention.

[0139] The pharmaceutical compositions provided herein can be in any form that allows the composition to be administered to a subject.The compositions can be used in methods for treating and / or preventing genetic disorders, conditions, or diseases.In certain embodiments, the pharmaceutical compositions are suitable for veterinary administration and / or human administration.

[0140] Provided herein is a pharmaceutical composition comprising an oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof.

[0141] In one embodiment, the composition comprises the oligonucleotide of the present invention in admixture with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier can be simply saline. The carrier can be isotonic or hypotonic.

[0142] In some embodiments, the pharmaceutical composition may include one or more other therapies in addition to the oligonucleotide of the present invention.

[0143] In some embodiments, the compositions of the present invention contain various buffer components (e.g., Tris-HCl, acetate, phosphate), diluents of pH and ionic strength, as well as additives such as detergents and solubilizers (e.g., Tween 80, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol), and bulking substances (e.g., lactose, mannitol). In some embodiments, materials can be incorporated into microparticle formulations of polymeric compounds such as polylactic acid, polyglycolic acid, or into liposomes. In some embodiments, hyaluronic acid can also be used. Such compositions may affect the physical state, stability, in vivo release rate, and / or in vivo clearance rate of the ASOs and derivatives of the present invention. In some embodiments, the compositions can be prepared in liquid form or as a dry powder, such as a lyophilized form.

[0144] In some embodiments, the pharmaceutical compositions described herein further comprise one or more salts, such as sodium chloride, calcium chloride, sodium phosphate, monosodium glutamate, and aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, alum (aluminum potassium sulfate), or mixtures of such aluminum salts). In other embodiments, the pharmaceutical compositions described herein do not comprise a salt.

[0145] The pharmaceutical compositions described herein can be included in a container, pack, or dispenser together with instructions for administration. Preventive and therapeutic use

[0146] The present invention generally describes the use of chemically modified oligonucleotides and / or compositions comprising them in medical settings.Specifically, it is for site-specific editing of target RNA (for example, by binding to target RNA via targeting sequence and recruiting deaminase to target site).The present invention describes chemically modified oligonucleotides and / or compositions for use in the treatment and prevention of genetic disorders, conditions, or diseases, and methods for treating or preventing genetic disorders, conditions, or diseases.Site-specific editing is carried out in vitro, in vivo, or ex vivo.

[0147] Chemically modified oligonucleotides or compositions comprising same can be used in the treatment and / or prevention of medical conditions. In one aspect, provided herein is the use of the oligonucleotides of the present invention and / or compositions comprising same in the treatment or prevention of genetic disorders, conditions, or diseases. In one embodiment, the genetic disorder, condition, or disease is selected from the group consisting of retinitis pigmentosa (RP), Stargardt macular degeneration, age-related macular degeneration (AMD), choroideremia, cone-rod dystrophy, cystic fibrosis (CF), Hurler syndrome, alpha-1-antitrypsin (A1AT) deficiency, Parkinson's disease, Alzheimer's disease, albinism, amyotrophic lateral sclerosis, asthma, beta-thalassemia, Cadasil syndrome, Charcot-Marie dental disease, chronic obstructive pulmonary disease (COPD), distal spinal muscular atrophy (DSMA), Duchenne / Becker muscular dystrophy, dystrophic epidermolysis bullosa, epidermolysis bullosa, Fabry disease, and factor V. Leiden-related disorders, familial adenomatous polyposis, polyposis, galactosemia, Gaucher disease, glucose-6-phosphate dehydrogenase, hemophilia, hereditary hematochromatosis, Hunter syndrome, Huntington's disease, inflammatory bowel disease (IBD), hereditary polymorphic aggregation syndrome, Leber congenital amaurosis (LCA), Lesch-Nyhan syndrome, Lynch syndrome, Marfan syndrome, mucopolysaccharidoses, muscular dystrophies, myotonic dystrophy types I and II, neurofibromatosis, Niemann-Pick disease types A, B and C, NY-eso1-associated cancers, Peutz-Jeghers syndrome, phenylketonuria, Pompe disease, primary ciliary body disease, prothrombin mutation-related disorders.

[0148] In one embodiment, the genetic disorder, condition, or disease is associated with a point mutation. For example, the SERPINA1 gene encodes the serine protease inhibitor alpha-I antitrypsin (A1AT). A1AT protects tissues from certain inflammatory enzymes, including neutrophil elastase. Deficiency of A1AT (alpha-1 antitrypsin deficiency, A1AD) leads to excessive degradation of lung elastin by neutrophil elastase. This can result in reduced lung elasticity and respiratory complications such as emphysema and chronic obstructive pulmonary disease (COPD). Mutant A1AT can also accumulate in the liver, causing cirrhosis and liver failure. Thus, in one embodiment, the genetic disorder, condition, or disease is associated with a G-to-A mutation in a gene selected from the following list: SERPINA1, PDE6A, LRRK2, and CRB1. In one embodiment, the mutation is selected from the list comprising: SERPINA1 E342K, PDE6A V685M, NLRP3 Y166, and CRB1 C948Y. In one embodiment, the mutation is a PiZZ mutation (alpha 1-antitrypsin deficiency).

[0149] The chemically modified oligonucleotide of the present invention or a composition comprising the same can be used to edit the adenosine base of wild-type allele (beneficial editing).In one embodiment, this editing regulates signal transduction, for example, JAK / STAT signal transduction.In one embodiment, editing introduces STAT1 Y701C mutation.In one embodiment, editing introduces NLRP3 Y166C mutation, thereby regulating inflammasome signal transduction.

[0150] The chemically modified oligonucleotide or (pharmaceutical) composition of the present invention may be administered orally in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions, or solutions, or parenterally, for example, by parenteral injection. In some embodiments, formulations suitable for parenteral administration include a sterile aqueous preparation of at least one embodiment of the present disclosure, which is approximately isotonic with the blood of the intended recipient. The amount, dosage, and administration regimen of the administered oligonucleotide or composition may vary depending on the cell type, the disease being treated, the target population, the mode of administration (e.g., systemic vs. local), the severity of the disease, and the acceptable level of side effects. In some embodiments, the amount of oligonucleotide administered in the pharmaceutical composition depends on the subject being treated, the subject's weight, and the mode of administration.

[0151] Various delivery systems can be used to deliver the oligonucleotides of the present invention.The oligonucleotides of the present invention can be delivered as they are (i.e., naked and / or isolated form) to individuals, organs (eyes), or especially cells.When administering oligonucleotides according to the present invention, it is preferred that the oligonucleotides are dissolved in a solution that is compatible with the delivery method.This delivery can be in vivo, in vitro, or ex vivo.Nanoparticles and microparticles that can be used for in vivo ASO delivery are well known in the art.Alternatively, plasmids can be provided by transfection using known transfection reagents.

[0152] In a preferred embodiment, the oligonucleotide of the present invention is administered and delivered "as is", also called "naked". Nevertheless, the art includes multiple methods for delivering oligonucleotides to cells, either in vitro, ex vivo, or in vivo. That is, the route of administration or delivery method may be selected depending on the disease, disorder, or infectious disease that needs to be treated, or the cell, tissue, or body part that needs to be reached by the oligonucleotide of the present invention (for example, in the case of beneficial editing). When the oligonucleotide is not delivered naked, examples of delivery agents or vehicles include nanoparticles such as polymer nanoparticles, liposomes, antibody-conjugated liposomes, cationic lipids, polymers, or cell-penetrating peptides.

[0153] The use of excipients or transfection reagents can aid in the delivery of each oligonucleotide or composition as defined herein to cells and / or within cells (preferably cells affected by G-to-A mutations or cells in which "beneficial editing" as outlined herein is achieved). Preferably, the excipients or transfection reagents capable of forming complexes, nanoparticles, micelles, vesicles, and / or liposomes that deliver each oligonucleotide or composition as defined herein are complexed or entrapped in the vesicles or liposomes through the cell membrane. Many of these excipients are known in the art. Suitable excipients or transfection reagents include polyethyleneimine (PEI; ExGen500 (MBI Fermentas)), LipofectAMINE™ 2000 (Invitrogen), lipofectin™, or derivatives thereof, and / or viral capsid proteins capable of self-assembly into particles capable of delivering each component as defined herein to target cells. Such vehicles have been shown to efficiently deliver oligonucleotides to a wide variety of cultured cells, and their high transfection potential is combined with low to moderate toxicity that is offset by overall cell viability.

[0154] The ASO of the present invention can be conjugated with a moiety that enhances the uptake of ASO in cells. Examples of such moieties include cholesterol, carbohydrates, vitamins, biotin, lipids, phospholipids, cell-penetrating peptides, including but not limited to, antigen-binding domains such as those provided by antennapedia, TAT, transportan, and positively charged amino acids such as oligoarginine, polyarginine, oligolysine, or polylysine, antibodies, Fab fragments, or single-chain antigen-binding domains such as cameloid single-domain antigen-binding domains. Thus, in some embodiments, ASOs are delivered using drug conjugates with antibodies, nanobodies, cell-penetrating peptides, and aptamers. In one embodiment, oligonucleotides are conjugated to antibodies, preferably Fab fragments.

[0155] In some embodiments, toxicity and therapeutic effect are measured, e.g., by LD 50 (the dose lethal to 50% of the population) and LD 50 The dose (therapeutically effective in 50% of the population) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. In some embodiments, the data obtained from cell culture assays or animal studies can be used in formulating a range of dosage for human use.

[0156] The oligonucleotides or compositions may be used as monotherapy or in combination with other medications, in particular in the treatment of retinitis pigmentosa (RP), Stargardt macular degeneration, age-related macular degeneration (AMD), cystic fibrosis (CF), Hurler syndrome, alpha-1-antitrypsin (A1AT) deficiency, Parkinson's disease, Alzheimer's disease, albinism, amyotrophic lateral sclerosis, asthma, beta-thalassemia, Cadasil syndrome, Charcot-Marie dental disease, chronic obstructive pulmonary disease (COPD), distal spinal muscular atrophy (DSMA), Duchenne / Becker muscular dystrophy, dystrophic epidermolysis bullosa, epidermolysis bullosa, Fabry disease, factor V Leiden-related disease, familial adenomatous polyposis, polyposis, galactosemia. In some embodiments, the present invention may be administered in combination with a pharmaceutical agent suitable for the treatment or prevention of: inflammatory bowel disease (IBD), Gaucher disease, glucose-6-phosphate dehydrogenase (GPD), hemophilia, hereditary hematochromatosis, Hunter syndrome, Huntington's disease, inflammatory bowel disease (IBD), hereditary polymorphic aggregation syndrome (PAS), Leber congenital amaurosis, Lesch-Nyhan syndrome, Lynch syndrome, Marfan syndrome, mucopolysaccharidoses, muscular dystrophies, myotonic dystrophy types I and II, neurofibromatosis, Niemann-Pick disease types A, B, and C, NY-eso1-associated cancer, Peutz-Jeghers syndrome, phenylketonuria, Pompe disease, primary ciliary body disease, prothrombin mutation-associated disease, and / or symptoms associated therewith. In a preferred embodiment, the oligonucleotide or composition may be administered as a monotherapy or in combination with additional medications for the treatment of any retinal disease, including, for example, inherited retinal diseases such as retinitis pigmentosa (RP), choroidopathy, Stargardt's disease, cone-rod dystrophy and / or Leber's congenital amaurosis (LCA).

[0157] In some embodiments, the provided oligonucleotide or composition is surprisingly effective compared to a reference oligonucleotide or composition. In some embodiments, the change is measured by an increase in the level of the desired mRNA and / or protein compared to a reference condition. In some embodiments, the change is measured by an increase or decrease in the editing effect mediated by the oligonucleotide or a composition comprising the same. In some embodiments, the change is measured by an increase in the stability of the oligonucleotide or a composition comprising the same.

[0158] Further provided herein is the method of targeting adenosine.Specifically provided herein is the method of targeting wild-type adenosine for beneficial and / or compensatory RNA editing.Provided herein is the method of targeting wild-type adenosine for beneficial editing.Provided herein is the method of targeting wild-type adenosine for compensatory editing.

[0159] Also provided herein is a method for treating a subject suffering from a genetic disorder, condition or disease, the method comprising administering to a subject in need thereof an effective amount of the chemically modified oligonucleotide of the present invention or the composition of the present invention.In one embodiment, the genetic disorder, condition or disease is associated with G-to-A mutation.Treating the disorder associated with G-to-A mutation can result in a reduction in the mortality rate of the treated subject population compared to the untreated population.

[0160] Provided herein is the use of the oligonucleotide of the present invention in therapy. Also provided herein is the use of the oligonucleotide of the present invention in the manufacture of a medicament for treating a condition, disorder or disease associated with G-to-A mutation. Also provided herein is the use of the oligonucleotide of the present invention in the manufacture of a medicament for treating a genetic disorder, condition or disease associated with G-to-A mutation. Also provided herein is the use of the oligonucleotide of the present invention in the manufacture of a medicament for treating a genetic disorder, condition or disease associated with G-to-A mutation.

[0161] The compositions of the present disclosure can be administered in many ways, depending on whether local or systemic treatment is desired and on the site to be treated. Administration can be by inhalation (e.g., via nebulization), nasal, oral, intravenous, subcutaneous, intracranial, intramuscular, intratracheal, intraperitoneal, or rectal injection or infusion, or by direct injection into a tumor or the like. Administration can be in solid form, powder form, tablet form, or any other form compatible with pharmaceutical use in humans. In some embodiments, the oligonucleotide construct can be delivered systemically. Number of patients

[0162] The oligonucleotide of the present invention and the composition comprising it can be administered to various groups of subjects or patients.In some embodiments, the patient needs treatment.In other embodiments, the patient does not need treatment (" beneficial editing "), that is, the subject receives the oligonucleotide or composition for editing the RNA from wild-type allele (not mutant allele), for example, to regulate the function of wild-type protein in a way that is useful for preventing or treating disease.

[0163] In some embodiments, the oligonucleotides or compositions comprising the oligonucleotides described herein are administered to naive subjects, i.e., subjects who do not have a disease or disorder. In one embodiment, the oligonucleotides or compositions comprising the oligonucleotides described herein are administered to a subject. In one embodiment, the oligonucleotides or compositions provided herein are administered to naive subjects who are at risk of developing a disease or disorder.

[0164] In certain embodiments, the oligonucleotides or compositions comprising the oligonucleotides described herein are administered to a patient diagnosed with a disease or disorder. In some embodiments, the oligonucleotides or compositions comprising the oligonucleotides described herein are administered to a patient before symptoms appear or before symptoms become severe.

[0165] In some embodiments, the oligonucleotide or the composition comprising the oligonucleotide described herein is administered to humans.In some embodiments, the human subject that is administered the oligonucleotide or the composition comprising the oligonucleotide described herein is any individual that is at risk of developing the disease or disorder associated with G-to-A mutation in genes.In one embodiment, the patient suffers from the disease or disorder associated with G-to-A mutation in genes.

[0166] In some embodiments, subjects or patients suitable for treatment of a condition, disorder, or disease associated with G-to-A mutation can be identified or diagnosed by a medical professional. In some embodiments, the symptoms of a condition, disorder, or disease associated with G-to-A mutation can be any condition, disorder, or disease that can benefit from A-to-I conversion.

[0167] Also provided herein is the method for treating the condition, disorder or disease associated with G-to-A mutation in subject.In some embodiments, the method of the present disclosure can be for treating the condition, disorder or disease associated with G-to-A mutation in subject, wherein the method comprises administering to subject a therapeutically effective amount of oligonucleotide or its pharmaceutical composition.

[0168] Also provided herein is the use of the oligonucleotide of the present invention in treatment.Also provided herein is the use of the oligonucleotide of the present invention in the manufacture of a medicament for treating a condition, disease and / or disorder associated with G-to-A mutation in a subject.Also provided herein is the use of the oligonucleotide of the present invention in the manufacture of a medicament for treating a condition, disease and / or disorder associated with G-to-A mutation.In one embodiment, the use of the oligonucleotide of the present invention is in the manufacture of a medicament for treating a condition, disease and / or disorder associated with G-to-A mutation.

[0169] The composition of the invention comprises an oligonucleotide of the invention. In a further aspect, the invention relates to a kit or kit-of-parts comprising an oligonucleotide of the invention and / or a (pharmaceutical) composition according to the invention. The kit further comprises instructions for use. How to edit

[0170] The present invention also relates to methods for editing a target adenosine in a target nucleic acid. For example, the present invention provides methods for editing a SERPINA1 polynucleotide, e.g., a SERPINA1 polynucleotide containing a single nucleotide polymorphism (SNP) associated with alpha I antitrypsin deficiency. Furthermore, the present invention relates to in vitro methods for editing a target adenosine in a target nucleic acid and for deaminating at least one specific adenosine present in a target RNA sequence in a cell.

[0171] In one aspect herein, there is provided an in vitro method for editing a target adenosine in a target nucleic acid, the method comprising contacting the target nucleic acid with an oligonucleotide of the invention.

[0172] In another aspect herein, there is provided an in vitro method for deaminating at least one specific adenosine present in a target RNA sequence in a cell, wherein the method comprises the steps of: (a) contacting a target nucleic acid with a chemically modified oligonucleotide of the present invention; (b) enabling the chemically modified oligonucleotide to be taken up into cells; (c) allowing the chemically modified oligonucleotide to anneal to the target RNA sequence; and (d) enabling a mammalian ADAR enzyme containing a naturally occurring dsRNA-binding domain found in a wild-type enzyme to deaminate a target adenosine in said target RNA sequence to inosine.

[0173] In one embodiment, the method comprises, after step (d), identifying the presence of inosine in the RNA sequence.

[0174] The editing reaction is preferably monitored or controlled by sequence analysis of the target RNA.

[0175] In addition, the chemically modified oligonucleotide or (pharmaceutical) composition of the present invention can be used for diagnosing genetic conditions, diseases or disorders. Wherein, the disease or disorder is preferably selected from the group consisting of infectious diseases, tumor diseases, cardiovascular diseases, autoimmune diseases, allergies and neurological diseases or disorders. In one embodiment, the genetic disorder, condition or disease is associated with G-to-A mutation.

[0176] The present invention uses an oligonucleotide that allows adenosine deaminase (ADAR) acting on RNA to convert adenosine to inosine, and is used to create desired changes in target sequences in cells or subjects through site-specific editing of nucleotides.As a result, the target sequence is edited through the adenosine deamination reaction mediated by ADAR, converting adenosine to inosine.In some embodiments, since I is recognized as G, deamination corrects the pathogenic mutation in the SERPINA1 gene, reverting the E342K mutation to wild type, and reversing or delaying the symptoms associated with A1AD experienced by patients.

[0177] The methods of the present invention can be used with cells from any organ, including, for example, skin, lung, heart, kidney, liver, pancreas, intestine, muscle, gland, eye, brain, and blood. The present invention is particularly suited to modifying cell, tissue, or organ arrays involved in disease states in (human) subjects. For example, such cells include, but are not limited to, hepatocytes, hepatocyte-like cells, and / or alveolar type II cells, neurons (PNS, CNS), retina, photoreceptor cells, Müller glia cells, RPE, immune cells, B cells, T cells, dendritic cells, macrophages, and the like.

[0178] The invention is explained in more detail by the following figures and examples. [Example]

[0179] The following examples are merely illustrative and provide a more detailed description of the present invention. These examples should not be construed as limiting the present invention. The sequences disclosed herein are also shown in the enclosed sequence listing. However, the sequence listing only shows the nucleotide sequence, and nucleotide modifications and inter-nucleotide bonds are not shown in the sequence listing. Modifications related to the sequences are partially disclosed in the following table and in the figures of this application.

[0180] For all experiments, editing efficiency is expressed as the percentage [%] of edited target sites observed among all detected target sites in the target transcript. Example 1. Editing efficacy and lysosomal stability of oligonucleotides targeting SERPINA1 E342K that increase DNA to RNA (DNA:RNA) ratio.

[0181] To investigate the effects of various DNA and RNA oligonucleotides on lysosomal stability, different versions of the construct targeting SERPINA1 E342K (v117.26 to v117.58) were generated and tested in vitro. The various ASOs differed in DNA and RNA content and are listed in Table 1. The results of Example 1 are shown in Figure 1. v117.26 served as a control.

[0182] To assess the effect on cells, 2.5x10 4HeLa cells (Cat. No.: ATCC CCL-2) were seeded in 24-well plates. 24 hours later, cells were forward transfected with a plasmid containing the human SERPINA1 E342K mutant cDNA. 300 ng of plasmid and 0.9 μl of FuGENE® 6 (Promega) were diluted in 50 μl of Opti-MEM and incubated for 5 minutes. After incubation, the mixture was combined and incubated for an additional 20 minutes. The medium was replaced, and the transfection mixture was evenly distributed per well. 24 hours after plasmid transfection, cells were forward transfected with 5 pmol of construct / well and 1.5 μl / well of Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific). 24 hours later, the medium was replaced. 48 hours after transfection, cells were harvested for RNA isolation and sequencing. Lysosomal degradation assays were performed using mixed-gender rat liver tritosomes (tebu-bio, product number 098R0610.LT). The tritosome solution was diluted with 20 mM Na-citrate solution (pH 5.0) to an acid phosphatase concentration of 0.1115 U / ml. For the experiment, 15 pmol of each ASO was diluted in the 0.1115 U / ml acid phosphatase tritosome solution. A mock sample contained 15 pmol of ASO diluted in PBS alone. All samples were incubated at 37°C for the indicated time, then frozen in liquid nitrogen and immediately stored at -80°C. Oligonucleotide degradation was visualized by denaturing urea-PAGE. Denaturation was completed by adding 7 μl of RNA loading dye (1:10 dilution of Rotiphorese® Sequencing Gel Buffer Concentrate in Rotiphorese® Sequencing Gel Diluent, Carl Roth) to each sample before incubating at 70°C for 2 min. Denatured samples were then loaded onto a urea (7 M) polyacrylamide (15%) electrophoresis (PAGE) gel and run at 1200 V for 4-6 h in 1x TBE (Tris-borate-EDTA) buffer.Bands were visualized using SYBR™ Gold Nucleic Acid Gel Stain (ThermoFisher Scientific) according to the manufacturer's instructions and analyzed using a Fujifilm FLA-5100 Fluorescent Image Analyzer at an excitation wavelength of λ. ex Scanning was performed at 473 nm. Half-lives were quantified by integrating the grayscale values ​​of individual full-length ASO bands using ImageJ. Construct half-lives were calculated relative to mock samples. [Table 2] JPEG2025541721000020.jpg169169

[0183] As shown in Figure 1A, incorporating more DNA into the oligonucleotide backbone increased the DNA:RNA ratio and resulted in an overall decrease in the editing yield of SERPINA1 E342K. Two control constructs, v117.26 and v117.39 (5.1% overall DNA), were used. These ASOs differ from each other, with v117.39 differing from v117.26 in that it contains additional phosphorothioate linkages at positions d, e, and f (Table 1). The addition of PS linkages to the CBT alone significantly reduced the lysosomal half-life (t) of the ASO. 50 The lysosomal half-life could be improved from 2 hours (v117.26) to 5 hours (v117.39). However, a longer lysosomal half-life would be more desirable from a therapeutic perspective. ASO v117.42 (39% DNA), in which all remaining RNA nucleosides were replaced with DNA, showed lower editing yields compared to the v117.26 control construct, which carried no DNA outside the CBT. Interestingly, constructs with a higher DNA:RNA ratio in these scaffolds (e.g., v117.53 (33.9%) to v117.58 (30.5%)) showed lower levels of RNA editing compared to constructs with a lower DNA:RNA ratio (e.g., v117.43 (23.7%) to v117.46 (22%)).

[0184] Furthermore, incorporating more DNA into the ASO backbone, increasing the DNA:RNA ratio of the ASO, increased its lysosomal stability (Figure 1B). This stability peaked when all remaining RNA nucleosides were replaced with DNA: ASO v117.42 (control DNA backbone; 39% DNA) showed the highest stability, with a half-life of over 31 days (t 50 Constructs v117.49 and v117.53 can tolerate 10% fetal bovine serum (FBS) and have a short half-life (t 50 ) showed high lysosomal stability up to about 7 days (7d) after 6 days. On the other hand, v117.39, which was not chemically modified at most 2'-hydroxyl groups of the nucleoside sugar moiety, was rapidly degraded by 6 hours (t 50 = 5 hours). The stability of both ASOs against degradation (t 50 ) improved compared to v117.39, but the editing efficiency decreased to 39.6% and 30.9%, respectively (see Figure 1A and Figure 1B).

[0185] Overall, these data indicate that in oligonucleotides containing 2'-OMe- and 2'-F-modifications at positions +2 and +3, respectively, where h and i are not PS-linked and no more than six consecutive nucleotides have the same 2'-modification, increasing the amount of DNA outside the CBT (DNA:RNA ratio) tends to decrease editing efficacy. At the same time, increasing the DNA:RNA ratio improves lysosomal stability and subsequently resistance to serum RNase degradation, thus extending the lifespan of different constructs in serum. However, introducing a very high degree of DNA hinders editing efficiency. Nevertheless, potentially useful embodiments can be created that contain significant amounts of 2'-unmodified ribonucleotides, which have a reasonable balance of stability versus editing efficiency, and while some 2'-F and 2'-O-methyl are usually necessary to stabilize ASOs (e.g., at least 10% of all nucleosides are 2'-F and at least 10% are 2'-O-methyl modified), the (potentially toxic) 2'-F content (e.g., 20% in v117.53) and 2'-O-methyl content (e.g., about 35% in v117.49) are relatively low. However, for many applications, further stabilization is desirable. Therefore, without being bound by any particular theory, the inventors believe that there must be an optimal balance of the mixture of 2'-F, 2'-OMe-nucleoside modifications, PS linkage modifications, and target editing efficacy and lysosomal stability of the oligonucleotide, which can be achieved by replacing RNA nucleosides with DNA nucleosides. Example 2. Editing Effect of Oligonucleotides Targeting SERPINA1 E342K with 2'-F- and 2'-OMe-Modifications and DNA Mixtures

[0186] To investigate the stabilizing effects of 2'-F- and 2'-OMe backbone modifications on and in combination with DNA content in more detail, different oligonucleotides were generated and assayed for RNA editing effects in vitro according to the protocol described in Example 1. The different ASO constructs tested are listed in Table 2, and the results are shown in Figure 2. As described for Example 1, v117.26 and v117.39 were used as controls.

[0187] As shown in Figure 2A, high levels of 2'-F-modifications are associated with reduced editing efficacy. Oligonucleotides containing high levels of DNA content (v117.42; 29.3%, containing 39% DNA) or 2'-F-modified nucleosides (v117.62; 7.2%, containing 58% 2'-F) showed significantly reduced editing efficacy. On the other hand, constructs containing more balanced amounts of 2'-F-modifications and DNA showed RNA editing efficiencies ranging from approximately 37.5% (v117.60) to 58.9% (v117.59), which was comparable to the RNA editing efficacy of the control. These data confirm that a balanced mix of 2'-F, 2'-OMe, and 2'-H modifications can be used to compensate for the replacement of 2'-OH modifications, and that an excess of a single type of modification, i.e., 2'-F (v117.62) or 2'-H (v117.42), has a detrimental effect on overall RNA editing. A certain preference for 2'-F over DNA was also observed. 2'-F-rich embodiments generally performed better than DNA-rich embodiments. For example, v117.65 (55.4% edited, 42% 2'-F, and 20% DNA) compared to v117.60 (37.5% edited, 32% 2'-F, and 31% DNA). Also, although containing 39% DNA and only 24% 2'-F, v117.42 still achieved 29.3% editing efficiency, which may be sufficient when combined with desirably high lysosomal stability (see Example 1). The 2'-OMe content of all embodiments remained between 32 and 37% (e.g., v117.99 and v117.59, respectively).

[0188] To further determine whether the 2'-sugar modifications outside the CBT and end-block termini are position-dependent or position-independent of the nature of the nucleotide (purine vs. pyrimidine), ASOs v117.99 and v117.100, both of which contain equal amounts of 2'-F-, 2'-OMe-, and 2'-H-modifications overall (25.4% DNA, 42.4% 2'-F, 32.2% 2'-OMe), were evaluated for RNA editing efficacy. The positions of all 2'-sugar modifications (2'-F, 2'-OMe, and DNA) were determined by the positions of the two termini ("end-blocks"), the CBT, and the N of the extension hotspot. +2 and N +3 Apart from the three modifications at each position (see, for example, Example 14 for extended hotspots), they were randomized. The modifications of the binding backbone were unchanged compared to all other embodiments. The results were then compared with the parent reference sequence v117.59. As shown in Figure 2A, v117.99 and v177.100 showed good editing (39.2% and 48.1%, respectively), indicating that the RNA editing effect is not significantly affected by the relative positioning of the 2'-modification. [Table 3] JPEG2025541721000022.jpg139169

[0189] In summary, the present data demonstrate that high levels of only one type of 2'-modification (e.g., high levels of 2'-F- or 2'-H-modification) have a detrimental effect on the overall editing efficiency of an oligonucleotide (see v117.42 and v117.62). In general, the most highly performing long (40 nt or longer, here 59 nt) embodiments (achieving greater than 45% editing) contained greater than 35% 2'-F-modifications and no more than 25% DNA content, indicating that the amount of 2'-modification is preferred. However, a certain tolerance for more DNA (up to about 40%) and fewer 2'-F-modifications (minimum about 25%) can be observed, which may depend on other factors, such as the nucleic acid sequence of the embodiment. The data also demonstrate that the overall amount of a particular 2'-modification has a stronger effect on the corresponding editing yield than the precise location of each 2'-modification within the oligonucleotide. Example 3. Editing Effect and Lysosomal Stabilization of Oligonucleotides Targeting STAT1 Y701

[0190] Similar to the evaluation of the RNA editing efficacy and lysosomal stability of ASOs targeting SERPINA1 E342K, the editing efficacy and lysosomal stability were determined for ASOs targeting various human STAT1 Y701. The different ASO construct sequences and their respective modifications are listed in Table 3. The results are shown in Figure 3. Constructs v117.28 and v117.29 (with 2'-F- and 2'-OMe-modifications but no 2'-H outside the CBT) were used as controls.

[0191] To evaluate the effect of cells, 1 x 10 5 HeLa cells were forward transfected using 25 pmol of construct and 1.5 μl of Lipofectamine RNAiMAX reagent (ThermoFisher Scientific) per 24-well according to the Lipofectamine RNAiMAX reagent protocol. After 24 hours, RNA was isolated and sequenced by Sanger sequencing. Lysosomal degradation assays were performed as described in Example 1.

[0192] This data shows a similar trend to that observed with the SERPINA1 E342K-specific oligonucleotides. Replacing 2'-OH groups (RNA) with 2'-H groups (DNA) and subsequently increasing the DNA:RNA ratio resulted in a general decrease in editing efficiency, reaching 2% for v117.30 and 2.3% for v117.31 (containing 44% and 22% DNA, respectively) (Figure 3A). Similarly, the inclusion of high levels of 2'-F modifications led to lower levels of RNA editing (v117.36 and v117.41 contained 41% and 63% 2'-F modifications, respectively). Similarly, v117.39, which has a relatively high 2'-H content compared to v117.83, for example, showed only an 11% editing yield. On the other hand, ASOs containing a combination of 2'-F, 2'-OMe, and 2'-H modifications showed enhanced editing ability. However, these embodiments performed similarly to the control only when they contained approximately twice the amount of 2'-F-modifications compared to DNA, as shown in construct v117.40 (containing 49.2% 2'-F, 18.6% DNA, and 32.2% 2'-OMe). V117.40 exhibited significantly improved editing efficiency (35.8%) and lysosomal stability (t) compared to v117.37. 50 >7d vs t 50 = 7d) (Figures 3A and B). [Table 4]

[0193] Similar to above, the data show that when 2'-F and 2'-OMe-modifications are combined with the 2'-H group in the ASO sugar moiety, it results in a 2'-O-alkyl-modification at the +2 position (N +2 ) and the 2'-F-modification is at the +3 position (N +3), suggesting that optimal editing efficiency is achieved when the ASO contains no more than six consecutive nucleotides with the same 2'-modification. While specific combinations of 2'-modifications resulted in the highest editing efficiency, there was a tendency to preferentially contain more 2'-F-modified nucleosides than DNA (v117.40 had 35.8% editing efficiency compared to v117.39, which had 11% editing efficiency). Furthermore, constructs targeting STAT1 Y701 tolerated higher 2'-F content (up to approximately 50% in v117.40) and less DNA (approximately 19% in v117.40) than constructs targeting SERPINA1 E342K.

[0194] Overall, this data supports the results observed for ASO targeting SERPINA1 E342K, showing that it should contain a certain level of 2'-F and 2'-OMe-modification, and should avoid blocks of consecutive uniform sugar modifications.In all embodiments that do not contain natural RNA, there is no block of more than 6 consecutive 2'-F, 2'-O-Me or DNA nucleosides.More importantly, these data show that specific modification patterns can be transferred to different targets (for example, STAT1 Y701 v117.37 pattern is taken from SERPINA1 E342K v117.59).In other words, although common modification patterns can be transferred to a suitable extent, these patterns may still require target-specific adaptation to ensure optimal editing ability of ASO. Example 4. Editing Effect and Lysosomal Stabilization of Oligonucleotides Targeting CRB1 C948Y

[0195] To further determine the combined effects of 2'-F-, 2'-OMe-, and 2'-H-modifications on the editing and stabilization of other gene targets, oligonucleotides targeting different human CRB1 C948Y were generated and tested. The different construct sequences and their modifications are shown in Table 4. The results of Example 4 are shown in Figure 4. Construct v117.20, which does not have 2'-H (DNA) outside the CBT, served as a control.

[0196] The effect of cells was evaluated by injecting 300 ng of plasmid (containing CRB1 C948Y cDNA) and 0.9 μl of FuGENE® 6 into 5 x 10 cells seeded in 24 wells 24 hours prior to transfection. 4 The constructs were evaluated by forward transfection into HeLa cells. The medium was changed every 24 hours. 24 hours after plasmid transfection, forward transfection was performed with 25 pmol of the construct in 1.5 μl of Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer's protocol. After 24 hours of incubation, cells were harvested for RNA isolation and Sanger sequencing. Lysosomal degradation assays were performed as described in Example 1. [Table 5]

[0197] As shown in Figure 4A, replacing some of the 2'OH groups in v117.20 with 2'H groups slightly reduced the editing efficiency of the ASO from 50.8% (v117.20, containing 5.1% DNA and 22% 2'-F) to 32.5% (v117.22, containing 25.4% DNA), but still maintained good editing. A similar observation was observed when replacing the 2'OH groups in v117.20 with 2'-F modifications (v117.23; 39.5%, with the 2'-F level increasing to 42.4%). Notably, the introduction of a mixture of 2'-F-, 2'-OMe-, and 2'-H modifications led to similar editing efficiencies (v117.24 and v117.25; containing 40.7% 2'-F, 22% DNA, 37.3% 2'-OMe, and 44.1% 2'-F, 25.4% DNA, 30.5% 2'-OMe, respectively). However, with regard to lysosomal stabilization, the introduction of a mixture of 2'-F-, 2'-OMe-, and 2'-H-modifications was observed to lead to an overall improvement. Construct v117.20 showed a 5-hour t 50 However, v117.24 has t 50 A SOA stability of >7 days was achieved, demonstrating that replacing all natural RNA nucleosides with a mix of 2'-F / 2'-OMe / DNA promotes ASO stabilization (Figure 4A).

[0198] These data again demonstrate that a balanced mix of 2'-F and 2'-OMe modifications (approximately 40-45% 2'-F, 22-25% DNA, and 31% 2'-OMe), avoiding blocks of consecutive uniform sugar modifications, confers efficient editing yields and lysosomal stabilization. Also, as already observed in Example 3, the length of uniform 2'-modification blocks should not exceed 6 nt, or even 3 nt. Furthermore, these data again suggest that this combination of 2'-modifications can be applied to different oligonucleotide sequences and can therefore be used regardless of the actual target (e.g., the CRB1 C948Y v117.24 pattern is derived from SERPINA1 E342K v117.59). Example 5. Editing Efficacy and Lysosomal Stability of Oligonucleotides Targeting LRRK2 G2019S

[0199] Several mutations in leucine-rich repeat kinase-2 (LRRK2) are associated with Parkinson's disease (PD), among which G2019S is a highly prominent mutation. To investigate the effects of 2'-F, 2'-OMe, and 2'-H-backbone modifications on oligonucleotides targeting LRRK2 G2019S, different modified constructs were prepared and tested for in vitro editing efficacy and lysosomal stability. The different human LRRK2 G2019S-targeting constructs used in Example 5 are listed in Table 5. The results are shown in Figure 5.

[0200] To evaluate the effect of cells, 5x10 4 HeLa cells were seeded in 24-well plates. 24 hours later, cells were forward transfected with 300 ng of a plasmid containing the human LRRK2 G2019S mutant cDNA and 0.9 μl of FuGENE® 6. 24 hours after plasmid transfection, 25 pmol of each construct was forward transfected in 1.5 μl of Lipofectamine RNAiMAX Reagent. After 24 hours of incubation, RNA was isolated and cells were harvested for Sanger sequencing. Lysosomal degradation assays were performed as described in Example 1. [Table 6]

[0201] As shown in Figure 5A, backbone modification (49.2% DNA, 16.9% 2'-F, 33.9% 2'-OMe) that simply replaced the 2'-OH group in v117.20 with a 2'-H group in v117.42 caused a strong decrease in editing efficiency (editing yield less than 10%). However, oligonucleotides combining 2'-F, 2'-OMe, and 2'-H modifications were able to restore the editing efficiency to that of the v117.20 construct. For example, v117.59, which contains a mixture of 42.4% 2'-F, 30.5% 2-OMe, and 27.1% 2'-H modifications, had an editing efficiency of around 40%. This editing efficiency was similar to that of v117.20. Indeed, v117.59 exhibited improved lysosomal stability (t) in addition to its normal editing effect. 50 Similarly, v117.60, which contained a mixture of 40.7% 2'-F, 37.3% 2-OMe, and 22% 2'-H modifications outside the CBT, not only showed a more stable editing effect compared to v117.20, but also improved lysosomal stability (t 50 >7 days) was also shown (Figure 5B).

[0202] These data support the observations made with constructs targeting SERPINA1 E342K (Example 2), STAT1 Y701 (Example 3), and CRB1 C948Y (Example 4), which indicate that certain levels of 2'-F, 2'-OMe, and 2'-H modifications should be included in the oligonucleotide backbone to provide good editing efficacy and lysosomal stability. As already observed in Example 3, the length of uniform 2'-modified blocks does not exceed 6 nt, or even 4 nt. As already observed in Example 2, embodiments including high levels of DNA (nearly 50% in LRRK2 G2019S v117.42) combined with low levels of 2'-F (just over 15% in LRRK2 G2019S v117.42) were shown to strongly enhance lysosomal stability but impair editing efficacy. Preferably, embodiments targeting LRRK2 G2019S contain approximately 40% 2'-F, approximately 25% DNA, and approximately 35% 2'-OMe. These data also demonstrate that this combination of 2'-modifications is applicable to different oligonucleotide sequences and can therefore be used regardless of the actual target (see LRRK2 G2019S v117.60 and SERPINA1 E342K v117.59).

[0203] This demonstrates that modification patterns can be transferred in a position-specific manner regardless of the target sequence. Although transferability between embodiments for SERPINA1 E342K and LRRK2 G2019S appears to be high, the corresponding embodiments may still require further target-specific adaptation to provide optimal editing effects to ASOs. Example 6. Editing effect of oligonucleotides targeting mouse PDE6A (mPDE6A) V685M

[0204] Mutations in the PDE6A gene can cause rod photoreceptor degeneration and are associated with retinitis pigmentosa (RP), a blinding disease (Sothilingham et al., 2015). RP animal models include the Pde6a V685M mutant mouse model. Therefore, to determine the effects of 2'-F, 2'-OMe, and 2'-H modifications on ASOs targeting mouse PDE6A (mPDE6A), constructs containing such modifications were prepared and the editing effects were tested in vitro. The different constructs used in Example 6 are listed in Table 6. The results are shown in Figure 6A. [Table 7]

[0205] Construct v117.21 contains a mixture of 2'-F, 2'-OMe, and 2'-OH (RNA) modifications (28.8% 2'F, 39% 2'OMe, and 27.1% 2'OH nucleoside modifications). This result demonstrates a stable editing efficiency of around 19%. Replacing the 2'-OH groups with 2-H groups and / or introducing 2'-F and 2'-OMe modifications yielded v117.27 (40.7% 2'F, 37.3% 2'OMe, and 22% 2'H), which showed similar editing efficiency.

[0206] These data support the above observations, which show that the combination of 2'-F and 2'-OMe-modifications with 2'-H groups can provide stable ASOs without adversely affecting their editing efficacy. As already observed in the above examples, the length of the uniform 2'-OH modified blocks does not exceed 6 nt, or even 3 nt. Example 7. Editing effect of oligonucleotides targeting NLRP3 Y166

[0207] The NLRP3 (nucleotide-binding domain, leucine-rich repeat containing family, pyrin domain containing-3) inflammasome has been reported to play an important role in retinal neurodegeneration and many other diseases. NLRP3 is involved in inflammasome formation, which is associated with various diseases, including inflammation, aging, heart and vascular disease, metabolic syndrome, gout, and autoimmune diseases. Once activated, it often contributes to the prolongation of disease duration. The target site, Y166, is a phosphorylation site essential for NLRP3 activation (Bittner et al., 2021). Through beneficial RNA editing, the phosphorylation site can be blocked, preventing NLRP3 from being activated or from being activated efficiently. Therefore, the editing effect of oligonucleotides targeting human NLRP3 Y166 was tested using a plasmid expression system (A) and genome-integrated oligonucleotide sequences (B). The sequences and modifications of the different NLRP3 Y166-targeting constructs used in Example 7 are listed in Table 7. The results are shown in Figure 7. "No ASO" was used as a negative control.

[0208] Plasmid-based approach (A): 5x10 4 HeLa cells (Cat. No.: ATCC CCL-2) were seeded in 24-well plates and transfected with 300 ng of wild-type NLRP3 cDNA containing plasmid and 0.9 μl of FuGENE® 6 (Promega). 24 hours after transfection, forward transfection was performed with 25 pmol of the construct in 1.5 μl of Lipofectamine RNAiMAX Reagent. After 24 hours of incubation at 37°C, cells were harvested for RNA isolation and Sanger sequencing.

[0209] Genome integration approach (B): 1x10 human wild-type NLRP3-mNeonGreen cDNA was stably integrated into the genome via the piggyBac transposase system. 5 HeLa cells were seeded in 24-well plates. 24 hours after seeding, each well was forward transfected with 25 pmol of construct and 1.5 μl of RNAiMAX. After 24 hours of incubation at 37°C, cells were harvested for RNA isolation and Sanger sequencing. [Table 8]

[0210] As shown in Figures 7A and 7B, no significant differences were observed between the plasmid-based approach (A) and the genome-integrated approach (B). Overall, the combination of 40.7% 2'-F, 37.3% 2'-OMe, and 22% 2'-H modifications was able to stabilize and maintain efficient editing yields (v117.20), demonstrating that this stabilization and maintenance was achieved without consecutive (≤6 nt) blocks of 2'-modifications (i.e., without any 2'-F, 2'-O-methyl, or DNA). Example 8. Adaptation of the lengths of the 5' and 3' ends of oligonucleotides targeting the GAPDH 3' UTR

[0211] Depending on the specific ASO type and / or sequence, and without being bound by any theory, we believe there is an optimal ASO length at which hybridization strength is optimal and ADAR utilization is at least sufficient for editing. Furthermore, shorter ASOs are expected to facilitate delivery (e.g., escape from endosomes), reduce the risk of aggregation, toxicity, and / or immunogenicity, and facilitate large-scale production and screening of such ASOs. Using the artificial SNAP-ADAR approach, we previously demonstrated that guide RNAs can be easily shortened to 14 nt while maintaining substrate duplex recognition and editing by the ADAR deaminase domain (data not shown). These latter findings differ from prior art, in which recruitment of endogenous ADARs to endogenous substrates was mediated by block-design ASOs with stereochemically pure PS / PN-modified backbones and lengths of 30–33 nt (Monian et al., 2022).

[0212] To determine the possible length range of ADAR-recruiting oligonucleotides, four embodiments targeting the 3'UTR of human GAPDH were evaluated for editing efficacy in vitro (see v121.10 to v121.13). The corresponding results from Example 8 are shown in Figure 8. The different ASO constructs and their modifications are shown in Table 8.

[0213] To evaluate the effect on cells, 1 x 10 cells were transfected according to the Lipofectamine RNAiMAX Reagent protocol. 5 HeLa cells were forward transfected with 25 pmol of construct per well and 1.5 μl of Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific). After 24 hours, RNA was isolated and sequenced by Sanger sequencing. [Table 9]

[0214] Overall, the dataset shows that efficient editing is possible even for very short embodiments (e.g., down to at least 25 nt). Example 9. Adaptation of the 5' and 3' end lengths of oligonucleotides targeting SERPINA1 E342K

[0215] To determine the significance and role of the 5' and 3' ends in RNA editing, several truncated versions of oligonucleotides targeting human SERPINA1 E342K were generated and their editing effects evaluated in vitro (see v117.80 to v117.83). The corresponding results in Example 9 are shown in Figure 9. Various 5'- and / or 3'-truncated ASO constructs and their modifications are listed in Table 9. A 31-nt-long oligonucleotide [Block Design_31nt] and a 40-nt-long oligonucleotide [Block Design_40nt] served as negative controls. [Table 10]

[0216] First, different 3'-truncated ASO constructs were tested for their ability to maintain RNA editing efficacy (Figure 9). Starting with a symmetric 59-nt ASO (v117.59), we sequentially truncated the ASO from its 3' end (while maintaining the 1-3x2'-OMe-modification at the 3' end) to obtain 3'-truncated ASOs of various lengths (v117.80 to v117.83) (Figure 9B). As shown in Figure 9A, shortening the 3' end of v117.59 to 50 nt (v117.80) or 45 nt (v117.81) did not alter the editing yield. v117.82 (40 nt) maintained its ability to efficiently edit SERPINA targets, albeit with a slight decrease. Most importantly, constructs v117.81 and v117.80 showed a slight increase in editing efficacy compared to v117.59 (Figure 9A).

[0217] However, simultaneous truncation of the 5' and 3' ends significantly reduced the editing effect (v117.83; 31 nt) (Figure 9A), near the detection limit of Sanger sequencing. While shortening the 3' end to 40 nt only slightly reduced the editing effect, shortening the 3' end, and especially the 5' end, of the ASO down to 31 nt (v117.83) almost completely eliminated the editing effect (Figure 9A). Without being bound by any theory, we believe that truncation from the 3' end may be better tolerated than truncation from the 5' end.

[0218] As a next step, to further determine the importance of the 5' and 3' ends in controlling the editing effect, we tested additional constructs with different combinations of 5' and 3' truncations. Specifically, to evaluate the editing effect of 5' and 3' double-truncated ASOs, we created different ASOs with one, two, three, four, or five nucleobase deletions at one or both ends. "No ASO" served as a negative control. The constructs targeting SERPINA1 E342K and their modifications are also listed in Table 9. The results are shown in Figure 10.

[0219] The 5'- and 3'-truncated ASOs (v117.83 and block design_31nt) showed reduced editing efficacy overall compared to the full-length constructs (v117.82 and block design_40nt) (Figure 10A). Interestingly, the two 3'-truncated constructs (v117.85 and v117.86) showed only a slight reduction compared to the full-length construct (v117.82), indicating that 3'-truncations are generally well tolerated, especially up to 5 nt outside the CBT. On the other hand, 5'-truncations had a significant impact on the editing efficacy of certain oligonucleotides (v117.87 and v117.88). Surprisingly, truncating the 5' end to just 25 or 24 nucleotides outside the CBT was particularly effective, especially when combined with a 3' end length of 5 nucleotides, resulting in ASOs v117.141 and v117.142 (33 nt and 32 nt long, respectively). As shown in Figure 10, the 5'- and 3'-truncated versions maintained similar editing efficacy to the 3'-truncated ASO.

[0220] Overall, these data support the results presented above, demonstrating that combining 2'-F and 2'-OMe modifications with 2'H groups can provide stable ASOs without adversely affecting editing efficacy. The observed range of 2'-F modifications was 37.5-42.4% (v117.141 and v117.82, respectively), the range of 2'-H modifications was 20-25% (v117.82 and v117.142, respectively), and the range of 2'-OMe modifications was 32.4%-40% (v117.91 and v117.88, respectively). Importantly, the data support the idea that, to obtain efficient editing yields, blocks of consecutive sugar modifications should be avoided and consistent levels of 2'-F and 2'-OMe modifications should be included, as all embodiments contain uniform 2'-modification blocks no longer than 6 nt, or even no longer than 3 nt. Furthermore, the data in Example 9 demonstrate that 3'-end truncation is generally more tolerated than 5'-end truncation. Conclusively, oligonucleotide truncation results in asymmetric positioning of the oligonucleotide around the CBT, for which specific preferred 5'-end and 3'-end lengths exist. That is, 3'-end truncation is tolerated up to a 3' length of about 5 nt of the CBT (e.g., v117.86, v117.141, v117.142), but 5'-end truncation significantly reduces the overall editing effect of the oligonucleotide (e.g., v117.88). Surprisingly, the data obtained from Example 9 show that when optimal 2'-modification, ideal internucleoside modification / PS pattern, and correct truncation are applied to the 5' and 3' ends, very short (33 nt and 32 nt) and highly effective oligonucleotides can be obtained. This was particularly the case for the 5'-24-3-5 symmetry (scheme: 5'-end-CBT-3'-end) of the 32 nt embodiment (v117.142) and the 5'-25-3-5 symmetry of the 33 nt embodiment (v117.141). Finally, the present data demonstrate that a high content of stereo-pure bond modifications is not necessarily required to obtain high editing yields with short, stable ASOs. Example 10. Precision screening of oligonucleotides targeting truncated SERPINA (33nt / 32nt)

[0221] To determine the combined effects of continuous and / or intermittent 2'-F- and 2'-OMe-modifications in combination with DNA on the editing efficacy of short (32nt or 33nt) asymmetric ASOs, several SERPINA-targeting constructs were prepared and used in additional screening experiments. The different 32nt and 33nt ASO designs and their respective backbone modifications are listed in Table 10. The corresponding results are shown in Figure 11. "No ASO" was used as a negative control. [Table 11]

[0222] As shown in Figure 11A, a 33-nt long construct (v117.141) containing a mixture of 42.4% 2'-F-modifications, 33.3% 2'-OMe-modifications, and 24.2% DNA (5 nt outside the CBT) showed better editing (47.3%) than v117.167, a prior art construct containing a contiguous block of 2'-F-modifications at the 5' end and 2'-OMe-modifications located 5' and 3' of the CBT (blocks of ≥ 5 nt) (Figure 11B). Interestingly, blocks of 2'-F- and 2'-OMe-modifications of limited size (e.g., ≤ 5 nt) were very well tolerated in the 33-nt construct (v117.168).

[0223] Similarly, in 32-nt-long constructs, disrupting consecutive 2'-F-regions at the 5' end with 2'-OMe-modifications and DNA led to an overall increase in editing efficiency (see v117.169 vs. v117.142 and v117.170) (Figure 11A). Both v117.168 and v117.170 demonstrate that replacing DNA from shorter (<40 nt) versions with 2'F increases editing yield. In particular, the increased editing efficiency observed with v117.170 suggests that blocks of consecutive 2'-F- and 2'-OMe-modifications of limited size (e.g., ≤6 nt) may be beneficial in short ASOs of 33 nt and 32 nt. Furthermore, we show that short ASOs may benefit from a reduced amount of DNA nucleosides (≤6 nt) outside the CBT (v117.141 vs. v117.168 and v117.142 vs. v117.170). Conclusively, this suggests that a higher 2'-F content (40.6-57.6%, v117.142 and v117.168, respectively) is well tolerated and even preferred for short embodiments (≤50 nt).

[0224] Overall, these findings were similar to the editing effects reported for previously described oligonucleotides (Monian et al., 2022). Without being bound by any theory, we believe that for effective editing, ASOs with a length of 30 nt (or a "sweet spot" around 33 nt / 32 nt) (see the results in Figure 11) depend on specific modification rules: the absence of a uniform block of 2'-F in the 5' half of the ASO (located 5' of the CBT) and the absence of a uniform block of 2'-OMe in the 3' half (located 3' of the CBT). This was also demonstrated in Example 9 for 40 nt reads, where a uniform solution (i.e., consecutive 2'-F modifications) was relatively inefficient in target editing.

[0225] Therefore, we believe that the editing efficacy and stability of oligonucleotides can be optimized by using a combination of chemical modifications. For example, using optimal 2'- and sterically random linkage modification patterns in oligonucleotides with optimized length and asymmetry may enable a large amount of sterically pure linkage modifications (or simply the introduction of sterically pure linkages). However, the present data demonstrate that efficient RNA editing is possible even with chemically modified oligonucleotides that do not contain a large amount of sterically pure internucleoside linkages, provided that the optimal modification pattern (maximum block size, e.g., ≤6 nt) and the optimal amount of 2'-F and 2'-O-alkyl (approximately 35-65% 2'-F and 30-35% 2'-O-alkyl) are present, and a natural and sterically random internucleoside modification pattern (e.g., linkages d and e are modified; linkages h and i are not phosphorothioate) is obtained. While this does not exclude the possibility that a small number of sterically pure linkage modifications (e.g., 10 or fewer, more preferably 5 or fewer) may further improve editing efficiency, it clearly demonstrates that the high level of sterically pure linkage modifications seen in the prior art is not absolutely essential. Furthermore, the present data demonstrate that ASO positioning, specifically the length of the asymmetric end including at least 4 nt 3' of the CBT and / or at least 16 nt 5' of the CBT, in combination with other modification rules, is crucial for obtaining highly efficient editing oligonucleotides of short length (e.g., less than 40 nt). Example 11. Adaptation of the 3' end length of oligonucleotides targeting STAT1 Y701

[0226] To determine the combined effects of 2'-F and 2'-OMe modifications, linkage modifications, and DNA on editing efficacy, similar cleavage experiments were performed on oligonucleotides targeting STAT1 Y701. The specific oligonucleotide construct sequences and their modifications used in Example 11 are shown in Table 12, and the corresponding results are shown in Figure 12. [Table 12]

[0227] A 59-nt ASO construct (v117.29) was used as the basis for introducing a mixture of 2'-F, 2'-OMe, and 2'-H backbone modifications and subsequently generating a 3'-truncated ASO (v117.42) (see Example 3). Construct v117.29, which contained mostly phosphorothioate (PS) linkages and no 2'-H modifications outside the CBT, had a lysosomal stability of 2 hours (data not shown) and an editing efficiency of approximately 38%. This editing efficiency was slightly reduced to 37.4% for construct v117.40 (59 nt), which contained a mixture of 49.2% 2'-F, 32.2% 2'-OMe, and 13.5% DNA (8 nt outside the CBT) modifications outside the CBT (Figure 12A). Furthermore, shortening the 3' end to a total oligonucleotide length of 40 nt (v117.42 and v117.44) improved editing yields compared to the parent construct v117.29, shifting the scope of 2'-OH modifications to 62.5% 2'F (v117.44), 30% 2'O-Me, and 0% DNA outside the CBT. The achieved editing yield (approximately 50%) was maintained even when the oligonucleotides were further shortened to 33 nt (v117.56 and v117.57). These embodiments include 60.6-69.7% 2'F and 9.1-0% DNA (up to 3 nt DNA outside the CBT, v117.56 and v117.57, respectively). Of particular interest is that these short embodiments (≦50 nt) also tolerate relatively low amounts of 2′-OMe-modification (21.2% for v117.56 and v117.57), demonstrating that full 2′-ribose modification through precise patterns of 2′-F- and 2′-OMe-modification, PS linkages, and DNA must be observed if reasonable editing efficacy is to be achieved.

[0228] In general, these findings suggest that a mixture of specific oligonucleotide modifications is necessary to maintain, improve, or restore the editing effect of ASOs. Specifically, this data not only shows that a mixture of 2'-F, 2'-OMe, and 2'-H modifications can be used to stabilize or maintain the editing effect of ASOs, but also shows that subsequently shortening these oligonucleotides to at least 33 nt, including such a mixture of 2'-F, 2'-OMe, and 2'-H modifications and PS linkages, does not adversely affect overall editing efficacy. Therefore, if reasonable editing effects are to be achieved, a mixture of 2'-F, 2'-OMe, and 2'-H modifications and PS linkage modifications is necessary. Furthermore, this data set presents embodiments showing that 2'-F block sizes of up to 6 nt are well tolerated, and that embodiments with shorter ASO lengths (e.g., ≦45 nt) may favor slightly higher 2'-F content (e.g., up to 70%) and benefit from reduced DNA content outside the CBT (to 0 nt). Example 12. Adaptation of the 3' end length of oligonucleotides targeting mCTNNB1 T41

[0229] To further test the hypothesis that a specific, optimal mix of 2'-F-, 2'-OMe-, and 2'-H- and PS-linked modifications is required to provide a stable and effective ASO, and to further validate the results obtained with the ASO targeting 3'-truncated STAT1 (Example 10), similar cleavage studies were performed using oligonucleotides targeting mCTNNB1 T41. Specific construct sequences and their modifications are shown in Table 12. Corresponding results are shown in Figure 13. "No ASO" served as a negative control. [Table 13]

[0230] The different ASOs were based on v117.22, a 59-nt construct containing a mixture of 2'-F- and 2'-OMe-modifications, RNA / DNA, and PS-linked modifications. Three 3'-truncated versions of this construct were generated, each containing a different mixture of 2'-F- and 2'-OM-modifications, RNA / DNA, and PS-linked modifications (Figure 13). Replacement of all natural RNA nucleosides with DNA and additional 2'-F- and 2'-OM-modifications initially reduced editing efficiency (v117.24, 15.2% and v117.25, 12.6%). Interestingly, a 37-nt construct (v117.29) containing 2'-F- and 2'-OMe-modifications, with a high overall DNA content of 21.6% (5 nt outside the CBT), showed 32.7% editing efficiency.

[0231] These data suggest that in 40-nt-long ASOs, a mix of 2'-F- and 2'-OMe-modifications, RNA / DNA content, and PS-linked modifications are important for editing. Notably, for these embodiments, up to six DNA nucleosides were tolerated outside the CBT in short oligonucleotides (≤50 nt, here 37-40 nt). Example 13. Adaptation of 5' and 3' end lengths of oligonucleotides targeting CRB1 C948Y

[0232] Similar to the generation of 5'- and 3'-truncated constructs specific for the SERPINA1 target (Example 8), 5'- and 3'-truncated versions of the CRB1 C948Y-specific oligonucleotide were generated and tested for RNA editing effects in vitro. The different constructs tested in Example 13 are shown in Table 13. The results are shown in Figure 14. "No ASO" served as a negative control. [Table 14]

[0233] As shown in Figures 14A and 14C, a 45-nt-long version (v120.2) containing 2'-F, 2'-OMe, and 2'-H modifications outside the CBT showed favorable RNA editing efficiencies (50.9% and 49.9%). Notably, truncation from the 5' end resulted in a gradual decrease in editing (v117.26 (15%) and v117.28 (18.7%)). Notably, extension of the 3' end led to an overall increase in editing efficiency (v117.24 (37.2%) and v117.25 (38.2%)) (Figure 14A). These data suggest that truncation of the 5' end of the oligonucleotide may have a significant impact on the editing efficiency of ASOs. Furthermore, the data indicate that a higher 2'-F content (>50%) in 40-nt-long ASOs correlates with higher editing values ​​(v117.27, containing 52.5% 2'-F). Notably, all embodiments do not include uniform modification blocks larger than 6 nt.

[0234] To assess whether there is a correlation between ASO length, overall 2' modification pattern, and editing efficacy, we tested shorter versions of the CRB1 C948Y-targeting construct. To do so, we created a set of shorter CRB1 C948Y-targeting constructs (v117.39 to v117.44) (Table 13) and tested them for in vitro editing efficacy. As shown in Figure 14C, editing was reduced at a DNA content of approximately 20% (five DNA nucleosides outside the CBT) in a 40-nt-long ASO (i.e., v117.26). However, by further shortening to a length of 38 nt (v117.44), we discovered a "38-nt sweet spot." For example, we found that for v117.44 (38 nt long), the editing yield increased to approximately 33% compared to the similar 40-nt embodiment v117.27 (editing yield 27.2%). As seen in Example 12, these short embodiments (≦50 nt) also tolerate very low amounts of 2′-OMe (21.2% for v117.40 and v117.41). Furthermore, we observed that certain short (e.g., ≦50 nt) embodiments, in which low levels of DNA (e.g., 3 nt outside the CBT) can improve editing yields compared to pure 2′-F and 2′-OMe versions, e.g., v117.27 (15% DNA / 3 DNA nucleosides outside the CBT) vs. v117.39 (7.5% DNA / no DNA nucleosides outside the CBT) and v117.40 (9.1% DNA / no DNA nucleosides outside the CBT) vs. v117.41 (18.2% DNA / 3 DNA nucleosides outside the CBT).

[0235] Overall, these data confirm that ASOs containing, for example, no more than six consecutive nucleotides with the same modification, as well as a mixture of 2'-F- and 2'-OMe-modifications, have good editing effects, but the data also show that certain levels of DNA may be better than pure 2'-F- and 2'-OMe-modified versions. Example 14. Modulation preference of ASOs targeting short (<45 nt) STAT1 Y701 after truncation

[0236] To determine whether replacing DNA with 2'-OMe-modifications affects RNA editing in a position-dependent manner, various ASOs were constructed and tested for STAT1 Y701 RNA editing effects. The different construct sequences and their modifications are shown in Table 14. The results are shown in Figure 15. "No ASO" served as a negative control. [Table 15]

[0237] The present data demonstrate that replacement of DNA with 2'-OMe-modifications affects editing in a position-dependent manner. As shown in Figure 16A, 2'-OMe-modifications are preferred over DNA close to the CBT (N -9 From N +9, v117.48 vs. v117.47). Furthermore, replacing all four DNA nucleotides outside the CBT (see v117.42) with 2'-F-modifications (as in v117.44) and keeping the largest block ≤6 nt improved the editing yield to a level of 64%, similar to that of construct v117.48 (65.2%) (Figure 16A), demonstrating that 2'-F modifications are generally well tolerated, and even better than the 59-nt ASO (see Figure 3, 17.3% editing efficiency for v117.41). However, 2'-F-modified nucleosides outside the CBT can also be replaced to some extent in short ASOs (≤50 nt) by 2'-OMe and / or DNA nucleosides (e.g., 2 nt of DNA, v117.48, without loss of editing efficiency) to, for example, reduce the overall 2' / F / PS content, which may cause toxicity in certain sequence contexts. Nevertheless, the impact of replacing 2'-F-modifications with other 2'-modifications on editing efficiency is highly position-specific. Again, it appears preferable to avoid blocks of uniform 2'-modifications (≤6 nt). Furthermore, the present data also show that higher levels of 2'-OMe (up to 40% in v117.43) are generally well tolerated and, if correctly positioned, have only a minor impact on editing yield. Example 15. Identification of hotspot sites 3' to the central base triplet (CBT) of ASOs targeting SERPINA1 E342K

[0238] As previously described by the inventors of the present application, the first 3' position of conventional CBT (structure [Am]-N -1 -N0-N +1 -N +2- The choice of nucleotide at position +2 in [Bn] (where N0 is the editing site) can have a significant impact on the target editing rate. To further investigate the effect of nucleotide modifications located 3' of the CBT, we modified the first two nucleotides located directly 3' of the CBT (i.e., positions +2 and +3). [ka] Constructs targeting different SERPINA1 were generated with nucleotide modifications at positions +2 and +3. Generally, the constructs targeting different SERPINA1 have the same base sequence, differing only at positions +2 and +3 (Table 15 and Figure 16B). The results are shown in Figure 16. The various SERPINA1-targeting ASOs and their modifications are listed in Table 15.

[0239] As shown in Figure 16A, the +2 position (N +2 ) with 2'-O-methyl modification at the +3 position (N +3 Construct v117.82, containing a 2'-fluoro modification at the +2 position, showed the highest level of editing efficiency, at 42.8%. On the other hand, replacing the 2'-O-methyl modification at the +2 position with either a 2'-F, 2'-H, or 2'-MOE modification gradually reduced SERPINA1 editing efficiency. Similarly, ASO constructs that received either a 2'-OMe, 2'-H, or 2'-MOE modification at the +3 position instead of the 2'-fluoro modification showed reduced SERPINA1 editing efficiency, down to editing levels as low as 0.4% (see v117.101, v117.102, v117.104, v117.106, v117.119, v117.120, and v117.124). Interestingly, 2'-MOE modifications at either the +2 position (v117.118 and v117.119) or the +2 and +3 positions (v117.120) resulted in the lowest editing efficacy (Figure 16A). These data suggest that a 2'-O-methyl modification at the +2 position and a 2'-F-modification at the +3 position are necessary to provide optimal editing efficacy. The +2 position is particularly important, followed by the +3 position.

[0240] Overall, these data suggest that CBT [ka] The first two nucleotides located directly 3' of [ka] These results suggest that the type of 2'-modification at the CBT plays an important role in determining the editing effect of ASOs. Interestingly, we have thus discovered modification-sensitive "hotspot" regions at positions +2 and +3, located directly 3' of the CBT. Therefore, we propose that the CBT, together with the first two nucleosides located directly 3' of the CBT, constitutes an "extended CBT." [ka] In this case, the best arrangement and / or modification to obtain efficient target editing is 5'-CBT-mN-fN-3'... and where CBT stands for central base triplet, mN is a 2'-O-methyl-modification, and fN is a 2'-fluoro-modification. The organization of this extended modification-sensitive "hotspot" is also shown in Figure 16B.

[0241] Interestingly, this extended CBT is also sensitive to internucleoside bond modifications, accepting PS modifications very well at bonds d and e, well at bonds f and / or g, but not at bonds h and i. Thus, in one embodiment, d and e are PS bond modifications. In one embodiment, f is a PS bond. In one embodiment, g is a PS bond. In some cases, h and i are not PS bond modifications. [Table 16]

[0242] To further explore the expanded hotspot region, ASOs targeting CTNNB1 T41A with 2'-O-methyl or 2'-F-modifications at positions +2 or +3 were evaluated for their in vitro editing efficacy. The sequence modifications of the different ASOs tested are shown in Table 16, and the results are shown in Figure 17. "No ASO" served as a negative control. [Table 17]

[0243] As shown in Figure 17A, construct v117.22, which has a 2'-O-methyl modification at the +2 position and a 2'-F-modification at the +3 position, showed an editing efficiency of 33.9%, which was higher than the editing efficiency of constructs with a 2'-OH at the +2 position and either a 2'-O-methyl modification at the +3 position (v117.20 (18.9%)) or a 2'-F-modification at the +3 position (v117.21 (27.5%)).

[0244] Thus, these data demonstrate and support that the optimal mix and placement of combinations for maintaining good RNA editing is defined by an extended hotspot with the structure 5'-CBT-mN-fN-3'. Example 16. Replacement of 2'-OMe-modified endblocks with 2'-MOE in ASOs targeting SERPINA1 E342K

[0245] 2'-MOE residues are frequently used in splice-switching oligonucleotides and are generally known to have very low toxicity. Because 2'-MOE residues are bulky and located in the minor groove, they are poorly tolerated in large quantities and are completely unacceptable at certain positions in ADAR-recruiting ASOs. To determine the effect of 2'-MOE modification on stability, toxicity, and editing efficacy, constructs targeting different SEPINA1 sites were generated and tested for editing efficacy and lysosomal stability. The different ASOs and their sequence modifications are listed in Table 17. The results are shown in Figure 18. "No ASO" served as a negative control. Toxicity was quantified using the CellTox™ Green Cytotoxicity Assay (Promega) according to the manufacturer's protocol and then normalized to the negative control ("No ASO") and the positive control (v117.59).

[0246] In the first study, we generated a 59-nt ASO containing a total of six 2'-MOE nucleotides, with three 2'-MOE nucleotides consecutively placed at each end (v117.68). This ASO variant was based on a 59-nt lead ASO (v117.59) with an 85% PS-modified backbone. Notably, introducing 2'-MOE modifications at the ends of the lead ASO did not affect the overall editing yield (Figure 18A). Similarly, the lysosomal half-life of the v117.68 variant was unaffected compared to the control, and both constructs exhibited a significant reduction in lysosomal half-life. 50 However, as shown in Figure 18C, the cytotoxicity of the test constructs was surprisingly reduced to only 26.6% when compared to the v117.59 control.

[0247] Similarly, 2'-MOE modifications were introduced into a 40-nt ASO in the same manner as v117.68. This ASO variant was based on a 40-nt lead ASO (v117.137), which also contained 85% PS-modified backbone. The results are shown in Figure 18E. The editing yield was somewhat lower in constructs containing 2'-MOE termini compared to those without. The reduced editing yield in v117.172 may result from the 2'-MOE modification being located closer to the CBT, a well-known contact site for ADAR enzymes. Without being bound by any theory, we suggest that the 2'-MOE modification should be positioned further away from known enzyme contact sites to prevent interference with protein binding through the bulky 2'-MOE-modified side chain. [Table 18]

[0248] These data demonstrate that the presence of 2'-MOE endblocks at the 5' and 3' ends of ASOs impacts overall ASO activity. For example, the findings suggest that 2'-MOE modification of the 5' and 3' ends of ASOs may aid in enhancing lysosomal stabilization. Furthermore, the data demonstrate that 2'-MOE endblocks at the 5' and 3' ends can be incorporated without affecting editing efficiency, while clearly reducing the cytotoxicity of long (>40 nt) embodiments. Shorter embodiments containing terminal 2'-MOE modifications exhibit a slight reduction in editing efficacy compared to embodiments containing less bulky 2'-modifications, such as 2'-OMe. Example 17. Reduced PS-linked backbone modifications in ASOs targeting SERPINA1 E342K

[0249] Phosphorothioate (PS) linkages can confer beneficial properties to ASOs by improving albumin binding, cellular uptake, endosomal escape, and protein binding. Furthermore, PS linkages have been reported to translocate ASOs from the cytoplasm to nuclear vesicles (Crooke et al., 2020). On the other hand, PS linkages tend to make ASOs sticky, which can lead to protein and / or ASO aggregation and toxicity. For example, increased PS-associated toxicity has been observed in ASOs rich in 2'-F modifications. Consequently, there is growing interest in learning how to modify ASOs with PS linkages and ultimately reduce the amount of PS linkages and / or 2'-F content within each ASO.

[0250] We tested four different ASO constructs with reduced PS content and used two different model systems [plasmid-based (A) and genome integration approach (B)] to investigate the effect of reduced PS-binding modifications on editing efficacy.

[0251] Plasmid transfection approach (A): 2.5 x 10 4HeLa cells were seeded in 24-well plates. 24 hours later, cells were forward transfected with a plasmid containing the human SERPINA1 E342K (PiZZ) mutant cDNA. Forward transfection was performed by diluting 300 ng of plasmid and 0.9 μl of FuGENE® 6 (Promega) in 50 μl of Opti-MEM, incubating for 5 minutes, then combining the two mixtures and incubating for an additional 20 minutes. The medium was replaced, and the transfection mixture was evenly distributed into one well. 24 hours after plasmid transfection, cells were forward transfected using 5 pmol of construct / well and 1.5 μl / well of Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific). Forward transfection of ASO constructs was performed by mixing the construct and Lipofectamine reagent in 50 μL of OptiMEM. After a 5-minute incubation, both solutions were combined and incubated for an additional 20 minutes. After changing the medium, the transfection mixture was evenly distributed into one well. 24 hours later, the medium was changed again. 48 hours after transfection, cells were harvested for RNA isolation and prepared for Sanger sequencing.

[0252] Genome integration approach (B): 1x10 cells containing the human SERPINA1 E342K mutant cDNA gene stably integrated into the genome via the piggyBac transposase system. 5 HeLa cells were seeded in 24-well plates. After 24 hours, cells were forward transfected using 25 pmol of construct / well and 1.5 μl / well of Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific) according to the Lipofectamine RNAiMAX protocol (described above). After 24 hours, cells were harvested for RNA isolation and Sanger sequencing.

[0253] The different test constructs were compared to a PS-rich lead construct (v117.59, 59 nt in length) with a PS content of approximately 90%. In the most extreme construct, PS bonds were contained only at the 5' and 3' ends (2x3 PS bonds each) and around the CBT (4 PS), resulting in an overall PS content of only approximately 17% (v117.71). This PS-poor ASO still had remarkable lysosomal stability (Figure 19C, t 50 >7 days), resulting in significant editing, which was significantly reduced compared to PS-rich reads (v117.59) (Figures 19A and 19B).

[0254] To further evaluate the effect of varying levels of PS content on ASO activity and stability, additional PS linkages were introduced at either the 5' or 3' of each DNA base across the ASO (v117.72 and v117.73) (see Table 18 and Figure 19D). This resulted in an overall PS content of approximately 30.5% (v117.72) and 32.2% (v117.73). As shown in Figures 19A and 19B, increasing the overall PS content clearly improved editing yields compared to v117.71, regardless of the approach used.

[0255] Furthermore, the reduction of the overall PS content within each ASO did not dramatically reduce the stability of the tritosome (t 50 >7 days). In particular, it was found that the PS bond 3' to the DNA is more important than the PS bond 5' to the DNA in terms of tritosome stability (Fig. 19C, v117.73, t 50 = 96 h). Introduction of 5' and 3' PS linkages to each DNA base generated ASOs with only 49.2% PS content (v 117.74), which was related to the low tritosome stability (t 50 >7 days) and editing efficiency were similar to the PS-rich read construct v117.59 (Figure 19). [Table 19]

[0256] These findings demonstrate that the PS content in fully modified ASOs can be significantly reduced without compromising editing efficacy. However, some reduction in editing efficacy may be encountered. A minimum of 15% bond modification is required. For longer embodiments (e.g., ≥40 nt), an overall internucleoside content in the range of 15-90% is desirable, and editing and lysosomal stability are maintained even with an internucleoside modification content as low as 30% (v117.72 and v117.73).

[0257] We further tested the effect of PS content on shorter ASOs (40 nt). The various test constructs are shown in Table 19. The results are shown in Figure 20. [Table 20]

[0258] The 40-nt short-read ASO contained 85% PS linkage content (v117.82). As with the longer ASOs, PS linkages were added directly to the 3', 5', or 3' and 5' ends of each DNA nucleotide, reducing the overall PS linkage content of the 40-nt short ASO to approximately 30% or 50%. Construct v117.96 is identical in sequence to v117.109, differing only in the last two nucleotides at the 3' end (v117.96 contains two 2'-OMe-modifications, whereas v117.109 does not) and PS content. It also served as a control oligonucleotide with 50% PS content because v117.82 also contains an endblock at the 3' end that is not present in the other tested versions. Constructs v117.107, v117.108, and v117.109 exhibited PS content of approximately 30%, 30%, and 47.5%, respectively. As shown in Figure 20, v117.109 was unable to fully restore the editing yield of PS-rich ASO reads. This suggests that the reduction in PS content of short ASO reads (40 nt) was significantly greater than that of long ASO reads (59 nt). This suggests that it may not be acceptable in comparison.

[0259] Furthermore, we found that for short ASOs (≦50 nt, e.g., 40 nt), a contiguous region (at least 10) of linkage modifications was more beneficial than randomly distributed linkage modifications in reducing overall PS content. The different SERPINA1 E342K-targeting constructs and their modifications are shown in Table 20. "No ASO" served as a negative control. [Table 21]

[0260] An ASO with three or fewer consecutive PS bonds (v117.107, 30% PS content) was tested and compared with an ASO with the same sequence but 10 (v117.132, 47.5% PS) or 16 (v117.133, 60% PS) consecutive PS bonds. As shown in Figure 21A, 10 consecutive PS bonds already increased the editing yield but failed to rescue the editing yield of the high-PS ASO, v117.82. However, 16 consecutive PS bonds achieved editing levels similar to those of the PS-rich read ASO (v117.82; 25 consecutive PS bonds, 85% PS). This highlights that while the content of bond modifications can be reduced (e.g., by at least 30%) in short embodiments (≦50 nt), a contiguous region of modified bonds (e.g., PS) is more beneficial than randomly spreading modified bonds (e.g., PS) throughout the ASO. Furthermore, the present data suggest that the PS content in these fully modified ASOs can be significantly reduced, which may be important if reduced toxicity is required.

[0261] Surprisingly, many ASOs known from the prior art (e.g., WO2021 / 071858 and WO2022 / 099159) contain extremely high levels of (sterically pure) phosphorothioate bond modifications to achieve similar ranges of editing yields. More importantly, these data are based on editing results in primary mouse hepatocytes, which generally result in high editing yields and may therefore provide a limited picture.

[0262] In particular, there tends to be a certain threshold for bond modification. Therefore, according to the present invention, in one embodiment, the bond modification content (e.g., PS) is at least 30% to obtain optimal editing yield. In one embodiment, the PS bond modification content (e.g., PS) is at least 50%. In one embodiment, the PS content is at least 60%. Without being bound by a particular theory, it appears that short ASOs prefer more than 10 consecutive bond modifications (e.g., PS) to dispersed patterns. Example 18. Improved editing effect and editing efficacy of ASO targeting LNA-modified SERPINA1 E342K

[0263] It was observed that when shortening the ASO, the editing yield and editing efficacy of the 40nt lead ASO may be slightly reduced. LNA is known in the art to increase stability against enzymatic degradation and improve specificity and binding affinity in base pairing. Therefore, a 40nt-long ASO (v117.82; "No LNA") with 85% PS content was modified by introducing LNA building blocks at the 5' and 3' ends or within the oligonucleotide sequence. The different ASOs used in Example 17 are listed in Table 21, and the respective results are shown in Figure 22. [Table 22]

[0264] As shown in Figure 22A, for construct v117.97, which contained a total of four terminal LNAs (two LNAs at each end separated by 2'-OMe), LNA modification had little effect on the overall editing yield at high ASO doses compared to v117.82. However, the potency of v117.97 was slightly improved, given that it exhibited significantly higher editing yields at ASO doses of 2.5 pmol and 1.25 pmol (Figure 22B). When compared to ASOs with LNA modifications only at the 5' and 3' ends, placing the LNA modifications within the ASO closer to the CBT appeared to hinder the editing effect and editing potency of the ASO (see v117.97 vs. v117.98).

[0265] To determine the combined effect of terminal LNA modification and ASO length on editing efficacy, LNA-modified versions (v117.129 to v117.131) of a short ASO (36 nt) with 85% PS content (v117.86; "No LNA") were generated to contain 2, 4, or 6 LNA-modified nucleotides at their 5' ends, as shown in Table 22 and depicted in Figure 23B. The results are shown in Figure 23A.

[0266] Two 5'-terminal LNAs were well tolerated, improving overall editing of the ASO from 29% v117.86 to 49.4% v117.129, but additional 5'-terminal LNAs hindered editing yield (v117.130 and v117.131). Thus, without being bound by any theory, we believe that short ASOs may benefit from fewer 5'-terminal LNAs.

[0267] Finally, we evaluated whether 40nt ASOs with reduced PS, with overall PS content of approximately 30% (v117.107 and v117.126) and 48% (v117.109 and v117.127), would benefit from terminal LNAs. Different SERPINA1 E342K-targeting constructs were generated to contain a total of four LNAs (two LNA modifications at each end interrupted by 2'-OMe). The different constructs and their modifications are shown in Table 22, and the results are shown in Figure 24A. [Table 23]

[0268] Interestingly, a 40-nt short ASO targeting SERPINA1 E342K with reduced PS benefited significantly from the addition of LNAs at the 3' and 5' ends (v117.127), achieving a higher editing yield than that achieved by a 40-nt lead ASO (v117.82) lacking terminal LNA modifications and possessing 85% PS. These data demonstrate that the overall editing efficacy and editing potency of ASOs can be improved through LNA modifications, more specifically by placing a certain number of LNAs (e.g., up to six, more preferably less than four) at the termini (e.g., preferably at the 5' end) of the ASO (preferably in short embodiments with lengths of ≤50 nt). More importantly, the data demonstrate that ASO performance improved through LNA base modifications despite reduced PS content. Without being bound by theory, the inventors believe that LNAs may help compensate for the low PS content in short embodiments (≤50 nt, e.g., ≤40 nt). Example 19. Disruption of consecutive blocks of 2'-modifications in constructs targeting SERPINA1 E342K and STAT1.

[0269] ASOs with long stretches (or "contiguous blocks") of the same sugar modification at the 2' position (e.g., long blocks of 2'-O-methyl interrupted only by CBTs, or long blocks of 2'-F combined with long blocks of 2'-O-methyl interrupted only at CBTs) are known in the art. Specifically, Monian et al. (2022) previously demonstrated that chemically modified oligonucleotides ("AIMers") with chimeric backbones containing consecutive 2'-fluoro- and / or 2'-OMe-modified backbones and highly (sterically pure) PS and PN linkages can mediate efficient in vitro target editing.

[0270] To determine the effect of disrupting such "contiguous blocks," e.g., contiguous regions of 2'-fluoro- and / or 2'-OMe-backbone modifications, different SERPINA 1 E342K- and STAT1 Y701-targeting constructs were generated and tested for editing efficacy in vitro. The constructs used in Example 18 are listed in Table 23 and Table 24, and the results are shown in Figures 25 and 26, respectively. "No ASO" served as a negative control. [Table 24]

[0271] As shown in Figure 25, for the construct targeting SERPINA1 E342K, disruption of consecutive blocks of 2'-modifications (e.g., 2'-F and / or 2'-O-methyl) increased the overall editing efficacy of the ASO. This data suggests a negative correlation between the block size of consecutive 2'-modification blocks and their respective editing yields. As shown in Figure 25A, a 40-nt control ASO (block design_40nt) containing a 2'-F block (e.g., 20 nt) and a 2'-OMe block (e.g., 8 nt) located 5' of the CBT and a 2'-OMe block (e.g., 9 nt) located 3' of the CBT showed a low editing efficiency of approximately 20%. In comparison, the highest editing yields were obtained for v117.123 and v117.158 (achieving approximately 60% editing yield), both of which contain interruptions of 2'-F blocks with a single 2'-O-methyl modification (e.g., 2'-F block sizes up to 5 nt) and interruptions of two 2'-O-methyl blocks with a single 2'-F-modification or small blocks of 2'-F-modifications (e.g., 2'-O-methyl block sizes up to 4 nt), suggesting that the interrupted blocks are crucial for optimal editing efficiency.

[0272] The data further demonstrate that DNA nucleosides can be used to disrupt either the 2'-F block or the 2'-O-methyl block (v117.82). In this case, the DNA may not have been positioned ideally, resulting in somewhat reduced editing efficiency, but still better results than the block design_40nt control. Furthermore, the data demonstrate that relatively large 2'-F and 2'-O-methyl blocks can be accommodated in the 5' half of the ASO (v117.155). However, the editing yield was significantly lower than that of constructs with smaller 2'-F and / or 2'-O-methyl blocks (e.g., v117.121, v117.123, and v117.158 vs. v117.153, v117.154, v117.158). The poor performance of constructs v117.152, v117.153, and v117.154 (e.g., editing yields of approximately 25-40%) compared to the two best embodiments in this dataset (e.g., v117.158 and v117.123, which have editing yields of approximately 60%) demonstrates the importance of combining block disruption in a large 2'-F block with disruption in two large 2'-OMe blocks around the CBT. These embodiments also demonstrate that high amounts of 2'-OMe (55% for v117.121) can be well tolerated, even improving editing yields when replacing DNA nucleosides (e.g., approximately 40% editing yield for v117.82 vs. approximately 60% editing yield for v117.121).

[0273] Similarly, ASOs targeting STAT1 Y701, which have been subjected to disruption of successive block modifications, were created and tested for RNA editing effects. The constructs targeting STAT1 Y701 are listed in Table 24, and the results are shown in Figure 26. "No ASO" served as a negative control. [Table 25]

[0274] The ASO targeting STAT1 Y701 was based on a short embodiment (≤50 nt, e.g., 40 nt), in which the ASO contained no more than three (≤3 nt) consecutive 2'-OMe-modifications and no more than six (≤6 nt) consecutive 2'-F-modifications (v117.44). This embodiment yielded the best editing results (42%) in the dataset. This was compared to a control ASO with a large 5'-terminal 2'-F block (20 nt) and two large 2'-O-methyl blocks located 3' (8 nt) and 5' (9 nt) of the CBT (see v117.53). This version performed very poorly, achieving only a low editing yield of 7%, close to the limit of sequence detection by Sanger sequencing. Using domain-swap experiments, we demonstrated that for this target sequence, block interruptions with 2'-O-methyl blocks are particularly important. This was particularly demonstrated for v117.54, which still contained a large 2′-F block and still performed well compared to v117.55, which contained block interruptions in both the 2′-F block and two large consecutive 2′-O-methyl blocks.

[0275] This demonstrates that large 2'-F blocks are likely to be more readily accepted than large 2'-O-methyl blocks. However, the best performing embodiment features a maximum contiguous block size of 6 nt (v117.44). Example 20. 2'-FANA modification within the CBT of constructs targeting CRB1 C948Y

[0276] 2'-FANA modification in CBT (WO2021 / 243023), especially N +1 It has previously been shown that 2'-FANA modification at this position has a positive effect on ASO-mediated on-target editing (Brinkman et al., 2022). To investigate the effect of 2'-FANA modification located within and 5' of the CBT, we performed 2'-FANA modification at the N position of a construct targeting CRB1 C948Y. -2 rank, N -1Rank, N0th place, and / or N +1 The various constructs and their modifications used in Example 19 are shown in Table 25. The corresponding results are shown in Figure 27. "No ASO" was used as a negative control. [Table 26]

[0277] As shown in Figure 27A, one or more 2'-FANA modifications located within and / or 5' of the CBT, along with a combination of 2'-F and / or 2'-OMe-modifications outside the CBT, significantly increased the overall editing effect when compared to the control, highlighting that FANA is more than just a substitute for DNA, 2'-F and 2'-O-methyl within the CBT in this embodiment.

[0278] The introduction of 2'-F, 2'-OMe, and 2'-H modifications alone resulted in a slight decrease in editing efficiency compared to the control containing 17 natural RNA nucleosides (see v117.29 vs. v120.2). However, the fully 2'-modified embodiment (v117.30; 2 x 2'-FANA in CBT) at the N0 and N1 positions showed a significant decrease in editing efficiency. +1 Introducing a 2'-FANA modification at this position increased the editing efficiency back to the level of the RNA-rich control ASO v120.2, thereby clearly surpassing the fully 2'-modified embodiment lacking FANA (v117.29; no 2'-FANA). -2 Position and N -1Addition of additional 2'-FANA modifications to positions 117.31 was well tolerated but did not further enhance the editing effect of ASO (v117.31) compared to v117.30 (two 2'-FANA modifications located in the CBT). Interestingly, compared to v117.30, a control ASO (v117.33), containing two additional nucleosides at the 5' end and three additional nucleotides at the 3' end, showed a dramatic reduction in editing efficiency below the detection limit of Sanger sequencing (less than 5%). This control construct was based on an embodiment disclosed in the prior art. That is, the 2'-sugar linkage modification pattern is identical to sequence KB-018-698 described in patent WO2021 / 243023. In this case, the nucleobase sequence was altered to match the CRB1 C948Y site by replicating the modification framework and sequence symmetry of KB-018-698 and transferring it to a different target transcript / site. As expected, the uniform 2'-O-methyl modification outside the CBT, characteristic of v117.33, strongly hinders the RNA editing efficiency effect and cannot be restored by the presence of FANA in the CBT or by the extended length of the ASO (50 nt). This again emphasizes the importance of combining optimal binding and 2'-modification patterns (2'-O-methyl, 2'-F, and / or DNA), proper positioning of the ASO (asymmetric for short embodiments ≤ 50 nt), and limiting the block size (e.g., ≤ 6 nt) of uniform 2'-modifications (e.g., 2'-O-methyl, but also 2'-F and DNA).

[0279] These data suggest that a combination of 2'-FANA modifications located in the CBT and 5' of the CBT, combined with 2'-F- and 2'-OMe-modifications, can be used to stabilize ASOs and maintain or increase their editing efficacy. Overall, ASOs containing a combination of 2'-F- and 2'-OMe-modifications with DNA were able to tolerate at least four 2'-FANA modifications in total.

[0280] In summary, we have demonstrated that a balanced mix of modifications at the 2'-position of the sugar moiety of an oligonucleotide (e.g., at least 10% (preferably 20-70%) 2'-F-, at least 10% (preferably 20-60%) 2'-OMe-, and optionally 2'-H (50% or less for long ASOs, e.g., ≥40 nt, and 6 or less for short ASOs, e.g., ≤50 nt), 2'-OH, etc.) allows for stability in the lysosomal periphery, thus protecting ASOs from nuclease degradation, e.g., during uptake, while maintaining high editing efficiency and efficacy. Specifically, we have successfully demonstrated that mixing such modifications with specifically positioned internucleoside linkage modifications (e.g., linkage modifications at linkages d and e, but no PS-modifications at linkages h and i) provides a method for generating effective and stable ASOs. In particular, we have shown that the positioning of these specific 2'-modifications at specific sites within an ASO can significantly affect the overall editing efficacy of the ASO, depending on the length of the ASO. Furthermore, we have identified extended hotspot regions (5'-CBT-mN-fN-3'). Where the positioning of the 2'-modification is less critical, the combination of chemical modifications is crucial. Furthermore, we have shown that a certain level of 2'-F- and 2'-OMe-modifications should be included ("extended hotspots"), but that contiguous blocks of uniform sugar modifications should clearly be avoided (e.g., ≤6 nt for 2'-F, 2'-O-methyl, and / or DNA, with 2'-F being most compatible in larger, uniform blocks). Furthermore, we have shown that the placement of these modifications is somewhat position-independent, and that specific modifications and / or modification patterns can be transferred, to some extent, in a position-specific manner to different ASOs with distinct target specificities. The inventors have shown that, at least for embodiments of the present invention, FANA modifications within CBTs are well-accepted at specific positions and can improve editing yields over CBTs constructed from DNA alone.Furthermore, the inventors have successfully shown that the PS content can be significantly reduced (up to 15%), especially for longer embodiments (≧40 nt); and that shorter embodiments (≦50 nt) benefit from a combination of (e.g., a stretch of ≧10) linkage modifications (e.g., PS) and / or (terminal) LNA modifications, or generally from long, continuous stretches of slightly higher degrees of linkage modification (e.g., at least 30%, e.g., PS).

[0281] Those skilled in the art will appreciate that the present disclosure can be modified in ways not specifically described herein. [Brief explanation of the drawings]

[0282] [Figure 1-1] FIG. 1 is a graph showing the editing efficacy and lysosomal stability of oligonucleotides targeting SERPINA. [Figure 1-2] FIG. 1 is a graph showing the editing efficacy and lysosomal stability of oligonucleotides targeting SERPINA. [Figure 2] FIG. 2 is a graph showing the SERPINA-editing effect of 2′-F-modified oligonucleotides. [Figure 3] FIG. 3 is a graph showing the editing efficacy and lysosomal stability of oligonucleotides targeting STAT1 Y701. [Figure 4] Figure 4 is a graph showing the editing efficacy and lysosomal stability of oligonucleotides targeting CRB1 C948Y. [Figure 5] FIG. 5 is a graph showing the editing efficacy and lysosomal stability of oligonucleotides targeting LRRK2 G2019S. [Figure 6] FIG. 6 is a graph showing the editing effect of oligonucleotides targeting mouse PDE6A (mPDE6A) V685M. [Figure 7]Figure 7 is a graph showing the editing effect of oligonucleotides targeting NLRP3 Y166 expressed from a plasmid (A) and expressed after integration into the genome. [Figure 8] FIG. 8 is a graph showing data regarding the editing effect of oligonucleotides targeting the GAPDH 3′UTR. [Figure 9] Figure 9 is a graph showing data on the editing effects of truncated variants (31 nt, 40 nt, 45 nt, 50 nt, 59 nt) of oligonucleotides targeting SERPINA. [Figure 10] FIG. 10 shows data on the editing effects of 5′ and / or 3′ truncated variants of SERPINA-targeting oligonucleotides. [Figure 11] FIG. 11 is a graph showing the editing effect of 32-nt and 33-nt long SERPINA-targeting oligonucleotides. [Figure 12] FIG. 12 is a graph showing the editing effect of oligonucleotides targeting 3′-truncated STAT1 Y701. [Figure 13] Figure 13 is a graph showing the editing effect of oligonucleotides targeting 3'-truncated CTNNB1 T41. [Figure 14-1] FIG. 14 is a graph showing the editing effect of oligonucleotides targeting 5′- and / or 3′-truncated CRB1 C948Y. [Figure 14-2] FIG. 14 is a graph showing the editing effect of oligonucleotides targeting 5′- and / or 3′-truncated CRB1 C948Y. [Figure 15] Figure 15 is a graph showing the editing effect of oligonucleotides targeting STAT1 Y701. [Figure 16] FIG. 16 is a graph showing the editing effect of oligonucleotides targeting SERPINA with modifications in the extended hotspot region 3′ to the CBT (optimal versions: +2 (2′-OMe) and +3 (2′-F)). [Figure 17]Figure 17 is a graph showing the editing effect of oligonucleotides targeting CTNNB1 T41 with modifications in the extended hotspot region 3' to the CBT (optimal versions: +2 (2'-OMe) and +3 (2'-F)). [Figure 18-1] FIG. 18 is a graph showing the editing efficacy, lysosomal stability, and relative toxicity of SERPINA-targeting oligonucleotides containing 2′-MOE end blocks. [Figure 18-2] FIG. 18 is a graph showing the editing efficacy, lysosomal stability, and relative toxicity of SERPINA-targeting oligonucleotides containing 2′-MOE end blocks. [Figure 19-1] Figure 19 is a graph showing the editing efficacy and lysosomal stability of a long SERPINA-targeting oligonucleotide (59 nt) with reduced PS binding using a genomic system (A) and a plasmid system (B). [Figure 19-2] Figure 19 is a graph showing the editing efficacy and lysosomal stability of a long SERPINA-targeting oligonucleotide (59 nt) with reduced PS binding using a genomic system (A) and a plasmid system (B). [Figure 20] FIG. 20 is a graph showing data on the editing effect of short SERPINA-targeting oligonucleotides (40 nt) with reduced PS-binding modifications. [Figure 21] FIG. 21 is a graph showing the editing effect of oligonucleotides targeting SERPINA containing a continuous region of PS binding. [Figure 22] Figure 22 is a graph showing the effect of LNA modifications on the editing effect and editing efficacy of SERPINA-targeting oligonucleotides. [Figure 23] FIG. 23 is a graph showing the effect of LNA modifications at the 5′ end of SERPINA-targeting oligonucleotides. [Figure 24] FIG. 24 is a graph showing 5′-terminal LNA modification of short SERPINA-targeted oligonucleotides. [Figure 25]FIG. 25 is a graph showing 5′ and 3′ end block disruption of SERPINA-targeting oligonucleotides. [Figure 26] FIG. 26 is a graph showing block disruption of short STAT1-targeted oligonucleotides. [Figure 27] FIG. 27 is a graph showing 2′-FANA modification of CBT of oligonucleotides targeting CRB1 C948Y.

Claims

1. a central nucleotide (N) directly opposite a target adenosine in said target RNA, said central nucleotide (N) comprising a sequence of 23 to 80 nucleotides in length capable of binding to a target sequence in said target RNA; ０ ) a central base triplet (CBT) of three nucleotides 【Chemistry 1】 1. A chemically modified oligonucleotide comprising the following core oligonucleotide: 【Chemistry 2】 Including; where: (a) at least two of the three nucleotides of said CBT are chemically modified at the 2' position of the sugar moiety, are deoxyribonucleosides, or a combination thereof, and d and e are internucleoside linkage modifications; (b) N ＋２ The nucleotide is 2'-O-alkyl-modified; ＋３ The nucleotides are 2'-fluoro (2'-F)-modified; (c) at least 10% of the nucleotides are 2'-F-modified and at least 10% of the nucleotides are 2'-O-alkyl-modified, where no more than six consecutive nucleotides have the same 2'-modification; (d) the internucleoside linkage modification content is at least 15%; and (e) A chemically modified oligonucleotide, wherein linkage h and linkage i are not phosphorothioate (PS) linkages.

2. The chemically modified oligonucleotide according to claim 1, wherein 20 to 100% of the nucleotides are deoxyribonucleosides or 2'-modified nucleotides, preferably 50 to 100% of the nucleotides are 2'-modified nucleotides.

3. 20-70% of the nucleotides are 2'-F-modified, preferably 35-65% of the nucleotides are 2'-F-modified, and / or 3. The chemically modified oligonucleotide according to claim 1 or 2, wherein 20-60% of the nucleotides are 2'-O-methyl (2'-OMe)-modified, preferably 25-55% of the nucleotides are 2'-OMe-modified.

4. (i) the internucleoside bond modifications do not exceed 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% of the bonds outside said CBT; or (ii) The chemically modified oligonucleotide according to any one of claims 1 to 3, wherein 15 to 90% of the linkages are internucleoside linkage modifications, preferably 40 to 80%, and most preferably 45 to 60% of the linkages are internucleoside linkage modifications.

5. The chemically modified oligonucleotide of any one of claims 1 to 4, wherein the oligonucleotide has a length of 28 to 70 nucleotides.

6. The oligonucleotide comprises: (i) a length of 28 to 60, 28 to 55, or 28 to 45 nucleotides; (ii) a length of 59 nucleotides; or (iii) a length of 45 nucleotides or less The chemically modified oligonucleotide of claim 5 , having:

7. 7. The chemically modified oligonucleotide of claim 6, wherein the oligonucleotide has a length of 45 nucleotides or less and, outside the CBT, no more than four nucleotides are deoxyribonucleotides.

8. a central nucleotide (N) directly opposite a target adenosine in the target RNA, comprising a sequence of 23 to 50 nucleotides in length capable of binding to a target sequence in the target RNA; ０ ) a central base triplet (CBT) of three nucleotides 【Transformation 3】 1. A chemically modified oligonucleotide comprising the following core oligonucleotide: 【Chemistry 4】 Including; where: (a) at least two of the three nucleotides of said CBT are chemically modified at the 2' position of the sugar moiety, are deoxyribonucleosides, or a combination thereof, and d and e are internucleoside linkage modifications; (b) N ＋２ The nucleotide is 2'-O-alkyl-modified; ＋３ The nucleotides are 2'-fluoro (2'-F)-modified; (c) at least 10% of the nucleotides are 2'-F-modified and at least 10% of the nucleotides are 2'-O-alkyl-modified, where no more than six consecutive nucleotides have the same 2'-modification; (d) the regions located 3' and 5' of the CBT do not contain a total of more than six deoxyribonucleosides; (e) Chemically modified oligonucleotides having an internucleoside linkage modification content of at least 30%.

9. Outside the CBT: (i) the oligonucleotide does not contain any deoxyribonucleosides, or (ii) The chemically modified oligonucleotide of claim 8, wherein no more than 1, 2, 3, or 4 nucleotides are deoxyribonucleotides.

10. The chemically modified oligonucleotide according to claim 8 or 9, wherein the internucleoside bond modification content is 30 to 90%.

11. a central nucleotide (N) directly opposite a target adenosine in said target RNA, said central nucleotide (N) comprising a sequence of 40 to 80 nucleotides in length capable of binding to a target sequence in said target RNA; ０ ) a central base triplet (CBT) of three nucleotides 【Transformation 5】 1. A chemically modified oligonucleotide comprising the following core oligonucleotide: 【Transformation 6】 Including; where: (a) at least two of the three nucleotides of said CBT are chemically modified at the 2' position of the sugar moiety, are deoxyribonucleosides, or a combination thereof, and d and e are internucleoside linkage modifications; (b) N ＋２ The nucleotide is 2'-O-alkyl-modified; ＋３ The nucleotides are 2'-fluoro (2'-F)-modified; (c) at least 10% of the nucleotides are 2'-F-modified and at least 10% of the nucleotides are 2'-O-alkyl-modified, where no more than six consecutive nucleotides have the same 2'-modification; (d) A chemically modified oligonucleotide, wherein the regions located 3' and 5' of the CBT have a total deoxyribonucleoside content of 5 to 50%.

12. 12. The chemically modified oligonucleotide of claim 11, wherein the deoxyribonucleoside content outside the CBT is 10-40%, more preferably 11-30%, and even more preferably 13-25%.

13. 13. The chemically modified oligonucleotide of any one of claims 1 to 12, comprising at least one internucleoside linkage modification selected from the group consisting of phosphorothioate (PS), 3'-methylene phosphonate, 5'-methylene phosphonate, 3'-phosphoramidate, 2'-5' phosphodiester, and phosphorylguanidine (PN).

14. 14. The chemically modified oligonucleotide of claim 13, wherein the at least one internucleoside linkage modification is PS.

15. A chemically modified oligonucleotide according to claim 8 or 11 or any claim dependent thereon, wherein bond h and bond i are not phosphorothioate (PS) bonds.

16. 16. The chemically modified oligonucleotide according to any one of claims 1 to 15, wherein bond h and bond i are phosphate (PO) bonds.

17. (i) not more than 4, 5, or 6 consecutive 2'-F-modified nucleotides; and / or (ii) not more than four, five, or six consecutive 2'-O-alkyl-modified nucleotides; A chemically modified oligonucleotide according to any one of claims 1 to 15.

18. 18. The chemically modified oligonucleotide of any one of claims 1 to 17, wherein fewer than 6, fewer than 5, fewer than 4, or fewer than 3 consecutive nucleotides have the same 2'-modification.

19. 19. The chemically modified oligonucleotide of claim 1, wherein the chemically modified oligonucleotide comprises one or more stereorandom internucleoside linkage modifications.

20. 20. The chemically modified oligonucleotide of any one of claims 1 to 19, wherein the oligonucleotide comprises no more than 10, preferably no more than 5, stereochemically pure internucleoside linkages.

21. 21. The chemically modified oligonucleotide of claim 20, wherein the sterically pure bond is a PS bond and / or a PN bond.

22. 22. The chemically modified oligonucleotide of claim 1, wherein the oligonucleotide does not contain a sterically pure PS bond and / or does not contain a sterically pure PN bond.

23. 23. The chemically modified oligonucleotide of claim 1, wherein the chemically modified oligonucleotide does not contain a sterically pure PS-linked modification.

24. The oligonucleotide comprises: (i) 2'-O-(2-methoxyethyl)-oligoribonucleotide (2'-MOE) end blocks at the 3'-terminus and 5'-terminus, wherein each end has no more than four nucleotides with 2'-MOE, and preferably no more than three nucleotides with 2'-MOE; and / or (ii) Terminal-locked nucleic acids (LNA), wherein the oligonucleotide comprises 2 to 6 LNAs at each end or at the 5' end; preferably, the oligonucleotide comprises 2 LNAs at each end or at the 5' end.

24. The chemically modified oligonucleotide of any one of claims 1 to 23, comprising:

25. 25. The chemically modified oligonucleotide of claim 1, wherein bond g is not a PS bond, preferably bond g is a phosphate (PO) bond.

26. 26. The chemically modified oligonucleotide of any one of claims 1 to 25, wherein d and e are PS linkage modifications, and optionally, f is an internucleoside linkage modification.

27. The modification at the 2' position of item (a) is: (i) 2'-O-alkyl-modified; (ii) a 2'-F-modification, or (iii) 2'-fluoroarabinoside (FANA) modification 27. The chemically modified oligonucleotide according to any one of claims 1 to 26, wherein:

28. 28. The chemically modified oligonucleotide of any one of claims 1 to 27, wherein the 2'-O-alkyl-modification is a 2'-OMe-modification.

29. Each of the three nucleosides of the CBT is as follows: (i) deoxyribonucleotides; and / or (ii) 2'-FANA modification; and / or (iii) a 2'-O-methyl modification; and / or (iv) 2'-F-modification, 29. The chemically modified oligonucleotide according to any one of claims 1 to 28, wherein:

30. (i) N －１ is 2'-F, 2'-FANA, DNA, or 2'-O-methyl; and / or (ii) N ０ is 2'-FANA or DNA; and / or (iii) N ＋１ The chemically modified oligonucleotide according to any one of claims 1 to 29, wherein is 2'-FANA, DNA, or 2'-O-methyl.

31. N ０ The chemically modified oligonucleotide according to any one of claims 1 to 30, wherein is deoxycytidine or FANA-cytidine.

32. 32. The chemically modified oligonucleotide of any one of claims 1 to 31, wherein the -5, -4 and -3 positions are 2'-O-alkyl-modified; and / or the -2 position is 2'-F-modified.

33. (i) at least four nucleotides 3' of said CBT; and / or (ii) at least 16 nucleotides 5' of said CBT; 33. The chemically modified oligonucleotide of any one of claims 1 to 32, having:

34. (i) at least 10 consecutive internucleoside linkage modifications; and / or (ii) modification of three consecutive internucleoside linkages at each end 34. The chemically modified oligonucleotide of any one of claims 1 to 33, comprising:

35. 35. The chemically modified oligonucleotide of any one of claims 1 to 34, wherein the oligonucleotide does not contain an ADAR recruitment motif of a loop hairpin structure.

36. The oligonucleotide may contain one or more mismatches, wobble bases, and / or bulges relative to its target, and / or N ０ 36. The chemically modified oligonucleotide of any one of claims 1 to 35, comprising a mismatch at

37. 37. A pharmaceutical composition comprising the oligonucleotide of any one of claims 1 to 36, or a pharmaceutically acceptable salt thereof.

38. A chemically modified oligonucleotide according to any one of claims 1 to 36 or a pharmaceutical composition according to claim 37 for use in the treatment or prevention of a genetic disorder, condition or disease.

39. The genetic disorders, conditions, or diseases described above include retinitis pigmentosa (RP), Stargardt macular degeneration, age-related macular degeneration (AMD), cystic fibrosis (CF), Hurler syndrome, alpha-1-antitrypsin (A1AT) deficiency, Parkinson's disease, Alzheimer's disease, albinism, amyotrophic lateral sclerosis, asthma, beta-thalassemia, Cadacill syndrome, Charcot-Marie-Tooth disease, chronic obstructive pulmonary disease (COPD), distal spinal muscular atrophy (DSMA), Duchenne / Becker muscular dystrophy, dystrophic epidermolysis bullosa, epidermolysis bullosa, Fabry disease, factor V Leiden-related disease, familial adenomatous polyposis, polyposis, galactosemia, Gaucher disease, glucose intolerance, and steroid use disorders.

39. The chemically modified oligonucleotide or pharmaceutical composition for use according to claim 38, wherein the oligonucleotide or pharmaceutical composition is selected from the group consisting of: -6-phosphate dehydrogenase, hemophilia, hereditary hematochromatosis, Hunter syndrome, Huntington's disease, inflammatory bowel disease (IBD), hereditary polymorphic aggregation syndrome, Leber congenital amaurosis (LCA), Lesch-Nyhan syndrome, Lynch syndrome, Marfan syndrome, mucopolysaccharidoses, muscular dystrophies, myotonic dystrophy types I and II, neurofibromatosis, Niemann-Pick disease types A, B, and C, NY-eso1-associated cancer, Peutz-Jeghers syndrome, phenylketonuria, Pompe disease, primary ciliary body disease, and prothrombin mutation-associated disease.

40. The chemically modified oligonucleotide or pharmaceutical composition for use according to claim 38 or 39, wherein said genetic disorder, condition or disease is associated with a G-to-A mutation in a gene selected from the list comprising SERPINA1, PDE6A, LRRK2, NLRP3 and CRB1.

41. 41. The chemically modified oligonucleotide or pharmaceutical composition for use according to claim 40, wherein the mutation is selected from the list comprising SERPINA1 E342K, PDE6A V685M, LRRK2 G2019S, and CRB1 C948Y.

42. 37. An in vitro method for editing a target adenosine in a target nucleic acid, the method comprising contacting the target nucleic acid with an oligonucleotide of any one of claims 1 to 36.

43. 1. An in vitro method for deaminating at least one specific adenosine present in a target RNA sequence in a cell, the method comprising the steps of: (a) contacting the target nucleic acid with the chemically modified oligonucleotide of any one of claims 1 to 36; (b) allowing the chemically modified oligonucleotide to be taken up by the cell; (c) allowing the chemically modified oligonucleotide to anneal to the target RNA sequence; and (d) enabling a mammalian ADAR enzyme containing a naturally occurring dsRNA binding domain found in the wild-type enzyme to deaminate a target adenosine in said target RNA sequence to inosine.

44. 44. The in vitro method for deaminating at least one specific adenosine present in a target RNA sequence in a cell described in Claim 43, wherein the method comprises, after step (d), a step of identifying the presence of inosine in the RNA sequence.

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