Chemical and sequence optimisation of antisense oligonucleotides for ADAR-mediated RNA editing

Abstract

The invention relates to modified antisense oligonucleotides suitable for editing an adenosine in a target nucleic acid to inosine (A-to-I editing), wherein the oligonucleotide comprises: (I) a nucleic acid sequence of 25 to 70 nucleotides (nt) that is an antisense sequence substantially complementary to the target nucleic acid, (II) a central base triplet (CBT) (5'- N+1 e N0 f N-1 g -3') with the central nucleotide (N0) directly opposite the target adenosine to be edited when the antisense oligonucleotide is hybridised to the target nucleic acid, and (III) the following core sequence: 5'- a N+4 b N+3 c N+2 d N+1 e N0 f N-1 g N-2 h N-3 i N-4 j N-5 k N-6 l N-7 m N-8 n N-9 -3';and wherein: (a) at least two of the three nucleotides of the CBT are chemically modified at the 2' position of the sugar moiety, are deoxyribonucleosides, or a combination thereof and wherein d and e are internucleoside linkage modifications; (b) the N-2 nucleotide carries a 2'-O-alkyl- modification; and wherein the N-3 nucleotide carries a 2´-fluoro (2'-F)-modification; (c) at least 10% of nucleotides of the oligonucleotide are 2'-F-modified and at least 10% of nucleotides of the oligonucleotide are 2'-O-alkyl-modified, wherein no more than 6 consecutive nucleotides have the same 2'-modification; and wherein the oligonucleotide comprises one or more nucleotide mismatches outside the CBT that is / are not complementary to the corresponding position in the target nucleic; and wherein the mismatch is: at position -7 (N-7), wherein there is a G or C directly 3' to the mismatch and an A or U directly 5' to the mismatch; at position -9 (N-9), wherein there is a G or C directly 3' to the mismatch and an A or U directly 5' to the mismatch; or at position -9 (N-9), wherein there is a G or C directly 3' to the mismatch and an A or U directly 5' to the mismatch, and at position +2 (N+2).

Description

CHEMICAL AND SEQUENCE OPTIMISATION OF ANTISENSE OLIGONUCLEOTIDES FOR ADAR-MEDIATED RNA EDITING FIELD OF THE INVENTION

[0001] The present invention relates to the field of site-directed RNA editing, whereby an RNA sequence is targeted by an antisense oligonucleotide (ASO) for RNA editing of a genetic mutation (“compensatory editing” for repair of disease- causing G-to-A mutations) or for editing of an RNA derived from a wildtype allele to introduce de-novo A-to-G variants (“beneficial editing”). BACKGROUND OF THE INVENTION

[0002] RNA editing is a natural process through which some cells can make discrete changes to specific nucleotide sequences within an RNA molecule in a site- specific way. Unlike DNA editing, the advantage of site-directed RNA editing is that it allows modification of the genetic information in a more precise and efficient manner. Contrary to DNA, RNA is generally quickly degraded and any errors introduced by off-target modifications will be washed out rather than permanently introduced into the modified DNA of a subject. RNA editing may also be less likely to cause an immune reaction since it is an editing mechanism naturally found in humans. Moreover, RNA editing might provide a more natural response than introducing an external, engineered gene.

[0003] Over the years, oligonucleotide therapeutics have been developed to silence, restore or modify the expression of disease-causing or disease-associated genes in, e.g., cancer and (other) genetic disorders. Such therapeutics include, e.g., antisense oligonucleotides (ASOs), small interfering RNA (siRNA) and microRNA (miRNA) that interfere with coding and noncoding RNAs in a sequence specific manner. The relative ease and accuracy with which ASOs can be customized allows virtually any gene to be targeted. As a result, ASOs are the most clinically developed, with several drugs already approved by the U.S. Food and Drug Administration (FDA) and in clinical trials (Cideciyan et al., 2019; Gagliardi and Ashizawa, 2021).

[0004] Site-Directed RNA Editing (SDRE) describes the alteration of an RNA sequence by introducing or removing nucleotides from an RNA or by changing the character of a nucleobase by deamination. To date, the two most useful and most studied types of RNA editing are cytidine (C) to uridine (U) (“C-to-U”) and adenosine(A) to inosine (I) (“A-to-I”) conversions. “A-to-I” editing was initially identified in Xenopus eggs (Bass and Weintraub, 1987; Rebagliati and Melton, 1987). Specifically, “A-to-I” editing changes the informational content of the RNA molecule, as inosine preferentially basepairs with cytidine and is therefore interpreted as guanosine (G) by the translational and splicing machinery. Notably, for therapeutic purposes in higher eukaryotes the most prevalent type of RNA editing is the “A-to-I” conversion, which is catalysed by the adenosine deaminases acting on RNA (ADARs) family. Therefore, ADARs have the effect of introducing a functional adenosine to guanosine mutation on the RNA level. Potentially, this approach may be used to repair genetic defects and alter genetic information at the RNA level.

[0005] Over the years, three vertebrate ADAR genes have been identified, which give rise to several ADAR proteins through alternative promoters and / or splicing (Wulff and Nishikura, 2010). ADAR proteins are expressed across various types of human tissues and can alter, inter alia, splicing and translation machineries, double- stranded RNA (dsRNA) structures as well as the binding affinity between RNA and RNA-binding proteins (Tomaselli et al., 2014). Of the three known ADAR genes, hADAR1 and hADAR2 are expressed in most tissues and encode active deaminases. Human ADAR3 (hADAR3) has been described to only be expressed in the central nervous system and reportedly has no deaminase activity in vitro. Accordingly, the ADAR may be hADAR1, hADAR2 or hADAR3, or any variant thereof. The ability of ADARs to alter the sequence of RNAs has also been used to artificially target RNAs in vitro in cells for RNA editing. Endogenous ADAR proteins can be directed to edit RNA targets of interest by providing short target specific antisense oligonucleotides (ASOs). However, the optimal design of ADAR recruiting ASOs remains to be determined. The present invention takes a targeted approach to optimize the stability of ASOs and their ability to effectively recruit ADARs for improved target editing. This was done by chemically optimizing the oligonucleotide, followed by sequence optimization. Hence, in some embodiments, the oligonucleotide sequence conforms to one or more generalizable rules.

[0006] ASOs are generally short single-stranded synthetic RNA or DNA molecules, which use Watson-Crick base pairing to bind sequence specifically to the target RNA. They can be broadly classified into 1st(Gen 1), 2nd(Gen 2), and 3rd(Gen 3) generation ASOs.

[0007] Gen 1 ASOs were initially employed to inhibit translation of Rous sarcoma virus ribosomal RNA (Stephenson and Zamecnik, 1978). They are characterised inhaving a modified backbone, wherein the nucleotide linkages are modified by sulphur, methyl or amine groups to generate phosphorothioates (PS), methyl- phosphonates, and phosphoramidates, respectively.

[0008] Gen 2 ASOs show increased nuclease stability and affinity for their RNA targets, which has translated to improved potency and therapeutic index in the clinic. Gen 2 ASOs are typically modified using PS backbone modification and additionally carry alkyl modifications at the 2’ position of the ribose. Such 2’-sugar modifications may include 2’-O-methyl (2’-OMe), 2’-fluoro (2’-F), 2’-O-methoxyethyl (2’-MOE) modifications. Hence, these Gen 2 ASOs tend to be less toxic than PS-modified ASOs and have a slightly higher affinity for their target.

[0009] Gen 3 ASOs tend to be even more heterogenous as they include a large number of chemical modifications that aim to further improve binding-affinity, stability, and pharmacokinetics (Quemener et al., 2019). Hence, the diversity of chemical modifications, together with the sequence of the ASO, offers considerable flexibility as relates to the therapeutic approach. That is, depending on their mechanism of action, ASOs can be used to degrade target mRNA, decrease protein levels, modify or correct splicing events, modulate RNA translation or target pathological coding or non-coding RNAs (Quemener et al., 2019).

[0010] To ensure specificity, their sequences are generally complementary or at least partially complementary to the target RNA. However, in the case of site- directed mutagenesis, i.e., “A-to-I” RNA editing, the ASO targeting domain contains a mismatch opposite the targeted adenosine. Further, it is to be noted that several endogenous substrates of ADAR contain mismatches and / or bulges (Thomas and Beal, 2017) and therefore could alter or even improve substrate recognition, if these features are mimicked in the ASO / resulting dsRNA. For instance, ADARs have a preference for adenosines within a certain local sequence context. While most efficient editing seems to occur at adenosines found opposite C (C-A), A-U pairs are also commonly edited (Wong et al., 2001).

[0011] The use of antisense oligonucleotides for site-directed RNA editing has been described (Vogel et al., 2014; Merkle et al., 2019) and ASO-based therapies have been gaining more traction over the past years for use in the treatment of different genetic disorders. Oligonucleotide constructs for site-directed RNA editing are described in WO 2016 / 097212 and WO 2017 / 010556, which utilise endogenous cellular pathways, i.e., endogenous ADAR, to edit endogenous RNA.

[0012] ASOs can be chemically modified to improve their properties. For instance, ASOs can be modified to protect them against nucleases and to increase their effectiveness. While phosphorothioate (PS) modifications seem to have a positive effect on the stability and pharmacokinetics of ASOs, the difference in chirality of PS linkages may have a substantial influence on the ASO's overall property. PS linkages can be found in two stereoisomers, Rp and Sp, and it is known from the art, that Rp and Sp linkages can influence properties such as, e.g., thermal stability, binding affinity, pharmacologic properties, etc., of the ASO.

[0013] New designs for nucleoside analogues are constantly being investigated. ASOs are typically very rich in 2’-F-modifications within the 5’ half, which are generally present as blocks of 2’-F-modifications and uniform block of 2’-O-Methyl- modifications within the 3’ terminus on either side of the CBT. That precise, site- specific RNA editing can be achieved by endogenous ADARs has previously been shown by Merkle et al. (2019). They were able to demonstrate that chemically optimized ASOs can be used to recruit endogenous ADARs to edit endogenous transcripts in a simple and programmable way with almost no off-target editing.

[0014] In WO 2018 / 041973, single-stranded RNA-editing oligonucleotides were provided that do not, in themselves, form an intramolecular hairpin- or stem-loop structure. Other prior art, such as WO 2021 / 071858, relates to RNA-editing oligonucleotides comprising a first and second domain, wherein the first domain comprises one or more 2’-F modifications and the second domain comprises one or more sugars that do not have a 2'-F modification. WO 2022 / 099159 relates to oligonucleotides with a first and second domain, wherein the domains comprise specific percentages of 2’-F modifications and aliphatic substitutions.

[0015] WO 2018 / 041973 relates to single-stranded RNA-editing oligonucleotides for the deamination of a target adenosine by an ADAR enzyme whereby the central base triplet (CBT) of three sequential nucleotides comprises a sugar modification and / or a base modification. It was found that deoxyribose at all three positions of the CBT is well tolerated and provides substantial stabilization against nuclease digestion. However, research in the field of ASO optimisation for A-to-I editing has led not only to the identification of a CBT but also to a more thorough investigation of the region immediate 5’ and 3’ to the CBT. For instance, WO 2021 / 243023 describes modifications 3’ to the CBT (at position +2 of an oligonucleotide comprising the structure [Am]-X1-X2-X3-X4-[Bn], wherein X4corresponds to the +2position). It was found that editing the +2 position can affect the editing rate of the target. Improved editing was observed with a 2’-F modification at the +2 position.

[0016] PCT / EP2017 / 065467 describes that additional mismatches in the formed dsRNA, caused by nucleotides in the oligonucleotide that do not form perfect base pairs with the target RNA are tolerable but not essential for specific targeted editing of the target RNA sequence. Accordingly, newer developments focus not only on the chemical modification of ASOs, but also aim to sequence optimise the oligonucleotide in order to enhance the potency of the oligonucleotide.

[0017] However, despite being a promising technology, few ASOs have been marketed due to difficulties pertaining to, for example, stability and cellular delivery, as well as off-target effects. The success of ASOs is, in part, defined by its ability to affect the target. Hence, to translate ASO-based therapies into a widespread clinical success, one aim is to improve selectivity towards the target, fewer off-target effects and improved ADAR recruitment. SUMMARY OF THE INVENTION

[0018] The present invention provides oligonucleotides (or antisense oligonucleotides, ASOs) with desirable properties for in vitro and in vivo use. The problem solved by the instant invention lies in the provision of improved, chemically and sequence modified ASOs that mediate a functional change from an adenosine (A) to a guanosine (G). Specifically, the invention relates to a modified antisense oligonucleotide suitable for editing an adenosine in a target nucleic acid to inosine (A-to-I editing).

[0019] The solution to the technical problem is achieved by the embodiments described herein and defined by the appended claims.

[0020] In a first aspect, the present invention provides a modified antisense oligonucleotide suitable for editing an adenosine in a target nucleic acid to inosine (A-to-I editing), wherein the oligonucleotide comprises: (I) a nucleic acid sequence of 25 to 70 nucleotides (nt) that is an antisense sequence substantially complementary to the target nucleic acid, (II)a central base triplet (CBT) (5’- N+1eN0fN-1g-3’) with the central nucleotide (N0) directly opposite the target adenosine to be edited when the antisense oligonucleotide is hybridised to the target nucleic acid, and (III) the following core sequence: 5’-aN+4bN+3cN+2dN+1eN0fN-1gN-2hN-3iN-4jN-5kN-6lN-7mN-8nN-9-3’ ;and wherein: (a) at least two of the three nucleotides of the CBT are chemically modified at the 2' position of the sugar moiety, are deoxyribonucleosides, or a combination thereof and wherein d and e are internucleoside linkage modifications; (b) the N-2nucleotide carries a 2’-O-alkyl-modification; and wherein the N-3nucleotide carries a 2´-fluoro (2’-F)-modification; (c) at least 10% of nucleotides of the oligonucleotide are 2’-F-modified and at least 10% of nucleotides of the oligonucleotide are 2’-O-alkyl-modified, wherein no more than 6 consecutive nucleotides have the same 2’-modification; and wherein the oligonucleotide comprises one or more nucleotide mismatches outside the CBT that is / are not complementary to the corresponding position in the target nucleic; and wherein the mismatch is: i. at position -7 (N-7), wherein there is a G or C directly 3’ to the mismatch and an A or U directly 5′ to the mismatch; ii. at position -9 (N-9),wherein there is a G or C directly 3’ to the mismatch and an A or U directly 5’ to the mismatch; or iii. at position -9 (N-9), wherein there is a G or C directly 3’ to the mismatch and an A or U directly 5’ to the mismatch, and at position +2 (N+2).

[0021] In a second aspect provided herein is a composition comprising the antisense oligonucleotide of the invention.

[0022] In a third aspect provided herein is a method for site-directed A-to-I editing of a target nucleic acid, comprising providing to a cell or subject an oligonucleotide according to the invention, or composition according to the invention.

[0023] In a fourth aspect provided herein is a method of treating a disease or condition associated with a point mutation in a subject by administering to the subject a therapeutically effective amount of an oligonucleotide according to the invention, or a composition according to the invention. BRIEF DESCRIPTION OF DRAWINGS

[0024] The figures shown in the following are merely illustrative and shall describe the present invention in a further way. The figures shall not be construed to limit the present invention thereto.

[0025] Fig. 1 represents graphs showing (A) the principle of chemistry and sequenced optimised oligonucleotide specific to the human SERPINA1 E342K RNA target. (B) In vitro editing efficacy of SERPINA1 E342K specific oligonucleotide. (C) Chemical modification pattern and nucleotide sequences of human SERPINA1E342K targeting, 59mer oligonucleotides. (D) Target editing yields (%) of SERPINA1 PIZZ of 59mer oligonucleotides.

[0026] Fig.2 shows (A) chemical modification pattern and nucleotide sequences of human SERPINA1 E342K targeting, 40mer oligonucleotides. (B) Target editing yields (%) of SERPINA1 E342K specific, 40mer oligonucleotides.

[0027] Fig. 3 represents (A) chemical modification pattern and nucleotide sequences of PDE6A targeting, 42mer oligonucleotides. (B) Target editing yields (%) of PDE6A targeting oligonucleotides.

[0028] Fig. 4 represents a graph showing the editing yields (%) of SCNA A53T targeting oligonucleotides. DETAILED DESCRIPTION Terminology

[0029] In order that the present invention may be more readily understood, certain terms are first defined. Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclature used in connection with, and techniques of, cell and tissue culture, biochemistry, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art.

[0030] 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 element" means one element or more than one element, e.g., a plurality of elements.

[0031] The terms "about" and "approximately" may be understood to permit standard variation as would be understood by those of ordinary skill in the art.

[0032] The term “including” or “having” is used herein to mean, and is used interchangeably with, the phrase “including, but not limited to” or “having, but not limited to”. Likewise, the term “comprising” is used herein to mean, and is used interchangeably with, the phrase “comprising, but not limited to".

[0033] The term “mismatch” as used herein refers to opposing nucleotides in a double stranded nucleic acid complex, which does not form perfect base pairs according to the Watson-Crick base pairing rules. Mismatch base pairs are G-A, C- A, U-C, A-A, G-G, C-C, U-U base pairs. Wobble base pairs are: G-U, l-U, l-A, and l-C base pairs. For the recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0034] As used herein, the term "nucleic acid" is intended to include any DNA molecules (e.g., cDNA or genomic DNA) and any RNA molecules (e.g., mRNA) and analogues of the DNA or RNA generated using nucleotide analogues. Oligonucleotides can be single-stranded (ss) or double-stranded (ds). A single- stranded oligonucleotide can have double-stranded regions (formed by portions of the single-stranded oligonucleotide). A double-stranded oligonucleotide can have single- stranded regions, for example, at regions where the two oligonucleotide chains are not complementary to each other. Each component of the DNA or RNA can be modified and categorized by modification of (1) the internucleoside linkage, (2) the deoxyribose / ribose, and / or (3) the nucleobase.

[0035] The term “nucleobase” or “base” refers to biological building blocks that can form nucleosides, which, in turn, may be components of nucleotides. Naturally occurring bases are generally guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and uracil (U), which are derivatives of purine or pyrimidine. Cytosine, thymine, and uracil are pyrimidine bases that are generally linked to the backbone through their 1 - nitrogen. Adenine and guanine are purine bases and generally linked to the backbone through their 9-nitrogen. It should be understood that naturally and non-naturally occurring base analogues are also included and that the term “nucleobase” also includes “modified nucleobases”.

[0036] Within the context of this invention, the term "modified nucleobase" and "modified base" may be used interchangeably with one another and / or with the term “nucleobase”. Hence, a nucleobase may be a nucleobase, which comprises a chemical modification. In one embodiment, the modified nucleobase is capable of increasing hydrogen bonding, base pair stacking interactions and / or stabilizing a nucleic acid complex. In one embodiment, the modified nucleobase is not able to form a traditional Watson-Crick base pairing. In some embodiments, a modified nucleobase is 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 oligonucleotides refer to a nucleobase that is not A, T, C, G or U. Modifications include but are not limited to nonstandard nucleobases 5-methyl-2’-deoxycytidine (m5C), pseudouridine(pU), dihydrouridine, inosine (I), and 7-methylguanosine. Other modifications may include nucleobase replacement by (N) heterocycles (e.g., nebularine) or aromatic rings that stack well in the RNA duplex, such as, e.g., a Benner’s base Z (and / or analogues) or 8-oxo-adenosine (8-oxo-A). As used herein, the term “Benner’s base Z” refers to the pyrimidine analogue 6-amino-5-nitro-3-(1′-β-D-2′-deoxyribofuranosyl)- 2(1H)-pyridone (dZ). In one embodiment, a modification includes the introduction of nucleobase analogues or simple heterocycles that boost editing. As used herein, and as commonly understood by the skilled person in the art, the expression “derivative thereof” refers to a derivative of a (modified) nucleobase, nucleoside or nucleotide. For example, a derivative may be a corresponding nucleobase, nucleoside or nucleotide that has been chemically derived from said nucleobase, nucleoside or nucleotide. For instance, a derivative of deoxycytidine may include fluoro-modified deoxycytidine, 5-methyl-2’-deoxycytidine (m5C), or ribocytidine.

[0037] The term "nucleoside(s)" refers to a moiety wherein a nucleobase or a modified nucleobase is covalently bound to a sugar or a modified sugar. In some embodiments, a “nucleoside” refers to a nucleoside unit in an oligonucleotide or a nucleic acid. The term "nucleoside(s)" encompasses all modified versions and derivatives “modified nucleobases”.

[0038] The term "nucleotide(s)" as used herein refers to a monomeric unit of a polynucleotide that consists 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 one specific embodiment, the modified linkage is a PS linkage. In some embodiments, a “nucleotide” refers to a nucleotide unit in an oligonucleotide or a nucleic acid. The term "nucleotide(s)" encompasses all modified versions and derivatives of “nucleosides” and “modified nucleobases”.

[0039] The term “oligonucleotide(s)“ as used herein is defined as is generally understood by the skilled person as a molecule including two or more covalently linked nucleosides. They can comprise DNA and / or RNA. The oligonucleotides may have a backbone comprising deoxyribonucleotides and / or ribonucleotides.

[0040] The term “internucleoside linkage” refers to a linkage between adjacent nucleosides. “Internucleoside linkage” and “linkage” may be used interchangeably. Linkages may be continuous (consecutive; several in a consecutive order) or discontinuous (interrupted). As used herein, the term “discontinuous” or “interrupted”means that there are not more than, e.g., 7, 8, 9, 10 or more consecutive internucleoside linkage modifications of the same modification. In some embodiments, the naturally occurring PO linkages are replaced by modified internucleoside linkages. In some embodiments, the linkage is a non-natural internucleoside linkage.

[0041] As used herein the term “stereopure” or “stereorandom” refers to chemically modified oligonucleotides. Specifically, the term “stereopure” refers to oligonucleotides that are chirally pure (or “stereochemically pure”). The term “stereorandom” refers to racemic (or “stereorandom”, “non-chirally controlled”) oligonucleotides. Hence, the oligonucleotides of the invention comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more stereorandom internucleoside linkages (mixture of Rp and Sp linkage phosphorus at the internucleoside linkage, e.g., from traditional non-chirally controlled oligonucleotide synthesis). In one embodiment, an internucleoside linkage is a phosphorothioate (PS) linkage. In one embodiment, an internucleoside linkage is a stereorandom PS linkage. In one embodiment, an internucleoside linkage is a chirally controlled PS linkage. In one embodiment, an internucleoside linkage is not chirally controlled. In one embodiment, an internucleoside linkage is not a chirally controlled PS linkage.

[0042] As used herein the term “antisense oligonucleotide” or “ASO” refers to a strand of nucleotide analogue that hybridizes with the complementary (target) RNA in a sequence-specific manner. This may be via Watson-Crick base pairing. The ASO may be chemically modified. The ASO may be sequence modified. The terms “antisense oligonucleotide” and “oligonucleotide” may be used interchangeably.

[0043] As used herein, the term “target RNA” refers to an RNA, which is subject to the editing process, and “targeted” by the respective ASO(s) or composition(s) of the invention.

[0044] As used herein, the term “off-target” or “off-targeting” refers to non-specific and / or unintended genetic modification(s) of the target. Off-target editing may include unintended point mutations, deletions, insertions, inversions, and translocations. For instance, off-target editing may arise from the promiscuous reactivity of the deaminase enzymes.

[0045] The term "modified sugar" refers to a moiety that can replace a naturally occurring sugar. A modified sugar mimics the spatial arrangement, electronic properties, or some other physicochemical property of a sugar. The naturally occurring sugar is the pentose (five carbon sugar) deoxyribose (to form DNA) orribose (to form RNA), though it should be understood that naturally and non-naturally occurring sugar analogues are also included. For example, other sugars may comprise, e.g., C4 sugars, C5 sugars and / or C6 sugars. In some embodiments, a modified sugar is substituted ribose or deoxyribose. In some embodiments, a modified sugar comprises a 2'-modification. Examples of useful 2’-sugar modifications, e.g., 2’- ribose, 2’-deoxyribose, 2’-arabinose etc., are widely used in the art and described herein. Those skilled in the art, after reading the present disclosure, will appreciate that various types of 2’-sugar modifications are known and can be utilized in accordance with the present disclosure. In some embodiments, a modified sugar is a bicyclic sugar (e.g., a sugar used in LNA, BNA, etc.). In some embodiments, a modified sugar is an LNA sugar. The term “locked nucleic acid” (LNA) or term “locked nucleic acids” (LNAs) are also known as bridged nucleic acid (BNA) and refers to modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon. In some embodiments, a sugar modification is 2’-OMe, 2'-O-methoxy-ethyl (2’-MOE), 2’-F, 5’-vinyl, or S-constrained ethyl (S-cEt). In one embodiment, a 2’-modification is a C2-stereoisomer of 2’-F-ribose. In one embodiment, a 2'-modification is 2’-F. In one embodiment, a 2'-modification is 2'- FANA. In one embodiment, a modified sugar is a sugar of morpholino. In one embodiment, the oligonucleotide comprises, e.g., an UNA (unlocked nucleic acid), a PMO (phosphorodiamidate linked morpholino) or a PNA (peptide nucleic acid). Hence, in one embodiment, the nucleic acid analogue is a PNA (peptide nucleic acid). In one embodiment, the nucleic acid analogue is PMO (phosphorodiamidate linked morpholino). In one embodiment, a 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, a 2’-modification is 2'-OMe. In some embodiments, a 2'-modification is 2'-MOE. In some embodiments, a 2'-modification is 2'-OR, wherein R is substituted C1-10 aliphatic. In some embodiments, in the context of oligonucleotides, a modified sugar is a sugar that is not ribose or deoxyribose as typically found in natural RNA or DNA (e.g., arabinose). In some instances, the 2’-O-alkyl modification is not a 2'-MOE.

[0046] The term “FANA” or “FANA-modified” refers to 2'-fluoroarabinoside modified nucleobases and / or oligonucleotides comprising such nucleobases. For example, the expression “FANA-cytidine” refers to a cytidine that comprises a 2'-fluoro-beta-D- arabinonucleic acid sugar modification. Within the context of this invention, the expression “a derivate thereof” refers to a corresponding nucleotide(s) oroligonucleotide(s) that has been chemically derived from said nucleotide or oligonucleotide(s).

[0047] As used herein, the term “complementary” or “substantially complementary” refer to nucleic acid sequences, which, due to their complementary nucleotides, are capable of specific intermolecular base-pairing. The oligonucleotide may comprise a nucleic acid sequence complementary to a target sequence, e.g., SERPINA1, or any other target sequence. The ASO may be self-complementary. The ASO may be complementary to a coding or non-coding sequence. Perfect (e.g., 100%) complementarity is not required and one or more wobbles (wobble base pairing), bulges, or mismatches, etc. are tolerated. The one or more wobbles, bulges, or mismatches, etc. may be within and / or outside the CBT. According to the invention, the oligonucleotide comprises one or more mismatches outside the CBT. In one embodiment, the oligonucleotide comprises one or more wobbles outside the CBT. The oligonucleotides of the invention also include a mismatch opposite the target adenosine that is to be edited. Hence, the overall complementarity of the ASOs is not 100%.

[0048] The term "mutation" as used herein, refers to a substitution of a residue with another residue within a sequence, e.g., a nucleic acid sequence or amino acid sequence, or to a deletion or insertion of one or more residues within a sequence, e.g., point mutation. Mutations are typically described herein by identifying the original residue and by identifying of the newly substituted residue. Notably, the invention is not limited to correcting mutations, as it may instead be useful to change a wildtype sequence into a mutated sequence using the ASOs of the invention. Various methods for making amino acid substitutions are well known in the art, and are provided by, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)).

[0049] As used herein, the term “beneficial editing” refers to the editing of a target sequence (or base) derived from a wildtype allele (not a mutated allele) in order to, e.g., modulate the function of a wildtype protein in a useful way to prevent or treat a disease. For example, beneficial editing may include sites, such as STAT1 Y701, that are not causes for genetic diseases but rather represent wildtype protein sites. These sites may be changed (no underlying G-to-A mutation) to alter the function of the wildtype protein.

[0050] The term “compensatory editing” refers to the modification of RNA nucleotides to change and correct one or more detrimental or unfavourable changesin the RNA sequence when compared to wildtype, e.g., a compensatory A-to-I change could help to functionally compensate for an otherwise non-editable mutation to ameliorate a disease phenotype.

[0051] The term "adenosine deaminase(s)" or “adenosine deaminase(s) acting on RNA” [ADAR(s)], as used herein, refers to any (poly)peptide, protein or protein domain or fragment thereof capable of catalysing the hydrolytic deamination of adenosine to inosine. The term thus not only refers to full-length and wild type ADARs but also to a functional fragment or a functional variant of an ADAR. In some embodiments, the ADAR is an (endogenous) adenosine deaminase catalysing the deamination of adenosine to inosine or deoxy-adenosine to deoxyinosine. In some embodiments, the ADAR catalyses the deamination of adenine or adenosine in deoxyribonucleic acid (DNA) or in ribonucleic acid (RNA). The ADAR may be a human ADAR. The ADAR may be an endogenous ADAR. Accordingly, in some embodiments, the ADAR is an endogenous human ADAR1, ADAR2 or ADAR3 (hADAR1, hADAR2 or hADAR3), or any fragment or isoform(s) thereof (e.g., hADAR1 p110 and p150).

[0052] 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 decreased by inducing an immune response in the subject through the administration of a pharmaceutical composition.

[0053] As used herein, the term “effective amount” in the context of administering a therapy to a subject refers to the amount of a therapy which has a prophylactic and / or therapeutic effect(s).

[0054] As used herein, the term “in combination” in the context of the administration of two or more therapies to a subject, refers to the use of more than one therapy (e.g., more than one prophylactic agent and / or therapeutic agent). The use of the term "in combination" does not restrict the order in which therapies are administered to a subject. For instance, one or more ASOs may be used in combination.

[0055] As used herein, the terms “prevent”, “preventing” and “prevention” refer to the inhibition of the development or onset of a disease or symptoms thereof. In one embodiment, it relates to the administration of the compound to a patient who is known to have an increased risk of developing a certain condition, disorder, or disease.

[0056] As used herein, the terms “treat”, “treatment”, and “treating” refer to the halting, ceasing the progression of, or (partially) reversing particular symptoms of a disease or disorder. Beneficial or desired clinical results include, but are not limitedto, alleviation of symptoms; diminishment of the extent of a condition, disorder, or disease stabilized (i.e., not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression; amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0057] The terms “subject” or “patient” are used interchangeable and relate to an animal (e.g., mammals) that may need administration of the compound of the invention in the field of human or veterinary medicine. In specific embodiments, the subject is a human. The subject may be administered the oligonucleotide of the invention for beneficial editing. The subject may be administered the oligonucleotide of the invention for compensatory editing.

[0058] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatine, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The formulation should suit the mode of administration.

[0059] 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 the present disclosure; other, suitable methods and materials known in the art can also be used. Oligonucleotides

[0060] Described herein are, inter alia, chemically and sequence modified antisense oligonucleotides (ASOs). The invention includes modifications of the 2’-sugar and internucleoside linkages of the oligonucleotide together with nucleotide sequence modifications to introduce one or more mismatches with the target RNA. These modifications are useful in stabilising ASOs, reducing their off-target editing, and improving their overall target editing efficacy. Since the one or more modificationscan be synthetically transferred to various oligonucleotide sequences, such modifications have the potential to improve the editing efficacy of oligonucleotides with different target specificities. The ASOs of the invention can thus be used for several purposes associated with “A-to-I” conversions, which means that the ASOs provided herein are not just limited to correcting G-to-A mutations but are also useful in changing a wildtype sequence into a mutated sequence to modulate protein expression and / or function (“beneficial editing”). Thus, the oligonucleotides may be used as active agents to treat genetic disorders or diseases associated with one or more G-to-A mutations or change wildtype sequences.

[0061] Described herein are ASOs with optimized editing efficiency for a variety of target RNA sequences. The oligonucleotides of the invention may comprise different types of chemical modifications, e.g., 2’-sugar modifications and internucleoside linkage modifications, along with nucleotide sequence modifications to introduce mismatches relative to the target RNA. Oligonucleotides according to the claims promote increased target editing yields.

[0062] The oligonucleotides according to the claimed invention may have a mixture of different modifications at the 2’-position of the sugar residue but preferably not stretches of more than 6 nucleotides with the same 2’-modification. Avoiding uniform blocks of more than 6 nucleotides with the same 2’-modification prevents a strong loss of editing activity with natural ADARs. In addition to this design feature, the modified oligonucleotides of the invention may have at least two of the three nucleotides of the CBT modified at the 2’-position of the sugar base or being deoxyribonucleosides, which permits added stabilization against nuclease digestion. Accordingly, it is preferred that a mixture of 2’-F- and 2’-O-alkyl-modifications is used and a minimum of 10% of each is preferred. According to the invention, the oligonucleotides comprise one or more mismatches outside of the CBT with respect to the target nucleic acid. Such oligonucleotides may show enhanced editing, specifically when the mismatches are at position N-7, at position N-9or at positions N-9and N+2and wherein mismatches at N-7or N-9are placed in the context of a G or C 3’ to the mismatch and an A or U 5’ to the mismatch. When combined, these features confer high levels of lysosomal stability and RNA editing efficacy of the oligonucleotides. Hence, the oligonucleotides of the invention are modified and designed accordingly.

[0063] The core oligonucleotide comprises the sequence: 5’- a N b c d +4 N+3 N+2 N+1 e N f 0 N g -1 N h -2 N i -3 N j -4 N k -5 N l -6 N m -7 N n -8 N-9 -3’ ; and contains a specific patternsof 2’-modification and internucleoside linkages, as well as nucleotide sequence modifications, which contribute to the advantageous properties of the oligonucleotides. Hence, according to the invention the oligonucleotide is suitable for editing an adenosine in a target nucleic acid to inosine (A-to-I editing), wherein the oligonucleotide comprises: (I) a nucleic acid sequence of 25 to 70 nucleotides (nt) that is an antisense sequence substantially complementary to the target nucleic acid, (II) a central base triplet (CBT) (5’- N+1eN0fN-1g-3’) with the central nucleotide (N0) directly opposite the target adenosine to be edited when the antisense oligonucleotide is hybridised to the target nucleic acid, and (III) the following core sequence: 5’-aN+4bN+3cN+2dN+1eN0fN-1gN-2hN-3iN-4jN-5kN-6lN-7mN-8nN-9 -3’ ; and wherein: (a) at least two of the three nucleotides of the CBT are chemically modified at the 2' position of the sugar moiety, are deoxyribonucleosides, or a combination thereof and wherein d and e are internucleoside linkage modifications; (b) the N-2nucleotide carries a 2’-O-alkyl-modification; and wherein the N-3nucleotide carries a 2´-fluoro (2’-F)-modification; (c) at least 10% of nucleotides of the oligonucleotide are 2’-F-modified and at least 10% of nucleotides of the oligonucleotide are 2’-O-alkyl-modified, wherein no more than 6 consecutive nucleotides have the same 2’-modification; and wherein the oligonucleotide comprises one or more nucleotide mismatches outside the CBT that is / are not complementary to the corresponding position in the target nucleic; and wherein the mismatch is: i. at position -7 (N-7), and wherein there is a G or C directly 3’ to the mismatch and an A or U directly 5′ to the mismatch; ii. at position -9 (N-9),and wherein there is a G or C directly 3’ to the mismatch and an A or U directly 5’ to the mismatch; or iii. at position -9 (N-9),and wherein there is a G or C directly 3’ to the mismatch and an A or U directly 5’ to the mismatch and at position +2 (N+2).

[0064] In one embodiment, the oligonucleotide is suitable for A-to-I editing and comprises: (I) a nucleic acid sequence of 25 to 70 nucleotides (nt) that is an antisense sequence substantially complementary to the target nucleic acid, (II) a central base triplet (CBT) (5’- N+1eN0fN-1g-3’) with the central nucleotide (N0) directly opposite thetarget adenosine to be edited when the antisense oligonucleotide is hybridised to the target nucleic acid, and (III) the following core sequence: 5’-aN+4bN+3cN+2dN+1eN0fN-1gN-2hN-3iN-4jN-5kN-6lN-7mN-8nN-9-3’ ; and wherein: (a) at least two of the three nucleotides of the CBT are chemically modified at the 2' position of the sugar moiety, are deoxyribonucleosides, or a combination thereof and wherein d and e are internucleoside linkage modifications; (b) the N-2 nucleotide carries a 2’-O-alkyl-modification; and wherein the N-3 nucleotide carries a 2´-fluoro (2’-F)-modification; (c) at least 10% of nucleotides of the oligonucleotide are 2’-F-modified and at least 10% of nucleotides of the oligonucleotide are 2’-O-alkyl-modified, wherein no more than 6 consecutive nucleotides have the same 2’-modification; and wherein the oligonucleotide comprises one or more nucleotide mismatches outside the CBT that is / are not complementary to the corresponding position in the target nucleic; and wherein the mismatch is at position -9 (N-9) and / or at position -1 (N-1). In one embodiment, the mismatch is at position -1 (N-1). In one embodiment, the target sequence is SNCA. In one embodiment, the target sequence is SNCA A53T.

[0065] The oligonucleotides of the invention do benefit from having a base level of internucleoside linkage modifications, and oligonucleotides having at least 15% modification are preferred as this is beneficial to achieve good RNA editing. Hence, in one embodiment, the linkage modification content is at least 15 %; and linkages h and i are not phosphorothioate (PS) linkages. In one embodiment, the regions 3’ and 5’ to the CBT do not contain more than a total of 6 deoxyribonucleosides and the internucleoside linkage modification content is at least 30%. In one embodiment, the regions 3’ and 5’ to the CBT have a total deoxyribonucleoside content of 5-50%.

[0066] Reversal of classical disease-causing point mutations require metabolically stable ASOs that reduce dosing frequency. While embodiments with low content of 2’- modification are well conceivable, in order to provide maximum stability, it is preferred that each RNA nucleoside is replaced by either a 2’-modified RNA or DNA. It is envisaged that, provided blocks of no more than 6 consecutive nucleotides have the same 2’-modification, the oligonucleotides can tolerate high percentages of 2’- modifications without detrimental loss of activity. Accordingly, in one embodiment, 20- 100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100% , 80-100%, or 90-100% of nucleotides are deoxyribonucleosides (DNA) or 2’-modified. In one embodiment, 20- 100% of nucleotides are DNA or 2’-modified. In one embodiment, 50-100% of nucleotides are DNA or 2’-modified nucleotides. In one embodiment, 100% of nucleotides are DNA or 2’-modified nucleotides. In one embodiment, 30-95%, 40- 95%, 40-90%, 50-95%, 50-90%, 60-95% or 60-90% of nucleotides are DNA or 2’-modified nucleotides. In one embodiment the above percentages are satisfied with only 2’-modified nucleotides and no DNA.

[0067] According to the invention, the oligonucleotides comprise modifications at the 2’-position on nucleotides using different modifying groups. In one embodiment, 20-70% of nucleotides are 2’-F-modified. In one embodiment, 35-65% of nucleotides are 2’-F-modified. In one embodiment, 20-60% of nucleotides are 2’-O-methyl (2’- OMe)-modified. In one embodiment, 25-55% of nucleotides are 2’-OMe-modified.

[0068] It is envisaged that the modified oligonucleotides of the invention do not require all of the internucleoside linkages to be modified, provided that a minimum level of internucleoside modification is incorporated and provided that linkages d and e of the core oligonucleotide sequence are modified. In one embodiment, the internucleoside linkage modification content is at least 15 %. In one embodiment, the internucleoside linkage modification content is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, or 90%. In one embodiment, no more than 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, or 30% of the linkages are internucleoside linkage modifications.

[0069] Internucleoside linkage modifications, such as PS linkages, tend to have a positive effect on the pharmacokinetics as well as stability, protein binding, and intracellular localization of ASOs. However, at the same time, it is desirable to reduce overall PS content to reduce, e.g., toxicity and non-specific protein binding. In one embodiment, (a) no more than 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30% or 20% of the linkages outside the CBT are internucleoside linkage modifications; or (b) 15-90% of the linkages are internucleoside linkage modifications, preferably wherein 40-80%, most preferably 45-60%, of the linkages are internucleoside linkage modifications. In one embodiment, no more than 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30% or 20% of the linkages outside the CBT are internucleoside linkage modifications. In one embodiment, 15-90% of the linkages are internucleoside linkage modifications, preferably wherein 40-80%, 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 no more than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20%. In one embodiment, the internucleoside linkage modification content is 10-90%, 15-90%, 15-80%, 15-70%, 15-60%, 20-90%, 10-80%, 20-80%, 25-80%, 30-80%, 30-90%, 40-90%, 40-80%, 40-70%, 45-90%, 45-85%, 45-75%, 45-70%, 45-60% or 45-55%. In one embodiment, 15-90% of the linkages are internucleoside linkage modifications. In one embodiment, 40-80% of the linkages are internucleoside linkage modifications. In one embodiment, 45-60% of the linkages are internucleoside linkage 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%.

[0070] Oligonucleotides of different lengths may require a different mixture of particular 2’-modifications and internucleoside linkage modifications in order to provide optimal RNA editing. The shorter the oligonucleotide, the better may be the endosomal escape and the lower the cytotoxicity. Also, shorter oligonucleotides may experience higher specificity. On the other hand, while longer oligonucleotides may bind stronger or faster to their respective RNA target, editing-boosting bulges, mismatches and wobbles may also work better in long oligonucleotides. As a result, there is a benefit and / or trade-off for both long and short oligonucleotides of the invention. Accordingly, the oligonucleotides of the invention may be of varying lengths. In some instances, the oligonucleotides may range from about 25-70nt in length, e.g., about 25-39nt or about 40-60nt in length. In one embodiment, the oligonucleotide has a length of 25-60nt. In one embodiment, the oligonucleotide has a length of 40-59nt. In one embodiment, the oligonucleotide has a length of 25-59nt. In one embodiment, the oligonucleotide has a length of 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, 70nt. In one embodiment, the oligonucleotide has a length of 25-30nt. In one embodiment, the oligonucleotide has a length of 25-45nt. In one embodiment, the oligonucleotide has a length of 25nt. In one embodiment, the oligonucleotide has a length of 40nt. In one embodiment, the oligonucleotide has a length of 42nt. In one embodiment, the oligonucleotide has a length of 59nt. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention. In one embodiment, the oligonucleotide has a length of 42 or 59 nucleotides and wherein outside of the CBT no more than 3 nucleotides are deoxyribonucleotides.

[0071] In one embodiment, the oligonucleotide comprises an internucleoside linkage modification selected from the group consisting of phosphorothioate (PS), 3'- methylenephosphonate, 5'-methylenephosphonate, 3'-phosphoroamidate, 2'- 5'phosphodiester, and phosphoryl guanidine (PN). In a preferred embodiment, theinternucleoside linkage modification is a PS linkage. In one embodiment, the internucleoside linkage modification is a 3'-methylenephosphonate linkage. In one embodiment, the internucleoside linkage modification is a 5'-methylenephosphonate linkage. In one embodiment, the internucleoside linkage modification is a 3'- phosphoroamidate linkage. In one embodiment, the internucleoside linkage modification is a 2'-5'-phosphodiester linkage. In one embodiment, the internucleoside linkage modification is a PN linkage. In one embodiment, the at least one internucleoside linkage modification is PS. In one embodiment, the oligonucleotide contains a continuous stretch of PS linkages. In one embodiment, the continuous stretch of PS linkages is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more linkages long. In one embodiment, the continuous stretch of PS linkages is 25 linkages long. In one embodiment, the continuous stretch of PS linkages is 6 linkages long. In one embodiment, the continuous stretch of PS linkages is 3 linkages long.

[0072] The positioning of internucleoside linkages within the oligonucleotide plays an important role when determining a balance between high editing yields and a long half-life. At the same time, the need for a carefully considered placement of backbone modifications reflects the sensitivity of the CBT and bases surrounding the CBT for subtle chemical modifications on the ribose, the nucleobase or the linkage. For example, positions h and i may be chemically modified or unmodified. However, it is preferred that PS linkages are not placed at positions h and i, which may be detrimental to the editing efficacy of the constructs. Accordingly, in one embodiment, linkage h and i are not chemically modified. In one embodiment, linkage h is not chemically modified. In one embodiment, linkage i is not chemically modified. In some embodiments, linkages h and i are phosphate (PO) linkages. In some embodiments, linkages h and i are not phosphorothioate (PS) linkages. In one embodiment, up to three linkages selected from the group consisting of linkages b, c, f, g and j are also PS linkages. It is, however, excluded that all linkages a to j are PS linkages. In especially preferred embodiments the linkage f is a PS linkage. In especially preferred embodiments, linkages a, d and e are PS linkages whereas linkages h and i are PO linkages.

[0073] The CBT (5’- N+1eN0fN-1 - 3’) may carry different modifications and permutations of the various modifications. In certain embodiments, the CBT is chemically modified. That is, positions N+1, N0and / or N-1may carry modifications at the 2’ position. In one embodiment, only one position within the CBT is chemicallymodified. In one embodiment, two positions within the CBT are chemically modified. In one embodiment, all positions within the CBT are chemically modified. According to one embodiment, each of the three nucleosides of the CBT is either singularly or a combination of: (a) a deoxyribonucleotide; and / or (b) 2’-FANA-modification; and / or (c) 2’-O-methyl-modification; 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.

[0074] In some embodiments, position N+1 is 2'-F-RNA, 2'-FANA or DNA. In one embodiment, N+1 is 2'-FANA. In one embodiment, N+1 is DNA. In some embodiments, position N0 is 2'-FANA or DNA. In some embodiments, N-1 is 2'-FANA or DNA. CBT modification may comprise any permutation of the modifications described above. In one embodiment, N0 is deoxycytidine or FANA-cytidine. In one embodiment, N0 is DNA. In one embodiment, N0 is deoxycytidine. In one embodiment, N0 is FANA- cytidine. Other modifications may include nucleobase replacement by (N) heterocycles or aromatic rings that stack well in the RNA duplex, such as, e.g., a Benner’s base Z (dZ) (and / or analogues) or 8-oxo-adenosine (8-oxo-A). Hence, in one embodiment, N0 is a Benner’s base. In one embodiment, N0 is 8-oxo-adenosine.

[0075] The oligonucleotides of the invention may be modified in a way to not include uniform blocks or a continuous stretch of the same 2’-sugar modification. For example, the oligonucleotides may be modified such that they do not comprise continuous stretches or uniform blocks of nucleotides carrying the same chemical modification (i.e., avoidance of a block-like modification structure).

[0076] In one embodiment, the oligonucleotide is not uniformly modified. In one embodiment, the oligonucleotide contains no uniform blocks and / or no block-like modification structure. In one embodiment, the oligonucleotide does not comprise continuous stretches or uniform blocks of nucleotides carrying the same chemicalmodification at the 2’ position of the sugar moiety. A “block” or “stretch” may, e.g., not comprise more than 4, 5 or 6 nucleotides with the same 2’-sugar modification. In some instances, the block or stretch may be shorter or longer. In one embodiment, the oligonucleotide contains only 1 block of no more than 6, 5, 4, or 3 nucleotides with the same 2’-sugar modification. In one embodiment, the oligonucleotide contains 2 blocks, separated by one or more oligonucleotides having a different 2’-sugar modification. In order embodiments, the oligonucleotides comprise at least 1 block of nucleotides with the same 2’-sugar modification. In another embodiment, the oligonucleotide comprises 1, 2, 3, or more blocks of nucleotides with the same 2’- sugar modification.

[0077] In one embodiment, no more than 6 consecutive nucleotides have the same 2’-modification. In one embodiment, no more than 5 consecutive nucleotides have the same modification. In one embodiment, no more than 4 consecutive nucleotides have the same modification. In one embodiment, no more than 3 consecutive nucleotides have the same modification. In one embodiment, no more than 2 consecutive nucleotides have the same modification.

[0078] In one embodiment, no more than 6 consecutive nucleotides are 2’-H (DNA) modified. In one embodiment, no more than 5 consecutive nucleotides are 2’-H- modified. In one embodiment, no more than 4 consecutive nucleotides are 2’-H- modified. The 2’-sugar modification may be 2’-ribose. In one embodiment, no more than 6 consecutive nucleotides are 2’-H (DNA) modified. In one embodiment, no more than 5 consecutive nucleotides are 2’-H-modified. In one embodiment, no more than 4 consecutive nucleotides are 2’-H-modified. In one embodiment, no more than 6 consecutive nucleotides are 2’-F-modified. In one embodiment, no more than 5 consecutive nucleotides are 2’-F-modified. In one embodiment, no more than 4 consecutive nucleotides are 2’-F-modified. In one embodiment, no more than 6 consecutive nucleotides are 2’-O-alkyl-modified. In one embodiment, no more than 5 consecutive nucleotides are 2’-O-alkyl-modified. In one embodiment, no more than 4 consecutive nucleotides are 2’-O-alkyl-modified, optionally wherein no more than 4 consecutive nucleotides are 2’-OMe-modified. In one embodiment, the oligonucleotide comprises 2, 3, 4, 5, or 6 consecutive nucleotides with the same 2’- modification, e.g., 5 consecutive nucleotides are 2’-F-modified.

[0079] The oligonucleotide of the invention may contain some “continuous stretch(es)” or “uniform block(s)” of a certain length. In one embodiment, the size or length of the “continuous stretch(es)” or “uniform block(s)” is 2, 3, 4, 5, or 6 nucleotideslong. In one embodiment, the size or length of the “continuous stretch(es)” or “uniform block(s)” is no more than 2, 3, 4, 5, or 6 nucleotides long. In one embodiment, the oligonucleotide comprises no more than 2, 3, 4, 5, or 6 consecutive nucleotides comprising a 2’-F modification. In one embodiment, the oligonucleotide comprises no more than 2, 3, 4, 5, or 6 consecutive nucleotides comprising a 2’-OMe modification. In one embodiment, one or more uniform blocks are interrupted. Interruption can take place 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 interrupted, preferably by 2’-OMe-modified nucleotides. In one embodiment, one or more uniform blocks of 2’-OMe-modified nucleotides are interrupted, preferably by 2’-F-modified nucleotides. In some embodiments the blocks are disrupted by DNA.

[0080] According to the invention, the oligonucleotides do not contain blocks of more than 6 continuous 2’-OMe-modified nucleotides. According to the invention, the oligonucleotides do not contain blocks of more than 6 continuous 2’-F-modified nucleotides. In one embodiment, the oligonucleotides do not contain blocks of more than 5, 4, or 3 continuous 2’-OMe-modified nucleotides. In one embodiment, the oligonucleotides do not contain blocks of more than 4 continuous 2’-OMe-modified nucleotides.

[0081] Linkage g may 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 (i) at least 10 continuous internucleoside linkage modifications; and / or (ii) 3 consecutive internucleoside linkage modifications at each terminus. In one embodiment, the oligonucleotide comprises at least 10 continuous 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 continuous PS linkages. In some embodiments, each terminus contains 3 consecutive internucleoside linkage modifications. In some embodiments, each terminus contains 4, 5, or 6 consecutive internucleoside linkage modifications. In some embodiments, each terminus contains no more than 8, 7, 6, 5, 4, or 3 consecutive internucleoside linkage modifications. In a preferred embodiment, each terminus contains 3 consecutive internucleoside linkage modifications. In some embodiments, the modification is a 3'-methylenephosphonate, 5'-methylenephosphonate, 3'- phosphoroamidate, 2'-5'phosphodiester, or a phosphoryl guanidine (PN) modification.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 ester bonds (3′-P-3′ and 5′-P-5′).

[0082] Moreover, the oligonucleotides of the invention may have a limited DNA content outside of the CBT. In one embodiment, DNA is located outside the CBT. In one embodiment, DNA is located 3’ and / or 5’ of the CBT. In one embodiment, DNA is located 3’ of the CBT. In one embodiment, DNA is located 5’ of the CBT. Shorter oligonucleotides (≤ 45 nt) may have a 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 25 nt. In one embodiment, the oligonucleotide has a length of 45 nt or less nucleotides (e.g., 25 nt) and no more than 3 nucleotides outside of the CBT are deoxynucleotides. In one embodiment, no more than 1, 2, 3 or 4 nucleotides outside of the CBT are deoxyribonucleotides. In one embodiment, the nucleotide has a length of 25-30, 25-40, 25-60, 28-60, 28- 55, or 28-45 nucleotides. In one embodiment, the nucleotide has a length of 25-60 and a deoxyribonucleoside content outside the CBT that is 10-40%, more preferably 11-30%, and even more preferably 13-25%. In one embodiment, the oligonucleotide does not contain any unmodified RNA nucleotides.

[0083] 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.

[0084] It is envisaged that the ideal asymmetry for each target might depend on the length and the specific underlying sequence of the particular oligonucleotide. It is known that ADAR works as an asymmetric dimer with a footprint of up to 50 bp. While some substrates are more efficiently edited by the deaminase domain alone than by the full-length protein, the opposite holds true for other substrates. This suggests that depending on the size of the target / drug RNA helix, ADAR might bind in different ways (monomer versus dimer) and registers (with no, one, two or up to six dsRBDs). This leads to a situation, wherein, depending on the length of the ASO, specific symmetries on the target adenosine and specific modifications patterns (e.g., ribose and linkage) are preferred. For an optimal binding of the deaminase, a short 3’ terminus seems to be sufficient (at least 4 nt beside the CBT). On the other hand, the 5’ terminus may provide binding space for the dsRBDs and thus typically requires more nucleotides (at least 16 nt). Hence, in certain embodiment the ASO may be asymmetric, i.e., thereare different numbers of nucleotides (nt) at the 3’ and 5’ end of the oligonucleotide. In one embodiment, there are a) at least 4 nucleotides 3’ of the CBT; or b) at least 16 nucleotides 5’ of the CBT.

[0085] Hairpin-loop structured oligonucleotides have previously been described (WO 2020 / 001793) and used successfully to harness ADARs with chemically modified oligonucleotides. Hence, the oligonucleotides may or may not include a recruitment motif for a deaminase. Instead, the chemically modified nucleic acids of the present invention form an RNA duplex to which the ADAR enzyme adheres, whereby the editing efficiency is increased. In one embodiment, the oligonucleotide comprises a hairpin-loop structured ADAR recruitment motif. In one embodiment, the oligonucleotide does not comprise a hairpin-loop structured ADAR recruitment motif. In one embodiment, the oligonucleotide comprises a recruitment motif for a deaminase.

[0086] The oligonucleotides of the invention contain specific patterns of 2’- modifications, internucleoside linkage modifications and nucleotide sequence modifications, which contribute, inter alia, to the advantageous properties of enhanced target editing of the oligonucleotides. The core sequence may have, e.g., PS linkages at positions d, e and optionally at position a. Notably, the regions particularly sensitive to the discovered linkage pattern are the CBT and the adjacent hotspot (“extended hotspot”), where very specific nucleotide modifications are required to adopt ideally to the ADAR´s enzyme active site for improved target editing efficacy. Oligonucleotides according to the invention may contain a mixture of 2’-F-, 2’-OMe, and 2’-H modifications, at least 15% internucleoside linkage modifications, no more than 6 consecutive nucleotides of the same 2’-modification, and a 2’-O-alkyl-modification at the N-2 and a 2’-F-modification at N-3 plus one or more changes in nucleotide sequence outside the CBT to create a mismatch with respect to the target nucleic acid. In one embodiment, positions +5, +4, and +3 are 2’-O-alkyl-modified; and / or position +2 is 2’-F-modified. In one embodiment, positions +5, +4, and +3 are 2’-O-alkyl-modified. In one embodiment, position +2 is 2’-F-modified.

[0087] Hence, the oligonucleotide typically comprises a nucleic acid sequence partially complementary to a target nucleic acid sequence. In some embodiments, the targeting sequence comprises a nucleic acid sequence complementary to at least 75%, 80%, 85%, 90%, or 95% of a nucleic acid sequence in the target RNA. In oneembodiment, complementarity is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%. In one embodiment, complementarity is 95%-97%.

[0088] In certain cases, the oligonucleotide comprises, one or more wobble bases or mismatches to create imperfect duplexes between the oligonucleotide and target RNA that may be more favourably bound and edited by ADAR than a perfect duplex between a fully complementary oligonucleotide and target sequence.

[0089] To compensate for the variability in the target sequence. That is, the less stringent base-pairing requirement of the wobble base (e.g., G-U, I-A, G-A, I-U, I-C, etc.) allows the ASO to pair with more than just one target nucleic acid. Accordingly, in some embodiments, mismatches and / or wobbles enable targeting of different target nucleic acids. In one embodiment, the oligonucleotide comprises one or more additional mismatches, wobble base and / or bulges. In some embodiments, the oligonucleotides contain bulges of 1, 2, 3 or more nucleotides. In one embodiment, the oligonucleotide comprises one or more mismatches, wobble base, and / or bulges with respect to its target. In one embodiment, the oligonucleotide comprises one or more mismatches, wobble base, and / or bulges with respect to its target, and a mismatch at N0. In one embodiment, the oligonucleotide comprises a mismatch at N0. In one embodiment, the oligonucleotide comprises one or more mismatches outside of the CBT.

[0090] In one embodiment, the ASO comprises 1, 2, 3, 4, 5 or more mismatches when aligned with the target nucleic acid. In one embodiment, the ASO comprises 2 or more mismatches when aligned with the target nucleic acid. In one embodiment, the ASO comprises 3 or more mismatches when aligned with the target nucleic acid.

[0091] According to the invention, the oligonucleotides comprise one or more mismatches outside of the CBT. In one embodiment, the ASOs comprise 4 mismatches or wobble bases outside the CBT. In one embodiment, the ASOs comprise 3 mismatches or wobble bases outside the CBT. In one embodiment, the ASOs comprise 2 mismatches or wobble bases outside the CBT. In one embodiment, the ASOs comprise 1 mismatch or wobble base outside the CBT.

[0092] In some embodiments, oligonucleotides comprise 0, 1, 2 or 3 mismatches outside of the CBT. In some embodiments, a single mismatch comprises a single nucleotide. In some embodiments, a single mismatch comprises several sequential nucleotides. In some embodiments, the mismatch is a paired G-A, C-A, U-C, A-A, G- G, C-C, U-U, or G-U mismatch. In a preferred embodiment, the mismatch is a C-to- C mismatch. In one embodiment, the mismatch is a C-C mismatch. In oneembodiment, the mismatch is a G-U mismatch. In one embodiment, the mismatch is a C-C mismatch or a G-U mismatch.

[0093] In one embodiment, the mismatch is located 3’ and / or 5’ to the nucleotide that is opposite to the target adenosine in the target RNA. The mismatch may comprise a single nucleotide or sequential nucleotides.

[0094] For instance, in one embodiment, chemically optimised oligonucleotides contain a C residue at position -9 (N-9) that is flanked by a 5’ C or G and 3’ A or U, thus creating a mismatch with the target nucleic acid (C-C mismatch) increases editing. In one embodiment, the mismatch is at position N-9. In some embodiments, the C-C mismatch at position N-9 is combined with other features, e.g., a mismatch at another position.

[0095] Alternatively, to facilitate ADAR-mediated A-to-G base replacement, in one embodiment, chemically optimised oligonucleotides contain a C residue at position - 7 (N-7), flanked by a 5’ G or C and a 3’ A or U. This leads to a mismatch with the target nucleic acid (C-C mismatch) that increases editing. In some embodiments, the C-C mismatch at position N-7 is combined with other features., e.g., a mismatch at another position. As shown in Examples 1 and 2, in some embodiments the target RNA encodes SERPINA1, carrying the E342K mutation associated with AAT deficiency.

[0096] Similarly, in some embodiments, the oligonucleotide comprises a change in nucleotide sequence at position +2 (N+2). Chemically optimised oligonucleotides containing a U at position N+2 across a G in the target RNA (G-U mismatch) increase editing. In one embodiment, the oligonucleotide comprises a U at position N+2. In some embodiments, the G-U mismatch at position N+2 is combined with other features. In one embodiment, the mismatches are at positions N+2 and N-9. In one embodiment, the mismatches are at positions N+2 and N-7.

[0097] Accordingly, in addition to the mismatch opposite the targeted adenosine, the oligonucleotides of the invention comprise one or more mismatches outside of the CBT. In one embodiment, the oligonucleotide comprises: (i) a C at position N-7 and optionally a U at position N+2; (ii) a U at position N+2; or (iii) a C at position N- 9 and optionally a U at position N+2.

[0098] Oligonucleotides of the invention may be specific to targeting SERPINA1. In one embodiment, the target nucleic acid comprises a sequence from a mutant SERPINA1 gene that encodes SERPINA1 having a E342K mutation, and wherein the nucleic acid sequence of the oligonucleotide is an antisense sequence that issubstantially complementary to the sequence from the mutant SERPINA1 gene that encodes SERPINA1 having the E342K mutation.

[0099] SERPINA1 targeting antisense oligonucleotides may contain one or more mismatches. In one embodiment, a mismatch is at position N-7. In one embodiment, this mismatch is a C-C mismatch. In one embodiment, the oligonucleotide comprises SEQ ID NO: 2. That is, in one embodiment, the oligonucleotide comprises the following sequence: 5’ - mC*mA*mU*fG*dG*fC*mC*fC*mC*fA*dG*fC*fA*dG*mC*fU*mU*fC*fA*dG*mU*fC*m C*fC*mU*mUmUfC*dT*dC*dImUfCfG*dA*mU*fC*dG*fU*mC*fA*dG*fC*fA*mC*dA*f G*fC*mC*fU*mU*dA*fU*fG*mC*dA*mC*mG*mG – 3‘ (SEQ ID NO: 2), wherein m = 2‘-O-methyl; f = 2‘-fluoro; I = inosine; * = phosphorothioate (PS) linkage; and d = deoxyribose (DNA) (see, Example 1). The CBT has been underlined.

[0100] The length of the oligonucleotide can be shortened without losing its editing efficacy provided the oligonucleotide comprises specific 2’-sugar and internucleoside linkage modifications. For instance, the oligonucleotide may be 40nt long. In one embodiment, the oligonucleotide is 40nt long and comprises a C at position N-7. In one embodiment, the oligonucleotide comprises SEQ ID NO: 4. That is, in one embodiment, the oligonucleotide comprises the following sequence: 5’ - mC*mA*mU*fG*dG*fC*mC*fC*mC*fA*dG*fC*fA*dG*mC*fU*mU*fC*fA*dG*mU* fC*mC*fC*mU*mUmUfC* dT*dC*dI mUfCfG*dA*mU*fC*mG*mU*mC – 3‘ (SEQ ID NO: 4), wherein m = 2‘-O-methyl; f = 2‘-fluoro; * = phosphorothioate (PS) linkage; and d = deoxyribose (DNA) (see, Example 2).

[0101] Mutations in the PDE6A gene can cause rod photoreceptors degeneration and have been associated with the blinding disease retinitis pigmentosa (RP) (Sothilingham et al., 2015). The oligonucleotides specific to targeting PDE6A can be chemically modified and sequence optimised, i.e., to include one or more mismatches outside the CBT in order to enhance target editing yields. Hence, in one embodiment, the target nucleic acid comprises a sequence from a mutant PDE6A gene that encodes PDE6A having a V685M mutation, and wherein the nucleic acid sequence of the oligonucleotide is an antisense sequence that is substantially complementary to the sequence from the mutant PDE6A gene that encodes PDE6A having the V685M mutation.

[0102] Oligonucleotides specific to the PDE6A target may be chemically modified to enhance editing. A 42nt long, chemically modified oligonucleotide that enhances editing of PDE6A RNA can provide stable ASOs without negatively impacting theirediting efficacy. This chemically optimised oligonucleotide, which is not an oligonucleotide according to the invention, comprises the following sequence: 5’ - mU*mU*mC*fA*fC*mU*fC*mU*fC*fA*mU*fA*fU*fG*mU*fC*mU*fU*fA *fG*fA*mC*fU*fG*fA*mUfCmC* dA*dC*dG mAfUmC*fU*mU*fU* mU*fG*mG*mA*mA – 3‘ (SEQ ID NO: 5), wherein m = 2‘-O-methyl; f = 2‘-fluoro; * = phosphorothioate (PS) linkage; and d = deoxyribose (DNA) (Table 3). This PDE6A (mPDE6A) V685M targeting oligonucleotide may be modified according to the invention to include one or more mismatches outside the CBT in order to enhance RNA editing yields (see, Example 3). Accordingly, in one embodiment, the oligonucleotide comprises SEQ ID NO: 6. In one embodiment, the oligonucleotide comprises the following sequence: 5’ - mU*mU*mC*fA*fC*mU*fC*mU*fC*fA*mU*fA*fU*fG*mU*fC*mU*fU*fA*fG*fA*mC* fU*fG*fA*mUfCmC* dA*dC*dG mA fUmC*fU*mU*fU* mU*fC*mG*mA*mA – ‘3 (SEQ ID NO: 6; TMR530).

[0103] In one embodiment, the oligonucleotide comprises SEQ ID NO: 7. In one embodiment, the oligonucleotide comprises the following sequence: 5’ - mU*mU*mC*fA*fC*mU*fC*mU*fC*fA*mU*fA*fU*fG*mU*fC*mU*fU*fA*fG*fA*mC*fU*f G*fA*mUfCmU* dA*dC*dG mAfUmC*fU*mU*fU* mU* fC*mG*mA*mA – 3‘ (SEQ ID NO: 7; TMR531).

[0104] Oligonucleotides of the invention may be specific to targeting SCNA. In one embodiment, the target nucleic acid comprises a sequence from a mutant SCNA gene that encodes SCNA having a A53T mutation. In one embodiment, the target nucleic acid comprises a sequence from a mutant SCNA gene that encodes SCNA having a A53T mutation, and wherein the nucleic acid sequence of the oligonucleotide is an antisense sequence that is substantially complementary to the sequence of the mutant SCNA gene that encodes SCNA having the A53T mutation.

[0105] SCNA targeting antisense oligonucleotides may contain one or more mismatches. In one embodiment, a mismatch is at position -9 (N-9), i.e., there is a mismatch at position N-9, when the ASO is hybridised to the target nucleic acid. In one embodiment, a mismatch is at position -1 (N-1), i.e., there is a mismatch at position N-1, when the ASO is hybridised to the target nucleic acid. In one embodiment, a mismatch is at position N-1and at position N-9. In one embodiment, the mismatch is a C-C mismatch. In one embodiment, the mismatch is a G-G mismatch. In one embodiment, a C-C mismatch is at N-9. In one embodiment, a G-G mismatch is at N-1. In one embodiment, a C-C mismatch is at N- 9and a G-G mismatch is at N-1.

[0106] In one embodiment, the oligonucleotide comprises a C at position -9 (N-9). In one embodiment, the oligonucleotide comprises a G atposition -1 (N-1).In one embodiment, the oligonucleotide comprises a C at position -9 (N-9) and a G at position -1 (N-1).

[0107] In one embodiment, the oligonucleotide comprises SEQ ID NO: 8. In one embodiment, the oligonucleotide comprises the following sequence: 5’ - mU*mU*mG*fC*mU*fC*mU*fU*mU*fG*fG*fU*mC*fU*mU*fC*mU*fC*fA*fG*mC*fC*fA *mC*fU*fGmUfU *dG*dC*dC mAfCfA*mC*fC*fA *mU*fG* mC*mA*mC – 3’ (SEQ ID NO: 8; TMR555), wherein m = 2‘-O-methyl; f = 2‘-fluoro; * = phosphorothioate (PS) linkage; and d = deoxyribose (DNA) (Table 4).

[0108] In one embodiment, the oligonucleotide comprises SEQ ID NO: 9. In one embodiment, the oligonucleotide comprises the following sequence: 5’ - mU*mU*mG*fC*mU*fC*mU*fU*mU*fG*fG*fU*mC*fU*mU*fC*mU*fC*fA*fG*mC*fC*fA *mC*fU*fGmUfU *dG*dC*dC mAfCfA*mC*fC*fA*mU*fC* mC*mA*mC – 3’ (SEQ ID NO: 9; TMR556), wherein m = 2‘-O-methyl; f = 2‘-fluoro; * = phosphorothioate (PS) linkage; and d = deoxyribose (DNA) (Table 4).

[0109] In one embodiment, the oligonucleotide comprises SEQ ID NO: 10. In one embodiment, the oligonucleotide comprises the following sequence: 5’ – mU*mU*mG*fC*mU*fC*mU*fU*mU*fG*fG*fU*mC*fU*mU*fC*mU*fC*fA*fG*mC*fC*fA *mC*fU*fGmUfU *dG*dC*dG mAfCfA*mC*fC*fA*mU*fG* mC*mA*mC – 3’ (SEQ ID NO: 10; TMR557), wherein m = 2‘-O-methyl; f = 2‘-fluoro; * = phosphorothioate (PS) linkage; and d = deoxyribose (DNA) (Table 4).

[0110] In one embodiment, the oligonucleotide comprises SEQ ID NO: 11. In one embodiment, the oligonucleotide comprises the following sequence: 5’ - mU*mU*mG*fC*mU*fC*mU*fU*mU*fG*fG*fU*mC*fU*mU*fC*mU*fC*fA*fG*mC*fC*fA *mC*fU*fGmUfU *dG*dC*dG mAfCfA*mC*fC*fA*mU*fC* mC*mA*mC – 3’ (SEQ ID NO: 11; TMR558), wherein m = 2‘-O-methyl; f = 2‘-fluoro; * = phosphorothioate (PS) linkage; and d = deoxyribose (DNA) (Table 4).

[0111] Targeted delivery of oligonucleotides to cells using N-acetylgalactosamine (GalNAc) has become a breakthrough approach in the therapeutic oligonucleotide field (Debacker et al., 2020). Hence, the oligonucleotides of the invention may be modified at their 5’ and / or 3’ termini. That is, in one embodiment, the oligonucleotide is conjugated to a moiety that enhances cellular uptake of the oligonucleotide, e.g., N-acetylgalactosamine (GalNAc). For instance, targeted delivery of oligonucleotides to liver hepatocytes using bi- or triantennary N-acetylgalactosamine (GalNAc)conjugates has previously described for, e.g., treating liver diseases, including Hepatitis B virus (HBV), non-alcoholic Fatty Liver Disease and genetic diseases (Debacker et al., 2020). The oligonucleotides may comprise one or more different linkers, tags or coupling agents at either one or both termini. For example, the oligonucleotides may comprise amino-linkers, preferably C6-amino-linkers. Hence, in some embodiments, the oligonucleotides comprise a C6-amino-linker at the 5’ terminus. In some embodiments, the oligonucleotides comprise a C6-amino-linker at the 3’ terminus. Hence, in some embodiments, the modified oligonucleotide comprises a moiety or is conjugated to a moiety that enhances cellular uptake of the oligonucleotide. In one embodiment, the moiety enhancing cellular uptake is a triantennary N-acetyl galactosamine (GalNAc3), which is preferably conjugated to the 3' terminus or to the 5' terminus of the oligonucleotide.

[0112] The modified oligonucleotides of the invention may be incorporated into compositions. Accordingly, provided herein is a composition containing one or more of the oligonucleotide(s) of the invention. In some embodiments, the compositions are pharmaceutical compositions. In the context of the invention, the term composition and pharmaceutic compositions are used interchangeably. In one embodiment, the composition contains one or more oligonucleotides of the invention. In some embodiments, provided herein is a composition comprising a plurality of oligonucleotides. As used herein, pharmaceutical composition means a substance or a mixture of substances suitable for administering to an individual. In some embodiments, a pharmaceutical composition comprises one or more other therapies in addition to an oligonucleotide of the invention. For example, a pharmaceutical composition may comprise one or more oligonucleotides of the invention, one or more active pharmaceutical agents and a sterile aqueous solution.

[0113] In one embodiment, a composition comprises an oligonucleotide of the invention. In one embodiment, a composition comprises a pharmaceutically acceptable salt of the composition of the invention. In one embodiment, a composition comprises an oligonucleotide of the invention in an admixture with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier can simply be a saline solution. This can be isotonic or hypotonic.

[0114] The amount of an oligonucleotide or composition which will be effective in the treatment and / or prevention of a disease or disorder will depend on the nature ofthe disease, and can be determined by standard clinical techniques. Exemplary doses for oligonucleotides range from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg oligonucleotide, e.g., RNA, per patient. In one embodiment, the concentration of the oligonucleotide is 10, 20, 30, 40, 50, or 60 nM. In one embodiment, the concentration of the oligonucleotide is between 10 and 60 nM. In one embodiment, the concentration of the oligonucleotide is more than 5, 10, 20, 30, 40, or 50 nM. In one embodiment, the oligonucleotide is used in the amount of 1 to 5 pmol or 1 to 2.5 pmol. In one embodiment, the oligonucleotide is used in the amount of 1.25, 2.5 or 5 pmol.

[0115] In certain embodiments, the compositions of the invention include diluents of various buffer content (e.g., Tris-HCI, acetate, phosphate), pH, and ionic strength, and additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol), and bulking substances (e.g., lactose, mannitol). In some embodiments, the material are incorporated into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. In some embodiments, hyaluronic acid is used. Such compositions may influence the physical state, stability, rate of in vivo release, and / or rate of in vivo clearance of the oligonucleotides and / or derivatives and / or pharmaceutically acceptable salt thereof. In some embodiments, the compositions are in liquid form or in dried powder, such as lyophilized form.

[0116] The oligonucleotides or compositions thereof can be tested for in vivo toxicity in animal models. For example, animal models, described herein and / or others known in the art, used to test the activities of active compounds can also be used to determine the in vivo toxicity of these compounds. For example, animals are administered a range of concentrations of active compounds. Subsequently, the animals are monitored over time for lethality, weight loss or failure to gain weight, and / or levels of serum markers that may be indicative of tissue damage. These in vivo assays may also be adapted to test the toxicity of various administration mode and / or regimen in addition to dosages.Uses

[0117] The invention describes the use of chemically and sequence modified oligonucleotides and compositions comprising the same in the medical setting, specifically, for site-directed editing of a target RNA (e.g., binding to the target RNAvia the targeting sequence and by recruiting a deaminase to the target site). The invention generally describes oligonucleotides, compositions and methods for treating of preventing a genetic disorder, condition, or disease using said oligonucleotides or compositions. This means, the invention is used to make desired changes in a target sequence in a cell or a subject by site-directed editing of nucleotides using an oligonucleotide that is capable of effecting an adenosine deaminase acting on RNA (ADAR)-mediated adenosine to inosine. As a result, the target sequence is edited through an adenosine deamination reaction mediated by ADAR, converting adenosines into inosine. In some embodiments, because I is recognized as G, the deamination correcting the pathogenic mutation reverses the mutation back to wild- type, reversing or slowing symptoms associated with the particular disease. Site- directed editing may take place in vitro, in vivo or ex vivo.

[0118] Provided herein is a method for site-directed A-to-I editing of a target RNA, comprising providing to a cell or subject an oligonucleotide according to the invention, or a composition of the invention.

[0119] A modified oligonucleotide or composition comprising the same may be used in the treatment and / or prevention of a medical condition. For instance, provided herein in a method of treating a monogenic disease or condition associated with a point mutation in a subject by administering to the subject a therapeutically effective amount of an oligonucleotide according to the invention, or a composition according to the invention. In one embodiment, the disease or condition is α(1)AT deficiency or an associated pathology or retinitis pigmentosa or an associated pathology.

[0120] Also provided is a modified oligonucleotide or a composition of the invention for use in therapy. In another aspect provided herein is an oligonucleotide of the invention or a composition comprising the same for use in the treatment or prevention of a genetic disorder, condition, or disease. In some embodiments, the disease or disorder is selected form the group consisting of liver or metabolic diseases and / or cardiac or cardiovascular diseases associated with a gain-of-function (GOF) or loss- of-function (LOF) mutation. In one embodiment, the genetic disorder, condition, or disease is associated with a point mutation.

[0121] In one embodiment, the disease or condition is A1AT (alpha 1 antitrypsin deficiency, A1AD) deficiency or an associated pathology, and the target transcript comprises the sequence from the mutant SERPINA1 gene. In one embodiment, SERPINA1 has the E342K mutation.

[0122] The oligonucleotides and / or compositions of the invention may be used to target the SERPINA1 gene, which encodes serine protease inhibitor alpha-I antitrypsin (A1AT). A1AT protects tissues from certain inflammatory enzymes, including neutrophil elastase. A deficiency in A1AT can lead to excessive break down of elastin in the lungs by neutrophil elastase. This may lead to reduced elasticity in the lungs and subsequent respiratory complications, including emphysema and chronic obstructive lung disease (COPD). Mutant A1AT can also build up in the liver, resulting in cirrhosis and liver failure. Accordingly, in one embodiment, the genetic disorder, condition or disease is associated with a G-to-A mutation in the SERPINA1 gene. In one embodiment, the mutation is SERPINA1 E342K. In one embodiment, the disease or disorder comprises the SERPINA1 gene or an alpha-1-antitrypsin deficiency (A1AD or AATD), optionally wherein the target protein is alpha-1 antitrypsin. In one embodiment, the mutation is referred to as the PiZZ mutation (α1-antitrypsin deficiency).

[0123] The oligonucleotides and / or compositions of the invention may be used to target the PDE6A gene, which encodes a subunit of the rod phosphodiesterase. Loss of enzyme function leads to a chronically elevated cGMP level, which causes an increased calcium inflow into the cell and thereby the hyperactivation of cell death pathways, leading to retinitis pigmentosa. In one embodiment, the disease or condition is retinitis pigmentosa or an associated pathology, and the target transcript comprises the sequence from the mutant PDE6A gene. In one embodiment, PDE6A has the V685M mutation.

[0124] For example, in other instances the target may be RNA encoding SNCA. The SNCA gene encodes the small protein called alpha-synuclein (aSyn), which is abundant in the brain, and to a lesser extent in the heart, muscles, and other tissues. Studies suggest that the SNCA protein plays an important role in multiple system atrophy, dementia with Lewy bodies and Parkinson’s disease (PD) (Pedersen et al., 2021). Specifically, the A53T mutation of the SNCA gene has been associated with familial PD (Polymeropoulos et al., 1997). In one embodiment, the target transcript encodes SNCA, carrying the A53T mutation. In one embodiment, an oligonucleotide and / or composition of the invention is used to target the SNCA transcript carrying the A53T mutation. In one embodiment, an oligonucleotide and / or composition of the invention is used to treat or prevent a disease or disorder associated with a A53T mutation. In one embodiment, the disease or disorder is multiple system atrophy,dementia or Parkinson’s disease (PD). In one embodiment, the disease or disorder is familial PD.

[0125] Provided herein are oligonucleotides for use in the treatment of a disease or disorder associated with the SNCA gene. In one embodiment, the antisense oligonucleotide is for use in the treatment or prevention of a disease or disorder, wherein the disease or disorder is associated with the SNCA gene and carries the A53T mutation. In one embodiment, the antisense oligonucleotide for use of the invention is any one of the oligonucleotides listed in Table 4, i.e., SEQ ID NO: 8 to 11. In one embodiment, the oligonucleotide is for use in the treatment or prevention of a disease, wherein the target transcript comprises the sequence from the mutant SNCA gene, optionally encoding SNCA having the A53T mutation.

[0126] Oligonucleotides of the invention may be linked to a moiety that enhances uptake of the ASO in cells. Examples of such moieties are cholesterols, carbohydrates, vitamins, biotin, lipids, phospholipids, amide derivates, cell- penetrating peptides including but not limited to antennapedia, TAT, transportan and positively charged amino acids such as oligoarginine, poly-arginine, oligolysine or polylysine, antigen-binding domains such as provided by an antibody, a Fab fragment, or a single chain antigen binding domain such as a cameloid single domain antigen- binding domain. Accordingly in some embodiments, the ASO is delivered using drug conjugates with antibodies, nanobodies, cell penetrating peptides and aptamers. In one embodiment, the oligonucleotide is conjugated to an antibody, preferably a Fab fragment.

[0127] The modified oligonucleotide of the invention or the (pharmaceutical) composition may be administered, for example, orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions, or solutions, or parenterally, e.g., by parenteral injection. In some embodiments, formulations suitable for parenteral administration comprise sterile aqueous preparations of at least one embodiment of the present disclosure, which are approximately isotonic with the blood of the intended recipient. The amount of oligonucleotide or composition to be administered, the dosage and the dosing regimen can vary from cell type to cell type, the disease to be treated, the target population, the mode of administration (e.g., systemic versus local), the severity of disease and the acceptable level of side activity. In some embodiments, the amount of oligonucleotides administered in a pharmaceutical composition is dependent on the subject being treated, the subject's weight, and the manner of administration.

[0128] Various delivery systems can be used to deliver the oligonucleotides of the invention. That is, the compositions provided herein can be in any form that allows for the composition to be administered to a subject. An oligonucleotide according to the invention can be delivered as is, i.e., naked and / or in isolated form to an individual, through an organ, e.g., mucosa of the eye, or directly to a cell. Hence, in a preferred embodiment, the oligonucleotide of the invention is administered and delivered ‘as is’, also referred to as ‘naked’. When administering an oligonucleotide of the invention, it is preferred that the oligonucleotide is dissolved in a solution that is compatible with the delivery method. Such delivery may be in vivo, in vitro or ex vivo. Hence, depending on the disease, disorder or infection that needs to be treated, or on the cell, tissue or part of the body that needs to be reached by the oligonucleotides (e.g., in case of beneficial editing), a different administration route or delivery method may be selected. Examples for delivery when an oligonucleotide is not delivered naked, are delivery agents or vehicles such as nanoparticles, like polymeric nanoparticles, microparticles, micelles, liposomes, antibody-conjugated liposomes, cationic lipids, polymers, or cell-penetrating peptides.

[0129] Different excipients or transfection reagents may be used in the delivery of each of the oligonucleotides or compositions to a cell and / or into a cell. Preferred are excipients or transfection reagents capable of forming complexes, nanoparticles, micelles, vesicles and / or liposomes that deliver each oligonucleotide or composition as defined herein. Many of these excipients are known in the art. Suitable exemplary excipients or transfection reagents comprise polyethylenimine (PEI; ExGen500 (MBI Fermentas)), LipofectAMINE™ 2000 (lnvitrogen), lipofectinTM, or derivatives thereof, and / or viral capsid proteins that are capable of self-assembly into particles that can deliver each constituent as defined herein to a target cell.

[0130] The oligonucleotide or composition may be administered as a monotherapy or in combination with a further different medicament. In particular, the oligonucleotide of the invention may be administered together with a medicament suitable for the treatment or prevention of alpha-1-antitrypsin (A1AT) deficiency or retinitis pigmentosa.

[0131] Compared to reference oligonucleotides or compositions, the provided oligonucleotides or compositions are surprisingly effective in target editing. In some embodiments, a change is measured by an increase of a desired mRNA and / or protein level compared to a reference sample or condition. In some embodiments, a change is measured by an increase in the editing efficacy (%) mediated by theoligonucleotide or composition of the invention. In some embodiments, a change is measured by an increase in stability of the oligonucleotide or composition comprising the same. In some embodiments, a change is measured in the levels of cytotoxicity, viability, apoptosis or immune activation. In some embodiments, a change is detected by means of luminescence and / or gene expression. In some embodiments, toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of the population) and the LD50 (the dose therapeutically effective in 50% of the population). In some embodiments, data obtained from the cell culture assays or animal studies can be used in formulating a range of dosage for use in humans.

[0132] The compositions of the present disclosure may be administered in a number of routes depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be by inhalation (e.g., through nebulization), intranasally, orally, by injection or infusion, intravenously, subcutaneously, intra-dermally, intra-cranially, intramuscularly, intra-tracheally, intra- peritoneally, intra-rectally, by direct injection into a tumour, and the like. Administration may be in solid form, in the form of a powder, a pill, or in any other form compatible with pharmaceutical use in humans. In some embodiments the oligonucleotide construct can be delivered systemically. Patient Population

[0133] The oligonucleotides of the invention of compositions comprising the same may be administered to various groups of subjects or patients. In certain embodiments, the subject is in need of treatment. In certain embodiments, the subject is in need of prevention. In other embodiments, the subject is not in need of treatment (“beneficial editing”). That is, the subject receives the oligonucleotide or composition to edit an RNA derived from a wildtype allele (not a mutated allele) in order to modulate the function of the wildtype protein in a useful way.

[0134] In one embodiment, an oligonucleotide or composition containing the same is administered to a subject. In some embodiments, an oligonucleotide or composition containing the same is administered to a mammal, preferably a human. In certain embodiments, an oligonucleotide or composition containing the same is administered to a naive subject, i.e., a subject that does not have a disease or disorder or has not previously received any oligonucleotide. In one embodiment, an oligonucleotide or composition containing the same is administered to a naive subject that is at risk of developing a disease or disorder. In some embodiments, anoligonucleotide or composition containing the same is administered to a patient before symptoms manifest or symptoms become severe. In certain embodiments, an oligonucleotide or composition containing the same is administered to a patient who has been diagnosed with a disease or disorder, e.g., alpha-1 antitrypsin deficiency or retinitis pigmentosa.

[0135] In some embodiments, the subject to be administered an oligonucleotide or composition containing the same is any individual at risk of developing a disease or disorder associated with a G-to-A mutation in genes. In one embodiment, the subject suffers from a disease or disorder associated with a G-to-A mutation in genes. In some embodiments, a symptom of a condition, disorder or disease associated with a G-to-A mutation can be any condition, disorder or disease that can benefit from an A- to-I conversion.

[0136] Also provided herein are methods of treating a disease or condition associated with a point mutation in a subject by administering to the subject a therapeutically effective amount of an oligonucleotide according to the invention or composition according to the invention. In one embodiment, the genetic disease or genetic disorder is associated with a G-to-A mutation in a subject. In one embodiment, an effective amount of the oligonucleotide of the invention or the composition of the invention is administered to the subject.

[0137] Also provided herein is the use of an oligonucleotide of the invention in therapy. Also, provided herein is a use of an oligonucleotide of the invention in the manufacture of a medicament for treating conditions, diseases and / or disorders associated with a G-to-A mutation. In certain embodiments, the use of an oligonucleotide of the invention is in the manufacture of a medicament for treating a condition, disease and / or disorder associated with a G-to-A mutation.

[0138] According to a further aspect, the invention relates to a kit or kit of parts comprising an oligonucleotide of the invention and / or the (pharmaceutical) composition of the invention. The kit additionally comprises instructions for use. Methods for editing

[0139] The present invention also relates to methods for editing a target adenosine in a target nucleic acid. For example, the present invention provides methods of editing a SERPINA1 polynucleotide, e.g., a SERPINA1 polynucleotide comprising asingle nucleotide polymorphism (SNP) associated with alpha I antitrypsin deficiency. Alternatively, the target may be human PDE6A or a variant thereof.

[0140] Alternatively, the target may be human SCNA or a variant thereof. In one embodiment, the method is for treating a disease or condition associated with a point mutation, wherein the point mutation is A53T. In one embodiment, the method is for treating multiple system atrophy, dementia with Lewy bodies and Parkinson’s disease (PD).

[0141] The methods of editing may be in vitro, in vivo, or ex vivo. Hence, the invention provides a method for site-directed A-to-I editing of a target RNA, comprising providing to a cell or subject an oligonucleotide or composition of the invention. In one embodiment, the method comprising a step of contacting a target RNA with the oligonucleotide of the invention or the composition of the invention. In one embodiment, the method comprises, after the step of contacting, the following steps: (a) allowing uptake by the cell of the modified antisense oligonucleotide; (b) allowing annealing of the oligonucleotide to the target RNA; and (c) allowing a mammalian ADAR enzyme comprising a natural dsRNA binding domain as found in the wild type enzyme to deaminate the target adenosine in the target RNA sequence to an inosine.

[0142] The invention also provides an in vitro method for deaminating at least one specific adenosine present in a target RNA sequence in a cell. Hence, in on aspect provided herein is 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 the target nucleic acid with a chemically modified oligonucleotide of the invention; (b) allowing uptake by the cell of the chemically modified oligonucleotide; (c) allowing annealing of the chemically modified oligonucleotide to the target RNA sequence; and (d) allowing a mammalian ADAR enzyme comprising a natural dsRNA binding domain as found in the wild type enzyme to deaminate the target adenosine in the target RNA sequence to an inosine.

[0143] In one embodiment, the method comprises after step (d), a step of identifying the presence of the inosine in the RNA sequence.

[0144] The editing reaction is preferably monitored or controlled by sequence analysis of the target RNA.

[0145] Also, a modified antisense oligonucleotide of the invention or a (pharmaceutical) composition may be used in the diagnosis of a genetic condition, disease or disorder. Therein, the disease or disorder is preferably selected from the group consisting of infectious diseases, tumour diseases, cardiovascular diseases, autoimmune diseases, allergies and neurological diseases or disorders. In one embodiment, the genetic disorder, condition or disease is associated with a G-to-A mutation.

[0146] The methods of the present invention can be used with cells from any organ, e.g., skin, lung, heart, kidney, liver, pancreas, gut, muscle, gland, eye, brain, blood and the like. The invention is particularly suitable for modifying sequences in cells, tissues or organs implicated in a diseased state of a (human) subject. For example, such cells may include, but are not limited, to hepatocytes, hepatocyte like cells, and / or alveolar type II cells, neurons (PNS, CNS), retina, photo receptors cells, Müller Glia cells, RPE, immune cells, B cells, T cells, dendritic cells, macrophages. EXAMPLES

[0147] The present invention shall be described in more detail by the following Examples. The examples shown in the following are merely illustrative and shall describe the present invention in a further way. These examples shall not be construed to limit the present invention thereto. The sequences disclosed herein are also shown in the enclosed sequence listing. However, the sequence listing shows only the sequence of nucleotides, whereas the modification of the nucleotides and of the bonds between the nucleotides is not shown in the sequence listing. The relevant modifications associated with the sequences are disclosed in the tables below and, to some extent, in the Figures of this application.

[0148] For all experiments, target editing efficacy is expressed as the percentage [%] of edited target sites found in all detected target sites in the target transcript. Example 1. Combining favourable chemical and sequence modifications improves potency in long SERPINA1 targeting Oligonucleotide

[0149] Chemical modifications have a stabilizing effect on target specific oligonucleotides, leading to an overall increase in editing efficacy (%) of target RNA. A balanced mixture of 2’-F-, 2’-OMe-, and 2’-H-modifications cancompensate for the replacement of 2’-OH modifications and oligonucleotides containing a mixture of 2’-F-, 2’-OMe-, and 2’-H / 2’-OH together with internucleoside linkage modifications, e.g., PS linkages, enhance target editing (PCT / EP2022 / 083943). The data in PCT / EP2022 / 083943 shows that there is a modification-sensitive “hot spot” region directly 3’ to the CBT at positions -2 and -3. For this “extended CBT” the best arrangement and / or modification to obtain efficient target editing is 5‘ – CBT - mN - fN – 3‘ , wherein CBT refers to the central base triplet, mN is a 2'-O-methyl-modification, and fN is 2’-fluoro- modification. Moreover, it was shown that blocks of continuous uniform sugar modifications should be avoided. Based on these data chemically optimised SERPINA1 targeting constructs with high editing efficacy were selected.

[0150] The present invention involves further improving the editing efficacy of these chemically optimised SERPINA1 targeting oligonucleotides, by introducing specific sequence modifications into the chemically optimised oligonucleotides to generate chemically and sequence optimised SERPINA1 targeting constructs. As shown in Figure 1(A) and summarised in Table 1, a chemically modified ASO v16 (SEQ ID NO: 1) was further sequence-optimised by introducing a single guanosine (G) to cytosine (C) (G ^ C) nucleotide change at position -7 (N-7) of the SERPINA1 E342K targeting construct. This optimized construct is reflected in SEQ ID NO: 2. Introducing this single G ^ C nucleotide change in the oligonucleotide leads to a C- C mismatch with respect to the target RNA. The optimised construct was subsequently assessed for its in vitro editing efficacy (%) of the SERPINA1 target RNA.

[0151] Editing Efficacy: To assess cellular editing efficacy, 1 x 104HeLa cells (Cat. No.: ATCC CCL-2), which were modified to constitutively express the SERPINA1 E342K mutated cDNA via the piggyBac genomic integration system (referred to as “SERPINA1 piggyBac HeLa cells”), were seeded in a 96-well plate. After 24 h, cells were forward transfected with the construct. For each 96-well, 5, 2.5 or 1.25 pmol construct (which corresponds to 50nM, 25nM or 12.5nM, respectively, in the total media volume per 96-well) and 0.3 µl Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific) were each diluted in 10 µl Opti-MEM and incubated for 5 min, then combined and incubated for an additional 20 min. The medium was changed, and the transfection mix evenly distributed into one well. After 24 h, medium was changed. 48 h post transfection, cells were harvested for RNA isolation and sequencing.The SERPINA1, 59nt long targeting constructs, sequences and modifications are listed in Table 1. Construct TMR291 (v16, no mismatch outside the CBT) is a chemically optimized, 59nt long oligonucleotide and served as control. Construct TMR305 (v16m) represents a chemically and sequence optimized version, carrying a G ^ C nucleotide change at position -7 (N-7). Results are shown in Figure 1. Table 1: SERPINA1 E342K targeting construct sequences including nucleobase and backbone modifications used in Example 1. mN = 2'-O-methyl (2’-OMe), fN = 2’-fluoro (2’-F), N = 2’-OH (ribose; RNA), dN = 2’-H (deoxyribose; DNA), * = phosphorothioate (PS) linkage. The CBT has been underlined.

[0152] As shown in Figure 1(B), there was an overall increase in SERPINA1 target editing with the sequence optimised oligonucleotide in vitro. Specifically, for optimised construct TMR305 (v16m), the single nucleotide change from G to C significantly improved target editing by 1.4-fold (at 1.25 pmol) and 1.2-fold (at 2.5 pmol) when compared to control. The fold-increase was more pronounced at the lower ASO concentration (1.25 pmol) and no further difference was observed at a concentration of 5 pmol. Further assessment of the TMR305 construct (-7C:C) showed maximum editing levels of close to 60%, which were similar to that of control construct TRM291 (Figure 1(D)). Consistent with an improved potency, the sequence modified TMR305 construct reached its maximum editing yields of around 60% at a concentration of about 25nM, while the TMR291 construct reached its maximum at 50 nM.

[0153] Overall, these results show that a combination of chemical and sequence modifications improve the overall target editing efficacy of the oligonucleotide and more than 1 nucleotide modifications can be tolerated. More specifically, the data show that introducing a C-C mismatch at position -7 (N-7) enhances editing yields of the 59nt long oligonucleotide and that the sequence modified construct needed a lower concentration than the control to reach its maximum editing yield.Example 2. Combining favourable chemical and sequence modifications improves potency in short SERPINA1 targeting Oligonucleotide

[0154] To determine whether the same approach could be taken for shorter oligonucleotides without decreasing the editing efficacy of the oligonucleotide, a shorter, 40nt long ASO was chemically optimised (TMR317, ASO v17) and subsequently sequence optimised by introducing a G ^ C nucleotide change at position -7 (N-7). This chemically and sequence optimized construct is referred to as TMR543 / v17m. This change in oligonucleotide sequence lead to a C-C mismatch with respect to the target RNA. The 40nt targeting constructs, sequences, and modification pattern are listed in Table 2. The results are shown in Figure 2. Editing efficacy (in %) of target RNA was determined as described under Example 1 above. Table 2: SERPINA1 E342K targeting construct sequences including nucleobase and backbone modifications used in Example 2. mN = 2'-O-methyl (2’-OMe), fN = 2’-fluoro (2’-F), N = 2’-OH (ribose; RNA), dN = 2’-H (deoxyribose; DNA), * = phosphorothioate (PS) linkage. The CBT has been underlined.

[0155] As shown in Figure 2(B), there was a pronounced increase in editing yields with construct TMR543 (ASO17m) when compared to control (TMR317, v17; no mismatch outside the CBT). Specifically, at a concentration of between 10 nM and 25 nM a significant increase and improvement in editing yields was observed. A plateau was reached at around 30 nM for TMR543, whereas TMR317 did not reach a plateau, even at the highest concentration tested (50 nM).

[0156] Overall, these results show that a combination of chemical and sequence modifications improve the overall target editing efficacy of the oligonucleotide. More specifically, the data show that introducing a C-C mismatch at position -7 (N-7) enhances editing yields of the short 40nt long oligonucleotide. Furthermore, these data corroborate the findings of Example 1.Example 3. Combining favourable chemical and sequence modifications improves potency in PDE6A specific Oligonucleotides

[0157] To assess whether the sequence rules learned from SERPINA1 can be applied to other targets, the inventors generated chemically modified and sequence optimized PDE6A V685M targeting constructs (Table 3). A chemically and sequence optimized construct TMR530 was generated carrying a G ^ C nucleotide change at position -9 (N-9), thus introducing a C-C mismatch (-9C:C) with respect to the target RNA. A second construct TMR531 was generated carrying a further C ^ U conversion at position +2 (N+2), which creates a G:U mismatch (+2G:U) with respect to the target RNA. TMR529 served as control (no mismatch outside the CBT). The PDE6A V685M targeting constructs, sequences and their modification pattern are listed in Table 3. The results are shown in Figure 3. Table 3: PDE6A V685M targeting construct sequences including nucleobase and backbone modifications used in Example 3. mN = 2'-O-methyl (2’-OMe), fN = 2’-fluoro (2’-F), N = 2’-OH (ribose; RNA), dN = 2’-H (deoxyribose; DNA), * =

[0158] As shown in Figure 3, there was an overall increase in target editing yields with the chemically and sequence optimised oligonucleotide. This increase was significantly higher for the chemically and sequence optimised constructs TMR531, which carries two nucleotide changes (+2G:U and -9C:C mismatch), than for the chemically optimised oligonucleotide TMR529 (no mismatch). Unexpectedly, for TMR530, which carries a single nucleotide change at position N-9to create a C-C mismatch, there was a slight decrease in editing yields when compared to the control. For the PDE6A specific construct, two nucleotide changes showed a robust improvement in target editing.Example 4. Combining chemical and sequence modifications improves potency of SNCA specific oligonucleotides

[0159] Chemically- and sequence-optimised oligonucleotides targeting SERPINA1 and PDE6A transcripts showed enhanced target editing efficacy (Examples 1 to 3). Hence, to determine whether the combination of chemical and sequence modifications also enhances the potency of SNCA-targeting oligonucleotides by promoting a stabilizing effect on target-specific sequences, thus increasing the editing efficacy, different chemically and sequence-optimised ASO constructs were generated.

[0160] The different SCNA targeting constructs, sequences and their modification pattern are listed in Table 4. Construct TMR555 (no mismatch inside and outside the CBT) is a chemically optimised, 42nt long oligonucleotide and served as control. Constructs TMR556, TMR557 and TMR558 represent a chemically and sequence optimised version, carrying a C ^ G nucleotide change at position -1 (N-1) and / or a G C nucleotide change at position -9 (N-9).

[0161] Specifically, as summarized in Table 4, the chemically modified ASO v.117.4 (SEQ ID NO: 8) was further sequence-optimised by introducing a single guanosine (G) to cytosine (C) (GC) nucleotide change at position -9 (N-9) and / or a single cytosine (C) to guanosine (G) (C ^ G) nucleotide change at position -1 (N- 1) of the SNCA A53T targeting construct. The optimised constructs are reflected in SEQ ID NOs: 9, 10, and 11. Introducing a single C ^ G nucleotide change at position -1 (N-1) in the oligonucleotide leads to a G-G mismatch with respect to the target RNA (- 1G:G), while a single G ^ C nucleotide change at position -9 (N-9) in the oligonucleotide leads to a C-C mismatch (-9C:C) with respect to the target RNA.

[0162] The optimised constructs were subsequently assessed for their in vitro editing efficacy (%) of SNCA target RNA. The results are shown in Figure 4. The efficacy of target editing is measured as the percentage of edited target sites among all detected target sites in the target transcript. Table 4: SNCA targeting constructs including nucleobase and backbone modifications used in Example 4. mN = 2'-O-methyl (2’-OMe), fN = 2’-fluoro (2’-F), N = 2’-OH (ribose; RNA), dN = 2’-H (deoxyribose; DNA), * = phosphorothioate (PS) linkage. The CBT has been underlined.

[0163] Editing Efficacy: To assess cellular editing efficacy, 1 x 104HeLa cells (Cat. No.: ATCC CCL-2), were seeded in a 96-well plate. After 24 h, cells were forward transfected by FuGene6 transfection reagent (Promega) with a SNCA A53T expression plasmid expressing SNCA A53T cDNA. For each 96-well, 60ng plasmid were diluted in 10µL Opti-MEM, and 0.18µL FuGene6 transfection reagent (Promega) were diluted in 10µL Opti-MEM, and then incubated for 5 min, then combined and incubated for an additional 20 min. The medium was changed, and the transfection mix evenly distributed within one well. 24 h post plasmid transfection, the cells were forward transfected with the construct. For each 96-well, 5 pmol construct (which corresponds to 50nM in the total media volume per 96-well) and 0.3μl Lipofectamine RNAiMAX Reagent10 (ThermoFisher Scientific) were each diluted in 10μl Opti-MEM and incubated for 5 min, then combined and incubated for an additional 20 min. The medium was changed, and the transfection mix evenly distributed into one well. After 24 h, cells were harvested for RNA isolation and sequencing.

[0164] As shown in Figure 4, for the optimised construct TMR556 (SEQ ID NO: 9), the single nucleotide changes from G to C (G ^ C) at position -9 (N-9) did not have a significant impact on the editing efficacy when compared to control. However, the single nucleotide changes from C to G at position 1 (N-1) in construct TMR557 (SEQ ID NO: 10), significantly improved target editing by 3.2-fold when compared to control. In addition, the combination of having a mismatch at position -9 (N-9) and at position - 1 (N-1), i.e., a nucleotide change from G to C at position -9 and a nucleotide change from C to G at position -1 in the ASO, improved target editing by 2.6-fold when compared to control (see, Figure 4).

[0165] The results show that the introduction of a G-G mismatch at position -1 (N- 1) combined with a C-C mismatch at position -9 (N-9) enhances editing yields of the 42nt oligonucleotide.

[0166] In summary, the data demonstrate and further confirm that combining chemical modifications and sequence modifications improves the overall targetediting efficacy of the oligonucleotide. That is, oligonucleotides that incorporate a balanced mixture of 2’-F, 2’-OMe, and 2’-H modifications are able to offset the loss of 2’-OH modifications. When combined with internucleoside linkage modifications, such as PS linkages, this further boosts target editing efficiency. Further, the data show that more than 1 nucleotide change can be tolerated.

[0167] Overall, the results demonstrate that introducing specific mismatches in a chemically modified oligonucleotide with respect to the target RNA can increase editing yields of the oligonucleotide. The results further show that the number of required nucleotide changes and their positioning may be target dependent.REFERENCES Bass, B.L.; Weintraub H. (1987) A developmentally regulated activity that unwinds RNA duplexes. Cell.48:607–13. Bittner, Z. A.; Liu, X.; Shankar, S.; Tapia-Abellán, A.; Kalbacher, H.; Andreeva, L.; Mangan, M.; Düwell, P.; Lovotti, M.; Bosch, K.; Dickhöfer, S.; Marcu, A.; etc. (2021). BTK operates a phospho-tyrosine switch to regulate NLRP3 inflammasome activity. JEM.218(11): e20201656. Cideciyan, A. V.; Jacobson, S. G.; Drack, A. V.; Ho, A. C.; Charng, J.; Garafalo, A. V.; et al. (2019). Effect of an intravitreal antisense oligonucleotide on vision in Leber congenital amaurosis due to a photoreceptor cilium defect. Nat. Med.25(2): 225-228. Debacker, A. J.; Voutila, J.; Catley, M.; Blkey, D.; and Habib, N. (2020). Delivery of Oligonucleotides to the Liver with GalNAc: From Research to Registered Therapeutic Drug. Molecular Therapy.28(8): 1759-1771. Doherty, E. E.; Wilcox, X. E.; van Sint Fiet, L.; Kemmel, C.; Turunen, J. J.; Klein, B.; Tantillo, D. J.; Fisher, A. J.; and Beal, P. A. (2021). Rational Design of RNA Editing Guide Strands: Cytidine Analogs at the Orphan Position. J. Am. Chem. Soc.143: 6865-6876. Gagliardi, M., and Ashizawa, A. T. (2021). The Challenges and Strategies of Antisense Oligonucleotide Drug Delivery. Biomedicines.9(4): 433. Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012). Merkle T.; Merz, S.; Reautschnig, P.; Blaha, A.; Li, Q.; Vogel, P.; Wettengel, J; Li, J. B.; Stafforst, T. (2019). Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides. Nature Biotechnol.37: 133-138. Monian, P.; Shivalila, C.; Lu, G.; Shimizu; M.; Boulay, D.; Bussow, K.; Byrne, M.; Bezigian, A.; Chatterjee, A.; Chew, D.; Desai, J.; Favaloro, F.; Godfrey, F.; Hoss, A.; et al. (2022). Endogenous ADAR-mediated RNA editing in non-human primates using stereopure chemically modified oligonucleotides. Nature Biotech.40: 1093–1102. . Pedersen, C., C.; J. Lange, M. G. Gunnarsdatter Førland; A. D. Macleod; G. Alves; and J. Maple-Grødem. (2021). A systematic review of associations between common SNCA variants and clinical heterogeneity in Parkinson’s disease. npj Parkinson's Disease.7(54): 1-10. . Polymeropoulos, M. H.; C. Lavedan; E. Leroy; S. E. Ide; et al. (1997). Mutation in the alpha- synuclein gene identified in families with Parkinson's disease.276(5321): 2045-2047. . Quemener, A. M.; Bachelot, L.; Forestier, A.; Donnou-Fournet, E.; Gilot, D.; Galibert, M.-D. (2019). The powerful world of antisense oligonucleotides: From bench to bedside. Wiley Interdiscip. Rev. RNA.11(5): e1594. . Quinones-Valdez, G.; Tran, S. S.; Jun, H.-I.; Bahn, J. H.; Yang, E.-W.; Zhan, L.; Brümmer, A.; Wei, X.; Van Nostrand, E. L.; Pratt, G. A.; Yeo, G. W.; Graveley, B. R.; Xiao, X. (2019). Regulation of RNA editing by RNA-binding proteins in human cells. Comm. Biol.2(19): 1-14. . Rebagliati, M.R.; Melton D. A. (1987) Antisense RNA injections in fertilized frog eggs reveal an RNA duplex unwinding activity. Cell.48: 599–605.Zinshteyn, B., and Nishikura, K. (2009). Adenosine-to-inosine RNA editing. Rev. Syst. Biol. Med.1(2): 202-209. . Sothilingham, V.; Garrido, M. G.; Jiao, K.; Buena-Atienza, E.; Sahaboglu, A. et al. (2015). Retinitis pigmentosa: impact of different Pde6a point mutations on the disease phenotype. Human Mol. Genetics.24(19): 5486-5499. . Stephenson, M. L; Zamecnik, P. C. (1978). Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc. Natl. Acad. Sci. USA.75(1): 285-288. . Thomas, J. M., and Beal, P. A. (2017). How do ADARs bind RNA? New protein-RNA structures illuminate substrate recognition by the RNA editing ADARs. Bioassays.39(4): 1-. . Tomaselli, S.; Locatelli, F.; Gallo, A. (2014). The RNA editing enzymes ADARs: mechanism of action and human disease. Cell and Tissue Research.356: 527–532. . Vogel, P.; Schneider, M. F.; Wettengel, J.; and Stafforst, T. (2014). Improving site-directed RNA editing in vitro and in cell culture by chemical modification of the guideRNA. Angew. Chem. Int. Ed. Engl.53(24): 6267-6271. . Wong, S. K., Sato, S., and Lazinski, D. W.. (2001) Substrate recognition by ADAR1 and ADAR2. RNA.7: 846-858. . Wulff, B.-E., and Nishikura, K. (2010). Substitutional A-to-I RNA editing. Interdiscip. Rev. RNA. 1(1): 90–101.

Claims

CLAIMS 1. A modified antisense oligonucleotide suitable for editing an adenosine in a target nucleic acid to inosine (A-to-I editing), wherein the oligonucleotide comprises: (I) a nucleic acid sequence of 25 to 70 nucleotides (nt) that is an antisense sequence substantially complementary to the target nucleic acid, (II) a central base triplet (CBT) (5’- N+1eN0fN-1g-3’) with the central nucleotide (N0) directly opposite the target adenosine to be edited when the antisense oligonucleotide is hybridised to the target nucleic acid, and (III) the following core sequence: 5’-aN+4bN+3cN+2dN+1eN0fN-1gN-2hN-3iN-4jN-5kN-6lN-7mN-8nN-9 -3’ ; and wherein: (a) at least two of the three nucleotides of the CBT are chemically modified at the 2' position of the sugar moiety, are deoxyribonucleosides, or a combination thereof and wherein d and e are internucleoside linkage modifications; (b) the N-2 nucleotide carries a 2’-O-alkyl-modification; and wherein the N-3 nucleotide carries a 2´-fluoro (2’-F)-modification; (c) at least 10% of nucleotides of the oligonucleotide are 2’-F-modified and at least 10% of nucleotides of the oligonucleotide are 2’-O-alkyl- modified, wherein no more than 6 consecutive nucleotides have the same 2’-modification; and wherein the oligonucleotide comprises one or more nucleotide mismatches outside the CBT that is / are not complementary to the corresponding position in the target nucleic; and wherein the mismatch is: i. at position -7 (N-7), wherein there is a G or C directly 3’ to the mismatch and an A or U directly 5′ to the mismatch; ii. at position -9 (N-9),wherein there is a G or C directly 3’ to the mismatch and an A or U directly 5’ to the mismatch; or iii. at position -9 (N-9),wherein there is a G or C directly 3’ to the mismatch and an A or U directly 5’ to the mismatch, and at position +2 (N+2).

2. The oligonucleotide according to claim 1, wherein(d) the internucleoside linkage modification content is at least 15 %; and linkages h and i are not phosphorothioate (PS) linkages; (e) the regions 3’ and 5’ to the CBT do not contain more than a total of 6 deoxyribonucleosides and the internucleoside linkage modification content is at least 30%; or (f) the regions 3’ and 5’ to the CBT have a total deoxyribonucleoside content of 5-50%.

3. The modified antisense oligonucleotide according to claim 1 or 2, wherein 20-100% of nucleotides are 2’-modified, preferably wherein 50-100% of nucleotides are 2’- modified nucleotides.

4. The modified antisense oligonucleotide according to any one of claims 1-3, wherein 20-70% of nucleotides are 2’-F-modified, preferably wherein 35-65% of nucleotides are 2’-F-modified; and / or wherein 20-60% of nucleotides are 2’-O-methyl (2’-OMe)-modified, preferably wherein 25-55% of nucleotides are 2’-OMe-modified.

5. The modified antisense oligonucleotide according to any one of claims 1-4, wherein (i) no more than 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30% or 20% of the linkages outside the CBT are internucleoside linkage modifications; or (ii) 15-90% of the linkages are internucleoside linkage modifications, preferably wherein 40-80%, most preferably 45-60%, of the linkages are internucleoside linkage modifications.

6. The modified antisense oligonucleotide according to any one of claims 1-5, wherein the oligonucleotide has a length of 25-60nt, preferably 35-60nt, more preferably a length of 40-59 nt.

7. The modified antisense oligonucleotide according to claim 6, wherein the oligonucleotide has a length of 40, 42 or 59 nucleotides and wherein outside of the CBT no more than 3 nucleotides are deoxyribonucleotides.

8. The modified antisense oligonucleotide according to any one of claims 1-7, comprising at least two internucleoside linkage modification selected from the group consisting of phosphorothioate (PS), 3'-methylenephosphonate, 5'- methylenephosphonate, 3'-phosphoroamidate, 2'-5'phosphodiester, andphosphoryl guanidine (PN), preferably wherein the at least one internucleoside linkage modification is PS.

9. The modified antisense oligonucleotide according to any one of claims 1-8, wherein (i) linkages h and i are not phosphorothioate (PS) linkages, and / or phosphate (PO) linkages; and / or (ii) linkage g is not a PS linkage, preferably wherein g is a phosphate (PO) linkage; and / or (iii) linkages d and e are PS linkage modifications; and / or (iv) linkage f is an internucleoside linkage modification.

10. The modified antisense oligonucleotide according to any one of claims claim 1-9, wherein (i) no more than 4, 5, or 6 consecutive nucleotides are 2’-F-modified; and / or (ii) no more than 4, 5, or 6 consecutive nucleotides are 2’-O-alkyl-modified.

11. The modified antisense oligonucleotide according to any one of claims 1-10, wherein less than 6, 5, 4, or 3 consecutive nucleotides have the same 2’- modification.

12. The modified antisense oligonucleotide according to any one of claims 1-11, wherein the 2’-O-alkyl-modification is a 2’-O-methyl (2’-OMe)-modification.

13. The modified antisense oligonucleotide according to any one of claims 1-12, wherein (i) N+1 is 2'-F, 2’-FANA, DNA, or 2´-O-methyl; and / or (ii) N0is 2'-FANA or DNA; and / or (iii) N-1is 2'-FANA, DNA, or 2’-O-methyl.

14. The modified antisense oligonucleotide according to any one of claims 1-13, wherein positions +5, +4, and +3 are 2’-O-alkyl-modified; and / or wherein position +2 is 2’-F-modified.

15. The modified antisense oligonucleotide according to any one of claims 1-14, wherein there are: (i) at least 4 nucleotides 3’ of the CBT; and / or (ii) at least 16 nucleotides 5’ of the CBT.

16. The modified antisense oligonucleotide of any one of claims 1-15, comprising (i) at least 10 continuous internucleoside linkage modifications; and / or (ii) 3 consecutive internucleoside linkage modifications at each terminus.

17. The modified antisense oligonucleotide according to any one of claims 1-16, wherein the oligonucleotide does not comprise a hairpin-loop structured ADAR recruitment motif.

18. The modified antisense oligonucleotide according to claim 1-17, wherein the mismatch is a paired G-A, C-A, U-C, A-A, G-G, C-C, U-U, or G-U mismatch, preferably wherein the mismatch is a paired C-C or G-U mismatch.

19. The modified antisense oligonucleotide according to claim 1-18, wherein the oligonucleotide comprises: (i) a C at position N-7 and optionally a U at position N+2; (ii) a U at position N+2; or (iii) a C at position N-9 and optionally a U at position N+2.

20. The modified antisense oligonucleotide according to any one of claims 1-19, wherein the target nucleic acid comprises a sequence from a mutant SERPINA1 gene that encodes SERPINA1 having a E342K mutation, and wherein the nucleic acid sequence of the oligonucleotide is an antisense sequence that is substantially complementary to the sequence from the mutant SERPINA1 gene that encodes SERPINA1 having the E342K mutation.

21. The modified antisense oligonucleotide according to claim 20, wherein the oligonucleotide comprises the following sequence: 5’ - mC*mA*mU*fG*dG*fC*mC*fC*mC*fA*dG*fC*fA*dG*mC*fU*mU*fC*fA*dG*mU*fC *mC*fC*mU*mUmUfC*dT*dC*dImUfCfG*dA*mU*fC*dG*fU*mC*fA*dG*fC*fA*mC* dA*fG*fC*mC*fU*mU*dA*fU*fG*mC*dA*mC*mG*mG – 3‘ (SEQ ID NO: 2); wherein m = 2‘-O-methyl; f = 2‘-fluoro; I = inosine; * = phosphorothioate (PS) linkage; andd = deoxyribose (DNA).

22. The modified antisense oligonucleotide according to claim 21, wherein the oligonucleotide comprises the following sequence: 5’ - mC*mA*mU*fG*dG*fC*mC*fC*mC*fA*dG*fC*fA*dG*mC*fU*mU*fC*fA*dG*mU*fC *mC*fC*mU*mUmUfC*dT*dC*dImUfCfG*dA*mU*fC*mG*mU*mC –3‘ (SEQ ID NO: 4); wherein m = 2‘-O-methyl; f = 2‘-fluoro; I = inosine; * = phosphorothioate (PS) linkage; and d = deoxyribose (DNA).

23. The modified antisense oligonucleotide according to any one of claims 1-19, wherein the target nucleic acid comprises a sequence from a mutant PDE6A gene that encodes PDE6A having a V685M mutation, and wherein the nucleic acid sequence of the oligonucleotide is an antisense sequence that is substantially complementary to the sequence from the mutant PDE6A gene that encodes PDE6A having the V685M mutation.

24. The modified antisense oligonucleotide according to claim 23, wherein the oligonucleotide comprises the following sequence: 5’ - mU*mU*mC*fA*fC*mU*fC*mU*fC*fA*mU*fA*fU*fG*mU*fC*mU*fU*fA*fG*fA*mC*f U*fG*fA*mUfCmU* dA*dC*dG mAfUmC*fU*mU*fU* mU* fC*mG*mA*mA – 3‘ (SEQ ID NO: 7); wherein m = 2‘-O-methyl; f = 2‘-fluoro; * = phosphorothioate (PS) linkage; and d = deoxyribose (DNA).

25. The modified antisense oligonucleotide according to any one of claims 1-24, wherein the oligonucleotide is conjugated to a moiety that enhances cellular uptake of the oligonucleotide.

26. A composition comprising the oligonucleotide according to any one of claims 1-25.

27. A method for site-directed A-to-I editing of a target RNA, comprising providing to a cell or subject an oligonucleotide according to any one of claims 1-25, or a composition according to claim 26.

28. A method of treating a disease or condition associated with a point mutation in a subject by administering to the subject a therapeutically effective amount of an oligonucleotide according to any one of claims 1-25, or a composition according to claim 26.

29. The method of treating a disease or condition associated with a point mutation according to claim 28, wherein the disease or condition is α(1)AT deficiency or an associated pathology or retinitis pigmentosa or an associated pathology.

30. The method according to claim 28 or 29, wherein the disease or condition is α(1)AT deficiency or an associated pathology, and the target transcript comprises the sequence from the mutant SERPINA1 gene, optionally encoding SERPINA1 having the E342K mutation.

31. The method according to claim 28 or 29, wherein the disease or condition is retinitis pigmentosa or an associated pathology, and the target transcript comprises the sequence from the mutant PDE6A gene, optionally encoding PDE6A having the V685M mutation.

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