Chemically modified antisense oligonucleotides (ASOS) and compositions for RNA editing

Abstract

The invention relates to chemically modified oligonucleotides for use in site-directed A-to-I editing of a target RNA inside a cell with endogenous adenosine deaminase acting on RNA (ADAR), the oligonucleotide comprising a sequence capable of binding to a target sequence in a target RNA and a central base triplet (CBT) of (3) nucleotides (N-1 N0 N+1), wherein N0 is the central nucleotide directly opposite to a target adenosine in the target RNA that is to be edited, wherein the oligonucleotide comprises at least one internucleoside linkage that is a methanesulfonyl (mesyl) linkage.

Description

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2025 / 036984 A1 20 February 2025 (20.02.2025) WIPO PCT (51) International Patent Classification: C12N 15 / 11 (2006.01) C12N 15 / 113 (2010.01) (21) International Application Number: PCT / EP2024 / 073031 (22) International Filing Date: 15 August 2024 (15.08.2024) (25) Filing Language: English (26) Publication Language: English (30) Priority Data: PCT / EP2023 / 072474 15 August 2023 (15.08.2023) EP (71) Applicant: AIRNA CORPORATION [US / US]; 238 Main Street c / o Bayer Co.Lab, Cambridge, Massachusetts 02142 (US). (71) Applicant (for MN only): AIRNA BIO GERMANY GMBH [DE / DE]; Rosentalstr. 5, 72070 Tübingen (DE). (72) Inventor: MERKLE, Tobias; c / o AIRNA Bio Germany GmbH, Rosentalstr. 5, 72070 Tübingen (DE). (74) Agent: GILL JENNINGS & EVERY LLP; The Broadgate Tower, 20 Primrose Street, London EC2A 2ES (GB). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW. BY, BZ. CA, CH, CL, CN, CO, CR, CU, CV, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IQ, IR, IS, IT, JM, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, MG, MK, MN, MU, MW, MX, MY, MZ, NA, NG, NI, NO. NZ, OM, PA, PE. PG. PH, PL. PT, OA, RO. RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, WS. ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, CV, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SC, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS. IT. LT. LU. LV. MC, ME, MK, MT, NL, NO, PL, PT, RO, RS, SE. SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Published: - with international search report (Art. 21(3)) - before the expiration of the time limit for amending the claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) - in black and white; the international application as filed contained color or greyscale and is available for download from PATENTSCOPE (54) Title: CHEMICALLY MODIFIED ANTISENSE OLIGONUCLEOTIDES (ASOS) AND COMPOSITIONS FOR RNA EDITING (B) (N-1 No N+1), (1) 100- 90- 2MP (24,-8) 200M 2Mesyl (24,-8) 4nM 80- 70- 60 (57) Abstract: The invention relates to chemically modified oligonucleotides for use in site-directed A-to-I editing of a target RNA inside a cell with endogenous adeno- sine deaminase acting on RNA (ADAR), the oligonucleotide comprising a sequence capable of binding to a target sequence in a target RNA and a central base triplet (CBT) of (3) nucleotides (N.1 No N+1), wherein NO is the central nucleotide direct- ly opposite to a target adenosine in the target RNA that is to be edited, wherein the oligonucleotide comprises at least one internucleoside linkage that is a methanesul- fonyl (mesyl) linkage. W O 2 0 2 5 / 0 3 6 9 8 4 A 1 Ed it in g (% ) Al -2 93 9 A1 -2 93 8 Figure 9 WO 2025 / 036984 1 PCT / EP2024 / 073031 CHEMICALLY MODIFIED ANTISENSE OLIGONUCLEOTIDES (ASOS) AND COMPOSITIONS FOR RNA EDITING FIELD OF THE INVENTION 5

[001] 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") or for editing of an RNA derived from a wildtype allele (“beneficial editing"). BACKGROUND OF THE INVENTION 10

[002] 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 that leads to a modified protein in a more 15 20 25 30 precise, efficient, and safe manner. Contrary to DNA, RNA is generally quickly degraded and any errors introduced by off-target modifications to other RNAs 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.

[003] 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).

[004] 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. RNA editing enzymes are known in the art. The first RNA editing process discovered in mammals was the deamination of cytidine (C) by APOBEC proteins to form uridine (U) (Zinshteyn and Nishikura, WO 2025 / 036984 2 PCT / EP2024 / 073031 5 10 15 2009). 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-l") conversions. Notably, for therapeutic purposes in higher eukaryotes the most prevalent type of RNA editing is the "A-to-l' conversion, which is catalysed by the adenosine deaminases acting on RNA (ADARs) family.

[005] Over the years, three vertebrate ADAR genes have been identified, which give rise to several ADAR proteins through alternative promoters 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; Zinshteyn and Nishikura, 2009). Of the three known ADAR genes, hADAR1 and hADAR2 are expressed in most tissues and encode active deaminases. Human ADAR3 (hADAR3) has been described to only be expressed in the central nervous system and reportedly has no deaminase activity in vitro. While all ADARs are multidomain proteins, comprising a targeting or dsRNA-binding domain (dsRBD) and a catalytic domain, ADAR1 proteins additionally comprise one or more Z binding domains, while splice variant ADAR2R and ADAR3 comprises an R domain (Zinshteyn and Nishikura, 2009; Wulff and Nishikura, 2010). Accordingly, the ADAR may be hADAR1, hADAR2 or hADAR3, or 20 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. 25 30 35

[006] "A-to-l' editing was initially identified in Xenopus eggs (Bass and Weintraub, 1987; Rebagliati and Melton, 1987). Human cDNA encoding "double stranded RNA adenosine deaminase" was first cloned by Kim et al. (1994) and "A-to-f' conversion activity of the protein confirmed by recombinant expression in insect cells. Specifically, “A-to-l" 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. 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.

[007] ASOs are generally short (approx.18 to 25 nucleobases in length) 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. Notably, ASO sequence and WO 2025 / 036984 3 PCT / EP2024 / 073031 design are the primary drivers that determine the pharmacological and toxicological properties of the oligonucleotide.

[008] Gen 1 ASOs were initially employed to inhibit translation of Rous sarcoma virus ribosomal RNA (Stephenson and Zamecnik, 1978). They are characterised in 5 having a modified backbone, wherein the nucleotide linkages are modified by sulphur, methyl or amine groups to generate phosphorothioates (PS), methyl- phosphonates (MP), and phosphoramidates, respectively. Hence, ASOs can be chemically modified to improve their properties. For instance, ASOs can be modified 10 15 20 25 30 to protect them against nucleases and to increase their effectiveness. While PS modifications seem to have a positive effect on ASOs stability and pharmacokinetics, the difference in chirality of PS linkages may have a substantial influence on the ASO's overall property (lwamoto et al., 2017; Crooke et al., 2020).

[009] 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.

[0010] In comparison, 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).

[0011] ASOs can work through many mechanisms depending, in part, on the region in the RNA sequence that is targeted and ASO design / chemical properties. 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-" RNA editing, the ASO targeting domain contains a mismatch opposite the targeted adenosine. It is to be noted that several endogenous substrates of ADAR contain mismatches and / or bulges (Thomas and WO 2025 / 036984 4 РCТ / EР2024 / 073031 Beal, 2017) and therefore could alter or even improve substrate recognition, if these features are mimicked in the ASO / resulting dsRNA.

[0012] Furthermore, ASOs can be chemically modified to improve their properties. For instance, ASOs can be modified to protect them against nucleases and to 5 increase their effectiveness. While phosphorothioate (PS) modifications seem to have a positive effect on ASOs stability and pharmacokinetics, 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. However, the10 15 20 benefit of Rp and Sp stereoisomers has been controversial (Iwamoto et al., 2017; Crooke et al., 2020).

[0013] The use of antisense oligonucleotides for site-directed RNA editing has previously been described (Vogel et al., 2014; Merkle et al., 2019) and ASO-based therapies have been gaining more and more traction over the past years for use in the treatment of different genetic disorders.

[0014] Loop-hairpin structured oligonucleotides have previously been described (WO 2020 / 001793) and have been used successfully to harness ADARs with chemically modified oligonucleotides. However, they are comparably large and - without being bound by any theory - the inventors believe that a more intelligent design of the ASO can form a substrate duplex that is also very well and quickly recognized by endogenous ADAR so that the large recruitment motifs can be omitted. For the delivery and manufacture this is a clear advantage as much shorter ASOs can be designed. 25

[0015] New designs for nucleoside analogues are constantly being investigated. These oligonucleotides typically are 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 central base triplet (CBT), wherein the CBT has the general structure (5'- N+1 No N-1 -3') and No is the 30 central nucleotide (No) directly opposite the target adenosine (A) to be edited, when the oligonucleotide is hybridized to the target RNA sequence. Further, some of these oligonucleotides contain almost complete stereopure PS-modified backbones and additional charge-neutral PN linkages (also stereopure), the latter of which is not yet applied in the clinics. That precise, site-specific RNA editing can be achieved by 35 recruiting endogenous ADARs with antisense oligonucleotides has previously been WO 2025 / 036984 5 PCT / EP2024 / 073031 5 shown by Merkle et al. (2019). They were able to demonstrate that chemically optimized ASOs can be used to recruit endogenous human ADARs to edit endogenous transcripts in a simple and programmable way with almost no off-target editing.

[0016] In WO 2020 / 001793, an artificial nucleic acid for site-directed "A-to-l" editing was provided, wherein the artificial nucleic acid comprised a targeting sequence and recruiting moiety. Similarly, WO 2018 / 041973 relates to ASOs that do not form an intramolecular hairpin or stem-loop structure. WO 2018 / 041973 specifically relates to chemically modified single-stranded RNA-editing oligonucleotides for the 10 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. 15

[0017] Other prior art, such as WO 2021 / 071858, relates to 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. 20

[0018] Research in the field of ASO optimisation for A-to- / editing has led not only to the identification of the CBT but also to a more thorough investigation of the region immediate 5' and 3' to the CBT. In addition to specifically looking at CBT modifications (e.g., 2'-F and 2'-FANA), WO 2021 / 243023 also mentions guide or 25 30 35 targeting domain modifications 3' to the nucleobase just outside the CBT (at position +2 of an oligonucleotide comprising the structure [Am]-X1-X2-X3-X4-[Bn], wherein X4 corresponds to the +2 position). 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.

[0019] However, despite being a promising technology, few ASOs have been marketed. This is due to difficulties pertaining to stability, cellular delivery, clinical efficacy, as well as off-target effects and / or preclinical toxicologic challenges. Hence, to translate ASO-based therapies into a widespread clinical success, it is crucial to overcome these different challenges. Accordingly, there is currently an unmet need for improved ASOs and effective therapies for the treatment of genetic disorders involving these improved ASOs. One aim of the invention is to provide ASOs with WO 2025 / 036984 6 PCT / EP2024 / 073031 improved properties, including stability to aid in vivo delivery, and improved A-to-l editing. SUMMARY OF THE INVENTION 5

[0020] The inventors found that chemically modified antisense oligonucleotides comprising one or more mesyl phosphoramidate (or mesyl) linkages can be synthesised and used as alternatives to oligonucleotides comprising traditional internucleoside linkage modifications such as, e.g., phosphonothioate linkages (PS) and / or methylphosphonate (MP) linkages. It was observed that mesyl linkages have 10 a beneficial effect and that by placing mesyl linkages at specific positions within the oligonucleotide, oligonucleotide stability and A-to-I target editing can be improved. For example, placement of mesyl linkages in the 5' and 3' flanking regions of the individual oligonucleotide enhanced editing. The inventors further identified key internal positions (e.g., position -2 and +13), where the mesyl linkage can be placed 15 to improve oligonucleotide stability and activity. 20 25 30

[0021] 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 modified ASOs capable of mediating a functional change from an adenosine (A) to a guanosine (G). Specifically, the invention relates to chemically modified oligonucleotides for use in site-directed A-to-l editing, comprising at least one linkage that is a methanesulfonyl (mesyl) linkage. To date, no prior art has been identified that teaches or suggests the oligonucleotides, compositions, and methods as provided herein, which are particularly effective in providing stable and less hydrophobic ASOs and compositions comprising the same for use in site-directed A- to- / editing of a target RNA.

[0022] The solution to the technical problem is achieved by the embodiments described herein and defined by the appended claims.

[0023] The present invention generally provides for chemically modified oligonucleotides for use in site-directed A-to- / editing of a target RNA inside a cell with endogenous adenosine deaminase acting on RNA (ADAR).

[0024] In a first aspect, the present invention provides a chemically modified oligonucleotide for use in site-directed A-to-l editing of a target RNA inside a cell with endogenous adenosine deaminase acting on RNA (ADAR), the oligonucleotide WO 2025 / 036984 7 PCT / EP2024 / 073031 5 10 15 comprising a sequence capable of binding to a target sequence in a target RNA and a central base triplet (CBT) of 3 nucleotides (5' - N+1 No N-1- 3'), wherein No is the central nucleotide directly opposite to a target adenosine in the target RNA that is to be edited, wherein the oligonucleotide comprises at least one linkage that is a methanesulfonyl (mesyl) linkage.

[0025] In a second aspect provided herein is a composition comprising the chemically modified oligonucleotide of the invention.

[0026] In a third aspect provided herein is a chemically modified oligonucleotide for therapeutic use.

[0027] In a fourth aspect provided herein is a chemically modified oligonucleotide of the invention or a composition of the invention for use in the treatment of a disease or disorder, where in the disease or disorder is selected form the group consisting of liver, metabolic, neurodegenerative, and / or cardiac or cardiovascular diseases associated with a gain-of-function (GOF) or loss-of-function (LOF) mutation.

[0028] In a fifth aspect provided herein is a method for treating a subject suffering from a genetic disease or genetic disorder, comprising administering an effective amount of the chemically modified oligonucleotide of the invention or the composition of the invention to the subject.

[0029] In a sixth aspect provided herein is an in vitro method for site-directed A-to-I 20 editing of a target RNA, the method comprising a step of contacting a target RNA with the chemically modified oligonucleotide of the invention or the composition of the invention.

[0030] The inventors found that chemically modified antisense oligonucleotides comprising one or more mesyl phosphoramidate (or mesyl) linkages can be 25 synthesised and used as alternatives to oligonucleotides comprising traditional internucleoside linkage modifications such as, e.g., phosphonothioate linkage (PS). It was observed that by placing mesyl linkages at specific positions within the oligonucleotide A-to-l target editing could be improved. Specially, placement of mesyl linkages in the flanking regions of the individual oligonucleotide improved 30 editing. The inventors further identified key internal positions in the ASO where the mesyl linkage should be placed to improve ASO activity. WO 2025 / 036984 8 PCT / EP2024 / 073031 5 10 15 20 25 30 BRIEF DESCRIPTION OF DRAWINGS

[0031] 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.

[0032] Fig. 1 represents graphs showing (A) different types of internucleoside linkage modifications and positioning of individual PO, mesyl and PN internucleoside linkages within the oligonucleotide. (B) Mesyl Walk: respective editing (in %) depending on the specific positioning of the mesyl linkage at 4nM and 20nM.

[0033] Fig. 2 presents a bar graph showing the editing efficacy (in %) of different chemically modified oligonucleotides having a length of 30nt to 38nt and comprising a combination of different chemical modifications at the 2'-position of the sugar residue.

[0034] Fig. 3 presents a bar graph showing the editing (in %) of ASO candidates of varying asymmetries and lengths (30nt, 34nt, 36nt, 38nt) carrying different combinations of 2'F, PN, mesyl and 2'MOE modifications.

[0035] Fig. 4 presents a bar graph showing the editing (in %) of ASO candidates of varying asymmetries and lengths (30nt, 34nt, 36nt, 38nt) comprising different combinations of 2'F and 2'MOE modifications.

[0036] Fig. 5 presents a graph showing the editing (in %) of various GaINAc- conjugated SERPINA1 targeting oligonucleotides.

[0037] Fig. 6 presents a bar graph showing the editing (in %) of GalNAc-conjugated SERPINA1 targeting oligonucleotides in ASO transfected Piz mouse hepatocytes at concentrations 0.8nM, 4nM and 20nM.

[0038] Fig. 7 shows (A) a layout of the in vivo study design and bar graphs showing (B) the editing (%) of the ASO candidates and (C) M-AAT (μM) levels.

[0039] Fig. 8 shows a bar graph depicting the results of a mesyl walk. The graph displays the editing (%) of various ASOs ("25-1-8" asymmetry) and a base mesyl- modified backbone ("+24, -2, -8") and 1 additional moving mesyl linkage.

[0040] Fig. 9 shows bar graphs comparing the in vitro ((A) and (B)) and in vivo ((C) and (D)) editing (in %) of ASO candidates comprising or 2 MP or 2 mesyl linkage modifications located at positions +24 and -8 (5' and 3' terminal position respectively) or 3 methylphosphonate (MP) or 3 mesyl linkage modifications located at positions +24, -2, and -8.

[0041] Fig. 10 shows (A) a layout of the in vivo study design and presents bar graphs 35 (B) and (C) showing target editing (in %). WO 2025 / 036984 9 PCT / EP2024 / 073031 5 10

[0042] Fig. 11 shows the in vivo editing (in %) in liver tissue of ASO candidates comprising 2'MOE modifications or mesyl linkage or PN linkage modifications.

[0043] Fig. 12 presents a bar graph showing the in vivo editing (in %) of ASO candidates of different asymmetries comprising mesyl linkages in the 5' and 3' flanking regions.

[0044] Fig. 13 presents bar graphs showing (A) the editing (in %) of ASOs of various asymmetries, and (B) the editing (in %) of ASOs carrying an additional mesyl linkage modification at position +13.

[0045] Fig. 14 presents a bar graph showing the editing efficacy of surrogate ASO candidates comprising mesyl linkages in the 5' and 3' flanking regions.

[0046] Fig. 15 presents a bar graph showing the editing efficacy (in %) of various hACTB targeting oligonucleotides at 4nM and 20 nM. 15 20 DETAILED DESCRIPTION Terminology

[0047] In order that the present invention may be more readily understood, certain terms are first defined.

[0048] 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.

[0049] The terms "about" and "approximately" may be understood to permit standard variation as would be understood by those of ordinary skill in the art.

[0050] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including, but not limited to". Likewise, the term "comprising" is used 25 herein to mean, and is used interchangeably with, the phrase "comprising, but not limited to".

[0051] As used herein, the expressions "mesyl phosphoramidate", "methansulfonyl phosphoramidate" and "methanesulfonyl (mesyl)" internucleoside linkage (modification) have the same meaning and can be used interchangeably. The 30 oligonucleotides of the invention contain at least one mesyl linkage, which means that at least one mesyl phosphoramidate linkage is incorporated into the ASO backbone instead of the natural phosphate (PO) linkage (i.e., phosphodiester group). WO 2025 / 036984 Ο O=P-O Base ό α X PCT / EP2024 / 073031 10 H3C S-N P Base OH X 10 15 Phosphodiester (PO) Mesyl phosphoramidate (mesyl) As used herein, a 'mesyl' linkage at position +24 (N+24) indicates that the nucleotide at position 24 is linked via its phosphate to a (NSO2CH3) group and that the mesyl 5 linkage is located between nucleotide 25 (N+25) and nucleotide 24 (N+24). Likewise, if it is indicated the mesyl position is located at "-8" (N-3), this means that the mesyl linkage is between the nucleotides located at position -7 (N-7) and -8 (N.g). As used herein, nucleotide positions that are underlined indicate the terminal or penultimate positions containing a mesyl linkage modification, e.g., mesyl modification pattern "+24, -2, -8". 20

[0052] As used herein the term "flanking region" refers to the 5' and / or 3' region on the oligonucleotide is adjacent or directly adjacent to the No on the 5' and / or 3' portion of the oligonucleotide. In one embodiment, the flanking region is located directly adjacent to No. Alternatively, in one embodiment, a flanking region is located anywhere upstream and / or anywhere downstream of No. In one embodiment, the flanking region is located at the far end of the 5' terminus and / or at the far end of the 3' terminus. The flanking region may comprise one or more nucleotide, i.e., a range of nucleotides. For instance, the flanking region my comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or more nucleotides 5' and / or 3' to No. That is, in some embodiments, the flanking region comprises the entire region 5' and / or 3' to No, in other embodiments the flanking region comprises the outermost 1, 2, or 3 nucleotides at the 5' and / or 3' terminus.

[0053] 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. 25 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 WO 2025 / 036984 11 PCT / EP2024 / 073031 5 10 15 modified and categorized by modification of (1) the internucleoside linkage, (2) the deoxyribose / ribose, and / or (3) the nucleobase.

[0054] 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".

[0055] Within the context of this invention, the term "modified nucleobase" and "modified base" may be used interchangeably with the term "nucleobase". A nucleobase may be a nucleobase, which comprises a modification. In some embodiments, a modified nucleobase is capable of at least one function of a nucleobase, e.g., forming a moiety in a polymer capable of base-pairing to a nucleic acid comprising an at least complementary sequence of bases. In one embodiment, the modified nucleobase is capable of increasing hydrogen bonding, base pair stacking interactions and / or stabilizing a nucleic acid complex. The modified 20 nucleobase (e.g., Benner's base) may be capable of mimicking the N3 protonated cytosine base. In some embodiments, a modified nucleobase is substituted A, T, С, 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- 25 methyl-2'-deoxycytidine (m³C), pseudouridine (pU), dihydrouridine, inosine (I), and 7- methylguanosine. In some embodiments, the modification is iso-uridine (SbU). 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, 30 the term "Benner's base Z" refers to the pyrimidine analogue 6-amino-5-nitro-3-(1'-B- 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, 35 nucleoside or nucleotide. For example, a derivative may be a corresponding WO 2025 / 036984 PCT / EP2024 / 073031 5 10 12 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 (m³C), or ribocytidine.

[0056] 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".

[0057] 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, 15 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".

[0058] The term "oligonucleotide(s)" as used herein is defined as is generally 20 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.

[0059] The term "internucleoside linkage" refers to a linkage between adjacent 25 30 35 nucleosides. "Internucleoside linkage" and "linkage" may be used interchangeably. Linkages may be continuous (consecutive) or discontinuous (interrupted). As used herein, the term "discontinuous" or "interrupted" means that there are not more than, e.g., 4, 5, 6, 7 or more consecutive internucleoside linkage modifications of the same modification. In some embodiments, the naturally occurring PO linkages are replaced by modified internucleoside linkages. Hence, in some embodiments, the linkage is a non-natural internucleoside linkage.

[0060] 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, WO 2025 / 036984 PCT / EP2024 / 073031 13 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 5 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.

[0061] As used herein the term "antisense oligonucleotide" or "ASO" refers to a strand 10 of nucleotide analogue that hybridizes with the complementary (target) RNA in a sequence-specific manner via Watson-Crick base pairing. The ASO may be chemically modified. The terms "antisense oligonucleotide" and "oligonucleotide" may be used interchangeably. 15 20 25

[0062] As used herein, the term "target RNA" refers to an RNA, which is subject to the editing process, and "targeted" by the respective ASOs of the invention.

[0063] 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.

[0064] The term "modified sugar" refers to a moiety that can replace a naturally occurring sugar. A modified sugar may mimic the spatial arrangement, electronic properties, or some other physicochemical property of a sugar. The naturally occurring sugar is generally the pentose deoxyribose or ribose, though it should be understood that naturally and non-naturally occurring sugar analogues are also included. For example, sugars may comprise C4 sugars, C5 sugars and / or C6 sugars. In some embodiments, a modified sugar is substituted. In some embodiments, 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 embodiments, a modified 30 sugar comprises a 2'-modification. Examples of useful 2'-sugar modifications include, e.g., 2'-ribose (RNA), 2'-deoxyribose (DNA), 2'-arabinose etc.. Those skilled in the art, will appreciate that various types of 2'-sugar modifications are known that can be used in accordance with the present disclosure. In one embodiment, the 2'-sugar modification is 2'-ribose. In one embodiment, the 2'-sugar modification is 2'- 35 deoxyribose. The term "locked nucleic acid" (LNA) or "locked nucleic acids" (LNAs) WO 2025 / 036984 РCТ / EР2024 / 073031 5 10 15 20 25 30 35 14 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 modified sugar is a bicyclic sugar, e.g., a sugar used in locked nucleic acid (LNA), BNA, etc.. In some embodiments, a modified sugar is an LNA sugar. In some embodiments, a modified sugar is an BNA sugar. In some embodiments, a sugar modification is 2'-OMe, 2'-O-methoxyethyl (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). 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).

[0065] The term "FANA” or "FANA-modified" refers to 2'-fluoroarabinoside modified nucleobases and / or oligonucleotides comprising such nucleobases. Forexample, the expression "FANA-сytidine" 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) or oligonucleotide(s) that has been chemically derived from said nucleotide or oligonucleotide(s).

[0066] As used herein, the term "complementary", "partially 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. As those skilled in the art appreciate, perfect (e.g., 100%) complementarity or pairing is not required and one or more wobbles (wobble base pairing), bulges, mismatches, etc. may be tolerated. The one or more wobbles, bulges, mismatches, etc. may be within or outside the CBT. Hence, in one embodiment, the ASOs comprise a wobble base outside the CBT. In one embodiment, the ASO comprises a mismatch outside the CBT. For example, the ASOs may include a mismatch opposite the target adenosine. Hence, the complementarity of the ASOs may be 100%, except at the nucleoside opposite to a target nucleoside to be edited. In one embodiment, WO 2025 / 036984 PCT / EP2024 / 073031 5 10 15 15 complementarity is at least 80%, 85%, 90%, 95%. In one embodiment, complementarity is 85%-99%. In one embodiment, the ASO comprises 1, 2, 3, 4, 5 or more mismatches when aligned with the target nucleic acid. In one embodiment, mismatchesone or more are independently a wobble base paring. In one embodiment, the ASOs comprise up to 4 mismatches or wobble bases outside the CBT. In one embodiment, the ASOs comprise up to 3 mismatches or wobble bases outside the CBT.

[0067] 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 followed by the position of the residue within the sequence and by the identity 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)).

[0068] As used herein, the term "beneficial editing" refers to the editing of a target 20 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, NLRP3 Y166 and CTNNB1 T41 that are not causes for genetic diseases but rather 25 30 35 represent wildtype protein sites. These sites may be changed (no underlying G-to-A mutation) to alter the function of the wildtype protein.

[0069] The term "compensatory editing" refers to the modification of RNA nucleotides to change and correct one or more detrimental or unfavourable changes in 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.

[0070] 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 WO 2025 / 036984 PCT / EP2024 / 073031 16 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 5 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).

[0071] The term "guide RNA" (gRNA) or "guide oligonucleotide" refers 10 15 to a piece of RNA or oligonucleotide (comprising RNA and / or DNA) that functions as a guide for enzymes, with which it forms complexes. The guide RNA or guide oligonucleotide may comprise endogenous and / or exogenous sequences. Guide RNAs bind to their target in a sequence-specific manner. Guides can be used in vitro and in vivo. For example, the guide RNA or guide oligonucleotide directs the base-modifying activity / editing function (e.g., ADAR) to the target to be edited in trans.

[0072] 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.

[0073] As used herein, the term "effective amount" in the context of administering a 20 therapy to a subject refers to the amount of a therapy which has a prophylactic and / or therapeutic effect(s). 25

[0074] 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 а subject. For instance, one or more ASOs may be used in combination.

[0075] 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 30 known to have an increased risk of developing a certain condition, disorder, or disease. 35

[0076] 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 limited to, alleviation of symptoms; diminishment of the extent of a condition, disorder, or WO 2025 / 036984 PCT / EP2024 / 073031 17 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 5 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.

[0077] The terms "subject" or "patient" are used interchangeable and relate to an 10 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. 15

[0078] 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, 20 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.

[0079] 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 25 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. 30 35 Oligonucleotides

[0080] Described herein are, inter alia, chemically modified antisense oligonucleotides (ASOs). While not intending to be bound by any particular theory of operation, it is believed that nucleobase and backbone linkage modifications are useful in stabilising ASOs, improving their editing efficacy, reducing their off-target editing, and / or hydrophobicity. Since the one or more modifications can be synthetically transferred to various oligonucleotide sequences, such modifications have the of oligonucleotides with differentpotential to improve the editing efficacy WO 2025 / 036984 PCT / EP2024 / 073031 5 10 15 20 25 30 35 18 target specificities. The ASOs of the invention can be used for several purposes associated with "A-to- / " conversions. That is, the ASOs of the invention are not just limited to correcting G-to-A mutations but are also useful in changing a wildtypе sequence into a mutated sequence in order 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-А mutations or to change wildtype sequences.

[0081] While the oligonucleotides of the invention comprise different types of internucleoside linkages, the inventors have shown oligonucleotides comprising at least one linkage that is a methanesulfonyl (mesyl) linkage have enhanced RNA editing. That is, the inventors have realised that the oligonucleotides of the invention do not require all of the internucleoside linkages to carry a mesyl linkage, provided that a minimum level of internucleoside modification is incorporated. Accordingly, oligonucleotides of the invention comprise at least one methanesulfonyl (mesyl) linkage modification.

[0082] The inventors have also realised that to provide shorter oligonucleotides for RNA editing, and to achieve a beneficial balance of high editing efficacy and low hydrophobicity, it is desirable to incorporate certain backbone linkage and nucleobase modifications and / or mixtures thereof into the oligonucleotides. In particular, depending on the length of the ASO, it is desirable that the ASOs have a mixture of different modifications at the 2'-position of the sugar residue. The inventors have specifically realised that introducing mesyl modifications into the core oligonucleotide backbone reduces overall hydrophobicity of the ASO as well as immune activation. Hence, according to the invention, the oligonucleotide comprises at least one internucleoside linkage that is a methanesulfonyl (mesyl) linkage.

[0083] Provided herein is a chemically modified oligonucleotide for use in site- directed A-to-l editing of a target RNA inside a cell with endogenous adenosine deaminase acting on RNA (ADAR), the oligonucleotide comprising a sequence capable of binding to a target sequence in a target RNA and a central base triplet (CBT) of 3 nucleotides (5' - N+1 No N-1 - 3'), wherein No is the central nucleotide directly opposite to a target adenosine in the target RNA that is to be edited, and wherein the oligonucleotide comprises at least one linkage that is a methanesulfonyl (mesyl) linkage.

[0084] The oligonucleotides of the invention benefit from having a base level of internucleoside linkage modifications, i.e., at least one linkage that is a WO 2025 / 036984 PCT / EP2024 / 073031 19 methanesulfonyl (mesyl) linkage. This will have a positive effect on, inter alia, the pharmacokinetics as well as stability, protein binding, intracellular localization, hydrophobicity and cytotoxicity of ASOs. The oligonucleotide of the invention may in addition to the mesyl linkage(s) comprise further internucleoside linkage 5 modifications. 10 15 20 25

[0085] The chemically modified oligonucleotides of the invention comprise at least one linkage that is a methanesulfonyl (mesyl) linkage. In one embodiment, the mesyl linkage content is at least 10% or 15%, that is at least 10% or 15% of the internucleoside linkages are methanesulfonyl (mesyl) linkages. In one embodiment, the mesyl linkage content is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, or 90%. In some embodiments, at least 10% of linkages are mesyl modified internucleoside linkages, optionally at least 20%, 30%, 40% or 50%.In one embodiment, no more than 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, or 30% of the linkages are mesyl linkages. In one embodiment, at least 5% of the internucleoside linkages are methanesulfonyl (mesyl) linkages. In one embodiment, at least 8% of the internucleoside linkages are methanesulfonyl (mesyl) linkages.

[0086] In one embodiment, the mesyl linkage content is 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 mesyl linkages. In one embodiment, 40-80% of the linkages are mesyl linkages. In one embodiment, 45-60% of the linkages mesyl linkages. In one embodiment, the mesyl linkages content is 20%, 30%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%. In one embodiment, the mesyl linkages content is 30%. In one embodiment, the mesyl linkages content is 15%.

[0087] In one embodiment, no more than 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30% or 20% of the linkages outside the CBT are mesyl linkages; or 15-90% of the linkages are mesyl linkages, preferably wherein 40-80%, most preferably 45-60%, of the linkages are mesyl linkages. In one embodiment, 15-90% of the linkages are 30 mesyl linkages, preferably wherein 40-80%, most preferably 45-60%, of the linkages are mesyl linkages. In one embodiment, the mesyl linkages content is at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In one embodiment, the mesyl linkages 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, 100% of WO 2025 / 036984 20 PCT / EP2024 / 073031 internucleoside linkages are mesylated. In one embodiment, the oligonucleotide is fully mesylated, i.e., all backbone linkages are mesyl linkages.

[0088] In one embodiment, the between 2 and 33 mesyl linkages are methanesulfonyl (mesyl) linkages. In one embodiment, the between 2 and 30, between 5 and 25, 5 between 50 and 20, or between 2 and 20 mesyl linkages are methanesulfonyl (mesyl) 10 linkages. In one embodiment, the oligonucleotide comprises no more than 8, 7, 6, 5, 4, or 3 mesyl linkages. In one embodiment, a) at least 5%, 8%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% the internucleoside linkages are methanesulfonyl (mesyl) linkages; b) between 2 and 20 mesyl linkages are methanesulfonyl (mesyl) linkages, preferably between 2 to 8; or c) the chemically modified oligonucleotide is fully mesylated. In one embodiment, the oligonucleotide contains 4 mesyl linkages (e.g., Al-3059). In one embodiment, the oligonucleotide contains 5 mesyl linkages (e.g., Al-3008, AI-2693). In one embodiment, the oligonucleotide contains 6 mesyl linkages (e.g., Al-1068, Al-1686, AI-1701). In one embodiment, the oligonucleotide 15 contains 8 mesyl linkages (e.g., Al-1691). The specificity sequence of the ASOs of the invention may be described as a 5' to 3' (antisense) oligonucleotide or polynucleotide sequence. The specificity sequence and target region will be described with reference to the target "A" (adenosine to be edited). The target A is located at the "zero position" within the target sequence. The specificity sequence site within the ASO that is 20 directly opposite the target "A" to be edited is referred to as the zero position (No). 25 30 35 The downstream positions (i.e., 3' to the No position) are marked -1, -2, -3, etc. (N-1, N-2, N-3, etc.), while the upstream (i.e., 5' to the No position) positions are numbered +1, +2, +3 (N+1, N+2, N+3, etc.). Accordingly, an oligonucleotide of the invention may have a general sequence of 5'-.....N+5 N+4 b N+3° N+2 d N+1 ° No N.1 9 N-2h N-3 N-4 N.5..... -3'.

[0089] Mesyl linkages may be located at any nucleotide position within the oligonucleotides of the invention. For instance, one or more mesyl linkage modifications may be located at internal positions anywhere along the entire length of the oligonucleotide or (only) at the 5' and / or 3' terminal ends of the oligonucleotide. Alternatively, in one embodiment, the mesyl linkage is located within a 5' and / or a 3' terminus flanking region(s) outside of the CBT (5' - N+1 No N-1 -3'), i.e., upstream of N+1 and / or downstream of N-1. In one embodiment, the mesyl linkage is located within the CBT, i.e., between position +1 and 0 and / or between positions 0 and -1. In one of N+1 (atembodiment, the mesyl linkage is directly (i.e., adjacent to) upstream position +2). In one embodiment, the mesyl linkage is directly downstream (i.e., WO 2025 / 036984 PCT / EP2024 / 073031 5 10 15 20 25 30 21 adjacent to) of N-1 (at position -2). In one embodiment, the oligonucleotide comprises a mesyl linkage within the flanking region 3' to No. In one embodiment, the oligonucleotide comprises a mesyl linkage within the flanking region 5' to No. In one embodiment, the oligonucleotide comprises a mesyl linkage within each of the 5' and 3' flanking regions. In one embodiment, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, or 8 mesyl linkages within the flanking regions 3' and / or 5' to No. In one embodiment, the oligonucleotide comprises between 1-20 mesyl linkages 5' to No. In one embodiment, the oligonucleotide comprises between 1-10 mesyl linkages 3' to No.

[0090] In one embodiment, the mesyl linkage is located within the 3' and / or 5' flanking region(s) outside of the CBT. Since oligonucleotides may vary in overall length, the length of the 3' and / or 5' terminal flanking regions of each oligonucleotide may vary in length accordingly. In one embodiment, the flanking regions at the 5' and 3' termini have the same length. In one embodiment, the flanking regions at the 5' and 3' termini have different lengths.

[0091] In one embodiment, the oligonucleotide comprises 2, 3, 4, 5, 6 or 7 mesyl modifications within a 3' and / or 5' flanking region(s) outside of the CBT. In one embodiment, the oligonucleotide comprises at least 2, 3, 4, 5, 6, or 7 mesyl modifications within a 3' and / or 5' flanking region(s) outside of the CBT. In one embodiment, the 5' terminus flanking region comprises the terminal 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide (s) of the oligonucleotide, preferably wherein the 5' terminus flanking region comprises the outermost 6, 5, 4, 3, 2, or 1 nucleotide(s). In one embodiment, the 3' terminus flanking region comprises the terminal 7, 6, 5, 4, 3, 2 or 1 nucleotide(s) of the oligonucleotide, preferably wherein the 3' terminus flanking region comprises the outermost 4, 3, 2, or 1 nucleotide(s).

[0092] In one embodiment, the oligonucleotide comprises 1, 2, or 3 mesyl linkages within the 3' and / or 5' terminus flanking region(s). In one embodiment, the 1, 2, or 3 mesyl linkages within the 3' and / or 5' terminus flanking region(s) are between the terminal 1, 2, 3 and 4 nucleotides of the 5' and / or 3' terminus. That is, in one embodiment, the 1, 2, or 3 mesyl linkages are located between the outermost 5 nucleotides of the 5' and / or outermost 4 nucleotides of the 3' terminus of the oligonucleotide. In one embodiment, the 1 or 2 mesyl linkages are located between the outermost 2 or 3 nucleotides of the 5' and / or outermost 2 or 3 nucleotides of the 3' terminus of the oligonucleotide. WO 2025 / 036984 PCT / EP2024 / 073031 22

[0093] In other embodiments, the oligonucleotide comprises a mesyl linkage between the terminal and penultimate nucleotide of the 5' terminus and a mesyl linkage between the terminal and penultimate nucleotide of the 3' terminus. In other embodiments, the oligonucleotide comprises 2 mesyl linkages at the 5' terminus, 5 which are placed between the terminal 3 nucleotides of the 5' terminus. In other embodiments, the oligonucleotide comprises 2 mesyl linkages at the 3' terminus, which are placed between the terminal 3 nucleotides of the 3' terminus. In one embodiment, the oligonucleotide comprises 2 mesyl linkages at the 5' terminus, which are placed between the terminal 3 nucleotides of the 5' terminus and 2 mesyl linkages 10 at the 3' terminus, which are placed between the terminal 3 nucleotides of the 3' 15 20 25 terminus.

[0094] In one embodiment, a mesyl linkage is located between any two of the nucleotide positions of the oligonucleotide. That is, in an oligonucleotide with a length of 34nt, a mesyl linkage may be located at any of the 34 positions of the oligonucleotide (e.g., at position 5' - +24, +23, +22, [... ],...O,... [...], -3, -4, -5, -6, -7, -8 - 3'). In one embodiment, a mesyl linkage is located between the outermost 1-5, 1-6, 1-7, 1-8, 1-9 or 1-10 nucleotides. In one embodiment, the mesyl linkage is located at one or more of positions + positions +28, +27, +26, +25, +24, +23, +22, +21, +20, +19, +14, +13, +12, +11, +10, +5, +4, +3, -2, -5, -7, -8, -9, -11, -12, -13, -14, -15, -16, -17, -18, and / or -19. In one embodiment, the mesyl linkage is positioned at one or more of the following positions +27, +26, +25, +24, +23, +22, +21, +20, +19, +13, +12, +11, +6, +5, +4, -2, -6, -7, and -8. In one embodiment, the mesyl linkage is located at one or more of the following positions selected from: +24, +23, +21, +13, +4, -2, -6, -7, and -8.

[0095] In some embodiments, there is no mesyl linkage modification at one or more of the following positions: +19, +18, +17, +16, +15, +14, +10, +9, +8, +7, +6, +3, +2, +1, 0, -1, -2, -3, -4, -5, -6, -7, -8, -9, and -10. In one embodiment, there is no mesyl linkage modification at one or more of the following positions: +18, +17, +16, +15, +9, +8, +7, +6, +2, +1, 0, -1, -3, -4, -6, -7, -8, and -10. In one embodiment, there is no 30 mesyl linkage modification at one or more of the following positions: +18, +17, +15, +8, +6, +1, -3, and -6.

[0096] The inventors have found that oligonucleotides comprising mesyl linkages show improved editing compared to those oligonucleotides that do not (e.g., Example 5). The oligonucleotides of the invention may thus contain internal mesyl linkages or 35 mesyl linkages at the 5' and / or 3' terminal ends. As used herein, "internal mesyl WO 2025 / 036984 PCT / EP2024 / 073031 5 10 15 20 25 30 23 linkages" are those linkages that are not located between the terminal two nucleotides of the 5' or 3' terminus. In one embodiment, the mesyl linkage is located at position - 6. In one embodiment, the mesyl linkage is located at position -5. In one embodiment, the mesyl linkage is located at position +18. In one embodiment, the mesyl linkage is located at position +19. In one preferred embodiment, the mesyl linkage is located at position -2. In one preferred embodiment, the mesyl linkage is located at position -7. In one preferred embodiment, the mesyl linkage is located at position +4. In one preferred embodiment, the mesyl linkage is located at position +13. In one preferred embodiment, the mesyl linkage is located at position +21. In one preferred embodiment, the mesyl linkage is located at position +23.

[0097] Accordingly, in one embodiment, the oligonucleotide comprises mesyl linkages at positions +24, +23, +13, -2, -7 and -8. In one embodiment, the oligonucleotide comprises mesyl linkages at positions +24, +21, +13, +4, -7 and -8. In one embodiment, the oligonucleotide comprises mesyl linkages at positions +24, +23, +21, +13, +4, -2, -7 and -8. In one embodiment, the oligonucleotide comprises mesyl linkages at positions +24, +23, -2, -7 and -8. In one embodiment, the oligonucleotide comprises mesyl linkages at positions +24, +23, -7 and -8. In one embodiment, the oligonucleotide comprises mesyl linkages at positions +24, -2, and -8. In one embodiment, the oligonucleotide comprises mesyl linkages at positions +24 and -8.

[0098] Alternatively, or additionally mesyl linkages may be located at the terminal nucleotides of the ASO of the invention, i.e., between the terminal and penultimate nucleotide of the 5' and / or 3' end of the ASO. In one embodiment, a mesyl linkage is located in the 5' and / or 3' flanking regions of the ASO. In one embodiment, a mesyl linkage is located at position +24 and / or at position -8.

[0099] The chemically modified oligonucleotides of the invention may be symmetrical, which means that the two nucleotide sequences adjacent to the CBT have the same length, or not symmetrical (asymmetrical or asymmetric design), which means that the two sequences flanking the CBT, i.e., the regions 5' and 3' to the CBT and / or position No, have different lengths. The asymmetric design enables a more flexible use of the sequence space around the target. Hence, in one embodiment, the the oligonucleotide comprises an asymmetric design. In one embodiment, the oligonucleotide has: (i) a length of 20 to 29nt located 5' to No, and (ii) a length of 5 to 20nt located 3' to No. WO 2025 / 036984 PCT / EP2024 / 073031 5 10 24

[00100] The oligonucleotides of the invention may be of any length suitable to achieve an edit. The oligonucleotides of the invention are preferably at least 22, more preferably at least 25 nucleotides (nt) long, at least 27 nucleotides long, at least 30 nucleotides long, at least 35 nucleotides long. In some instances, the oligonucleotides may range from about 25-80nt in length, e.g., about 25-39nt, about 40-60nt or about 61-80nt in length. In one embodiment, the oligonucleotide has a length of 25-80nt. In one embodiment, the oligonucleotide has a length of 25-50nt. 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, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80nt. In one embodiment, the oligonucleotide has a length of 27nt. In one embodiment, the oligonucleotide has a length of 30nt. In one embodiment, the oligonucleotide has a length of 33nt. In one embodiment, the oligonucleotide has a length of 34nt. In one embodiment, the oligonucleotide has a length of 38nt. In one embodiment, the 15 oligonucleotide has a length of 44nt. In one embodiment, the oligonucleotide has a length of 45nt. In certain embodiments, the oligonucleotide has a length of 27, 30, 32, 34, 36, or 38nt. In one embodiment, the oligonucleotide has a length of 30-40 nt. In some embodiments, the oligonucleotide has a length of 30-38nt. In some embodiments, the oligonucleotide has a length of 30-34nt. In some embodiments, 20 25 30 35 the oligonucleotide has a length of 34-38nt or 36-38nt. In one embodiment, the oligonucleotide has a length of no more than 30, 31, 32, 33, 34, 35, 36, 37, or 38nt. In one embodiment, the oligonucleotide has a length of no more than 38, 39, 40, 41, 42, 43, 44, or 45nt. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

[00101] Without being bound by any theory, inventors submit that the ideal asymmetry for each target might depend on the length and the specific underlying sequence of the particular oligonucleotide. The inventors previously showed that for asymmetric ASOs, a shorter overall oligoribonucleotide is sufficient for high editing efficacy compared to the symmetric design (WO 2022 / 253810). 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 rather 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. This leads to a situation, wherein, depending on the length of the ASO, specific (a)symmetries on the target adenosine and specific modifications patterns (e.g., sugar WO 2025 / 036984 PCT / EP2024 / 073031 5 10 15 25 and internucleoside linkage modifications) are preferred. For an optimal binding of the deaminase, a short 3' terminus seems to be sufficient (at least 5 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 19 nt beside the CBT).

[00102] Some well-working embodiments of asymmetries provided herein and identified by the inventors of the instant application are listed in Table A below. As previously described, the oligonucleotides of the invention have the following structural scheme: (length of 5' terminus) - (1) - (length of 3' terminus), wherein 1 corresponds to the central nucleotide of the CBT opposite of the target A. For example, an ASO of the invention with a length of 38nt and an asymmetry of "29-1- 8", has a 5' terminus that is 29nt long and a 3' terminus that is 8nt long. In one embodiment, the oligonucleotide is asymmetric. In one embodiment, the oligonucleotide has any one of the asymmetries listed in Table A. Table A: Asymmetries of exemplary ASO designs according to the invention. Exemplary Length (nt) Structural scheme / Asymmetry SEQ ID NO: Construct (5' terminus - 1 - 3' terminus) AI-0067 38 29-1-8 40 Al-0083 36 23-1-12 36 Al-0949 34 25-1-8 146 AI-0099 32 25-1-6 14 Al-0107 30 23-1-6 8 AI-1685 27 20-1-6 204 20 25

[00103] According to the invention, the ASO may be asymmetric. Hence, in one embodiment, the chemically modified oligonucleotide of the invention comprises an asymmetric design, wherein there is a different number of nucleotides 5' and 3' of No. For instance, there may be 20-30nt at the 5' terminus (5' to No) and 5-20nt at the 3' terminus (3' to No). Alternatively, in one embodiment, there are 26nt 5' to No, and 6nt 3' to No. In some embodiments, there are up to 29nt 3' of the CBT. In some embodiments, there are no more than 29nt 3' of the CBT. In some embodiments, the 3' terminus is shortened to a length of 5nt 3' of the CBT. In some embodiments, the 3' terminus is shortened to a length of 4nt 3' of the CBT. In one embodiment, the region 3' to the CBT contains 4, 5, or 6nt. In some embodiments, there are 4-30nt 5' of the CBT. In one embodiment, there are no more than 30nt 5' of the CBT. In one embodiment, the 5' terminus is shortened to a length of 28nt 5' of the CBT. In one embodiment, the region 5' to the CBT contains 22, 23, 24, 25, or 26nt. WO 2025 / 036984 PCT / EP2024 / 073031 5 10 15 26

[00104] The oligonucleotides of the invention may have specific asymmetries. In some embodiments, the oligonucleotide has an asymmetry as listed in Table A. In a preferred embodiment, the oligonucleotide has an asymmetry of 25-1-8 in a 5' to 3' direction.

[00105] The inventors have further realised that the length of the oligonucleotide can be shortened without losing its editing efficacy provided the oligonucleotide comprises additional 2'-sugar and internucleoside linkage modifications. The oligonucleotide may have a length of 26nt to 38nt. In one embodiment, the oligonucleotide comprises a length of 27nt to 35nt. In a preferred embodiment, the oligonucleotide has a length of 27nt, 30nt, 33nt, or 34nt. In one embodiment, the oligonucleotide comprises an asymmetric design, wherein at least 20nt are 5' to No, and wherein at least 5nt are 3' to No. In one embodiment, the oligonucleotide comprises an asymmetry of: a) 25-1-8; b) 29-1-8; c) 27-1-6; d) 26-1- 6; e) 23-1-6; or f) 20-1-6 and wherein the oligonucleotide comprises at least four 2'-F modifications. In one embodiment, the oligonucleotide comprises an asymmetric design, wherein at least 20nt are 5' to No, and wherein at least 5nt are 3' to No. In one embodiment, the oligonucleotide has an asymmetry of 25-1-8 and comprises between 5 and 20 2'-F modifications.

[00106] In one preferred embodiment, the oligonucleotide has an asymmetry 20 of 25-1-8 in a 5' to 3' direction. In one preferred embodiment, the oligonucleotide has an asymmetry of 25-1-8 in a 5' to 3' direction, and wherein the mesyl linkage is located at positions +24, -2, and -8. In one preferred embodiment, a further mesyl linkage is located at position +4 and / or position +13. In one preferred embodiment, a further mesyl linkage is located at position +21, and optionally at position -7 and -23. 25

[00107] Furthermore, the ASO of the invention may comprise 2'-fluoro (2'-F) and / or 2'Ome modifications. In one embodiment, at least 20%, 30%, 40%, 50% or 60% nucleotides are fluoro (F)-modified at the 2' position of the sugar residue. In one embodiment, the oligonucleotide comprises 5 to 20 2'-F modifications. In one embodiment, the oligonucleotide comprises 12 2'-F modifications. In one 30 embodiment, a 2'-F modification is located at one or more of the following positions selected from the group consisting of: +22, +21, +19, +17, +16, +15, +14, +13, +11, +9, +8 +7, +6, +5, +3, +2, +1, and -3. In one embodiment, the 2'-F modification is located at position +22, +21, +19, +16, +15, +13, +11, +9, +7, +5, +2, and -3. 1 INTERNATIONAL SEARCH REPORT International application No PCT / EP2024 / 073031 A. CLASSIFICATION OF SUBJЕСТ МАTTER INV. C12N15 / 11 C12N15 / 113 ADD. According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) C12N Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) EPO-Internal, WPI Data, BIOSIS, CHEM ABS Data, Sequence Search, EMBASE C. DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Y Y WO 2020 / 201406 A1 (PROQR THERAPEUTICS II BV [NL]) 8 October 2020 (2020-10-08) page 22 - page 23; claims 1-16; figures 1,6,8; examples 1-4 PATUTINA OLGA A. ET AL: "Mesyl phosphoramidate backbone modified antisense oligonucleotides targeting miR-21 with enhanced in vivo therapeutic potency", PNAS, vol. 117, no. 51, 7 December 2020 (2020-12-07), pages 32370-32379, XP093021468, DOI: 10.1073 / pnas.2016158117 page 32370, left-hand column; figure 1; table 1 - / -- ☑ See patent family annex. Relevant to claim No. 1-66 1-66 ☑ Further documents are listed in the continuation of Box C. Special categories of cited documents: "A" document defining the general state of the art which is not considered to be of particular relevance "E" earlier application or patent but published on or after the international filing date "L" document which may throw doubts on priority claim(s) or which is cited to establish the publication date of another citation or other special reason (as specified) "O" document referring to an oral disclosure, use, exhibition or other means "P" document published prior to the international filing date but later than the priority date claimed Date of the actual completion of the international search "T" later document published after the international filing date or priority date and not in conflict with the application but cited to understand the principle or theory underlying the invention "Y" document of particular relevance:; the claimed invention cannot be considered novel or cannot ot be considered to involve an inventive step when the document is taken alone "Y" document of particular relevance;; the claimed invention cannot be considered to involve an inventive step when the document is combined with one or more other such documents, such combination being obvious to a person skilled in the art "&" document member of the same patent family Date of mailing of the international search report 11 December 2024 Name and mailing address of the ISA / European Patent Office, P.B. 5818 Patentlaan 2 NL - 2280 HV Rijswijk Tel. (+31-70) 340-2040, Fax: (+31-70) 340-3016 Form PCT / ISA / 210 (second sheet) (April 2005) 02 / 01 / 2025 Authorized officer Bucka, Alexander page 1 of 4 INTERNATIONAL SEARCH REPORT International application No PCT / EP2024 / 073031 C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT Category* A A A A Citation of document, with indication, where appropriate, of the relevant passages MONIAN PRASHANT ET AL: "Endogenous ADAR-mediated RNA editing in non-human primates using stereopure chemically modified oligonucleotides", NATURE BIOTECHNOLOGY 7 March 2022 (2022-03-07), XP055911929, New York ISSN: 1087-0156, DOI: 10.1038 / s41587-022-01225-1 Retrieved from the Internet: URL:https: / / www.nature.com / articles / s41587 -022-01225-1.pdf figures 1,2,6 WO 2023 / 099494 A1 (UNIV EBERHARD KARLS TUEBINGEN [DE]) 8 June 2023 (2023-06-08) claims 1-25; sequence 7 HAMMOND SUZAN M. ET AL: "Mesyl Phosphoramidate Oligonucleotides as Potential Splice-Switching Agents: Impact of Backbone Structure on Activity and Intracellular Localization", NUCLEIC ACID THERAPEUTICS, vol. 31, no. 3, 4 June 2021 (2021-06-04), pages 190-200, XP093071905, US ISSN: 2159-3337, DOI: 10.1089 / nat.2020.0860 fiqure 1 ZHANG LINGDI ET AL: "The Combination of Mesyl-Phosphoramidate Inter-Nucleotide Linkages and 2'- O -Methyl in Selected Positions in the Antisense Oligonucleotide Enhances the Performance of RNaseH1 Active PS-ASOs", Relevant to claim No. 1-66 1-66 1-66 1-66 NUCLEIC ACID THERAPEUTICS, vol. 32, no. 5, 14 October 2022 (2022-10-14), pages 401-411, XP093129225, US ISSN: 2159-3337, DOI: 10.1089 / nat.2022.0005 figures 1,2 - / -- 1 Form PCT / ISA / 210 (continuation of second sheet) (April 2005) page 2 of 4 INTERNATIONAL SEARCH REPORT International application No PCT / EP2024 / 073031 C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT Category A A A Citation of document, with indication, where appropriate, of the relevant passages MIROSHNICHENKO S. K. ET AL: "Mesyl phosphoramidate antisense oligonucleotides as an alternative to phosphorothioates with improved biochemical and biological properties", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 116, no. 4, 8 January 2019 (2019-01-08), pages 1229-1234, XP093021469, ISSN: 0027-8424, DOI: 10.1073 / pnas.1813376116 figure 1; table 1 ANDERSON BROOKE A ET AL: "Towards next generation antisense oligonucleotides: mesylphosphoramidate modification improves therapeutic index and duration of effect of gapmer antisense oligonucleotides", NUCLEIC ACIDS RESEARCH vol. 49, no. 16 20 September 2021 (2021-09-20), pages 9026-9041, XP093036509, GB ISSN: 0305-1048, DOI: 10.1093 / nar / gkab718 Retrieved from the Internet: URL:https: / / watermark.silverchair.com / gkab 718.pdf?token=AQECAHi208BE490oan9kkhW_Ercy 7Dm3ZL_9Cf3qfKAc485ysgAAAtYwggLSBgkqhkiG9w OBBwagggLDMIICvWIBADCCArgGCSqGSIb3DQEНАТАе BglghkgBZQMEAS4WEQQM-A0MsthJORb0IJH8AgEQgI ICiYQnek19YNnEFTQL71SubpCXfeHMc_RFWulASJcl Velh-zCI3ZFmRZf0xxEZhYl-bXHhmHwjBsvbk600VХ vDFlliukuU figures 1,4,5 DOHERTY ERIN E. ET AL: "Oligonucleotide-directed RNA editing in primates", MOLECULAR THERAPY, vol. 30, no. 6, 22 April 2022 (2022-04-22) US pages 2117-2119, XP093129628, ISSN: 1525-0016, DOI: 10.1016 / j.ymthe.2022.04.005 figure 1 Relevant to claim No. 1-66 1-66 1-66 - / -- 1 Form PCT / ISA / 210 (continuation of second sheet) (April 2005) page 3 of 4 INTERNATIONAL SEARCH REPORT International application No PCT / EP2024 / 073031 C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT Category* A Citation of document, with indication, where appropriate, of the relevant passages BRINKMAN HANNAH F. ET AL: "Nucleoside analogs in ADAR guide strands targeting 5'-UA sites", RSC CHEMICAL BIOLOGY, vol. 4, no. 1, 31 October 2022 (2022-10-31), pages 74-83, XP093099702, DOI: 10.1039 / D2CB00165A figures 2-4,10 Relevant to claim No. 1-66 A WO 2018 / 156056 A1 (STETSENKO DMITRIJ ALEKSANDROVICH [RU] ET AL.) 30 August 2018 (2018-08-30) claims 1-4; figure 2 1-66 A WO 2021 / 030778 A1 (IONIS PHARMACEUTICALS INC [US]) 18 February 2021 (2021-02-18) claims 1-4,35 1-66 X,P WO 2024 / 013361 A1 (PROQR THERAPEUTICS II BV [NL]; UNIV CALIFORNIA [US]) 18 January 2024 (2024-01-18) page 15; claims 1-17 1-66 X,P WO 2024 / 013360 A1 (PROOR THERAPEUTICS II BV [NL]) 18 January 2024 (2024-01-18) page 10; claims 1-14 1-66 E WO 2024 / 200278 A1 (PROQR THERAPEUTICS II BV [NL]) 3 October 2024 (2024-10-03) claims 1-12; examples 1-4 1-66 1 Form PCT / ISA / 210 (continuation of second sheet) (April 2005) page 4 of 4 INTERNATIONAL SEARCH REPORT International application No. PCT / EP2024 / 073031 Box No. I Nucleotide and / or amino acid sequence(s) (Continuation of item 1.c of the first sheet) 1. With regard to any nucleotide and / or amino acid sequence disclosed in the international application, the international search was carried out on the basis of a sequence listing: a. ☐ forming part of the international application as filed. 2. b. ☑ furnished subsequent to the international filing date for the purposes of international search (Rule 13ter.1(a)). X accompanied by a statement to the effect that the sequence listing does not go beyond the disclosure in the international application as filed. ☐ With regard to any nucleotide and / or amino acid sequence disclosed in the international application, this report has been established to the extent that a meaningful search could be carried out without a WIPO Standard ST.26 compliant sequence listing. 3. Additional comments: Form PCT / ISA / 210 (continuation of first sheet (1)) (July 2022) INTERNATIONAL SEARCH REPORT Information on patent family members International application No PCT / EP2024 / 073031 Patent document Publication cited in search report date Patent family member(s) Publication date WO 2020201406 A1 08-10-2020 AU 2020250895 A1 11-11-2021 CA 3132180 A1 08-10-2020 CN 113748206 A 03-12-2021 EP 3947679 A1 09-02-2022 IL 286395 A 31-10-2021 JP 2022527814 A 06-06-2022 US 2022340900 A1 27-10-2022 WO 2020201406 A1 08-10-2020 WO 2023099494 A1 08-06-2023 AU 2022399905 A1 23-05-2024 CA 3238033 A1 08-06-2023 CN 118355119 A 16-07-2024 EP 4441216 A1 09-10-2024 IL 312762 A 01-07-2024 JP 2024544067 A 27-11-2024 KR 20240111752 A 17-07-2024 US 2023235329 A1 27-07-2023 WO 2023099494 A1 08-06-2023 WO 2018156056 A1 30-08-2018 NONE WO 2021030778 A1 18-02-2021 CN 114555621 A 27-05-2022 EP 4013767 A1 22-06-2022 JP 2022544587 A 19-10-2022 KR 20220062517 A 17-05-2022 US 2022186222 A1 16-06-2022 US 2024002851 A1 04-01-2024 WO 2021030778 A1 18-02-2021 WO 2024013361 A1 18-01-2024 NONE WO 2024013360 A1 18-01-2024 NONE WO 2024200278 A1 03-10-2024 NONE Form PCT / ISA / 210 (patent family annex) (April 2005) 摘要 本發明涉及用於在細胞內利用內源性腺苷脫氨酶（ADAR）對目標 RNA進行定點 A-to-I 編輯的化學修飾寡核苷酸，該寡核苷酸包含能夠與目標 RNA中的目標序列結合的序列 以及由（3）個核苷酸（N-1 N0 N+1）組成的中心鹼基三聯體（CBT），其中 N0是與待 編輯的靶 RNA中的靶腺苷直接相對的中心核苷酸，並且該寡核苷酸包含至少一個核苷 間連接，該核苷間連接是甲磺酰基（mesy1）連接。

Claims

CLAIMS 1. A chemically modified oligonucleotide for use in site-directed A-to-I editing of a target RNA inside a cell with endogenous adenosine deaminase acting on RNA (ADAR), the oligonucleotide comprising a sequence capable of binding to a target sequence in a target RNA and a central base triplet (CBT) of 3 nucleotides (5’ - N+1N0N-1- 3’), wherein N0is the central nucleotide directly opposite to a target adenosine in the target RNA that is to be edited, and wherein the oligonucleotide comprises at least one internucleoside linkage that is a methanesulfonyl (mesyl) linkage.

2. The chemically modified oligonucleotide of claim 1, wherein the oligonucleotide has a length of at least 25 nucleotides (nt), optionally 25 to 80nt, preferably 25 to 50nt, more preferably 30 to 40 nt.

3. The chemically modified oligonucleotide of claim 1 or 2, wherein: a) at least 5%, 8%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% the internucleoside linkages are methanesulfonyl (mesyl) linkages; b) between 2 and 20 mesyl linkages are methanesulfonyl (mesyl) linkages, preferably between 2 to 8; or c) the chemically modified oligonucleotide is fully mesylated.

4. The chemically modified oligonucleotide of any one of claims 1-3, wherein the oligonucleotide comprises no more than 8, 7, 6, 5, 4, or 3 mesyl linkages.

5. The chemically modified oligonucleotide any one of claims 1-4, wherein the mesyl linkage is located within a 5’ and / or a 3’ terminus flanking region(s) outside of the CBT.

6. The chemically modified oligonucleotide of claim 5, wherein (i) the 5' terminus flanking region comprises the terminal 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide(s) of the oligonucleotide, preferably wherein the 5' terminus flanking region comprises the outermost 6, 5, 4, 3, 2, or 1 nucleotide(s); and / or(ii) the 3' terminus flanking region comprises the terminal 7, 6, 5, 4, 3, 2 or 1 nucleotide(s) of the oligonucleotide, preferably wherein the 3' terminus flanking region comprises the outermost 4, 3, 2, or 1 nucleotide(s).

7. The chemically modified oligonucleotide of any one of claims 1-6, wherein the oligonucleotide comprises 1, 2, or 3 mesyl linkages within the 3’ and / or 5’ terminus flanking region(s).

8. The chemically modified oligonucleotide of claim 7, wherein the 1, 2, or 3 mesyl linkages are located between the outermost 5 nucleotides of the 5’ and / or outermost 4 nucleotides of the 3’ terminus of the oligonucleotide.

9. The chemically modified oligonucleotide of any one of claims 1-8, wherein the oligonucleotide comprises a mesyl linkage between the terminal and penultimate nucleotide of the 5’ terminus and a mesyl linkage between the terminal and penultimate nucleotide of the 3’ terminus.

10. The chemically modified oligonucleotide of any one of claims 1-9, wherein the oligonucleotide comprises an asymmetric design, optionally wherein the oligonucleotide has: (i) a length of 20 to 29nt located 5’ to N0, and (ii) a length of 5 to 20nt located 3’ to N0.

11. The chemically modified oligonucleotide of any one of claims 1-10, having a length of 26nt to 38nt, optionally wherein the oligonucleotide comprises a length of 30nt to 38nt, preferably a length of 33nt, 34nt, or 38nt.

12. The chemically modified oligonucleotide of any one of claims 1-11, wherein the oligonucleotide has a length of 34nt.

13. The chemically modified oligonucleotide of any one of claims 1-11, wherein the oligonucleotide comprises an asymmetry of: a) 25-1-8; b) 29-1-8; c) 27-1-6;d) 26-1-6; e) 23-1-6; or f) 20-1-6.

14. The chemically modified oligonucleotide of claim 13, wherein the mesyl linkage is located at one or more of positions +28, +27, +26, +25, +24, +23, +22, +21, +20, +19, +14, +13, +12, +11, +10, +5, +4, +3, -2, -5, -7, -8, -9, -11, -12, -13, -14, -15, -16, -17, -18, and / or - 19, more preferably at one or more of positions +27, +26, +25, +24, +23, +22, +21, +20, +19, +13, +12, +11, +9, +6, +5, +4, -2, -6, -7, and -8.

15. The chemically modified oligonucleotide of any one of claims 1-14, wherein the mesyl linkage is located at one or more of the following positions selected from: +24, +23, +21, +13, +4, -2, -6, -7, and -8.

16. The chemically modified oligonucleotide of any one of claims 1-15, wherein the mesyl linkage is located at position +24.

17. The chemically modified oligonucleotide of any one of claims 1-16, wherein the mesyl linkage is located at position +4.

18. The chemically modified oligonucleotide of any one of claims 1-17, wherein the mesyl linkage is located at position +13.

19. The chemically modified oligonucleotide of any one of claims 1-18, wherein the mesyl linkage is located at position -2.

20. The chemically modified oligonucleotide of any one of claims 1-19, wherein the mesyl linkage is located at position -6.

21. The chemically modified oligonucleotide of any one of claims 1-20, wherein the mesyl linkage is located at position -8.

22. The chemically modified oligonucleotide of any one of claims 1-21, wherein a mesyl linkage is located between the terminal and penultimate nucleotide of the 5’ and / or 3’ endof the ASO, optionally wherein the mesyl linkage is at position +24 and at position -8, respectively. ^ 23. The chemically modified oligonucleotide of any of claims 1-22, wherein the oligonucleotide has an asymmetry of 25-1-8 in a 5' to 3' direction. ^ 24. The chemically modified oligonucleotide of any one of claims 1-23, wherein the oligonucleotide has an asymmetry of 25-1-8 in a 5' to 3' direction, and wherein the mesyl linkage is located at positions +24, -2, and -8. ^ 25. The chemically modified oligonucleotide of claim 24, wherein a further mesyl linkage is located at position +4 and / or position +13.

26. The chemically modified oligonucleotide of claim 25, wherein a further mesyl linkage is located at position +21, and optionally at position -7 and -23.

27. The chemically modified oligonucleotide of any one of claims 1-26, wherein at least 20%, 30%, 40%, 50% or 60% nucleotides are fluoro (F)-modified at the 2’ position of the sugar residue, or wherein the oligonucleotide comprises 5 to 20, optionally 12, 2’-F modifications.

28. The chemically modified oligonucleotide of any one of claims 1-27, wherein at least 20%, preferably 30-70%, more preferably 40-60% of the chemical modifications outside the CBT are 2´-O-methyl substituents.

29. The chemically modified oligonucleotide of claim 27 or 28, wherein a 2’-F modification is located at one or more of the following positions selected from the group consisting of: +22, +21, +19, +17, +16, +15, +14, +13, +11, +9, +8 +7, +6, +5, +3, +2, +1, and -3; preferably wherein the 2’-F modification is located at position +22, +21, +19, +16, +15, +13, +11, +9, +7, +5, +2, and -3.

30. The chemically modified oligonucleotide of any one of claims 1-29, wherein the oligonucleotide comprises one or more internucleoside linkages selected from the group consisting of phosphoryl guanidine (PN), phosphodiester (PO) and phosphorothioate (PS).

31. The chemically modified oligonucleotide of claim 30, wherein the one or more internucleoside linkage is a PS linkage, optionally wherein at least 40% of linkages are PS linkages, preferably wherein between 40% and 65% of linkages are PS linkages.

32. The chemically modified oligonucleotide of any one of claims 1-31, wherein (i) less than 60%, 50%, 45%, 40%, preferably less than 30% of the internucleoside linkages are PO linkages; and / or (ii) no more than 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% of the internucleoside linkages are PS linkages.

33. The chemically modified oligonucleotide of any one of claims 30-32, wherein the oligonucleotide comprises a PO linkage at position +22, +17, +16, +11, +8, +5, +3, +2 and / or -3.

34. The chemically modified oligonucleotide of any one of claims 1-33, wherein there is a) no mesyl linkage at position -3; and / or b) no PN linkage at position +24, +21, +13, +4, -2, and / or -8.

35. The chemically modified oligonucleotide of any one of claims 1-34, wherein at least one of the three nucleotides of the CBT is chemically modified at the 2' position of the sugar residue, a deoxyribonucleoside, or a combination thereof.

36. The chemically modified oligonucleotide of any one of claims 35, wherein the chemical modification at the 2’ position is one or more of the following: (i) N+1 is 2’-fluoro (2’-F), 2’-fluoroarabinoside (2’-FANA), deoxyribonucleic acid (DNA), 2‘-O-Methoxyethyl (2’-MOE) or 2'-O-Methyl (2’-OMe); and / or (ii) N0is 2'-FANA or DNA; and / or (iii) N-1is 2'-FANA, DNA or 2’-OMe.

37. The chemically modified oligonucleotide of claim 36, wherein: (i) N+1is 2‘-O-Methoxyethyl (2’-MOE); (ii) N0is DNA; and (iii) N-1is DNA.

38. The chemically modified oligonucleotide of any one of claims 1-37, wherein N-1 is inosine (I).

39. The chemically modified oligonucleotide of any one of claims 1-38, wherein (i) N0comprises no 2’-sugar modification, preferably wherein N0comprises no 2’-alkyl modification, most preferably, no 2’-methyl-modification, and / or (ii) the CBT comprises no cytosine analogues.

40. The chemically modified oligonucleotide of any one of claims 1-39, wherein the oligonucleotide comprises the following core sequence: 5’- …..N+5aN+4bN+3cN+2dN+1eN0fN-1gN-2hN-3iN-4jN-5 ….. -3’, wherein at least linkages d and e are modified, optionally wherein (i) d and e are phosphorothioate (PS) linkages and whereby at least 2 linkages are phosphate (PO) linkages; and / or (ii) linkage h is not a PS linkage; and / or (iii) f and j are a PS linkage, and / or (iv) b is a PO or a PS linkage.

41. The chemically modified oligonucleotide of claim 40, wherein linkage h is a PO linkage.

42. The chemically modified oligonucleotide of claim 40 or 41, wherein linkage i is a PS linkage.

43. The chemically modified oligonucleotide of any one of claims 40-42, wherein linkage d and e are PS linkages, linkage h is a PO linkage, linkage i is a PS linkage, and linkage g is a mesyl linkage.

44. The chemically modified oligonucleotide of any one of claims 40-43, wherein linkage b and h are PO linkages.

45. The chemically modified oligonucleotide of any one of claims 1-44, wherein the oligonucleotide comprises one or more 2’-sugar modifications, optionally wherein no more than 6 consecutive nucleotides have the same 2’-sugar modification.

46. The chemically modified oligonucleotide of any one of claims 1-45, wherein at least 50%, more preferably at least 80% of the nucleotides outside the CBT are modified independently from another at the 2’ position of the sugar residue, optionally wherein the 2’-sugar modification is selected from 2’-F, 2’-FANA, 2’-O- alkyl, 2’-O-methoxyethyl (2’-MOE), and / or locked nucleic acid (LNA), optionally wherein the 2’-O-alkyl modification is a 2’-OMe modification.

47. The chemically modified oligonucleotide of claim 46, wherein there is a 2’-MOE at position +12 (N+12).

48. The chemically modified oligonucleotide of any one of claims 1-47, wherein the oligonucleotide comprises an iso-uridine (SbU) modification, optionally wherein the SbU modification is at position zero (0; N0).

49. The chemically modified oligonucleotide of any one of claims 1-48, wherein the oligonucleotide comprises a moiety, which enhances cellular uptake of the artificial nucleic acid, optionally wherein the moiety enhancing cellular uptake is N-acetyl galactosamine (GalNAc).

50. The chemically modified oligonucleotide of claim 49, wherein the GalNAc is conjugated to the 5’ terminus or to the 3’ terminus of the oligonucleotide.

51. The chemically modified oligonucleotide of any one of claims 1-50, wherein not all nucleotides comprise a 2’-alkyl modification.

52. The chemically modified oligonucleotide of any one of claims 1-51, wherein the oligonucleotide does not comprise independently controlled chiral phosphates.

53. The chemically modified oligonucleotide of any one of claims 1-52, wherein the oligonucleotide does not comprise any 2’-O-methoxyethyl (2’-MOE) modifications at the outermost three nucleotides of the 3’ terminus and / or the 5’ terminus.

54. The chemically modified oligonucleotide of any one of claims 1-53, wherein the oligonucleotide does not comprise any PN modifications at the outermost three nucleotides of the 3’ terminus and / or the 5’ terminus.

55. The chemically modified oligonucleotide of any one of claims 1-54, wherein the oligonucleotide does not comprise any methylphosphonate (MP) linkage modifications.

56. The chemically modified oligonucleotide of any one of claims 1-55, wherein the oligonucleotide does not comprise a hairpin-loop structured ADAR recruiting moiety.

57. The chemically modified oligonucleotide of any one of claims 1-56, wherein the oligonucleotide is selected from the group consisting of the sequences listed in Tables 1- 14; optionally wherein the ASO is selected from the group consisting of: a) AI-0731 (SEQ ID NO: 151), AI-0860 (SEQ ID NO: 107), AI-0943 (SEQ ID NO: 150), AI-0946 (SEQ ID NO: 147), AI-0947 (SEQ ID NO: 144), AI-0948 (SEQ ID NO: 148), AI-0949 (SEQ ID NO: 146), AI-0950 (SEQ ID NO: 149), AI-0991 (SEQ ID NO: 145); b) AI-1068 (SEQ ID NO: 192), AI-1071 (SEQ ID NO: 200), AI-1207 (SEQ ID NO: 197), AI-1208 (SEQ ID NO: 198), AI-1209 (SEQ ID NO: 199); c) AI-1442 (SEQ ID NO: 191), AI-1443 (SEQ ID NO: 193), AI-1444 (SEQ ID NO: 194), AI-1445 (SEQ ID NO: 195), AI-1446 (SEQ ID NO: 196); d) AI-1685 (SEQ ID NO: 204), AI-1686 (SEQ ID NO: 212), AI-1687 (SEQ ID NO: 205), AI-1902 (SEQ ID NO: 209), AI-1699 (SEQ ID NO: 201), AI-1700 (SEQ ID NO: 202); e) AI-1849 (SEQ ID NO: 210), AI-1850 (SEQ ID NO: 211), AI-1952 (SEQ ID NO: 214), AI-1953 (SEQ ID NO: 215), AI-1954 (SEQ ID NO: 216), AI-1701 (SEQ ID NO: 217); f) AI-1684 (SEQ ID NO: 203), AI-1689 (SEQ ID NO: 206), A-1690 (SEQ ID NO: 207), AI-1691 (SEQ ID NO: 208), AI-1901 (SEQ ID NO: 213), AI-3163 (SEQ ID NO: 218), AI-3166 (SEQ ID NO: 219); g) AI-2940 (SEQ ID NO: 190), AI-2938 (SEQ ID NO: 188), AI-2934 (SEQ ID NO: 184), AI-2936 (SEQ ID NO: 186); and h) AI-2691 (SEQ ID NO: 220), AI-2692 (SEQ ID NO: 221), AI-2693 (SEQ ID NO: 222), AI-2694 (SEQ ID NO: 223), AI-2695 (SEQ ID NO: 224), AI-2696 (SEQ ID NO: 225), AI-2584 (SEQ ID NO: 226), AI-2698 (SEQ ID NO: 227), AI-2699 (SEQ ID NO: 228), AI-2703 (SEQ ID NO: 229), AI-2704 (SEQ ID NO: 230), AI-2705 (SEQ ID NO: 231), AI-2706 (SEQ ID NO: 232), AI-3008 (SEQ ID NO: 233), and AI-3059 (SEQ ID NO: 234).

58. A composition comprising the chemically modified oligonucleotide of any one of claims 1-57, wherein the oligonucleotide is present at a concentration of 0.8nM to 100nM, optionally between 4nM and 25nM, preferably at 4nM or 20nM.

59. A chemically modified oligonucleotide of any one of claims 1-57 or a composition of claim 58 for use in therapy.

60. A chemically modified oligonucleotide of any one of claims 1-57 or a composition of claim 58, for use in the treatment of a disease or disorder, where in the disease or disorder is selected form the group consisting of liver, metabolic, neurodegenerative and / or cardiac or cardiovascular diseases or disorders, optionally wherein the disease or disorder is associated with a gain-of-function (GOF) or loss-of-function (LOF) mutation.

61. The chemically modified oligonucleotide or the composition for use of claim 59, wherein 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, 62. The chemically modified oligonucleotide or the composition for use of claim 61, wherein the disease or disorder comprises the SERPINA1 gene and wherein the mutation is SERPINA1 E342K.

63. A method for treating a subject suffering from a genetic disease or genetic disorder, comprising administering an effective amount of the chemically modified oligonucleotide as defined in any of claims 1 to 57 or the composition of claim 58 to the subject.

64. The method of claim 63, wherein the genetic disease or disorder is a liver or metabolic diseases and / or cardiac or cardiovascular diseases associated with a gain-of-function (GOF) or loss-of-function (LOF) mutation, optionally wherein the disease or disorder comprises the SERPINA1 gene.

65. An in vitro method for site-directed A-to-I editing of a target RNA, the method comprising a step of contacting a target RNA with the chemically modified oligonucleotide of any one of claims 1 to 57 or the composition of claim 58.

66. The in vitro method of claim 65, comprising, after the step of contacting, the following steps: (a) allowing uptake by the cell of the chemically modified oligonucleotide; (b) allowing annealing of the chemically modified 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.

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