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WO2025083268A1 - Oligonucléotides antisens (asos) chimiquement modifiés et compositions les contenant pour l'édition de l'arn - Google Patents

Oligonucléotides antisens (asos) chimiquement modifiés et compositions les contenant pour l'édition de l'arn Download PDF

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WO2025083268A1
WO2025083268A1 PCT/EP2024/079598 EP2024079598W WO2025083268A1 WO 2025083268 A1 WO2025083268 A1 WO 2025083268A1 EP 2024079598 W EP2024079598 W EP 2024079598W WO 2025083268 A1 WO2025083268 A1 WO 2025083268A1
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chemically modified
modified oligonucleotide
nucleotide
oligonucleotide according
nucleotides
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Piotr PITAJ
Juri EYBERG
Dirk LINDENBLATT
Tobias MERKLE
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Airna Bio Germany GmbH
Airna Corp
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Airna Bio Germany GmbH
Airna Corp
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04004—Adenosine deaminase (3.5.4.4)
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/336—Modified G
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/344—Position-specific modifications, e.g. on every purine, at the 3'-end

Definitions

  • 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”).
  • ASO antisense oligonucleotide
  • ADAR adenosine deaminase acting on RNA
  • the present invention advantageously provides oligonucleotides (or antisense oligonucleotides, ASOs) with desirable properties for in vitro and in vivo use.
  • the problem solved by the present invention lies in the provision of improved chemically modified ASOs capable of mediating a functional change from an adenosine (A) to a guanosine (G).
  • the invention provides chemically modified oligonucleotides that provide enhanced site-directed A-to-l RNA editing.
  • the chemically modified oligonucleotides are for use in site-directed A-to-l editing of a target RNA inside a cell with endogenous adenosine deaminase acting on RNA (ADAR).
  • ADAR endogenous adenosine deaminase acting on RNA
  • ASOs chemically modified antisense oligonucleotides
  • the ASOs of the invention are not just limited to correcting G-to-A mutations but are also useful in changing a wildtype sequence into a mutated sequence in order to modulate protein expression and/or function (“beneficial editing").
  • the chemically modified oligonucleotides according to the present invention may be used as active agents to treat disorders or diseases, such as genetic disorders or diseases, associated with one or more G-to-A mutations or to change wildtype sequences.
  • 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 chemically modified oligonucleotide comprising a sequence capable of binding to a target sequence in a target RNA, and a central base triplet (CBT) of three nucleotides (... N-1, No, N+1...) where No is the central nucleotide directly opposite to a target adenosine in the target RNA that is to be edited, wherein the chemically modified oligonucleotide comprises the following sequence:
  • nucleotide is independently chemically modified with a moiety that is positively charged under physiological conditions, wherein the least one nucleotide is selected from N-6, N-5, N. 4 , N+6, N +7 , N+s, N +9 , N+w and where present N+n, N+12, N+13, N+ 14 , N+23, and N+ 24 .
  • composition comprising 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 chemically modified oligonucleotide comprising a sequence capable of binding to a target sequence in a target RNA, and a central base triplet (CBT) of three nucleotides (... N-i, No, N+1...) where No is the central nucleotide directly opposite to a target adenosine in the target RNA that is to be edited, wherein the chemically modified oligonucleotide comprises the following sequence:
  • 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 chemically modified oligonucleotide comprising a sequence capable of binding to a target sequence in a target RNA, and a central base triplet (CBT) of three nucleotides (... N-1, No, N+1...) where No is the central nucleotide directly opposite to a target adenosine in the target RNA that is to be edited, wherein the chemically modified oligonucleotide comprises the following sequence:
  • nucleotide is independently chemically modified with a moiety that is positively charged under physiological conditions, wherein the least one nucleotide is selected from N-6, N-5, N.
  • a composition comprising 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 chemically modified oligonucleotide comprising a sequence capable of binding to a target sequence in a target RNA, and a central base triplet (CBT) of three nucleotides (... N-1, No, N+1...) where No is the central nucleotide directly opposite to a target adenosine in the target RNA that is to be edited, wherein the chemically modified oligonucleotide comprises the following sequence:
  • N-6 his N-4 his biz N-1 NO N+1 N +2 N +3 N +4 N +5 N +6 N +7 N +8 N +9 N+ W . . . . - 5’ and at least one nucleotide is independently chemically modified with a moiety that is positively charged under physiological conditions, wherein the least one nucleotide is selected from N-6, N-5, N. 4 , N+6, N+ 7 , N+s, N+ 9 , N+w and where present N+n, N+12, N+13, N+ 14 , N+23, and N+ 24 .
  • 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 chemically modified oligonucleotide comprising a sequence capable of binding to a target sequence in a target RNA, and a central base triplet (CBT) of three nucleotides (... N-1, No, N+1...) where No is the central nucleotide directly opposite to a target adenosine in the target RNA that is to be edited, wherein the chemically modified oligonucleotide comprises the following sequence:
  • nucleotide is independently chemically modified with a moiety that is positively charged under physiological conditions, wherein the least one nucleotide is selected from N-6, N-5, N.
  • a composition comprising 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 chemically modified oligonucleotide comprising a sequence capable of binding to a target sequence in a target RNA, and a central base triplet (CBT) of three nucleotides (... N-1, No, N+1...) where No is the central nucleotide directly opposite to a target adenosine in the target RNA that is to be edited, wherein the chemically modified oligonucleotide comprises the following sequence:
  • N-6 his N-4 his biz N-1 NO N+1 N +2 N +3 N +4 N +5 N +6 N +7 N +8 N +9 N+ W . . . . - 5’ and at least one nucleotide is independently chemically modified with a moiety that is positively charged under physiological conditions, wherein the least one nucleotide is selected from N-6, N-5, N. 4 , N+6, N+ 7 , N+s, N+ 9 , N+w and where present N+n, N+12, N+13, N+ 14 , N+23, and N+ 24 .
  • a method of treating or preventing a disease or disorder in a subject in need thereof comprising administering to the subject an effective amount of: 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 chemically modified oligonucleotide comprising a sequence capable of binding to a target sequence in a target RNA, and a central base triplet (CBT) of three nucleotides (... N-1, No, N+1...) where No is the central nucleotide directly opposite to a target adenosine in the target RNA that is to be edited, wherein the chemically modified oligonucleotide comprises the following sequence:
  • nucleotide is independently chemically modified with a moiety that is positively charged under physiological conditions, wherein the least one nucleotide is selected from N-6, N-5, N.
  • a composition comprising 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 chemically modified oligonucleotide comprising a sequence capable of binding to a target sequence in a target RNA, and a central base triplet (CBT) of three nucleotides (... N-1, No, N+1...) where No is the central nucleotide directly opposite to a target adenosine in the target RNA that is to be edited, wherein the chemically modified oligonucleotide comprises the following sequence:
  • N-6 his N-4 his biz N-1 NO N+1 N +2 N +3 N +4 N +5 N +6 N +7 N +8 N +9 N+ W . . . . - 5’ and at least one nucleotide is independently chemically modified with a moiety that is positively charged under physiological conditions, wherein the least one nucleotide is selected from N-6, N-5, N. 4 , N+6, N+ 7 , N+s, N+ 9 , N+w and where present N+n, N+12, N+13, N+ 14 , N+23, and N+ 24 .
  • an in vitro method for site- directed A-to-l editing of a target RNA comprising a step of contacting a target RNA with: 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 chemically modified oligonucleotide comprising a sequence capable of binding to a target sequence in a target RNA, and a central base triplet (CBT) of three nucleotides (... N-1, No, N+1...) where No is the central nucleotide directly opposite to a target adenosine in the target RNA that is to be edited, wherein the chemically modified oligonucleotide comprises the following sequence:
  • nucleotide is independently chemically modified with a moiety that is positively charged under physiological conditions, wherein the least one nucleotide is selected from N-6, N-5, N.
  • composition comprising 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 chemically modified oligonucleotide comprising a sequence capable of binding to a target sequence in a target RNA, and a central base triplet (CBT) of three nucleotides (...
  • ADAR endogenous adenosine deaminase acting on RNA
  • CBT central base triplet
  • N.1, N o , N+1 wherein the chemically modified oligonucleotide comprises the following sequence:
  • N-6 his N-4 his biz N-1 NO N+1 N +2 N +3 N +4 N +5 N +6 N +7 N +8 N +9 N+ W . . . . - 5’ and at least one nucleotide is independently chemically modified with a moiety that is positively charged under physiological conditions, wherein the least one nucleotide is selected from N-6, N-5, N. 4 , N+6, N+ 7 , N+s, N+ 9 , N+w and where present N+n, N+12, N+13, N+ 14 , N+23, and N+ 24 .
  • an in vitro method for deaminating at least one specific adenosine present in a target RNA sequence in a cell comprising the steps of:
  • nucleotide is independently chemically modified with a moiety that is positively charged under physiological conditions, wherein the least one nucleotide is selected from N-6, N-5, N. 4 , N+6, N+ 7 , N+ 8 , N+ 9 , N+10 and where present N+n, N+12, N+ 13 , N+ 14 , N+ 23 , and N+ 24 ;
  • Fig. 1 shows a calculated vacuum electrostatic surface representation of ADAR1 , as obtained according to Example 1 .
  • Negative (red) and positive (blue) regions of the surface are shaded, and three negatively charged areas (‘pockets’) were identified. These positions correspond to positions, +7, +8, +9, and +10; -1 and -2, and -4, -5 and -6, of the bound oligonucleotide and are labelled in Fig.1.
  • these represent positions where the introduction of positive charges to the binding nucleic acid can increase editing.
  • Fig. 2 shows a 2’-amino (NH 2 ) pyrimidine walk, demonstrating the RNA editing % of SERPINA1 ASOs transfected in Hela-SERPINA E342K cells with 0.3pL Lipofectamine RNAiMAX in 96-well plate.
  • RNA editing was determined 24h after transfection, in accordance with the methods set out in Example 4.
  • the tested ASOs are set out in Table 1 in the examples. Nucleotides were modified at the 2’ position with an amine group (-NH2).
  • Fig. 3A shows a modified internucleoside linkage between position +9 and +10, modified according to formula (Vc) described herein, wherein L is 3.
  • the terminal NH 3 + group is shown to interact with three acidic residues in close proximity, indicated by dashed lines. The respective distances are presented in angstrom [A] (3.3, 2.9, 3.4 and 3.7).
  • Fig. 3B shows a nucleotide having a 2’ modification at position -4, modified according to formula (lllf) described herein, wherein n is 1.
  • the terminal NH 3 + group is shown to interact with a nearby aspartic acid residue, as indicated by the dashed line. The distance is presented in angstrom [A] (3.3).
  • Fig. 3C shows a modified internucleoside linkage between position -4 and -5, modified according to formula (Ve) described herein, wherein L is 2.
  • the terminal NH 3 + group is shown to interact with two acidic residues in close proximity, indicated by dashed lines. The respective distances are presented in angstrom [A] (3.0 and 3.2).
  • Fig. 3D shows a nucleotide having a 2’ modification (-RNH2) at position +6, where R is CH2CH2CH2.
  • the modification is a 2’-propylamino (-CH2CH2CH2NH2) modification.
  • the terminal NH 3 + group is shown to interact with a nearby glutamic acid residue, as indicated by the dashed lines.
  • the respective distances are presented in angstrom [A] (2.9 and 3.5).
  • the propylamino chain is stabilised by interaction with proline.
  • Fig. 4 shows a 2’-amino (-NH 2 ) pyrimidine walk, demonstrating the RNA editing % of SERPINA1 ASOs transfected in Hela-SERPINA E342K cells with 0.3pL Lipofectamine RNAiMAX in 96-well plate.
  • RNA editing was determined 24h after transfection, in accordance with the methods set out in Example 5.
  • the tested ASOs are set out in Table 2 in the examples. Nucleotides were modified at the 2’ position with an amine group (-NH 2 ).
  • Fig. 5 shows a 2’-propylamino (-CH2CH2CH2NH2) pyrimidine walk, demonstrating the RNA editing % of SERPINA1 ASOs transfected in Hela-SERPINA E342K cells with 0.3pL Lipofectamine RNAiMAX in 96-well plate.
  • RNA editing was determined 24h after transfection, in accordance with the methods set out in Example 6.
  • the tested ASOs are set out in Table 3 in the examples. Nucleotides were modified at the 2’- position with a propylamino group (-CH2CH2CH2NH2).
  • RNA editing is a natural process through which some cells can make discrete changes to specific nucleotide sequences within an RNA molecule in a site-specific way. Unlike DNA editing, the advantage of site-directed RNA editing is that it allows modification of the genetic information in a more precise and efficient manner. Contrary to DNA, RNA is generally quickly degraded, and any errors introduced by off-target modifications will be washed out rather than permanently introduced into the modified DNA of a subject. RNA editing may also be less likely to cause an immune reaction since it is an editing mechanism naturally found in humans. Moreover, RNA editing might provide a more natural response than introducing an external, engineered gene.
  • 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.
  • ASOs antisense oligonucleotides
  • siRNA small interfering RNA
  • miRNA microRNA
  • RNA editing enzymes are known in the art.
  • C cytidine
  • U uridine
  • A-to-l adenosine
  • I inosine
  • RNA editing is the “A-to-l” conversion, which is catalysed by the adenosine deaminases acting on RNA (ADARs) family.
  • ADARs adenosine deaminases acting on RNA
  • 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).
  • dsRNA double-stranded RNA
  • hADARI 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.
  • ADAR1 proteins additionally comprise one or more Z binding domains
  • splice variant ADAR2R and ADAR3 comprises an R domain (Zinshteyn and Nishikura, 2009; Wulff and Nishikura, 2010).
  • the ADAR may be hADARI , hADAR2 or hADAR3, or any variant thereof.
  • the ability of ADARs to alter the sequence of RNAs has also been used to artificially target RNAs in vitro in cells for RNA editing.
  • 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-l" 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.
  • ASOs are generally short (approx.18 to 45 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 1 st (Gen 1), 2 nd (Gen 2), and 3 rd (Gen 3) generation ASOs. Notably, ASO sequence and design are the primary drivers that determine the pharmacological and toxicological properties of the oligonucleotide.
  • Gen 1 ASOs were initially employed to inhibit translation of Rous sarcoma virus ribosomal RNA (Stephenson and Zamecnik, 1978). They are characterised in having a modified backbone, wherein the nucleotide linkages are modified by sulphur, methyl or amine groups to generate phosphorothioates (PS), methyl-phosphonates, and phosphoramidates, respectively.
  • PS phosphorothioates
  • ASOs can be chemically modified to improve their properties. For instance, ASOs can be modified to protect them against nucleases and to increase their effectiveness.
  • 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.
  • these Gen 2 ASOs tend to be less toxic than PS-modified ASOs and have a slightly higher affinity for their target.
  • 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).
  • 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).
  • ASOs can work through many mechanisms depending, in part, on the region in the RNA sequence that is targeted and ASO design/chemical properties.
  • the ASO targeting domain typically 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 Beal, 2017) and therefore could alter or even improve substrate recognition, if these features are mimicked in the ASO/resulting dsRNA.
  • ASOs can be chemically modified to improve their properties.
  • ASOs can be modified to protect them against nucleases and to increase their effectiveness.
  • PS phosphorothioate
  • 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.
  • Rp and Sp can influence properties such as, e.g., thermal stability, binding affinity, pharmacologic properties, etc., of the ASO.
  • Rp and Sp stereoisomers has been controversial (Iwamoto et al., 2017; Crooke et al., 2020).
  • RNA editing systems to specifically recruit endogenous adenosine deaminases have previously been described.
  • Oligonucleotide constructs for site-directed RNA editing are described in WO 2016/097212 and WO 2017/010556, which utilise endogenous cellular pathways, i.e., endogenous ADAR, to edit endogenous RNA.
  • Loop-hairpin structured oligonucleotides have previously been described (WO 2020/001793) and have been used successfully to harness ADARs with chemically modified oligonucleotides.
  • 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 CBT. Further, these oligonucleotides contain massively 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 recruiting endogenous ADARs with antisense oligonucleotides has previously been 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.
  • 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.
  • 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 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.
  • CBT central base triplet
  • 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.
  • WO 2021/243023 also mentions guide or targeting domain modifications 3’ to the nucleobase just outside the CBT (at position +2 of an oligonucleotide comprising the structure [Am]-X 1 -X 2 -X 3 -X 4 -[Bn], wherein X 4 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.
  • the present inventors have advantageously determined that the presence of positive charge in ASOs enhances their interaction with the endogenous adenosine deaminase acting on RNA (ADAR), enabling enhanced site-directed A-to-l RNA editing capabilities of the ASOs.
  • ADAR endogenous adenosine deaminase acting on RNA
  • the presence of positive charge enables the ASOs to conform to, and interact with, the ADAR to a significantly greater extent.
  • the present invention is thus directed towards 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 chemically modified oligonucleotide comprising a sequence capable of binding to a target sequence in a target RNA, and a central base triplet (CBT) of three nucleotides (... N-i, No, N+1...) where No is the central nucleotide directly opposite to a target adenosine in the target RNA that is to be edited, wherein the chemically modified oligonucleotide comprises the following sequence:
  • nucleotide is independently chemically modified with a moiety that is positively charged under physiological conditions, wherein the at least one nucleotide is selected from N-6, N-5, N. 4 , N+6, N +7 , N+s, N +9 , N+w and where present N+n,N+I2 , N+13, N+ 14 , N+ 23 , and N+ 24 .
  • the present inventors consider that the presence of a moiety that is positively charged under physiological conditions at at least one of the nucleotides selected from N.6, N-5, N.4, N+6, N+7, N+s, N+9, N+w and where present N+n, N+12, N+13, N+14, N+23, and N +2 4 enhances the interaction of the chemically modified oligonucleotide of the present invention with the ADAR through enhanced electrostatic interactions and/or hydrogen bonding. Such enhanced interactions enable the chemically modified oligonucleotide to conform to the shape, surface structure and/or charge profile of the ADAR, enabling enhancing site-directed A-to-l editing.
  • At least one of the nucleotides N+s and N.6 is chemically modified with a moiety that is positively charged under physiological conditions.
  • the positioning of a moiety that is positively charged under physiological conditions in the chemically modified oligonucleotide of the present invention i.e. at at least one of the nucleotides selected from N.6, N-5, N.4, N+6, N+7, N+s, N+g, N+w and where present N+1i, N+w, N+W, N+14, N+23, and N+24, reflects the regions of negative charge in the charge profile of the ADAR. These regions of negative charge in the ADAR can be identified, for example using alphafold software, and without being bound by theory, are thought to be caused by acidic groups having negative charge (deprotonation) under physiological conditions.
  • the present inventors consider that the ADAR has different ‘pockets’ of negative charge, corresponding to N.6 to N.4, and N+6 to N+14 nucleotide regions of the chemically modified oligonucleotide of the present invention.
  • another ‘pocket’ of negative charge may be found corresponding to the N-1 and N-2 positions of the chemically modified oligonucleotide.
  • modification of the oligonucleotide at either of these positions N-1 or N-2 may be detrimental as these positions are at the active site of the ADAR enzyme upon interaction of the chemically modified oligonucleotide therewith.
  • the chemically modified oligonucleotide of the present invention therefore has a stronger affinity for the ADAR.
  • the present inventors consider that the presence of a moiety that is positively charged under physiological conditions at nucleotides N +2 3 and/or N +2 4 of the chemically modified oligonucleotide of the present invention is beneficial. Without being bound by theory, the present inventors consider this likely due to additional stabilisation and/or altered protein binding achieved.
  • the chemically modified oligonucleotide of the present invention has the following core sequence:
  • the chemically modified oligonucleotide of the present invention may have any suitable length, as further discussed herein.
  • the chemically modified oligonucleotide of the present invention may have a length of at least 25 nucleotides (N), optionally 25 to 80 N, preferably 25 to 50 N, and more preferably 30 to 40 N.
  • N+n,N+I2 , N+13, N+14, N +2 3, and N +2 4 means that, when any of the nucleotides in question (N+n and/orN+I2 and/or N+13 and/or N+14 and/or N +2 3 and/or N +2 4) are present in the oligonucleotide, the nucleotide(s) is included in the list of nucleotides from which the at least one nucleotide modified with a moiety that is positively charged under physiological conditions is selected.
  • the at least one nucleotide modified with a moiety that is positively charge under physiological conditions may be selected from N-6, N-5, N-4, N+6, N+ 7 , N+s, N+ 9 , and N+w, and also N+1i,N+I2 , N+13, and N+14, i.s. N-6, N-5, N-4, N+6, N+ 7 , N+s, N+ 9 , N+10, N+n, ,N N++I213, and N+14.
  • flanking region refers to the 5’ and/or 3’ region a region on the oligonucleotide is adjacent or directly adjacent to the No on the 5' and/or 3’ portion of the oligonucleotide.
  • nucleic acid is intended to include any DNA molecules (e.g., cDNA or genomic DNA) and any RNA molecules (e.g., mRNA) and analogues of the DNA or RNA generated using nucleotide analogues.
  • Oligonucleotides can be single-stranded (ss) or double-stranded (ds).
  • a single-stranded oligonucleotide can have double-stranded regions (formed by portions of the single-stranded oligonucleotide).
  • a double-stranded oligonucleotide can have single-stranded regions, for example, at regions where the two oligonucleotide chains are not complementary to each other.
  • Each component of the DNA or RNA can be modified and categorized by modification of (1) the internucleoside linkage, (2) the deoxyribose/ribose, and/or (3) the nucleobase.
  • nucleobase 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 (II), 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”.
  • a nucleobase may be a nucleobase, which comprises a modification.
  • a modified nucleobase may be 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.
  • the modified nucleobase may be capable of increasing hydrogen bonding, base pair stacking interactions and/or stabilizing a nucleic acid complex.
  • the modified nucleobase (e.g., Benner’s base) may be capable of mimicking the N3 protonated cytosine base.
  • a modified nucleobase may be a substituted A, T, C, G, or II, or a substituted tautomer of A, T, C, G, or II.
  • a modified nucleobase in the context of oligonucleotides may refer to a nucleobase that is not A, T, C, G or II. Modifications include but are not limited to nonstandard nucleobases 5-methyl-2’-deoxycytidine (m5C), pseudouridine (pll), dihydrouridine, inosine (I), and 7-methylguanosine.
  • nucleobase replacement by (N) heterocycles e.g., nebularine
  • aromatic rings that stack well in the RNA duplex
  • a Benner’s base Z and/or analogues
  • 8-oxo-adenosine (8-oxo-A 8-oxo-A
  • Benner’s base Z refers to the pyrimidine analogue 6-amino-5-nitro-3-(1 '-p- D-2'-deoxyribofuranosyl)-2(1 H)-pyridone (dZ).
  • a modification may include the introduction of nucleobase analogues or simple heterocycles that boost editing.
  • a derivative thereof refers to a derivative of a (modified) nucleobase, nucleoside or nucleotide.
  • a derivative may be a corresponding nucleobase, nucleoside or nucleotide that has been chemically derived from said nucleobase, nucleoside or nucleotide.
  • a derivative of deoxycytidine may include fluoro-modified deoxycytidine, 5-methyl-2’-deoxycytidine (m5C), or ribocytidine.
  • nucleoside(s) refers to a moiety wherein a nucleobase or a modified nucleobase is covalently bound to a sugar or a modified sugar.
  • a “nucleoside” may refer to a nucleoside unit in an oligonucleotide or a nucleic acid.
  • nucleoside(s) encompasses all modified versions and derivatives “modified nucleobases”.
  • nucleotide(s) 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. The linkage may be an internucleoside linkage as described herein. The modified linkage may be a PS linkage.
  • a “nucleotide” may refer 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”.
  • oligonucleotide(s)“ as used herein is defined as is generally understood by the skilled person as a molecule including two or more covalently linked nucleosides. They can comprise DNA and/or RNA. The oligonucleotides may have a backbone comprising deoxyribonucleotides and/or ribonucleotides.
  • internucleoside linkage refers to a linkage between adjacent nucleosides. “Internucleoside linkage” and “linkage” may be used interchangeably. Linkages may be continuous (consecutive) or discontinuous (interrupted).
  • 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.
  • the naturally occurring PO linkages may be replaced by modified internucleoside linkages.
  • the linkage may be a non-natural internucleoside linkage.
  • 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.
  • the oligonucleotides of the invention comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more stereorandom internucleoside linkages (mixture of Rp and Sp linkage phosphorus at the internucleoside linkage, e.g., from traditional non-chirally controlled oligonucleotide synthesis).
  • An internucleoside linkage may be a phosphorothioate (PS) linkage.
  • An internucleoside linkage may be a stereorandom PS linkage.
  • An internucleoside linkage may be a chirally controlled PS linkage.
  • An internucleoside may not be chirally controlled.
  • An internucleoside linkage may not be a chirally controlled PS linkage.
  • antisense oligonucleotide refers to a strand 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.
  • antisense oligonucleotide and oligonucleotide may be used interchangeably.
  • target RNA refers to an RNA, which is subject to the editing process, and “targeted” by the respective ASOs of the invention.
  • off-target 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.
  • off-target editing may arise from the promiscuous reactivity of the deaminase enzymes.
  • 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.
  • sugars may comprise C4 sugars, C5 sugars and/or C6 sugars.
  • a modified sugar may be substituted.
  • a modified sugar may be a sugar that is not ribose or deoxyribose as typically found in natural RNA or DNA (e.g., arabinose).
  • a modified sugar may comprise a 2'-modification.
  • Examples of useful 2’-sugar modifications include, e.g., 2’-ribose (RNA), 2’-deoxyribose (DNA), 2’-arabinose etc..
  • RNA 2’-ribose
  • DNA 2’-deoxyribose
  • 2’-arabinose 2’-arabinose
  • LNA locked nucleic acid
  • LNAs locked nucleic acids
  • BNA bridged nucleic acid
  • a modified sugar may be a bicyclic sugar, e.g., a sugar used in locked nucleic acid (LNA), BNA, etc..
  • a modified sugar may be an LNA sugar.
  • a modified sugar may be an BNA sugar.
  • a sugar modification may be 2’-OMe, 2'-O-methoxyethyl (2’-MOE), 2’- F, 5’-vinyl, or S-constrained ethyl (S-cEt).
  • a 2’-modification may be a C2-stereoisomer of 2’-F-ribose.
  • a 2'-modification may be 2’-F.
  • a 2'-modification may be 2'-FANA.
  • a modified sugar may be a sugar of morpholino.
  • the oligonucleotide may comprise, e.g., an UNA (unlocked nucleic acid), a PMO (phosphorodiamidate linked morpholino) or a PNA (peptide nucleic acid).
  • the nucleic acid analogue may be a PNA (peptide nucleic acid).
  • the nucleic acid analogue may be PMO (phosphorodiamidate linked morpholino).
  • FANA or “FANA-modified” refers to 2'-fluoroarabinoside modified nucleobases and/or oligonucleotides comprising such nucleobases.
  • FANA-cytidine refers to a cytidine that comprises a 2'-fluoro-beta-D- arabinonucleic acid sugar modification.
  • a derivate thereof refers to a corresponding nucleotide(s) or oligonucleotide(s) that has been chemically derived from said nucleotide or oligonucleotide(s).
  • 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.
  • ASOs may comprise a wobble base outside the CBT.
  • the ASO may comprise a mismatch outside the CBT.
  • the ASOs may include a mismatch opposite the target adenosine.
  • the complementarity of the ASOs may be 100%, except at the nucleoside opposite to a target nucleoside to be edited.
  • Complementarity may be at least 80%, 85%, 90%, 95%.
  • Complementarity may be 85%-99%.
  • the ASO may comprise 1 , 2, 3, 4, 5 or more mismatches when aligned with the target nucleic acid.
  • One or more mismatches may be independently a wobble base paring.
  • the ASOs may comprise up to 4 mismatches or wobble bases outside the CBT.
  • the ASOs may comprise up to 3 mismatches or wobble bases outside the CBT.
  • mutation 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.
  • beneficial editing refers to the editing of a target sequence (or base) derived from a wildtype allele (not a mutated allele) in order to, e.g., modulate the function of a wildtype protein in a useful way to prevent or treat a disease.
  • beneficial editing may include sites, such as STAT1 Y701 , NLRP3 Y166 and CTNNB1 T41 that are not causes for genetic diseases but rather represent wildtype protein sites. These sites may be changed (no underlying G-to-A mutation) to alter the function of the wildtype protein.
  • RNA 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- 1 change could help to functionally compensate for an otherwise non-editable mutation to ameliorate a disease phenotype.
  • adenosine deaminase(s) or “adenosine deaminase(s) acting on RNA” [ADAR(s)] 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.
  • the ADAR may be an (endogenous) adenosine deaminase catalysing the deamination of adenosine to inosine or deoxy-adenosine to deoxyinosine.
  • the ADAR may catalyse the deamination of adenine or adenosine in deoxyribonucleic acid (DNA) or in ribonucleic acid (RNA).
  • the ADAR may be a human ADAR.
  • the ADAR may be an endogenous ADAR. Accordingly, the ADAR may be an endogenous human ADAR1 , ADAR2 or ADAR3 (hADARI , hADAR2 or hADAR3), or any fragment or isoform(s) thereof (e.g., hADARI p110 and p150).
  • guide RNA refers 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.
  • the base-modifying activity/editing function e.g., ADAR
  • the terms “disease” or “disorder” are used interchangeably to refer to a condition in a subject.
  • the condition may be a disease in a subject, the severity of which may be decreased by inducing an immune response in the subject through the administration of a pharmaceutical composition.
  • the condition typically impairs physiological function and may be associated with specific symptoms.
  • the term “effective amount” defines an amount that can be administered to a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, but one that is sufficient to provide the desired effect, e.g. the treatment or prophylaxis manifested by a permanent or temporary improvement in the subject's condition.
  • the amount will vary from subject to subject, depending on the age and general condition of the individual, mode of administration and other factors. Thus, while it is not possible to specify an exact effective amount, those skilled in the art will be able to determine an appropriate "effective" amount in any individual case using routine experimentation and background general knowledge.
  • a therapeutic result in this context includes eradication or lessening of symptoms, reduced pain or discomfort, prolonged survival, improved mobility and other markers of clinical improvement. A therapeutic result need not be a complete cure alone and can be used in combination with other agents.
  • the term “in combination” in the context of the administration of two or more therapies to a subject refers to the use of more than one therapy (e.g., more than one prophylactic agent and/or therapeutic agent).
  • the use of the term “in combination” does not restrict the order in which therapies are administered to a subject.
  • one or more ASOs may be used in combination.
  • the terms “prevent”, “preventing” and “prevention” refer to the inhibition of the development or onset of a disease or symptoms thereof. The term may relate to the administration of the compound to a subject who is known to have an increased risk of developing a certain disorder, or disease.
  • 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 disease stabilized (i.e., not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression; amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease.
  • Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
  • subject or “patient” are used interchangeable and relate to an animal (e.g., mammals) that may need administration of the compound of the invention in the field of human or veterinary medicine.
  • the subject may be 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.
  • the term "pharmaceutically acceptable” means approved by a regulatory agency.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable excipients include starch, glucose, lactose, sucrose, gelatine, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the formulation should suit the mode of administration.
  • physiological conditions takes its normal definition in the art.
  • the term refers to the extracellular and/or intracellular environment occurring in nature for an organism, tissue or cell system, in particular in a mammalian cell, for example a human cell within the human body.
  • Physiological conditions include a physiological pH of from 5 to 8, such as 6 to 8, typically 6.5 to 7.5, most typically 7.4.
  • Physiological conditions include a temperature of from 20 to 40 °C. It will be appreciated that physiological conditions can be replicated in an in vitro environment.
  • C1-4alkyl denotes a straight or branched alkyl group having from 1 to 4 carbon atoms. For parts of the range C1-4alkyl all subgroups thereof are contemplated such as C1-3alkyl, C1-2alkyl, C2-4alkyl, C2-3alkyl and C3-4alkyl. Examples of said C1-4alkyl include methyl (Me), ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert- butyl.
  • Ci-salkylene denotes a straight or branched divalent saturated hydrocarbon chain having from 1 to 5 carbon atoms.
  • the Ci-salkylene chain may be attached to the rest of the molecule through one carbon within the chain or through any two carbons within the chain.
  • Examples of Ci-salkylenes include methylene [-CH2- ], 1 ,2-ethylene [-CH2-CH2-], 1 , 1 -ethylene [-CH(CH 3 )-J, 1 ,2-propylene [-CH 2 -CH(CH 3 )-] and 1 ,3-propylene [-CH2-CH2-CH2-].
  • Ci-salkylene all subgroups thereof are contemplated, such as C1-2alkylene, C1-3alkylene or C2-3alkylene.
  • heterocyclyl and “heterocyclic ring” denote a non-aromatic, fully saturated or partially unsaturated, preferably fully saturated, monocyclic ring system having from 4 to 7 ring atoms (unless otherwise specified), especially 5 to 7, or 5 or 6 ring atoms, in which one or more of the ring atoms are other than carbon, such as nitrogen, sulphur or oxygen.
  • the said ring system may be attached to the rest of the molecule through either a heteroatom or a carbon atom of the ring system.
  • heterocyclic groups include but are not limited to piperidinyl, morpholinyl, homomorpholinyl, azepanyl, piperazinyl, oxo-piperazinyl, diazepinyl, tertahydropyridinyl, tetrahydropyranyl, pyrrolidinyl, tertrahydrofuranyl, and dihydropyrrolyl.
  • Particular examples include morpholine, piperidine (e.g. 1 -piperidinyl, 2-piperidinyl, 3-piperidinyl and 4-piperidinyl), pyrrolidine (e.g.
  • thiomorpholine and its S-oxide and S,S-dioxide particularly thiomorpholine
  • Still further examples include azetidine, piperidone, piperazone, and /V-alkyl piperidines such as /V-methyl piperidine.
  • heteroaryl and “heteroaromatic ring” denote a monocyclic heteroaromatic ring comprising 5 to 6 ring atoms in which one or more of the ring atoms are other than carbon, such as nitrogen, sulphur or oxygen.
  • the heteroaryl ring will contain up to 4 heteroatoms, more typically up to 3 heteroatoms, more usually up to 2, for example a single heteroatom.
  • the said heteroaromatic ring may be attached to the rest of the molecule through either a heteratom or a carbon atom of the ring system.
  • heteroaryl groups include but are not limited to furyl, pyrrolyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, oxatriazoly, thiazolyl, isothiazolyl, tetrazolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl and thiadiazolyl.
  • the heteroaryl ring may contain at least one ring nitrogen atom.
  • the nitrogen atoms in the heteroaryl rings can be basic, as in the case of an imidazole or pyridine, or essentially non-basic as in the case of a pyrrole nitrogen.
  • the number of basic nitrogen atoms present in the heteroaryl group, including any amino group substituents of the ring, will be less than five.
  • the term “fully saturated” refers to rings where there are no multiple bonds between ring atoms.
  • nitrogen-containing refers to monocyclic ring system in which at least one of the ring atoms is nitrogen.
  • the group is attached to the rest of the molecule via the nitrogen atom or through a carbon atom on the ring system.
  • One or more of the remaining ring atoms may be other than carbon, such as nitrogen, sulphur or oxygen.
  • the ring system comprises only nitrogen and carbon atoms. Examples of such groups include piperidine (1 -piperidinyl), pyrrolidine (1-pyrrolidinyl), pyrrolidone, morpholine or piperazine.
  • a chemical group(s) or moiety is attached to the rest of the molecule by the atom or group listed first.
  • the feature also denotes the attachment of chemical groups or moieties to each other, or to the rest of the molecule.
  • the feature of a wave line also denotes the attachment of a chemical group(s) or moiety to each other, or to the rest of the molecule.
  • one or more substituents preferably refers to one or two substituents.
  • the at least one nucleotide may be selected from N_ 6 , N_ 5 , and N. 4 , preferably N_ 6 .
  • the at least one nucleotide may be selected from N +6 , N +7 , N +8 , N +9 , and N+w, preferably N +7 , N +8 , N +9 , and N+w, more preferably N +8, N+ 9 , and N+w.
  • the at least one nucleotide may be selected from N +6 , N +7 , N +8 , N +9 , N+w, N+n, N+I2, N+ 13 , and N+ 14 , preferably from N +7 , N +8 , N +9 , N+w, N+n, N+w, N+w, and N+w, and more preferably from N+8, N+9, N+10, N+11, N+12, N+13, and N+14.
  • the at least one nucleotide may be selected from N+ 23 and N +24 .
  • the at least one nucleotide may be selected from N.6, N.s, N. 4 , N+ 7 , N+s, N+ 9 , and N+w.
  • the at least one nucleotide may be selected from:
  • N.6, N-5, and N. 4 preferably N_ 6 ;
  • N+6, N+ 7 , N+S, N+9, and N+w preferably N +7 , N +8 , N +9 , and N+w; more preferably N +8 , N+9, and N+w; and/or N+11, N+w, N+W, and N+ 14 ; and/or
  • the at least one nucleotide may be selected from:
  • N.6, N-5, and N. 4 preferably N. 8 ;
  • N+6, N+ 7 , N+ 8 , N+9, N+W, N+11, N+W, N+W, and N+ 14 preferably N+ 7 , N+ 8 , N+ 9 , N+10, N+n, N+w, N+W, and N+ 14 , more preferably N+ 8 , N+ 9 , N+w, N+n, N+w, N+w, and N+ 14 ; and/or
  • the chemically modified oligonucleotide of the present invention may include modification with a moiety that is positively charged under physiological conditions at any combination of the above-mentioned nucleotides N_ 6 , N-5, N-4, N+6, N+ 7 , N+ 8 , N+ 9 , N+w and where present N+n, N+w, N+w, N+w, N+ 23 , and N+ 24 .
  • two or more of the nucleotides selected from N_ 6 , N_ 5 , N_ 4 , N +6 , N +7 , N +8 , N+9, N+IO and where present N+n, N+12, N+13, N+14, N+23, and N+24 are independently chemically modified with a moiety that is positively charged under physiological conditions.
  • no more than eight, optionally no more than six, of the above nucleotides are chemically modified with a moiety that is positively charged under physiological conditions.
  • the two or more nucleotides may be selected from N.6, N.s, N. 4 , preferably N.6.
  • the two or more nucleotides may be selected from N+6, N+ 7 , N+s, N+9, and N+10, preferably from N+ 7 , N+8, N+9, and N+10, and more preferably from N+s, N+9, and N+10.
  • the two or more nucleotides may be selected from N+6, N+ 7 , N+s, N+9, N+10, N+n, N+12, N+13, and N+14, preferably from N+ 7 , N+s, N+9, N+10, N+n, N+12, N+13, and N+14, and more preferably from N+8, N+9, N+10, N+11, N+12, N+13, and N+14.
  • the two or more nucleotides may be selected from N+23 and N+24.
  • the two or more of the nucleotides may be selected from N.6, N.s, N. 4 , N+ 7 , N+s, N+9 and N+10.
  • the two or more nucleotides may be selected from:
  • N.6, N-5, and N. 4 preferably N.e;
  • N+6, N+ 7 , N+S, N+9, and N+10 preferably N+ 7 , N+s, N+9, and N+10, more preferably N+s, N+9, and N+10; and/or
  • the two or more nucleotides may be selected from:
  • N-6, N-5, and N. 4 preferably N_ 6 ;
  • N+6, N+7, N+8, N+9, N+IO, N+II , N+12, N+13, and N+14 preferably N+7, N+ 8 , N+ 9 , N+10, N+n, N+12, N+13, and N+ 14 , more preferably N +8 , N +9 , N+w, N+n, N+w, N+w, and N+ 14 ; and/or
  • the two or more nucleotides may include: at least one nucleotide selected from N_ 6 , N_ 5 and N. 4 , preferably N_ 6 ; and at least one nucleotide selected from N +6 , N +7 , N +8 , N +9 , and N+w, preferably N +7 , N +8 , N+g, and N+w, more preferably N +8 , N +9 , and N+w.
  • the two or more nucleotides may include: at least one nucleotide selected from N. 8 , N.s and N. 4 , preferably N. 8 ; and at least one nucleotide selected from N +8 , N+7, N+ 8 , N+ 9 , N+w, N+n, N+12, N+13, and N+ 14 , preferably N+7, N+ 8 , N+ 9 , N+w, N+n, N+12, N+w, and N+ 14 , more preferably N+ 8 , N+ 9 , N+w, N+11, N+12, N+W, and N+ 14 .
  • the two or more nucleotides may include: at least one nucleotide selected from N +6 , N +7 , N +8 , N +9 , and N+w, preferably N +7 , N +8 , N+9, and N+w, more preferably N +8 , N +9 , and N+w; and at least one nucleotide selected from N +28 and N +2 4.
  • the two or more nucleotides may include: at least one nucleotide selected from N +8 , N +7 , N +8 , N +9 , N+w, N+n, N+,I2 N+13, and N+14, preferably N +7 , N +8 , N +9 , N+w, N+n, N+I2, N+I 8 , and N+14, more preferably N +8 , N +9 , N+w, N+1i,N+I2 , N+13, and N+14; and at least one nucleotide selected from N +28 and N +2 4.
  • the two or more nucleotides may include: at least one nucleotide selected from N. 8 , N.s and N-4, preferably N. 8 ; at least one nucleotide selected from N +8 , N +7 , N +8 , N +9 , and N+w, preferably N +7 , N +8 , N+9, and N+w, more preferably N +8 , N +9 , and N+w; and at least one nucleotide selected from N +28 and N +2 4.
  • the two or more nucleotides may include: at least one nucleotide selected from N. 8 , N.s and N-4, preferably N. 8 ; at least one nucleotide selected from N +8 , N +7 , N +8 , N +9 , N+w, N+n, N+,I2 N+13, and N+14, preferably N +7 , N +8 , N +9 , N+w, N+n, N+I2, N+13, and N+14, more preferably N +8 , N +9 , N+w, N+1i,N+I2 , N+13, and N+14; and at least one nucleotide selected from N +28 and N +2 4.
  • each nucleotide may have a different modification.
  • the at least one nucleotide selected from N.6, N.s, N.4, N+6, N+7, N+s, N+g, N+w and where present N+n, N+12 , N+w, N+14, N+23, and N+24 may be independently chemically modified with a moiety that is positively charged under physiological conditions at the 2’ position of its sugar residue.
  • one or two modifications can occur at the 2’ position of a nucleotide, carbon being capable of forming 4 bonds.
  • the modification involves a single modification at the 2’ position, i.e., the introduction of a single moiety/group as a modification at this position of the sugar residue.
  • the modification may be independently selected from -NH2 (2’-amino), -RNH2 where R is Ciwalkylene, and the following modifications (I), (II), (III), and (IV):
  • X is selected from N(R X )2 or S-CH3; each L is independently a linker group selected from a direct bond and Ci-salkylene, each R1 is independently selected from H, N(R X )2, a monocyclic 5- to 7- membered heterocyclyl group, a monocyclic 5- or 6-membered heteroaryl group, and NHC(NH)NH 2 ; wherein when R1 is a monocyclic 5- to 7- membered heterocyclyl group, or a monocyclic 5- or 6-membered heteroaryl group one or more of the ring carbon atoms may be optionally substituted with one or more groups selected from halo, Ci-4alkyl, NH 2 and OH;
  • R 2 is N(R X ) 2 H; n is 1 to 5;
  • R3 is selected from N(R X )2H + and a 5- or 6-membered heteroaryl optionally substituted with -L-R1; wherein when R 3 is a monocyclic 5- or 6-membered heteroaryl group optionally substituted with -L-Ri, one or more of the ring carbon atoms may be optionally substituted with one or more groups selected from halo, Ci-4alkyl, NH2 and OH;
  • R 4 is -CH(L-N(R X ) 2 ) 2 ; and each R x is independently selected from H and Ci ⁇ alkyl.
  • the wave line of formulae (I) to (IV) denotes the attachment of the modification to the 2’ position of the sugar residue of the nucleotide in question.
  • R is preferably a straight chain alkylene.
  • R is Ci-4alkylene, such as Ci - salkylene. More preferably, when the modification is -RNH 2 , the modification is - CH 2 CH 2 CH 2 NH 2 (propylamino).
  • the modification of the 2’ position of the sugar residue is preferably selected from -NH 2 (2’-amino), and -RNH 2 , where R is Ci-ealkylene.
  • R is a straight chain alkylene. More preferably, the modification is selected from -NH 2 (2’-amino), and -RNH 2 where R is Ci-4alkylene, such as -CH 2 CH 2 CH 2 NH 2 (propylamino).
  • each instance of the group R x is preferably, independently H or methyl. Most preferably, each instance of R x is H.
  • the group N(R X ) 2 may be -N(CH3)2, -N(CH3)H, or -NH 2 , any of which may be in their protonated form, i.e. - NH(CH 3 )2 + , -N(CH3)H 2 + , or -NH3 + , depending on the conditions.
  • a counterion such as an anion
  • the counterion may be any suitable anion, for example, iodide or chloride.
  • each L is preferably selected from a direct bond and Ci-4alkylene.
  • each L may be Ci-3alkylene, e.g. unbranched Ci - salkylene.
  • each L is preferably selected from a direct bond and Ci - salkylene.
  • L is unbranched.
  • the monocyclic 5- to 7- membered heterocyclyl group of Ri is preferably a nitrogen-containing 5- to 7- membered heterocyclyl group. More preferably, the monocyclic 5- to 7- membered heterocyclyl group of Ri is a nitrogen - containing 5- or 6-membered heterocyclyl group, for example selected from piperidine, pyrrolidine, pyrrolidone, morpholine or piperazine. More preferably, the monocyclic 5- to 7- membered heterocyclyl group of Ri is piperidine.
  • the monocyclic 5- or 6-membered heteroaryl group of Ri is preferably a nitrogen-containing 5- or 6-membered heteroaryl group, for example selected from pyrrole, imidazole, triazole, tetrazole, pyrazole, pyridine, pyrimidine, pyridazine, pyrazine, and triazine. More preferably, the monocyclic 5- or 6-membered heteroaryl group of Ri is selected from pyridine and imidazole.
  • the 5- or 6-membered heteroaryl of Rs is preferably a nitrogen - containing 5-membered heteroaryl group, for example selected from pyrrole, imidazole, triazole, tetrazole, and pyrazole. More preferably, the monocyclic 5- or 6- membered heteroaryl group of Ri is triazole.
  • the 5-membered heteroaryl of Rs is preferably substituted with -L-Ri.
  • Ri is preferably selected from, N(R x )s, a monocyclic 5- to 7- membered heterocyclyl group, a monocyclic 5- or 6-membered heteroaryl group, and NHC(NH)NH2, and more preferably R x at each instance is H.
  • n is preferably 1 to 3. In some embodiments, n is 1 or 3.
  • one or more of the ring carbon atoms of the monocyclic 5- to 7- membered heterocyclyl group of Ri, the monocyclic 5- or 6-membered heteroaryl group of Ri, and the 5- or 6-membered heteroaryl of R 3 may be optionally substituted with one or more groups selected from halo (preferably fluoro or chloro), Ci-4alkyl,
  • NH2 and OH preferably said optional substituents are selected from fluoro, chloro, methyl, ethyl and NH2.
  • substituents on a given Ri or R3 group preferably one.
  • the modification may be selected from the following (Ila) to (He):
  • L is a direct bond.
  • L is Ci-salkylene, more preferably Ci-4alkylene.
  • the modification may be selected from the following (Illa) to (lllf):
  • L is a direct bond.
  • L is Ci-salkylene, more preferably Ci-4alkylene, most preferably Ci-3alkylene.
  • n is 1 to 5, more preferably 1 to 3.
  • each L is independently selected from a direct bond and Ci-4alkylene.
  • each nucleotide may have a different modification detailed above at the 2’ position that is positively charged under physiological conditions.
  • the at least one nucleotide selected from N.6, N.s, N-4, N+6, N+7, N+s, N+9, N+10 and where present N+n, N+12, N+13, N+14, N+23, and N+24 may be independently chemically modified with a moiety that is positively charged under physiological conditions at an internucleoside linkage linking the nucleotide with an adjacent nucleotide.
  • an internucleoside linkage links the 3’ position of the sugar residue of one nucleotide with the 5’ position of the sugar residue of the next nucleotide.
  • a nucleotide may thus have two internucleoside linkages associated therewith, one associated with the 3’ position of its sugar residue and the other with the 5- position of its sugar residue.
  • the independent chemical modification of the at least one nucleotide selected from N-6, N.s, N-4, N+6, N+7, N+s, N+9, N+10 and where present N+n, N+12, N+ 13 , N+14, N+23, and N+24 at the internucleoside linkage linking the nucleotide with an adjacent nucleotide is preferably the independent chemical modification of the internucleoside linkage associated with the 3’ position of the sugar residue of the at least one nucleotide selected from N-6, N.s, N-4, N+6, N+7, N+s, N+9, N+10 and where present N+n, N+12, N+ 13 , N+14, N+ 23 , and N+24.
  • the modification of the internucleoside linkage of the at least one nucleotide selected from N-6, N-5, N-4, N+6, N+7, N+S, N+g, N+W and where present N+n, N+12, N+13, N+14, N+23, and N+24 linking the nucleotide with an adjacent nucleotide with a moiety that is positively charged under physiological conditions may be the modification of a phosphodiester (PO) or phosphorothioate (PS) linkage.
  • PO phosphodiester
  • PS phosphorothioate
  • the modification of the internucleoside linkage of the at least one nucleotide selected from N-6, N-5, N-4, N+6, N+7, N+s, N+9, N+10 and where present N+n, N+12, N+13, N+14, N+23, and N+24 linking the nucleotide with a moiety that is positively charged under physiological conditions with an adjacent nucleotide is selected from the following modifications (V), (VI) and (VII):
  • Z is selected from (-OCi-4alkyl-) n , 0, S, and NH, where n is from 1 to 5;
  • L is a linker group selected from a direct bond or Ci-salkylene
  • R 5 is selected from N(R X ) 2 H + , N(R X )3 + , a monocyclic 5- to 7- membered heterocyclyl group, a monocyclic 5- or 6-membered heteroaryl group, NHC(NH)NH 2 , and -CH(L- NH 2 ) 2 ; wherein when R 5 is a monocyclic 5- to 7- membered heterocyclyl group or a monocyclic 5- or 6-membered heteroaryl group, one or more of the ring carbon atoms may be optionally substituted with one or more groups selected from halo, Ci-4alkyl, NH 2 and OH; and each R x is independently selected from H and Ci-4alkyl.
  • the wave line of formulae (V) to (VII) denotes the attachment of the internucleoside linkage modification to the 3’ position of the sugar residue of one nucleotide and the attachment of the internucleoside linkage modification to the 5’ position of the sugar residue of the adjacent nucleotide.
  • each instance of the group R x is preferably, independently H or methyl. Most preferably, each instance of R x is H.
  • the group N(R X ) 2 may be -N(CH3) 2 , -N(CH3)H, or -NH 2 , any of which may be in their protonated form, i.e. -NH(CH3) 2 + , -N(CH3)H 2 + , or -NH3 + , depending on the conditions.
  • a counterion such as an anion
  • the counterion may be any suitable anion, such as iodide or chloride.
  • L is Ci-salkylene, more preferably Ci-3alkylene.
  • L is unbranched.
  • the monocyclic 5- to 7- membered heterocyclyl group of Rs is preferably a nitrogen-containing 5- to 7- membered heterocyclyl group. More preferably, the monocyclic 5- to 7- membered heterocyclyl group of Rs is a nitrogen - containing 5- or 6-membered heterocyclyl group, for example selected from piperidine, pyrrolidine, pyrrolidone, morpholine or piperazine. More preferably, the monocyclic 5- to 7- membered heterocyclyl group of Rs is piperazine.
  • the monocyclic 5- or 6-membered heteroaryl group of Rs is preferably a nitrogen-containing 5- or 6-membered heteroaryl group, for example selected from pyrrole, imidazole, triazole, tetrazole, pyrazole, pyridine, pyrimidine, pyridazine, pyrazine, and triazine. More preferably, the monocyclic 5- or 6-membered heteroaryl group of Ri is selected from pyridine and imidazole.
  • one or more of the ring carbon atoms of the monocyclic 5- to 7- membered heterocyclyl group or the monocyclic 5- or 6-membered heteroaryl group of Rs may be optionally substituted with one or more groups selected from halo (preferably fluoro or chloro), Ci-4alkyl, NH2 and OH, preferably said optional substituents are selected from fluoro, chloro, methyl, ethyl and NH2. In some embodiments, if present, there may be one or two, such substituents on a given Rs group, preferably one.
  • Rs is N(CH3)3 + , or NHs + , preferably NHs + .
  • the modification may be selected from the following:
  • L is Ci-salkylene. More preferably, L is Ci-3alkylene.
  • n 1 to 3.
  • the modification is preferably (Via):
  • L is Ci-salkylene. More preferably, L is Ci-3alkylene.
  • the modification is preferably (Vila) or (Vllb):
  • L is Ci-salkylene. More preferably, L is Ci-3alkylene.
  • L is Ci-salkylene.
  • the modification is more preferably (Vila).
  • nucleotide selected from N.6, N.s, N.4, N+6, N+7, N+8, N+9, N+IO and where present N+n, N+12, N+13, N+14, N+23, and N+24 is independently modified in the above-mentioned way at the internucleoside linkage linking the nucleotide with an adjacent nucleotide with a positively charged moiety at physiological conditions, the modification of each nucleotide is made independently.
  • each nucleotide may have a different internucleoside linkage modification as outlined herein.
  • the at least one nucleotide selected from N.6, N.s, N-4, N+6, N+7, N+s, N+9, N+10 and where present N+n, N+12, N+13, N+14, N+23, and N+24 may be independently chemically modified at the 2’ position of its sugar residue and/or at the internucleoside linkage linking the nucleotide with an adjacent nucleotide, with a moiety that is positively charged under physiological conditions.
  • the same nucleotide (at least one nucleotide selected from N_ 6 , N_ 5 , N. 4 , N +6 , N +7 , N+ 8 , N+9, N + 10 and where present N+n,N+I2 , N+1 3 , N+ 14 , N +2 3, and N +24 ) may be independently chemically modified at the 2’ position of its sugar residue and at the internucleoside linkage linking the nucleotide with an adjacent nucleotide, with a moiety that is positively charged under physiological conditions. This may be the case for more than one of the nucleotides selected from N.6, N.s, N. 4 , N+6, N+ 7 , N+s, N+9, N+w and where present N+n,N+I2 , N+13, N+ 14 , N+ 23 , and N+ 24 .
  • the modification(s) is independent to the modification(s) made to the other of the nucleotides selected from N.6, N.s, N. 4 , N+6, N+ 7 , N+s, N+9, N+10 and where present N+n, N+I 2 , N+13, N+ 14 , N+ 23 , and N+ 24 .
  • the nucleotides selected from N.6, N.s, N. 4 , N+6, N+ 7 , N+S, N+9, N+10 and where present N+n,N+I2 , N+13, N+ 14 , N+ 23 , and N+ 24 is independent to the modification(s) made to the other of the nucleotides selected from N.6, N.s, N. 4 , N+6, N+ 7 , N+s, N+9, N+10 and where present N+n, N+I 2 , N+13, N+ 14 , N+ 23 , and N+ 24 .
  • the nucleotide may be independently chemically modified at the 2’ position of its sugar residue and/or at the internucleoside linkage linking the nucleotide with an adjacent nucleotide, with a moiety that is positively charged under physiological conditions. Any number of the nucleotides selected from N.6, N.s, N.
  • N.6 may be modified at the 2’ position of its sugar residue with a moiety that is positively charged under physiological conditions
  • N. 4 may be modified with a moiety that is positively charged under physiological conditions at the internucleoside linkage linking it with an adjacent nucleotide
  • Ns may be modified with a moiety that is positively charged under physiological conditions at both the 2’ position on its sugar residue and the internucleoside linkage linking it with an adjacent nucleotide.
  • N_ 6 , N_ 5 , N-4, N+6, N+7, N+8, N+9, N+IO and where present N+n, N+I2, N+ 13 , N+ 14 , N +2 3, and N +24 is independently modified in the above-mentioned way at both or one of the 2’ position of its sugar residue and/or the internucleoside linkage linking the nucleotide with an adjacent nucleotide, with a moiety that is positively charged under physiological conditions, the modification of each nucleotide is made independently. Accordingly, if two or more of the nucleotides selected from N.6, N.s, N.
  • each nucleotide may have a different modification(s). All combinations of modifications of the individual nucleotides selected from N.6, N.s, N. 4 , N+6, N+ 7 , N+s, N+9, N+10 and where present N+n, N+12, N+13, N+14, N+23, and N+ 24 are contemplated.
  • any of the nucleotides selected from N.6, N.s, N. 4 , N+6, N+ 7 , N+s, N+9, N+10 and where present N+n, N+I2 , N+ 13 , N+ 14 , N+ 23 , and N+ 24 of the chemically modified oligonucleotide of the present invention that are not modified with a moiety that is positively charged under physiological conditions, may be unmodified nucleotides, or may be modified nucleotides, independently chemically modified with alternative modifications as discussed in more detail below.
  • nucleotide may be modified at the other of those (i.e. the internucleoside linkage or the 2’ position of its sugar residue) with the alternative modifications discussed in more detail below.
  • the chemically modified oligonucleotide of the present invention is for use in the site-directed A-to-l editing of a target RNA inside a cell with endogenous adenosine deaminase acting on RNA (ADAR).
  • ADAR endogenous adenosine deaminase acting on RNA
  • the ADAR is ADARI .
  • the other nucleotides of the chemically modified oligonucleotide of the present invention may be modified as outlined further below. Further, as noted above, for any of the nucleotides selected from N-6, N-5, N.
  • nucleotide may be modified as outlined further below. Still further, as noted above, for any of the nucleotides selected from N. 8 , N.s, N.
  • nucleotide may be modified as outlined further below. All combinations of modifications of individual nucleotides are contemplated by the present invention.
  • the chemically modified oligonucleotides of the present invention may comprise at least one nucleotide of the CBT modified at the 2’-position of the sugar base or being deoxyribonucleosides, which permits added stabilization against nuclease digestion.
  • the CBT may be chemically modified.
  • the CBT (3’...- N-1-N0-N+1 -...5’) may carry different modifications and permutations of the various modifications. That is, positions N+1, No and/or N-1 may carry modifications at the 2’ position. Only one position within the CBT may be chemically modified, or two positions within the CBT may be chemically modified, or all positions within the CBT may be chemically modified.
  • At least one of the three oligonucleotides of the CBT may be a deoxyribonucleotide. At least one of the three oligonucleotides may be 2’-FANA-modified. At least one of the three oligonucleotides may be 2’-O-methyl-modified. At least one of the three oligonucleotides may be 2’-F-modified. At least one of the three nucleotides of the CBT may be chemically modified at the 2'-position of the sugar residue, a deoxyribonucleoside, or a combination thereof.
  • the chemical modification at the 2’ position may be one or more of the following: (i) N+1 is 2’-fluoro (2’-F), 2’- fluoroarabinoside (2’-FANA), deoxyribonucleic acid (DNA) (i.e. the sugar residue of N+1 is deoxyribose), or 2'-O-Methyl (2’-OMe); and/or (ii) N o is 2'-FANA or DNA; and/or (iii) N-i is 2'-FANA, DNA or 2’-OMe. N.i may be 2’-OMe.
  • At least two of the three nucleotides of the CBT may be chemically modified at the 2'-position of the sugar residue, a deoxyribonucleoside, or a combination thereof.
  • N+1 may be 2'-F, 2’-FANA, DNA, or 2’-OMe; and/or No may be 2'-FANA or DNA; and/or N.i may be 2'-FANA, DNA, or 2’-O-methyl.
  • N+1 may be DNA.
  • N+1 may be 2’-F.
  • N+1 may be 2’-FANA.
  • No may be 2'-FANA.
  • No may be DNA.
  • N.i may be 2'-FANA.
  • N. i may be DNA.
  • Each of the three nucleosides of the CBT may be either singularly or a combination of:
  • the middle or centre nucleotide (N o ) of the CBT may not comprise a 2’-sugar modification.
  • N o may not comprise a 2’-alkyl modification.
  • N o may not comprise a 2’- OMe modification.
  • the chemically modified oligonucleotide of the present invention may comprise various amounts and combinations of 2’-sugar modifications.
  • the chemically modified oligonucleotides benefit from having at least one nucleotide selected from N.6, N.s, N.4, N+6, N+7, N+s, N+g, N+w and where present N+n, N+12, N+13, N+14, N+23, and N+24 independently chemically modified with a moiety that is positively charged under physiological conditions, and internucleoside linkage modifications and modified 2’-positions of the nucleotides.
  • the chemically modified oligonucleotide of the present invention may comprise modifications at the 2’-position of the nucleotides and these modifications are composed of different groups.
  • the chemically modified oligonucleotide may comprise a mixture of 2’-O-alkyl, 2’-F, 2’- MOE, 2'-FANA and/or LNA modifications.
  • the oligonucleotides may comprise any permutation of these 2’-sugar modifications.
  • a 2’-sugar modification may be a 2’-O-alkyl modification, such as an 2’-OMe, 2’-O- ethyl, or 2’-O-propyl modification.
  • a 2’-sugar modification may be a 2 -MOE modification.
  • a 2'-sugar modification may be 2'-OR, wherein R is substituted C1-10 aliphatic.
  • a 2’-sugar modification may be 2’-F or 2’-FANA.
  • a mixture of 2’-F- and 2’-O-alkyl-modifications may be beneficial to editing and preferably, a minimum of 10% of each is desirable.
  • the chemically modified oligonucleotide of the present invention may comprise a mixture of 2’-F- and 2’-O- alkyl-modifications and a minimum of 15% of each 2’-F- and 2’-O-alkyl-modifications.
  • the chemically modified oligonucleotide may comprise a mixture of 2’-F- and 2’-O- alkyl-modifications and a minimum of 20% of each 2’-F- and 2’-O-alkyl-modifications.
  • the chemically modified oligonucleotide may comprise a mixture of 2’-F- and 2’-O- alkyl-modifications and a combined minimum of 15%-20%, 20-30%, 30%-40%, 40- 50% or 40-60% of 2’-F- and 2’-O-alkyl-modifications.
  • the chemically modified oligonucleotide of the present invention may comprise at least 10% of 2’-F, 2’-OMe, 2’-MOE and/or 2'-FANA modifications.
  • the chemically modified oligonucleotide may comprise at least 15%, 20%, 25%, 30%, 35%, 40% of 2’-F, 2’-OMe, 2’-MOE or 2'-FANA modifications.
  • the chemically modified oligonucleotide may comprise at least 15%, 20%, 25%, 30%, 35%, 40% of 2’-F, 2’- OMe, 2’-MOE and 2'-FANA modifications.
  • the chemically modified oligonucleotide of the present invention may comprise internucleoside linkages such as PS or PN linkages.
  • the chemically modified oligonucleotide may further comprise one or more internucleoside linkages selected from the group consisting of phosphoryl guanidine (PN), phosphodiester (PO) and phosphorothioate (PS).
  • PN phosphoryl guanidine
  • PO phosphodiester
  • PS phosphorothioate
  • the internucleoside linkage may be a PN linkage, optionally wherein the PN linkage is located within the 3’ and/or 5’ flanking region of the chemically modified oligonucleotide.
  • the chemically modified oligonucleotides of the present invention may comprise RNA and/or DNA. Also, apart from any modification made with a moiety that is positively charged under physiological conditions at the 2’ position of the sugar residue at any of the nucleotides selected from N.6, N.s, N.4, N+6, N+7, N+ 8 , N+g, N+w and where present N+1i, N+w, N+W, N+14, N+23, and N+24, all of the other nucleotides, including any of the nucleotides selected from N.6, N.s, N.4, N+6, N+7, N+s, N+g, N+w and where present N+n, N+w, N+W, N+14, N+23, and N+24 not modified with a moiety that is positively charged under physiological conditions at the 2’ position of the sugar residue, of the chemically modified oligonucleotide of the present invention, may be otherwise modified at the 2’-position of the sugar residue.
  • nucleotides including any of the nucleotides selected from N_ 6 , N_ 5 , N_ 4 , N+ 6 , N+ 7 , N+ 8 , N+g, N+w and where present N+n, N+w, N+W, N+14, N+23, and N+24 not modified with a moiety that is positively charged under physiological conditions at the 2’ position of the sugar residue
  • 20-100%, 30- 100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, or 90-100% may be DNA or 2’-modified.
  • nucleotides including any of the nucleotides selected from N_ 6 , N_ 5 , N_ 4 , N+ 6 , N+ 7 , N+ 8 , N+g, N+w and where present N+n, N+w, N+w, N+14, N+23, and N+24 not modified with a moiety that is positively charged under physiological conditions at the 2’ position of the sugar residue
  • nucleotides including any of the nucleotides selected from N_ 6 , N_ 5 , N_ 4 , N+ 6 , N+ 7 , N+ 8 , N+g, N+w and where present N+n, N+w, N+w, N+14, N+23, and N+24 not modified with a moiety that is positively charged under physiological conditions at the 2’ position of the sugar residue
  • no more than 4 nucleotides outside the CBT are deoxynucleotides. In one embodiment, no more than 3 nucleotides outside the CBT are deoxynucleotides. In one embodiment, no more than 5 nucleotides outside the CBT are deoxynucleotides. In one embodiment, no more than 6 nucleotides outside the CBT are deoxynucleotides. In some embodiment, no more than 7 nucleotides outside the CBT are deoxynucleotides.
  • the chemically modified oligonucleotides of the present invention may specifically comprise 2’-F and/or 2’-OMe modifications.
  • the chemically modified oligonucleotide may comprise one or more 2’-F modifications.
  • no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of nucleotides are 2’-F-modified.
  • At least 5%, 10%, 20%, 30%, 40%, 50%, or 60% of nucleotides may be 2’-F-modified.
  • no more than 35% of nucleotides are 2’-F modified.
  • 30-60% of nucleotides may be 2’-F-modified. 20-70%, preferably 30-45%, of nucleotides may be 2’-F-modified. 35-65% of nucleotides may be 2’-F-modified.
  • the chemically modified oligonucleotides of the present invention may also comprise 2’-O-methyl (2’-OMe) modifications.
  • the chemically modified oligonucleotides may comprise one or more 2’-OMe modifications. In one embodiment, no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of nucleotides are 2’-OMe-modified. 20- 60% of nucleotides may be 2’-OMe-modified. 5-55%, preferably 25-55% of nucleotides may be 2’-OMe-modified.
  • the chemically modified oligonucleotide of the present invention may comprise one or more internucleoside linkages selected from the group consisting of phosphoryl guanidine (PN), phosphodiester (PO), methanesulfonyl (mesyl) and phosphorothioate (PS).
  • the internucleoside linkage modification may be a 3’-3’ or 5’-5’ phosphate ester bonds (3 -P-3' and 5 -P-5').
  • the natural 3’-5’ phosphodiester linkage may be replaced by modified internucleoside linkages.
  • the naturally occurring one or more PO linkages may be replaced by modified internucleoside linkages in order to introduce one or more PS linkages or non-phosphorus derived internucleoside linkages.
  • An internucleoside linkage may be a PS linkage.
  • An internucleoside linkage may be a stereorandom PS linkage.
  • An internucleoside linkage may be a chirally controlled PS linkage.
  • An internucleoside linkage may be a PN linkage.
  • An internucleoside linkage may not be a chirally controlled PS linkage.
  • the chemically modified oligonucleotide of the present invention comprises the following sequence:
  • CBT Central Base Triplet
  • nucleotide of formula I is flanked at the 5'-end (adjacent to nucleotide +10) and at the 3'-end (adjacent to nucleotide -6) with further oligonucleotide sequences, which may have either the same length or different lengths.
  • linkage a may be a PS linkage.
  • Linkages d and e may be PS linkage modifications, optionally wherein f is an internucleoside linkage modification.
  • Linkages d and e may be PS linkage modifications.
  • Linkage f may be a PS linkage. At least linkages d and e may be modified.
  • Linkages d and e may be phosphorothioate (PS) linkages, and/or at least two linkages may be phosphate (PO) linkages, and/or linkage h may not be a PS linkage, and/or linkages f and j may be a PS linkage, and/or linkage b may be a PO or a PS linkage.
  • linkage g is a mesyl linkage and/or linkage a is a mesyl linkage.
  • Linkage a may be a mesyl or PS linkage.
  • Linkages d and e may be PS linkage modifications, optionally wherein linkage f is an internucleoside linkage modification.
  • Linkages d and e may be PS linkage modifications.
  • Linkage f may be a PS linkage.
  • the inventors have found that placement of PO linkages at specific positions within the oligonucleotide stabilise the oligonucleotide and contributes to enhanced target editing.
  • linkages b and h may be PO linkages.
  • Linkage h may not be chemically modified, i.e., linkage h may be a PO.
  • Linkage h may be a PO linkage.
  • Linkage i may be chemically modified.
  • Linkage i may be a PS linkage.
  • Linkage h may be a PO linkage and linkage i may be a PS linkage.
  • Linkage i may be a PS linkage.
  • Up to three linkages may be selected from the group consisting of linkages b, c, f, g and j are also PS linkages.
  • linkages a to j are PS linkages.
  • the linkage f is a PS linkage. More preferably, linkages d and e are PS linkages whereas linkage h is a PO linkage.
  • Linkage b may be a PO or a PS linkage.
  • Linkages b and h may be PO linkages.
  • linkage a is a mesyl linkage.
  • At least linkages a, d, and e may be PS linkages and at least 2 linkages may be phosphate (PO) linkages.
  • PS linkages should preferably be avoided at position h of the sequence. PS linkages at such positions were found to impair editing strongly.
  • linkages h and i are not PS linkages, optionally wherein h and i are PO linkages.
  • linkage f, j, g and/or c are/is a PS linkage(s).
  • linkage g is a phosphate (PO) linkage.
  • linkage g is a 3',5'-phosphodiester linkage.
  • linkage g is a mesyl linkage.
  • the chemically modified oligonucleotide of the present invention may comprise the following sequence:
  • linkages d and e are modified, optionally wherein (i) a, 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, optionally wherein h and i are PO linkages; and/or (iii) linkages f and j are a PS linkage, optionally wherein linkages f, j, g and/or c are/is a PS linkage; and/or (iv) linkage b is a PO or PS linkage.
  • linkage g is a mesyl linkage and/or linkage a is a mesyl linkage.
  • Chemically modified oligonucleotides of different lengths may require a different mixture of particular 2’-modifications and internucleoside linkage modifications in order to provide optimal RNA editing.
  • cytotoxicity of the particular chemically modified oligonucleotide may also depend on its length.
  • shorter chemically modified oligonucleotides may experience higher specificity.
  • longer chemically modified oligonucleotides may bind stronger or faster to their respective RNA target, editing-boosting bulges, mismatches and wobbles may also work better in long chemically modified oligonucleotides. As a result, there is a benefit and/or trade-off for both long and short chemically modified oligonucleotides of the invention.
  • the chemically modified oligonucleotides of the present invention may be of varying lengths.
  • the chemically modified oligonucleotides of the invention may be at least 25 nucleotides (N) long.
  • the oligonucleotides may range from about 25-80N in length, e.g., about 25-39N, about 40-60N or about 61-80N in length.
  • the chemically modified oligonucleotide may have 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 80N.
  • the chemically modified oligonucleotide may have a length of 40N, or 44 N or 45N.
  • the chemically modified oligonucleotide may have a length of 30, 32, 34, 36, or 38N.
  • the chemically modified oligonucleotide may have a length of 30-38N, such as a length of 30-34N or 36-38N.
  • the chemically modified oligonucleotide may have a length of 34-38N or 36-38N.
  • the chemically modified oligonucleotide may have a length of 25-80N, more preferably a length of 25-50N. In one embodiment, the oligonucleotide has a length of no more than 30, 31 , 32, 33, 34, 35, 36, 37, or 38N.
  • the oligonucleotide has a length of no more than 38, 39, 40, 41 , 42, 43, 44, or45N. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.
  • the chemically modified oligonucleotide of the present invention may have a length of at least 25 nucleotides (N), optionally 25 to 80 N, preferably 25 to 50 N, and more preferably 30 to 40 N.
  • nucleotides of the present invention may be fluoro (F)-modified at the 2’ position of the sugar residue, optionally wherein the 2’-F modification is at one or more of the following positions: 29, 28, 25, 23, 21 , 17, 15, 14, 13, 9, 7, 6, 5, 4, 3, 1 , -3, -6, -7, -8, - 10, -12, -13, -14, and -15.
  • the 2’-F modification may be at one or more of the following positions: 29, 28, 25, 23, 21 , 17, 15, 14, 13, 9, 7, 6, 5, 4, 3, 1 , -3, -6, -7, -8, -10, -12, - 13, -14, and -15.
  • the 2’-F modification may be at one or more of the following positions: 29, 28, 23, 21 , 15, 9, 7, 6, 5, 3, 1 , -10, -13, -14, and -15.
  • the 2’- F modification may be at one or more of the following positions: 28, 23, 21 , 9, 1 , -13 and -14.
  • about 10%-20%, 20%-30%, 30%-40%, or 50%-60% nucleotides may be F-modified at the 2’ position of the sugar residue.
  • the chemically modified oligonucleotide of the present invention may have a length of 30-40N and 4- 20 2’-F modifications.
  • the chemically modified oligonucleotide may have a length of 30-38N and 2-19 2’-F modifications.
  • the chemically modified oligonucleotide may have a length of 38N and 17 2’-F modifications.
  • the chemically modified oligonucleotide may have a length of 34N and 19 2’-F modifications.
  • the oligonucleotide may have a length of 33N and 19 2’-F modifications.
  • the chemically modified oligonucleotides of the present invention may be modified in a way to avoid such interference.
  • the chemically modified oligonucleotides may be modified such that they do not comprise continuous stretches or uniform blocks of nucleotides carrying the same chemical modification (/.e., avoidance of a block-like modification structure). Avoiding uniform blocks of more than 6 nucleotides with the same 2’-modification prevents a strong loss of editing activity with natural ADARs. Hence, the chemically modified oligonucleotide may not be uniformly modified.
  • the oligonucleotide contains no uniform blocks and/or no block-like modification structure. In one embodiment, the oligonucleotide does not comprise continuous stretches or uniform blocks of nucleotides carrying the same chemical modification at the 2’ position of the sugar moiety.
  • a “block” or “stretch” may, e.g., not comprise more than 4, 5 or 6 nucleotides with the same 2’-sugar modification. In some instances, the block or stretch may be shorter or longer.
  • the chemically modified oligonucleotide may contain only 1 block of no more than 6, 5, 4, or 3 nucleotides with the same 2’-sugar modification.
  • the chemically modified oligonucleotide may contain 2 blocks, separated by one or more nucleotides having a different 2’-sugar modification.
  • the chemically modified oligonucleotides may comprise at least 1 block of nucleotides with the same 2’-sugar modification.
  • the chemically modified oligonucleotide may comprise 1 , 2, 3, or more blocks of nucleotides with the same 2’-sugar modification.
  • the chemically modified oligonucleotides of the present invention may be modified to not include uniform blocks or a continuous stretch of the same 2’-sugar modification.
  • the chemically modified oligonucleotide may comprises one or more 2’-sugar modifications, optionally wherein no more than 6 consecutive nucleotides have the same 2’-modification. In one embodiment, no more than 5 consecutive nucleotides have the same modification. In one embodiment, no more than 4 consecutive nucleotides have the same modification.
  • the 2’-sugar modification may be 2’-deoxyribose (DNA).
  • no more than 6 consecutive nucleotides are 2’-H (DNA) modified.
  • no more than 5 consecutive nucleotides are 2’-H-modified.
  • no more than 4 consecutive nucleotides are 2’-H-modified.
  • the 2’-sugar modification may be 2’- ribose.
  • no more than 6 consecutive nucleotides are 2’-H (DNA) modified. In one embodiment, no more than 5 consecutive nucleotides are 2’-H- modified. In one embodiment, no more than 4 consecutive nucleotides are 2’-H- modified. In one embodiment, no more than 6 consecutive nucleotides are 2’-F- modified. In one embodiment, no more than 5 consecutive nucleotides are 2’-F- modified. In one embodiment, no more than 4 consecutive nucleotides are 2’-F- modified. In one embodiment, no more than 6 consecutive nucleotides are 2’-O-alkyl- modified. In one embodiment, no more than 5 consecutive nucleotides are 2’-O-alkyl- modified.
  • no more than 4 consecutive nucleotides are 2’-O-alkyl- modified, optionally wherein no more than 4 consecutive nucleotides are 2’-OMe- modified.
  • the chemically modified oligonucleotide may comprise 2, 3, 4, 5, or 6 consecutive nucleotides with the same 2’-modification, e.g., 5 consecutive nucleotides are 2’-F-modified.
  • the chemically modified oligonucleotide of the present invention may contain some “continuous stretch(es)” or “uniform block(s)” of a certain length.
  • the size or length of the “continuous stretch(es)” or “uniform block(s)” may be 2, 3, 4, 5, or 6 nucleotides long.
  • the size or length of the “continuous stretch(es)” or “uniform block(s)” may be no more than 2, 3, 4, 5, or 6 nucleotides long.
  • the chemically modified oligonucleotide may comprise no more than 2, 3, 4, 5, or 6 consecutive nucleotides comprising a 2’- F modification.
  • the oligonucleotide comprises no more than 2, 3, 4, 5, or 6 consecutive nucleotides comprising a 2’-OMe modification.
  • One or more uniform blocks may be interrupted. Interruption can take place by any other chemical modification (e.g., DNA, RNA, 2’-F, 2’-OMe, 2’-MOE, LNA, etc.).
  • One or more uniform blocks of 2’-F-modified nucleotides may be interrupted, preferably by 2'-OMe- modified nucleotides.
  • One or more uniform blocks of 2'-OMe-modified nucleotides may be interrupted, preferably by 2’-F-modified nucleotides. The blocks may be disrupted by DNA.
  • the targeting sequence of the artificial nucleic acid typically comprises a nucleic acid sequence complementary or at least partially complementary to a nucleic acid sequence in the target RNA.
  • the targeting sequence may comprise a nucleic acid sequence complementary or at least 60%, 70%, 80%, 90%, 95% or 99% of a nucleic acid sequence in the target RNA.
  • the chemically modified oligonucleotides of the present invention may be modified at their 5’ and/or 3’ termini.
  • the chemically modified oligonucleotides may comprise one or more different linkers, tags or coupling agents at either one or both termini.
  • the chemically modified oligonucleotides may comprise a moiety, which enhances cellular uptake of the oligonucleotide, e.g., N-acetylgalactosamine (GalNAc).
  • GalNAc N-acetylgalactosamine
  • the chemically modified oligonucleotide may comprise a moiety or be conjugated to a moiety that enhances cellular uptake of the oligonucleotide.
  • the moiety enhancing cellular uptake may be a triantennary N-acetyl galactosamine (GalNAc3), which is preferably conjugated to the 3' terminus or to the 5' terminus of the oligonucleotide.
  • GalNAc3 triantennary N-acetyl galactosamine
  • targeted delivery of chemically modified oligonucleotides to liver hepatocytes using bi- or triantennary N-acetylgalactosamine (GalNAc) conjugates has previously described for, e.g., treating liver diseases, including Hepatitis B virus (HBV), non-alcoholic Fatty Liver Disease and genetic diseases (Debacker et al., 2020).
  • the chemically modified oligonucleotides of the present invention show increased hydrophobicity, stability against degradation and an optimal chemical modification pattern to bind ADARs.
  • the chemically modified oligonucleotides of the present invention preferably do not require a loop-hairpin structured recruiting moiety specifically for recruiting a deaminase.
  • the chemically modified oligonucleotides of the present invention may or may not comprise a loop-hairpin structure.
  • the chemically modified oligonucleotides may not comprise a loop-hairpin structured recruiting moiety.
  • an internucleoside linkage is not chirally controlled.
  • an internucleoside linkage is not a chirally controlled PS linkage.
  • the oligonucleotide does not comprise independently controlled chiral phosphates.
  • one or more internucleoside linkage is not independently chirally controlled. In some embodiments, at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or all internucleoside linkages are not chiral internucleoside linkages.
  • the chemically modified oligonucleotides according to any of the embodiments of the present invention described above may further comprise internucleoside linkages selected from phosphoryl guanidine (PN), phosphodiester (PO), phosphorothioate (PS) and methanesulfonyl (mesyl) linkages.
  • internucleoside linkages selected from phosphoryl guanidine (PN), phosphodiester (PO), phosphorothioate (PS) and methanesulfonyl (mesyl) linkages.
  • the chemically modified oligonucleotide according to the above embodiment may comprise a 3’ and/or 5’ flanking region comprising PN internucleoside linkages located with the 3’ and/or 5’ flanking region. Also provided is a chemically modified oligonucleotide according to either of the previous embodiments, wherein:
  • a chemically modified oligonucleotide according to any previous embodiment, wherein at least 10%, preferably 20%, 30%, 40%, 50% or 60%, of the nucleotides of the chemically modified oligonucleotide are fluoro (F)-modified in the 2’ position of the sugar residue (2’-F), optionally wherein the 2’-F modification is at one or more of the following nucleotides: 29, 28, 25, 23, 21 , 17, 15, 14, 13, 9, 7, 6, 5, 4, 3, 1 , -3, -6, -7, -8, -10, -12, -13, -14, and -15.
  • a chemically modified oligonucleotide according to any previous embodiment wherein at least one of the three nucleotides N-i, No and N+1 of the CBT is independently selected to be chemically modified at the 2’ position of its sugar residue, or its sugar residue is a deoxyribose.
  • the chemical modification at the 2’ position of the sugar residue of the nucleotide N+1 is 2’fluoro (2’-F), 2’fluoroarabinoside (2’FANA), 2’-O- Methoxyethyl (2’-MOE) or 2’-O-Methyl (2’-OMe), or the sugar residue of N+1 is deoxyribose; and/or
  • N o comprises no 2’-modification of its sugar residue.
  • oligonucleotide according to any previous embodiment, wherein No is selected from cytidine, deoxycytidine, uridine, and deoxy uridine, preferably wherein No is selected from cytidine and deoxycytidine.
  • a chemically modified oligonucleotide according to any previous embodiment, wherein at least 50%, more preferably at least 80%, of the nucleotides outside the CBT are chemically modified independently from another at the 2’ position of their sugar residue, preferably wherein the modification is selected from 2’-F, 2’- FANA, 2’-O-alkyl such as 2’-OMe, 2’-O-methoxyethyl (2’-MOE), and/or locked nucleic acid (LNA).
  • the chemically modified oligonucleotide of the present invention may be incorporated into a composition.
  • composition comprising 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 chemically modified oligonucleotide comprising a sequence capable of binding to a target sequence in a target RNA, and a central base triplet (CBT) of three nucleotides (... N.i, No, N+1...) where No is the central nucleotide directly opposite to a target adenosine in the target RNA that is to be edited, wherein the chemically modified oligonucleotide comprises the following sequence:
  • composition may be a pharmaceutical composition.
  • composition and ‘pharmaceutical composition’ are used interchangeably.
  • the composition may contain one or more chemically modified oligonucleotides of the present invention.
  • present invention therefore provides for a composition comprising a plurality of chemically modified oligonucleotides, each of the present invention.
  • a pharmaceutical composition refers to a substance, or mixture of substances, suitable for administering to subject.
  • a pharmaceutical composition may comprise one or more active pharmaceutical agents (such as a chemically modified oligonucleotide of the present invention) and a sterile aqueous solution.
  • active pharmaceutical agents such as a chemically modified oligonucleotide of the present invention
  • sterile aqueous solution a sterile aqueous solution.
  • the composition of the present invention can be in any form that allows for the composition to be administered to a subject.
  • the compositions may be used in methods of treating and/or preventing a disorder or disease, such as a genetic disease or disorder.
  • composition of the present invention may comprise a chemically modified oligonucleotide of the present invention.
  • composition of the present invention may comprise a chemically modified oligonucleotide of the present invention in admixture with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier may be a saline solution. This can be isotonic or hypotonic.
  • composition of the present invention may be for veterinary and/or human administration.
  • composition of the present invention may be used in conjunction with one or more other therapies.
  • the amount of the chemically modified oligonucleotide of the present invention, or the composition of the present invention, which will be effective in the treatment and/or prevention of a disease or disorder, such as a genetic disease or genetic disorder, will depend on the nature of the disease or disorder, and may be determined by standard clinical techniques.
  • Exemplary doses for chemically modified oligonucleotides of the present invention range from about 10ng to 1g, 100ng to 100mg, 1 pg to 10mg, or 30- 300pg oligonucleotide, e.g., RNA, per patient.
  • the chemically modified oligonucleotide may be present at a concentration of 4nM to 100nM, optionally at 20nM or 25nM. Alternatively, the chemically modified oligonucleotide may be present at a concentration of 0.8nM, or at a concentration of 4nM.
  • the composition of the present invention may comprise diluents, additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol), and bulking substances (e.g., lactose, mannitol).
  • the diluents may be of various buffer content (e.g., Tris-HCI, acetate, phosphate), pH, and ionic strength.
  • the composition of the present invention may comprise, or be incorporated into, particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc.
  • compositions may influence the physical state, stability, rate of in vivo release, and/or rate of in vivo clearance of the chemically modified oligonucleotide of the present invention.
  • the composition of the present invention may be in liquid form or a dried powder, such as in lyophilized form.
  • composition of the present invention may comprise one or more salts, e.g., sodium chloride, calcium chloride, sodium phosphate, monosodium glutamate, and aluminium salts (e.g., aluminium hydroxide, aluminium phosphate), alum (potassium aluminium sulfate), or a mixture of such aluminium salts).
  • salts e.g., sodium chloride, calcium chloride, sodium phosphate, monosodium glutamate
  • aluminium salts e.g., aluminium hydroxide, aluminium phosphate), alum (potassium aluminium sulfate), or a mixture of such aluminium salts.
  • composition of the present invention can be included in a container, pack, or dispenser together with instructions for administration.
  • the present invention describes the use of chemically modified oligonucleotides of the present invention, and compositions of the present invention, in the medical setting, specifically, for site-directed editing of a target RNA (e.g., binding to the target RNA via the targeting sequence and by recruiting to the target site a deaminase).
  • the present invention describes chemically modified oligonucleotides of the present invention, and compositions of the present invention, for use in the treatment or prevention of a disorder or disease, such as a genetic disorder or disease, as well as methods for treating or preventing a disorder or disease, such as a genetic disorder or disease.
  • Site-directed editing may take place in vitro, in vivo or ex vivo.
  • a chemically modified oligonucleotide of the present invention or a composition of the present invention, for therapeutic use.
  • a chemically modified oligonucleotide of the present invention or a composition of the present invention, for use in the treatment or prevention of a disease or disorder.
  • a method of treating or preventing a disease or disorder in a subject in need thereof comprising administering to the subject an effective amount of a chemically modified oligonucleotide of the present invention, or a composition of the present invention.
  • an in vitro method for site- directed A-to-l editing of a target RNA comprising a step of contacting a target RNA with a chemically modified oligonucleotide of the invention or a composition of the invention.
  • a chemically modified oligonucleotide of the present invention, or a composition of the present invention may be used in the treatment and/or prevention of a disease or disorder, such as a genetic disease or genetic disorder.
  • the disease or disorder may be selected from liver, metabolic, neurodegenerative, and/or cardiac or cardiovascular diseases or disorders.
  • liver or metabolic disease or disorders and/or cardiac or cardiovascular diseases or disorders are selected from liver, metabolic, neurodegenerative, and/or cardiac or cardiovascular diseases or disorders.
  • the disease or disorder may be associated with a gain-of-function (GOF) or loss-of- function (LOF) mutation.
  • GAF gain-of-function
  • LEF loss-of- function
  • the disease or disorder is a genetic disease or genetic disorder.
  • the disease or disorder may comprise the SERPINA1 gene or an alpha-1 -antitrypsin deficiency (A1AD or AATD), optionally wherein the target protein is alpha-1 antitrypsin.
  • the disease or disorder comprises the SERPINA1 gene.
  • the disease or disorder comprises the SERPINA1 gene and the mutation is SERPINA1 E342K.
  • the genetic disease or genetic disorder is selected from liver or metabolic diseases and/or cardiac or cardiovascular diseases associated with a gain-of-function (GOF) or loss-of-function (LOF) mutation.
  • the genetic disorder or disease may be associated with a point mutation.
  • the SERPINA1 gene encodes serine protease inhibitor alpha-l antitrypsin (A1AT).
  • A1AT protects tissues from certain inflammatory enzymes, including neutrophil elastase.
  • neutrophil elastase including neutrophil elastase.
  • a deficiency in A1AT alpha 1 antitrypsin deficiency, A1 AD
  • A1 AD can lead to excessive break down of elastin in the lungs by neutrophil elastase.
  • the genetic disorder or genetic disease may be associated with a G-to-A mutation in the SERPINA1 gene.
  • the mutation may be selected from SERPINA1 E342K.
  • the genetic disease or genetic disorder may comprise the SERPINA1 gene or an alpha-1-antitrypsin deficiency (A1AD or AATD), optionally wherein the target protein is alpha-1 antitrypsin.
  • the mutation may be the PiZ mutation (a1 -antitrypsin deficiency).
  • the chemically modified oligonucleotide of the present invention, or the composition of the present invention may be administered, for example, orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions, or solutions, or parenterally, e.g., by parenteral injection.
  • Formulations suitable for parenteral administration may comprise sterile aqueous preparations of a chemically modified oligonucleotide of the present invention, or composition comprising such, which is approximately isotonic with the blood of the intended recipient.
  • the amount of chemically modified oligonucleotide or composition to be administered, the dosage and the dosing regimen can vary from cell type to cell type, the disease to be treated, the target population, the mode of administration (e.g., systemic versus local), the severity of disease and the acceptable level of side activity.
  • the amount of chemically modified oligonucleotide(s) administered in a composition of the present invention is dependent on, for example, the subject being treated, the subject's weight, and the manner of administration.
  • a chemically modified oligonucleotide of the present invention or composition of the present invention may be delivered as is, i.e., naked and/or in isolated form to a subject, through an organ, e.g., mucosa of the eye, or directly to a cell.
  • the chemically modified oligonucleotide is dissolved in a solution that is compatible with the delivery method.
  • Such delivery may be in vivo, in vitro or ex vivo.
  • a different administration route or delivery method may be selected depending on the disease or disorder that needs to be treated, or on the cell, tissue or part of the body that needs to be reached by the chemically modified oligonucleotide (e.g., in case of beneficial editing).
  • excipient or transfection reagent may be used in the delivery of the chemically modified oligonucleotide of the present invention, or the composition of the present invention, to a cell and/or into a cell (preferably a cell affected by a G-to-A mutation or that wherein “beneficial editing” is to be achieved as outlined herein).
  • excipients or transfection reagents capable of forming complexes, nanoparticles, micelles, vesicles and/or liposomes that deliver the chemically modified oligonucleotide or composition as defined herein, complexed or trapped in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art.
  • Suitable excipients or transfection reagents comprise polyethylenimine (PEI; ExGen500 (MBI Fermentas)), LipofectAMINETM 2000 (Invitrogen), lipofectinTM, or derivatives thereof, and/or viral capsid proteins that are capable of self-assembly into particles that can deliver each constituent as defined herein to a target cell.
  • PEI polyethylenimine
  • ExGen500 MBI Fermentas
  • LipofectAMINETM 2000 Invitrogen
  • lipofectinTM or derivatives thereof
  • viral capsid proteins that are capable of self-assembly into particles that can deliver each constituent as defined herein to a target cell.
  • the chemically modified oligonucleotides of the present invention may be linked to a moiety that enhances uptake of the ASO in cells.
  • moieties are cholesterols, carbohydrates, vitamins, biotin, lipids, phospholipids, cell-penetrating peptides including but not limited to antennapedia, TAT, transportan and positively charged amino acids such as oligoarginine, poly-arginine, oligolysine or polylysine, antigen-binding domains such as provided by an antibody, a Fab fragment, or a single chain antigen binding domain such as a cameloid single domain antigen-binding domain.
  • the ASO may be delivered using drug conjugates with antibodies, nanobodies, cell penetrating peptides and aptamers.
  • the chemically modified oligonucleotide may be conjugated to an antibody, preferably a Fab fragment.
  • the chemically modified oligonucleotide of the present invention, or composition of the present invention may be administered as a monotherapy or in combination with a different medicament, particularly a medicament suitable for the treatment or prevention of alpha-1-antitrypsin (A1AT) deficiency.
  • A1AT alpha-1-antitrypsin
  • the chemically modified oligonucleotides of the present invention or compositions of the present invention are surprisingly effective.
  • a change may be measured by an increase of a desired mRNA and/or protein level compared to a reference sample or condition. Additionally or alternatively, a change may be measured by an increase in the editing efficacy (%) mediated by the chemically modified oligonucleotide of the present invention or composition comprising the same. Additionally or alternatively, a change may be measured by an increase in stability of the chemically modified oligonucleotide of the present invention, or composition of the present invention.
  • a change may be measured in the levels of cytotoxicity, viability, apoptosis or immune activation. Additionally, a change may be detected by means of luminescence and/or gene expression. Toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). Data obtained from the cell culture assays or animal studies can be used in formulating a range of dosage for use in humans.
  • an in vitro method for site-directed A-to-l editing of a target RNA comprising a step of contacting a target RNA with the chemically modified oligonucleotide of the present invention, or the composition of the present invention.
  • the method may be for beneficial and/or compensatory RNA editing. That is, the method may be for targeting wildtype adenosines for beneficial editing or for targeting wildtype adenosines for compensatory editing.
  • compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be by inhalation (e.g., through nebulization), intranasally, orally, by injection or infusion, intravenously, subcutaneously, intra - dermally, intra-cranially, intramuscularly, intra-tracheally, intra-peritoneally, intra - rectally, by direct injection into a tumour, and the like. Administration may be in solid form, in the form of a powder, a pill, or in any other form compatible with pharmaceutical use in humans.
  • the chemically modified oligonucleotides of the present invention, or compositions of the present invention may be administered to various groups of subjects.
  • the subject may be in need of treatment.
  • the subject may not be in need of treatment (“beneficial editing”). That is, the subject receives the chemically modified oligonucleotide of the present invention, or composition of the present invention, to edit an RNA derived from a wildtype allele (not a mutated allele) in order to modulate the function of the wildtype protein in a useful way.
  • the chemically modified oligonucleotides of the present invention, or compositions of the present invention may be administered to a subject.
  • the subject may be a mammal, preferably a human.
  • the chemically modified oligonucleotide of the present invention, or composition of the present invention may be administered to a naive subject, i.e., a subject that does not have a disease or disorder, such as a genetic disease or disorder.
  • a chemically modified oligonucleotide of the present invention, or a composition of the present invention may be administered to a naive subject that is at risk of developing a disease or disorder, such as a genetic disease or disorder.
  • the chemically modified oligonucleotide of the present invention or composition of the present invention may be administered to a subject before symptoms manifest or symptoms become severe.
  • the chemically modified oligonucleotide of the present invention or composition of the present invention may be administered to a subject who has been diagnosed with a disease or disorder, such as a genetic disease or disorder.
  • the subject may be any individual at risk of developing a disease or disorder, such as a genetic disease or disorder, associated with a G-to-A mutation in genes.
  • the subject may suffer from a disease or disorder, such as a genetic disease or disorder, associated with a G-to-A mutation in genes.
  • the disease or disorder such as a genetic disease or disorder, may be associated with a G-to-A mutation in a subject.
  • the disease or disorder such as a genetic disease or disorder, may be a liver or metabolic disease or disorder and/or cardiac or cardiovascular disease or disorder associated with a gain-of-function (GOF) or loss-of-function (LOF) mutation, optionally wherein the disease or disorder comprises the SERPINA1 gene.
  • GAF gain-of-function
  • LEF loss-of-function
  • a chemically modified oligonucleotide of the present invention in the manufacture of a medicament for treating or preventing a disease or a disorder, such as a genetic disease or genetic disorder, associated with a G-to-A mutation.
  • kits or kit of parts comprising a chemically modified oligonucleotide of the present invention and/or a composition of the present invention.
  • the kit typically additionally comprises instructions for use.
  • the present invention also relates to methods for editing a target adenosine in a target nucleic acid.
  • the present invention provides methods of editing a SERPINA1 polynucleotide, e.g., a SERPINA1 polynucleotide comprising a single nucleotide polymorphism (SNP) associated with alpha I antitrypsin deficiency.
  • the target may be human beta actin (hACTB) or a variant thereof.
  • the in vitro method for site-directed A-to-l editing of a target RNA of the present invention may comprise, after the step of contacting, the following steps:
  • an in vitro method for deaminating at least one specific adenosine present in a target RNA sequence in a cell there is further provided an in vitro method for deaminating at least one specific adenosine present in a target RNA sequence in a cell, wherein the method comprises the steps of:
  • the method may comprise, after step (d), a step of identifying the presence of the inosine in the RNA sequence.
  • the editing reaction is preferably monitored or controlled by sequence analysis of the target RNA.
  • a chemically modified oligonucleotide of the present invention may be used in the diagnosis of a disease or disorder, such as a genetic disease or disorder.
  • a disease or disorder such as a genetic disease or disorder.
  • the disease or disorder is preferably selected from the group consisting of infectious diseases, tumour diseases, cardiovascular diseases, autoimmune diseases, allergies and neurological diseases or disorders.
  • the disorder or disease, such as a genetic disease or disorder may be associated with a G-to-A mutation.
  • the present invention may be used to make desired changes in a target sequence in a cell or a subject by site-directed editing of nucleotides using an oligonucleotide that is capable of effecting an adenosine deaminase acting on RNA (ADAR)-mediated adenosine to inosine.
  • ADAR adenosine deaminase acting on RNA
  • the target sequence is edited through an adenosine deamination reaction mediated by ADAR, converting adenosines into inosine.
  • I is recognized as G
  • the deamination correcting the pathogenic mutation in the SERPINA1 gene reverses the E342K mutation back to wild-type, reversing or slowing symptoms associated with A1AD experienced by the subject.
  • the methods of the present invention for example the in vitro method for site-directed A-to-l editing of a target RNA, can be used with cells from any organ, e.g., skin, lung, heart, kidney, liver, pancreas, gut, muscle, gland, eye, brain, blood and the like.
  • the present invention is particularly suitable for modifying sequences in cells, tissues or organs implicated in a diseased state of a (human) subject.
  • such cells may include, but are not limited, to hepatocytes, hepatocyte like cells, and/or alveolar type II cells, neurons (PNS, CNS), retina, photo receptors cells, Muller Glia cells, RPE, immune cells, B cells, T cells, dendritic cells, macrophages.
  • PNS neurotrophic factor
  • CNS corthelial growth factor receptors
  • the electrostatic potential surface of the double stranded RNA-specific adenosine deaminase ADAR1 was created with PyMOL (The PyMOL Molecular Graphics System, Version 2.5.4 Schrodinger, LLC). The calculations were based on the alphafold structure prediction model AF P55265 F1. Conformational corrections of this model were made using the program Coot (Emsley P, Cowtan K (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr. D60, 2126-2132; version 0.9.8.1) and the map coefficients of PDB entry 8E4X. The sequence of ADAR1 was n terminally truncated to the amino acid region 711-1226 which contains the deaminase domain and a single double-strand binding domain.
  • the modelled nucleic acid in complex with ADAR1 was adopted from PDB entry 8e4x and positioned based on the used 2fo-fc electron density map.
  • nucleotides in oligonucleotide sequences were modified according to the present invention in order to provide positive charges, and the interaction of these positive charges with ADAR1 was modelled. The results are demonstrated in Fig. 3A, B, and C.
  • Fig. 3A shows a modified internucleoside linkage between position +9 and +10, modified according to formula (Vc) described herein, wherein L is 3.
  • Fig. 3B shows a nucleotide having a 2’ modification at position -4, modified according to formula (lllf) described herein, wherein n is 1.
  • Fig. 3C shows a modified internucleoside linkage between position -4 and -5, modified according to formula (Ve) described herein, wherein L is 2.
  • Fig. 3D shows a nucleotide having a 2’ modification (-RNH 2 ) at position +6, where R is -CH2CH2CH2-.
  • the modification is a 2’-propylamino (-CH2CH2CH2NH2) modification.
  • Example oligonucleotides were synthesised using the following protocols. Further oligonucleotides of the present invention may be synthesised by analogy to the methods set out below.
  • Oligonucleotides were synthesized on a MerMade48 oligonucleotide synthesizer, by means of standard solid-phase oligonucleotide synthesis using the following reagents: 3% dichloroacetic acid in DCM for deblocking, 0.25 M ETT in acetonitrile as activator for amidite couplings, 20% acetic anhydride in THF and 10% 1- methylimidazole in THF/pyridine for capping, 0.02M iodine in THF/water/pyridine for oxidation and 0.1 M xanthane hydride in pyridine:acetonitrile 1 :1 (v:v) for thiolation. Syntheses were carried out in a DMT-ON mode, on a 200 nmol scale and using 1000A CPG supports from Glen Research: either standard or universal (loading of ca. 30 pmol/g).
  • the phosphoramidate linkages were obtained via Staudinger reaction, which was carried out with 0.5 M solution of an appropriate azide in dry acetonitrile or methyl- tertbutyl ether (MTBE) for 15 min at ambient temperature.
  • MTBE methyl- tertbutyl ether
  • mesyl phosphoramidate linkages mesyl azide (Aurum Pharmatech) was used while for guanidine phosphoramidate linkages 2-azido-1 ,3- dimethylimidazolinium hexafluorophosphate (abcr GmbH) was used.
  • oligonucleotides were cleaved from CPG and deprotected at room temperature in 28%-30% ammonium hydroxide and/or 50%/50% mixture of 28%- 30% ammonium hydroxide/40% aqueous methylamine (AMA) for 36 hours or 2 h, respectively.
  • Deprotected oligonucleotides were directly adsorbed on GlenPak cartridges and purified DMT-ON.
  • oligonucleotides were dried down, desalted by precipitation from NaCI/ethanol, quantified by means of UV-Vis spectrophotometry and reconstituted in 1xPBS for use in biological experiments.
  • Oligonucleotides containing 2’propargyl functional groups were synthesized DMT-ON without any changes of experimental conditions described above, followed by deprotection at room temperature in 28%-30% ammonium hydroxide for 36 hours. After standard Glen-Pak cartridge purification, oligonucleotides were lyophilized to dryness. Click chemistry reactions were then carried out in a liquid phase: oligonucleotides were redissolved in RNAse-free water to an initial concentration of 5 mmol. Subsequently, 10 eq. of an appropriate azide, 10 eq. of CuBr x Me2S complex (ThermoFisher) dissolved in DMSO, 10 eq.
  • Tris((1-benzyl-4-triazolyl)methyl)amine (TBTA, Merck) ligand dissolved in DMSO and 50 eq. of DI PEA were added. The reaction mixture was agitated for 3h at room temperature. Afterwards, 200pl of 0.5M EDTA solution were added to complexate copper ions and the mixture was agitated for additional 10 min. at room temperature. Oligonucleotides were precipitated from the mixture with NaCI/ethanol and collected as pellets after centrifugation. The pellets were then redissolved in LC-MS grade water and analyzed by LC-MS to confirm their identity and evaluate degree of conversion of propargyl residues.
  • TBTA Tris((1-benzyl-4-triazolyl)methyl)amine
  • oligonucleotides were quantified by means of UV-Vis spectrophotometry and reconstituted in 1xPBS for use in biological experiments.
  • 4-azidobutyric acid, (2-azidoethyl)phosphonic acid, 3-(azidomethyl)benzoic acid, 2-methylbenzene-1 -sulfonyl azide, 3-azido-4,5-dihydroisoxazole, 1 H-imidazole-1- sulfonylazide hydrochloride and propylsulfamoyl azide were obtained from Enamine Ltd. 6-azido-L-lysine hydrochloride was obtained from Apollo Scientific.
  • HeLa cells with genomically integrated SERPINA1 E342K were cultured in DM EM supplemented with 10% FBS (both Gibco) at 37°C and 5% CO2, and passaged every 3-4 days. Upon 80% confluence, cells were dissociated with Trypsin-ETDA (0,25%) and seeded at 7,500 cells/ well in 96-well plates. After 24 hours, cells were transfected with example ASOs set out in Table 1 and Fig. 2 at the indicated final concentrations using 0.3 pl Lipofectamine RNAiMAX (Invitrogen) per well in OptiMEM (Gibco).
  • Transfection mix was prepared by mixing equal volumes of 10x concentrated ASO and transfection reagent, and 20 pl of transfection mix was transferred to cells containing 80 pl fresh culture medium. If not stated otherwise, cells were washed with PBS and harvested 24 hours after transfection in 125 pl/well lysis buffer (Dynabeads mRNA direct kit, Invitrogen). Lysates of 96-well plates were transferred to a 384-plate and mRNA was isolated using the Dynabeads mRNA direct kit and an automated plate washer (Cytena C.Wash).
  • RNA was heated to 90°C for 2 min with an excess of a sense primer prior to RT.
  • a reverse transcription and cDNA amplification was performed with Luna Universal One-Step RT-qPCR mix (NEB) in a 10 pl reaction in a 384-well plate. Both the forward and reverse primer had an overhang to enable a second PCR with primers that bind to that overhang.
  • the samples were pooled and the DNA library was purified with the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel), diluted and sequenced together with a PhiX library on an iSeq 100 (Illumina). Results were analysed using a Python script. Demultiplexed reads were filtered by quality, length and position before editing percentages were calculated by dividing the number of G reads by the sum of the number of G reads and A reads at the respective target site. Data are represented as mean percentage of editing ⁇ standard deviation (SD).
  • SD standard deviation
  • the ASOs tested are set out in Table 1 below. These ASOs tested contained 2’- amino modifications at the indicated nucleotides. The editing results for these ASOs are shown in Fig.2. As can be seen, the addition of 2’-amino modifications at, for instance, nucleotides N-6, N+6, N+7, N+8, N+9, N+12, N+13, N+ 14 , N +2 3 and N +2 4 showed increased editing.
  • N is the nucleobase, e.g. A, G, C, U.
  • Example 5 Cell culture, transfection and mRNA isolation This was carried out as described in Example 4. NGS amplicon sequencing
  • the ASOs tested are set out in Table 2 below. These ASOs tested contained 2’- amino modifications at the indicated nucleotides. The editing results for these ASOs are shown in Fig.4.
  • N 2'0Me-N
  • fN 2'F-N
  • dN deoxy-N
  • * PS
  • n2rN 2'NH2-N
  • pNH2N 2'propylamino -N (2'CH2CH2CH2NH2)-N.
  • N is the nucleobase, e.g. A, G, C, U.
  • Example 6 Cell culture, transfection and mRNA isolation This was carried out as described in Example 4.
  • N is the nucleobase, e.g. A, G, C, U.
  • N-6 his N-4 his biz N-1 NO N + I N +2 N +3 N +4 N +5 N +6 N +7 N +8 N +9 N +W . . . . - 5’ and at least one of the nucleotides N_ 6 , N_ 5 , N. 4 , N. 2 , N.i , N +7 , N +8 , N +9 and N+w is independently chemically modified with a moiety that is positively charged under physiological conditions.
  • the chemically modified oligonucleotide according to embodiment 1 wherein the at least one nucleotide N. 8 , N.s, N. 4 , N.
  • N-i, N +7 , N +8 , N +9 and N+w is independently chemically modified with a moiety that is positively charged under physiological conditions at the 2’ position of its sugar residue.
  • N.i, N +7 , N +8 , N +9 and N+w is independently chemically modified with a moiety that is positively charged under physiological conditions at the 2’ position of its sugar residue with a modification independently selected from independently selected from -NH 2 (2’-amino) and the following modifications (I), (II), (III), and (IV): wherein, in formulae (I), (II), (III), and (IV):
  • X is selected from N(R X ) 2 or S-CH 3 ; each L is independently a linker group selected from a direct bond and Ci - salkylene; each Ri is independently selected from H, N(R X )2, a monocyclic 5- to 7- membered heterocyclyl group, a monocyclic 5- or 6-membered heteroaryl group, and NHC(NH)NH 2 ; wherein when Ri is a monocyclic 5- to 7- membered heterocyclyl group, or a monocyclic 5- or 6-membered heteroaryl group, one or more of the ring carbon atoms may be optionally substituted with one or more groups selected from halo, Ci-4alkyl, NH 2 and OH;
  • R 2 is N(R X ) 2 H; n is 1 to 5;
  • R 3 is selected from N(R X ) 2 H + and a 5- or 6-membered heteroaryl optionally substituted with -L-Ri; wherein when R 3 is a monocyclic 5- or 6-membered heteroaryl group optionally substituted with -L-Ri, one or more of the ring carbon atoms may be optionally substituted with one or more groups selected from halo, Ci- 4 alkyl, NH 2 and OH; R 4 is -CH(L-N(R X ) 2 ) 2 ; and each R x is independently selected from H and Ci- 4 alkyl.
  • the monocyclic 5- to 7- membered heterocyclyl group of Ri is a nitrogen - containing 5- to 7- membered heterocyclyl group, preferably a nitrogen - containing 5- or 6-membered heterocyclyl group, and more preferably piperidine; or
  • the monocyclic 5- or 6-membered heteroaryl group of Ri is a nitrogen - containing 5- or 6-membered heteroaryl group, preferably selected from pyridine and imidazole.
  • the modification is selected from the following modifications (Ila) to (He):
  • the modification is selected from the following (Illa) to (lllf): (Hla);
  • the 5-membered heteroaryl of R3 is a nitrogen-containing 5-membered heteroaryl group, preferably triazole.
  • the chemically modified oligonucleotide according to any of embodiments 1 to 6, wherein the at least one nucleotide N.6, N.s, N-4, N-2, N.i, N+?, N+s, N+g and N+1o is independently chemically modified with a moiety that is positively charged under physiological conditions at an internucleoside linkage linking the nucleotide with an adjacent nucleotide; preferably wherein said internucleoside linkage is a modified phosphodiester (PO) or phosphorothioate (PS) linkage.
  • PO phosphodiester
  • PS phosphorothioate
  • Z is selected from (-OCi-4alkyl-) n , O, S, and NH, where n is from 1 to 5; each L is independently a linker group selected from a direct bond or Ci - salkylene;
  • Rs is selected from N(R X )2H + , a monocyclic 5- to 7- membered heterocyclyl group, a monocyclic 5- or 6-membered heteroaryl group, -NHC(NH)NH2, and -CH(L-NH 2 ) 2 ; wherein when R 5 is a monocyclic 5- to 7- membered heterocyclyl group or a monocyclic 5- or 6-membered heteroaryl group, one or more of the ring carbon atoms may be optionally substituted with one or more groups selected from halo, Ci-4alkyl, NH 2 and OH; and each R x is independently selected from H and Ci ⁇ alkyl. 9.
  • the modification is selected from the following (Va) to (Vf), (Via) and (Vila):
  • a pharmaceutical composition comprising the chemically modified oligonucleotide according to any of embodiments 1 to 11.
  • the genetic disease or genetic disorder is selected from liver or metabolic diseases and/or cardiac or cardiovascular diseases associated with a gain- of-function (GOF) or loss-of-function (LOF) mutation; or
  • the genetic disease or genetic disorder comprises the SERPINA1 gene or an alpha-1-antitrypsin deficiency (A1AD or AATD), optionally wherein the target protein is alpha-1 antitrypsin.
  • An in vitro method for site-directed A-to-l editing of a target RNA comprising a step of contacting a target RNA with the chemically modified oligonucleotide according to any of embodiments 1 to 11 or the composition according to embodiment 12; preferably wherein the method comprises, after the step of contacting, the following steps:
  • BTK operates a phospho-tyrosine switch to regulate NLRP3 inflammasome activity. JEM. 218(11): e20201656.

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Abstract

La présente invention concerne un oligonucléotide chimiquement modifié destiné à être utilisé dans l'édition dirigée A-en-I (Adénosine en Inosine) vers le site d'un ARN cible à l'intérieur d'une cellule avec de l'adénosine désaminase endogène agissant sur l'ARN (ADAR), l'oligonucléotide chimiquement modifié comprenant une séquence apte à se lier à une séquence cible dans un ARN cible, un triplet de base central (CBT) de trois nucléotides (...N-1, N0, N+1...) où N0 est le nucléotide central directement opposé à une adénosine cible dans l'ARN cible qui doit être édité, et des liaisons internucléosidiques entre des nucléotides adjacents, l'oligonucléotide chimiquement modifié comprenant la séquence suivante : 3' -.... N-6 N-5 N-4 N-3 N-2 N-1 N0 N+1 N+2 N+3 N+4 N+5 N+6 N+7 N+8 N+9 N+10.... -5' et au moins un nucléotide étant chimiquement modifié indépendamment par une fraction chargée positivement dans des conditions physiologiques, dans lequel l'au moins un nucléotide est choisi parmi N-6, N-5, N-4, N+6, N+7, N+8, N+9, N+10 et où sont présents N+11, N+12, N+13, N+14, N+23 et N+24.
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