Antisense oligonucleotides for the treatment of liver disease

Abstract

The present invention relates to an antisense oligonucleotide (AON), which can mediate RNA editing by binding to a target RNA nucleic acid molecule, preferably an RNA transcript molecule, in a cell and recruiting an endogenous deaminationase in the cell to deaminate one or more target adenosine nucleotides in the target RNA molecule to inosine. The target RNA molecule is Na + This is a transcript molecule derived from the SLC10A1 gene that encodes taurocholic acid cotransport polypeptide (NTCP). RNA editing of one or more target adenosines results in loss of function of the NTCP protein, which in turn reduces the uptake of bile acids from the portal circulation into the liver, thereby reducing the risk of developing disorders associated with bile accumulation in the liver.

Description

[Technical Field]

[0001] This disclosure relates to the field of medicine, specifically to the field of (chronic) liver diseases such as non-alcoholic fatty liver disease (NAFLD) and cirrhosis caused by bile accumulation. This disclosure relates to the encoding of Na by mediated nucleotide-specific RNA editing in the SLC10A1 gene transcript. + This article describes antisense oligonucleotides that induce amino acid changes in taurocholic acid cotransport polypeptides (NTCPs), and how these changes affect NTCP activity. [Background technology]

[0002] Cholestatic disorders are caused by the accumulation of bile acids in the liver due to bile duct dysfunction, which leads to damage to liver cells. The consequences of these disorders are catastrophic and can significantly impact the patient's quality of life. Symptoms include itching, dry skin, fatigue, pain, and weight loss. If left untreated, the damage progresses through various stages from fibrosis to cirrhosis, ultimately leading to liver failure and an increased risk of liver cancer. Primary sclerosing cholangitis (PSC) and biliary atresia (BA) are two forms of cholestatic disorders with high unmet medical needs, often requiring liver transplantation. PSC is an inflammatory condition usually diagnosed in people aged 30-40, and more commonly in men (66%). In North America and Europe, it is estimated that approximately 80,000 people have PSC, with a prevalence of 1-9 per 100,000 people. This condition causes fibrosis and hardening of the bile ducts, leading to the toxic accumulation of bile acids in the liver. Biliary pulmonary artery disease (BA) is a childhood disease that develops in the newborn and is caused by the absence or defect of the bile ducts. This condition causes harmful bile acids to accumulate in the liver, leading to rapid progression to cirrhosis in early childhood. It is estimated that approximately 20,000 people in North America and Europe suffer from BA, with a prevalence of 1 in 10,000 to 15,000 newborns in the Western world.

[0003] Currently, there are no approved drugs to treat PSC or BA. For PSC, liver transplantation is the only treatment option for which there is evidence to extend survival. However, PSC can recur in 20-40% of patients who undergo liver transplantation, and the median survival without transplantation is only about 21 years. For BA, surgery within the first few weeks of life is the standard treatment. However, the majority of patients who undergo this surgery still require a liver transplant in early childhood.

[0004] Hepatocytes in the liver primarily obtain bile acids from enterohepatic circulation. The process of bile acid uptake from portal circulation into hepatocytes is mainly due to Na + This is carried out by a transporter protein called taurocholic acid cotransport polypeptide (NTCP), which is encoded by the SLC10A1 gene. The NTCP protein has attracted considerable attention because it has been identified as a major protein involved in the recognition and entry of hepatitis B virus (HBV) and hepatitis D virus (HDV). However, the normal function of NTCP is at least related to the uptake of conjugated bile acids from the circulation into hepatic parenchymal cells. As mentioned above, the accumulation of bile acids in the liver is associated with a wide variety of liver diseases. Furthermore, it has been shown that inhibiting NTCP can improve liver function by reducing intracellular levels of toxic bile acids in the hepatocyte parenchyma, and that it can also improve liver function by preventing increased liver damage, as indicated by specific available markers such as fibrosis, cholangioproliferation, alkaline phosphatase (ALP), alanine aminotransferase (ALT), and cytokines (Slijepcevic D and Van de Graaf 2017. Dig Dis. 35(3):251-258; Slijepcevic D et al. 2018. Hepatology 68(3):1057-1069).

[0005] This disclosure relates to a completely different approach to reducing NTCP activity, namely, providing a loss-of-function NTCP protein by specifically targeting and modifying one or more nucleotides in the SLC10A1 transcript using antisense oligonucleotides (AONs) and the cell's own nucleic acid editing mechanisms, thereby reducing the ability of hepatocytes to take up bile acids from the portal circulation, and thereby treating diseases associated with bile accumulation in the liver. The technology to which this disclosure relates is commonly referred to as "RNA editing."

[0006] RNA editing is a natural process in which eukaryotic cells alter the sequence of their RNA molecules, often in a site-specific and precise manner, thereby increasing the repertoire of RNA encoded in the genome by several orders of magnitude. RNA editing enzymes have been reported in eukaryotic species throughout the animal and plant kingdoms, and these processes play a crucial role in maintaining cellular homeostasis in metazoans, from the simplest organisms (e.g., Caenorhabditis elegans) to humans. Examples of RNA editing include the conversion of adenosine (A) to inosine (I) and cytidine (C) to uridine (U), which occur through enzymes called adenosine deaminationase (ADAR) and APOBEC / AID (cytidine deaminationase acting on RNA), respectively.

[0007] ADARs are multi-domain proteins containing a catalytic domain and 2-3 double-stranded RNA recognition domains depending on the enzyme in question. Each recognition domain recognizes a specific double-stranded RNA (dsRNA) sequence and / or conformation. The catalytic domain also plays a role in recognizing and binding to a portion of the dsRNA helix, but the primary function of the catalytic domain is to convert A to I by deamination of the nucleic acid base at a (pre-defined) location near the target RNA. Inosine is read as guanosine by the cell's translation mechanism, meaning that if edited adenosine is present in the coding region of mRNA or pre-mRNA, it can recode the protein sequence. The A-to-I conversion can also occur in the 5′ non-coding sequence of the target mRNA, generating a new translation initiation site upstream of the original start site, which produces a protein with an extended N-terminus, or it can occur in the 3′ untranslated region (UTR) or other non-coding portions of the transcript, which can affect RNA processing and / or stability. In addition, the conversion from A to I can occur in splice elements within introns or exons of pre-mRNA, thereby altering the splicing pattern. As a result, exons may be incorporated or skipped. Enzymes that catalyze adenosine deamination belong to the ADAR enzyme family, which includes the human deaminationases hADAR1, hADAR2, and hADAR3. However, deaminationase activity has not been demonstrated in hADAR3.

[0008] The use of oligonucleotides for editing target RNA by applying adenosine deaminationase is described below (e.g., Woolf et al. Proc Natl Acad Sci USA 1995, 92:8298-8302; Montiel-Gonzalez et al. Proc Natl Acad Sci USA 2013, 110(45):18285-18290; Vogel et al. Angewandte Chemie Int 2014, Ed 53:267-271; Montiel-Gonzalez et al. (2013)). A drawback of the method described is that it requires a fusion protein consisting of the box B recognition domain of the bacteriophage λN protein and the adenosine deaminationase domain of the truncated native ADAR protein. This method requires the target cells to be transduction with the fusion protein (which is a major obstacle), or to be transfected with a nucleic acid construct encoding a modified adenosine deaminationase fusion protein for expression. The system described by Vogel et al. (2014) suffers from similar drawbacks, where it is unclear how to apply the system without first genetically modifying ADAR and then transfecting or transforming cells containing the target RNA to supply the modified protein to the cells. A similar system is also described in US9,650,627. (Woolf et al. (1995)) The oligonucleotide was 100% complementary to the target RNA sequence, but suffered from a severe lack of specificity: that is, almost all adenosine in the target RNA strand complementary to the AON was edited out.

[0009] ADARs are known to act on any dsRNA. Through a process sometimes called "promiscuous editing," these enzymes edit multiple A molecules within the dsRNA. Therefore, there has been a need for methods and means to bypass such promiscuous editing and target only specific adenosines within the target RNA molecule for therapeutic applications. Vogel et al. (2014) showed that such off-target editing can be suppressed by using a 2′-O-methyl (2′-OMe) modified nucleoside at the oligonucleotide opposite the adenosine that should not be edited, and using an unmodified nucleoside at the position directly opposite the adenosine that is specifically targeted on the target RNA. However, it has not been shown that a specific editing effect at the target nucleotide can be achieved without using recombinant ADAR enzymes that have a covalent bond with AON. Multiple studies have shown that recruitment of endogenous ADARs (and therefore without requiring exogenous and / or recombinant sources) is feasible while maintaining specificity in targeting a single adenosine within a target RNA molecule and deaminating it to inosine. WO2016 / 097212 discloses an AON for targeted RNA editing, characterized by a sequence complementary to the target RNA sequence (referred to as the “targeting portion” in the same study) and the presence of a stem-loop / hairpin structure (referred to as the “recruitment portion” in the same study) (which is preferably non-complementary to the target RNA). Such oligonucleotides are referred to as “self-looping AONs.” The recruitment portion recruits a cellular (endogenously present) native ADAR enzyme to a dsRNA formed by hybridization with the targeting portion of the target sequence. The presence of the mobilization portion eliminates the need for a conjugate or modified recombinant ADAR enzyme.WO2016 / 097212 describes the recruiting moiety as a stem-loop structure that mimics a native substrate (e.g., the GluB receptor) or a Z-DNA structure (known to be recognized by the dsRNA-binding domain or Z-DNA-binding domain of ADAR enzymes). The stem-loop structure can be an intermolecular stem-loop structure formed by two separate nucleic acid strands, or an intramolecular stem-loop structure formed within a single nucleic acid strand. The aforementioned stem-loop structure of the recruiting moiety is an intramolecular stem-loop structure formed within the AON itself and is thought to attract (endogenous) ADAR. Systems containing similar stem-loop structures for RNA editing have since been described in WO2017 / 050306, WO2020 / 001793, WO2017 / 010556, US11,390,865, WO2020 / 246560, and WO2022 / 078995.

[0010] WO2017 / 220751 and WO2018 / 041973 describe next-generation AONs that do not contain such stem-loop structures but appear to be (almost perfectly) complementary to the target region and still capable of attracting endogenous ADAR enzymes. In one embodiment, one or more mismatched nucleotides, wobbles, or bulges are present between the oligonucleotide and the target sequence. While the mismatch may be present only at the nucleoside site opposite the target adenosine, in other embodiments, AONs (or “RNA-editing oligonucleotides”—often abbreviated as “EONs”—even though the deamination reaction is carried out by ADAR enzymes) having multiple bulges and / or wobbles upon attachment to the target sequence region have been described. It appeared possible to achieve in vitro, ex vivo, and in vivo RNA editing using AONs lacking stem-loop structures and endogenous ADAR enzymes, provided that the AON sequence was carefully selected to attract / mobilize ADAR. An "orphan nucleoside" is defined as a nucleoside in the AON located directly opposite the target adenosine in the target RNA molecule, but which is a nucleotide with an unmodified cytosine nucleic acid base and lacks 2′-OMe modification. The orphan nucleoside may also be a deoxyribonucleoside (DNA), and the rest of the AON may still have 2′-O-alkyl modification (e.g., 2′-OMe) in the sugar portion, or the nucleotide directly surrounding the orphan nucleoside may contain chemical modification (e.g., DNA compared to RNA), resulting in further improved RNA editing efficiency and / or improved resistance to nucleases. Such effects can be further enhanced by the use of sense oligonucleotides (SONs) that "protect" AONs from degradation during delivery to cells (as described in WO2018 / 134301 and US11,274,300).

[0011] The use of chemical modifications and specific structures in oligonucleotides that can be used for ADAR-mediated editing of specific adenosines within target RNA has been the subject of numerous disclosures in this art, such as: WO2019 / 111957, WO2019 / 158475, WO2020 / 165077, WO2020 / 201406, WO2020 / 211780, WO2021 / 008447, WO2 021 / 020550, WO2021 / 060527, WO2021 / 117729, WO2021 / 136408, WO2021 / 182474, WO2021 / 216853, WO2021 / 242778, WO2021 / 242870, WO2021 / 242889, WO2022 / 007803, WO2022 / 018207, WO2022 / 026928, and WO2022 / 124345. The use of specific sugar moieties is disclosed, for example, in WO2020 / 154342, WO2020 / 154343, WO2020 / 154344, WO2022 / 103839, and WO2022 / 103852.On the other hand, the use of sterically defined linker moieties (generally for oligonucleotides that can be used for exon skipping, gapmers, siRNA, or more specifically for RNA editing oligonucleotides associated with a wide variety of target sequences) is described below: WO2011 / 005761, WO2014 / 010250, WO2014 / 012081, WO2015 / 107425, WO2017 / 01 5575(HTT), WO2017 / 062862, WO2017 / 160741, WO2017 / 192664, WO2017 / 192679(DMD), WO2017 / 198775, WO2017 / 210647 , WO2018 / 067973, WO2018 / 098264, WO2018 / 223056(PNPLA3), WO2018 / 223073(APOC3), WO2018 / 223081(PNPLA3), WO201 8 / 237194, WO2019 / 032607(C9orf72), WO2019 / 055951, WO2019 / 075357(SMA / ALS), WO2019 / 200185(DM1), WO2019 / 217 784(DM1), WO2019 / 219581, WO2020 / 118246(DM1), WO2020 / 160336(HTT), WO2020 / 191252, WO2020 / 196662, WO2020 / 219 981(USH2A), WO2020 / 219983(RHO), WO2020 / 227691(C9orf72), WO2021 / 071788(C9orf72), WO2021 / 071858, WO2021 / 178237(MAPT), WO2021 / 234459, WO2021 / 237223, WO2022 / 099159, WO2021 / 030778, WO2022 / 174053, and WO2023 / 278589. In addition to these disclosures, there is a great many publications relating to the targeting of specific RNA target molecules, or specific adenosines within such RNA target molecules, for the repair of mutations resulting in premature stop codons, or other disease-causing mutations.Examples of such disclosures targeting adenosine within specific RNA target molecules include: WO2020 / 157008 and WO2021 / 136404 (USH2A); WO2021 / 113270 (APP); WO2021 / 113390 (CMT1A); WO2021 / 209010 (IDUA, Hurler syndrome); WO2021 / 231673 and WO2021 / 242903 (LRRK2); WO2021 / 231675 (ASS1); WO2021 / 23 1679(GJB2);WO2019 / 071274 and WO2021 / 231680(MECP2);WO2021 / 231685 and WO2021 / 231692(OTOF, autosomal recessive non-symptomatic hearing loss);WO2021 / 231691(XLRS);WO2021 / 231698(argininosuccinate lyase deficiency);WO2021 / 130313 and WO2021 / 231830(ABCA4);and WO2021 / 243023(SERPINA1).

[0012] The present invention aims to provide one or more alternative and / or improved compounds or compositions for use in the treatment of liver diseases, such as cholestatic disorders caused by the accumulation of bile acids in the liver. [Overview of the project]

[0013] An antisense oligonucleotide (AON) is disclosed herein, wherein the AON is capable of recruiting an endogenous ADAR enzyme in human cells after the AON forms a double-stranded complex with a region of an intracellular target RNA nucleic acid molecule, the region comprising target adenosine, the nucleotide in the AON opposite the target adenosine being a lone nucleotide, the ADAR enzyme being able to deaminate the target adenosine to inosine after binding to the double-stranded complex, and the target RNA nucleic acid molecule being Na + / A transcript molecule of the human SLC10A1 gene encoding a taurocholic acid cotransport polypeptide (NTCP). Preferably, the transcript molecule is a pre-mRNA or mRNA molecule. Preferably, the cell is a hepatocyte, more preferably a hepatocyte. In one embodiment, the SLC10A1 gene is wild-type, and the target adenosine is selected from the group consisting of: (i) Adenosine in the CAG codon encoding glutamine (Q) at position 68 of the NTCP protein, wherein the adenosine is deaminated to change the amino acid to arginine (R); (ii) The adenosine is the first adenosine in the CAA codon encoding glutamine (Q) at position 261 of the NTCP protein, wherein the adenosine is deaminated to change the amino acid to arginine (R); (iii) The adenosine is located in the GAG ​​codon encoding glutamic acid (E) at position 257 of the NTCP protein, and the adenosine is deaminated to change the amino acid to glycine (G); and (iv) The adenosine is the first adenosine in the AAG codon encoding lysine (K) at position 314 of the NTCP protein, wherein the adenosine is deaminated to glutamic acid (E); Here, the deamination of the target adenosine results in an NTCP protein whose function of transporting bile acids from the portal circulation into the cell is impaired. In one embodiment, the solitary nucleotide is deoxycytidine or deoxyuridine. In one embodiment, the solitary nucleotide is a cytidine analog, for example, a deoxynucleotide containing the 6-amino-5-nitro-3-yl-2(1H)-pyridone nucleic acid base. In one embodiment, the solitary nucleotide is a uridine analog, for example, a deoxynucleotide containing the isouracil nucleic acid base. In one embodiment, the nucleotide numbering in the AON is such that the solitary nucleotide is number 0, and the nucleotides further increase positively (+) toward the 5′ end and negatively (-) toward the 3′ end, and the first nucleotide (-1) 3′ from the solitary nucleotide is deoxyinosine if the nucleotide opposite this position is cytidine in the target RNA nucleic acid molecule.

[0014] AON is disclosed, the AON comprising one or more modifications at the binding site, the modifications being independently selected from phosphorothioate (PS), phosphonoacetate, phosphorodithioate, methylphosphonate (MP), sulfonyl phosphoramidate, (1,3-dimethylimidazolidinedine-2-ylidene)phosphoamidate (PNdmi) bond, or PNms bond having a structure according to formula (I), where: X = O or S; and R = aryl, substituted aryl, heterocycle, substituted heterocycle, aromatic heterocycle, substituted aromatic heterocycle, C1-C6 alkoxy, substituted C1-C6 alkoxy, C1-C 20 Alkyl, substituted C1-C 20 These are alkyl groups, C1-C6 alkenyl groups, substituted C1-C6 alkenyl groups, C1-C6 alkynyl groups, substituted C1-C6 alkynyl groups, or conjugated groups.

[0015] An AON is disclosed, wherein the numbering of nucleoside-to-nucleoside bonds in the AON is such that bond number 0 is a bond 5′ away from the isolated nucleotide, and the bond positions within the oligonucleotide increase positively (+) toward the 5′ end and negatively (-) toward the 3′ end, and bond position -2 is an MP bond.

[0016] An AON is disclosed, which comprises one or more nucleotides including one or two substitutions at the 2′, 3′, and / or 5′ positions of ribose, each independently selected from the group consisting of: -OH; -F; substituted or unsubstituted, linear or branched lower (C1-C) 10 ) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, which may be interrupted by one or more heteroatoms; -O-, S-, or N-alkyl; -O-, S-, or N-alkenyl; -O-, S-, or N-alkynyl; -O-, S-, or N-allyl; -O-alkyl-O-alkyl; -methoxy; -aminopropoxy; -methoxyethoxy; -dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy. In one embodiment, the AON is (non)covalently bonded to the GalNAc moiety.

[0017] Pharmaceutical compositions are disclosed herein and comprise an AON disclosed herein, or a vector disclosed herein, and a pharmaceutically acceptable carrier. AONs for use in the treatment of diseases caused by the accumulation of bile in the liver, such as cholestasis, primary sclerosing cholangitis (PSC), biliary atresia (BA), and cirrhosis are disclosed herein. AONs for use in the manufacture of therapeutic pharmaceuticals for diseases caused by the accumulation of bile in the liver, such as cholestasis, PSC, BA, and cirrhosis are also disclosed herein.

[0018] A method of editing a human SLC10A1 polynucleotide in a cell, preferably a hepatocyte, is disclosed herein, wherein the human SLC10A1 polynucleotide is a pre-mRNA or mRNA molecule, and the method comprises contacting the SLC10A1 polynucleotide with an AON capable of inducing ADAR-mediated deamination of adenosine to inosine, thereby editing the SLC10A1 polynucleotide to encode an NTCP protein with attenuated, reduced, or lost function in bile acid uptake.

[0019] A method of treating, ameliorating, or delaying the progression of a disease caused by bile accumulation in the liver, such as cholestasis, PSC, BA, and cirrhosis, in a human subject who needs it is disclosed herein, the method comprising administering to the subject an AON disclosed herein, a vector disclosed herein, or a pharmaceutical composition disclosed herein, thereby contacting the SLC10A1 polynucleotide in the cells of the subject with an AON capable of causing ADAR-mediated deamination of adenosine to inosine, thereby editing the SLC10A1 polynucleotide to encode an NTCP protein with attenuated, reduced, or lost function in bile acid uptake, thereby treating the subject.

[0020] [Brief Description of the Drawings] One or more embodiments of the present invention will be described below by way of example only, with reference to the accompanying drawings:

Brief Description of the Drawings

[0021] [Figure 1]A portion of the 5′→3′ sequence of the human SLC10A1 mRNA transcript is shown, in bold the CAG codon encoding glutamine (Q) at position 68 in the NTCP protein (SEQ ID NO: 1). The underlined adenosine is the target of the RNA editing disclosed herein, which, after editing, becomes a codon (CIG / CGG) encoding an arginine (R) residue at this position. Below the target sequence, the 5′→3′ sequences of the first 30 AONs designed to target the target adenosine in SEQ ID NO: 1 are shown. AONs T1-01 to T1-30 are SEQ ID NOs 5 to 34, as shown. The chemical modifications are as follows: Ae and Ge are 2′-MOE modified adenosine and guanosine, respectively; Cm, Am, Um, and Gm are 2′-OMe modified cytidine, adenosine, uridine, and guanosine, respectively; Gf, Cf, Af, and Uf are 2′-F modified guanosine, cytidine, adenosine, and uridine, respectively; m5Ce is 2′-MOE modified 5-methylcytidine; Zd (isolated nucleotide) is a deoxynucleotide (deoxycytidine analog) with Benner's base. Id is deoxyinosine; Te (i.e., m5Ue) is 2′-MOE modified thymidine (identical to 5-methyluridine with a 2′-MOE substitution); "!" refers to a PNdmi bond; "^" refers to an MP bond; "*" refers to a PS bond. All other internucleoside bonds are phosphodiester (PO) bonds. [Figure 2]A portion of the sequence of the human SLC10A1 mRNA transcript is shown, in bold, where the CAA codon encoding glutamine (Q) at position 261 in the NTCP protein is shown (SEQ ID NO: 2). The underlined adenosine is the target of the RNA editing disclosed herein, which, after editing, becomes a codon (CIA / CGA) encoding an arginine (R) residue at this position. Below the target sequence, the 5′→3′ sequences of the first 30 AONs designed to target the target adenosine in SEQ ID NO: 2 are shown. AONs T2-01 to T2-30 are SEQ ID NOs 35 to 64, as shown. Chemical modifications are as shown in Figure 1. [Figure 3] A portion of the sequence of the human SLC10A1 mRNA transcript is shown, in bold the GAG ​​codon encoding glutamic acid (E) at position 257 in the NTCP protein (SEQ ID NO: 3). The underlined adenosine is the target of the RNA editing disclosed herein, which, after editing, becomes a codon (GIG / GGG) encoding a glycine (G) residue at this position. Below the target sequence are the 5′→3′ sequences of the first 30 AONs designed to target the target adenosine in SEQ ID NO: 3. AONs T3-01 to T3-30 are SEQ ID NOs 65 to 94, as shown. Chemical modifications are as shown in Figure 1. [Figure 4] A portion of the sequence of the human SLC10A1 mRNA transcript is shown, in bold the AAG codon encoding lysine (K) at position 314 in the NTCP protein (SEQ ID NO: 4). The underlined adenosine is the target of the RNA editing disclosed herein, which, after editing, becomes a codon (IAG / GAG) encoding a glutamic acid (E) residue at this position. Below the target sequence, the 5′→3′ sequences of the first 30 AONs designed to target the target adenosine in SEQ ID NO: 4 are shown. AONs T4-01 to T4-30 are SEQ ID NOs 95 to 124, as shown. Chemical modifications are as shown in Figure 1. [Figure 5]An additional set of AONs (Q68R) designed to target the target adenosine in SEQ ID NO: 1 is shown. Each SEQ ID NO is as shown. Chemical modifications are as given in Figure 1, where the symbol "#" represents a PNms bond and the symbol "θ" represents a PO bond. [Figure 6] An additional set of AONs designed to target the target adenosine in SEQ ID NO: 3 is shown (E257G). Each SEQ ID NO is as shown. Chemical modifications are given in Figure 1. [Figures 7A-7B] For c.203A>G editing (Q68R), the editing percentages are shown in two independent experiments, each performed in primary human hepatocytes (PHH) after co-treatment with AG1856 saponin using the 16 AONs listed. Two unrelated AONs (RM4777 and RM4266), AG1856 alone, and untreated samples were used as negative controls. [Figures 8A-8B] For c.770A>G editing (E257G), the editing percentages are shown in two independent experiments, each measured in PHH after co-treatment with AG1856 saponin using the 17 AONs listed. Two unrelated AONs (RM4777 and RM4266), AG1856 alone, and untreated samples were used as negative controls. [Figure 9A-9B] For c.203A>G editing (Q68R), the editing percentages are shown for each of the 18 AONs listed, measured in two independent gymnotic uptake experiments performed in PHH in the absence of saponins. Two unrelated AONs (RM4777 and RM4266) and an untreated sample were used as negative controls. [Figure 10A-10B]For each of the c.770A>G edits (E257G), the edit percentages are shown, measured in two independent gymnotic uptake experiments performed in PHH in the absence of saponins, using the 16 AONs indicated. Two unrelated AONs (RM4777 and RM4266) and an untreated sample were used as negative controls. [Figure 11] Regarding c.203A>G editing (Q68R), the editing percentages measured in two experiments were performed on liver spheroids using the 16 AONs shown: after co-treatment with AG1856 saponin (Figure 11A) and without co-treatment with saponin (Figure 11B). Untreated samples and AG1856 alone were used as negative controls. [Figure 12] Regarding c.770A>G editing (E257G), the editing percentages measured in two experiments were performed on liver spheroids using the 17 AONs shown: after co-treatment with AG1856 saponin (Figure 12A) and without co-treatment with saponin (Figure 12B). Untreated samples and AG1856 alone were used as negative controls. [Figure 13] Regarding c.203A>G editing (Q68R), the editing percentages are shown in experiments conducted in HepG2 cells (HepG2NTCP) overexpressing human NTCP after co-treatment with AG1856 saponin using the 11 AONs listed. Untreated samples were used as negative controls. [Figure 14] The following shows the editing percentages for c.770A>G editing (E257G) measured in HepG2NTCP cells after co-treatment with AG1856 saponin using the 10 AONs listed. Untreated samples and AG1856 alone were used as negative controls. [Figure 15]For c.203A>G editing (Q68R), the editing percentages are shown in additional experiments performed in PHH after co-treatment with AG1856 saponin, using the 25 asymmetric AONs shown (Figure 15A) and the 34 symmetric AONs shown (Figure 15B). An unrelated AON (RM4777) and untreated samples were used as negative controls. [Figure 16] For c.770A>G editing (E257G), the editing percentages are shown in additional experiments performed in PHH after co-treatment with AG1856 saponin, using the 21 asymmetric AONs shown (Figure 16A) and the 38 symmetric AONs shown (Figure 16B). An unrelated AON (RM4777) and untreated samples were used as negative controls. [Figure 17] The pmolal uptake of radiolabeled taurocholic acid (TCA; bile acid) in human U2OS cells is shown after (independent) transfection with plasmids expressing the seven human NTCP variants shown, followed by treatment with 1.0 μM TCA (Figure 17A) and 10 μM TCA (Figure 17B). Plasmids expressing wild-type NTCP were used as positive controls, and untreated samples were used as negative controls. [Figure 18] This shows an additional set of AONs (Q68R) designed to target the target adenosine in SEQ ID NO: 1, using various bonds that substitute for the bonds in the previous set of AONs. Each SEQ ID NO is shown in the second column. The chemical modifications are given in Figure 1, where the symbol "#" represents a PNms bond and the symbol "θ" represents a PO bond. [Figure 19] The edit percentages in c.203A>G editing (Q68R) experiments performed in PHH after transfection with lipofectamine are shown for the 12 AONs (details in Figure 18). An unrelated AON (RM4777), lipofectamine alone, and untreated (NT) PBS samples were used as negative controls. [Figure 20]The editing percentages in c.203A>G editing (Q68R) experiments performed in PHH after transfection with lipofectamine are shown for the 24 AONs (details in Figure 18). Irrelevant AONs (control), lipofectamine alone (Lipo2000), and untreated (NT) PBS samples were used as negative controls. [Figure 21] The editing percentages in c.203A>G editing (Q68R) experiments performed in PHH after co-treatment with AG1856 using the nine AONs shown (details in Figure 18) are presented. Irrelevant AONs (control ON), saponins alone (AG1856), and untreated (NT) samples were used as negative controls. [Figure 22] The editing percentages in c.203A>G editing (Q68R) experiments performed in PHH after transfection with lipofectamine are shown for the 26 AONs (details in Figure 18). An unrelated control (RM4777), lipofectamine only (mock Lipo), and untreated (NT) samples were used as negative controls. [Modes for carrying out the invention]

[0022] The AONs disclosed herein can recruit deaminationases such as ADAR1 and / or ADAR2 that are endogenously present in cells. After binding to a target RNA molecule, the AONs disclosed herein can mediate RNA editing of target adenosine present in the target RNA molecule. This is because the deaminationases are recruited to the double-stranded AON / target RNA molecule complex and subsequently deaminate the target adenosine to inosine.

[0023] Oligonucleotides are abbreviated as "AON" herein, but are sometimes referred to as "editing oligonucleotides" or "EONs," even though the RNA editing event is carried out by deaminationases and the action of oligonucleotides merely induces RNA editing. There is always a need to improve the pharmacokinetic properties of AONs without adversely affecting the efficiency of editing of target adenosine in target RNA and / or adversely affecting the stability of AONs themselves, which are always easily degraded by nucleases present in native cells. Many chemical modifications are available for the production of AONs (and many are applied in the art). However, many of these properties are not always compatible with the desire to achieve efficient RNA editing. In the search for better pharmacokinetic properties, it has been previously found that 2′-O-methoxyethyl (i.e., 2′-methoxyethoxy, or 2′-MOE) modification of ribose at some (but not all) nucleotides appears to be surprisingly compatible with efficient ADAR involvement and editing (WO2019 / 158475). Similarly, it has been previously found that PS binding at some (but not all) nucleoside-to-nucleoside bonds appears to be surprisingly compatible with efficient ADAR involvement and editing (WO2019 / 219581). Furthermore, it has been previously found that phosphonoacetate binding modification and / or unlocked nucleic acid (UNA) ribose modification at some (but not all) positions in AON appears to be compatible with efficient involvement of enzymes with nucleotide deamination activity and subsequent deamination (WO2020 / 165077). While the properties of phosphonoacetate and UNA modification were known, the involvement of enzymes with nucleotide deamination activity, and the fact that these can coexist with the deamination reaction, were unknown.

[0024] AONs that can provide (mediate, cause, or induce) RNA editing of a target adenosine in a target transcript molecule, such as pre-mRNA and / or mRNA, are disclosed herein. The target transcript molecule may be encoded by a mutant gene, the mutant being the cause of the disease, and the editing may reverse the mutant, resulting in a wild-type protein or a protein with wild-type function (for example, if the mutant amino acid is changed to an amino acid that does not cause the disease or gives an improved phenotype). As disclosed in more detail herein, the target transcript molecule may also be encoded by a wild-type gene, for example, in a preferred embodiment of this disclosure, the target nucleic acid molecule is a transcript from the wild-type human SLC10A1 gene shown herein, and RNA editing causes the encoded NTCP protein to lose function, but the disease condition being treated is improved.

[0025] Non-limiting examples of transcript molecules targeted using RNA editing for various therapeutic purposes include: SERPINA1 (for the treatment of α1-antitrypsin (A1AT) deficiency; see, e.g., WO2016 / 097212, WO2017 / 220751, WO2018 / 041973, and WO2021 / 243023), IDUA (for the treatment of Hurler syndrome; see, e.g., WO2017 / See 220751, WO2018 / 041973, and WO2021 / 209010), LRRK2 (for the treatment of Parkinson's disease; see WO2016 / 097212, WO2017 / 220751, WO2018 / 041973, WO2021 / 231673, and WO2021 / 242903), ABCA4 (for the treatment of Stargardt disease; see, e.g., WO2021 / 130313 and WO202 (See 1 / 231830), USH2A (for the treatment of Usher syndrome; e.g., WO2020 / 157008, WO2020 / 219981, and WO2021 / 136404), APP (e.g., WO2021 / 113270), CMT1A (e.g., WO2021 / 113390), ASS1 (e.g., WO2021 / 231675), GJB2 (e.g., WO2021 / 231679) MECP2 (for the treatment of Rett syndrome; e.g., WO2019 / 071274 and WO2021 / 231680), OTOF (for the treatment of autosomal recessive non-syndromic hearing loss; e.g., WO2021 / 231685 and WO2021 / 231692), XLRS (e.g., WO2021 / 231691), and PCSK9 (for the treatment of hypercholesterolemia; e.g., WO2023 / 152371).

[0026] This disclosure relates to an AON that mediates RNA editing of one or more adenosines present in the transcript of the SLC10A1 gene using an endogenous (naturally occurring) ADAR enzyme in a host cell, preferably a hepatocyte. The two-dimensional structure of the NTCP protein in the cell membrane of hepatocytes is known from the prior art (Ho RH et al. 2004; J Biol Chem. 279(8): 7213-7222). The AON disclosed herein aims to reduce bile acid reabsorption in the liver by inhibiting NTCP function. Several loss-of-function variants of NTCP have been identified in the prior art. Therefore, these mutations occur spontaneously in a limited number of individuals, but do not cause any symptoms associated with cholestasis, despite relatively high circulating bile acid concentrations (Vaz et al. 2015; Hepatology. 61(1):260-267; Schneider et al. 2022; Clin Res Hepatol Gastroenterol. 46(3):101824). Prior art has described how multiple amino acid substitutions in the sodium-binding pocket lead to loss-of-function variants of the NTCP protein, resulting in impaired bile acid transport without impairing protein expression or localization (Huan Yan et al. 2014; J Virology. 88(6): 3273-3284). This finding suggests that treating subjects with bile accumulation-related disorders using the AONs disclosed herein may result in a reduction of such accumulation of toxic bile acids in the liver. Furthermore, generating loss-of-function variants of NTCP is also expected to promote the elimination of bile acids from the body by increasing their excretion in feces and urine. This process, known as bile acid sulfation, increases the solubility of bile acids and reduces their absorption in the intestinal tract.This disclosure relates to various AONs aimed at deamination of various adenosines in the SLC10A1 transcript, each of which is thought to independently cause loss of function in the NTCP protein, and each of which is thought to independently be used to treat the bile acid storage disorders disclosed herein. However, it is not ruled out that two or more adenosines may be targeted for deamination in a single treatment. While we do not wish to be bound by theory, it is thought that combining the AONs disclosed herein to target multiple adenosines, and thus multiple amino acids, within a single NTCP protein may yield synergistic or additional effects, thereby increasing the therapeutic effect.

[0027] Cholestasis can cause inflammation and lead to the development of hepatic fibrosis, cirrhosis, hepatocellular carcinoma, and / or liver failure. The AONs disclosed herein are designed to reduce inflammation and mitigate or prevent fibrosis and cirrhosis by reducing the toxic accumulation of bile acids, thereby protecting hepatocytes and function.

[0028] Cholestatic disease can be chronic or acute. Chronic liver disease causes fibrosis and cirrhosis (scarring) of liver tissue, which impairs liver function. Chronic conditions may include: infections caused by hepatitis viruses (hepatitis B virus, hepatitis C virus, hepatitis D virus, etc.); alcohol-induced hepatitis; autoimmune and autoinflammatory diseases (including primary sclerosing cholangitis, primary biliary cholangitis, and autoimmune hepatitis); metabolic diseases (including non-alcoholic fatty liver disease, metabolism-related fatty liver disease, and non-alcoholic fatty liver disease); and genetic diseases (including Wilson's disease, hereditary hemochromatosis, and α1-antitrypsin disorders). Acute hepatitis and cholestasis can be caused by: infections (including hepatitis viruses, infectious mononucleosis, HIV, cytomegalovirus, sepsis, and gallbladder infection); alcoholism or toxic hepatitis; liver cancer or lymphoma; the use of medications (including contraceptives, anabolic steroids, penicillin antibiotics (including amoxicillin), azathioprine, imipramine, estradiol, cimetidine, chlorpromazine, prochlorperazine, tolbutamide, and terbinafine); and pregnancy-related cholestasis.

[0029] Cholestasis can also result from extrahepatic causes, including, for example, bile duct obstruction or stricture: gallstones in the common bile duct, cystic duct, or Hartmann's capsule; pancreatic cysts and pseudocysts; extrahepatic bile duct tumors; chronic pancreatitis; pancreatic cancer; cholangiocarcinoma; cholangitis; biliary atresia; and past injury or surgery. Furthermore, cholestasis can also occur in newborns. The causes may also include: infectious agents (e.g., viruses, bacteria, spirochetes, parasites); toxins from drugs, endotoxins, total parenteral nutrition-related cholestasis, or herbal products; metabolic causes (including hypothyroidism or panhypopituitarism); immune-related pregnancy-related alloimmune liver disease; anatomical obstruction (including biliary atresia, common bile duct cysts, cholelithiasis, biliary sludge, concentrated bile, spontaneous perforation of the common bile duct, or tumors); idiopathic neonatal hepatitis (transient neonatal cholestasis), cardiovascular and circulatory disorders, hemophagocytic lymphohistiocytosis, malignant tumors, or congenital lupus-related cholestasis; or hereditary and metabolic etiologies (α1 -Including antitrypsin (A1AT) deficiency, Alagille syndrome, joint contracture-renal dysfunction-cholestasis syndrome (ARC syndrome), Calori disease, congenital hepatic fibrosis, chromosomal trisomy 21, Turner syndrome, citrin deficiency, cystic fibrosis, bile acid synthesis disorders, bile acid conjugation disorders, fatty acid oxidation disorders, galactosemia, glycogen storage disorder type IV, hereditary fructose intolerance, mitochondrial respiratory chain complex deficiency, neonatal ichthyosis-sclerosing cholangitis syndrome, neonatal sclerosing cholangitis, Niemann-Pick disease type C, peroxisomal disorders, progressive familial intrahepatic cholestasis, lipid storage disorders, tyrosinemia, or urea cycle disorders). The AONs disclosed herein are designed to be used for all of the above disorders by preventing the accumulation of bile acids in the liver and the resulting cholestasis, in all the pharmaceutical formulations, uses, and therapeutic methods disclosed herein. AONs disclosed herein may also be used to treat infections caused by hepatitis B virus and hepatitis D virus by altering the NTCP structure to block uptake by hepatocytes.

[0030] [Definition] Whenever oligonucleotides, oligos, ONs, ASOs, oligonucleotide compositions, antisense oligonucleotides, AONs, (RNA)-edited oligonucleotides, EONs, and RNA (antisense) oligonucleotides are mentioned, they always mean both oligoribonucleotides and deoxyribonucleotides unless otherwise specified in the context. In some cases, oligonucleotides may be completely devoid of RNA and DNA nucleotides (in their naturally occurring forms) and consist of fully modified nucleotides. Whenever "oligoribonucleotides" are mentioned, they may contain the bases A, G, C, U, or I. Whenever "deoxyribonucleotides" are mentioned, they may contain the bases A, G, C, T, or I. However, AONs disclosed herein may contain mixtures of ribonucleotides and deoxyribonucleotides. When deoxyribonucleotides are used, i.e., without modification at the 2′ position of the sugar, the nucleotides are often abbreviated as dA (or Ad), dC (or Cd), dG (or Gd), or T, where "d" represents the deoxy status of the nucleoside. On the other hand, normal RNA or ribonucleosides modified at the 2′ position are often abbreviated without the "d" and often abbreviated with their respective modifications, as described herein.

[0031] The term "nucleoside" refers to a nucleic acid base bonded to a (deoxy)ribose that does not have a phosphate group. A "nucleotide" consists of a nucleoside and one or more phosphate groups. Thus, the term "nucleotide" refers to each nucleic acid base-(deoxy)ribose-phospholinker and any chemical modification of the ribose moiety or phospho group. Thus, this term includes: nucleotides containing a locked ribose moiety (containing a methylene group or any other group, including a 2′-4′ crosslink); unlocked nucleic acid (UNA); threose nucleic acid (TNA); phosphodiesters; phosphonoacetates; phosphotryesters; PS; phosphoro(di)thioates; MP (or MeP); methylthiophosphonates; phosphoramidate bonds; PNdmi; and nucleotides containing linkers containing bonds according to the structure of formula (I) as described herein. The terms nucleic acid base, nucleoside, and nucleotide may be used interchangeably unless the context clearly requires otherwise (for example, when a nucleoside is bonded to an adjacent nucleoside and the bond between these nucleosides is modified). As stated herein, a nucleotide is a nucleoside plus one or more phosphate groups. The terms “ribonucleoside” and “deoxyribonucleoside,” or “ribose” and “deoxyribose,” are used as they are used in the art.

[0032] The terms adenosine and adenine, guanosine and guanine, cytidine and cytosine, uracil and uridine, thymine and thymidine / uridine, inosine and hypoxanthine are sometimes used interchangeably, with one referring to the corresponding nucleic acid base and the other to the nucleoside or nucleotide. The nucleic acid base thymine (T) is 5-methyluracil (m 5 Also known as 5-methyluracil (U), it is a derivative of uracil (U); thymine and 5-methyluracil are interchangeable throughout this document. Similarly, nucleotide thymidine, also known as 5-methyluridine, is a derivative of uridine; thymidine and 5-methyluridine are interchangeable throughout this document.

[0033] Whenever a nucleotide is mentioned in relation to oligonucleotides, for example, cytosine, it includes 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-acetylcytosine, 5-hydroxycytosine, and β-D-glucosyl-5-hydroxymethylcytosine. Whenever adenine is mentioned, it includes N6-methyladenine, 8-oxo-adenine, 2,6-diaminopurine, and 7-methyladenine. Whenever uracil is mentioned, it includes dihydrouracil, isouracil, N3-glycosylated uracil, pseudouracil, 5-methyluracil, N1-methylpseudracil, 4-thiouracil, and 5-hydroxymethyluracil. Whenever guanine is mentioned, it includes 1-methylguanine, 7-methylguanosine, N2,N2-dimethylguanosine, N2,N2,7-trimethylguanosine, and N2,7-dimethylguanosine. Whenever nucleosides or nucleotides are mentioned, this includes ribofuranose derivatives such as 2′-deoxy, 2′-hydroxy, and 2′-O-substituted variants (e.g., 2′-O-methyl(2′-OMe)), as well as other modifications including 2′-4′ crosslinking variants. Whenever oligonucleotides are mentioned, one or more bonds may be naturally occurring phosphodiester bonds, while the remaining bonds between the two nucleotides may be modified bonds. Examples of such modified bonds include: phosphonoacetates, phosphotriesters, PS, phosphoro(di)thioates, MP, phosphoramidate bonds, phosphorylguanidine, thiophosphorylguanidine, sulfonophosphoramidates, PNdmi, and bond structures following formula (I), which are described in more detail below.

[0034] The term "comprising" encompasses "including" and "consisting of," for example, a composition "containing X" may consist only of X or it may include something additional (e.g., X + Y). The term "about" in relation to a numerical value x is arbitrary and means, for example, x ± 10%.

[0035] The term "substantially" does not exclude "completely"; for example, a composition "substantially free of Y" does not necessarily have to be completely free of Y. Where relevant, the term "substantially" may be omitted from the definitions of this invention.

[0036] The terms "conducive to" or "mediate" can be used interchangeably with the term "capable of facilitating." When used in the context of an AON that contributes to (or can mediate) ADAR editing, this means that after entering the cell, the AON interacts with the target RNA sequence, thereby forming a double-stranded structure that is recognized by the ADAR enzyme, which can then deaminate the target adenosine to inosine. Thus, although the AON itself does not possess the enzymatic function (of the ADAR enzyme), after binding to the target RNA molecule, it can induce, guide, cause, organize, mediate, donate, confer, produce, facilitate, and bring about RNA editing.

[0037] As used herein, the term “mismatch” refers to opposing nucleotides in a double-stranded RNA complex that do not form a complete base pair according to the Watson-Crick base pairing rules. In the traditional sense, mismatched nucleotides are GA, CA, UC, AA, GG, CC, and UU pairs. In some embodiments, the AONs disclosed herein include fewer than four mismatches with the target sequence, e.g., 0, 1, or 2 mismatches. “Wobble” base pairs are GU, IU, IA, and IC base pairs. When U is positioned opposite target A, there is no mismatch, and the AON can be 100% complementary. When C is positioned opposite target A, there is at least one mismatch between the AON and the target sequence. While G:G pair formation is considered a mismatch, this does not necessarily mean the interaction is unstable; in other words, the term "mismatch" may be somewhat outdated, given current disclosures that even when Hoogsteen base pairing is considered a mismatch based on nucleotide origin, it is still relatively stable. Solitary G:G pair formation within double-stranded RNA, for example, can be very stable but is still defined as a mismatch. Analysis of native targets of ADAR enzymes has shown that these generally contain mismatches between the two strands forming the RNA helix edited by ADAR1 or 2. It has been suggested that these mismatches enhance the specificity of the editing reaction (Stefl et al. 2006. Structure 14(2):345-355; Tian et al. 2011. Nucleic Acids Res 39(13):5669-5681). Characterizing the optimal pattern of pairing / mismatched nucleotides between AONs and target RNA also appears important for the development of efficient ADAR-based AON therapies.

[0038] As used herein, the term “complementary” refers to the fact that AON hybridizes with a second nucleic acid chain under physiological conditions. Examples include (i) AON forming a heterodouble-stranded RNA editing oligonucleotide complex with a second complementary nucleic acid chain (in vitro) as the first nucleic acid chain (= guide oligonucleotide), or (ii) AON forming a double-stranded complex with a target RNA molecule. This term does not necessarily mean that each nucleotide in the nucleic acid chain will perfectly pair with the opposing nucleotide in the opposing sequence. In other words, AON may be complementary to the target sequence, but mismatches, fluctuations, and / or bulges may exist between AON and the target sequence, while under physiological conditions AON may still hybridize with the target sequence, as a result allowing intracellular RNA editing enzymes to deaminate the target adenosine to inosine. Therefore, the term “substantially complementary” also means that, even if mismatches, fluctuations, and / or bulges are present, the AON has nucleotides that adequately match the target sequence, and as a result, the AON can hybridize with the target RNA molecule under physiological conditions. As shown herein, an AON may be complementary, but if the AON can hybridize with its target under physiological conditions, it may contain one or more mismatches, fluctuations, and / or bulges with the target sequence.

[0039] The term “orphan nucleotide” refers to a nucleotide in an AON that directly opposes a target adenosine, which is the adenosine deaminated by a deaminationase. The orphan nucleotide may be a natural cytidine or deoxycytidine, or a uridine or deoxyuridine. The orphan nucleotide may also be a chemically modified nucleotide, as will be described in more detail below, or a known or chemically modified analog of a natural (deoxy)cytidine (e.g., a nucleotide having Benner’s base), or a known or chemically modified analog of a natural (deoxy)uridine (e.g., isouridine), as will be described in more detail below.

[0040] A "nucleotide analog" refers to an analog of a nucleic acid nucleotide. Nucleotide analogs are analogs of adenosine, guanosine, cytidine, thymidine, uridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine, or deoxyuridine.

[0041] The term "downstream" in relation to nucleic acid sequences means continuing further along the sequence in the 3' direction; the term "upstream" means the opposite. Therefore, in any sequence encoding a polypeptide, the start codon is upstream of the stop codon in the sense strand but downstream of the stop codon in the antisense strand. The same applies to AONs disclosed herein. Nucleotides upstream of a solitary nucleotide in an antisense oligonucleotide are located toward the 5' end, and nucleotides downstream of a solitary nucleotide are located toward the 3' end.

[0042] The "numbering" of nucleotides in AONs disclosed herein is such that the lone nucleotide is number 0, and the nucleotide 5′ away from the lone nucleotide is number +1. The numbering further increases positively (+) toward the 5′ end and negatively (-) toward the 3′ end, where the first nucleotide 3′ away from the lone nucleotide is number -1. The numbering of nucleoside bonds in AONs is such that the number 0 bond is the bond 5′ away from the lone nucleotide, and the bond positions within the oligonucleotide increase positively (+) toward the 5′ end and negatively (-) toward the 3′ end.

[0043] References to "hybridization" typically refer to specific hybridization, excluding nonspecific hybridization. Specific hybridization can occur under experimental conditions selected using techniques known in the art, ensuring that the most stable interaction between the probe and target occurs when the probe and target have at least 70%, preferably at least 80%, and more preferably at least 90% sequence identity.

[0044] The term "splice mutation" refers to a mutation in a gene encoding pre-mRNA, where the splicing mechanism is disrupted, resulting in a malfunction in the splicing of introns from exons. This abnormal splicing causes subsequent translation to frame out, leading to the premature termination of the encoded protein. Such shortened proteins are often rapidly degraded and lack any functional activity.

[0045] Whenever the term "naked" form is referred to with respect to AONs disclosed herein, it means that the AON is manufactured in a laboratory or manufacturing facility and, throughout the process, is generally chemically modified to prevent rapid degradation after it has entered the body, tissues, or cells of a mammal via administration. Therefore, the naked form of AON is distinct from the form in which AON is encoded (and delivered) by a viral genome or within a plasmid vector. When such a viral or plasmid vector is administered, the encoded AON is expressed from the viral vector genome or plasmid within the cell to which the viral or plasmid vector is delivered. As a result, the AON consists only of naturally occurring RNA nucleotides that are subsequently unmodified.

[0046] The lengths of AONs disclosed herein when delivered in their naked form are preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides. However, when the AONs disclosed herein are delivered through the expression of a viral vector, the AONs may be longer, for example, 70, 80, 90, 100, 150, or 200 nucleotides or more.

[0047] The term "HEON" refers to a heteroduplex double-stranded complex molecule in which the AON disclosed herein is hybridized to a partially or completely complementary and partially or completely overlapping sense oligonucleotide. The AON disclosed herein often has specific chemical modifications different from those on the sense strand, so that the two strands form such a heteroduplex RNA editing oligonucleotide complex. The sense strand may be chemically modified almost entirely, and the modifications may be similar to or different from those performed on the AON disclosed herein, for example, by conferring a nucleotide containing a ribose moiety with a 2′-OMe substitution, a 2′-F substitution, or a 2′-MOE substitution. It should be understood that the sense strand present in the HEON is a distinct entity from the target RNA molecule in the cell. The sense strand within HEON is preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides long. HEON is often generated in vitro and used as a delivery tool to protect AON from degradation when administered to cells. In other words, HEON is preferably formed before AON is administered to cells.

[0048] AON is disclosed herein, wherein the AON is capable of recruiting an endogenous ADAR enzyme in human cells after the AON forms a double-stranded complex with a region of an intracellular target RNA nucleic acid molecule, the region comprising target adenosine, the nucleotide in the AON opposite the target adenosine being a lone nucleotide, the ADAR enzyme being able to deaminate the target adenosine to inosine after binding to the double-stranded complex, and the target RNA nucleic acid molecule being a transcript molecule of the human SLC10A1 gene encoding NTCP. Preferably, the transcript molecule is a pre-mRNA or mRNA molecule. Preferably, the cell is a hepatocyte, more preferably a hepatocyte. In one embodiment, the nucleotide numbering within the AON is such that the solitary nucleotide is number 0, and the nucleotides further increase positively (+) toward the 5′ end and negatively (-) toward the 3′ end, and the solitary nucleotide is a deoxynucleotide comprising cytosine, a cytosine analog, uracil, or isouracil. In one embodiment, the solitary nucleotide is a deoxynucleotide comprising a cytosine analog, the cytosine analog being a 6-amino-5-nitro-3-yl-2(1H)-pyridone nucleic acid base (also known as Benner's base). In one embodiment of the AON disclosed herein, the first nucleotide 3′ from the solitary nucleotide is deoxyinosine if the nucleotide opposite this position is cytidine in the target RNA nucleic acid molecule. In such cases, the target adenosine may be: a) located in the codon encoding glutamine at position 68, b) the first adenosine in the codon encoding glutamine at position 261, or c) the first adenosine in the codon encoding lysine at position 314. In one embodiment, the AON is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides long.In one embodiment, the AON comprises one or more modifications at the binding site, each independently selected from the group consisting of: PS, phosphonoacetate, phosphorodithioate, MP, sulfonylphosphoramidate, PNdmi, and PNms. In one embodiment, the numbering of the nucleoside bond in the AON is such that the 0th bond is a 5′ bond from the solitary nucleotide, and the binding positions within the oligonucleotide increase positively (+) toward the 5′ end and negatively (-) toward the 3′ end, and the -2nd binding position is an MP bond or a PNms bond. In one embodiment, the bond between the two terminal nucleotides at the 5′ and / or 3′ ends of the AON is a PNdmi bond or a PNms bond. In one embodiment, the AON comprises one or more nucleotides including one or two substitutions at the 2′, 3′, and / or 5′ positions of ribose, each independently selected from the group consisting of: -OH; -F; substituted or unsubstituted, linear or branched lower (C1-C). 10)alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, which may be interrupted by one or more heteroatoms;-O-, S-, or N-alkyl;-O-, S-, or N-alkenyl;-O-, S-, or N-alkynyl;-O-, S-, or N-allyl;-O-alkyl-O-alkyl;-methoxy;-aminopropoxy;-methoxyethoxy;-dimethylaminooxyethoxy; and-dimethylaminoethoxyethoxy. In one embodiment, the AON is covalently or noncovalently bound to the GalNAc moiety, directly or through a linker. Those skilled in the art can select the appropriate linker and the need for covalent or noncovalent binding of the GalNAc moiety when it is necessary to deliver the AON to hepatocytes, particularly hepatic parenchymal cells. In one embodiment, the AON is covalently or noncovalently bound to a triterpene glycoside, preferably AG1856, directly or through a linker. As described in PCT / EP2024 / 051278 (unpublished), when AON is linked to a saponin in a 1:1 ratio, particularly when the saponin is AG1856, it is highly efficient in increasing RNA editing. Therefore, in order to increase the endosomal release of AON (intracellularly) and make it available for hybridization with RNA targets, it is preferable that the AON be attached (covalently or noncovalently) to AG1856 before being administered to cells or therapeutic targets.

[0049] AON is disclosed herein, wherein the AON is capable of recruiting an endogenous ADAR enzyme in human cells after the AON forms a double-stranded complex with a region of an intracellular target RNA nucleic acid molecule, the region comprising target adenosine, the nucleotide in the AON opposite the target adenosine being a lone nucleotide, the ADAR enzyme being capable of deaminating the target adenosine to inosine after binding to the double-stranded complex, the SLC10A1 gene being wild-type, and the target adenosine being selected from the group consisting of: (i) Adenosine in the CAG codon encoding glutamine (Q) at position 68 of the NTCP protein, wherein the adenosine is deaminated to change the amino acid to arginine (R); (ii) The first adenosine in the CAA codon encoding glutamine (Q) at position 261 of the NTCP protein, wherein the adenosine is deaminated to change the amino acid to arginine (R); (iii) Adenosine in the GAG ​​codon encoding glutamic acid (E) at position 257 of the NTCP protein, wherein the adenosine is deaminated to change the amino acid to glycine (G); and (iv) The first adenosine in the AAG codon encoding lysine (K) at position 314 of the NTCP protein, wherein the adenosine is deaminated to glutamic acid (E); Here, the deamination of the target adenosine results in an NTCP protein whose function of transporting bile acids from the portal circulation into the cell is impaired. In a preferred embodiment, the target adenosine is located in the CAG codon encoding glutamine at position 68 of the NTCP protein, and the AON includes or consists of sequences and modifications selected from the group consisting of the following sequence numbers: 150, 151, 152, 154, 156, 158, 159, 163, 164, 165, 166, 1127, 1128, 1129, 1130, 1131, 1133, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1 150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1180, 1181, 1182, 1183, 1260, 1283, 1284, 1285, 1286, 1287, 1288, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, and 1306. In another preferred embodiment, the target adenosine is located within a GAG codon encoding glutamic acid at position 257 of the NTCP protein, and the AON includes or comprises sequences and modifications selected from the group consisting of the following sequence numbers: 1193, 1194, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210 , 1211, 1212, 1213, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1253, and 1254.This disclosure also relates to vectors, preferably viral vectors, and more preferably adeno-associated virus (AAV) vectors, which include nucleic acid molecules encoding the AON disclosed herein. When an encoding vector is applied and the AON is encoded by a viral vector (genome), the lone nucleotide is cytidine and the nucleotide at position -1 is guanosine, when the opposing nucleotide in the target sequence is cytidine. Furthermore, since the AON is transcribed from a viral vector (genome), there are no chemical modifications to the backbone, binding, and sugar ribose. This disclosure also relates to nanoparticle delivery vehicle formulations, which include the AON disclosed herein. In a preferred embodiment, the nanoparticle delivery vehicle is lipid nanoparticles (LNPs). LNPs that may be used in the context of the AON of this disclosure have been used in the art for the delivery of small and large RNA molecules, and are applied to the delivery of mRNA-based vaccines, such as those for the Covid-19 coronavirus. LNPs that have been applied to the delivery of other types of RNA, such as siRNA, may also be applied to the delivery of the AON disclosed herein. Even when an LNP or other similar type of carrier is applied, AON is still considered naked because it is not transcribed from the encoding polynucleotide (for example, in the case of a plasmid or vector, AON is not considered "naked"). Therefore, even if a chemically modified AON is encapsulated by a carrier, preferably an LNP, it is still considered naked because it is produced in that form in a laboratory setting and subsequently encapsulated in a carrier using methods known to those skilled in the art.This disclosure also relates to a delivery medium, preferably LNP, which includes “bare” and chemically modified AONs, disclosed herein, more preferably in any of the following sequence numbers: 150, 151, 152, 154, 156, 158, 159, 163, 164, 165, 166, 1127, 1128, 1129, 1130, 1131, 1133, 1137, 1138, 1139, 1 140, 1141, 1142, 1143, 1144, 1145, 1146, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1180, 1181, 1182, 1183, 1260, 12 83, 1284, 1285, 1286, 1287, 1288, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1193, 1194, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 12 13, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1253, and 1254. This disclosure also relates to pharmaceutical compositions comprising AONs, vectors, or nanoparticle delivery medium formulations disclosed herein, and pharmaceutically acceptable carriers. In one embodiment, the AONs disclosed herein are in a naked form. In one embodiment, the AONs disclosed herein are in a cyclic form. In one embodiment, the AON disclosed herein is expressed from the genome of a viral vector, rather than in a naked form. In another embodiment, the AON disclosed herein is expressed from an expression vector such as a plasmid, rather than in a naked form.In one embodiment, when the AON disclosed herein is not in a naked form, the AON is 15 to 60 nucleotides long as described above, or in another embodiment, 61 to 300 nucleotides long. It should be noted that when the AON is delivered via a vector (e.g., an AAV vector), the AON acting on the target RNA molecule is not chemically modified. While it is preferred to use a “naked” AON having chemical modifications as outlined herein, AONs delivered by other means (e.g., via AAV vector expression), or edited molecules having a circular or hairpin structure (recruited portion, e.g., disclosed in WO2016 / 097212, WO2017 / 050306, WO2020 / 001793, WO2017 / 010556, WO2020 / 246560, and WO2022 / 078995) are also included in this disclosure. This is because these can also be applied to edit adenosine in the target SLC10A1 RNA molecule to produce a deactivated NTCP protein. Those skilled in the art will understand that even when a delivery portion or adduct to the AON (e.g., a GalNAc portion targeting hepatocytes in the liver) is used, or when the GalNAc-AON is encapsulated in a delivery medium such as an LNP, the AON is still considered naked. This disclosure also relates to AONs disclosed herein for use in the treatment of diseases caused by the accumulation of bile in the liver, such as cholestasis, primary sclerosing cholangitis (PSC), biliary atresia (BA), and cirrhosis. This disclosure also relates to the use of AONs disclosed herein for use in the manufacture of therapeutic pharmaceuticals for diseases caused by the accumulation of bile in the liver, such as cholestasis, PSC, BA, and cirrhosis.This disclosure also relates to a method for editing a human SLC10A1 pre-mRNA or mRNA molecule in hepatocytes, preferably hepatic parenchymal cells, the method comprising the step of contacting the SLC10A1 pre-mRNA or mRNA molecule with an AON capable of inducing ADAR-mediated adenosine-to-inosine deamination, thereby editing the SLC10A1 pre-mRNA or mRNA molecule to encode an NTCP protein whose function in bile acid uptake is attenuated, reduced, or lost, and the AON is disclosed herein. This disclosure also relates to a method for treating, improving, or delaying the progression of diseases caused by the accumulation of bile in the liver, such as cholestasis, PSC, BA, and cirrhosis, in human subjects in need thereof, the method comprising the step of administering to the subject an AON, vector, or nanoparticle delivery medium formulation disclosed herein, thereby hybridizing the AON with a complementary portion of a region containing target adenosine in the subject's cells, thereby causing ADAR-mediated deamination of the target adenosine from adenosine to inosine, thereby altering the SLC10A1 pre-mRNA or mRNA molecule to encode an NTCP protein whose function in bile acid uptake is weakened, reduced, or lost, thereby treating the subject. RNA editing can, in principle, be forced to occur at various locations in the SLC10A1 transcript. Examples of target adenosines are disclosed herein, with preference being adenosine in the CAG codon encoding glutamine (Q) at position 68 of the NTCP protein, a first adenosine in the CAA codon encoding glutamine (Q) at position 261, adenosine in the GAG ​​codon encoding glutamic acid (E) at position 257, and a first adenosine in the AAG codon encoding lysine (K) at position 314. Particularly preferred are the AONs disclosed herein and methods of using these AONs that induce Q68R and / or E257G changes in the human NTCP protein.Furthermore, in vitro, ex vivo, or in vivo methods for deaminating a target adenosine within a human SLC10A1 pre-mRNA or mRNA molecule in hepatocytes, preferably hepatenchymal cells, are disclosed, said methods comprising each of the following steps:. (i) providing to said cells an AON disclosed herein; (ii) allowing said cells to take up said AON; (iii) annealing said AON to said SLC10A1 pre-mRNA or mRNA molecule; (iv) allowing an endogenous ADAR enzyme to deaminate said target adenosine within said SLC10A1 pre-mRNA or mRNA molecule to inosine; Furthermore, (v) may include identifying the presence of said inosine within said SLC10A1 pre-mRNA or mRNA molecule using functional readout. In one aspect, the methods disclosed herein include administering a triterpenoid glycoside before, after, or simultaneously with the administration of said AON, where in a preferred aspect, said triterpenoid glycoside is AG1856. However, in a preferred aspect, said triterpenoid glycoside (or, in many cases, what is referred to as a "saponin") is physically bound to said AON. In one aspect, the AONs disclosed herein include a linking moiety having a structure according to formula (I): JPEG2026518820000001.jpg53166where: X = O or S; and R = aryl, substituted aryl, heterocycle, substituted heterocycle, aromatic heterocycle, substituted aromatic heterocycle, C1-C6 alkoxy, substituted C1-C6 alkoxy, C1-C 20 alkyl, substituted C1-C 20The group is an alkyl, C1-C6 alkenyl, substituted C1-C6 alkenyl, C1-C6 alkynyl, substituted C1-C6 alkynyl, or conjugated group. In preferred embodiments, X=O and R=methyl, and the bond modification is referred to as a mesylphosphoramidate (MsPA or "PNms"). In one embodiment, the PNms bond is used instead of the MP and / or PNdmi bond.

[0050] [Chemical modification] Various chemical structures and modifications that can be readily used in accordance with the present invention are known in the field of oligonucleotides. All chemical modifications listed herein that can be used for AONs disclosed herein may also be used for a sense strand complementary to AON when AON and complementary strands form a HEON complex as described in GB2215614.5 (unpublished) and disclosed above, except that the opposing sense strand does not have a lone nucleotide. Thus, modifications relating to lone nucleotides relate only to AONs disclosed herein, while all other modifications relate to AONs disclosed herein and any (protective) sense oligonucleotides that can be used with AON in pharmaceuticals. This includes the use of hydrophobic moieties (e.g., tocopherol and cholesterol) and cell-specific ligands (e.g., GalNAc moieties), which are also described in detail herein and in PCT / EP2023 / 079290 (unpublished) and can be bound to AON or its opposing strand, or both. Preferred GalNAc portions that may be used in the context of AON disclosed herein are disclosed in WO2022 / 271806.

[0051] Those skilled in the art will know that oligonucleotides such as AONs outlined herein generally consist of repeating monomers. Such monomers are, in most cases, nucleotides or chemically modified nucleotides. The most common naturally occurring nucleotides in RNA are adenosine monophosphate (A), cytidine monophosphate (C), guanosine monophosphate (G), and uridine monophosphate (U). These consist of a pentose sugar (ribose), a 5′-linked phosphate group (which is linked via a phosphate ester), and a 1′-linked base. This sugar is often referred to as the "scaffold" of the nucleotide because it links the base and the phosphate group. Thus, modifications in pentose sugars are often referred to as "scaffold modifications." The original pentose sugar can be completely replaced by another moiety that similarly links the base and the phosphate group. Thus, it is understood that while pentose sugars are often scaffolds, scaffolds do not necessarily have to be pentose sugars. Examples of scaffold modifications applicable to the monomers of AON disclosed herein are disclosed in WO2020 / 154342, WO2020 / 154343, and WO2020 / 154344.

[0052] The nucleosides in AONs disclosed herein may be natural nucleosides (deoxyribonucleosides or ribonucleosides) or unnatural nucleosides. In RNA editing, since double-stranded RNA generally serves as a substrate for enzymes with deamination activity (e.g., ADAR), ribonucleosides are considered "natural," while deoxyribonucleosides may, for convenience of discussion, be considered unnatural or modified. This is because DNA does not exist in the RNA-RNA double-stranded (natural) substrate configuration. Those skilled in the art understand that even if a nucleotide has a natural ribose moiety, the base and / or binding may be unnaturally modified.

[0053] Common limiting factors in oligonucleotide-based therapies are recognized in the art as the ability of oligonucleotides to be taken up by cells (when delivered "naked" without the application of a delivery medium such as a viral vector or plasmid), their biodistribution, and their resistance to nuclease-mediated degradation. Those skilled in the art recognize that various chemical modifications can help overcome such limitations, and these have been reported in detail in the art. Examples of such chemical modifications currently in common use include the 2′-OMe, 2′-F, and 2′-MOE modifications of sugars, and the use of PS bonds between nucleosides, as disclosed herein.

[0054] [Scaffolding modification (ribose)] The ribose 2′ group in all nucleotides of the AON disclosed herein may be independently selected from the following, except for the ribose sugar moiety of isolated nucleotides, which has certain limitations with respect to compatibility with RNA editing: 2′-H (i.e., DNA), 2′-OH (i.e., RNA), 2′-OMe, 2′-MOE, 2′-F, or 2′-4′ bond (e.g., LOK nucleic acid (LNA)), or other 1′, 2′, 3′, 4′, or 5′ ribose substitutions. Isolated nucleotides in the AON that do not contain ribose sugar, base, or other chemical modifications to the bond preferably do not have 2′-OMe or 2′-MOE substitutions, but may have 2′-F, 2′,2′-difluoro (diF), or 2′-ara-F (FANA) substitutions, or may be DNA, when the nucleic acid base is naturally occurring cytosine. WO2024 / 013360 discloses the modification of the 2′ position of the ribose sugar moiety of an isolated nucleotide by a 2′,2′-disubstituted modification such as diF, which is also applicable to those disclosed herein. The 2′-4′ bond can be selected from many linkers known in the art, such as methylene linkers, amide linkers, or restricted ethyl linkers (cEt).

[0055] The AONs disclosed herein may comprise one or more nucleotides having a 2′-MOE ribose modification. Alternatively, the AONs disclosed herein may comprise one or more nucleotides not having a 2′-MOE ribose modification, or the 2′-MOE ribose modification may be located at a position that does not interfere with the deamination of target adenosine by an enzyme having adenosine deaminationase activity. The AONs disclosed herein may comprise a 2′-OMe ribose modification at a position that does not contain a 2′-MOE ribose modification. The AONs disclosed herein may comprise a deoxyribonucleotide at a position that does not contain a 2′-MOE or 2′-OMe ribose modification, or any other 2′-ribose substitution. The AONs disclosed herein may comprise one or more nucleotides comprising 2′ substitutions including 2′-MOE, 2′-OMe, 2′-OH, 2′-deoxy, TNA, 2′-fluoro(2′-F), 2′,2′-difluoro(diF) modification, 2′-fluoro-2′-C-methyl modification, or 2′-4′ linkages (i.e., crosslinked nucleic acids such as locked nucleic acids (LNA, i.e., examples described in WO2018 / 007475, for example). Other nucleic acid monomers that may be used in the AONs disclosed herein include, for example, arabino nucleic acids and 2′-deoxy-2′-fluoroarabino nucleic acids (FANA) for improved affinity. The 2′-4′ linkages may be selected from linkers known in the art, such as methylene linkers or restricted ethyl linkers. A wide variety of 2′ modifications that may be present in AONs disclosed herein include, but are not limited to, those known in the art and described in detail below: WO2016 / 097212, WO2017 / 220751, WO2018 / 041973, WO2018 / 134301, WO2019 / 219581, WO2019 / 158475, and WO2022 / 099159. In all cases, the modification must be compatible with RNA editing, and as a result the AON acts as an oligonucleotide that forms a double-stranded complex with the target RNA and, by generating this double-stranded nucleic acid complex, recruits a deaminationase so that the enzyme can subsequently deaminate the target adenosine.If a monomer in an AON disclosed herein contains an unlocked nucleic acid (UNA) ribose modification, the monomer may have a 2′ position containing the same modifications described above, such as 2′-MOE, 2′-OMe, 2′-OH, 2′-deoxy, 2′-F, 2′,2′-diF, 2′-fluoro-2′-C-methyl, arabino nucleic acid, FANA, or a 2′-4′ bond (i.e., a cross-linked nucleic acid such as Loc nucleic acid (LNA)). In one embodiment, an AON disclosed herein comprises at least one nucleotide containing a threose nucleic acid (TNA) ribose modification. In one embodiment, an AON disclosed herein comprises at least one nucleotide having a sugar moiety containing a 2′-fluoro(2′-F) modification. A preferred position for the nucleotide having the 2′-F modification is the -3 position in the AON, which may be present together with the same 2′ modifications described above in the isolated nucleotide.

[0056] [Base modification] Nucleic acid bases (sometimes called nucleic acid bases) are generally adenine, cytosine, guanine, thymine, or uracil, or their derivatives. Nucleic acid bases are defined as moieties that can bind to another nucleic acid base through hydrogen bonds, polar bonds (e.g., through the CF moiety), or aromatic electron interactions. Cytosine, thymine, and uracil are pyrimidine bases and are generally bound to the scaffold via their 1-nitrogen. Adenine and guanine are purine bases and are generally bound to the scaffold via their 9-nitrogen. As used herein, the terms “adenine,” “guanine,” “cytosine,” “thymine,” “uracil,” and “hypoxanthine” refer to the nucleic acid bases themselves. The terms “adenosine,” “guanosine,” “cytidine,” “thymidine,” “uridine,” and “inosine” refer to nucleic acid bases bound to (deoxy)ribose. The nucleic acid bases in the AON disclosed herein may be adenine, cytosine, guanine, thymine, or uracil, or any other moiety capable of interacting with another nucleic acid base through hydrogen bonds, polar bonds (e.g., CF), or aromatic electron interactions.Any nucleic acid base at any position within AON disclosed herein may be a modified form of adenine, cytosine, guanine, or uracil, such as: hypoxanthine (nucleic acid base in inosine), pseudouracil, pseudocytosine, isouracil, N3-glycosylated uracil, 1-methylpseuduracil, orotic acid, agmatidine, lysidine, 2-thiouracil, 2-thiothymine, 5-substituted pyrimidines (e.g., 5-halouracil, 5-halomethyluracil, 5-trifluoromethyluracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-formyluracil, 5-aminomethylcytosine, 5-formylcytosine), 5-hydroxymethylcytosine, 7-deazaguanine, 7-deazaadenine, 7 -Deaza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, 8-oxo-adenine, 3-deazapurine (e.g., 3-deaza-adenosine), pseudoisocytosine, N4-ethylcytosine, N2-cyclopentylguanine, N2-cyclopentyl-2-aminopurine, N2-propyl-2-aminopurine, 2,6-diaminopurine, 2-aminopurine, G-clamp and its derivatives, Super A, Super T, Super G, amino-modified nucleic acid bases or their derivatives; and denatured or universal bases (e.g., 2,6-difluorotoluene), or deletions such as abasic sites (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose, azaribose). Modified bases include synthetic and native bases, such as inosine, xanthine, hypoxanthine, and other -aza, deaza, -hydroxy, -halo, -thio, thiol, -alkyl, -alkenyl, -alkynyl, and thioalkyl derivatives of pyrimidine and purine bases, which are known or will be known in the art. Purine nucleic acid bases and / or pyrimidine nucleic acid bases may be modified, for example, by heterocyclic amination or deamination, to alter their properties.The exact chemical structure and form may vary from oligonucleotide construct to construct and application to application, and can be resolved according to the wishes and preferences of those skilled in the art.

[0057] Scaffold modifications refer to the presence of modified forms of naturally occurring ribose moieties (i.e., pentose moieties) in RNA, such as bicyclic sugars, tetrahydropyrans, hexoses, morpholinos, 2′-modified sugars, 4′-modified sugars, 5′-modified sugars, and 4′-substituted sugars. Examples of appropriate modifications include, but are not limited to, 2′-O-modified RNA monomers, e.g., 2′-O-alkyl or 2′-O-(substituted)alkyl (e.g., 2′-OMe, 2′-O-(2-cyanoethyl), 2′-MOE, 2′-O-(2-thiomethyl)ethyl, 2′-O-butyryl, 2′-O-propargyl, 2′-O-allyl, 2′-O-(2-aminopropyl), 2′-O-(2-(dimethylamino)propyl) Pyr), 2′-O-(2-amino)ethyl, 2′-O-(2-(dimethylamino)ethyl)); 2′-deoxy(DNA); 2′-O-(haloalkyl)methyl (e.g., 2′-O-(2-chloroethoxy)methyl (MCEM), 2′-O-(2,2-dichloroethoxy)methyl (DCEM)); 2′-O-alkoxycarbonyl (e.g., 2′-O-[2-(methoxycarbonyl)ethyl](MOCE), 2′-O-[2- N-methylcarbamoyl)ethyl](MCE), 2′-O-[2-(N,N-dimethylcarbamoyl)ethyl](DCME)); 2′-halo(e.g., 2′-F,FANA); 2′-O-[2-(methylamino)-2-oxoethyl](NMA); scaffold modifications of bicyclic or cross-linked nucleic acids (BNAs), e.g., conformation-restricted nucleotide (CRN) monomers, lock nucleic acid (LNA) monomers, xylo-LNA monomers, α-LNA mono BNA monomer, α-l-LNA monomer, β-d-LNA monomer, 2′-amino-LNA monomer, 2′-(alkylamino)-LNA monomer, 2′-(acylamino)-LNA monomer, 2′-N-substituted 2′-amino-LNA monomer, 2′-thio-LNA monomer, (2′-O,4′-C)-restricted ethyl (cEt)BNA monomer, (2′-O,4′-C)-restricted methoxyethyl (cMOE)BNA monomer, 2′,4′-BNA NC(NH) monomer, 2′,4′-BNA NC (NMe) monomer, 2′,4′-BNA NC (NBn) monomer, ethylene-bridged nucleic acid (ENA) monomer, carba-LNA (cLNA) monomer, 3,4-dihydro-2H-pyran nucleic acid (DpNA) monomer, 2′-C-bridged bicyclic nucleotide (CBBN) monomer, oxo-CBBN monomer, heterocyclic-bridged BNA monomer (e.g., triazolyl or tetrazolyl linked), amide-bridged BNA monomer (e.g., AmNA), urea-bridged BNA monomer, sulfonamide-bridged BNA monomer, bicyclic carbocyclic nucleotide monomer, TriNA monomer, α-l-TriNA monomer, bicyclic DNA (bcDNA) monomer, F-bcDNA monomer, tricyclic DNA (tcDNA) monomer, F-tc DNA monomers, α-anomeric bicyclic DNA (abcDNA) monomers, oxetane nucleotide monomers, locked PMO monomers derived from 2′-amino-LNA, guanidine-bridged nucleic acid (GuNA) monomers, spirocyclopropylene-bridged nucleic acid (scpBNA) monomers, and their derivatives; cyclohexenyl nucleic acid (CeNA) monomers, altriol nucleic acid (ANA) monomers, hexitol nucleic acid (HNA) monomers, fluorinated HNA (F-HNA) monomers, pyranosyl RNA (p-RNA) monomers, 3′-deoxypyranosyl DNA (p-DNA), unlocked nucleic acid (UNA); inverted forms of any of the above monomers. All of these modifications are known to those skilled in the art.

[0058] [isolated nucleotides] Mutagenesis studies of human ADAR2 have shown that a single mutation from glutamic acid to glutamine at residue 488 (E488Q) increases the deamination rate constant by 60-fold compared to the wild-type enzyme (Kuttan and Bass. Proc Natl Acad Sci USA 2012. 109(48):3295-3304). During the deamination reaction, ADAR flips the base to be edited from its RNA double helix and places it in the enzyme active site (Matthews et al. Nat Struct Mol Biol. 2016. 23(5):426-433). When ADAR2 edits adenosine in a preferred context (A:C mismatch), the nucleotide opposite the target adenosine is often referred to as a "lone nucleotide" (or sometimes "lone cytidine"), as described above. The crystal structure of ADAR2 E488Q bound to double-stranded RNA (dsRNA) revealed that the glutamine (Gln;Q) side chain at position 488 can donate a hydrogen bond to the N3 position of isolated cytidine, which leads to an improvement in the catalytic rate of ADAR2 E488Q. In the wild-type enzyme, glutamic acid (Glu;E) is present at position 488 instead of glutamine (Gln), and the amide group of glutamine is absent, replaced by a carboxylic acid. Therefore, in order to obtain the same contact as with isolated cytidine using the E488Q mutant in the wild-type environment, protonation is necessary for this contact to occur. When using endogenously expressed ADAR2 to correct disease-associated mutations, it is important to maximize the editing efficiency of the wild-type ADAR2 enzyme present in the cell. WO2020 / 252376 discloses the use of AON, specifically with modified RNA bases at the isolated cytidine position, to mimic the hydrogen bonding pattern observed by the E488Q ADAR2 mutant. It was hypothesized that the same contact, which is thought to improve catalytic rate in the mutant enzyme, could be stabilized by replacing the nucleotide opposite the target adenosine in the AON with a cytidine analog that functions as a hydrogen bond donor at N3.Two cytidine analogs were of particular interest: pseudoisocytidine (also known as "piC"; Lu et al. J Org Chem 2009. 74(21):8021-8030; Burchenal et al. (1976) Cancer Res 36:1520-1523) and Benner's base Z (also known as "dZ"; Yang et al. Nucleic Acid Res 2006. 34(21):6095-6101), which were initially selected because they donate a hydrogen bond at N3 with minimal perturbation to the shape of the nucleic acid base. Benner's base is also known as the 6-amino-5-nitro-3-yl-2(1H)-pyridone nucleic acid base. The presence of cytidine analogs in AONs can occur in addition to modifications to the ribose 2′ group. The ribose 2′ group within an isolated nucleotide can be independently selected from: 2′-H (i.e., DNA), 2′-OH (i.e., RNA), 2′-OMe, 2′-MOE, 2′-F, or a 2′-4′ bond (i.e., a crosslinked nucleic acid such as a locked nucleic acid (LNA)), or other 2′ substituents. The 2′-4′ bond can be selected from linkers known in the art, such as a methylene linker or a restricted ethyl linker.

[0059] The isolated nucleotides in the AONs disclosed herein are preferably cytidine or its analogues (e.g., nucleotides having Benner's base), or uridine or its analogues (e.g., isouridine). Whether the isolated nucleotide is cytidine or its analogue, or uridine or its analogue, it preferably contains deoxyribose (2′-H;=DNA), but may also contain a diF modification at the 2′ position of the sugar. In some embodiments, at least one of the adjacent (directly adjacent) nucleotides flanking the isolated nucleotide, and in other embodiments, both, do not contain the 2′-OMe modification. Complete modification of the oligonucleotide, in which all nucleotides (including the isolated nucleotide) have the native base while retaining the 2′-OMe modification, results in an oligonucleotide that does not function with respect to RNA editing (as is known in the art), presumably because it interferes with ADAR activity at the target site. Generally, adenosine within target RNA can be protected from editing by providing a 2′-OMe group to the opposing nucleotide (at least if no other chemical substitutions or modifications exist within the nucleotide), or by providing guanine or adenine as the opposing base, because these two nucleic acid bases can also reduce the editing of the opposing adenosine.

[0060] [Bond modification] Nucleosides are generally linked to adjacent nucleosides through the condensation of their 5′-phosphate moiety to the 3′-hydroxyl moiety of the adjacent nucleotide monomer. Similarly, their 3′-hydroxyl moiety is generally linked to the 5′-phosphate of the adjacent nucleotide monomer. This forms a phosphodiester bond. The phosphodiester and scaffold form an alternating copolymer. Bases are bonded to this copolymer, i.e., to the scaffold portion. Because of this characteristic, the alternating copolymer formed by the linked scaffolds of oligonucleotides is often called the "backbone" of the oligonucleotide. Since phosphodiester bonds link adjacent monomers, these are often referred to as "backbone linkages." Even when the phosphate group is modified and replaced with an analogous portion such as a phosphorothioate, such a portion is still understood to be a backbone linkage of the monomer. This is called a "backbone linkage modification." In a general sense, the backbone of an oligonucleotide contains alternating scaffolds and backbone linkages.

[0061] As described in detail herein, the naked AONs disclosed herein include at least one, preferably more, binding modifications. Generally, the AONs disclosed herein more preferably include binding modifications at most, and possibly all, positions if the AON can mediate RNA editing by deaminationases when it binds to a target RNA nucleic acid molecule. The binding modifications may be, but are not limited to, modified forms of phosphodiesters present in RNA, such as: phosphorothioates (PS), chiral pure PS, (R)-PS, (S)-PS, methylphosphonates (MP or MeP), chiral pure MP, (R)-MP, (S)-MP, phosphorylguanidine (e.g., PNdmi), chiral pure phosphorylguanidine, (R)-phosphorylguanidine, (S)-phosphorylguanidine, phosphorodithioates (PS2), and phosphonacetates. (PACE), phosphonoacetamide (PACA), thiophosphonoacetate, thiophosphonoacetamide, methylphosphorothioate, methylthiophosphonate, PS prodrug, alkylated PS, H-phosphonate, ethyl phosphate, ethyl PS, boranophosphate, borano PS, methylboranophosphate, methylborano PS, methylboranophosphonate, methylboranophosphothioate, phosphate, phosphotriester, aminoalkylphosphotriester, and derivatives thereof. Other modifications include: phosphoramidites, phosphoramidates, N3′→P5′ phosphoramidates, phosphorodiamidates, phosphorothiodiamidates, sulfamates, diethylene sulfoxides, amides, sulfonates, siloxanes, sulfides, sulfones, formacetyl, alkenyls, methylenehydrazinos, sulfonamides, triazoles, oxalyls, carbamates, methyleneiminos (MMIs), and thioacetamide nucleic acids (TANAs); and their derivatives. Various salts, mixed salts, deprotonated, protonated, tautomers, and free acid forms are also included, as are 3′→3′ and 2′→5′ links.The AONs disclosed herein may also include one or more binding modifications following the structure of formula (I), (II), (III), (IV), or (V).

[0062] In a preferred embodiment, the AON disclosed herein comprises an internucleoside bond of the structure of formula (I), where X=O and R=CH3, the bond being commonly referred to herein as a PNms bond. In another preferred embodiment, R is equal to one of the following structures (a), (b), (c), (d), (e), (f), (g), (h), or (i): JPEG2026518820000002.jpg156166

[0063] The one or more PN bonds shown in formula (I) present in the AON disclosed herein are independent of each other, R P or S P It may have chirality, or it may be three-dimensionally disordered.

[0064] In the AONs disclosed herein, one or more PN bonds illustrated in formula (I) may be tautomers and / or pH-dependent (de)protonated forms, including but not limited to structures (A), (B), (C), (D), and (E): JPEG2026518820000003.jpg53166JPEG2026518820000004.jpg57166JPEG2026518820000005.jpg51166JPEG2026518820000006.jpg55166JPEG2026518820000007.jpg56166Here, X and R are as shown above for equation (I).

[0065] AON is disclosed herein, which is capable of mediating the deamination of adenosine by recruiting a deaminationase in the cell after the AON forms a double-strand complex with a region of a target RNA nucleic acid molecule in the cell, wherein the region comprises target adenosine, the deaminationase is capable of deaminating the target adenosine to inosine, and the AON comprises a portion having a structure according to formula (II) at one or both ends: JPEG2026518820000008.jpg42166 where: X=O or S; Y=O - or S - and R = aryl, substituted aryl, heterocycle, substituted heterocycle, aromatic heterocycle, substituted aromatic heterocycle, C1-C6 alkoxy, substituted C1-C6 alkoxy, C1-C 20 Alkyl, substituted C1-C 20 The group is an alkyl, C1-C6 alkenyl, substituted C1-C6 alkenyl, C1-C6 alkynyl, substituted C1-C6 alkynyl, or conjugated group. In preferred embodiments, X=O and R=methyl. A preferred nucleoside bond modification used in the AON disclosed herein has the structure of formula (III): JPEG2026518820000009.jpg54166 This is also known as PNms merging. The application of PNms binding, its use within oligonucleotides, and its use as an alternative to PS binding are described below (Chelobanov BP et al. Russian J Bioorganic Chemistry. 2017. 43(6):664-668; DOI: 10.1134 / S1068162017060024; Klabenkova K et al. Molecules. 2021. 26(17):5420; Miroshnichenko SK et al. Proc Natl Acad Sci USA. 2019. 116(4):1229-1234). For example, its application within oligonucleotides that can provide splice switching is described below (Hammond SM et al. Nucleic Acid Ther. 31(3):190-200).

[0066] The AONs disclosed herein may include the substitution of one non-crosslinked oxygen in the phosphodiester bond. This modification slightly destabilizes base pairing but confers significant resistance to nuclease degradation. Preferred nucleotide analogs or equivalents include: PS, phosphonoacetates, phosphorodithioates, phosphotryesters, aminoalkylphosphotryesters, H-phosphonates, methyl and other alkylphosphonates (including 3′-alkylenephosphonates, 5′-alkylenephosphonates, and chiral phosphonates), phosphinates, phosphoramidates (including 3′-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotryesters, selenophosphates, or boranophosphates. Particularly preferred are nucleoside bonds modified to include PS. Particularly preferred are nucleoside bonds modified to include PNms. Particularly preferred are nucleoside-to-nucleoside bonds modified to include PNdmi. Conventional nucleoside-to-nucleoside bonds between nucleotides can be altered by mono- or dithiolation of phosphodiester bonds to produce PS esters or phosphorodithioate esters, respectively. Other modifications of nucleoside-to-nucleoside bonds are possible, including amidation and peptide linkers. Those skilled in the art can determine which binding modifications the AON should include at each of the AON binding sites disclosed herein for which target RNA nucleic acid molecules, in order to produce the most effective and stable oligonucleotide compounds.

[0067] Many unnatural binding modifications (e.g., PS) are chiral. This means that Rp and Sp configurations exist, which are known to those skilled in the art. In one embodiment, the chirality of the PS binding is controlled, meaning that each binding is either the preferred Rp or Sp configuration. The choice of Rp or Sp configuration at a particular binding site may depend on the target sequence, binding efficiency, and induction efficiency that causes RNA editing of the target adenosine. However, if such is not particularly desired, the composition may contain an AON having both Rp and Sp configurations at a particular binding site as the active compound. Mixtures of AONs are also possible in which one configuration is preferred at some sites, but not at others. In one embodiment, the AON disclosed herein comprises one or more (chiral pure or chiral mixed) PS bindings. In one embodiment, the AON disclosed herein comprises one or more (chiral pure or chiral mixed) phosphoramidate (PN) bindings. In one embodiment, the AON disclosed herein comprises one or more (chiral pure or chiral mixed) PNms bonds. In one embodiment, the PN bond ligates the terminal 2 nucleotides at each end of the AON. The AON disclosed herein may also comprise bond modifications at all chiral-uncontrolled positions. The AON disclosed herein may also comprise one or more naturally occurring nucleoside bonds. The selection and number of modified bonds may depend on the specific target, sequence, length, and stability of the AON observed in the particular cell type of interest, which can be evaluated by methods known to those skilled in the art. In one embodiment, at least one, at least two, at least three, or at least four nucleoside bonds present between 2, 3, 4, or 5 nucleosides at the 5′ and / or 3′ ends of the AON disclosed herein are modified nucleoside bonds. In one embodiment, the AON disclosed herein comprises at least one MP nucleoside bond following the structure of formula (IV): JPEG2026518820000010.jpg41166

[0068] As has been pointed out in the art, the preferred site for MP binding in AON is the -2 binding site, which links the nucleoside at position -1 with the nucleoside at position -2. In preferred embodiments, this site in the AON disclosed herein includes a binding modification according to the structure of formula (I), more preferably according to the structure of formula (III), instead of MP binding. WO2020 / 201406 discloses the use of MP binding modification at specific sites around isolated nucleotides in a first nucleic acid chain. While the presence of MP binding is compatible with RNA editing by human ADAR enzymes, introducing MP binding during the production of oligonucleotides is difficult considering the additional production (purification) steps in the binding and detachment processes. In one embodiment, the AON does not contain MP binding.

[0069] In one embodiment, the AON disclosed herein comprises at least one PNdmi bond, preferably linking the two terminal nucleosides at the 5′ and / or 3′ ends of the AON. The PNdmi bond that may be used in the AON disclosed herein has the structure of formula (V): JPEG2026518820000011.jpg65166

[0070] In one embodiment, inverted deoxy-T or dideoxy-T nucleotides are incorporated at one or both ends of the AON disclosed herein. Other nucleoside bonds that may be used in the AON disclosed herein are disclosed in WO2023 / 278589.

[0071] In one embodiment, the AON disclosed herein comprises at least one phosphonoacetate and / or at least one phosphonoacetamide nucleoside interbonding.

[0072] [Chemical structure of the conjugate] In one embodiment, a sense chain that can be annealed with an AON (as disclosed herein) or (in a HEON as disclosed herein) before entering a target cell is bound to a hydrophobic moiety, such as palmityl or its analogue, cholesterol or its analogue, or tocopherol or its analogue. This is preferably bound to the 5′ end. If the hydrophobic moieties are bound to the 5′ and 3′ ends, they may be the same or different. The hydrophobic moiety bound to the oligonucleotide may be directly bound or indirectly mediated by another substance. If the hydrophobic moiety is directly bound, it is sufficient that the moiety is bound via a covalent bond, ionic bond, hydrogen bond, etc. If the hydrophobic moiety is indirectly bound, it may be bound via a linker. The linker may be cleavable or non-cleavable. A cleavable linker refers to a linker that can be cleaved under physiological conditions, for example, intracellularly or in an animal (for example, in a human). Cleavage linkers are selectively cleaved by endogenous enzymes such as nucleases, or by physiological conditions specific to a part of the body or cell, such as pH or a reducing environment (e.g., glutathione concentration). Examples of cleavage linkers include, but are not limited to, amides, esters, phosphodiesters (one or both), phosphoesters, carbamates, and disulfide bonds, and the natural DNA linker. Cleavage linkers also include self-immolative linkers. Non-cleavage linkers are those that do not cleave under physiological conditions, or cleave much slower than cleavage linkers, and include, for example, linkers consisting of PS bonds, modified or unmodified deoxyribonucleosides linked by PS bonds, spacers linked via PS bonds, and modified or unmodified ribonucleosides. When the linker is a nucleic acid such as DNA or oligonucleotide, there is no limit to its chain length. However, they can usually be 2-20 bases, 3-10 bases, or 4-6 bases in length.There are no restrictions on the length or composition of the spacer linking the ligand and the oligonucleotide, and may include, for example, ethylene glycol, triethylene glycol (TEG), HEG, alkyl chains, propyl, 6-aminohexyl, or dodecyl. In one embodiment, the GalNAc moiety is linked to the AON disclosed herein via a TEG linker. One or more other types of molecules may be linked to the AON via one or more linkers, including peptides, sugars, vitamins, polymers, aptamers, antibodies (fragments of antibodies), and small molecules.

[0073] [General] In addition to certain preferred chemical modifications at specific positions in the compounds disclosed herein, the AONs disclosed herein may include one or more (additional) modifications to the nucleic acid bases, scaffolds, and / or skeletal bonds, which may or may not be present within the same monomer (e.g., at the 3′ and / or 5′ positions). In one embodiment, the AONs disclosed herein include at least one internucleoside bond according to the structure of formula (I), and / or the AON further includes at least one nucleotide having a sugar moiety including a 2′-OMe modification, and / or the AON includes at least one nucleotide having a sugar moiety including a 2′-MOE modification, and / or the AON includes at least one nucleotide having a sugar moiety including a 2′-F modification, and / or the AON includes a solitary nucleotide having a 2′-H in the sugar moiety and therefore referred to as a DNA nucleotide, but additional modifications may be present in the bond to its base and / or its adjacent nucleoside. In one embodiment, the solitary nucleotide has a 2′-F in the sugar moiety. In one embodiment, the solitary nucleotide has a diF substitution in the sugar moiety. In one embodiment, the solitary nucleotide has 2′-F and 2′-C-methyl in the sugar moiety. In one embodiment, the solitary nucleotide contains 2′-F(FANA) in an arabinose configuration in the sugar moiety.

[0074] In one embodiment, AON is an antisense oligonucleotide capable of forming a double-stranded nucleic acid complex with a target RNA molecule, wherein the double-stranded nucleic acid complex can recruit an adenosine deaminationase to deaminate target adenosine in the target RNA molecule, the nucleotide in the AON facing the target adenosine is a solitary nucleotide, and the solitary nucleotide has the structure of formula (VI): JPEG2026518820000012.jpg40166 Here: X is O, NH, OCH2, CH2, Se, or S; B is a nitrogenous base selected from the group consisting of: cytosine, uracil, isouracil, N3-glycosylated uracil, pseudoisocytosine, 8-oxo-adenine, and 6-amino-5-nitro-3-yl-2(1H)-pyridone; R1 and R2 are each independently selected from H, OH, F, or CH3; R3 is part of the AON located on the 5′ side of the solitary nucleotide and consists of 7 to 30 nucleotides; and R4 is part of the AON located on the 3′ side of the solitary nucleotide and consists of 4 to 25 nucleotides. The nucleotides on the 3′ side and / or 5′ side of the solitary nucleotide may be DNA, more preferably the nucleotide at the 3′ side (position -1).

[0075] Other chemical modifications of AON disclosed herein include substituting one or more arbitrary hydrogen atoms with deuterium or tritium, examples of which can be found, for example, in WO2014 / 022566 or WO2015 / 011694. Again, in all cases, the modification must be compatible with editing, and as a result, the AON acts as an oligonucleotide that, after binding to its target sequence, enables the recruitment of adenosine deaminationase by the resulting double-stranded nucleic acid complex. In all aspects of this disclosure, the enzyme having adenosine deaminationase activity is preferably ADAR1, ADAR2, or ADAT.

[0076] The AONs disclosed herein preferably do not contain 5′-terminal O6-benzylguanosine or 5′-terminal amino modification, and preferably are not covalently bonded to a SNAP tag domain (modified O6-alkylguanosine-DNA-alkyltransferase). The AONs disclosed herein preferably do not contain a boxB RNA hairpin sequence. In one embodiment, the AONs disclosed herein contain 0, 1, 2, or 3 fluctuation base pairs with the target sequence and / or 0, 1, 2, 3, 4, 5, 6, 7, or 8 mismatch base pairs with the target RNA sequence. If the isolated nucleotide is uridine, no mismatch exists, but if the isolated nucleotide is a uridine analog or derivative, it may be defined differently. One alternative to uridine is to place isouridine opposite the target adenosine, which would not pair in the same way as the G-U pair. Preferably, the target adenosine in the target sequence forms a mismatch base pair with the nucleoside in the AON directly opposite the target adenosine.

[0077] As outlined above, the AONs disclosed herein utilize specific nucleotide modifications at predetermined locations to ensure stability and proper ADAR binding and activity. As described in detail herein, these modifications may vary and may include modifications in the AON backbone, in the sugar portion of nucleotides, and in nucleic acid base or phosphodiester bonds. These may also be variably distributed throughout the AON sequence. Certain modifications may be necessary to support interactions with various amino acid residues within the RNA-binding domain of the ADAR enzyme and with those within the deaminationase domain. For example, PS bonds between nucleotides or 2′-OMe or 2′-MOE modifications may be permissible in some parts of the AON, but should be avoided in other parts so as not to interfere with important interactions between the enzyme and the phosphate and 2′-OH groups. Certain nucleotide modifications may also be necessary to enhance editing activity for substrate RNAs whose target sequences are not optimal for ADAR editing. Previous studies have established that certain sequence contexts are more susceptible to editing. For example, the target sequence 5′-UAG-3′ (with target A in the center) contains the most preferred nearest neighbor nucleotide for ADAR2, while the 5′-CAA-3′ target sequence is undesirable (Schneider et al. 2014. Nucleic Acids Res 42(10):e87). Structural analysis of the ADAR2 deaminationase domain suggests the possibility of promoting editing by carefully selecting the nucleotide opposite the target trinucleotide. For example, the 5′-CAA-3′ target sequence pairs with the 3′-GCU-5′ sequence on the opposite strand (forming an AC mismatch in the center), which is undesirable because the guanosine base sterically collides with the amino acid side chain of ADAR2. In such a situation, the guanosine opposite C (i.e., at position -1 in AON) is preferably substituted with inosine, more preferably with deoxyinosine.

[0078] Unlike what is described herein regarding siRNA or gapmers and their relationship to ribonuclease degradation, and the use of such gapmers in double-stranded complexes (see, for example, EP 3954395 A1), the AONs disclosed herein do not contain a continuous region of DNA nucleotides that would make the target sequence (or sense nucleic acid strand) a target for ribonuclease-mediated degradation. It is undesirable for the target transcript molecule to be degraded through the binding of the AON to the transcript molecule. In one embodiment, the AON does not contain four or more consecutive DNA nucleotides at any position in its sequence. In one embodiment, the AON is composed of as many (chemically) modified nucleotides as possible to enhance resistance to ribonuclease-mediated degradation, while at the same time being as efficient as possible in producing the RNA editing effect. This means that while the isolated nucleotides and other multiple nucleotides in the AON may be DNA, there are no continuous regions of four or more consecutive DNA nucleotides in the AON. Therefore, the AONs disclosed herein are not gapmers. Gapmers reduce the expression of a target transcript but do not cause RNA editing of a specific adenosine within the target transcript. Gapmers are, in principle, single-stranded nucleic acids, consisting of a central region (a DNA gap region having at least four consecutive deoxyribonucleotides) and wing regions directly located at its 5′ end (5′ wing region) and 3′ end (3′ wing region). In contrast, the AONs disclosed herein may be any oligonucleotides that produce an RNA editing effect by deaminating a target adenosine in a target RNA molecule to inosine, and are therefore as resistant as possible to ribonuclease-mediated degradation in order to achieve this effect and to enable the mRNA transcript to be translated into a protein.

[0079] The AONs disclosed herein may also be administered in the presence of adjuvants that increase the entry of the AON into target cells and / or its endosomal extrusion immediately after entering the cell. The portion that may be applied to such uses is a set of chemical compounds (generally purified from nature) referred to, for example, “saponins” or “triterpene glycosides.” A preferred saponin that may be used in the methods disclosed herein is AG1856, disclosed in WO2021 / 122998 and further described in PCT / EP2024 / 051278 (unpublished) for use in combination with RNA editing oligonucleotides.

[0080] Furthermore, pharmaceutical compositions are disclosed herein, comprising the AON disclosed herein and further comprising pharmaceutically acceptable carriers, solvents, diluents, and / or other additives (e.g., saponins or triterpene glycosides such as AG1856 (above), which may actually be administered separately from the AON), which may be dissolved in pharmaceutically acceptable organic solvents, etc. The dosage form in which the AON or the pharmaceutical composition is administered may depend on the disorder to be treated and the tissue to be targeted, and may be selected according to general procedures in the art. The pharmaceutical composition may be administered by a single dose or by multiple doses, which may be administered daily or at appropriate time intervals, the time intervals may be determined using general knowledge in the art, and may be adjusted based on the disorder and the efficacy of the active ingredient.

[0081] In a preferred embodiment, the AON disclosed herein is a single-stranded oligonucleotide comprising a lone nucleotide opposite a target adenosine, wherein the lone nucleotide is chemically modified as disclosed herein, and the remainder of the oligonucleotide is chemically modified as also disclosed herein to prevent degradation by nucleases. In another embodiment, any type of oligonucleotide or heterodouble-stranded oligonucleotide complex is disclosed, wherein the complex may or may not be bound to a hairpin structure (internally or terminally), may be bound to an ADAR or its catalytic domain, or the oligonucleotide may be cyclic. In a preferred embodiment, the AON disclosed herein is a “naked” oligonucleotide comprising various chemical modifications to the ribose sugar and / or base of one or more nucleotides in the sequence, preferably comprising at least one linkage following the structure of formula (I) disclosed herein, and can hybridize with a target transcript or a portion thereof containing target adenosine, and can mobilize endogenous (naturally occurring) ADAR within the target cell for deamination of target adenosine. In another embodiment, the AONs disclosed herein, delivered in a “naked” form, do not include a stem-loop structure for the recruitment of deaminationases, thereby enabling shorter AONs and improved intracellular delivery and kinetics.

[0082] It is known in the art that RNA editors (e.g., human ADAR enzymes) edit dsRNA structures with varying specificities and in a manner dependent on multiple factors. One important factor is the degree of complementarity of the two strands constituting the dsRNA sequence. When the two strands are perfectly complementary, the catalytic domain of human ADAR typically reacts with all adenosine it encounters, causing indiscriminate deamination of adenosine. The specificity of hADAR1 and hADAR2 can be enhanced by introducing chemical modifications and / or ensuring multiple mismatches within the dsRNA, which is presumably useful for positioning the dsRNA-binding domain in a manner not yet clearly defined. Furthermore, the deamination reaction itself may be facilitated by providing oligonucleotides containing mismatches opposite the adenosine to be edited. By following the instructions of this application, those skilled in the art can design complementary portions of oligonucleotides according to their needs.

[0083] Those skilled in the art will understand that the extent to which intracellular editing enzymes are redirected to other target sites can be regulated by altering the affinity of the primary nucleic acid chain to the recognition domain of the editing enzyme. The modification itself can be determined through some trial and error and / or through calculation methods based on the structural interaction between AON and the recognition domain of the editing enzyme. In addition, or alternatively, the degree of recruitment and redirection of intracellular editing enzymes can be regulated by the AON dosage and administration regimen. This is typically determined by the experimenter (in vitro) or clinician, usually in Phase I and / or Phase II clinical trials.

[0084] Site-directed editing of target adenosine within RNA sequences in eukaryotes, preferably metazoans, more preferably mammals, more preferably human cells, more preferably human hepatocytes, and most preferably human hepatocytes, is disclosed herein. Target cells may be in vitro, ex vivo, or in vivo. One advantage of the AONs disclosed herein is that they can be used in vivo on cells, but also on cultured cells. In some embodiments, cells are treated ex vivo and then introduced into vivo (e.g., reintroduced into the vivo from which they originally originated). The AONs disclosed herein may also be used to edit target RNA sequences in cells derived from grafts or in so-called organoids (e.g., liver tissue organoids). Organoids are thought of as three-dimensional in vitro derived tissues, but are driven to generate individual, isolated tissues using specific conditions. In a therapeutic context, organoids are useful because they can be generated in vitro from the patient's cells and then reintroduced into the patient as autologous material that is less likely to be rejected than conventional grafts.

[0085] While we do not wish to be bound by theory, it is thought that RNA editing by human ADAR2 occurs on primary transcripts, for example, during transcription or splicing in the nucleus, or in the cytoplasm, at sites where mature mRNA, miRNA, or ncRNA can be edited. Generally speaking, RNA editing can be used to create RNA sequences with different properties. Such properties may be coding properties (creating proteins with different sequences or lengths, altering the properties or function of a protein) or binding properties (causing inhibition or overexpression of the RNA itself or its target or binding partner; the entire expression pathway may be altered by recoding their corresponding sequences on the miRNA or target RNA). Protein function or localization can be freely altered by functional domains or recognition motifs (including, but not limited to, signal sequences, targeting or localization signals, recognition sites for proteolytic cleavage or co-translation or post-translational modification, enzyme catalytic sites, binding sites for binding partners, signals for degradation or activation, etc.). These and other forms of RNA and protein "engineering" are included in this disclosure as diagnostic, preventive, therapeutic, research tools, or otherwise in medicine or biotechnology, whether for the purpose of preventing, delaying, or treating disease, or for any other purpose.

[0086] The amount, dosage, and administration regimen of AON administered may vary between cell types, depending on the disease being treated, target population, administration method (e.g., systemic or topical), disease severity, and tolerance level for side effects, and these may and should be evaluated through trial and error in in vitro, preclinical, and clinical trials. Testing is particularly straightforward if the modified sequence causes readily detectable phenotypic changes or changes in specific biomarkers (levels or activity) (e.g., plasma levels of bile acids). Higher doses of AON may compete for binding to ADAR enzymes within cells, thereby depleting the amount of enzyme free to participate in RNA editing; however, such effects with respect to a given AON and a given target would be revealed through routine administration studies.

[0087] One appropriate testing technique involves delivering AON to a cell line or test organism and then collecting biopsy samples at various time points. The sequence of the target RNA in the biopsy samples can be evaluated, and the percentage of cells with modification can be easily tracked. As described above, the plasma concentration of bile acids in samples from treated subjects is a suitable biomarker for evaluating the function of specific proteins in a subject, before and after treatment, or with or without treatment of the subject with the AON disclosed herein. Once this test is performed, the knowledge is retained, and subsequent delivery can be carried out without the need to collect biopsy samples. Thus, the methods disclosed herein may include a step of identifying the presence of a desired change in the target RNA sequence of a cell, thereby verifying that the target RNA sequence has been modified. This step typically includes sequencing the relevant portion of the target RNA, or its cDNA copy (or, if the target RNA is pre-mRNA, a cDNA copy of its splicing product), as described above, so that the sequence change can be easily verified. Alternatively, as described above, the changes may be evaluated by protein function before, during, and / or after treatment, or by other potential markers, which are preferably performed in vitro on samples obtained from the treated subject.

[0088] After RNA editing occurs within a cell, the modified RNA may be diluted over time due to factors such as cell division or the finite half-life of the edited RNA. Therefore, from a practical therapeutic standpoint, the methods disclosed herein may include repeatedly delivering AON until sufficient target RNA has been modified in order to provide a tangible benefit to the patient and / or to maintain that benefit over time.

[0089] The AONs disclosed herein are particularly suitable for therapeutic applications, and therefore, pharmaceutical compositions comprising the AONs disclosed herein and pharmaceutically acceptable carriers, solvents, or diluents are also disclosed. In some embodiments, the pharmaceutically acceptable carrier may simply be a salt solution, which may be useful to be isotonic or hypotonic, particularly for pulmonary delivery. The AONs disclosed herein are appropriately administered in aqueous solutions (e.g., salt solutions) or suspensions and may contain additives, excipients, and other components suitable for pharmaceutical applications, with concentrations ranging from 1 ng / ml to 1 g / ml, preferably 10 ng / ml to 500 mg / ml, more preferably 100 ng / ml to 100 mg / ml. Appropriate dosages may range from about 1 μg / kg to about 100 mg / kg, preferably about 10 μg / kg to about 10 mg / kg, more preferably about 100 μg / kg to about 1 mg / kg. Administration may be by inhalation (e.g., via nebulizer), nasal, oral, infusion or infusion, intravenous, subcutaneous, intradermal, intramuscular, intratracheal, intraperitoneal, intrarectal, intrathecal, cisterna magna, or parenteral. Administration may be in the form of solid dosage forms, powders, tablets, gels, solutions, sustained-release formulations, or any other form suitable for human pharmaceutical use.

[0090] In one embodiment, the step of identifying whether editing has occurred, depending on the final deamination effect of the conversion from A to I, includes the following steps: sequencing the target RNA; assessing the presence or absence of a protein lacking or reduced function; assessing whether the deamination has altered the splicing of the pre-mRNA; or using a functional readout. This is because the deaminated target RNA should encode a protein with reduced or deleted function, or conversely, a protein with increased or restored function. Identification of deamination to inosine may be done using a functional readout with appropriate biomarkers. Functional evaluation is generally performed according to methods known to those skilled in the art. A suitable method for identifying the presence of inosine after deamination of target adenosine is, of course, dPCR and even sequencing using methods well known to those skilled in the art. However, those skilled in the field of liver disease prefer to apply tests to monitor specific biomarkers related to liver function.

[0091] In one embodiment, the method disclosed herein comprises the following steps: administering to the subject an AON or pharmaceutical composition disclosed herein; causing a double-stranded nucleic acid complex to form in the cells within the subject between the AON and a specific and complementary target nucleic acid molecule; enabling an existing endogenous adenosine deaminationase such as ADAR1 or ADAR2 to be involved; and causing the enzyme to deaminate target adenosine in the target nucleic acid molecule to inosine, thereby alleviating, treating, improving or delaying the progression of a disease.

[0092] RNA editing molecules present in cells are typically proteinaceous in nature, such as ADAR enzymes found in metazoans, including mammals. Of particular interest are human ADARs, namely hADAR1 and hADAR2, and any of their isoforms. RNA editing enzymes known in the art, to which the oligonucleotide constructs disclosed herein can be conveniently designed, include adenosine deaminationases (ADARs) that act on RNA, such as hADAR1 and hADAR2 in human or human cells, and cytidine deaminationases. Two isoforms of hADAR1 are known: a longer 150 kDa interferon-inducible form and a shorter 110 kDa form produced from a common pre-mRNA via alternative splicing. Consequently, the level of the 150 kDa isoform available in cells can be influenced by interferons, particularly interferon-γ (IFN-γ). hADAR1 is also induced by TNF-α. This provides an opportunity to develop combination therapies in which IFN-γ or TNF-α and the AON disclosed herein are administered to the patient simultaneously or sequentially (in any order), either as a combination product or as separate products. In certain disease conditions, elevated levels of IFN-γ or TNF-α may already be present in certain tissues of the patient, which presents further opportunities for more specific editing of diseased tissue. It will be understood by those skilled in the art that the extent to which intracellular edits are redirected to other target sites can be modulated by altering the affinity of the first nucleic acid strand to the recognition domain of the editing molecule.

[0093] The AONs disclosed herein can specifically edit target adenosine in a target RNA sequence by utilizing endogenous cellular pathways and naturally occurring ADAR enzymes. The AONs disclosed herein can recruit ADAR to form a complex with it, which then promotes the deamination of a (single) specific target adenosine nucleotide in the target RNA sequence to which it binds. Ideally, only one adenosine is deaminated. When the AONs disclosed herein form a complex with ADAR, it is preferable that this results in the deamination of a single target adenosine.

[0094] The AONs disclosed herein, particularly in their naked form, are typically longer than 10 nucleotides, preferably exceeding 11, 12, 13, 14, 15, and 16, and more preferably exceeding 17 nucleotides. In one embodiment, the AONs disclosed herein are longer than 20 nucleotides. The AONs disclosed herein are preferably shorter than 100 nucleotides, more preferably shorter than 60 nucleotides, and even more preferably shorter than 50 nucleotides. In a preferred embodiment, the AONs disclosed herein comprise 18 to 70 nucleotides, more preferably 18 to 60 nucleotides, and even more preferably 18 to 50 nucleotides. Accordingly, in a particularly preferred embodiment, the AON disclosed herein comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides. In one embodiment, the AON is 27, 28, 29, or 30 nucleotides in length. [Examples]

[0095] [Example 1. RNA editing of SLC10A1 transcript using various AONs.] To target each of the four target adenosines in the human SLC10A1 transcript (individually), a first set of 4 × 30 AONs was designed. The designs and chemical modifications of these 120 AONs are shown in Figures 1, 2, 3, and 4. Subsequently, a large additional set of AONs was designed to target adenosine in the CAG codon of SEQ ID NO: 1 encoding glutamine (Q) and reverse it to the CGG codon encoding arginine (R), thereby introducing the c.203A>G(Q68R) mutation. These additional AONs, along with their respective chemical modifications, are shown in Figure 5. A large additional set of AONs was also designed to target adenosine in the GAG ​​codon of SEQ ID NO: 3 encoding glutamate (E) and reverse it to the GGG codon encoding glycine (G), thereby introducing the c.770A>G(E257G) mutation. These additional AONs, along with their respective chemical modifications, are shown in Figure 6.

[0096] For initial screening of AON, the human hepatoblastoma cell line HuH-6 (Cell Lines Service) expressing SLC10A1 mRNA is transfected with 100 nM AON using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's specifications. In addition, HuH-6 cells are subjected to gymnotic exposure to different concentrations of AON with and without chemical agents. After 72 hours of incubation, the medium is removed and total RNA is isolated using the RNeasy Micro kit (Qiagen). cDNA is synthesized using the Maxima Reverse Transcriptase kit with a mixture of random hexamer and oligo(dT) primers. The percentage of ADAR-mediated A to I conversion is determined by quantitative digital PCR (dPCR) assays designed for different target sites using the primers and probes shown in Table 1. The percentage is calculated by dividing the guanidine-containing cDNA species by the total number of target copies and multiplying by 100. A control dPCR is performed upstream or downstream of the transcript for the purpose of standardization.

[0097] JPEG2026518820000013.jpg192166

[0098] [Example 2. RNA editing of two adenosines in human SLC10A1 transcript in primary human hepatocytes using saponin as a transfection adjuvant.] Primary human hepatocytes (PHH) were cultured and seeded to approximately 700,000 cells per well, and treated with 5 μM AON in the presence of 0.5 μM AG1856 for 72 hours. Cells were then harvested, and RNA was isolated as described above. Subsequently, dPCR was performed using the respective primers to determine the editing percentages for the A>G change at position 203 of human SLC10A1 mRNA (associated with the Q68R mutation in the protein) and the A>G change at position 770 of human SLC10A1 mRNA (associated with the E257G mutation in the protein). Both experiments were performed twice. Figures 7A and 7B show the editing percentages for c.203A>G editing (Q68R), measured separately in two experiments using the 16 AONs shown (given in Figure 5). Although the percentages were slightly lower in the second experiment, the editing percentages reached a maximum level of 65%, with RM106622, RM106624, RM106626, RM106631, RM106632, RM106633, and RM106634 performing best. Figures 8A and 8B show the editing percentages measured separately in two experiments for c.770A>G editing (E257G) using the 17 AONs shown (given in Figure 6). Although the percentages were slightly lower in the second experiment, the editing percentages reached a maximum level of 55%, with four asymmetric AONs (where the length of the 5′ side portion of the AON calculated from the isolated position is significantly longer than the 3′ side portion of the AON) RM106580, RM106581, RM106582, and RM106583 performing best as shown.

[0099] [Example 3. RNA editing of two adenosines in human SLC10A1 transcript in primary human hepatocytes using gymnotic uptake.] The experiment in Example 2 was repeated in PHH cells, except without the use of AG1856 saponin as an adjuvant during incubation. This entry of oligonucleotides into cells is also called "gymnotic uptake" or "gymnosis." The incubation was again 72 hours, with 10 μM AON and ~200,000 cells per well. A total of 18 AONs were tested for c.203A>G(Q68R), and a total of 16 AONs were tested for c.770A>G(E257G). Figures 9A and 9B show the editing percentages measured separately in two gymnotic uptake experiments for c.203A>G editing (Q68R) using the 18 AONs shown (given in Figure 5). In the second experiment, although the percentages were again slightly lower, the editing percentages reached a maximum level of 2%, with RM106620, RM106622, RM106873, and RM106874 performing best. Figures 10A and 10B show the editing percentages measured separately in two gymnotic incorporation experiments for c.770A>G editing (E257G) using the 16 AONs shown (given in Figure 6). Here again, although the percentages were slightly lower in the second experiment, the editing percentages reached a maximum level of 1.7%, with RM106867 and four asymmetric AONs (where the length of the 5′ side portion of the AON calculated from the isolated position is significantly longer than the 3′ side portion of the AON) RM106580, RM106581, RM106582, and RM106583 again performing remarkably well, as shown.

[0100] [Example 4. RNA editing of two adenosines in human SLC10A1 transcript in liver spheroids using saponin-assisted uptake and gymnotic uptake.] The experiments described in Examples 2 and 3 were repeated for both target sites, but this time using liver spheroid organoids generated from PHH cells using culture conditions known to those skilled in the art. Eight spheroids were used per incubation and incubated for 120 hours in each case with 5 μM AON, with or without 0.5 μM AG1856. RNA purification, cDNA synthesis, and dPCR were performed as described above. Figures 11A and 11B show the editing percentages measured in the experiment with respect to c.203A>G editing (Q68R) in the presence of AG1856 and in the absence of saponin, respectively, using the 16 AONs shown (given in Figure 5). In these liver organoids as well, the editing percentage reached a maximum level of 75% in the presence of saponins, with RM106622, RM106873, RM10631, RM106632, RM106633, and RM106634 showing the best results. On the other hand, when saponins were not applied, i.e., without any cell entry assistance, RM106622 outperformed all other AONs, reaching a level of almost 8%. Figures 12A and 12B show the editing percentages measured in experiments with AG1856 and in the absence of saponins, respectively, for c.770A>G editing (E257G) using the 17 AONs shown (given in Figure 5). In these liver organoids as well, the edit percentage reached a maximum level of 55% in the presence of saponins, with the "asymmetrical" AONs RM106580, RM106581, RM106582, and RM106583 showing the best results, and also reaching a level of approximately 4.5% even without saponin application.

[0101] [Example 5. RNA editing of two adenosines in human SLC10A1 transcript in human HepG2 cells overexpressing NTCP using saponin-assisted uptake.] Human HepG2 cells (HepG2NTCP) stably expressing wild-type human NTCP were cultured and seeded to approximately 100,000 cells per well. They were treated with 5 μM AON in the presence of 0.5 μM AG1856 for 72 hours. Subsequently, cells were harvested and RNA was isolated as described above. Subsequently, dPCR was performed using each primer to determine the editing percentage for the A>G change at position 203 of human SLC10A1 mRNA (associated with the Q68R mutation in the protein) and the A>G change at position 770 of human SLC10A1 mRNA (associated with the E257G mutation in the protein). Both experiments were performed twice. Figure 13 shows the editing percentages measured for c.203A>G editing (Q68R) using the 11 AONs shown (given in Figure 5). Edit percentages reached a maximum level of 25%, with RM106631, RM106632, RM106633, and RM106634 performing best. Figure 14 shows the edit percentages measured for c.770A>G editing (E257G) using the 10 AONs shown (given in Figure 6). Edit percentages reached a maximum level of 25%, with the four asymmetric AONs (where the length of the 5′ side portion of the AON calculated from the isolated position is significantly longer than the 3′ side portion of the AON) RM106580, RM106581, RM106582, and RM106583 performing best as shown.

[0102] [Example 6. RNA editing of two adenosines in the human SLC10A1 transcript in primary human hepatocytes using saponin-assisted uptake.] PHH cells were cultured in a smaller form, seeded up to ~200,000 cells per well in 12-well plates, and treated with 5 μM AON in the presence of 0.5 μM AG1856 for 72 hours. Cells were then harvested, and RNA was isolated as described above. Subsequently, dPCR was performed using the respective primers to determine the editing percentages for the A>G change at position 203 of human SLC10A1 mRNA (associated with the Q68R mutation in the protein) and the A>G change at position 770 of human SLC10A1 mRNA (associated with the E257G mutation in the protein). Figure 15A shows the editing percentages measured for c.203A>G editing (Q68R) using the 25 asymmetric AONs shown (given in Figure 5). Edit percentages reached a maximum level of 45%, with RM107341, RM107346, RM107350, and RM107354 performing best. Figure 15B shows the edit percentages measured for c.203A>G editing (Q68R) using the 32 symmetric AONs shown (given in Figure 5). Edit percentages reached a maximum level of 45%, with RM107357, RM107362, RM107377, RM107381, RM107382, and RM107385 performing best. Figure 16A shows the edit percentages measured for c.770A>G editing (E257G) using the 21 asymmetric AONs shown (given in Figure 6). Editing percentages reached a maximum level of 35%, with RM107275, RM107276, RM107278, RM107284, RM107291, and RM107293 showing the best results. Figure 16B shows the editing percentages measured for c.770A>G editing (E257G) using the 38 symmetric AONs shown (given in Figure 6). Editing percentages reached a maximum level of 15%, with RM107295 and RM107298 showing the best results as illustrated. These experiments appear to support the above prior findings that c.203A>G editing (Q68R) is more efficient with symmetric AONs, while c.770A>G editing (E257G) is more efficient with asymmetric AONs.

[0103] [Example 7. Expression of loss-of-function mutants of NTCP on the cell membrane of transfected U2OS cells] To test whether the hypothetical NTCP variants outlined herein actually result in loss of function in bile transport across the cell membrane, human U2OS cells were cultured in 48-well plates and allowed to adhere overnight. The following day, the cells were transfected with plasmids encoding wild-type human NTCP protein (as a positive control) and seven mutant human NTCP: E257G, Q68R, I223V, K314E, Q261R, I279V, and T268A. The experiment was repeated three times. After transfection, the cells were treated with 1 μM or 10 μM radiolabeled taurocholic acid (TCA) for 48 hours. TCA is a primary bile acid in humans. After TCA treatment, the bile acid and medium were washed away, the cells were washed multiple times, and then the radioactivity of each well (an indicator of TCA uptake in these cells) was measured. Figures 17A and 17B show TCA uptake in pmol units in different cells treated with different expression plasmids, where Figure 17A shows results with 1 μM treatment and Figure 17B shows results with 10 μM treatment. When cells were not transfected, no bile acid uptake was observed, indicating the absence of NTCP protein in U2OS cells. "Normal" TCA uptake levels using wild-type NTCP expression plasmids were approximately 1.6% and 7.6%, respectively. These levels were also achieved with mutants I223V, K314E, I279V, and T268A. However, after expressing mutants E257G, Q68R, and Q261R, a significant deficiency in bile acid uptake was observed, providing a rationale for introducing one or more of these mutations into the human NTCP transcript, thereby reducing the cell membrane-mediated bile acid transport capacity in hepatocytes.

[0104] [Example 8. c.203A>G editing (Q68R) of the SLC10A1 transcript in primary human hepatocytes during high-throughput screening.] Subsequently, as described herein, a set of 960 EONs was designed (represented by EON numbers RM108942 to RM109900; SEQ ID NOs 167 to 1126, see Figure 5), which included various chemical and other modifications, all targeting the human SLC10A1 transcript at position c.203A (Q68R). These EONs were tested as follows: On day 0, PHH (5.0 × 10⁻⁶) 4 Cells (per well) were transfected with EON three times using Lipofectamine® RNAiMAX reagent simultaneously with seeding, according to the manufacturer's protocol. Plates containing cells, medium, and EON were kept at 37°C, 5% CO2 for 72 hours, during which time the medium was refreshed 24 hours after transfection / seeding.

[0105] On day 3 (72 hours after transfection / seeding), the supernatant was discarded, and subsequent analysis was performed as follows. Cells were harvested and used for RNA isolation using the RNeasy 96 kit (Qiagen-74182) according to the manufacturer's instructions. The extracted RNA was treated with deoxyribonuclease I (ThermoFisher-EN0521) according to the manufacturer's protocol. The samples were incubated at 37°C for 30 minutes, then 1 μL of 50 mM EDTA was added, and the cells were incubated at 60°C for a further 2 minutes. The total RNA was then reverse transcribed using the Maxima Reverse Transcriptase (Thermo-EP0742) kit with oligo-dT primers, random hexamer primers, and a dNTP mix (10 mM each). Subsequently, quantitative PCR was performed using a Digital PCR system (Bio-Rad, QX200) in 22 μl aliquots of a reaction mixture containing cDNA, appropriate primer pairs, and ddPCR Supermix for Probes (no dUTP) (Bio-Rad-1863024). Primers for this Q68R target (Table 1) were used, and the PCR program was as follows: 10 minutes at 95°C; 40 repetitions of 30 seconds at 94°C and 60 seconds at 63°C; 10 minutes at 98°C; and a hold step at 4°C. The plate was then placed on a QX200 droplet reader, and the number of positive droplets was measured. The edit percentage was calculated by pooling all A and G counts for each transfection, repeating three times, and then scored as follows: Score = Total (G) / Total (A + G) × 100

[0106] The p-value (p=0.05) represents the probability that three treated replicas are different from three untreated replicas. EON editing scores were ranked from highest to lowest editing percentage. Of the 960 EONs tested, 415 showed editing scores greater than 0%. These scores are shown in Table 2 below. The remaining 545 EONs all showed 0% editing and are not shown in Table 2. Unexpectedly, several EONs performed remarkably well, with RM109899, ​​RM108970, RM109900, RM109571, RM108832, RM109040, RM109898, RM109306, and RM108977 being the best performers, all with percentages greater than 12%, and some replicas exceeding 21%.

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[0110] [Example 9. c.203A>G editing (Q68R) in SLC10A1 transcripts in PHH during small-scale transfection screening.] Several additional experiments outlined in Example 8 were performed in PHH using Lipofectamine transfection or AG1856 combined treatment with various additional AONs, including various chemical structures with alternative bindings. Details of the additionally tested AONs are shown in Figure 18, showing substitutions of multiple MP and / or PNdmi bindings by PNms bindings (indicated by the hashtag # symbol), and various repositionings by 2′-F and 2′-MOE substitutions. Transfection, incubation, RNA isolation, cDNA synthesis, and dPCR were performed as described above. Figure 19 shows experiments using RM107361, RM107362, RM107363, RM107364, RM107365, RM107376, RM107377, RM107378, RM107379, RM107380, RM107382, and RM107385 (with RM4777 as a negative control), clearly showing the preferred chemical structure of, for example, RM107378 (which is equivalent to RM107362 except that PNms bonds are present instead of PNdmi and MP bonds). Figure 20 shows experiments using RM108820 to RM108843, suggesting that the absence of 2′-MOE substituted nucleotides on the 5′ arm of AON and the low abundance of 2′-F substituted nucleotides (RM108826 to RM108831) negatively affect editing of the Q68R NTCP target. In contrast, the use of 2′-MOE and 2′-F patterns in RM108838 to RM108843 appears to contribute to editing efficiency, with RM108839 (along with RM108821, which contains a significantly large amount of 2′-F substituted nucleotides on the 5′ arm of AON and no 2′-MOE substituted nucleotides) showing the best results.Figure 21 shows additional experiments using RM108821, RM108826, RM108827, RM108836, RM108838, RM108839, and RM108840, compared with the previously tested (and relatively inferior) RM107352 and RM107368 AONs, with AG1856 applied in combination (as described above). This clearly demonstrates that the edited percentages obtained with RM108821 (sequence number 1284) and RM108839 (sequence number 1302) were reproducibly high, reaching levels above 55%. Figure 22 shows a comparison of RM108839 with various other AONs (RM117635-RM117647 and RM117837-RM117846) using lipofectamine transfection, which again shows that the editing level obtained with RM108839 was significantly higher, and RM117635 (SEQ ID NO: 1260) also performed well. By combining the findings disclosed herein, the AON of SEQ ID NO: 1302 can be further modified.

[0111] [Example 10. c.203A>G editing (Q68R) of SLC10A1 transcripts in non-human primates using AON encapsulated in lipid nanoparticles.] In the following experiment, it was investigated whether the most promising AONs from in vitro screening could induce editing at the c.203A position in the endogenous wild-type SLC10A1 transcript using endogenous ADAR enzymes in the liver of non-human primates (NHPs), providing a model for therapeutically relevant Q68R alterations in humans. For this purpose, RM107377 (SEQ ID NO: 1173), RM107378 (SEQ ID NO: 1174), and RM107385 (SEQ ID NO: 1181) (see Figures 5, 15B, and 19) were encapsulated in various lipid nanoparticles (LNPs) using standard methods known to those skilled in the art. The AONs were administered at four time points in the following doses: 1 mg / kg, 2 mg / kg, 2 mg / kg, and finally 4 mg / kg. Liver biopsies were taken at different time points after administration, and RNA editing was confirmed using ddPCR over a period of more than one month, generally following the protocol described above. Multiple negative controls are used in combination (unrelated AONs and untreated subjects). Bile acid concentrations are assessed as functional readouts in the plasma of treated NHPs.

Claims

1. It is an antisense oligonucleotide (AON), The AON is capable of recruiting endogenous ADAR enzymes in human cells after the AON forms a double-stranded complex with the region of a target RNA nucleic acid molecule within the cell. The region comprises target adenosine, The nucleotide in the AON facing the target adenosine is a solitary nucleotide. The ADAR enzyme can deaminate the target adenosine to inosine after binding it to the double-strand complex. and The aforementioned target RNA nucleic acid molecule is Na ＋ / This is a transcript molecule of the human SLC10A1 gene that encodes a taurocholic acid cotransport polypeptide (NTP). Antisense oligonucleotide.

2. AON as described in claim 1, The aforementioned transcript molecule is a pre-mRNA or mRNA molecule. AON.

3. AON as described in claim 1 or 2, The cells are liver cells, preferably hepatocytes. AON.

4. AON as described in any one of claims 1 to 3, Here, the nucleotides are numbered such that the isolated nucleotide is number 0, and the nucleotides increase positively (+) toward the 5' end and negatively (-) toward the 3' end. and The isolated nucleotide is a deoxynucleotide comprising cytosine, a cytosine analog, uracil, or isouracil. AON.

5. AON as described in claim 4, The aforementioned isolated nucleotide is a deoxyribonucleotide containing a cytosine analog. The cytosine analog is a 6-amino-5-nitro-3-yl-2(1H)-pyridone nucleic acid base. AON.

6. AON as described in any one of claims 1 to 5, The first nucleotide 3' from the isolated nucleotide is deoxyinosine if the nucleotide opposite this position is cytidine within the target RNA nucleic acid molecule. AON.

7. AON as described in any one of claims 1 to 6, The AON has a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides. AON.

8. AON as described in any one of claims 1 to 7, The AON includes one or more modifications in the bonding portion, The modifications are independently selected from the group consisting of phosphorothioates (PS), phosphonoacetates, phosphorodithioates, methylphosphonates (MP), sulfonyl phosphoramidates, (1,3-dimethylimidazolidinedine-2-ylidene)phosphoamidates (PNdmi), and mesylphosphoamidates (PNms). AON.

9. AON as described in claim 8, The numbering of nucleoside bonds in the AON is such that bond number 0 is the bond on the 5' side from the isolated nucleotide, and the binding positions within the oligonucleotide increase positively (+) toward the 5' end and negatively (-) toward the 3' end. and - The second bonding position is either an MP bond or a PNms bond. AON.

10. AON as described in claim 8 or 9, The bond between the two terminal nucleotides at the 5′ and / or 3′ ends of the AON is a PNdmi bond or a PNms bond. AON.

11. AON as described in any one of claims 1 to 10, The AON comprises one or more nucleotides, including one or two substitutions at the 2', 3', and / or 5' positions of ribose. The aforementioned substitutions consist of the following group: -OH; -F; substituted or unsubstituted, linear or branched lower (C) １ -C １０ ) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, which may be interrupted by one or more heteroatoms; -O-, S-, or N-alkyl; -O-, S-, or N-alkenyl; -O-, S-, or N-alkynyl; -O-, S-, or N-allyl; -O-alkyl-O-alkyl; -methoxy; -aminopropoxy; -methoxyethoxy; -dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy; Therefore, each is selected independently. AON.

12. An AON as described in any one of claims 1 to 11, The AON is bonded to the GalNAc portion covalently or noncovalently, directly or through a linker. AON.

13. AON as described in any one of claims 1 to 12, The AON is covalently or noncovalently bonded to a triterpene glycoside, preferably AG1856, directly or through a linker. AON.

14. AON as described in any one of claims 1 to 13, The aforementioned SLC10A1 gene is wild-type, and The target adenosine consists of the following group: - Adenosine within the CAG codon encoding glutamine (Q) at position 68 of the NTP protein, wherein the adenosine is deaminated to change the amino acid to arginine (R); - The first adenosine in the CAA codon encoding glutamine (Q) at position 261 of the NTP protein, wherein the adenosine is deaminated to change the amino acid to arginine (R); - Adenosine in the GAG ​​codon encoding glutamic acid (E) at position 257 of the NTP protein, wherein the adenosine is deaminated to change the amino acid to glycine (G); and - The first adenosine in the AAG codon encoding lysine (K) at position 314 of the NTP protein, wherein the adenosine is deaminated to glutamic acid (E); Selected from, Deamination of the target adenosine results in an NTP protein whose function of transporting bile acids from the portal circulation into the cell is impaired. AON.

15. AON as described in claim 14, The aforementioned target adenosine is located in the CAG codon encoding glutamine at position 68 of the NTP protein. and The AON includes or consists of sequences and modifications selected from the group consisting of the following sequence numbers: 150, 151, 152, 154, 156, 158, 159, 163, 164, 165, 166, 1127, 1128, 1129, 1130, 1131, 1133, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1150, 1151, 1152, 1153, 1154, 1155, 115 6, 1157, 1158, 1159, 1160, 1161, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1180, 1181, 1182, 1183, 1260, 1283, 1284, 1285, 1286, 1287, 1288, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, and 1306, AON.

16. AON as described in claim 14, The aforementioned target adenosine is located in the GAG ​​codon encoding glutamate at position 257 of the NTP protein. and The AON includes or consists of sequences and modifications selected from the group consisting of the following sequence numbers: 1193, 1194, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1215, 1216, 1217, 121 8, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1253, and 1254, AON.

17. A nucleic acid molecule encoding AON as described in claim 1, A vector, preferably a viral vector, more preferably an adeno-associated virus (AAV) vector.

18. A nanoparticle delivery vehicle formulation comprising AON as described in any one of claims 1 to 16.

19. A nanoparticle delivery medium formulation according to claim 18, Here, the nanoparticle delivery medium is lipid nanoparticles (LNPs). Nanoparticle delivery medium formulation.

20. A pharmaceutical composition comprising an AON as described in any one of claims 1 to 16, a vector as described in claim 17, or a nanoparticle delivery medium formulation as described in claim 18 or 19, and a pharmaceutically acceptable carrier.

21. AON as described in any one of claims 1 to 16, For use in the treatment of diseases caused by the accumulation of bile in the liver, such as cholestasis, primary sclerosing cholangitis (PSC), biliary atresia (BA), and cirrhosis. AON.

22. A use of AON as described in any one of claims 1 to 16, In the manufacture of pharmaceuticals for the treatment of diseases caused by the accumulation of bile in the liver, such as cholestasis, PSC, BA, and cirrhosis, use.

23. A method for editing human SLC10A1 pre-mRNA or mRNA molecules in hepatocytes, preferably hepatic parenchymal cells, The method comprises the step of contacting the SLC10A1 pre-mRNA or mRNA molecule with an AON capable of inducing ADAR-mediated deamination from adenosine to inosine, thereby editing the SLC10A1 pre-mRNA or mRNA molecule to encode an NTP protein whose function in bile acid uptake is weakened, reduced, or lost. and The AON is one of those described in any one of claims 1 to 16. method.

24. A method for treating, improving, or delaying the progression of diseases caused by the accumulation of bile in the liver, such as cholestasis, PSC, BA, and cirrhosis, in human subjects who require it, The method comprises the step of administering to the subject an AON described in any one of claims 1 to 16, a vector described in claim 17, or a nanoparticle delivery medium formulation described in claim 18 or 19, thereby contacting the SLC10A1 pre-mRNA or mRNA molecule in the subject's cells, thereby causing ADAR-mediated deamination from adenosine to inosine, thereby editing the SLC10A1 pre-mRNA or mRNA molecule to encode an NTP protein whose function in bile acid uptake is weakened, reduced, or lost, thereby treating the subject. method.

25. An in vitro, ex vivo, or in vivo method for deaminating human SLC10A1 pre-mRNA or target adenosine within an mRNA molecule in hepatocytes, preferably hepatic parenchymal cells, The above method involves the following steps: (i) Providing the cells with the AON described in any one of claims 1 to 16; (ii) The step of allowing the cells to take up the AON; (iii) The step of annealing the AON to the SLC10A1 pre-mRNA or mRNA molecule; (iv) A step of deaminating the target adenosine in the SLC10A1 pre-mRNA or mRNA molecule to inosine using an endogenous ADAR enzyme; Includes, and further (v) Step of identifying the presence of the inosine in the SLC10A1 pre-mRNA or mRNA molecule using a functional readout. It may include, method.

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