Antisense oligonucleotides for the treatment of aldehyde dehydrogenase 2 deficiency

Abstract

The present invention relates to the field of diseases caused by alcohol intolerance, such as those resulting from the ALDH2*2 mutation in the human ALDH2 gene. The invention includes oligonucleotides and their use in RNA editing methods that target adenosines in endogenous human ALDH2 transcripts in cells, particularly in the liver, such as the G>A mutation in transcript molecules of the mutant ALDH2 gene encoding the mutant p.E504K ALDH2 protein.

Description

[Technical Field]

[0001] The present invention relates to the field of medicine. The present invention relates to the field of diseases caused by mutant aldehyde dehydrogenase 2 (ALDH2) proteins. The present invention involves the use of nucleotide editing technology to target ALDH2 gene transcripts and create amino acid changes that restore the normal function of the ALDH2 protein in regulating ethanol metabolism. [Background technology]

[0002] Mitochondrial aldehyde dehydrogenase 2 (ALDH2) in the liver eliminates toxic aldehydes, including acetaldehyde, an intermediate in ethanol metabolism, and is therefore essential for alcohol detoxification. The well-known alcohol-induced flushing reaction is caused by mutations in the structural gene for the ALDH2 protein (Yoshida A et al. 1984. Proc Natl Acad Sci USA. 81:258-261). ALDH2 is also involved in the conversion and elimination of the reactive aldehyde 4-hydroxynonenal (HNE). At low concentrations, this compound is beneficial, but at high concentrations, it is toxic. A variant allele of ALDH2, designated ALDH2*2, encodes a glutamic acid to lysine substitution at residue 504 of the mature enzyme. The wild-type allele is designated ALDH2*1. The wild-type protein is often referred to as ALDH2E, while the mutant protein is often referred to as ALDH2K. Other known mutant alleles include ALDH2*3 and ALDH2*4 (Chen CH et al. 2020. EBioMedicine 55:102753). Approximately 40-45% of East Asians (approximately 8% of the world's population) inherit the inactive ALDH2*2 variant and exhibit a characteristic alcohol flushing response after alcohol consumption.

[0003] Alcohol flushing syndrome is not benign. Clear epidemiological data and meta-analyses have consistently shown that alcohol consumption in ALDH2*2 carrier populations significantly increases the risk of multiple cancers, particularly upper gastrointestinal cancers. ALDH2K activity is partially dominant-negative relative to wild-type activity due to the structural changes introduced into the ALDH2 tetramer complex by the mutation. As a result, individuals with heterozygous genotypes have less than half the wild-type activity, and mutant homozygotes have negligible residual activity.

[0004] Acetaldehyde binds to intracellular proteins and DNA, potentially leading to DNA damage and organ dysfunction. Specifically, endogenous aldehydes are toxic to hematopoietic stem cells, which have defective DNA repair in Fanconi anemia. This leads to accelerated disease progression in Fanconi anemia patients with the ALDH2*2 allele. Additionally, ALDH2*2 increases the risk of gastrointestinal cancers, including gastric, esophageal, and colon cancers. Mutant mice carrying a glutamic acid to lysine mutation (equivalent to the human E504K mutation) recapitulate essentially all human phenotypes, including impaired acetaldehyde clearance, increased susceptibility to acute or chronic alcohol-induced toxicity, and reduced ALDH2 expression due to the dominant-negative effect of the mutation. When treated with chemical carcinogens, mutant mice exhibited an increased DNA damage response in hepatocytes, significant liver injury, and accelerated hepatocellular carcinoma progression (Jin S et al. 2015. Proc Natl Acad Sci USA. 112(29):9088-9093), supporting the notion that common human ALDH2*2 variants are significant risk factors for liver carcinogenesis. Aldehyde toxicity is not limited to cancer; it has also been implicated in many other diseases for which ALDH2*2 populations are at increased risk, ranging from osteoporosis, cardiovascular disease, Alzheimer's disease, and rare genetic disorders such as Fanconi anemia, as mentioned above.

[0005] The ALDH2*2 variant has so far been characterized as an East Asian-specific polymorphism. Extensive global geographic and demographic mapping, based on data from over 80,000 individuals from 366 population samples, has confirmed that the ALDH2*2 allele is highly concentrated in southeastern China, Japan, Korea, Taiwan, Singapore, and Vietnam. In Taiwan, the prevalence of ALDH2*2 carriers is as high as 49%, affecting half of the population of a single country (Luo HR et al. 2009. Gene. 435(1-2):96-103).

[0006] A potential therapeutic agent known as Alda-1 (AD-6626) was developed as a small molecule therapeutic for the treatment of alcohol intoxication because it improved outcomes in animal models of myocardial infarction, stroke, radiation dermatitis, and pain. It was thought to form a molecular patch that restored the enzymatic activity of mutant ALDH2 proteins (Chen CH et al. 2008. Science 321(5895):1493-1495). However, this molecule does not appear to have been further developed for the treatment of human ALDH2*2 subjects. WO2014 / 160185 discloses various small molecules (such as Alda-1) that function as modulators of ALDH2 activity. WO2019 / 092282, WO2019 / 143621, WO2020 / 206350, and WO2022 / 104366 disclose nucleic acid-based therapeutic agents aimed at reducing the expression of ALDH2 in subjects suffering from alcohol use disorder (AUD), a disorder for which ALDH2*2 subjects, who are generally less prone to high alcohol intake due to alcohol intolerance issues caused by ALDH2*2 mutations, appear to be at lower risk.

[0007] Fomepizole (Antizol) was approved by the FDA in 1997 for the treatment of acute ethylene glycol and methanol poisoning. However, this product does not target ALDH2 and does not address ethanol poisoning.

[0008] The present invention aims to provide one or more alternative (and / or improved) compounds or compositions for use in the treatment of alcohol intolerance caused by ALDH2K mutant proteins. Summary of the Invention

[0009] The present specification discloses an RNA editing oligonucleotide (EON) capable of forming a double-stranded complex with a region of an endogenous human ALDH2 transcript molecule in a cell, wherein the region of the ALDH2 transcript molecule contains a target adenosine, and the double-stranded complex can recruit an endogenous ADAR enzyme to deaminate the target adenosine to inosine, thereby editing the ALDH2 transcript molecule.Preferably, the ALDH2 transcript molecule is a pre-mRNA or mRNA molecule.In one embodiment, the cell is a human liver cell, preferably a hepatocyte.A preferred target adenosine is the adenosine caused by a G>A mutation in the human ALDH2 gene, resulting in a mutant p.E504K ALDH2 protein. In some embodiments, the EON comprises at least one nucleotide comprising one or more non-natural chemical modifications in the ribose moiety, linker moiety, or base moiety, or one or more additional non-natural chemical modifications, provided that the isolated nucleotide in the EON, which is the nucleotide directly opposite the target adenosine, is not a cytidine comprising a 2'-OMe ribose substitution. Also disclosed is a vector, preferably a viral vector, more preferably an adeno-associated viral (AAV) vector, comprising a nucleic acid molecule encoding the EON disclosed herein. Also disclosed is a pharmaceutical composition comprising the EON disclosed herein or the vector disclosed herein and a pharmaceutically acceptable carrier.

[0010] Disclosed herein is an EON, vector, or pharmaceutical composition disclosed herein for use in treating a disorder caused by ALDH2 deficiency, preferably caused by ALDH2*2. Disclosed herein is the use of the disclosed EON or vector in the manufacture of a medicament for treating ALDH2*2-induced alcohol intolerance, such as alcohol intoxication, alcohol poisoning, or symptoms of alcohol consumption. Symptoms of alcohol consumption include hangover symptoms, such as dehydration, fatigue, headache, body aches, vomiting, diarrhea, flatulence, weakness, increased body temperature and heart rate, excessive salivation, difficulty concentrating, sweating, anxiety, discomfort, irritability, sensitivity to light and noise, abnormalities in motor function, difficulty sleeping, intense hunger, bad breath, and lack of depth perception.

[0011] Disclosed is a method for treating a disorder caused by ALDH2 deficiency, preferably ALDH2*2, in a patient in need thereof, the method comprising contacting an ALDH2 polynucleotide in the subject's cells with an EON capable of causing ADAR-mediated adenosine-to-inosine conversion of an adenosine associated with the ALDH2 deficiency, thereby treating the patient. Also disclosed is a method for treating a disorder caused by ALDH2*2, the method comprising administering a therapeutically effective amount of the disclosed EON, the disclosed vector, or the disclosed pharmaceutical composition to a patient in need thereof. Also disclosed is a method for editing an ALDH2 polynucleotide, the method comprising contacting the ALDH2 polynucleotide with an EON capable of causing RNA-acting adenosine deaminase (ADAR)-mediated adenosine-to-inosine conversion of an adenosine associated with alcohol intolerance, thereby editing the ALDH2 polynucleotide. Preferably, the ALDH2 transcript is derived from an ALDH2*2 mutant gene. Also disclosed is a method for treating ALDH2*2-induced alcohol intolerance or a disorder caused by said alcohol intolerance in a patient in need thereof, the method comprising contacting an ALDH2 polynucleotide in a subject's cells with an EON capable of causing ADAR-mediated adenosine to inosine conversion associated with alcohol intolerance or a disorder caused by said alcohol intolerance, such as symptoms of alcohol intoxication, alcohol poisoning, or alcohol use, thereby treating the patient.

[0012] [Brief description of the drawing] One or more embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings in which: [Brief explanation of the drawings]

[0013] [Figure 1]The human ALDH2 target RNA sequence (5'→3'; SEQ ID NO: 52) is shown at the top. The target adenosine is shown in bold, and the lysine codon is underlined. Below the target sequence are the sequences (also 5'→3') of the initial 51 EONs (SEQ ID NOs: 1-51; as indicated) originally designed to edit the target adenosines. Some EONs have two names separated by a backslash. The chemical modifications in EON are as follows: m5Ce is 2'-MOE-modified 5-methylcytidine; m5Ue is 2'-MOE-modified 5-methyluridine (identical to thymidine with a 2'-MOE substitution); Ge and Ae are 2'-MOE-modified guanosine and adenosine, respectively; Cm, Am, Um, and Gm are 2'-OMe-modified cytidine, adenosine, uridine, and guanosine, respectively; Gf, Cf, Af, and Uf are 2'-OMe-modified cytidine, adenosine, uridine, and guanosine, respectively. They are 2'-F modified guanosine, cytidine, adenosine, and uridine, respectively; Zd (isolated nucleotide) is a deoxynucleotide (deoxycytidine analog) with a Benner base; C2f (isolated nucleotide) is a cytidine with a 2',2'-difluoro modification; Cd (isolated nucleotide) is a deoxycytidine; Ad is a deoxyadenosine; "!" indicates a PNdmi bond; "^" indicates an MP bond; "*" indicates a PS bond. All other internucleoside linkages are phosphodiester bonds. [Figure 2] Percent editing of human ALDH2 transcripts after gymnotic uptake (GU) of the indicated EONs (black bars) and after co-administration of saponin (gray bars) is shown. (A) Shows total percentage, including background signal from the wild-type allele. (B) Shows the same results after normalization to the non-treated (NT) sample, thereby removing the signal from the wild-type allele. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present inventors have recognized that another approach may be possible: targeting the E504K mutation in ALDH2 to generate wild-type ALDH2 protein and potentially restore normal alcohol metabolism. This would prevent, alleviate, or treat disorders associated with toxic aldehyde accumulation or alcohol intolerance. This technology is generally referred to as RNA editing. Disclosed herein are oligonucleotides that can be used to specifically deaminate specific target adenosines in the transcripts of (human) mutant ALDH2 transcripts (pre-mRNA and / or mRNA) in vivo, preferably using endogenous deaminating enzymes to produce ALDH2 proteins with restored function of converting acetaldehyde to acetate during ethanol metabolism. While the E504K mutation described above is by far the most common mutation found in the ALDH2 gene, the RNA editing technology disclosed herein can also be applied to target other target adenosines in ALDH2, thereby restoring their function or even causing a gain-of-function effect. The E504K mutation results from a GAA codon (encoding glutamic acid; Glu; E) being mutated to an AAA codon (encoding lysine; Lys; K). The specific RNA editing disclosed herein converts the first adenosine of the mutated codon to an inosine, which is then read as guanosine by the translational machinery (AAA>IAA>GAA).

[0015] RNA editing is a natural process by which eukaryotic cells alter the sequences of their RNA molecules, often in a site-specific and precise manner, thereby expanding the genome-encoded RNA repertoire 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 critical role in managing 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 deamidase acting on RNA (ADAR) and APOBEC / AID (cytidine deamidase acting on RNA), respectively.

[0016] ADARs are multidomain proteins containing a catalytic domain and two to three 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. While the catalytic domain also plays a role in recognizing and binding portions of the dsRNA helix, its primary function is to convert A to I by deaminating nucleobases at nearby (predefined) locations in the target RNA. As mentioned above, inosine is read as guanosine by the cellular translation machinery, meaning that the edited adenosine can recode a protein sequence if present in the coding region of an mRNA or pre-mRNA. A to I conversion can also occur in the 5' non-coding sequence of the target mRNA, generating a new translation start site upstream of the original start site, which results in an N-terminally extended protein, or it can occur in the 3' UTR or other non-coding portion of the transcript, which can affect RNA processing and / or stability. Additionally, A-to-I conversion can occur at splice elements within introns or exons of pre-mRNA, thereby altering the splicing pattern, resulting in either inclusion or skipping of the exon. Enzymes that catalyze adenosine deamination belong to the ADAR enzyme family, which includes the human deaminases hADAR1, hADAR2, and hADAR3. However, hADAR3 has not demonstrated deaminating activity.

[0017] The use of oligonucleotides to apply adenosine deaminase to edit target RNA has been described (e.g., Woolf et al. 1995. PNAS 92:8298-8302; Montiel-Gonzalez et al. PNAS 2013, 110(45):18285-18290; Vogel et al. 2014. Angewandte Chemie Int Ed 53:267-271). A drawback of the method described in Montiel-Gonzalez et al. (2013) is that it requires a fusion protein consisting of a genetic fusion of the boxB recognition domain of the bacteriophage lambda N protein with the adenosine deaminase domain of a truncated native ADAR protein. This method requires target cells to be transduced with the fusion protein (a major obstacle) or transfected with a nucleic acid construct encoding the modified adenosine deaminase fusion protein for expression. The system described by Vogel et al. (2014) suffers from a similar drawback: it is unclear how to apply this system without first genetically modifying ADAR and then transfecting or transforming cells harboring the target RNA to deliver the genetically modified protein. A similar system is also described in US 9,650,627. Woolf et al.'s (1995) oligonucleotides were 100% complementary to the target RNA sequence but suffered from a severe lack of specificity: nearly every adenosine in the target RNA strand complementary to the antisense oligonucleotide was edited.

[0018] ADARs are known to act on any dsRNA. Through a process sometimes referred to as "promiscuous editing," these enzymes edit multiple A residues within dsRNA. Therefore, methods and tools were needed to circumvent such promiscuous editing and target specific adenosines within target RNA molecules for therapeutic applications. Vogel et al. (2014) demonstrated that such off-target editing can be suppressed by using 2'-O-methyl (2'-OMe)-modified nucleosides in oligonucleotides opposite adenosines that should not be edited, and by using unmodified nucleosides in oligonucleotides directly opposite the specifically targeted adenosines on the target RNA. However, specific editing at the target nucleotide has not been demonstrated without the use of recombinant ADAR enzymes covalently linked to AONs. Several publications have shown that it is possible to recruit endogenous ADAR (thus not requiring exogenous and / or recombinant sources) while maintaining specificity in targeting a single adenosine in a target RNA molecule and deaminating it to inosine. WO2016 / 097212 discloses an antisense oligonucleotide (AON) for targeted editing of RNA, characterized by a sequence complementary to the target RNA sequence (referred to as "targeting portion" in the publication) and the presence of a stem-loop / hairpin structure (referred to as "recruitment portion" in the publication), which is preferably non-complementary to the target RNA. Such oligonucleotides are referred to as "self-looping AONs". The recruitment portion acts to recruit the natural ADAR enzyme present in cells to the dsRNA formed by hybridization of the target sequence with the targeting portion. The presence of the recruitment moiety does not require the presence of the conjugate or modified recombinant ADAR enzyme.WO2016 / 097212 describes the recruitment moiety as a stem-loop structure that mimics a natural substrate (e.g., 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 stem-loop structure of the recruitment moiety is an intramolecular stem-loop structure formed within the AON itself, which is thought to attract (endogenous) ADAR. Systems containing similar stem-loop structures for RNA editing are described in WO2017 / 050306, WO2020 / 001793, WO2017 / 010556, WO2020 / 246560, and WO2022 / 078995.

[0019] WO2017 / 220751 and WO2018 / 041973 describe next-generation AONs, which do not contain such stem-loop structures but are (almost perfectly) complementary to target regions.In one embodiment, one or more mismatched nucleotides, wobbles, or bulges exist between the oligonucleotide and the target sequence.Sometimes, the mismatch only exists at the nucleoside site opposite the target adenosine, while in other embodiments, AONs (or RNA editing oligonucleotides, abbreviated as "EONs") have multiple bulges and / or wobbles when added to target sequence regions.It appears that if the sequence of EONs is carefully selected to attract / recruit ADAR, it may be possible to achieve RNA editing in vitro, ex vivo, and in vivo using EONs that lack stem-loop structures and endogenous ADAR enzymes. An "orphan nucleoside" is defined as a nucleoside within an EON directly opposite a target adenosine in a target RNA molecule, but which did not have a 2'-OMe modification. The orphan nucleoside may be a deoxyribonucleoside (DNA), with the remainder of the EON still having a 2'-O-alkyl modification (e.g., 2'-OMe) in the sugar moiety, or the nucleotides immediately surrounding the orphan nucleoside may contain chemical modifications (e.g., DNA compared to RNA), resulting in further improved RNA editing efficiency and / or increased resistance to nucleases. Such effects can be further improved by the use of sense oligonucleotides (SONs) to "protect" the EON from degradation (as described in WO 2018 / 134301).The use of chemical modifications and specific structures in oligonucleotides that can be used for ADAR-mediated editing of specific adenosines in target RNA has been the subject of numerous publications in the field, for example, 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 certain 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 example, for exon skipping, gapmers, siRNA, or specifically for RNA editing oligonucleotides related to a wide variety of target sequences) has been described in WO2011 / 005761, WO2014 / 010250, WO2014 / 012081, WO2015 / 107425 , WO2017 / 015575(HTT), WO2017 / 062862, WO2017 / 160741, WO2017 / 192664, WO2017 / 192679(DMD), WO2017 / 19877 5, WO2017 / 210647, WO2018 / 067973, WO2018 / 098264, WO2018 / 223056(PNPLA3), WO2018 / 223073(APOC3), WO2018 / 223081(PNPLA3), WO2018 / 237194, WO2019 / 032607(C9orf72), WO2019 / 055951, WO2019 / 075357(SMA / ALS), WO2 019 / 200185(DM1), WO2019 / 217784(DM1), WO2019 / 219581, WO2020 / 118246(DM1), WO2020 / 160336(HTT), WO2020 / 191252, WO2020 / 196662, WO2020 / 219981 (USH2A), WO2020 / 219983 (RHO), WO2020 / 227691 (C9orf72), WO2021 / 071788 (C9orf72), WO2021 / 071858, WO2021 / 178237 (MAPT), WO2021 / 234459, WO2021 / 237223, and WO2022 / 099159. In addition to these disclosures, there are numerous publications relating to targeting specific RNA target molecules, or specific adenosines within such RNA target molecules, to repair mutations that result in premature stop codons or other disease-causing mutations.Examples of such disclosures targeting adenosines 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-syndromic hearing loss); WO2021 / 231691 (XLRS); WO2021 / 231698 (argininosuccinate lyase deficiency); WO2021 / 130313 and WO2021 / 231830 (ABCA4); and WO2021 / 243023 (SERPINA1).

[0020] Disclosed herein is an EON capable of RNA editing of a target adenosine in human ALDH2 transcripts (pre-mRNA and / or mRNA), thereby restoring the resulting ALDH2 protein to its wild-type function: converting acetaldehyde to acetate in ethanol metabolism. In a preferred embodiment, the EON causes deamination of the adenosine present at position 1459 of the mutant mRNA, thereby producing inosine. In other words, the AAA codon (mutant) encoding lysine at amino acid position 504 is converted to an IAA codon, which is read as GAA (wild-type), encoding glutamic acid. In other embodiments, the EON according to the present invention causes deamination of another adenosine present in the ALDH2 transcript, which may be any adenosine that, when deaminated to inosine, results in an ALDH2 protein with wild-type or gain-of-function. Other mutations may exist in the ALDH2 gene (and transcript), which can be targeted through RNA editing, thereby restoring normal ALDH2 function. The preferred targeted mutation is a G>A mutation at position 1510 in the transcript, resulting in the p.Glu504Lys ALDH2 protein mutation (c.1510G>A). This protein mutation is often referred to as E487K, but is also referred to as E504K, while the mutant allele is commonly referred to as ALDH2*2. In the literature, this mutation is also referred to as p.Glu487Lys, rs671, c.1510G>A, and p.E504K, but the "c.1459G>A" designation has not been found. In this specification, the transcript number of the human (Homo sapiens) ALDH2 protein and gene published on ensemble.org is used (transcript ENST00000261733.7), which means that this mutation is referred to as E504K in the protein and c.1510G>A in the transcript.

[0021] In a preferred embodiment, the EON of the present invention is a single-stranded oligonucleotide comprising an isolated nucleotide opposite a target adenosine, the isolated nucleotide being chemically modified as disclosed herein, and the remainder of the oligonucleotide being chemically modified as disclosed herein to prevent degradation by nucleases; however, in another embodiment, the present invention relates to any kind of oligonucleotide or heteroduplex oligonucleotide complex, which may or may not be bound to a hairpin structure (internally or at one or more ends), may bind to ADAR or its catalytic domain, or the oligonucleotide may be expressed through a vector such as an adeno-associated virus (AAV), or may be circular. It should be understood that any kind of oligonucleotide-based RNA editing is encompassed by the present invention as long as it is associated with a mutation that causes deamination of a nucleotide in the ALDH2 transcript, preferably E504K, and restores ALDH2 function. In preferred embodiments, the EONs of the present invention are "naked" oligonucleotides, which contain various chemical modifications to the ribose sugar, base, and / or internucleoside linkage of one or more nucleotides within the sequence, and are capable of hybridizing to an ALDH2 transcript or portion thereof containing a target adenosine, and are capable of recruiting endogenous ADARs for deamination of the target adenosine.

[0022] The present invention relates to an EON capable of forming a double-stranded complex with a region of an endogenous human ALDH2 transcript molecule in a cell, the region of the ALDH2 transcript molecule containing a target adenosine, and the double-stranded complex can recruit an endogenous ADAR enzyme to deaminate the target adenosine to inosine, thereby editing the ALDH2 transcript molecule. The endogenous ADAR enzyme is preferably ADAR2. The cell is preferably a human hepatocyte, more preferably a human hepatocyte. The ALDH2 transcript molecule is preferably a pre-mRNA or mRNA molecule. The EON of the present invention preferably targets for deamination an adenosine that causes dysfunction of the ALDH2 protein. Although several mutations that cause dysfunction of the ALDH2 protein are known, a preferred mutation targeted by the EON disclosed herein is an adenosine derived from a G>A mutation in the ALDH2 gene, resulting in a mutant p.E504K ALDH2 protein. The EONs disclosed herein can deaminate the first adenosine in a lysine-encoding AAA codon, thereby generating an IAA codon, which is read as GAA and therefore translated into glutamic acid. In one embodiment, an EON disclosed herein comprises or consists of any one of the EON sequences shown in Figure 1. In certain embodiments, an EON of the invention comprises or consists entirely of the chemical modifications shown in Figure 1.

[0023] In one embodiment, an EON according to the present invention comprises at least one nucleotide comprising one or more non-natural chemical modifications or one or more additional non-natural chemical modifications in the ribose moiety, linker moiety, or base moiety, provided that the isolated nucleotide in the EON directly opposite the target adenosine is not a cytidine comprising a 2'-OMe ribose substitution. In one embodiment, the one or more additional modifications in the linker moiety are each independently selected from phosphorothioate (PS), phosphonoacetate, phosphorodithioate, methylphosphonate (MP), sulfonyl phosphoramidate, or PNdmi internucleotide linkage. Preferably, the one or more additional modifications in the ribose moiety are mono- or di-substitutions at the 2', 3', and / or 5' positions of the ribose, each independently selected from the group consisting of: -OH; -F; substituted or unsubstituted, linear or branched lower alkyl (C1-C2) nucleotides. 10 ) alkyl, alkenyl, alkynyl, alkaryl, aryl, 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-aryl; -O-alkyl-O-alkyl; -methoxy; -aminopropoxy; -methoxyethoxy; -dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy. In some embodiments, the EON contains one or more mismatches, wobbles, or bulges, and a single mismatch may be present when the target adenosine has an opposing cytidine in the EON. When the isolated nucleotide is a cytidine, the cytidine does not contain a 2'-OMe ribose substitution.

[0024] Disclosed herein is a vector, preferably a viral vector, more preferably an adeno-associated viral (AAV) vector, comprising a nucleic acid molecule encoding the EON disclosed herein. Also disclosed herein is a pharmaceutical composition comprising the disclosed EON or the disclosed vector and a pharmaceutically acceptable carrier.

[0025] In one embodiment, the EON disclosed herein, the vector disclosed herein, or the pharmaceutical composition disclosed herein is disclosed for use in treating a disorder caused by ALDH2 deficiency, preferably caused by ALDH2*2, such as symptoms of alcohol intoxication, alcoholism, or alcohol consumption. In one embodiment, the EON disclosed herein or the vector disclosed herein is disclosed for use in the manufacture of a medicament for treating one or more symptoms of ALDH2*2-induced alcohol intolerance, such as alcohol intoxication, alcoholism, or alcohol consumption.

[0026] In one embodiment, a method for editing an ALDH2 polynucleotide is disclosed, the method comprising contacting the ALDH2 polynucleotide with an EON capable of causing an adenosine to inosine conversion mediated by adenosine deaminase acting on RNA (ADAR) associated with alcohol intolerance, thereby editing the ALDH2 polynucleotide.In one embodiment, a method for treating an ALDH2 deficiency, such as ALDH2*2-induced alcohol intolerance or intoxication, or a disorder caused by the alcohol intolerance or intoxication, in a patient in need thereof, the method comprising contacting an ALDH2 polynucleotide in a subject's cells with an EON capable of causing an adenosine to inosine conversion mediated by ADAR associated with alcohol intolerance or a disorder caused by the alcohol intolerance, such as alcohol intoxication, alcoholism, or symptoms of alcohol use, thereby treating the patient. A preferred mutation targeted in the methods and uses disclosed herein is a G>A mutation, which results in a glutamic acid to lysine change in the mature protein at position 504. In one embodiment, a method is disclosed for treating alcohol intolerance or a disorder caused by said alcohol intolerance, the method comprising administering to a patient in need thereof a therapeutically effective amount of an EON disclosed herein, a vector disclosed herein, or a pharmaceutical composition disclosed herein.

[0027] In one embodiment, a method for deaminating target adenosine in ALDH2 pre-mRNA or mRNA molecules in cells is disclosed, the method comprising the following steps: (i) providing the cell with EON or vector disclosed herein; (ii) allowing the cell to incorporate the EON or vector, respectively; (iii) allowing the EON to anneal with the ALDH2 pre-mRNA or mRNA molecule; (iv) allowing endogenous ADAR enzyme (such as ADAR2) to deaminate the target adenosine in the target RNA molecule to inosine; and (v) identifying the presence of the inosine in the target RNA molecule.Preferred target adenosine is G>A mutation in the transcript molecule of mutant ALDH2 gene encoding mutant p.E504K ALDH2 protein. Preferably, step (v) comprises: a) determining the sequence of said ALDH2 pre-mRNA or mRNA molecule; b) assessing the presence of wild-type ALDH2 protein; or c) using a functional readout, such as assessing the level of alcohol in a serum or plasma sample, or any other biomarker related to the function of ALDH2 known to those skilled in the art.

[0028] [Definition] The term "nucleoside" refers to a nucleic acid base linked to a (deoxy)ribose without a phosphate group. A "nucleotide" is composed of a nucleoside and one or more phosphate groups. The term "nucleotide" therefore refers to the respective nucleic acid base-(deoxy)ribose-phospholinker and any chemical modification of the ribose moiety or phospho group. The term therefore includes: nucleotides containing locked ribose moieties (including 2'-4' bridges containing methylene groups or any other groups), unlocked nucleic acids (UNA), threose nucleic acids (TNA), nucleotides containing linkers containing phosphodiesters, phosphonoacetates, phosphotriesters, PS, phosphoro(di)thioates, MP, methylthiophosphonates, phosphoramidate bonds, etc. The terms adenosine and adenine, guanosine and guanine, cytidine and cytosine, uracil and uridine, thymine and thymidine / uridine, and inosine and hypoxanthine are sometimes used interchangeably to refer to the corresponding nucleobases on the one hand and the nucleosides or nucleotides on the other hand. Thymine (T) is a nucleotide that is substituted with 5-methyluracil (m 5 Also known as 5-methyluridine, thymine is a derivative of uridine; thymine, 5-methyluridine, and uridine are used interchangeably throughout the document. The terms nucleobase, nucleoside, and nucleotide are sometimes used interchangeably unless the context clearly requires otherwise (e.g., when a nucleoside is linked to adjacent nucleosides and the bond between these nucleosides is modified). As used 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.

[0029] Whenever oligonucleotides, oligos, ONs, ASOs, oligonucleotide compositions, antisense oligonucleotides, AONs, (RNA) editing oligonucleotides, EONs, and RNA (antisense) oligonucleotides are mentioned, unless the context dictates otherwise, to refer to both oligoribonucleotides and deoxyoligoribonucleotides. In some cases, oligonucleotides may be completely devoid of RNA or DNA nucleotides (as they naturally occur) and consist entirely of modified nucleotides. Whenever "oligoribonucleotides" are mentioned, they may contain the bases A, G, C, U, or I. Whenever "deoxyoligoribonucleotides" are mentioned, they may contain the bases A, G, C, T, or I. However, EONs disclosed herein may contain a mixture of ribonucleosides and deoxyribonucleosides. When deoxyribonucleotides are used, i.e., when there is no modification at the 2' position of the sugar, the nucleotide is often abbreviated as dA, dC, dG, or T, where "d" denotes the deoxy nature 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 the respective modification, as described herein.

[0030] Whenever nucleotide is mentioned in oligonucleotide, for example, cytosine 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-methylpseudouracil, 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 a nucleoside or nucleotide is referred to, ribofuranose derivatives such as 2'-deoxy, 2'-hydroxy, and 2'-O-substituted variants (e.g., 2'-OMe) are included, as well as other modifications, including 2'-4' bridged variants. Whenever an oligonucleotide is referred to, the linkage between two mononucleotides may be a phosphodiester bond, and modifications thereof include phosphonoacetate, phosphotriester, PS, phosphoro(di)thioate, MP, phosphoramidate linker, phosphorylguanidine, thiophosphorylguanidine, sulfonophosphoamidate, and the like.

[0031] The term "comprising" encompasses "including" and "consisting of," e.g., a composition "comprising X" may consist solely of X or may include something additional (e.g., X+Y). The term "about" in reference to a numerical value x is optional, and means, for example, x±10%.

[0032] The word "substantially" does not exclude "completely", for example a composition that is "substantially free of Y" may be completely free of Y. Where relevant, the word "substantially" may be omitted from the definition of the invention.

[0033] The term "complementary" as used herein refers to the fact that an EON hybridizes with a second nucleic acid strand under physiological conditions (e.g., when an oligonucleotide, as a first nucleic acid strand (=guide oligonucleotide), forms a heteroduplex RNA-editing oligonucleotide complex (i.e., HEON) with another complementary nucleic acid strand, or when it forms a double-stranded complex with a target RNA sequence). This term does not necessarily mean that each nucleotide in a nucleic acid strand perfectly pairs with the opposite nucleotide in the opposite sequence. In other words, an EON can be complementary to a target sequence, but there may be mismatches, wobble, and / or bulges between the oligonucleotide and the target sequence, while the EON still hybridizes with the target sequence under physiological conditions, allowing RNA-editing enzymes in cells to edit the target adenosine. Thus, the term "substantially complementary" also means that the EON has sufficient nucleotide matches between the EON and the target sequence such that the EON can hybridize to the target RNA under physiological conditions, even if mismatches, wobble, and / or bulges are present. As provided herein, an EON can be complementary but contain one or more mismatches, wobble, and / or bulge with the target sequence, provided that the EON can hybridize to its target under physiological conditions.

[0034] The term "downstream," with respect to a nucleic acid sequence, means further along the sequence in the 3' direction; the term "upstream" means the opposite. Thus, in any sequence that encodes a polypeptide, the start codon is upstream of the stop codon on the sense strand, but downstream of the stop codon on the antisense strand.

[0035] References to "hybridization" typically refer to specific hybridization and exclude non-specific hybridization, which may occur under selected experimental conditions using techniques well known in the art to ensure that the most stable interaction between probe and target is when the probe and target have at least 70%, preferably at least 80%, and more preferably at least 90% sequence identity.

[0036] As used herein, the term "mismatch" refers to opposing nucleotides in a double-stranded RNA complex that do not form a perfect 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 first nucleic acid strand of the present invention contains 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. Although G:G pairing is considered a mismatch, this does not necessarily mean that the interaction is unstable; the term "mismatch" may be somewhat outdated based on the current invention, which considers Hoogsteen base pairings to be relatively stable even when they are considered mismatches based on the origin of the nucleotides. An isolated G:G pair within a duplex RNA, for example, may be very stable but is still defined as a mismatch.

[0037] The term "splice mutation" refers to a mutation in a gene encoding a pre-mRNA in which the splicing machinery is dysfunctional in the sense that splicing of an intron from an exon is disrupted and this aberrant splicing causes subsequent translation to frame out, resulting in premature termination of the encoded protein. Such truncated proteins are often rapidly degraded and lack any functional activity.

[0038] The EONs disclosed herein (and the complementary nucleic acid strands when two oligonucleotides form an HEON) can be chemically modified throughout, for example, by providing ribose sugar moieties with 2'-OMe, 2'-F, or 2'-O-methoxyethyl (2'-MOE) substitutions at the nucleotides. The isolated nucleotides in the EON are preferably cytidine or analogs thereof (e.g., nucleotides with a benign base), or uridine or analogs thereof (e.g., isouridine), and / or, in one embodiment, contain a diF modification at the 2' position of the sugar, or in another embodiment, deoxyribose (2'-H, DNA). In yet another embodiment, at least one, and in another embodiment, both, of the two adjacent nucleotides flanking the isolated nucleotide do not contain a 2'-OMe modification. Full modification, in which all nucleotides of an oligonucleotide have natural bases but retain 2'-OMe modifications, results in an oligonucleotide that does not function in RNA editing (as is known in the art), presumably because it prevents ADAR activity at the target position. Generally, an adenosine in a target RNA can be protected from editing by providing a 2'-OMe group at the opposing nucleotide (at least in the absence of other chemical substitutions or modifications within the nucleotide) or by providing guanine or adenine as the opposing base, as these two nucleobases can also reduce editing of the opposing adenosine.

[0039] A variety of chemical techniques and modifications are known in the field of oligonucleotides that can be readily used in accordance with the present invention. The normal internucleoside linkage between nucleotides can be altered by mono- or dithiolation of the phosphodiester bond to produce PS esters or phosphorodithioate esters, respectively. Other modifications of the internucleoside linkage are possible, including amidation and peptide linkers.

[0040] In certain embodiments, the EONs of the present invention comprise 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. Of note, when EONs are delivered via a (viral) vector, their length may be longer than 60 nucleotides. However, when EONs are delivered directly without a vector (also referred to as "naked form"), the length of the EON is limited to 15-60 nucleotides to reduce the risk of degradation. Furthermore, in the naked form, the EON is preferably chemically modified as described herein to reduce the risk of degradation.

[0041] It is known in the art that RNA editors (e.g., human ADAR enzymes) edit dsRNA structures with varying specificity, depending on several factors. One important factor is the degree of complementarity between the two strands that make up the dsRNA sequence. When the two strands are perfectly complementary, the catalytic domain of human ADAR typically reacts with every adenosine it encounters, indiscriminately deaminating adenosines. The specificity of hADAR1 and 2 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 yet-to-be-defined manner. Furthermore, the deamination reaction itself can be accelerated by providing an oligonucleotide containing a mismatch opposite the adenosine to be edited. By following the instructions of the present application, those skilled in the art can design the complementary portion of the oligonucleotide according to their needs.

[0042] The most interesting intracellular RNA editing protein that can be used with EON of the present invention is human ADAR2.Those skilled in the art will understand that the degree to which intracellular editing bodies are redirected to other target sites can be adjusted by changing the affinity of the first nucleic acid strand to the recognition domain of editing molecules.The modification itself can be determined by trial and error and / or by calculation methods based on the structural interaction between EON and the recognition domain of editing molecules.In addition, or alternatively, the degree of intracellular editing bodies recruitment and redirection can be adjusted by the dosage and administration regimen of EON.This is determined by experimenters (in vitro) or clinicians, usually in phase I and / or phase II clinical trials.

[0043] The present invention relates to the modification of target RNA sequences in eukaryotic organisms, preferably metazoans, more preferably mammals, even more preferably human cells, most preferably human liver cells such as hepatocytes. The present invention is particularly suitable for modifying RNA sequences in cells and tissues where ALDH2K is expressed and where this protein acts. Because ALDH2 is mainly produced in hepatocytes and plays an important role in ethanol metabolism, the preferred target cells of the EON of the present invention are hepatocytes, more preferably hepatocytes. Target cells can be located in vitro, ex vivo, or in vivo. One advantage of the present invention is that it can be used on cells in situ in vivo, but it can also be used on cultured cells. In some embodiments, cells are treated ex vivo and then introduced into a living body (e.g., reintroduced into the organism from which they originally originated). The present invention can also be used to edit target RNA sequences in transplanted cells or cells within so-called organoids (e.g., liver tissue organoids). Organoids are considered to be three-dimensional in vitro-derived tissues, but can be driven to generate individual, isolated tissues using specific conditions. In a therapeutic setting, organoids are useful because they are generated in vitro from a patient's cells and then reintroduced into the patient as autologous material, which is less likely to be rejected than a regular transplant.

[0044] Without wishing to be bound by theory, it is believed that RNA editing through human ADAR2 occurs on the primary transcript either in the nucleus during transcription or splicing, or in the cytoplasm, where, for example, mature mRNA, miRNA, or ncRNA may be edited.

[0045] It should be clear that targeted editing according to the present invention can be applied to any adenosine in the ALDH2 transcript, so long as deamination of that adenosine results in the improvement or restoration of ALDH2 protein function, but as outlined herein, it is preferred to target the first adenosine present in the mutated codon at position 504 of the mature protein.

[0046] Generally speaking, RNA editing can be used to create RNA sequences with different properties. Such properties can be coding properties (creating proteins with different sequences or lengths, altering the protein's properties or function) or binding properties (causing inhibition or overexpression of the RNA itself or its target or binding partner; entire expression pathways can be altered by recoding their corresponding sequences on the miRNA or target RNA). Protein function or localization can be freely modified by functional domains or recognition motifs (including, but not limited to, signal sequences, targeting or localization signals, recognition sites for proteolytic cleavage or co- or post-translational modification, catalytic sites for enzymes, binding sites for binding partners, signals for degradation or activation, etc.). These and other forms of RNA and protein "engineering" are encompassed by the present invention, whether for the purposes of disease prevention, delay, or treatment, or for other purposes, in medicine or biotechnology, as diagnostics, prophylactics, therapeutics, research tools, or otherwise. Therefore, any RNA editing of a target adenosine in the ALDH2 transcript that results in the improvement or restoration of ALDH2 protein function is encompassed by the present invention. The present invention opens up an entirely new field of treating alcohol intoxication using gene editing technology.

[0047] The amount, dosage, and administration regimen of administered EON may vary depending on cell type, disease to be treated, target population, mode of administration (e.g., systemic or local), severity of disease, and tolerance level of side effects, but these may and should be evaluated by trial and error during in vitro studies, preclinical and clinical trials.Testing is particularly easy when the modified sequence causes easily detectable phenotypic changes or changes in specific biomarkers (levels or activity).Higher doses of EON may compete for binding to ADAR in cells, thereby depleting the amount of entities free to participate in RNA editing, but regular administration tests will reveal such effects, if any, for a given EON and a given target.

[0048] One suitable testing technique involves delivering EONs to a cell line or test organism and then taking biopsies at various time points. The sequence of the target RNA in the biopsy sample can be assessed to easily track the proportion of cells with the modification. Additionally, the percentage of alcohol in a blood sample is a suitable biomarker for assessing the function of ALDH2 protein in a particular subject before and after treatment, or with or without treatment of the subject with an EON or vector disclosed herein. Once this testing is performed, knowledge is retained, and subsequent deliveries can be performed without the need for biopsies. Thus, the methods of the present invention can include a step of identifying the presence of a desired change in the target RNA sequence in a cell, thereby verifying that the target RNA sequence has been modified. This step typically involves 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 assessed based on protein function, for example, by measuring or assessing serum or plasma ethanol concentrations before, during, and / or after treatment, or assessing other potential markers, preferably performed in vitro on samples obtained from the treated subject.

[0049] After RNA editing occurs in a cell, the modified RNA may be diluted over time, e.g., due to cell division, the finite half-life of the edited RNA, etc. Thus, in terms of practical treatment, the methods of the invention may involve repeated delivery of EONs until enough of the target RNA is modified to provide a tangible benefit to the patient and / or to maintain that benefit over time.

[0050] The EON of the present invention is particularly suitable for therapeutic use, and therefore the present invention also relates to a pharmaceutical composition comprising the EON of the present invention, or a vector or plasmid encoding the EON of the present invention, and a pharmaceutically acceptable carrier. In some embodiments of the present invention, the pharmaceutically acceptable carrier may simply be a saline solution, which may be isotonic or hypotonic, particularly for pulmonary delivery. The present invention also provides a delivery device (e.g., syringe, inhaler, nebulizer) comprising the pharmaceutical composition of the present invention.

[0051] The present invention also provides an EON of the present invention for use in a method for repairing a mutation in a target ALDH2 RNA sequence in a mammalian, preferably human, hepatocyte, as described herein. Similarly, the present invention provides use of an EON of the present invention in the manufacture of a medicament for altering a target ALDH2 RNA sequence in a mammalian, preferably human, hepatocyte, as described herein, thereby treating, preventing, or ameliorating a disease associated with alcohol intoxication, such as a disease caused by ALDH2*2.

[0052] The present invention also relates to a method for deaminating at least one specific target adenosine present in a target ALDH2 RNA sequence in a cell, the method comprising the following steps: providing an EON according to the present invention to the cell; allowing the EON to be taken up by the cell; annealing the EON to a target RNA molecule; allowing a mammalian ADAR enzyme comprising a native dsRNA-binding domain found in a wild-type enzyme to deaminate the target adenosine in the target RNA molecule (preferably the first adenosine of the codon encoding lysine at position 504 in the mature protein) to inosine; and optionally identifying the presence of the inosine in the RNA sequence.

[0053] The present invention also relates to a method for deaminating at least one specific target adenosine present in a target ALDH2 RNA sequence in a cell, the method comprising the following steps: providing a vector or plasmid encoding an EON according to the present invention to the cell; allowing the cell to take up the vector or plasmid; annealing the EON to a target RNA molecule; allowing a mammalian ADAR enzyme comprising a native dsRNA-binding domain found in the wild-type enzyme to deaminate the target adenosine in the target RNA molecule (preferably the first adenosine of the codon encoding lysine at position 504 in the mature protein) to inosine; and optionally identifying the presence of the inosine in the RNA sequence.

[0054] In a preferred embodiment, depending on the final deamination effect of the conversion of A to I, the identifying step includes the following steps: sequencing the target RNA; assessing the presence or absence of a functional protein; assessing whether the deamination alters the splicing of pre-mRNA; or using a functional readout, since the target RNA after deamination should encode a functional protein. An example is assessing the ethanol and / or acetaldehyde concentration in the (blood) sample after RNA editing. Therefore, identifying deamination to inosine can be a functional readout using an appropriate biomarker. The functional assessment of alcohol intoxication referred to herein generally follows methods known to those skilled in the art. Naturally, a highly suitable method for identifying the presence of inosine after deamination of target adenosine is dPCR or even sequencing, using methods well known to those skilled in the art. However, those skilled in the art of liver disease can apply tests to monitor certain biomarkers related to alcohol intoxication, as described above.

[0055] The EONs of the present invention are suitably administered in aqueous solution (e.g., saline) or suspension, which may contain additives, excipients, and other ingredients suitable for pharmaceutical use, at a concentration ranging from 1 ng / ml to 1 g / ml, preferably from 10 ng / ml to 500 mg / ml, and more preferably from 100 ng / ml to 100 mg / ml. A suitable dosage range may be from about 1 μg / kg to about 100 mg / kg, preferably from about 10 μg / kg to about 10 mg / kg, and more preferably from about 100 μg / kg to about 1 mg / kg. Administration may be by inhalation (e.g., via a nebulizer), nasal, oral, injection or infusion, intravenous, subcutaneous, intradermal, intramuscular, intratracheal, intraperitoneal, rectal, intrathecal, intracisternal, parenteral, or the like. Administration may be in the form of a solid, powder, tablet, gel, solution, sustained-release formulation, or any other form suitable for human pharmaceutical use.

[0056] In one embodiment, the method of the present invention comprises the steps of: administering to a subject an EON or pharmaceutical composition of the present invention; allowing a double-stranded nucleic acid complex between the EON and its specific, complementary target nucleic acid molecule to form in cells of the subject; allowing an endogenous adenosine deaminase, such as ADAR2, to participate; and allowing the enzyme to deaminate the target adenosine in the target nucleic acid molecule to inosine, thereby reducing, preventing, or ameliorating a disorder associated with alcohol intoxication. The disorders that can be treated by this method are preferably, but are not limited to, the genetic disorders listed herein, and may also be any other disorder in which deamination of adenosine in ALDH2 transcripts restores protein function in a subject in need thereof.

[0057] The RNA editing molecules present in cells are usually proteinaceous in nature, for example, ADAR enzymes found in metazoans, including mammals.Preferably, the editing molecules in cells are enzymes, more preferably adenosine deaminase or cytidine deaminase, even more preferably adenosine deaminase.These are enzymes with ADAR activity.One of the most interesting is human ADAR, namely, hADAR1 and hADAR2, including any isoforms thereof.RNA editing enzymes known in the art that can be conveniently designed for the oligonucleotide construct of the present invention include adenosine deaminase (ADAR) acting on RNA, such as hADAR1 and hADAR2 in humans or human cells, and cytidine deaminase.It is known that there are two isoforms of hADAR1: a long interferon-induced form of 150 kDa, and a shorter form of 100 kDa produced through alternative splicing from a common pre-mRNA. As a result, the level of the 150 kDa isoform available in cells can be affected by interferon, 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 EON of the present invention are administered to patients as a combined product or as separate products, simultaneously or sequentially (in any order). In some disease states, elevated levels of IFN-γ or TNF-α may already exist in certain tissues of patients, which creates an additional opportunity for more specific editing of diseased tissues. Those skilled in the art will understand that the degree to which the editing product inside the cell is redirected to other target sites can be adjusted by changing the affinity of the first nucleic acid strand for the recognition domain of the editing molecule.

[0058] [Chemical modification] All of the chemical modifications listed below that can be used with the EONs of the present invention can also be used with the sense strand complementary to the EON when the EON and the complementary strand form a so-called heteroduplex RNA-editing oligonucleotide (HEON) complex, as described in GB2215614.5 (unpublished). The exception is that the opposing sense strand does not have an isolated nucleotide. Therefore, modifications involving an isolated nucleotide relate only to the EONs of the present invention, while all other modifications relate to the EONs of the present invention and any (protected) sense oligonucleotides that can be used with the EONs 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 GB2215614.5 (unpublished), and can be attached to the EON, its opposing strand, or both.

[0059] The internucleoside bond in the oligonucleotide of the present invention may comprise one or more natural internucleoside bond(s) and / or modified internucleoside bond(s).Without limitation, at least one, at least two, or at least three internucleoside bond(s) from the 5'-end and / or 3'-end of the EON are preferably modified internucleoside bond(s).Preferably, the modified internucleoside bond(s) is / are PS bond(s).In one embodiment, all internucleoside bond(s) of the EON are modified internucleoside bond(s).In one embodiment, the EON comprises a PNdmi bond connecting the most terminal nucleoside at the 5'-end and / or 3'-end and the second nucleoside at each of these ends, respectively.The PNdmi bond preferably used in the EON of the present invention has the following formula structure: JPEG2025539381000001.jpg49166

[0060] Common limiting factors in oligonucleotide-based therapeutics include the ability of the oligonucleotide to be taken up by cells (when delivered "naked" without the application of a delivery vehicle), biodistribution, and resistance to nuclease-mediated degradation. Those skilled in the art recognize that various chemical modifications can help overcome such limitations, and these have been extensively reported in the art. Examples of such currently commonly used chemical modifications include 2'-O-methyl (often abbreviated as 2'-OMe or 2'-O-Me), 2'-F, and 2'-O-methoxyethyl (often referred to as 2'-methoxyethoxy or 2'-MOE) sugar modifications, and the use of PS linkages between nucleosides. WO2020 / 201406 discloses the use of MP linkage modifications at specific positions around isolated nucleotides within a first nucleic acid strand. The ribose 2' group in all nucleotides of an EON, except for the ribose sugar moiety of an isolated nucleotide, which has certain limitations with respect to compatibility with RNA editing, can be independently selected from the following: 2'-H (i.e., DNA), 2'-OH (i.e., RNA), 2'-OMe, 2'-MOE, 2'-F, or 2'-4' linkage (e.g., locked nucleic acid (LNA)), or other ribose 1'-, 2'-, 3'-, 4'-, or 5'-substitutions. Isolated nucleotides in an EON that do not contain other chemical modifications in the ribose sugar, base, or linkage preferably do not have 2'-OMe or 2'-MOE substitutions, and may have 2'-F, 2',2'-difluoro (diF), or 2'-ara-F (FANA) substitutions, or may be DNA. GB2214347.3 (unpublished) describes the modification of the 2' position of the ribose sugar moiety of isolated nucleotides with a 2',2'-disubstituted modification such as diF, which is also applicable to the invention described herein. The 2'-4' bond can be selected from many linkers known in the art, such as a methylene linker, an amide linker, or a constrained ethyl linker (cEt).

[0061] The present invention relates to an EON for use in deaminating a target nucleotide (preferably adenosine) in a target RNA, wherein the EON is complementary to a continuous region of nucleotides in the target RNA that includes the target adenosine, and the nucleotide directly opposite the target nucleotide in a first nucleic acid strand is an isolated nucleotide. When the target nucleotide is adenosine, the isolated nucleotide preferably contains a base with an NH moiety at a position similar to the cyclic nitrogen, or a modified base, or a base analog (e.g., Benner's base Z). Nucleotides in the EON are numbered such that the isolated nucleotide is numbered 0 and the nucleotide 5' to the isolated nucleotide is numbered +1. Numbers increase positively (+) toward the 5' end and negatively (-) toward the 3' end, with the first nucleotide 3' to the isolated nucleotide being numbered -1. The numbering of internucleoside linkages in EON is such that linkage 0 is the 5' linkage of the lone nucleotide, and the linkage positions within the oligonucleotide increase in positive (+) order toward the 5' end and decrease in negative (-) order toward the 3' end.

[0062] Preferably, the EON comprises one or more (chirally pure or chirally mixed) PS bonds. In one embodiment, the PS bonds link the terminal 3, 4, 5, 6, 7, or 8 nucleotides at each end of the first nucleic acid strand. In one embodiment, the EON comprises one or more phosphoramidate (PN) bonds. In one embodiment, the PN bonds link the terminal 2 nucleotides at each end of the EON.

[0063] Nucleosides in EONs may be natural nucleosides (deoxyribonucleosides or ribonucleosides) or unnatural nucleosides. In RNA editing, double-stranded RNA generally serves as a substrate for enzymes with deaminase activity (e.g., ADAR), so ribonucleosides are considered "natural," while deoxyribonucleosides may be considered unnatural or modified for the sake of discussion, since DNA does not exist in an RNA-RNA double-stranded substrate structure. Those skilled in the art will understand that even if a nucleotide has a natural ribose moiety, the base and / or linkage may be unnaturally modified.

[0064] In addition to the specific preferred chemical modifications at certain positions in the compounds of the present invention, the compounds of the present invention may also contain or consist of one or more (additional) modifications to the nucleobase, scaffold, and / or backbone linkages. These may or may not be present in the same monomer, for example, at the 3' and / or 5' positions. Scaffold modifications refer to the presence of modified forms of the ribose moiety (i.e., pentose moiety) naturally occurring in RNA, such as bicyclic sugars, tetrahydropyrans, hexoses, morpholinos, 2'-modified sugars, 4'-modified sugars, 5'-modified sugars, and 4'-substituted sugars.Examples of suitable modifications include, but are not limited to, 2'-O-modified RNA monomers, such as 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- 2'-O-[2-(N,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); bicyclic or bridged nucleic acid (BNA) scaffold modifications, e.g., conformationally restricted nucleotide (CRN) monomers, locked nucleic acid (LNA) monomers, xylo-LNA monomers, α-LNA mono mer, 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) constrained ethyl (cEt) BNA monomer, (2′-O,4′-C) constrained methoxyethyl (cMOE) BNA monomer, 2′,4′-BNA. NC (NH) monomer, 2′,4′-BNA NC (NMe) monomer, 2′,4′-BNA NC(NBn) monomers, ethylene-bridged nucleic acid (ENA) monomers, carba-LNA (cLNA) monomers, 3,4-dihydro-2H-pyran nucleic acid (DpNA) monomers, 2'-C-bridged bicyclic nucleotide (CBBN) monomers, oxo-CBBN monomers, heterocyclic bridged BNA monomers (e.g., triazolyl- or tetrazolyl-linked), amide-bridged BNA monomers (e.g., AmNA), urea-bridged BNA monomers, sulfonamide-bridged BNA monomers, bicyclic carbocyclic nucleotide monomers, TriNA monomers, α-l-TriNA monomers, bicyclic DNA (bcDNA) monomers, F-bcDNA monomers, tricyclic DNA (tcDNA) monomers, 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, altritol 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); and inverted versions of any of the above monomers. All of these modifications are known to those skilled in the art.

[0065] The base sequence of the EON herein is complementary to a portion of the base sequence of the target ALDH2 transcript, including at least the target adenosine to be deaminated to inosine, and therefore can anneal (or hybridize) with the target transcript. The complementarity of the base sequence can be determined using a BLAST program or the like. Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which the double strands can hybridize, taking into account the complementarity between the strands.

[0066] In contrast to what has been described with respect to gapmers and their relationship to ribonuclease degradation, and the use of such gapmers in double-stranded complexes (see, for example, EP 3954395 A1), the EONs of the present invention do not contain consecutive stretches of DNA nucleotides that would render the target sequence (or sense nucleic acid strand) targetable for ribonuclease-mediated degradation. In one embodiment, the EON does not contain four or more consecutive DNA nucleotides at any position within its sequence. In certain embodiments, the EON is composed of as many (chemically) modified nucleotides as possible to increase resistance to ribonuclease-mediated degradation, while at the same time being as efficient as possible in producing an RNA editing effect. This means that isolated nucleotides and other multiple nucleotides within the EON may be DNA, but also means that there are no consecutive stretches of four or more consecutive DNA nucleotides within the EON. Thus, the EONs of the present invention are not gapmers. Gapmers reduce the expression of target transcripts but do not effect RNA editing of specific adenosines within the target transcript. A gapmer is essentially a single-stranded nucleic acid, consisting of a central region (a DNA gap region having at least four consecutive deoxyribonucleotides) and wing regions located directly at its 5'-end (5' wing region) and 3'-end (3' wing region). In contrast, an EON according to the present invention may be any oligonucleotide that produces an RNA editing effect by deaminating a target adenosine to inosine in a target RNA molecule, and therefore is as resistant as possible to ribonuclease-mediated degradation to achieve this effect.

[0067] In one embodiment, the EON or the sense strand that can anneal to it before entering the target cell is conjugated to a hydrophobic moiety, such as palmityl or its analog, cholesterol or its analog, or tocopherol or its analog. This is preferably conjugated to the 5'-end. When the hydrophobic moieties are conjugated to the 5'-end and the 3'-end, the hydrophobic moieties may be the same or different. The hydrophobic moiety conjugated to the oligonucleotide may be directly conjugated or indirectly conjugated via another substance. When the hydrophobic moiety is directly conjugated, it is sufficient that the moiety is conjugated via a covalent bond, ionic bond, hydrogen bond, or the like. When the hydrophobic moiety is indirectly conjugated, it may be conjugated 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, within a cell or an animal body (e.g., the human body). Cleavable linkers are selectively cleaved by endogenous enzymes such as nucleases or by physiological conditions specific to a portion of the body or cell, such as pH or a reducing environment (e.g., glutathione concentration). Examples of cleavable linkers include, but are not limited to, amides, esters, phosphodiesters (either one or both), phosphoesters, carbamates, and disulfide bonds, as well as natural DNA linkers. Cleavable linkers also include self-immolative linkers. A non-cleavable linker refers to a linker that is not cleaved under physiological conditions or is cleaved much more slowly than a cleavable linker, such as a linker consisting of a PS bond, a modified or unmodified deoxyribonucleoside linked by a PS bond, a spacer linked through a PS bond, and a modified or unmodified ribonucleoside. When the linker is a nucleic acid such as DNA or an oligonucleotide, there is no limit to its chain length. However, typically, it can be 2 to 20 bases in length, 3 to 10 bases in length, or 4 to 6 bases in length. The length or composition of the spacer linking the ligand and the oligonucleotide is not limited, and can include, for example, ethylene glycol, TEG, HEG, an alkyl chain, propyl, 6-aminohexyl, or dodecyl.

[0068] The present invention also relates to a pharmaceutical composition comprising an EON according to the present invention, further comprising a pharmaceutically acceptable carrier and / or other additives, and optionally dissolved in a pharmaceutically acceptable organic solvent or the like. The dosage form in which the EON 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 in a single dose or multiple doses. It may be administered daily or at appropriate time intervals determined using general knowledge in the art, and may be adjusted based on the disorder and the effectiveness of the active ingredient.

[0069] In one embodiment, an EON comprises at least one nucleotide having a sugar moiety comprising a 2'-OMe modification. In one embodiment, an EON comprises at least one nucleotide having a sugar moiety comprising a 2'-MOE modification. In one embodiment, an EON comprises at least one nucleotide having a sugar moiety comprising a 2'-F modification. In one embodiment, an isolated nucleotide has a 2'-H in the sugar moiety, and is therefore referred to as a DNA nucleotide, although additional modifications may be present in its base and / or its linkage to the adjacent nucleoside. In one embodiment, an isolated nucleotide has a 2'-F in the sugar moiety. In one embodiment, an isolated nucleotide has a diF substitution in the sugar moiety. In one embodiment, an isolated nucleotide has 2'-F and 2'-C-methyl in the sugar moiety. In one embodiment, an isolated nucleotide comprises a 2'-F in the arabinose configuration (FANA) in the sugar moiety. In one embodiment, the EON 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 is capable of recruiting adenosine deaminase to deaminate a target adenosine in a target ALDH2 RNA molecule, wherein the nucleotide opposite the target adenosine in the EON is an isolated nucleotide, and the isolated nucleotide has the following structure: JPEG2025539381000002.jpg39166 where: 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-2(1H)-pyridone; R1 and R2 are each independently selected from H, OH, F, or CH3; R3 is a portion of the EON located 5' to the isolated nucleotide and consisting of 7 to 30 nucleotides; and R4 is a portion of the EON located 3' to the isolated nucleotide and consisting of 4 to 25 nucleotides. The nucleotides on the 3' and / or 5' sides of the isolated nucleotide may be DNA, more preferably the nucleotide on the 3' side (-1 position).

[0070] In one embodiment, the first nucleic acid strand comprises at least one MP internucleoside linkage according to the following structure: JPEG2025539381000003.jpg39166

[0071] A preferred position for MP attachment in an EON according to the present invention is the -1 attachment position, thereby linking the nucleoside at the -1 position with the nucleoside at the -2 position, although MP attachment at other positions is not expressly excluded.

[0072] In one embodiment, the EON 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 position -3 in the EON, which may occur in conjunction with the same 2' modification in an isolated nucleotide, as described above.

[0073] In one embodiment, the EON comprises at least one phosphonoacetate or phosphonoacetamide internucleoside linkage.

[0074] In one embodiment, the EON comprises at least one nucleotide that comprises a locked nucleic acid (LNA) ribose modification or an unlocked nucleic acid (UNA) ribose modification. In certain embodiments, the EON comprises at least one nucleotide that comprises a threose nucleic acid (TNA) ribose modification.

[0075] Those skilled in the art know that oligonucleotides, such as the EONs outlined herein, are generally composed of repeating monomers. These monomers are most often 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). They consist of a pentose sugar (ribose), a 5'-linked phosphate group (which is linked via a phosphate ester), and a 1'-linked base. The sugar is often referred to as the "scaffold" of the nucleotide because it connects the base and the phosphate group.

[0076] Therefore, modifications to the pentose sugar are often referred to as "scaffold modifications." The original pentose sugar may be completely replaced by another moiety that similarly links the base and phosphate group. Thus, while a pentose sugar is often the scaffold, it is understood that a scaffold does not necessarily have to be a pentose sugar. Examples of scaffold modifications that can be applied to the monomers of the EONs of the present invention are disclosed in WO2020 / 154342, WO2020 / 154343, and WO2020 / 154344.

[0077] In one embodiment, an EON of the present invention may comprise one or more nucleotides having a 2'-MOE ribose modification. In another embodiment, an EON comprises one or more nucleotides without a 2'-MOE ribose modification, and the 2'-MOE ribose modification is located at a position where an enzyme with adenosine deaminase activity does not prevent deamination of the target adenosine. In another embodiment, an EON comprises a 2'-OMe ribose modification at a position where it does not comprise a 2'-MOE ribose modification, and / or wherein the oligonucleotide comprises a deoxynucleotide at a position where it does not comprise a 2'-MOE ribose modification. In one embodiment, the EON comprises one or more nucleotides containing a 2'-position that includes 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 a 2'-4' linkage (i.e., a bridged nucleic acid such as a locked nucleic acid (LNA, i.e., examples described in WO 2018 / 007475)). In another embodiment, other applicable nucleic acid monomers include arabinonucleic acid and 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), for example, to improve affinity. The 2'-4' linkage can be selected from linkers known in the art, such as a methylene linker or a constrained ethyl linker. A wide variety of 2' modifications are known in the art. Further examples are disclosed in more detail in, for example, WO2016 / 097212, WO2017 / 220751, WO2018 / 041973, WO2018 / 134301, WO2019 / 219581, WO2019 / 158475, and WO2022 / 099159. In all cases, the modification must be compatible with editing, such that the EON serves as an editing-initiating oligonucleotide that forms a double-stranded complex with the target RNA and recruits a deaminating enzyme that can then deaminate the target adenosine.When a monomer includes an unlocked nucleic acid (UNA) ribose modification, the monomer can have a 2′ position that includes the same modifications as those described above, such as 2′-MOE, 2′-OMe, 2′-OH, 2′-deoxy, 2′-F, 2′,2′-diF, 2′-fluoro-2′-C-methyl, arabinonucleic acid, FANA, or a 2′-4′ linkage (i.e., a bridged nucleic acid such as a locked nucleic acid (LNA)).

[0078] A base (sometimes called a nucleobase) is generally adenine, cytosine, guanine, thymine, or uracil, or a derivative thereof. A base (sometimes called a nucleobase) is defined as a moiety that can bind to another nucleobase through hydrogen bonds, polar bonds (e.g., through a CF moiety), or aromatic electron interactions. Cytosine, thymine, and uracil are pyrimidine bases, generally linked to the scaffold through their 1-nitrogen. Adenine and guanine are purine bases, generally linked to the scaffold through their 9-nitrogen. As used herein, the terms "adenine," "guanine," "cytosine," "thymine," "uracil," and "hypoxanthine" refer to the nucleobase itself. The terms "adenosine," "guanosine," "cytidine," "thymidine," "uridine," and "inosine" refer to a nucleobase linked to (deoxy)ribose.

[0079] The nucleobases in the EONs of the present invention can be adenine, cytosine, guanine, thymine, or uracil, or any other moiety capable of interacting with another nucleobase through hydrogen bonding, polar bonding (e.g., C—F), or aromatic electronic interactions. Nucleobases at any position within the nucleic acid strand can be modified forms of adenine, cytosine, guanine, or uracil, such as, for example, hypoxanthine (nucleobase in inosine), pseudouracil, pseudocytosine, isouracil, N3-glycosylated uracil, 1-methylpseudouracil, 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-deazapurines (e.g., 3-deaza-adenosine), pseudoisocytosine, N4-ethylcytosine, N2-cyclopentylguanine, N2-cyclopentyl-2-aminopurine, N2-propyl-2-aminopurine, 2,6-diaminopurine, 2-aminopurine, G-clamps and their derivatives, super A, super T, super G, amino-modified nucleobases or their derivatives; and modified 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).

[0080] In some embodiments, the nucleotide analog is an analog of a nucleic acid nucleotide. In some embodiments, the nucleotide analog is an analog of adenosine, guanosine, cytidine, thymidine, uridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine, or deoxyuridine. In some embodiments, the nucleotide analog is not guanosine or deoxyguanosine. In some embodiments, the nucleotide analog is not a nucleic acid nucleotide. In some embodiments, the nucleotide analog is not adenosine, guanosine, cytidine, thymidine, uridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine, or deoxyuridine.

[0081] Nucleotides are typically linked to adjacent nucleotides through condensation of their 5'-phosphate moiety to the 3'-hydroxyl moiety of the adjacent nucleotide monomer. Similarly, their 3'-hydroxyl moiety is typically linked to the 5'-phosphate of the adjacent nucleotide monomer. This forms a phosphodiester bond. The phosphodiester and scaffold form an alternating copolymer. The bases are attached to this copolymer, i.e., the scaffold moiety. Because of this characteristic, the alternating copolymer formed by the linked scaffolds of an oligonucleotide is often referred to as the "backbone" of the oligonucleotide. Because phosphodiester bonds connect adjacent monomers, they are often referred to as "backbone linkages." It is understood that even if the phosphate group is modified and replaced with an analogous moiety, such as PS, such moiety is still referred to as the backbone linkage of the monomer. This is referred to as "backbone linkage modification." In a general sense, the backbone of an oligonucleotide comprises alternating scaffolds and backbone linkages.

[0082] EONs according to the present invention may include linkage modifications, such as, but not limited to, modified forms of phosphodiesters present in RNA, including, but not limited to, PS, chiral pure PS, (R)-PS, (S)-PS, methyl phosphonate (MP), chiral pure methyl phosphonate, (R)-methyl phosphonate, (S)-methyl phosphonate, phosphoryl guanidine (e.g., PNdmi), chiral pure phosphoryl guanidine, (R)-phosphoryl guanidine, (S)-phosphoryl guanidine, phosphorodithioate (PS2), phosphonoacetate, e) (PACE), phosphonoacetamide (PACA), thiophosphonoacetate, thiophosphonoacetamide, methyl phosphorothioate, methylthiophosphonate, PS prodrugs, 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, formacetyls, alkenyls, methylenehydrazinos, sulfonamides, triazoles, oxalyls, carbamates, methyleneiminos (MMIs), and thioacetamido nucleic acids (TANAs); and derivatives thereof. Various salts, mixed salts, and free acid forms are also included, including 3'→3' and 2'→5' linkages.

[0083] In one embodiment, the EON contains a substitution of one of the non-bridging oxygens in the phosphodiester bond. This modification slightly destabilizes base pairing but confers significant resistance to nuclease degradation. Preferred nucleotide analogs or equivalents include PS, phosphonoacetate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl and other alkylphosphonates (including 3'-alkylenephosphonate, 5'-alkylenephosphonate, and chiral phosphonate), phosphinate, phosphoramidate (including 3'-aminophosphoramidate and aminoalkylphosphoramidate), thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate, or boranophosphate. Particularly preferred are internucleoside linkages modified to contain PS. Many of these non-natural bond modifications (e.g., PS) are chiral, meaning that they have both Rp and Sp configurations, and are known to those skilled in the art. In one embodiment, the chirality of the PS bond is controlled, meaning that each bond has either the preferred Rp or Sp configuration. The selection of the Rp or Sp configuration at a particular bond position may depend on the target sequence and the binding efficiency and induction efficiency resulting in RNA editing. However, if this is not particularly desired, the composition may contain an AON having both the Rp and Sp configurations at a particular bond position as an active compound. A mixture of EONs is also possible, where one configuration is preferred at some positions but not at other positions.

[0084] Again, in all cases, the modification must be compatible with editing, so that the EON serves as an editing oligonucleotide, and when bound to its target sequence, the properties of dsRNA are generated, thereby recruiting adenosine deaminase.In all aspects of the present invention, the enzyme with adenosine deaminase activity is preferably ADAR1, ADAR2, or ADAT.In a highly preferred embodiment, the EON is an RNA editing oligonucleotide that targets pre-mRNA or mRNA, wherein the target nucleotide is adenosine in the target RNA, and the adenosine is deaminated to inosine, which is read as guanosine by the translational machinery.The present invention also relates to a pharmaceutical composition comprising the EON characterized herein and a pharmaceutically acceptable carrier.

[0085] Other chemical modifications of EONs according to the present invention include replacing any one or more hydrogen atoms with deuterium or tritium, examples of which can be found, for example, in WO2014 / 022566 or WO2015 / 011694.

[0086] The present invention relates to an EON according to the present invention or a pharmaceutical composition comprising an EON according to the present invention, for use in the treatment or prevention of a disorder associated with alcohol intolerance or alcohol drunkenness, preferably caused by an ALDH2*2 mutant. In one embodiment, the present invention relates to an EON according to the present invention or a pharmaceutical composition comprising an EON according to the present invention, for use in the treatment or prevention of a disorder associated with alcohol intolerance or alcohol drunkenness, preferably caused by an ALDH2*2 mutant. In one embodiment, the present invention relates to an EON according to the present invention or a pharmaceutical composition comprising an EON according to the present invention, for use in the treatment or prevention of alcohol intolerance or alcohol drunkenness, preferably caused by an ALDH2*2 mutant.

[0087] The EONs of the present invention preferably do not contain a 5'-terminal O6-benzylguanosine or a 5'-terminal amino modification, and preferably do not contain a SNAP tag domain (modified O6-alkylguanosine-DNA-alkyltransferase) covalently linked to it. The EONs of the present invention preferably do not contain a boxB RNA hairpin sequence. In one embodiment, the EONs of the present invention contain 0, 1, 2, or 3 wobble 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. When the isolated nucleotide is a uridine, no mismatch occurs. One alternative to a uridine is to place an isouridine opposite the target adenosine, which likely does not pair like G and U do. Preferably, the target adenosine in the target sequence forms a mismatch base pair with the nucleoside in the EON directly opposite the target adenosine.

[0088] It should be noted that when EON is delivered through vector (for example, AAV vector), there is no chemical modification in the EON that acts on target RNA molecule.Although it is preferable to use "naked" EON with chemical modification as outlined herein, EON delivered through other means (for example, through AAV vector expression), or editing molecules that have circular or hairpin structure (recruitment part, for example, disclosed in WO2016 / 097212, WO2017 / 050306, WO2020 / 001793, WO2017 / 010556, WO2020 / 246560 and WO2022 / 078995) are also encompassed by the present invention.Because they can also be applied to edit adenosine in target ALDH2 RNA molecule to generate ALDH2 protein with restored function.

[0089] EON of the present invention can utilize endogenous cellular pathway and naturally occurring ADAR enzyme to specifically edit target adenosine in target RNA sequence.EON of the present invention can recruit ADAR and form complex with it, and then promote the deamination of (single) specific target adenosine nucleotide in target RNA sequence.Ideally, only one adenosine is deaminate.Preferably, when EON of the present invention forms complex with ADAR, it causes the deamination of single target adenosine.

[0090] Analysis of natural targets of ADAR enzymes has shown that they generally contain mismatches between the two strands that form the RNA helices edited by ADAR1 or 2. These mismatches have been suggested to 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). Characterization of the optimal pattern of paired / mismatched nucleotides between EONs and target RNAs is also believed to be important for the development of effective ADAR-based EON therapies.

[0091] As outlined above, the EONs of the present invention 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 include modifications in the EON backbone, in the sugar moiety of the nucleotide, and in the nucleobase or phosphodiester bond. They may also be variably distributed throughout the sequence of the EON. Specific modifications may be required to support interactions with various amino acid residues in the RNA-binding domain of the ADAR enzyme and those in the deaminase domain. For example, internucleotide PS bonds or 2'-OMe or 2'-MOE modifications may be tolerated in some EONs, but should be avoided in others to avoid interfering with the important interactions between the enzyme and the phosphate and 2'-OH groups. Specific nucleotide modifications may also be required 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 a central target A) contains the most favorable nearest neighbor nucleotide for ADAR2, while the 5'-CAA-3' target sequence is unfavorable (Schneider et al. 2014. Nucleic Acids Res 42(10):e87). Structural analysis of the ADAR2 deaminase domain suggests the possibility of enhancing 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 a central AC mismatch), but this is unfavorable because the guanosine base sterically clashes with the amino acid side chain of ADAR2. The present invention relates to RNA-editing oligonucleotides (commonly referred to herein as EONs) that can cause deamination of adenosines in ALDH2 transcripts, resulting in a fully functional ALDH2 protein in ethanol metabolism.This means that the present invention is not strictly limited to deamination of the adenosine of mutant ALDH2*2, but other adenosines (single or multiple) may be targeted, potentially resulting in increased ALDH2 protein function. Other adenosines may be identified, for example, by population genetic screening or in silico analysis, that are important (or may be more important) for ALDH2 function and can be targeted through RNA editing according to the teachings of the present invention. All RNA events and oligonucleotides that can be used for such targeting are encompassed by the present invention, regardless of the exact nucleic acid molecule or EON.

[0092] Mutagenesis studies of human ADAR2 revealed that a single mutation at residue 488 (E488Q) from glutamic acid to glutamine increased 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 duplex and places it in the enzyme's active site (Matthews et al. 2016). When ADAR2 edits an adenosine in a favorable context (A:C mismatch), the nucleotide opposite the target adenosine is often referred to as a "lone cytidine." The crystal structure of ADAR2 E488Q bound to double-stranded RNA (dsRNA) revealed that the glutamine (Gln) side chain at position 488 can donate a hydrogen bond to the N3 position of the lone cytidine, leading to an improved catalytic rate of ADAR2 E488Q. In the wild-type enzyme, a glutamic acid (Glu) exists at position 488 instead of a glutamine (Gln), and the amide group of the glutamine is absent, becoming a carboxylic acid instead. Therefore, to achieve the same contact with the lone cytidine using the E488Q mutant in the wild-type context, protonation is required 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 cells. WO 2020 / 252376 discloses the use of EONs, specifically modified RNA bases at the lone cytidine position, to mimic the hydrogen bonding pattern observed with the E488Q ADAR2 mutant. It was hypothesized that by replacing the nucleotide opposite the target adenosine in the EON with a cytidine analogue that functions as a hydrogen bond donor at N3, it would be possible to stabilize the same contacts that would improve catalytic rates in the mutant enzyme.Two cytidine analogs were of particular interest: pseudoisocytidine (also referred to 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 referred to as "dZ"; Yang et al. Nucl Acid Res 2006. 34(21):6095-6101), which were initially selected because they donate a hydrogen bond at N3 with minimal perturbation to the geometry of the nucleobase. Benner's base is also referred to as 6-amino-5-nitro-2(1H)-pyridone. The presence of cytidine analogs in AONs can be in addition to modifications to the ribose 2' group. The ribose 2' groups in the AON can be independently selected from the following: 2'-H (i.e., DNA), 2'-OH (i.e., RNA), 2'-OMe, 2'-MOE, 2'-F, or a 2'-4' linkage (i.e., bridged nucleic acids such as locked nucleic acids (LNAs)), or other 2' substituents. The 2'-4' linkage can be selected from linkers known in the art, such as a methylene linker or a constrained ethyl linker.

[0093] In one embodiment, the nucleotide analog or equivalent in EON contains one or more base modifications or substitutions.Modified bases include synthetic and natural bases, such as inosine, xanthine, hypoxanthine, and other aza, deaza, hydroxy, halo, thio, thiol, alkyl, alkenyl, alkynyl, thioalkyl derivatives of pyrimidine and purine bases, which are known or will be developed in the art.Purine nucleobases and / or pyrimidine nucleobases can be modified to change their properties, for example, by amination or deamination of heterocycles.The exact chemical structure and format can vary from oligonucleotide construction to oligonucleotide construction and application to application, and can be determined according to the wishes and preferences of those skilled in the art.

[0094] An EON according to the present invention is typically longer than 10 nucleotides, preferably greater than 11, 12, 13, 14, 15, or 16 nucleotides, and more preferably greater than 17 nucleotides. In one embodiment, an AON according to the present invention is longer than 20 nucleotides. An oligonucleotide according to the present invention is preferably shorter than 100 nucleotides, more preferably shorter than 60 nucleotides, and even more preferably shorter than 50 nucleotides. In a preferred embodiment, an oligonucleotide according to the present invention comprises 18 to 70 nucleotides, more preferably 18 to 60 nucleotides, and even more preferably 18 to 50 nucleotides. Thus, in particularly preferred embodiments, oligonucleotides of the invention comprise 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.

[0095] In one embodiment, an inverted deoxyT or dideoxyT nucleotide is incorporated at each or both ends of an EON according to the invention.

[0096] As mentioned above, in some embodiments, the present invention provides EON for forming a double-stranded complex with human ALDH2 RNA molecules in human hepatocytes.Therefore, it is preferable that the therapeutic effect is extended to human hepatocytes in vivo.Of course, the method can also be carried out in vitro or ex vivo.

[0097] The present invention provides an EON of the present invention or a pharmaceutical composition of the present invention for use in treating a disease. The present invention also provides the use of an EON of the present invention or a pharmaceutical composition of the present invention in the manufacture of a medicament for treating a disease. The present invention also provides a method for treating a disease in a patient, comprising administering a therapeutically effective amount of an EON of the present invention or a pharmaceutical composition of the present invention. Preferably, the disease is a disease caused by the E504K mutation in ALDH2. EON is administered therapeutically or prophylactically (after genetic counseling), as both forms of treatment can be beneficial.

[0098] After RNA editing occurs in a cell, the modified RNA may be diluted over time, e.g., due to cell division, the finite half-life of the edited RNA, etc. Thus, in terms of practical treatment, the methods of the invention may involve repeated delivery of AONs until enough of the target RNA is modified to provide a tangible benefit to the patient and / or to maintain that benefit over time. [Example]

[0099] Example 1. Editing of target adenosines in human ALDH2 target RNA molecules using an in vitro biochemical editing assay. First, the first set of ALDH2-targeting EONs (shown in Figure 1) was tested for editing of human ALDH2 target (pre-)mRNA in an in vitro biochemical editing assay. To obtain ALDH2 target RNA, PCR was performed using the ALDH2 G-block (IDT) containing the T7 promoter sequence and (part of) the ALDH2 sequence as a template, with the forward primer 5'-CTC GAC GCA AGC CAT AAC AC-3' (SEQ ID NO: 53) and the reverse primer 5'-TGG ACC GAC TGG AAA CGT AG-3' (SEQ ID NO: 54). The 5'→3' G-block sequence (SEQ ID NO: 55) is as follows, in which the target adenosine is underlined and bold, and the primer sequence is underlined:

[0100] JPEG2025539381000004.jpg34166

[0101] The PCR product is then used as a template for in vitro transcription using the MEGAscript T7 Transcription Kit. RNA is purified on a urea gel and then extracted in 50 mM Tris-Cl pH 7.4, 10 mM EDTA, 0.1% SDS, 0.3 M NaCl buffer, followed by phenol-chloroform purification. The purified RNA is used as a target in biochemical editing assays.

[0102] First, EON ALDH2-01, 02, 05, 06, 09, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, and 46 were annealed with the ALDH2 target RNA in a buffer solution (5 mM Tris-Cl pH 7.4, 0.5 mM EDTA, and 10 mM NaCl) at a target RNA to oligonucleotide ratio of 1:3 (600 nM oligonucleotide and 200 nM target). The sample was heated to 95°C for 3 minutes and then slowly cooled to room temperature. The editing reaction then took place. The annealed oligonucleotide / target RNA was mixed with protease inhibitors (cOmplete™ Mini, EDTA-free Protease I, Sigma-Aldrich), ribonuclease inhibitor (RNasin, Promega), polyA (Qiagen), tRNA (Invitrogen), and editing reaction buffer (15 mM Tris-Cl pH 7.4, 1.5 mM EDTA, 3% glycerol, 60 mM KCl, 0.003% NP-40, 3 mM MgCl2, and 0.5 mM DTT) to a final concentration of 6 nM oligonucleotide and 2 nM target RNA. The reaction was initiated by adding purified ADAR2 (GenScript) to the mixture to a final concentration of 6 nM and incubated at 37°C for a predetermined time. Each reaction was stopped by adding 95 μl of 95°C 3 mM EDTA solution. A 6 μl aliquot of the stopped reaction mixture is then used as a template for cDNA synthesis using the Maxima Reverse Transcriptase Kit (Thermo Fisher Scientific) with random hexamer primers (Thermo Fisher Scientific). Initial denaturation of the RNA is carried out in the presence of primers and dNTPs at 95°C for 5 min, followed by slow cooling to 10°C, after which first-strand synthesis is carried out according to the manufacturer's instructions in a total volume of 20 μl using an extension temperature of 62°C.For pyrosequencing analysis, the products were amplified by PCR using the Amplitaq Gold 360 DNA Polymerase kit (Applied Biosystems) according to the manufacturer's instructions, using 1 μl of cDNA as a template. PCR was then performed using the following thermocycling protocol: initial denaturation at 95°C for 5 minutes, followed by 40 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds, and a final extension at 72°C for 7 minutes.

[0103] During cDNA synthesis in the reverse transcription reaction, inosine base pairs with cytidine, so the nucleotide incorporated into the edited portion during PCR is guanosine. The percentage of guanosine (edited) and adenosine (unedited) is determined by pyrosequencing. Pyrosequencing of PCR products and data analysis are performed using a PyroMark Q48 Autoprep instrument (QIAGEN) according to the manufacturer's instructions, using 10 μl of PCR product input and 4 μM sequencing primers. Analysis performed by the instrument provides results for selected nucleotides as the percentage of adenosine and guanosine detected at that position. Therefore, the degree of A to I editing at a selected position is measured by the percentage of guanosine at that position.

[0104] Example 2: Editing of target adenosines in human ALDH2 target RNA molecules using patient-derived skin fibroblasts We investigated whether the modified EONs shown in Figure 1 (each with an RM number) could edit target adenosines in human ALDH2*2 transcript RNA containing the c.1510G>A mutation in cells. For this purpose, non-transformed human dermal fibroblasts (AG11369; Corriell, USA) heterozygous for the c.1510G>A mutation were used. The drawback of using heterozygous mutant cells is the significant background signal from the unedited transcript (wild-type and transcribed from the wild-type allele). Approximately 50,000 cells per 24-well plate were seeded 24 hours before administration of each EON, either alone at 100 nM or in combination with 5 μM saponin (AG1856; see WO2021 / 122998). Cells were incubated with EONs ± saponin for 3 days and then harvested. RNA was extracted using the Direct-zol RNA MicroPrep (Zymo Research) kit according to the manufacturer's instructions, and cDNA was prepared using the Maxima reverse transcriptase kit (ThermoFisher) with random hexamers according to the manufacturer's instructions. The cDNA was used as a template for digital PCR (dPCR) with 200 ng of RNA input per reaction. The dPCR assay for absolute quantification of nucleic acid target sequences was performed using Qiagen's QIAcuity 4 Digital PCR system. 1.2 μl of undiluted cDNA from the reverse transcription cDNA synthesis reaction was used in a total reaction mixture of 12 μl, which included 4x concentrated QIAcuity Probe Mastermix (Qiagen), Taqman SNP genotyping assays using the following forward and reverse primers in combination with the following gene-specific probes:

[0105] Forward primer: 5'-TGGTGGCTACAAGATGTCGG-3' (SEQ ID NO: 56) Reverse primer: 5'-TTATGAGTTCTTCTGAGGCACT-3' (SEQ ID NO: 57) Wild-type probe (FAM NFQ labeled): 5'- / 56-FAM / A+CAGTT+TTCACTT+C+A+GTGTATGCC / 3IABkFQ / -3' (SEQ ID NO: 58) Mutant probe (HEX NFQ labeled): 5'- / 5HEX / A+CAGTT+TTCACTT+T+A+GTGTATGCCC / 3IABkFQ / -3' (SEQ ID NO: 59)

[0106] A total volume of 12 μl of PCR mixture containing cDNA was loaded into a QIAcuity Nanoplate (Qiagen) using a multichannel pipette. After sealing the plate with a Nanoplate seal, it was placed in a QIAcuity 4 dPCR instrument, where fractionation, PCR amplification, and fluorescence measurement were performed fully automatically. The PCR program was as follows: 1 cycle of enzyme activation at 95°C for 2 minutes, 40 cycles of denaturation at 95°C for 15 seconds, and annealing / extension at 60°C for 30 minutes. After PCR, the plate was imaged and analyzed in the QIAcuity 4.

[0107] The results, shown in Figure 2, show that the percentage of editing observed in the absence of saponin (gymnotic uptake, or "GU") was similar for most EONs tested, and that the percentage of wild-type ALDH2 transcript reached levels exceeding 80%. However, as noted above, these values ​​are affected by the presence of the wild-type allele in the heterozygous cells used. Normalization to the untreated sample (NT), which "removes" the percentage of wild-type background signal, showed that editing levels reached approximately 30% when EONs were administered to cells in the presence of AG1856, demonstrating that we were able to edit the ALDH2*2 c.1510G>A mutation to wild-type in human cells. EON RM4740 performed best.

Claims

1. an RNA-editing oligonucleotide (EON) capable of forming a double-stranded complex with a region of an endogenous human ALDH2 transcript molecule in a cell, the region of the ALDH2 transcript molecule includes a target adenosine; and The double-stranded complex can recruit an endogenous ADAR enzyme to deaminate the target adenosine to inosine, thereby editing the ALDH2 transcript molecule. RNA-editing oligonucleotides.

2. 2. The EON according to claim 1, The ALDH2 transcript molecule is a pre-mRNA or mRNA molecule. EON.

3. 3. The EON according to claim 1 or 2, The cells are human liver cells, preferably hepatocytes. EON.

4. An EON according to any one of claims 1 to 3, the endogenous ADAR enzyme is ADAR2; EON.

5. An EON according to any one of claims 1 to 4, The target adenosine is the c.1510G>A mutation in the ALDH2 transcript. EON.

6. An EON according to any one of claims 1 to 5, The EON comprises or consists of any one of the EON sequences selected from the group consisting of SEQ ID NOs: 1 to 51; EON.

7. An EON according to any one of claims 1 to 6, wherein at least one nucleotide comprises one or more non-natural chemical modifications in the ribose moiety, the linker moiety, or the base moiety, or one or more additional non-natural chemical modifications; provided that the lone nucleotide in the EON directly opposite the target adenosine is not a cytidine containing a 2'-OMe ribose substitution. EON.

8. 8. The EON according to claim 7, the one or more additional modifications in the linking moiety are each independently selected from phosphorothioate (PS), phosphonoacetate, phosphorodithioate, methylphosphonate (MP), sulfonyl phosphoramidate, or PNdmi internucleotide linkages; EON.

9. 9. An EON according to claim 7 or 8, The one or more additional modifications in the ribose moiety are mono- or di-substitutions at the 2', 3', and / or 5' positions of the ribose and are selected from the group consisting of: -OH; -F; Substituted or unsubstituted, straight or branched lower (C １ -C １０ ) alkyl, alkenyl, alkynyl, alkaryl, aryl, 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; each independently selected from EON.

10. A nucleic acid molecule encoding an EON according to any one of claims 1 to 6, A vector, preferably a viral vector, more preferably an adeno-associated viral (AAV) vector.

11. A pharmaceutical composition comprising the EON according to any one of claims 1 to 9 or the vector according to claim 10, and a pharmaceutically acceptable carrier.

12. An EON according to claims 1 to 9, a vector according to claim 10, or a pharmaceutical composition according to claim 11, For use in the treatment of disorders caused by ALDH2 deficiency, preferably caused by ALDH2*2, EON, vector, or pharmaceutical composition.

13. Use of an EON according to any one of claims 1 to 9 or a vector according to claim 10, In the manufacture of a medicament for the treatment of a disorder caused by ALDH2 deficiency, preferably caused by ALDH2*2, more preferably caused by alcohol intoxication, alcohol poisoning, or symptoms of alcohol consumption, use.

14. 1. A method for editing an ALDH2 polynucleotide, comprising: The method includes contacting the ALDH2 polynucleotide with an EON capable of causing an adenosine deaminase (ADAR)-mediated conversion of an adenosine associated with alcohol intolerance to an inosine, thereby editing the ALDH2 polynucleotide. method.

15. 1. A method of treating a disorder caused by ALDH2 deficiency, preferably caused by ALDH2*2, in a patient in need thereof, comprising: The method includes contacting an ALDH2 polynucleotide in a cell of a subject with an EON capable of causing an ADAR-mediated adenosine-to-inosine conversion of an adenosine associated with the ALDH2 deficiency, thereby treating the patient. method.

16. A method for treating a disorder caused by ALDH2*2, comprising: The method comprises administering to a patient in need thereof a therapeutically effective amount of an EON according to any one of claims 1 to 9, a vector according to claim 10, or a pharmaceutical composition according to claim 11. method.

17. 1. A method for deaminating a target adenosine in an ALDH2 pre-mRNA or mRNA molecule in a cell, comprising: The method includes the following steps: (i) providing the cell with an EON according to any one of claims 1 to 9; (ii) allowing the EON to be taken up by the cells; (iii) annealing the EON to the ALDH2 pre-mRNA or mRNA molecule; (iv) allowing the endogenous ADAR enzyme to deaminate said target adenosine to inosine within the target RNA molecule; and (v) identifying the presence of said inosine within said target RNA molecule. may include method.

18. 18. The method of claim 17, The target adenosine is the c.1510G>A mutation in the ALDH2 transcript. method.

19. 19. The method according to claim 17 or 18, wherein step (v) is: a) determining the sequence of the ALDH2 pre-mRNA or mRNA molecule; b) assessing the presence of wild-type ALDH2 protein; or c) using a functional readout, preferably assessing the level of alcohol in a serum or plasma sample; Including, method.

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