Antisense oligonucleotides for treating acetaldehyde dehydrogenase 2 deficiency

Abstract

The invention relates to the field of diseases caused by alcohol intolerance, for example, diseases caused by ALDH2 * 2 mutation in the human ALDH2 gene. The present invention relates to oligonucleotides and their use in RNA editing methods for targeting a target adenosine in an endogenous human ALDH2 transcript in a cell, for example, Ggt in a mutant ALDH2 gene transcription molecule encoding a mutant p.E504K ALDH2 protein; a mutation, especially in the liver.

Description

Technical Field

[0001] The present invention relates to the medical field, particularly to diseases caused by mutant aldehyde dehydrogenase 2 (ALDH2) proteins. The present invention involves using nucleotide editing technology to target ALDH2 gene transcripts, thereby causing amino acid changes and restoring the normal function of the ALDH2 protein in regulating ethanol metabolism. Background Art

[0002] The mitochondrial aldehyde dehydrogenase 2 (ALDH2) enzyme in the liver eliminates toxic aldehydes, including acetaldehyde. Acetaldehyde is an intermediate in ethanol metabolism and is therefore crucial for alcohol detoxification. The well-known alcoholic flush 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 also plays a role in the conversion and elimination of 4-hydroxynonenal (HNE), a reactive aldehyde. At low levels, this compound is beneficial, but at high levels, it is toxic. The variant allele of ALDH2, designated ALDH2*2, encodes a lysine substitution for glutamic acid at residue 504 of the mature enzyme. The wild-type allele is designated ALDH2*1. The wild-type protein is commonly referred to as ALDH2E, while the mutant protein is often referred to as ALDH2K. Other variants of this allele are known as ALDH2*3, ALDH2*4, and others (Chen CH et al., 2020. EBioMedicine 55:102753). Approximately 40% to 45% of East Asians (approximately 8% of the world's population) inherit the inactive ALDH2*2 variant and experience a characteristic alcohol flush reaction after drinking alcohol.

[0003] Alcohol-induced flushing syndrome is not benign. Clear epidemiological data and meta-analyses consistently demonstrate that alcohol consumption in ALDH2*2 carriers significantly increases the risk of various cancers, particularly upper gastrointestinal cancers. Due to structural alterations in the ALDH2 tetramer complex caused by the mutation, ALDH2K activity is partially dominant-negative compared to the wild-type version. Consequently, heterozygous individuals exhibit less than half the activity of the wild-type version, while homozygous individuals exhibit very low residual activity.

[0004] Acetaldehyde binds to cellular proteins and DNA, potentially leading to DNA damage and organ damage. Specifically, endogenous aldehydes are harmful to hematopoietic stem cells with defective DNA repair function in Fanconi anemia, which accelerates the progression of the disease in patients with Fanconi anemia carrying the ALDH2*2 allele. In addition, ALDH2*2 also increases the risk of gastrointestinal cancers (such as gastric cancer, esophageal cancer, and colon cancer). Mutant mice carrying a lysine-to-glutamate substitution mutation (equivalent to the human E504K mutation) essentially reproduce all human phenotypes, including impaired acetaldehyde clearance, increased sensitivity to acute or chronic alcohol intoxication, and reduced ALDH2 expression due to the dominant negative effect of the mutation. When treated with chemical carcinogens, the DNA damage response of the liver cells of mutant mice was enhanced, liver damage was obvious, and the development of hepatocellular carcinoma was accelerated (Jin S et al., 2015. Proc Natl Acad Sci USA. 112(29):9088-9093), which supports the view that the common human ALDH2*2 mutation is an important risk factor for the occurrence of liver cancer. Aldehyde toxicity is not limited to cancer; it is also associated with a host of other diseases to which ALDH2*2 subjects have increased vulnerability, including osteoporosis, cardiovascular disease, Alzheimer's disease, and rare genetic disorders such as Fanconi anemia mentioned above.

[0005] The ALDH2*2 variant has been identified as a polymorphism unique to East Asians. Extensive global geographic and demographic analyses based on data from over 80,000 individuals across 366 population samples have confirmed that the ALDH2*2 allele is highly concentrated in Southeast China, Japan, South Korea, Singapore, and Vietnam. In Taiwan, the prevalence of ALDH2*2 carriers is as high as 49%, affecting half of the population (Luo HR et al. 2009. Gene. 435(1-2):96-103).

[0006] A potential treatment called Alda-1 (AD-6626) is being developed as a small molecule for the treatment of alcohol intoxication because it improves efficacy in animal models of myocardial infarction, stroke, radiation dermatitis, and pain. It is thought to construct a molecular patch that restores 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 a series of small molecules (such as Alda-1) that can act as modulators of ALDH2 activity. WO2019 / 092282, WO2019 / 143621, WO2020 / 206350, and WO2022 / 104366 disclose nucleic acid-based therapies designed to reduce ALDH2 expression in subjects with alcohol use disorder (AUD). AUD is a disorder that is at lower risk in ALDH2*2 subjects, who are generally less likely to consume large amounts of alcohol due to alcohol intolerance caused by the ALDH2*2 mutation.

[0007] In 1997, the FDA approved fomepizole (Antizol) for the treatment of acute ethylene glycol and methanol poisoning. However, this product does not target ALDH2 and cannot treat ethanol poisoning.

[0008] The present invention aims to provide one or more alternative and / or improved compounds or compositions for treating alcohol intolerance caused by ALDH2K mutant protein. Summary of the Invention

[0009] Disclosed herein is 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 comprises a target adenosine, and wherein the double-stranded complex is capable of recruiting endogenous ADAR enzymes to deaminate the target adenosine to inosine, thereby editing the ALDH2 transcript molecule. Preferably, the ALDH2 transcript molecule is a precursor mRNA (pre-mRNA) or mRNA molecule. In one embodiment, the cell is a human liver cell, preferably a hepatocyte. The preferred target adenosine is an adenosine with a G>A mutation in the human ALDH2 gene that results in the mutant p.E504K ALDH2 protein. In one embodiment, the EON comprises at least one nucleotide that comprises one or more non-naturally occurring chemical modifications in the ribose, linkage, or base moiety, or one or more additional non-naturally occurring chemical modifications, provided that the orphan nucleotide, i.e., the nucleotide in the EON that is opposite the target adenosine, is not a cytidine comprising a 2'-OMe ribose substitution. The present invention also discloses a vector, preferably a viral vector, more preferably an adeno-associated virus (AAV) vector, comprising a nucleic acid molecule encoding the EON disclosed herein. The present invention also discloses a pharmaceutical composition comprising the EON disclosed herein or the vector disclosed herein and a pharmaceutically acceptable carrier.

[0010] The present invention discloses an EON, a vector, or a pharmaceutical composition for treating a disease caused by ALDH2 deficiency, preferably a disease caused by ALDH2*2 deficiency. Disclosed herein is the use of an EON or a vector in the preparation of a medicament for treating ALDH2*2-induced alcohol intolerance, such as 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, restlessness, irritability, sensitivity to light and noise, motor instability, difficulty sleeping, severe hunger, bad breath, and loss of depth perception.

[0011] The present invention discloses a method for treating a disease caused by ALDH2 deficiency, preferably a disease caused by ALDH2*2 deficiency, in a patient in need thereof. The method comprises contacting an ALDH2 polynucleotide in a cell of a subject with an EON capable of effecting ADAR-mediated conversion of adenosine to inosine, thereby treating the patient. The adenosine is associated with ALDH2 deficiency. The present invention also discloses a method for treating a disease caused by ALDH2*2, comprising administering a therapeutically effective amount of an EON, a vector, or a pharmaceutical composition disclosed herein to a patient in need thereof. The present invention also discloses a method for editing an ALDH2 polynucleotide, comprising contacting an ALDH2 polynucleotide with an EON capable of effecting RNA-acting adenosine deaminase (ADAR)-mediated conversion of adenosine to inosine, thereby editing the ALDH2 polynucleotide. The adenosine is associated with alcohol intolerance. Preferably, the ALDH2 transcript is from a mutant ALDH2*2 gene. The present invention also discloses a method for treating ALDH2*2-induced alcohol intolerance or a disease caused by such alcohol intolerance in a patient in need thereof, the method comprising contacting an ALDH2 polynucleotide in a cell of the subject with an EON capable of achieving ADAR-mediated conversion of adenosine to inosine, thereby treating the patient, wherein the adenosine is associated with alcohol intolerance or a disease caused by such alcohol intolerance (e.g., alcohol intoxication, alcohol poisoning, or drinking symptoms). BRIEF DESCRIPTION OF THE DRAWINGS

[0012] 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:

[0013] Figure 1The human ALDH2 target RNA sequence (5' to 3'; SEQ ID NO: 52) is shown at the top, with the target adenosine in bold and the lysine codon underlined. Below the target sequence, the sequences (also 5' to 3') of the initial 51 EONs (shown in the figure, SEQ ID NOs: 1 to 51) originally designed to edit the target adenosine are given. 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 (same as 2'-MOE-substituted thymidine); 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'-F-modified guanosine, cytidine, adenosine, and uridine, respectively; Zd (orphan nucleotide) is a deoxynucleotide bearing a Benner base (a deoxycytidine analog); C2f (orphan nucleotide) is a cytidine with a 2',2'-difluoro modification; Cd (orphan nucleotide) is deoxycytidine; Ad is deoxyadenosine; "!" indicates a PNdmi linkage; "^" indicates an MP linkage; and "*" indicates a PS linkage. All other internucleoside linkages are phosphodiester linkages.

[0014] Figure 2 shows the percentage editing of the human ALDH2 transcript following naked uptake (GU) of the indicated EONs (black bars) and co-administration of saponin (grey bars). (A) Shown is the total percentage including background signal from the wild-type allele. (B) Shows the same results after normalization using untreated (NT) samples to remove the wild-type allele signal. DETAILED DESCRIPTION

[0015] The inventors of the present invention have recognized an alternative approach to targeting the E504K mutation in ALDH2, generating wild-type ALDH2 protein and potentially restoring normal alcohol metabolism, thereby preventing, ameliorating, or treating conditions associated with the accumulation of toxic aldehydes or alcohol intolerance. This technique is generally referred to as RNA editing. Disclosed herein are oligonucleotides that can be used to specifically deaminize specific target adenosines within (human) mutant ALDH2 transcripts (pre-mRNA and / or mRNA) in vivo, preferably using an endogenous deaminase, to generate an ALDH2 protein that restores its function in converting acetaldehyde to acetate during ethanol metabolism. While the most common mutation in the ALDH2 gene to date is the aforementioned E504K mutation, the RNA editing technology disclosed herein is also applicable to other target adenosines within ALDH2 that can be targeted to restore its function or even lead to gain-of-function effects. The E504K mutation is a mutation from a GAA codon (encoding glutamate; Glu; E) to an AAA codon (encoding lysine; Lys; K). The specific RNA editing disclosed herein converts the first adenosine of the mutant codon to inosine, which is then read by the translation machinery as guanosine (AAA>IAA>GAA).

[0016] RNA editing is a natural process by which eukaryotic cells alter the sequence of their RNA molecules, often in a site-specific and precise manner, thereby increasing the pool of genomically encoded RNAs by orders of magnitude. RNA editing enzymes have been described in eukaryotes from both the animal and plant kingdoms, and these processes play an important role in maintaining cellular homeostasis in metazoans, from the simplest life forms such as Caenorhabditis elegans to humans. Examples of RNA editing include the conversion of adenosine (A) to inosine (I), and the conversion of cytidine (C) to uridine (U), respectively, by adenosine deaminases (ADEs) acting on RNA. A denosine D eaminases acting on R This is achieved by the cytidine deaminases NA, ADAR) and APOBEC / AID (cytidine deaminases acting on RNA).

[0017] ADARs are multidomain proteins consisting of a catalytic domain and two or 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. The catalytic domain also participates in recognizing and binding to a portion of the dsRNA helix, although its key function is to convert an A to an I at a nearby predetermined position in the target RNA through deamination of the nucleobase. As mentioned above, the cell's translational machinery reads inosine as guanosine, meaning that if the edited adenosine is located in the coding region of an mRNA or pre-mRNA, it can recode the protein sequence. A-to-I conversions can also occur within the 5' noncoding sequence of the target mRNA, creating a new translation start site upstream of the original start site, resulting in an N-terminally extended protein; or within the 3' UTR or other non-coding regions of the transcript, potentially affecting RNA processing and / or stability. Furthermore, A-to-I conversions can occur within splicing elements within introns or exons of the pre-mRNA, altering splicing patterns. As a result, exons may be added or skipped. Enzymes that catalyze adenosine deamination belong to the ADAR enzyme family, which includes human deaminases hADAR1 and hADAR2, as well as hADAR3. However, hADAR3 has not been shown to have deaminase activity.

[0018] There have been reports of using adenosine deaminase to edit target RNA using oligonucleotides (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 disadvantage of the method described by Montiel-Gonzalez et al. (2013) is that it requires a fusion protein composed of the boxB recognition domain of the bacteriophage λN protein and the adenosine deaminase domain of a truncated native ADAR protein. This requires either transduction of the target cells with the fusion protein, which is a major obstacle, or transfection of the target cells with a nucleic acid construct encoding the engineered adenosine deaminase fusion protein for expression. The system described by Vogel et al. (2014) suffers from similar shortcomings, as it is unclear how to apply the system without first genetically engineering the ADAR and then transfecting or transforming cells containing the target RNA to provide the engineered protein. A similar system is described in US 9,650,627. The oligonucleotides of Woolf et al. (1995) are 100% complementary to the target RNA sequence but suffer from a severe lack of specificity: nearly all adenosines in the target RNA strand complementary to the antisense oligonucleotide are edited.

[0019] ADARs are known to act on any dsRNA. Through a process sometimes referred to as “promiscuous editing,” the enzymes edit multiple A’s in dsRNA. Therefore, methods and means to circumvent this promiscuous editing and target only specific adenosines in target RNA molecules are needed for therapeutic applications. Vogel et al. (2014) demonstrated that this off-target editing could be inhibited by using 2’-O-methyl (2’-OMe)-modified nucleosides in oligonucleotides opposite the adenosines that should not be edited, and using unmodified nucleosides directly opposite the specific targeted adenosines on the target RNA. However, it has not been shown whether the specific editing effect at the target nucleotide occurs without the use of a recombinant ADAR enzyme that forms a covalent bond with the AON. Several publications have shown that it is feasible to recruit endogenous ADARs (thus eliminating the need for exogenous and / or recombinant sources) while maintaining specificity, whereby a single adenosine in a target RNA molecule can be targeted and deaminated to inosine. WO2016 / 097212 discloses antisense oligonucleotides (AONs) for targeted editing of RNA, wherein the AON is characterized in that its sequence is complementary to the target RNA sequence (referred to herein as the "targeting portion") and has a stem-loop / hairpin structure (referred to herein as the "recruiting portion"), which is preferably not complementary to the target RNA. Such oligonucleotides are referred to as "self-loop AONs". The role of the recruiting portion is to recruit the natural ADAR enzyme present in the cell to the dsRNA formed by hybridization of the target sequence and the targeting portion. Due to the recruiting portion, there is no need for the presence of a conjugated entity or a modified recombinant ADAR enzyme. WO2016 / 097212 describes the recruiting portion as a stem-loop structure that simulates 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 the ADAR enzyme. The stem-loop structure can be an intermolecular stem-loop structure formed by two independent nucleic acid chains, or it can be an intramolecular stem-loop structure formed by one nucleic acid chain. The stem-loop structure of the recruitment moiety is an intramolecular stem-loop structure formed within the AON itself and is thought to attract (endogenous) ADARs. Similar stem-loop systems for RNA editing are described in WO2017 / 050306, WO2020 / 001793, WO2017 / 010556, WO2020 / 246560, and WO2022 / 078995.

[0020] WO2017 / 220751 and WO2018 / 041973 describe a new generation of AONs that do not contain such a stem-loop structure but are (almost completely) complementary to the target region. In one embodiment, there are one or more mismatched nucleotides, wobbles or protrusions between the oligonucleotide and the target sequence. The only mismatch may be located at the nucleoside site opposite the target adenosine, but in other embodiments, AONs (or RNA editing oligonucleotides, abbreviated as "EONs") are described as having multiple protrusions and / or wobbles when connected to the target sequence region. When the sequence of the EON is carefully selected to attract / recruit ADARs, it seems that RNA editing in vitro, in vitro and in vivo can be achieved using EONs that lack a stem-loop structure and endogenous ADAR enzymes. "Orphan nucleosides" refer to nucleosides in the EON that are located opposite the target adenosine in the target RNA molecule and do not carry 2'-OMe modifications. The orphan nucleoside can be a deoxyribonucleoside (DNA), in which the rest of the EON can still carry a 2'-O-alkyl modification (e.g., 2'-OMe) on the sugar entity, or the nucleotides directly surrounding the orphan nucleoside contain chemical modifications (e.g., DNA compared to RNA), thereby further improving RNA editing efficiency and / or enhancing resistance to nucleases. Such effects can even be further enhanced by using sense oligonucleotides (SONs) that "protect" the EON from degradation (see WO2018 / 134301). The use of chemical modifications and specialized structures in oligonucleotides for ADAR-mediated editing of specific adenosines in target RNAs has been the subject of numerous publications in this field, such as WO2019 / 111957, WO2019 / 158475, WO2020 / 165077, WO2020 / 201406, WO2020 / 211780, WO2021 / 008447, WO2021 / 020550, WO2021 / 060527、WO2021 / 117729、WO2021 / 136408、WO2021 / 182474、WO2021 / 216853、WO2021 / 242778、WO2 021 / 242870, WO2021 / 242889, WO2022 / 007803, WO2022 / 018207, WO2022 / 026928 and WO2022 / 124345.For example, the use of specific sugar moieties is disclosed in WO2020 / 154342, WO2020 / 154343, WO2020 / 154344, WO2022 / 103839 and WO2022 / 103852, while WO2011 / 005761, WO2014 / 010250, WO2014 / 012081, WO2015 / 107425, WO2017 / 015575 (HTT), WO2017 / 062862, WO2017 / 160741, WO20 17 / 192664、WO2017 / 192679(DMD)、WO2017 / 198775、WO2017 / 210647、WO2018 / 067973、WO2018 / 098264、WO2018 / 2230 56(PNPLA3), WO2018 / 223073(APOC3), WO2018 / 223081(PNPLA3), WO2018 / 237194, WO2019 / 032607(C9orf72), WO2019 / 055951、WO2019 / 075357(SMA / ALS)、WO2019 / 200185(DM1)、WO2019 / 217784(DM1)、WO2019 / 219581、WO2020 / 118246 (DM1), WO2020 / 160336(HTT), WO2020 / 191252, WO2020 / 196662, WO2020 / 219981(USH2A), WO2020 / 219983(RHO), WO20 The use of stereo-defined linker moieties (generally for oligonucleotides, e.g., useful for exon skipping, gapmers, siRNAs, or specifically for RNA editing oligonucleotides, associated with a variety of target sequences) is described in WO 20 / 227691 (C9orf72), WO 2021 / 071788 (C9orf72), WO 2021 / 071858, WO 2021 / 178237 (MAPT), WO 2021 / 234459, WO 2021 / 237223, and WO 2022 / 099159. In addition to these disclosures, a large number of publications relate to targeting specific RNA target molecules or specific adenosines in such RNA target molecules, whether for the purpose of repairing mutations that lead to premature stop codons or other disease-causing mutations.Examples of such disclosures targeting adenosine in specific target RNA molecules are: 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 / 231679 (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).

[0021] Disclosed herein are EONs capable of RNA-editing a target adenosine in human ALDH2 transcripts (pre-mRNA and / or mRNA). This allows the resulting ALDH2 protein to restore its wild-type function: converting acetaldehyde to acetate in ethanol metabolism. In one preferred aspect, the EONs cause the deamination of adenosine at position 1459 in the mutant mRNA, thereby generating inosine. In other words, the AAA codon encoding the amino acid lysine at position 504 (the mutant form) is converted to an IAA codon, which is interpreted as GAA, encoding glutamate (the wild-type form). In another embodiment, EONs according to the present invention cause the deamination of another adenosine present in the ALDH2 transcript. This adenosine can be any adenosine that, when deaminated to inosine, produces an ALDH2 protein with wild-type function or gain-of-function. Additional mutations may exist in the ALDH2 gene (and its transcripts) that can be targeted by RNA editing to restore normal ALDH2 function. The preferred targeted mutation is the G>A mutation at position 1510 of the transcript, resulting in the p.Glu504Lys ALDH2 protein mutation (c.1510G>A). This protein mutation is often referred to as E487K, also as E504K, and the mutant allele is often referred to as ALDH2*2. In the literature, this mutation is sometimes referred to as p.Glu487Lys, rs671, c.1510G>A, and p.E504K, but no reference to "c.1459G>A" has been found. This article uses the transcript number for the human (Homo sapiens) ALDH2 protein and gene from ensemble.org (transcript ENST00000261733.7), meaning that the mutation is referred to as E504K in the protein and c.1510G>A in the transcript.

[0022] Although in a preferred embodiment, the EON of the present invention is a single-stranded oligonucleotide comprising an orphan nucleotide opposite the target adenosine, wherein the orphan nucleotide is chemically modified as disclosed herein, and wherein the remainder of the oligonucleotide is also chemically modified as disclosed herein to prevent its degradation by nucleases, in another embodiment, the present invention relates to any type of oligonucleotide or heteroduplex oligonucleotide complex, which may or may not be associated with a hairpin structure (internal or terminal), may be associated with an ADAR or its catalytic domain, or wherein the oligonucleotide is expressed by a vector (e.g., adeno-associated virus (AAV)), or wherein the oligonucleotide is in a circular form. It should be understood that any oligonucleotide-based RNA editing that involves deamination of a nucleotide in the ALDH2 transcript (preferably resulting in the mutation E504K) and results in restoration of ALDH2 function is encompassed by the present invention. In a preferred aspect, the EON of the present invention is a "naked" oligonucleotide having a sequence in which one or more nucleotides contain various chemical modifications in the ribose, base, and / or internucleoside linkage, which can hybridize to the ALDH2 transcript or a portion thereof containing the target adenosine and recruit endogenous ADARs to deaminize the target adenosine.

[0023] The present invention relates to an EON that can form 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 endogenous ADAR enzymes 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 liver cell, 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 adenosine that causes ALDH2 protein dysfunction for deamination. Although it is known that a variety of mutations can cause ALDH2 protein dysfunction, the preferred mutation targeted by the EON disclosed herein is the adenosine produced by the G>A mutation in the ALDH2 gene, which results in a mutant p.E504K ALDH2 protein. The EON disclosed herein can deaminate the first adenosine in the AAA codon encoding lysine, thereby generating an IAA codon, which is read as GAA and is translated into glutamate. In one embodiment, the EON disclosed herein comprises Figure 1 In one embodiment, the EON of the present invention comprises Figure 1 The chemical modifications shown are present in the examples, or consist entirely of the same.

[0024] In one embodiment, an EON according to the present invention comprises at least one nucleotide comprising one or more non-naturally occurring chemical modifications in the ribose, linker or base moiety, or one or more additional non-naturally occurring chemical modifications, provided that the orphan nucleotide (i.e., the nucleotide directly opposite the target adenosine in the EON) 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), sulfonylphosphoramidate or PNdmi internucleotide linkages. 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 substituent being independently selected from: -OH; -F; substituted or unsubstituted, linear or branched lower (C1-C 10 ) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, which may be interrupted by one or more heteroatoms; -O-, S-, or N-alkyl; -O-, S-, or N-alkenyl; -O-, S-, or N-alkynyl; -O-, S-, or N-allyl; -O-alkyl-O-alkyl; -methoxy; -aminopropoxy; -methoxyethoxy; -dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy. In one embodiment, the EON comprises one or more mismatches, wobble, or bulges, wherein one mismatch may be present when the target adenosine has a corresponding cytidine in the EON. If the orphan nucleotide is a cytidine, the cytidine does not comprise a 2'-OMe ribose substitution.

[0025] Disclosed herein is a vector, preferably a viral vector, more preferably an adeno-associated virus (AAV) vector, comprising a nucleic acid molecule encoding an EON disclosed herein. Also disclosed herein is a pharmaceutical composition comprising an EON disclosed herein or a vector disclosed herein, and a pharmaceutically acceptable carrier.

[0026] In one embodiment, disclosed herein are EONs, vectors, or pharmaceutical compositions for use in treating diseases caused by ALDH2 deficiency, preferably diseases caused by ALDH2*2, such as intoxication, alcoholism, or symptoms of alcohol consumption. In one embodiment, disclosed herein are EONs or vectors for use in preparing a medicament for treating alcohol intolerance caused by ALDH2*2, such as intoxication, alcoholism, or one or more symptoms of alcohol consumption.

[0027] In one embodiment, a method for editing an ALDH2 polynucleotide is disclosed, the method comprising contacting the ALDH2 polynucleotide with an EON capable of achieving an adenosine deaminase acting on RNA (ADAR)-mediated conversion of adenosine to inosine, thereby editing the ALDH2 polynucleotide. The adenosine is associated with alcohol intolerance. In one embodiment, a method for treating an ALDH2 deficiency (e.g., alcohol intolerance or intoxication caused by ALDH2*2, or a disease caused by the alcohol intolerance or intoxication) in a patient in need thereof is disclosed, the method comprising contacting the ALDH2 polynucleotide in a subject's cells with an EON capable of achieving an ADAR-mediated conversion of adenosine to inosine, thereby treating the patient, the adenosine being associated with alcohol intolerance, or being associated with a disease caused by the alcohol intolerance (e.g., intoxication, alcoholism, or drinking symptoms). The mutation preferably targeted in the methods and uses disclosed herein is a G>A mutation, which results in a change of glutamic acid at position 504 of the mature protein to lysine. In one embodiment, disclosed is a method for treating alcohol intolerance or a disease caused by 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.

[0028] In one embodiment, a method for deaminating a target adenosine in an ALDH2 pre-mRNA or mRNA molecule in a cell is disclosed, comprising the following steps: (i) providing an EON or vector disclosed herein to the cell; (ii) allowing the cell to take up the EON or vector, respectively; (iii) annealing the EON to the ALDH2 pre-mRNA or mRNA molecule; (iv) allowing an endogenous ADAR enzyme (e.g., ADAR2) to deaminate the target adenosine in the target RNA molecule to inosine; and optionally (v) confirming the presence of inosine in the target RNA molecule. A preferred target adenosine is a G>A mutation in a mutant ALDH2 gene transcript encoding a mutant p.E504K ALDH2 protein. Preferably, step (v) comprises: 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, such as assessing alcohol content in a serum or plasma sample, or any other biomarker associated with ALDH2 function known to those skilled in the art.

[0029] definition

[0030] The term "nucleoside" refers to a nucleobase linked to a (deoxy)ribosyl sugar, without a phosphate group. A "nucleotide" consists of a nucleoside and one or more phosphate groups. Thus, "nucleotide" refers to the corresponding nucleobase-(deoxy)ribosyl-phosphate linker, as well as any chemical modification of the ribose moiety or the phosphate group. Thus, the term includes nucleotides containing a locked ribosyl moiety (comprising a 2'-4' bridge containing a methylene or any other group), unlocked nucleic acids (UNA), threose nucleic acids (TNA), and nucleotides containing the following linkages: including phosphodiester, phosphonoacetate, phosphotriester, PS, (di)phosphorothioate, MP, methyl thiophosphonate, phosphoramidate, and the like. Sometimes, the terms adenosine and adenine, guanosine and guanine, cytidine and cytosine, uracil and uridine, thymine and thymidine / uridine, inosine and hypoxanthine are used interchangeably to refer to the corresponding nucleobase on the one hand and to the nucleoside or nucleotide on the other hand. Thymine (T) is also called 5-methyluracil (m 5 U), is a derivative of uracil (U); thymine, 5-methyluracil and uracil are used interchangeably throughout this document. Similarly, thymidine, also known as 5-methyluridine, is a derivative of uridine; thymidine, 5-methyluridine and uridine are used interchangeably throughout this document. Sometimes, unless the context clearly requires otherwise, such as when a nucleoside is linked to an adjacent nucleoside and the linkage between these nucleosides is modified, the terms nucleobase, nucleoside and nucleotide are used interchangeably. As described herein, a nucleotide is a nucleoside plus one or more phosphate groups. The terms "ribonucleoside" and "deoxyribonucleoside", or "ribose" and "deoxyribose" are used as in the art.

[0031] Unless the context indicates otherwise, when referring to oligonucleotides, oligonucleotides, ONs, ASOs, oligonucleotide compositions, antisense oligonucleotides, AONs, (RNA) editing oligonucleotides, EONs and RNA (antisense) oligonucleotides, all refer to oligoribonucleotides and deoxyoligoribonucleotides. An oligonucleotide may be completely devoid of RNA or DNA nucleotides (as they appear in nature) or may be composed entirely of modified nucleotides. When referring to "oligoribonucleotides", it may contain the bases A, G, C, U or I. When referring to "deoxyoligoribonucleotides", it may contain the bases A, G, C, T or I. However, the EONs disclosed herein may contain a mixture of ribonucleosides and deoxyribonucleosides. When deoxyribonucleotides are used, since the 2' position of the sugar is unmodified, the nucleotides are often abbreviated as dA, dC, dG, or T, where the "d" represents the deoxy nature of the nucleoside, while normal RNA or ribonucleosides modified at the 2' position are often abbreviated without the "d" and are often abbreviated with the respective modification, as described herein.

[0032] When a nucleotide is mentioned in an oligonucleotide, for example, cytosine, 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-acetylcytosine, 5-hydroxycytosine, and β-D-glucosyl-5-hydroxymethylcytosine are all included. When adenine is mentioned, it includes N6-methyladenine, 8-oxo-adenine, 2,6-diaminopurine, and 7-methyladenine. When uracil is mentioned, it includes dihydrouracil, isouracil, N3-glycosylated uracil, pseudouracil, 5-methyluracil, N1-methylpseudouracil, 4-thiouracil, and 5-hydroxymethyluracil. When guanine is mentioned, it includes 1-methylguanine, 7-methylguanosine, N2,N2-dimethylguanosine, N2,N2,7-trimethylguanosine, and N2,7-dimethylguanosine. When referring to nucleosides or nucleotides, furanose derivatives are included, such as 2'-deoxy, 2'-hydroxy and 2'-O-substituted variants (e.g., 2'-OMe), as well as other modifications, including 2'-4' bridged variants. When referring to oligonucleotides, the linkage between the two mononucleotides can be a phosphodiester linkage and modifications thereof, including phosphonoacetate, phosphotriester, PS, (di)thiophosphate, MP, phosphoramidate linker, phosphoguanidine, thiophosphoguanidine, sulfonophosphoramidate, etc.

[0033] The term "comprising" encompasses "including" as well as "consisting of", e.g., a composition "comprising X" may consist solely of X or may also include other components, e.g., X + Y. The term "about" in relation to a value x is optional and means, e.g., x ± 10%.

[0034] The word "substantially" does not exclude "completely", for example, a composition "substantially free of Y" may be completely free of Y. Where applicable, the word "substantially" may be omitted from the definition of the present invention.

[0035] As used herein, the term "complementary" refers to the hybridization of an EON to 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, or HEON, with another complementary nucleic acid strand), or when it forms a double-stranded complex with a target RNA sequence. The term does not necessarily mean that every nucleotide in the nucleic acid strand is perfectly paired with the relative nucleotide in the relative sequence. In other words, although the EON may be complementary to the target sequence, there may be mismatches, wobbles, and / or bulges between the oligonucleotide and the target sequence, and under physiological conditions, the EON still hybridizes to the target sequence, allowing the cellular RNA editing enzyme to edit the target adenosine. Therefore, the term "substantially complementary" also means that, despite the presence of mismatches, wobbles, and / or bulges, the EON still has enough matching nucleotides between the EON and the target sequence so that the EON can hybridize to the target RNA under physiological conditions. As shown herein, an EON can be complementary to a target sequence if it can hybridize to its target under physiological conditions, but may also contain one or more mismatches, wobbles, and / or bulges.

[0036] With respect to nucleic acid sequences, the term "downstream" refers to a position further along the sequence in the 3' direction; "upstream" refers to the opposite direction. Thus, in any sequence encoding a polypeptide, the start codon is upstream of the stop codon in the sense strand but downstream of the stop codon in the antisense strand.

[0037] "Hybridization" generally refers to specific hybridization and does not include non-specific hybridization. Using techniques well known in the art, specific hybridization can be performed under selected experimental conditions to ensure that the most stable interaction between the probe and the target occurs where the probe and the target have at least 70%, preferably at least 80%, and more preferably at least 90% sequence identity.

[0038] The term "mismatch" refers to relative nucleotides in a double-stranded RNA complex that cannot form perfect base pairs according to the Watson-Crick base pairing rules. Traditionally, mismatched nucleotides are GA, CA, UC, AA, GG, CC, and UU pairs. In some embodiments, the mismatch between the first nucleic acid chain of the present invention and the target sequence is less than four, such as 0, 1, or 2 mismatches. "Wobble" base pairs are GU, IU, IA, and IC base pairs. Although G:G pairing is considered to be a mismatch, this does not necessarily mean that the interaction is unstable, which means that based on the current invention, the term "mismatch" may be somewhat outdated because Hoogsteen base pairing may be considered a mismatch based on the source of the nucleotide, but is still relatively stable. For example, a single G:G pairing in a double-stranded RNA may be very stable, but is still defined as a mismatch.

[0039] The term "splicing mutation" refers to a mutation in a gene encoding pre-mRNA in which the splicing machinery malfunctions, resulting in disordered splicing of introns from exons. Due to the abnormal splicing, subsequent translation will be out of frame, leading to premature termination of the encoded protein. This shortened protein is often rapidly degraded and loses any functional activity.

[0040] The EONs disclosed herein (and the complementary nucleic acid strands when two oligonucleotides form an HEON) can be almost completely chemically modified, for example by providing the nucleotides with 2'-OMe substitutions, 2'-F substitutions, or 2'-O-methoxyethyl (2'-MOE) substitutions on the ribose moiety. The orphan nucleotides in the EON are preferably cytidine or an analog thereof (e.g., a nucleotide bearing a Benner base), or uridine or an analog thereof (e.g., isouridine), and / or, in one embodiment, contain a diF (diF) modification at the 2' position of the sugar; in another embodiment, contain a deoxyribose (2'-H, DNA); in yet another embodiment, at least one of the two adjacent nucleotides flanking the orphan nucleotide does not contain a 2'-OMe modification; in another embodiment, neither of the two adjacent nucleotides flanking the orphan nucleotide contains a 2'-OMe modification. Full modification, i.e., all nucleotides of the oligonucleotide carry a 2'-OMe modification and have natural bases, renders the oligonucleotide nonfunctional with respect to RNA editing (as known in the art), presumably because it hinders ADAR activity at the targeted site. Typically, adenosine in the target RNA can be protected from editing by providing a counter nucleotide with a 2'-OMe group (at least when there are no other chemical substitutions or modifications in the nucleotide); or by providing a guanine or adenine (because these two nucleobases can also reduce editing relative to adenosine) as the counter base.

[0041] A variety of chemical reactions and modifications are known in the art of oligonucleotides and can be readily employed in accordance with the present invention. Conventional internucleoside linkages between nucleotides can be altered by mono-thioation or di-thioation of the phosphodiester bond to generate PS esters or phosphorodithioates, respectively. Other modifications of the internucleoside linkage are also possible, including amidation and peptide linkers.

[0042] In one embodiment, EON of the present invention comprises 15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59 or 60 nucleotides.It should be noted that when EON is delivered by (virus) vector, its length may increase, exceed 60 nucleotides.However, when EON is delivered with former state (not through vector delivery, also referred to as " naked form "), the length of EON is limited to 15 to 60 nucleotides, to reduce the risk of degradation.In addition, the EON of naked form is preferably chemically modified in the manner outlined herein, to reduce the risk of degradation.

[0043] RNA editing entities known in the art (such as human ADAR enzymes) edit dsRNA structures with different specificities, which depends on a variety of factors. One of the important factors is the degree of complementarity of the two chains that constitute the dsRNA sequence. The perfect complementarity of the two chains usually causes the catalytic domain of human ADAR to deaminize adenosine in a non-discriminatory manner and react with any adenosine encountered. The specificity of hADAR1 and 2 can be improved by introducing chemical modifications and / or ensuring multiple mispairings in dsRNA, which may help to locate the dsRNA binding domain in a manner not yet clear. In addition, the deamination reaction itself can be enhanced by providing oligonucleotides comprising mispairings relative to adenosine to be edited. According to the description in this application, those skilled in the art will be able to design the complementary parts of oligonucleotides as needed.

[0044] The most relevant RNA editing protein present in cells used together with EON of the present invention is human ADAR2. It will be understood by those of ordinary skill in the art that the degree of redirection of editing entities in cells to other target sites can be adjusted by changing the affinity of the first nucleic acid chain to the editing molecule recognition domain. Exact modification can be determined by repeated trials and / or calculation methods based on structural interactions between EON and editing molecule recognition domains. In addition, or, the degree of recruiting and redirecting editing entities residing in cells can be regulated by the dosage and dosage regimen of EON. This is usually determined by experimenters (in vitro) or clinicians in Phase I and / or Phase II clinical trials.

[0045] The present invention relates to the modification of target RNA sequences in eukaryotic organisms, preferably metazoans, more preferably mammalian cells, even more preferably human cells, and most preferably human liver cells (e.g., hepatocytes). The present invention is particularly suitable for modifying RNA sequences in cells and tissues in which ALDH2K is expressed and in which the protein functions. Since ALDH2 is primarily produced in liver cells and plays an important role in ethanol metabolism, the preferred target cells for the EONs of the present invention are liver cells, more preferably hepatocytes. The target cells can be located in vitro, ex vivo, or in vivo. One advantage of the present invention is that it can be used for both in situ cells in a living organism and for cultured cells. In some embodiments, cells are treated ex vivo and then introduced into a living organism (e.g., reintroduced into the organism from which they originally came). The present invention can also be used to edit target RNA sequences in cells derived from a transplant or in cells within so-called organoids (e.g., liver tissue organoids). Organoids can be considered three-dimensional in vitro-derived tissues, but require the use of specific conditions to generate a single, isolated tissue. They are very useful in therapeutic settings because they can be derived from patient cells in vitro and the organoids can then be reintroduced into the patient as autologous material, which is less likely to be rejected than normal transplants.

[0046] Without being bound by theory, RNA editing by human ADAR2 is believed to occur on primary transcripts in the nucleus (during transcription or splicing) or in the cytoplasm (e.g., where mature mRNA, miRNA, or ncRNA can be edited).

[0047] It should be understood that targeted editing according to the present invention can be applied to any adenosine in the ALDH2 transcript if deamination of adenosine results in enhanced or restored ALDH2 protein function. However, as described herein, targeting the first adenosine in the mutated codon at position 504 of the mature protein is preferred.

[0048] In general, RNA editing can be used to create RNA sequences with different properties. These properties can be coding properties (creating proteins with different sequences or lengths, resulting in altered protein properties or function) or binding properties (resulting in inhibition or overexpression of the RNA itself, its target, or its binding partner; by recoding miRNA or its homologous sequences on the target RNA, the entire expression pathway can be altered). Protein function or localization can be arbitrarily altered via functional domains or recognition motifs, including but not limited to signal sequences, targeting or localization signals, recognition sites for proteolysis or co-translational or post-translational modification, catalytic sites for enzymes, binding sites for binding partners, degradation or activation signals, and the like. The present invention encompasses these and other forms of RNA and protein "engineering," whether for the prevention, delay, or treatment of disease, or for any other purpose in medicine or biotechnology, such as diagnostics, prophylaxis, therapy, research tools, or other uses. Therefore, any RNA editing directed to the target adenosine in the ALDH2 transcript that improves or restores ALDH2 protein function is encompassed by the present invention. This invention opens up a whole new field of therapeutic applications for alcohol intoxication using gene editing technology.

[0049] The amount, dose, and dosing regimen of the EON to be administered may vary depending on the cell type, the disease to be treated, the target population, the route of administration (e.g., systemic versus local), disease severity, and acceptable side effect levels, but these can and should be evaluated through trial and error in in vitro studies, preclinical, and clinical trials. Trials are particularly straightforward when the modified sequence results in easily detectable phenotypic changes or changes in specific biomarkers (levels or activity). Higher doses of EON may compete for binding to ADARs within the cell, thereby depleting the number of entities free to participate in RNA editing, but conventional dosing trials will reveal any such effects for a given EON and a given target.

[0050] One suitable experimental technique involves delivering EONs to a cell line or test organism, followed by biopsy samples collected at various time points thereafter. The sequence of the target RNA can be assessed in the biopsy sample, and the proportion of cells with the modification can be easily tracked. Furthermore, alcohol levels in blood samples are suitable biomarkers for assessing ALDH2 protein function in a given subject (before and after treatment, or with or without treatment with EONs or a vector as described herein). Conducting such an experiment once preserves relevant knowledge and eliminates the need for biopsy samples for future deliveries. Therefore, the methods of the present invention can include a step of confirming the presence of the desired change in the cellular target RNA sequence, thereby verifying whether the target RNA sequence has been modified. This step typically involves sequencing the relevant portion of the target RNA or a cDNA copy thereof (or, if the target RNA is a pre-mRNA, a cDNA copy of its splicing product), as described above, thus allowing for easy verification of sequence changes. Alternatively, as described above, protein function can be assessed, for example, by measuring or assessing serum or plasma ethanol concentrations before, during, and / or after treatment, or by assessing any other potential markers. These measurements are preferably performed in vitro on samples obtained from the treated subject.

[0051] After RNA editing occurs in cells, the modified RNA will be diluted over time, for example due to cell division, the limited half-life of the edited RNA, etc. Therefore, in practical treatment, the methods of the present invention may involve repeated delivery of EONs until enough target RNAs are modified to provide tangible benefits to the patient and / or to maintain the benefits over time.

[0052] 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 be simply a saline solution. This may be isotonic or hypotonic, and is particularly suitable for pulmonary delivery. The present invention also provides a delivery device (e.g., a syringe, inhaler, nebulizer) comprising the pharmaceutical composition of the present invention.

[0053] As described herein, the present invention also provides a method for using an EON of the present invention to repair a target ALDH2 RNA sequence mutation in a mammal (preferably a human liver cell). Similarly, as described herein, the present invention also provides the use of an EON of the present invention in the preparation of a medicament for altering a target ALDH2 RNA sequence in a mammal (preferably a human liver cell), thereby treating, preventing, or ameliorating alcohol-related disorders, such as disorders caused by ALDH2*2.

[0054] 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 steps of: providing an EON according to the present invention to the cell; allowing the cell to take up the EON; annealing the EON to a target RNA molecule; allowing a mammalian ADAR enzyme comprising a native dsRNA binding domain identical to that of the wild-type enzyme to deaminate the target adenosine in the target RNA molecule (preferably the first adenosine in the codon encoding lysine at position 504 in the mature protein) to inosine; and optionally confirming the presence of inosine in the RNA sequence.

[0055] 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 steps of: providing a vector or plasmid encoding an EON according to the present invention to a 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 identical to that of the wild-type enzyme to deaminate a target adenosine (preferably the first adenosine in the codon encoding lysine at position 504 in the mature protein) in the target RNA molecule to inosine; and optionally confirming the presence of inosine in the RNA sequence.

[0056] In a preferred aspect, depending on the final deamination effect of the A to I conversion, the confirmation step comprises the following steps: sequencing the target RNA; assessing the presence or absence of a functional protein; assessing whether the splicing of the pre-mRNA is altered due to deamination; or using a functional readout, since the target RNA after deamination should encode a functional protein. For example, the concentration of ethanol and / or acetaldehyde in the (blood) sample after RNA editing is assessed. Therefore, confirmation of deamination to inosine can be a functional readout using a suitable biomarker. The functional assessment of intoxication described herein generally uses methods known to those skilled in the art. After the target adenosine is deaminated, the best way to confirm the presence of inosine is of course to perform dPCR or even sequencing using methods well known to those skilled in the art. However, those skilled in the art of liver disease can also apply detection methods to monitor certain biomarkers associated with intoxication, as described above.

[0057] The EON of the present invention is suitably administered in the form of an aqueous solution (e.g., normal saline) or a suspension, optionally containing additives, excipients, and other ingredients compatible with pharmaceutical use, in a concentration range of 1 ng / ml to 1 g / ml, preferably 10 ng / ml to 500 mg / ml, more preferably 100 ng / ml to 100 mg / ml. The dosage range is suitably from about 1 μg / kg to about 100 mg / kg, preferably from about 10 μg / kg to about 10 mg / kg, more preferably from about 100 μg / kg to about 1 mg / kg. Administration can be by inhalation (e.g., by atomization), intranasal, oral, injection or infusion, intravenous, subcutaneous, intradermal, intramuscular, intratracheal, intraperitoneal, intrarectal, intrathecal, intracerebellar medullary cisterna magna, parenteral, etc. It can be administered in the form of a solid, powder, pill, gel, solution, sustained-release formulation, or any other suitable human medication.

[0058] In one embodiment, the method of the present invention comprises the following steps: administering an EON or pharmaceutical composition according to the present invention to a subject, allowing the EON to form a double-stranded nucleic acid complex with its specifically complementary target nucleic acid molecule in the subject's cells; engaging an endogenously present adenosine deaminase (e.g., ADAR2); and causing the enzyme to deaminate adenosine in the target nucleic acid molecule to inosine, thereby alleviating, preventing, or ameliorating a disorder associated with alcohol intoxication. Diseases treatable according to this method are preferably, but not limited to, the genetic diseases listed herein, as well as any other disease in which deamination of adenosine in the ALDH2 transcript can restore protein function in a patient in need thereof.

[0059] The RNA editing molecules present in the cell are typically protein in nature, such as the ADAR enzymes found in metazoans (including mammals). Preferably, the cell editing entity is an enzyme, more preferably an adenosine deaminase or a cytidine deaminase, more preferably an adenosine deaminase. These are enzymes with ADAR activity. The most interesting are human ADARs, hADAR1 and hADAR2, including any isoforms thereof. RNA editing enzymes known in the art (for which oligonucleotide constructs according to the present invention can be conveniently designed) include adenosine deaminases (ADARs) that act on RNA, such as hADAR1 and hADAR2 in humans or human cells, and cytidine deaminases. It is known that hADAR1 exists in two isoforms; a longer 150 kDa interferon-induced version and a shorter 100 kDa version, which are produced by alternative splicing of a common pre-mRNA. Therefore, the level of the 150 kDa isoform available in the cell may be affected by interferons, particularly interferon gamma (IFN-γ). hADAR1 can also be induced by TNF-α. This provides an opportunity to develop combination therapies in which the EONs of the present invention and IFN-γ or TNF-α can be administered to patients simultaneously or subsequently in any order as a combination product or as separate products. Certain disease conditions may have occurred simultaneously with elevated levels of IFN-γ or TNF-α in certain tissues of the patient, creating an opportunity for further more specific editing of the diseased tissue. One of ordinary skill in the art will appreciate that the extent to which the intracellular editing entity is redirected to other target sites can be regulated by changing the affinity of the first nucleic acid chain for the editing molecule recognition domain.

[0060] Chemical modification

[0061] All chemical modifications listed below that can be used for the EON of the present invention can also be used for the sense strand complementary to the EON when the EON forms a so-called heteroduplex RNA editing oligonucleotide (HEON) complex with the complementary strand, as described in GB 2215614.5 (unpublished), but the opposing sense strand does not contain an orphan nucleotide. Therefore, modifications related to orphan nucleotides are only relevant to the EON of the present invention, but all other modifications are relevant to the EON of the present invention and any (protective) sense oligonucleotide that can be used in conjunction with the EON for pharmaceuticals. This includes the use of hydrophobic moieties (such as tocopherol and cholesterol) and cell-specific ligands (such as GalNAc moieties), which have also been described herein and described in detail in GB 2215614.5 (unpublished), which can be bound to the EON or its opposing strand, or both.

[0062] The internucleoside linkages in the oligonucleotides of the present invention may comprise one or more naturally occurring internucleoside linkages and / or modified internucleoside linkages. Without limitation, at least one, at least two or at least three internucleoside linkages at the 5' and / or 3' ends of the EON are preferably modified internucleoside linkages. Preferred modified internucleoside linkages are PS linkages. In one embodiment, all internucleoside linkages of the EON are modified internucleoside linkages. In one embodiment, the EON comprises a PNdmi linkage, which connects the terminal nucleoside at the 5' and / or 3' ends, and the preceding nucleoside at the two ends, respectively. The PNdmi linkage preferably used in the EON of the present invention has the following structure:

[0063]

[0064] A common limiting factor for oligonucleotide therapy is the ability of the oligonucleotide to be taken up by cells (when delivered as such, or “naked” without a delivery vehicle), its biodistribution, and its resistance to nuclease-mediated degradation. It is known to those skilled in the art, and a variety of chemical modifications have been described in detail in the art to help overcome these limitations. Examples of currently commonly used chemical modifications include 2'-O-methyl (commonly abbreviated as 2'-OMe or 2'-O-Me), 2'-F and 2'-O-methoxyethyl (also commonly referred to as 2'-methoxyethoxy or 2'-MOE) modifications of sugars, and the use of PS linkages between nucleosides. WO2020 / 201406 discloses the use of MP linkage modifications at certain positions around orphan nucleotides in a first nucleic acid chain. The ribose 2' group of all nucleotides in EONs, with the exception of orphan nucleotides, which have certain limitations regarding their compatibility with RNA editing, can be independently selected from 2'-H (i.e., DNA), 2'-OH (i.e., RNA), 2'-OMe, 2'-MOE, 2'-F, or 2'-4'-linkages (e.g., locked nucleic acids (LNA)), or other ribose 1'-, 2'-, 3'-, 4'-, or 5'-substitutions. In EONs without other chemical modifications to the ribose sugar, base, or linkage, orphan nucleotides preferably do not carry 2'-OMe or 2'-MOE substitutions, but may carry 2'-F, 2',2'-difluoro (diF), or 2'-ara-F (FANA) substitutions, or may be DNA. GB 2214347.3 (unpublished) describes modification of the 2' position of the ribose moiety of orphan nucleotides by 2',2'-disubstitution (e.g., diF), which is also applicable to the invention described herein. The 2'-4' linker can be selected from a number of linkers known in the art, such as a methylene linker, an amide linker, or a constrained ethyl linker (cEt).

[0065] The present invention relates to an EON for deaminating a target nucleotide (preferably adenosine) in a target RNA, wherein the EON is complementary to a stretch of nucleotides comprising the target adenosine in the target RNA, wherein the nucleotide directly opposite the target nucleotide in the first nucleic acid strand is an orphan nucleotide; when the target nucleotide is adenosine, the orphan nucleotide preferably comprises a base, modified base, or base analog with an NH moiety located in a position similar to a ring nitrogen (e.g., Benner base Z). Nucleotides in the EON are numbered as follows: the orphan nucleotide is numbered 0, and the nucleotide at the 5' end of the orphan nucleotide is numbered +1. Nucleotides are numbered in a positive (+) direction toward the 5' end and a negative (-) direction toward the 3' end, with the first nucleotide at the 3' end of the orphan nucleotide being numbered -1. Internucleoside linkages in the EON are numbered as follows: linkage number 0 is the linkage at the 5' end of the orphan nucleotide, and linkage positions in the oligonucleotide increase in a positive (+) direction toward the 5' end and in a negative (-) direction toward the 3' end.

[0066] Preferably, the EON comprises one or more (chirally pure or chirally mixed) PS linkages. In one embodiment, the PS linkages connect 3, 4, 5, 6, 7, or 8 nucleotides at both ends of the first nucleic acid strand. In one embodiment, the EON comprises one or more phosphoramidate (PN) linkages. In one embodiment, the PN linkages connect the terminal two nucleotides at both ends of the EON.

[0067] The nucleosides in EONs can be natural nucleosides (deoxyribonucleosides or ribonucleosides) or non-natural nucleosides. It should be noted that for RNA editing, double-stranded RNA is often a substrate for enzymes with deamination activity (e.g., ADARs), so ribonucleosides are considered "natural," while deoxyribonucleosides may (for ease of discussion) be considered non-natural or modified simply because DNA does not exist in the RNA-RNA double-stranded substrate configuration. Those skilled in the art understand that when a nucleotide has a natural ribose moiety, it can still be non-naturally modified in the base and / or linkage.

[0068] In addition to specific preferred chemical modifications at certain positions in the compounds of the invention, the compounds of the invention may also comprise or consist of one or more (additional) modifications to the nucleobases, scaffolds and / or backbone linkages (these may or may not be present in the same monomer, for example at the 3' and / or 5' positions). Backbone modifications refer to the presence of modified forms of ribosyl moieties (i.e., pentose moieties) naturally occurring in RNA (e.g., bicyclic sugars, tetrahydropyranoses, 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, such as 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), 2'-O -(2-amino)ethyl, 2'-O-(2-(dimethylamino)ethyl); 2'-deoxy(DNA); 2'-O-(haloalkyl)methyl, such as 2'-O-(2-chloroethoxy)methyl (MCEM), 2'-O-(2,2-dichloroethoxy)methyl (DCEM); 2'-O-alkoxycarbonyl, such as 2'-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), 2'-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DCME); 2'-halogen, such as 2'-F, FANA; 2'-O-[2-(methylamino)-2-oxoethyl] (NMA); bicyclic or bridged nucleic acid (BNA) backbone modifications, such as conformationally restricted nucleotide (CRN) monomers, locked nucleic acid (LNA) monomers, xylose-LNA (xylo-LNA) monomers, α-LNA monomers, 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-pyranose nucleic acid (DpNA) monomers, 2'-C-bridged bicyclic nucleotide (CBBN) monomers, oxo-CBBN monomers, heterocyclic bridged BNA monomers (e.g., triazole- 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 NA) monomers, F-tcDNA monomers, α-anomeric bicyclic DNA (abcDNA) monomers, oxetane nucleotide monomers, locked PMO monomers derived from 2'-amino LNA, guanidine bridged nucleic acid (GuNA) monomers, spirocyclopropene bridged nucleic acid (scpBNA) monomers, and derivatives thereof; cyclohexenyl nucleic acid (CeNA) monomers, ahtriol nucleic acid (ANA) monomers, hexitol nucleic acid (HNA) monomers, fluorinated HNA (F-HNA) monomers, pyranosyl-RNA (p-RNA) monomers, 3'-deoxypyranosyl DNA (p-DNA), non-locked nucleic acid UNA); and reverse versions of any of the above monomers. All of these modifications are known to those skilled in the art.

[0069] Here, the base sequence of the EON is complementary to a portion of the base sequence of the target ALDH2 transcript, which contains at least the target adenosine to be deaminated to inosine, and is therefore capable of annealing (or hybridizing) with the target transcript. Base sequence complementarity can be determined using a BLAST program or similar programs. Those skilled in the art can readily determine the conditions (temperature, salt concentration, etc.) under which the two strands can hybridize based on the complementarity between the strands.

[0070] Unlike previously described gapmers and their relationship to RNase degradation and the use of such gapmers in double-stranded complexes (e.g., see EP 3954395 A1), the EONs according to the present invention do not contain a stretch of DNA nucleotides that renders the target sequence (or sense nucleic acid strand) a target for RNase-mediated degradation. In one embodiment, the EONs do not contain four or more consecutive DNA nucleotides anywhere in their sequence. In one embodiment, the EONs are composed of as many (chemically) modified nucleotides as possible to enhance resistance to RNase-mediated degradation while producing the RNA editing effect as efficiently as possible. This means that the orphan nucleotides and several other nucleotides in the EONs may be DNA, but there are no stretches of four or more consecutive DNA nucleotides in the EONs. Therefore, the EONs according to the present invention are not gapmers. Gapmers reduce the expression of target transcripts but do not produce RNA editing of specific adenosines in the target transcripts. A gapmer is, in principle, a single-stranded nucleic acid consisting of a central region (a DNA gap region with at least four consecutive deoxyribonucleotides) and wings located 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, wherein a target adenosine in a target RNA molecule is deaminated to inosine and is therefore as resistant as possible to RNase-mediated degradation to produce this effect.

[0071] In one embodiment, the EON or its sense strand to which it may anneal prior to entry into the target cell is bound to a hydrophobic moiety, such as palmitoyl or its analogs, cholesterol or its analogs, tocopherol or its analogs. It is preferably bound to the 5' end. If the hydrophobic moiety is bound to both the 5' end and the 3' end, these hydrophobic moieties may be the same or different. The hydrophobic moiety bound to the oligonucleotide may be directly bound or indirectly mediated by another substance. When the hydrophobic moiety is directly bound, the moiety is bound by a covalent bond, an ionic bond, a hydrogen bond, or the like. When the hydrophobic moiety is indirectly bound, the binding may be through a linking group (linker). The linker may be cleavable or non-cleavable. A cleavable linker refers to a linker that is cleavable under physiological conditions (e.g., in a cell or animal body, such as the human body). A cleavable linker can be selectively cleaved by endogenous enzymes (e.g., nucleases) or by a physiological environment specific to the body or cell site (e.g., pH or reducing environment, such as glutathione concentration). Examples of cleavable linkers include, but are not limited to, one or both esters and disulfide bonds among amides, esters, phosphodiesters, phosphates, and carbamates, as well as natural DNA linkers. Cleavable linkers also include self-destructive linkers. Non-cleavable linkers refer to linkers that do not crack under physiological conditions or that crack very slowly compared to cleavable linkers, such as in PS linkers, modified or unmodified deoxyribonucleosides connected by PS linkers, spacers connected by PS bonds, and linkers consisting of modified or unmodified ribonucleosides. When the linker is a nucleic acid (e.g., DNA) or an oligonucleotide, the chain length is not limited. However, the length is typically 2 to 20 bases, 3 to 10 bases, or 4 to 6 bases. There is no limit on the length or composition of the spacer connecting the ligand and the oligonucleotide, and may include, for example, ethylene glycol, TEG, HEG, an alkyl chain, a propyl group, a 6-aminohexyl group, or a dodecyl group.

[0072] The present invention also relates to a pharmaceutical composition comprising an EON according to the present invention and further comprising a pharmaceutically acceptable carrier and / or other additives, and being soluble in a pharmaceutically acceptable organic solvent, etc. The dosage form of the EON or pharmaceutical composition depends on the disease to be treated and the tissue to be targeted, and can be selected according to conventional methods in the art. The pharmaceutical composition can be administered in a single dose or in multiple doses. The administration method can be daily or at appropriate time intervals, which can be determined using common knowledge in the art and can be adjusted according to the disease and the efficacy of the active ingredient.

[0073] In one embodiment, the EON comprises at least one nucleotide comprising a 2'-OMe modified sugar moiety. In one embodiment, the EON comprises at least one nucleotide comprising a 2'-MOE modified sugar moiety. In one embodiment, the EON comprises at least one nucleotide comprising a 2'-F modified sugar moiety. In one embodiment, the orphan nucleotide carries 2'-H in the sugar moiety and is therefore referred to as a DNA nucleotide, even though other modifications may exist in its base and / or linkage to adjacent nucleosides. In one embodiment, the orphan nucleotide carries 2'-F in the sugar moiety. In one embodiment, the orphan nucleotide carries a double F substitution in the sugar moiety. In one embodiment, the orphan nucleotide carries 2'-F and 2'-C-methyl in the sugar moiety. In one embodiment, the orphan nucleotide comprises 2'-F in the arabinose configuration (FANA) in the sugar moiety. In one embodiment, the EON is an antisense oligonucleotide that can form a double-stranded nucleic acid complex with a target RNA molecule, wherein the double-stranded nucleic acid complex can recruit adenosine deaminase to deaminate the target adenosine in the target ALDH2 RNA molecule, wherein the nucleotide opposite the target adenosine in the EON is an orphan nucleotide, and wherein the orphan nucleotide has the following structure:

[0074]

[0075] wherein: 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-oxoadenine, and 6-amino-5-nitro-2(1H)-pyridone; R1 and R2 are independently selected from H, OH, F, or CH3; R3 is the portion of the EON located 5' to the orphan nucleotide, consisting of 7 to 30 nucleotides; R4 is the portion of the EON located 3' to the orphan nucleotide, consisting of 4 to 25 nucleotides. The nucleotides at the 3' and / or 5' position of the orphan nucleotide may be DNA, more preferably the 3' nucleotide (position -1).

[0076] In one embodiment, the first nucleic acid strand comprises at least one MP internucleoside linkage according to the following structure:

[0077]

[0078] The preferred position for the MP linker in the EON according to the present invention is the linker-1 position, thereby linking the nucleoside at the -1 position to the nucleoside at the -2 position, but MP linkers at other positions are not specifically excluded.

[0079] In one embodiment, the EON comprises at least one nucleotide comprising a 2'-fluoro (2'-F) modified sugar moiety. The preferred position of the nucleotide with the 2'-F modification is the -3 position in the EON, which can be present simultaneously with the same 2' modification in the orphan nucleotide as described above.

[0080] In one embodiment, the EON comprises at least one phosphonoacetate or phosphonoacetamide internucleoside linkage.

[0081] In one embodiment, the EON comprises at least one nucleotide with a locked nucleic acid (LNA) ribose modification or an unlocked nucleic acid (UNA) ribose modification. In one embodiment, the EON comprises at least one nucleotide comprising a threose nucleic acid (TNA) ribose modification.

[0082] It is known to those skilled in the art that oligonucleotides, such as EON as described herein, are generally composed of repeating monomers. Such monomers are generally nucleotides or chemically modified nucleotides. The most common natural nucleotides in RNA are adenosine monophosphate (A), cytidine monophosphate (C), guanosine monophosphate (G) and uridine monophosphate (U). These nucleotides are composed of pentose, ribose, a 5'-connected phosphate group and a 1'-base connected by a phosphate ester. Sugar connects base and phosphate and is therefore commonly referred to as the "backbone" of nucleotides.

[0083] Therefore, modification of pentoses is often referred to as "backbone modification." The original pentose can be completely replaced by another moiety that similarly connects a base and a phosphate. Therefore, although a pentose is often the backbone, the backbone is not necessarily a pentose. WO2020 / 154342, WO2020 / 154343, and WO2020 / 154344 disclose examples of backbone modifications of monomers that can be used in the EONs of the present invention.

[0084] In one embodiment, the EON of the present invention may include one or more nucleotides with 2'-MOE ribose modifications. In addition, in one embodiment, the EON includes one or more nucleotides without 2'-MOE ribose modifications, and wherein the 2'-MOE ribose modification is located at a position that does not prevent an enzyme with adenosine deaminase activity from deaminating the target adenosine. In another embodiment, the EON includes a 2'-OMe ribose modification at a position that does not include a 2'-MOE ribose modification, and / or wherein the oligonucleotide includes a deoxynucleotide at a position that does not include a 2'-MOE ribose modification. In one embodiment, the EON includes one or more nucleotides that include 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 or an example mentioned in WO2018 / 007475)). In another embodiment, the other nucleic acid monomers used are arabinose nucleic acid and 2'-deoxy-2'-fluoroarabinose nucleic acid (FANA), for example, for the purpose of improving affinity. The 2'-4' linker can be selected from linkers known in the art, such as methylene linkers or restricted ethyl linkers. Various 2' modifications are known in the art. For example, more examples are disclosed in more detail in WO2016 / 097212, WO2017 / 220751, WO2018 / 041973, WO2018 / 134301, WO2019 / 219581, WO2019 / 158475 and WO2022 / 099159. In all cases, the modification should be compatible with editing so that EON plays its role as an editor to produce oligonucleotides that can form double-stranded complexes with the target RNA and recruit deaminases, subsequently deaminating the target adenosine. When a monomer comprises an unlocked nucleic acid (UNA) ribose modification, the 2' position of the monomer may comprise the same modifications described above, such as 2'-MOE, 2'-OMe, 2'-OH, 2'-deoxy, 2'-F, 2',2'-difluoro, 2'-fluoro-2'-C-methyl, arabinonucleotide, FANA, or a 2'-4'-linkage (i.e., a bridged nucleic acid, such as a locked nucleic acid (LNA)).

[0085] Base, also sometimes referred to as nuclear base, is typically adenine, cytosine, guanine, thymine or uracil, or its derivatives. Base, also sometimes referred to as nuclear base, refers to a portion that can be bound to another nuclear base by hydrogen bonds, polarized bonds (e.g., by CF moieties) or aromatic electron interactions. Cytosine, thymine and uracil are pyrimidine bases, typically connected to the backbone by their 1-nitrogen. Adenine and guanine are purine bases, typically connected to the backbone by their 9-nitrogen. The terms "adenine," "guanine," "cytosine," "thymine," "uracil," and "hypoxanthine" used herein refer to the nuclear base itself. The terms "adenosine," "guanosine," "cytidine," "thymidine," "uridine," and "inosine" refer to the nuclear bases connected to (deoxy) ribosyl sugars.

[0086] The nucleobase in the EON of the present invention can be adenine, cytosine, guanine, thymine or uracil, or any other moiety capable of interacting with another nucleobase via hydrogen bonding, polarized bonding (e.g., CF) or aromatic electronic interactions. The nucleobase at any position in the nucleic acid chain can be a modified form of adenine, cytosine, guanine or uracil, such as hypoxanthine (the nucleobase in inosine), pseudouracil, pseudocytosine, isouracil, N3-glycosylated uracil, 1-methylpseudouracil, orotic acid, agmatidine, lysine nucleoside (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-amino methylcytosine, 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-oxoadenine, 3-deazapurine (e.g., 3-deazaadenosine), pseudoisocytosine, N4-ethylcytosine, N2-cyclopentylguanine, N2-cyclopentyl-2-aminopurine, N2-propyl-2-aminopurine, 2,6-diaminopurine, 2-aminopurine, G-clamp and its derivatives, Super A, Super T, Super G, amino-modified nucleobases or their derivatives; and degenerate bases or universal bases, such as 2,6-difluorotoluene, or deleted base sites (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose, azaribose).

[0087] In one embodiment, the nucleotide analog is an analog of a nucleic acid nucleotide. In one embodiment, the nucleotide analog is an analog of adenosine, guanosine, cytidine, thymidine, uridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine, or deoxyuridine. In one embodiment, the nucleotide analog is not guanosine or deoxyguanosine. In one embodiment, the nucleotide analog is not a nucleic acid nucleotide. In one embodiment, the nucleotide analog is not adenosine, guanosine, cytidine, thymidine, uridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine, or deoxyuridine.

[0088] Nucleotide is usually connected to adjacent nucleotides by condensation of its 5'-phosphate moiety with the 3'-hydroxyl portion of adjacent nucleotide monomers. Similarly, its 3'-hydroxyl moiety is usually connected to the 5'-phosphate of adjacent nucleotide monomers. This forms a phosphodiester bond. Phosphodiester and backbone form an alternating copolymer. Bases are grafted onto this copolymer, i.e., grafted onto the backbone portion. Due to this characteristic, the alternating copolymer formed by the connection backbone of oligonucleotides is commonly referred to as the "main chain" of oligonucleotides. Because the phosphodiester bond connects adjacent monomers together, they are commonly referred to as "main chain connection bonds". It is understandable that when the phosphate group is modified into a similar group (such as PS), the group is still referred to as the main chain connection bond of the monomer. This is referred to as "main chain connection bond modification". Generally speaking, the main chain of an oligonucleotide is composed of alternating backbone and main chain connection bonds.

[0089] The EON according to the present invention may include linkage modifications. The linkage modifications may be, but are not limited to, modified forms of phosphodiesters present in RNA, such as PS, chirally pure PS, (R)-PS, (S)-PS, methylphosphonate (MP), chirally pure methylphosphonate, (R)-methylphosphonate, (S)-methylphosphonate, phosphoguanidine (e.g., PNdmi), chirally pure phosphoguanidine, (R)-phosphoguanidine, (S)-phosphoguanidine, phosphorodithioate (PS2), phosphonoacetate (PACE), phosphonoacetamide (PACA), thiophosphonoacetate, thiophosphonoacetamide, methylphosphorohioate, methylphosphonothioate, PS prodrug, alkylated PS, H-phosphonate, ethyl phosphate, ethyl PS, boranophosphate, borane PS, methylboranophosphate, methylborane PS, methylboranephosphonate, borylphosphorothioate, phosphates, phosphotriesters, aminoalkylphosphotriesters, and derivatives thereof. Other modifications include phosphoramidite, phosphoramidate, N3'→P5' phosphoramidate, phosphorodiamidate, phosphorothioate, sulfamate, diethyl sulfoxide, amide, sulfonate, siloxane, sulfide, sulfone, formacetyl, alkenyl, methylenehydrazine, sulfonamide, triazole, oxalyl, carbamate, methyleneimino (MMI) and thioacetamide nucleic acid (TANA); and their derivatives. In addition, various salts, mixed salts and free acid forms are included, as well as 3'→3' and 2'→5' linkages.

[0090] In one embodiment, the EON comprises a substitution of a non-bridging oxygen in a phosphodiester linkage. This modification slightly destabilizes base pairing but significantly enhances resistance to nuclease degradation. Preferred nucleotide analogs or equivalents include PS, phosphonoacetate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H-phosphonate, methylphosphonate and other alkylphosphonates (including 3'-alkylenephosphonates, 5'-alkylenephosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate), thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate. Particularly preferred are internucleoside linkages modified to include PS. Many such non-naturally occurring linker modifications (such as PS) are chiral, which means that there are Rp and Sp configurations known to those skilled in the art. In one embodiment, the chirality of the PS linker is controlled, which means that each linker is either in Rp configuration or in Sp configuration, whichever is preferred. Selecting Rp or Sp configuration at a specific linker position may depend on the target sequence and the efficiency of binding efficiency and induction RNA editing. However, if not particularly required, at a specific linker position, the composition can include an AON with Rp and Sp configuration as an active compound. The mixture of such EONs is also feasible, wherein some positions preferably have one of these configurations, and is insignificant for other positions.

[0091] Likewise, in all cases, the modification should be compatible with editing so that the EON plays its role as an editing-generating oligonucleotide that, when attached to a target sequence, can recruit adenosine deaminase due to its dsRNA properties. In all aspects of the invention, the enzyme having adenosine deaminase activity is preferably ADAR1, ADAR2 or ADAT. In a highly preferred embodiment, the EON is an RNA editing oligonucleotide targeting a precursor mRNA or mRNA, wherein the target nucleotide is adenosine in the target RNA, wherein adenosine is deaminated to inosine, and the translation mechanism reads it as guanosine. The present invention also relates to a pharmaceutical composition comprising an EON as described herein and a pharmaceutically acceptable carrier.

[0092] Other chemical modifications of the EON according to the present invention include replacing one or more hydrogen atoms with deuterium or tritium, as described, for example, in WO 2014 / 022566 or WO 2015 / 011694.

[0093] The present invention relates to an EON according to the invention or a pharmaceutical composition comprising an EON according to the invention for use in treating or preventing a disease associated with alcohol intolerance or intoxication, preferably a disease caused by an ALDH2*2 mutant. In one embodiment, the present invention relates to an EON according to the invention or a pharmaceutical composition comprising an EON according to the invention for use in treating or preventing a disease associated with alcohol intolerance or intoxication, preferably a disease caused by an ALDH2*2 mutant. In one embodiment, the present invention relates to an EON according to the invention or a pharmaceutical composition comprising an EON according to the invention for use in treating or preventing an alcohol intolerance or intoxication, preferably a disease caused by an ALDH2*2 mutant.

[0094] The EONs of the present invention preferably do not contain a 5'-terminal O6-benzylguanosine or a 5'-terminal amino modification, and preferably are not covalently linked to a SNAP-tag domain (an engineered O6-alkylguanosine-DNA-alkyltransferase). The EONs of the present invention preferably do not contain a boxB RNA hairpin sequence. In one embodiment, the EONs of the present invention have 0, 1, 2, or 3 wobble base pairs with the target sequence and / or 0, 1, 2, 3, 4, 5, 6, 7, or 8 mismatched base pairs with the target RNA sequence. When the orphan nucleotide is uridine, there are no mismatches. An alternative to uridine is to place isouridine opposite the target adenosine, which may not pair like G with U. Preferably, the target adenosine in the target sequence forms a mismatched base pair with the nucleoside in the EON that is directly opposite the target adenosine.

[0095] It is important to note that when EONs are delivered via a vector (e.g., an AAV vector), no chemical modifications are present in the EONs acting on the target RNA molecule. Although it is preferred to use "naked" EONs with the chemical modifications outlined herein, EONs delivered by other means, such as expression via an AAV vector, or editing of circular or hairpin molecules (recruiting moieties, e.g., as disclosed in WO2016 / 097212, WO2017 / 050306, WO2020 / 001793, WO2017 / 010556, WO2020 / 246560, and WO2022 / 078995) are also encompassed by the present invention, as these EONs can also be used to edit adenosine in the target ALDH2 RNA molecule to generate ALDH2 protein with restorative function.

[0096] The EONs according to the present invention can utilize endogenous cellular pathways and naturally occurring ADAR enzymes to specifically edit target adenosines in target RNA sequences. The EONs of the present invention are capable of recruiting and complexing with ADARs, which then promote the deamination of a (single) specific target adenosine nucleotide in the target RNA sequence. Ideally, only one adenosine is deaminated. When complexed with ADARs, the EONs of the present invention preferably result in the deamination of a single target adenosine.

[0097] Analysis of the natural targets of ADAR enzymes has shown that these typically include mismatches between the two strands that form the RNA helix edited by ADAR1 or ADAR2. Studies have shown that these mismatches enhance the specificity of the editing reaction (Stefl et al. 2006. Structure 14 (2): 345-355; Tian et al. 2011. Nucleic Acids Res 39 (13): 5669-5681). Characterization of the optimal pattern of nucleotide pairing / mismatching between EON and target RNA is also important for developing effective EON therapies based on ADARs.

[0098] As mentioned above, the EON of the present invention utilizes specific nucleotide modifications at predetermined sites to ensure stability and appropriate ADAR binding and activity. These changes may be different and may include modifications of the main chain of the EON, modifications of the nucleotide sugar moiety, and modifications of the core base or phosphodiester linkage, as described herein. They may also be distributed in different ways throughout the EON sequence. It may be necessary to carry out specific modifications to support the interaction of different amino acid residues within the RNA binding domain of the ADAR enzyme and in the deaminase domain. For example, in some parts of the EON, PS linkages or 2'-OMe or 2'-MOE modifications between nucleotides are acceptable, while in other parts of the EON, these modifications should be avoided to avoid destroying the key interactions of the enzyme with the phosphate group and the 2'-OH group. When the target sequence is not suitable for ADAR editing, it may also be necessary to carry out specific nucleotide modifications to enhance the editing activity to the substrate RNA. Previous studies have confirmed that certain sequence environments are more suitable for editing. For example, the target sequence 5'-UAG-3' (target A is in the middle) contains the most preferred nearest neighbor nucleotide for ADAR2, while the 5'-CAA-3' target sequence is not favored (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 is not favored due to the steric clash between the guanosine base and the amino acid side chain of ADAR2 due to the steric clash between the guanosine base and the amino acid side chain of ADAR2. The present invention relates to RNA editing oligonucleotides (collectively referred to herein as EONs) that are capable of deaminating adenosine in ALDH2 transcripts, thereby producing an ALDH2 protein that is fully functional in ethanol metabolism. This means that the present invention is not strictly limited to the deamination of adenosine in mutant ALDH2*2, but can also target other (single or multiple) adenosines, which may also enhance the function of the ALDH2 protein. Other adenosines that are equally important (or potentially more important) for ALDH2 function can also be identified, for example, by population genetic screening or computer simulation, and these adenosines can also be targeted by RNA editing according to the teachings of the present invention. All such RNA events and oligonucleotides that can be used for such targeting are included in the present invention, regardless of the specific nucleic acid molecule or EON.

[0099] Mutagenesis studies of human ADAR2 have shown that a single mutation from glutamic acid to glutamine at residue 488 (E488Q) increases the rate constant of the deamination reaction 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 edited base out of its RNA duplex and into the enzyme active site (Matthews et al., 2016). When ADAR2 edits adenosine under preferred conditions (A:C mismatch), the nucleotide opposite the target adenosine is often referred to as an "orphan cytidine". The crystal structure of ADAR2 E488Q bound to double-stranded RNA (dsRNA) shows that the glutamine (Gln) side chain at position 488 can provide a hydrogen bond to the N3 position of the orphan cytidine, thereby increasing the catalytic rate of ADAR2 E488Q. In the wild-type enzyme, position 488 is glutamic acid (Glu) instead of glutamine (Gln), so the amide group of glutamine does not exist, but a carboxylic acid. In order for the orphan cytidine to obtain the same contact with the E488Q mutant, protonation is required for this contact to occur in the wild-type case. In order to use endogenously expressed ADAR2 to correct disease-associated mutations, the editing efficiency of the wild-type ADAR2 enzyme present in the cell must be maximized. WO2020 / 252376 discloses the use of EONs that modify RNA bases, especially at the position of orphan cytidine, to mimic the hydrogen bond pattern observed in the E488QADAR2 mutant. By replacing the nucleotide opposite to the target adenosine in the EON with a cytidine analog that acts as an H-bond donor at N3, it is conceivable that the same contact can be stabilized, which is thought to increase the catalytic rate of the mutant enzyme. Two cytidine analogs are of particular interest: pseudoisocytidine (also known as "piC"; Lu et al. J Org Chem 2009. 74(21):8021-8030; Burchenal et al. (1976) Cancer Res 36:1520-1523) and Benner base Z (also known as "dZ"; Yang et al. Nucl Acid Res 2006. 34(21):6095-6101), which were initially chosen because they provide hydrogen bond donation at N3 while minimally perturbing the shape of the nucleobase. Benner bases are also known as 6-amino-5-nitro-2(1H)-pyridone. In addition to modifications of the ribose 2' group, cytidine analogs may also be present in AONs. The ribose 2' groups in the AON can be independently selected from 2'-H (ie DNA), 2'-OH (ie RNA), 2'-OMe, 2'-MOE, 2'-F or 2'-4' linkages (ie bridged nucleic acids such as locked nucleic acids (LNA)) or other 2' substitutions.The 2'-4' linker may be selected from linkers known in the art, such as a methylene linker or a constrained ethyl linker.

[0100] In one embodiment, the nucleotide analogs or equivalents in the EON comprise one or more base modifications or substitutions. Modified bases include synthetic and natural bases, such as inosine, xanthine, hypoxanthine, and other -aza, denitrogenation, -hydroxyl, -halogen, -thio, thiol, -alkyl, -alkenyl, -alkynyl, sulfanyl derivatives of pyrimidine and purine bases known or to be known in the art. Purine and / or pyrimidine nucleobases can be modified to change their properties, such as by amination or deamination of heterocycles. The exact chemical properties and form may vary depending on the oligonucleotide construct and application, and can be designed according to the wishes and preferences of those skilled in the art.

[0101] The EON according to the present invention is typically longer than 10 nucleotides, preferably longer than 11, 12, 13, 14, 15, 16 nucleotides, more preferably longer than 17 nucleotides. In one aspect, the AON according to the present invention is longer than 20 nucleotides. The oligonucleotide according to the present invention is preferably shorter than 100 nucleotides, more preferably shorter than 60 nucleotides, more preferably shorter than 50 nucleotides. In a preferred aspect, the oligonucleotide according to the present invention comprises 18 to 70 nucleotides, more preferably 18 to 60 nucleotides, even more preferably 18 to 50 nucleotides. 55, 56, 57, 58, 59, or 60 nucleotides.

[0102] In one aspect, inverted deoxy-T or dideoxy-T nucleotides are introduced at either or both ends of the EON according to the present invention.

[0103] As described above, in some embodiments, the present invention provides an EON for forming a double-stranded complex with a human ALDH2 RNA molecule in human liver cells. Therefore, the therapeutic effect is preferably achieved in vivo on human liver cells. Of course, the method can also be performed in vitro or ex vivo.

[0104] 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 use of the EON of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease. The present invention also provides a method of treating a disease in a patient, comprising administering a therapeutically effective amount of the EON of the present invention or the pharmaceutical composition of the present invention. Preferably, the disease is caused by the E504K mutation in ALDH2. The EON can be administered therapeutically or prophylactically (following genetic counseling), as both treatment modalities may be beneficial.

[0105] After RNA editing occurs in cells, the modified RNA will be diluted over time, for example due to cell division, the limited half-life of the edited RNA, etc. Therefore, in practical treatment, the methods of the present invention may involve repeated delivery of AONs until enough target RNA is modified to provide tangible benefits to the patient and / or maintain the benefits over a long period of time.

[0106] Example

[0107] Example 1. Editing of target adenosine in human ALDH2 target RNA molecules using an in vitro biochemical editing assay.

[0108] First, an initial set of EONs targeting ALDH2 (e.g. Figure 1 ) to edit human ALDH2 target (precursor) mRNA in an in vitro biochemical editing assay. To obtain ALDH2 target RNA, ALDH2 G-block (IDT) containing the T7 promoter sequence and (partial) ALDH2 sequence was used as a template, and PCR was performed using the forward primer 5'-CTC GAC GCA AGC CAT AACAC-3' (SEQ ID NO: 53) and the reverse primer 5'-TGG ACC GAC TGG AAACGT AG-3' (SEQ ID NO: 54). The 5' to 3' G-block sequence (SEQ ID NO: 55) is as follows, where the target adenosine is underlined and bold, and the primer sequence is underlined:

[0109]

[0110] The PCR product was then used as a template for in vitro transcription. The reaction was performed using the MEGAscript T7 transcription kit. RNA was purified from a urea gel and then extracted with 50 mM Tris-Cl pH 7.4, 10 mM EDTA, 0.1% SDS, and 0.3 M NaCl buffer, followed by phenol-chloroform purification. The purified RNA was used as a target for biochemical editing experiments.

[0111] 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 to the ALDH2 target RNA. The annealing step was performed in a buffer (5 mM Tris-Cl pH 7.4, 0.5 mM EDTA, and 10 mM NaCl) with a target RNA to oligonucleotide ratio of 1:3 (600 nM oligonucleotide and 200 nM target). The sample was heated at 95°C for 3 minutes and then slowly cooled to room temperature. Next, the editing reaction was performed. The annealed oligonucleotide / target RNA was mixed with protease inhibitors (cOmplete TM , Mini, EDTA-free proteinase I, Sigma-Aldrich), RNase inhibitor (RNasin, Promega), poly A (Qiagen), tRNA (Invitrogen) and editing reaction buffer (15mMTris-Cl pH 7.4, 1.5mM EDTA, 3% glycerol, 60mM KCl, 0.003% NP-40, 3mM MgCl2 and 0.5mM DTT) were mixed to a final concentration of 6nM oligonucleotide and 2nM target RNA. The reaction was initiated by adding purified ADAR2 (GenScript) to the mixture to a final concentration of 6nM and incubated at 37°C for the predetermined time points. Each reaction was terminated by adding 95μl of 95°C 3mM EDTA solution. A 6μl aliquot of the terminated reaction mixture was then used as a template for cDNA synthesis using a Maxima reverse transcriptase kit (Thermo Fisher) and random hexamer primers (ThermoFisher Scientific). RNA was initially denatured at 95°C for 5 minutes in the presence of primers and dNTPs, then slowly cooled to 10°C. First-strand synthesis was then performed in a total volume of 20 μl using an extension temperature of 62°C, according to the manufacturer's instructions. PCR amplification of 1 μl of cDNA was performed using the Amplitaq Gold 360 DNA Polymerase Kit (Applied Biosystems) according to the manufacturer's instructions, using 1 μl of cDNA as a template for pyrosequencing analysis. PCR was then performed using the following thermal cycling 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, followed by a final extension at 72°C for 7 minutes.

[0112] Because inosine base pairs with cytidine during cDNA synthesis in the reverse transcription reaction, the nucleotide inserted at the edited position during PCR will be guanosine. The percentage of guanosine (edited) to adenosine (unedited) was determined by pyrosequencing. 10 μl of PCR product and 4 μM sequencing primer were input, and pyrosequencing and data analysis were performed using a PyroMark Q48 Autoprep instrument (QIAGEN) according to the manufacturer's instructions: The analysis performed by the instrument provides a result for the selected nucleotide, that is, the percentage of adenosine and guanosine detected at that position. Therefore, the extent of A-to-I editing at the selected position is measured by the percentage of guanosine at that position.

[0113] Example 2. Editing target adenosine in human ALDH2 target RNA molecules using patient-derived skin fibroblasts.

[0114] This study examined Figure 1 Whether the modified EONs shown in (each with an RM number) are able to edit target adenosines in human ALDH2*2 transcript RNA carrying the c.1510G>A mutation in cells. To this end, untransformed human skin fibroblasts (AG11369; Corriell, USA) heterozygous for the c.1510G>A mutation were used. The disadvantage of using heterozygous mutant cells is that there is a significant background signal of unedited transcripts, which are wild-type and transcribed from the wild-type allele. Approximately 50,000 cells were seeded per 24-well plate 24 hours before application of the corresponding EON. Application of the corresponding EON can be performed using 100nM EON alone or in the context of 5μM saponin (AG1856; see WO2021 / 122998). The cells were harvested after incubation with EON±saponin for 3 days. RNA was extracted using the Direct-zol RNA MicroPrep (Zymo Research) kit according to the manufacturer's instructions, and cDNA was prepared with random hexamers using the Maxima Reverse Transcriptase Kit (Thermo Fisher) according to the manufacturer's instructions. Digital PCR (dPCR) was performed using cDNA as a template, with 200 ng of RNA input per reaction. dPCR assays were performed using Qiagen's QIAcuity 4 Digital PCR System for absolute quantification of nucleic acid target sequences. 1.2 μl of undiluted cDNA obtained from the RT cDNA synthesis reaction was used in a total mixture of 12 μl reaction mixtures, including 4× concentrated QIAcuity Probe Mastermix (Qiagen), Taqman SNP Genotype Assays with the following forward and reverse primers and the following gene-specific probes:

[0115] Forward primer:

[0116] 5'-TGGTGGCTACAAGATGTCGG-3'(SEQ ID NO:56)

[0117] Reverse primer:

[0118] 5'-TTATGAGTTCTTCTGAGGCACT-3'(SEQ ID NO:57)

[0119] Wild-type probe (FAM NFQ labeled):

[0120] 5'- / 56-FAM / A+CAGTT+TTCACTT+C+A+GTGTATGCC / 3IABkFQ / -3'(SEQ ID NO: 58)

[0121] Mutation probe (HEX NFQ labeled)

[0122] 5'- / 5HEX / A+CAGTT+TTCACTT+T+A+GTGTATGCCC / 3IABkFQ / 3'(SEQ ID NO:59)

[0123] A total volume of 12 μl of the PCR mixture (including cDNA) was added to a QIAcuity Nanoplate (Qiagen) using a multichannel pipette. After sealing the plate with a Nanoplate seal, it was placed in a QIAcuity 4dPCR device, where dispensing, PCR amplification, and fluorescence measurement are fully automated. 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.

[0124] The results in Figure 2 show that for most of the EONs tested, similar editing percentages were observed in the absence of saponin (gymnotic uptake, or "GU"), and the percentage of wild-type ALDH2 transcripts reached levels exceeding 80%. However, as described above, these values were affected by the presence of the wild-type allele in the heterozygous cells used. Normalizing to the untreated sample (NT), i.e., the percentage of wild-type background signal "removed," the results showed that when EONs were applied to cells in the presence of AG1856, editing levels reached nearly 30%, indicating that the inventors were able to edit the ALDH2*2c.1510G>A mutation to wild-type in human cells, with EON RM4740 performing 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, wherein the region of the ALDH2 transcript molecule comprises a target adenosine, and wherein the double-stranded complex is capable of recruiting an endogenous ADAR enzyme to deaminize the target adenosine to inosine, thereby editing the ALDH2 transcript molecule. 2 . The EON of claim 1 , wherein the ALDH2 transcript molecule is a pre-mRNA or mRNA molecule.

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

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

5. The EON of any one of claims 1 to 4, wherein the target adenosine is a c.1510G>A mutation in the ALDH2 transcript. 6 . The EON according to claim 1 , wherein the EON comprises or consists of any one of the EON sequences selected from SEQ ID NOs: 1 to 51. 7 .

7. The EON according to any one of claims 1 to 6, wherein at least one nucleotide comprises one or more non-naturally occurring chemical modifications in the ribose, linkage or base moiety, or one or more additional non-naturally occurring chemical modifications, provided that the nucleotide opposite the target adenosine in the EON, i.e., the orphan nucleotide, is not a cytidine comprising a 2'-OMe ribose substitution.

8. The EON of claim 7, wherein the one or more additional modifications in the linker moiety are each independently selected from phosphorothioate (PS), phosphonoacetate, phosphorodithioate, methylphosphonate (MP), sulfonylphosphoramidate, or PNdmi internucleotide linkages.

9. The EON according to claim 7 or 8, wherein 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 substituent being independently selected from the group consisting of: -OH; ·F； Substituted or unsubstituted, linear or branched lower (C1-C 10 ) alkyl, alkenyl, alkynyl, alkaryl, allyl or aralkyl, which may be interrupted by one or more heteroatoms; -O-, S-, or N-alkyl; -O-, S- or N-alkenyl; -O-, S- or N-alkynyl; -O-, S- or N-allyl; ●-O-alkyl-O-alkyl; ●-methoxy; ●-aminopropoxy; -methoxyethoxy; -dimethylaminooxyethoxy; and ·-Dimethylaminoethoxyethoxy.

10. A vector, preferably a viral vector, more preferably an adeno-associated virus (AAV) vector, comprising a nucleic acid molecule encoding the EON according to any one of claims 1 to 6. 11 . A pharmaceutical composition comprising the EON according to any one of claims 1 to 9 or the carrier according to claim 10 , and a pharmaceutically acceptable carrier.

12. The EON according to claims 1 to 9, the vector according to claim 10, or the pharmaceutical composition according to claim 11, for use in treating a disease caused by ALDH2 deficiency, preferably a disease caused by ALDH2*2.

13. Use of the EON according to any one of claims 1 to 9 or the vector according to claim 10 in the preparation of a medicament for treating a disease caused by ALDH2 deficiency, preferably caused by ALDH2*2, more preferably drunkenness, alcoholism, or symptoms of drinking.

14. A method for editing an ALDH2 polynucleotide, the method comprising contacting the ALDH2 polynucleotide with an EON capable of effecting adenosine deaminase acting on RNA (ADAR)-mediated conversion of adenosine to inosine, thereby editing the ALDH2 polynucleotide, wherein the adenosine is associated with alcohol intolerance.

15. A method of treating a disease caused by ALDH2 deficiency, preferably a disease caused by ALDH2*2, in a patient in need thereof, the method comprising contacting an ALDH2 polynucleotide in a cell of the subject with an EON capable of effecting ADAR-mediated conversion of adenosine to inosine, thereby treating the patient, wherein the adenosine is associated with ALDH2 deficiency. 16 . A method for treating a disease caused by ALDH2*2, comprising administering a therapeutically effective amount of the EON according to any one of claims 1 to 9 , the vector according to claim 10 , or the pharmaceutical composition according to claim 11 to a patient in need thereof.

17. A method for deaminating a target adenosine in an ALDH2 pre-mRNA or mRNA molecule in a cell, the method comprising the steps of: (i) providing the cell with an EON according to any one of claims 1 to 9; (ii) allowing the cells to take up the EON; (iii) annealing the EON to the ALDH2 pre-mRNA or mRNA molecule; (iv) allowing endogenous ADAR enzymes to deaminize the target adenosine in the target RNA molecule to inosine; and optionally (v) confirming the presence of inosine in the target RNA molecule.

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

19. The method according to claim 17 or 18, wherein step (v) comprises: 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 alcohol content in a serum or plasma sample.

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