Nucleotides encoding acetaldehyde dehydrogenase 2 (ALDH2) and uses thereof

Abstract

The invention relates to an RNA (Ribonucleic Acid) medicine, which is used for coding ALDH2 (Alcohol Dehydrogenase 2) protein, can efficiently express ALDH2 and quickly and durably reduce the content of acetaldehyde in a body, and is used for treating acute alcoholism caused by acetaldehyde accumulation as well as diseases such as metabolism, cardiovascular diseases, neurological diseases, malignant tumors and the like.

Description

Technical Field

[0001] The present application relates to the field of nucleotide technology applications. Specifically, the present application relates to a nucleotide for encoding acetaldehyde dehydrogenase (ALDH) and its pharmaceutical use. Background Art

[0002] Alcohol (ethanol) is a neuroactive and toxic substance. Alcohol consumption is the leading cause of death among young and middle-aged people worldwide, and alcohol abuse has become a major threat to public health. According to a 2018 WHO report, there are approximately 2.3 billion drinkers worldwide. In 2016, over 3 million people died from harmful alcohol use, accounting for 5% of all deaths worldwide. In Europe, excessive alcohol consumption is the most common cause of liver cirrhosis; in China, alcohol is associated with 50% of liver diseases. The incidence of acute alcohol poisoning has been increasing worldwide in recent years, resulting in increasing social and medical costs. Currently, the treatment of acute alcohol poisoning still relies primarily on supportive therapies, primarily to maintain airway patency and normal cardiac function, supplemented by fluid replacement to maintain water, electrolyte, blood sugar, and acid-base balance. There is currently a lack of medications specifically targeting ethanol metabolism.

[0003] In the body, ethanol metabolism and breakdown primarily occur in the liver, through a two-step enzymatic reaction: alcohol dehydrogenase (ADH) rapidly converts ethanol into the toxic intermediate acetaldehyde. Aldehyde dehydrogenase (ALDH), primarily ALDH2, then converts acetaldehyde into less toxic acetic acid. Finally, acetic acid enters the tricarboxylic acid cycle, decomposing into carbon dioxide and water before being excreted. Therefore, after consuming ethanol, blood acetaldehyde concentrations rise significantly, and the rate of acetaldehyde metabolism depends primarily on the concentration and activity of ALDH2 in the body.

[0004] ALDH2 is the most widely distributed and highly expressed of the 19 known ALDHs in humans. It is a tetrameric protein with identical subunits composed of 517 amino acids encoded by the ALDH2 gene located on chromosome 12. Each subunit contains three domains: a catalytic domain, an NAD+ binding domain, and an oligomerization domain. The ALDH2 gene exhibits genetic diversity, with mutation frequencies varying across different regions, ethnicities, and races. In the Chinese population, approximately 30%-50% of the population harbors the ALDH2*2 mutation. Heterozygous individuals with the mutation have less than 50% of the wild-type ALDH2 enzyme activity, while homozygous individuals have less than 1-4% of the wild-type ALDH2 enzyme activity.

[0005] Acetaldehyde, a bioactive molecule with strong redox activity, can bind to DNA and proteins to form adducts, affecting protein and DNA function and inducing intracellular oxidative stress. This stress produces large amounts of reactive oxygen species, which in turn damage liver cells, cardiomyocytes, neurons, and other cells. It can also promote cancer cell formation by inducing DNA damage. Alcohol consumption can lead to acute acetaldehyde exposure, resulting in symptoms of acute alcohol poisoning such as dizziness, headache, and vomiting. Carriers of the ALDH2*2 mutation are also at increased risk for metabolic, cardiovascular, and neurological diseases, as well as malignant tumors. Therefore, ALDH2 plays a crucial role in protecting the body from oxidative stress caused by aldehydes. Improving ALDH2 expression and activity is crucial, especially for the large number of carriers of the mutation. Summary of the Invention

[0006] The present invention relates to an RNA drug that encodes ALDH2 protein, can efficiently express ALDH2, and quickly and persistently reduce acetaldehyde content in the body, providing a new treatment solution for acute alcohol poisoning caused by acetaldehyde accumulation, as well as metabolic, cardiovascular, neurological, and malignant tumor diseases.

[0007] In one aspect, the present invention provides a nucleotide encoding human ALDH2, wherein the nucleotide encoding human ALDH2 is a circular RNA (circRNA) or a linear RNA.

[0008] In another aspect, the present invention provides a pharmaceutical composition comprising the nucleotide encoding human ALDH2 of the present invention and a pharmaceutically acceptable excipient.

[0009] In another aspect, the present invention provides use of a nucleotide encoding human ALDH2, a genetically engineered expression vector or a pharmaceutical composition thereof in the preparation of a drug for treating a disease by supplementing ALDH2 protein. Detailed Description of the Invention

[0011] In one aspect, the present invention provides a nucleotide encoding human ALDH2, comprising any one of SEQ ID NOs: 7-13, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% homology to any one of SEQ ID NOs: 7-13, or a combination thereof.

[0012] In some embodiments, the nucleotide sequence encoding human ALDH2 is a circRNA, which includes an internal ribosome entry site (IRES) and a protein coding region, wherein the protein coding region is selected from any one of the nucleotide sequences in SEQ ID NOs: 7-13, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% homology to any one of the nucleotide sequences in SEQ ID NOs: 7-13, or a combination thereof.

[0013] In some embodiments, the internal ribosome entry site (IRES) is selected from: coxsackievirus B3 (CVB3) IRES, enterovirus 71 (EV71) IRES, encephalomyocarditis virus (EMCV) IRES, picornavirus (PV) IRES, hepatitis C virus (HCV) IRES, adenovirus (AdV) IRES, human papillomavirus type 31 (HPV31) IRES, human herpesvirus (HHV) IRES, Rous sarcoma virus (RSV) IRES, classical swine fever virus (CSFV) IRES, FGF9 IRES, SLC7A1 IRES and RUNX1 IRES; preferably, the internal ribosome entry site (IRES) is CVB3 IRES; more preferably, the IRES is the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% homology to the nucleotide sequence of SEQ ID NO: 2.

[0014] In some embodiments, the circRNA is selected from the circularized nucleotide sequences described in SEQ ID NOs: 17-19, or circularized nucleotide sequences having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% homology to the nucleotide sequences of SEQ ID NOs: 17-19, or a combination thereof.

[0015] In some embodiments, the nucleotide sequence encoding human ALDH2 is a linear RNA comprising a 5'UTR, a nucleotide sequence of any one of SEQ ID NOs: 7-13, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% homology to any one of SEQ ID NOs: 7-13, or a combination thereof, a 3'UTR, and poly(A). In some embodiments, the linear RNA is mRNA.

[0016] In some embodiments, the 5'UTR is selected from the nucleotide sequence of SEQ ID NO: 3. The 3'UTR is selected from the nucleotide sequence of SEQ ID NO: 4.

[0017] In some embodiments, the linear RNA encoding human ALDH2 is selected from the nucleotide sequence of any one of SEQ ID NOs: 14-16, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% homology to the nucleotide sequence of any one of SEQ ID NOs: 14-16, or a combination thereof.

[0018] In some embodiments, the linear RNA encoding human ALDH2 further includes a 5'CAP, wherein the 5'CAP is preferably one or more selected from 7-methyl-guanosine-5'-triphosphate-5'-adenosine, guanosine-5'-triphosphate-5'-adenosine, 7-methyl-guanosine-5'-triphosphate-5'-guanosine, guanosine-5'-triphosphate-5'-guanosine and m7G(5')(2'-OMeA)pG.

[0019] In some embodiments, the number of A bases in the poly(A) sequence of the linear RNA encoding human ALDH2 is 60-120.

[0020] In some embodiments, the nucleotides encoding human ALDH2 further include a Flag tag nucleotide sequence. In some embodiments, the Flag tag nucleotide sequence is selected from the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the nucleotides encoding human ALDH2 are selected from the linear nucleotide sequence of SEQ ID NO: 20 or the circularized nucleotide sequence of SEQ ID NO: 21, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% homology to the nucleotide sequence of SEQ ID NO: 20 or 21.

[0021] In another aspect, the present invention provides a genetically engineered expression vector capable of transcribing a nucleotide encoding human ALDH2 of the present invention. In some embodiments, the genetically engineered expression vector is preferably a plasmid vector.

[0022] In another aspect, the present invention provides a pharmaceutical composition. The pharmaceutical composition comprises a nucleotide encoding human ALDH2 as described in any of the preceding embodiments and a pharmaceutically acceptable excipient. In some embodiments, the composition is a lipid nanoparticle (LNP). In some embodiments, the lipid nanoparticle (LNP) comprises an SM-102 aminocationic lipid.

[0023] In another aspect, the present invention provides use of a nucleotide encoding human ALDH2, a genetically engineered expression vector, or a pharmaceutical composition according to any of the aforementioned embodiments in the preparation of a drug for a disease treated by supplementing ALDH2 protein.

[0024] In some embodiments, the disease treated by supplementing ALDH2 protein is selected from the group consisting of: treating metabolic diseases, cardiovascular diseases, neurological diseases, and cancer.

[0025] In some embodiments, the disease treated by supplementing ALDH2 protein is selected from the group consisting of alcoholic liver disease, non-alcoholic fatty liver disease, hepatitis, myocardial ischemia-reperfusion injury, acute ischemic stroke, arrhythmia, Parkinson's disease, Alzheimer's disease, liver cancer, colorectal cancer, gastric cancer, esophageal cancer, lung cancer, bladder cancer, pulmonary hypertension, Fanconi anemia, or osteoporosis.

[0026] definition

[0027] Listed below are definitions of various terms used to describe the nucleic acid combinations and compositions disclosed herein. These definitions apply to the terms used throughout this specification and claims, except where otherwise limited in specific cases, and these terms are used either individually or as part of a larger group.

[0028] As used herein, the terms "optional" and "optionally" include both selection and non-selection. For example, "optionally modified" includes both modification and non-modification.

[0029] As used herein, the terms "a," "an," and "the" are generally construed to cover both the singular and the plural.

[0030] The term "include" as used herein means and can be used interchangeably with the phrase "including (but not limited to)". The term "comprising" as used herein means and can be used interchangeably with the phrase "including (but not limited to)". The technical solutions using "include" or "comprising" in this patent can be further limited to "consisting of" or "composed of".

[0031] Throughout this specification, reference to "one embodiment," "some embodiments," "an embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the aforementioned phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0032] As used herein, the terms "circRNA," "circularRNA," or "cyclic polyribonucleotide" or "circular RNA" are used interchangeably and refer to polyribonucleotides that form a circular structure through covalent bonds.

[0033] As used herein, "vector" refers to a segment of DNA that is synthesized (e.g., using PCR) or extracted from a virus, plasmid, or cell of a higher organism, into which foreign DNA segments can be or have been inserted for cloning and / or expression purposes.

[0034] As used herein, the term "homology" refers to the overall relatedness between polymer molecules, for example, between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Typically, the term "homology" means the evolutionary relationship between two molecules. Thus, two homologous molecules will have a common evolutionary ancestor. In the context of the present disclosure, the term homology includes identity and similarity. Polymer molecules are considered to be "homologous" to each other if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the monomers in the molecule are identical (identical monomers) or similar (conservative substitutions). The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0035] As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes at least one target peptide or polypeptide and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded target peptide or polypeptide. mRNA has been transcribed from a DNA sequence by RNA polymerase and interacts with ribosomes to synthesize the genetic information encoded by the DNA. In some embodiments, the linear RNA is mRNA. mRNA can be synthesized in a cell-free environment, for example, by in vitro transcription (IVT).

[0036] An "in vitro transcription template" refers to a deoxyribonucleic acid (DNA) suitable for use in an IVT reaction to produce messenger RNA (mRNA) or circRNA. For circRNA, an IVT template can encode an IRES and an open reading frame. In some embodiments, the IVT template encodes a 5' untranslated region (UTR), containing an open reading frame, and a 3' untranslated region (UTR) and a poly(A) tail. The specific nucleotide sequence composition and length of the IVT template will depend on the target mRNA or circRNA encoded by the template.

[0037] "5' untranslated region (5'UTR)" refers to the region of an mRNA just upstream (ie, 5') of the start codon (ie, the first codon of the mRNA transcript translated by the ribosome) that does not encode a protein or peptide.

[0038] "3' untranslated region (3'UTR)" refers to the region of an mRNA just downstream (ie, 3') of the stop codon (ie, the codon that signals the termination of translation) that does not encode a protein or peptide.

[0039] The poly(A) "poly A tail" is a region of an mRNA located downstream of the 3' UTR, for example, just downstream (i.e., 3'), which contains multiple consecutive adenosine monophosphates. In relevant biological environments (e.g., in cells, in vivo, etc.), the poly(A) tail serves to protect the mRNA from enzymatic degradation, for example, in the cytoplasm, and facilitates transcription termination, mRNA export from the nucleus, and translation. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 shows the construction strategy of linear RNA ( Figure 1A ) and in vitro transcription results ( Figure 1B ).

[0041] Figure 2 shows the construction strategy of circular RNA ( Figure 2A ) and circular RNA preparation results ( Figure 2B ).

[0042] Figure 3 shows the protein expression results of linear RNA and circular RNA: Figure 3A It showed that all three linear RNA sequences were expressed, with the linear ALDH2-2 sequence having a higher expression level; Figure 3B It showed that all three circular RNA sequences were expressed, with the circular ALDH2-3 sequence having a higher expression level; Figure 3C The expression of circular RNA was higher than that of linear RNA.

[0043] Figure 4 shows the in vivo expression detection results of linear and circular RNA: Figure 4A 、 4C It showed that both linear and circular RNAs could be detected in vivo 5 hours after administration; Figure 4B 、 4D showed that linear and circular RNAs did not affect endogenous ALDH2 expression; Figure 4E showed that linear and circular RNA can express ALDH2 protein in vivo; Figure 4F It was shown that cyclic ALDH2-3 could be detected in the liver of Aldh2- / - knockout homozygous animals 5 hours after administration. Figure 4G It was shown that cyclic ALDH2-3 was expressed in mouse liver.

[0044] Figure 5The results of the in vivo efficacy of linear ALDH2-2 and cyclic ALDH2-3 in Aldh2+ / - gene knockout heterozygous animals showed that both linear ALDH2-2 and cyclic ALDH2-3 could inhibit the accumulation of acetaldehyde in the serum of animals after alcohol ingestion, and the inhibitory effect of circular RNA was better than that of linear RNA.

[0045] Figure 6 shows the efficacy of cyclic ALDH2-3 in Aldh2- / - knockout homozygous animals: Figure 6A The results showed that the injection of cyclic ALDH2-3 could inhibit the accumulation of acetaldehyde in the serum of animals after alcohol ingestion; Figure 6B The results showed that the total time taken to cross the balance beam was reduced in mice administered cyclic ALDH2-3 during the balance beam test. Figure 6C It showed that the walking time of mice administered with cyclic ALDH2-3 was reduced on the balance beam. Figure 6D The results showed that the number of times mice administered cyclic ALDH2-3 slipped on the balance beam decreased. All three indicators showed that after the administration of cyclic ALDH2-3, the coordinated movement ability of mice that consumed high doses of alcohol was significantly improved. DETAILED DESCRIPTION

[0046] The embodiments of the present disclosure will be described in detail below with reference to the examples. However, it will be understood by those skilled in the art that the following examples are merely illustrative of the present disclosure and should not be construed as limiting the scope of the present disclosure. Where specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, all are commercially available conventional products.

[0047] Example 1 Sequence construction and preparation of linear RNA

[0048] In order to use RNA drugs to replenish ALDH2 protein and realize its physiological function in vivo, we designed a linear ALDH2 RNA sequence: its protein sequence is human ALDH2 protein, and its nucleotide sequence includes 5' non-coding region, protein coding region, 3' non-coding region and Poly A tail ( Figure 1A). The linear ALDH2 plasmid was cut with the restriction endonuclease BspQ1 and treated at 50°C for 2 hours to linearize the plasmid. The enzyme-digested product was purified using a DNA purification kit and eluted with enzyme-free water to obtain the linearized linear ALDH2 plasmid. After measuring the DNA concentration with Nanophotometer NP80, in vitro transcription was performed using the linearized plasmid as a template and T7 RNA polymerase. After the reaction was completed, DNase I was added and allowed to stand at 37°C for 15 minutes to digest the plasmid template. The transcription product was purified using an RNA column purification kit and eluted with enzyme-free water to obtain linear ALDH2 RNA. The size of the in vitro transcription product was detected using agarose gel. It can be seen from the agarose gel that the three sequences we designed, linear ALDH2-1 (SEQ ID NO.14), linear ALDH2-2 (SEQID NO.15), and linear ALDH2-3 (SEQ ID NO.16), can successfully achieve in vitro transcription of RNA and obtain linear RNA molecular products ( Figure 1B ).

[0049] Example 2: Sequence construction and preparation of circular RNA

[0050] The unique closed structure of circular RNA can help RNA molecules resist degradation by nucleases and prolong the half-life of RNA drugs. Therefore, we designed a circular ALDH2 RNA sequence: its protein sequence is human ALDH2 protein, and its nucleotide sequence contains the internal ribosome entry site and protein coding region ( Figure 2A), wherein the nucleotide sequence encoding ALDH2 was obtained using an independent algorithm optimization method. The circular ALDH2 plasmid was cleaved with the restriction endonuclease BspQ1 and linearized by treatment at 50°C for 2 hours. The digestion product was purified using a DNA purification kit and eluted with enzyme-free water to obtain the linearized circular ALDH2 plasmid. The DNA concentration was measured using a Nanophotometer NP80. In vitro transcription was then performed using T7 RNA polymerase using the linearized plasmid as a template. After the reaction, DNase I was added and the DNA template was incubated at 37°C for 15 minutes to digest the DNA template. The circular RNA precursor was purified using an RNA column purification kit and eluted with enzyme-free water to obtain the circular RNA precursor. The circular RNA precursor was ligated using T4 RNA ligase II at 25°C for 2 hours and purified using an RNA column purification kit to obtain the circular RNA ligation product. The ligation product was digested with RNase R and purified using an RNA column purification kit. The circular RNA was then identified by urea-denaturing polyacrylamide gel electrophoresis. The electrophoresis process is as follows: prepare the gel solution and pour the gel between two fixed glass plates, let it stand and wait for the gel to solidify; add an equal volume of 2X RNA loading buffer to the digestion product, heat it at 70°C for 10 minutes, and then immediately place it on ice; load the digestion product into the gel comb hole, adjust the voltage for electrophoresis, and determine whether to terminate the electrophoresis based on the position of the indicator. After the electrophoresis, the gel was stained with YeaRed nucleic acid dye and imaged using the ChemiDoc MP imaging system. From the electrophoresis results, it can be seen that the cyclic ALDH2-1 (SEQ ID NO.17), cyclic ALDH2-2 (SEQ ID NO.18), and cyclic ALDH2-3 (SEQ ID NO.19) designed by us can all be successfully transcribed and connected in vitro, and a high proportion of circular RNA ( Figure 2B ).

[0051] Example 3: Transfection and expression of linear RNA and circular RNA

[0052] In order to detect the expression of linear and circular RNA at the cellular level in vitro, we performed in vitro transfection on the RNA obtained in Examples 1 and 2. Before transfection, 3e5 293T cells were seeded into a 24-well cell culture plate and 500 μL complete medium / 10% fetal bovine serum / 1% penicillin-streptomycin double antibody was added. When the cell confluence reached 70%-80% after 12 hours, 500 ng of linear RNA or circular RNA was transfected into the cells using Lipofectamine RNAiMAX. After 16 hours of transfection, the culture supernatant was removed, the cells were resuspended and lysed with cell lysis buffer, and protein blotting was performed using ALDH2 antibody. From the in vitro expression results, it can be seen that the protein expression levels of the three linear ALDH2 sequences (SEQ ID NO.14-16) and the three circular ALDH2 sequences (SEQ ID NO.17-19) were significantly higher than those of the control group. Both linear ALDH2 and circular ALDH2 can express ALDH2 protein ( Figure 3A , Figure 3B ); To further compare the in vitro expression levels of linear RNA and circular RNA, linear ALDH2-2 and circular ALDH2-3 were transfected simultaneously, and it was found that the expression level of circular RNA was higher than that of linear RNA ( Figure 3C ).

[0053] Example 4: In vivo expression detection of linear and circular RNA

[0054] To detect drug expression in wild-type animals, we added Flag tags to linear ALDH2 and circular ALDH2, and used the SM-102 formulation of LNP encapsulation as an in vivo delivery method for linear and circular RNA molecules. Wild-type mice were injected with 1 mg / kg of LNP-encapsulated circular, linear RNA, or placebo via the tail vein. Five hours after injection, 30 mg of mouse liver tissue samples were collected and subjected to fluorescence quantitative reverse transcription PCR and Western blot detection. The results showed that RNA molecules could be detected in the mouse liver 5 hours after linear and circular RNA injection ( Figure 4A , 4C), and the administration did not affect the expression of endogenous ALDH2 ( Figure 4B , 4D). At the same time, we used Western blotting to detect the protein expression level of the drug in the liver, using Flag-specific antibodies for detection. The results are as follows Figure 4E As shown, significant protein expression was detected in three mice in each of the linear ALDH2-Flag group and the cyclic ALDH2-Flag group.

[0055] Transfection and expression assays in 293T cells demonstrated significant protein expression of cyclic ALDH2-3 (SEQ ID NO. 19). To test drug expression in transgenic animals, we introduced Aldh2- / - knockout mice, homozygous for complete ALDH2 deficiency, to examine whether cyclic ALDH2-3 RNA could replenish the target protein in vivo. We employed LNP encapsulation with the SM-102 formulation as an in vivo delivery strategy for cyclic ALDH2-3 RNA. Aldh2- / - knockout mice were injected via the tail vein with a 1 mg / kg dose of LNP-encapsulated cyclic ALDH2-3 (SEQ ID NO. 19) or an equal volume of placebo. Five hours after injection, 30 mg of mouse liver tissue samples were obtained and frozen in liquid nitrogen for subsequent analysis. Furthermore, we confirmed the detectability of cyclic RNA in mice following cyclic ALDH2-3 injection. RNA was extracted from 30 mg of mouse liver tissue sample using RNeasy Kit, and the obtained RNA was reverse transcribed using HiScriptIII RTSuperMix kit. Real-time fluorescence quantitative reverse transcription PCR was performed using 480SYBR GreenIMaster to detect the level of circular RNA in the liver. The results showed that 5 hours after the injection of cyclic ALDH2-3, the presence of cyclic ALDH2-3 RNA molecules could be detected in the liver of mice ( Figure 4F ). After verifying that cyclic ALDH2-3 was successfully delivered to mice, in order to further detect whether the protein expression in mice increased after administration. Add 300μl of lysis solution and an appropriate amount of grinding beads to 30mg of mouse liver tissue samples, obtain tissue homogenate by shaking and grinding, take 250μl of homogenate 12000g, centrifuge at 4℃ for 10 minutes, and retain the supernatant as protein lysate. The total protein concentration in the lysate was determined by BCA protein quantification kit, 30μg of protein was loaded, and protein blotting was performed using ALDH2-specific antibodies. It can be seen from the test results that significant target protein expression was detected in the liver of mice 5 hours after cyclic ALDH2-3 injection ( Figure 4G ), demonstrating that cyclic ALDH2-3 has sufficient in vivo expression capacity and can achieve protein replenishment in Aldh2- / - gene knockout homozygous mice.

[0056] Example 5: Linear and cyclic ALDH2 alleviates acetaldehyde accumulation in serum of Aldh2+ / - knockout heterozygous mice after alcohol intake

[0057] The above results demonstrate that ALDH2 RNA injection can produce the target protein in the mouse liver. We then tested whether the ALDH2 protein expressed in vivo could affect acetaldehyde metabolism. Studies have shown that Aldh2+ / - knockout heterozygous mice have impaired acetaldehyde metabolism due to a partial loss of endogenously expressed ALDH2. Therefore, they were used to test the effects of linear ALDH2-2 (SEQ ID NO. 15) and cyclic ALDH2-3 (SEQ ID NO. 19) on acetaldehyde metabolism. Aldh2+ / - knockout heterozygous mice were administered a 1 mg / kg dose of LNP-encapsulated linear ALDH2-2, LNP-encapsulated cyclic ALDH2-3, or an equal volume of placebo via tail vein injection. Four hours after injection, the mice were gavage-administered 4 g / kg of ethanol. One hour after gavage, the mouse serum was collected for acetaldehyde analysis.

[0058] Acetaldehyde levels in mouse serum were detected using the Acetaldehyde Assay Kit. 10 μl of serum was added to a clear-bottom 96-well plate containing 80 μl of detection reagent or blank reagent, and the volume was made up to 100 μl with enzyme-free water. A gradient-diluted acetaldehyde standard solution was used as a control. The samples were incubated at room temperature for 30 minutes in the dark. The OD 565 nm was read using a Spark Multimode Microplate Reader, and the acetaldehyde content in the serum samples was calculated using the standard curve. The results showed that mice given a placebo showed a certain amount of acetaldehyde accumulation in their serum 1 hour after alcohol ingestion. The serum acetaldehyde accumulation levels of mice in the linear ALDH2-2 and cyclic ALDH2-3 administration groups showed a certain degree of reduction ( Figure 5 ), demonstrating that both linear ALDH2-2 and cyclic ALDH2-3 molecules can promote the metabolism of acetaldehyde in Aldh2+ / - gene knockout heterozygous mice and alleviate the accumulation of acetaldehyde in mice, and that cyclic ALDH2-3 has a better inhibitory effect on acetaldehyde accumulation than linear ALDH2-2.

[0059] Example 6: Cyclic ALDH2-3 inhibits acetaldehyde accumulation in serum of Aldh2- / - knockout homozygous mice after alcohol intake

[0060] Cyclic ALDH2-3 (SEQ ID NO. 19) exhibited an alleviating effect on acetaldehyde accumulation in heterozygous mice, and thus its effect on acetaldehyde metabolism in Aldh2- / - knockout homozygous mice that consumed alcohol was further tested. Aldh2- / - knockout homozygous mice were administered a 1 mg / kg dose of LNP-encapsulated cyclic ALDH2-3 or an equal volume of placebo via tail vein injection. Four hours after the injection, the mice were gavaged with 4 g / kg of alcohol, and the acetaldehyde content in the serum of the mice was measured one hour after gavage, consistent with the heterozygous mice. The detection method was consistent with that described in Example 5. The homozygous knockout Aldh2- / - mice that were given a placebo showed significant acetaldehyde accumulation in their serum one hour after consuming alcohol, while the acetaldehyde accumulation level in the serum of the mice in the cyclic ALDH2-3 administration group was reduced ( Figure 6A ), demonstrating that cyclic ALDH2-3 can also inhibit the accumulation of acetaldehyde in Aldh2- / - gene knockout homozygous mice.

[0061] After consuming high doses of alcohol, mice enter a state similar to intoxication and develop motor disorders. A behavioral balance beam test can be used to assess the mice's coordinated motor abilities and determine their degree of intoxication. Cyclic ALDH2-3 (SEQ ID NO. 19) has been shown to reduce acetaldehyde levels in the serum of Aldh2- / - knockout mice. To verify whether cyclic ALDH2-3 can improve the coordinated motor abilities of mice after alcohol ingestion, we performed a behavioral balance beam test on mice. Aldh2- / - knockout mice were administered a 1 mg / kg dose of LNP-encapsulated cyclic ALDH2-3 or an equal volume of placebo via tail vein injection. Four hours after the injection, the mice were gavage-administered with 2 g / kg of alcohol, and their behavioral performance in the balance beam test was assessed one hour after alcohol ingestion.

[0062] Behavioral analysis of mice revealed that the total time taken by mice in the cyclic ALDH2-3 administration group to cross the balance beam ( Figure 6B ) and walking time on the balance beam ( Figure 6C ) were lower than those in the placebo group, and the number of times the mice in the drug-treated group slipped on the balance beam ( Figure 6D ) was significantly lower than that in the placebo group. All three indicators showed that the behavioral performance of mice in the cyclic ALDH2-3 administration group was better than that in the placebo group. The coordinated movement ability of Aldh2- / - gene knockout homozygous mice administered with cyclic ALDH2-3 was significantly improved after ingesting a high dose of alcohol, proving that cyclic ALDH2-3 administration can not only reduce serum acetaldehyde levels by replenishing ALDH2 protein, but also alleviate the drunken behavior of mice to a certain extent.

Claims

1. A nucleotide encoding human ALDH2, characterized in that Comprising any one of the nucleotide sequences of SEQ ID NOs: 7-13 or a combination thereof.

2. The nucleotide encoding human ALDH2 according to claim 1, characterized in that The nucleotide is a circRNA, which includes an internal ribosome entry site (IRES) and a protein coding region, and the protein coding region is selected from any one of nucleotide sequences in SEQ ID NO: 7-13 or a combination thereof.

3. The nucleotide encoding human ALDH2 according to claim 2, characterized in that The internal ribosome entry site (IRES) is selected from: coxsackievirus B3 (CVB3) IRES, enterovirus 71 (EV71) IRES, encephalomyocarditis virus (EMCV) IRES, picornavirus (PV) IRES, hepatitis C virus (HCV) IRES, adenovirus (AdV) IRES, human papillomavirus type 31 (HPV31) IRES, human herpesvirus (HHV) IRES, Rous sarcoma virus (RSV) IRES, classical swine fever virus (CSFV) IRES, FGF9 IRES, SLC7A1IRES and RUNX1 IRES; preferably, the internal ribosome entry site (IRES) is CVB3IRES; more preferably, the IRES is the nucleotide sequence of SEQ ID NO:

2.

4. The nucleotide encoding human ALDH2 according to claim 2, characterized in that The circRNA is selected from the circularized nucleotide sequences described in SEQ ID NO: 17-19 or a combination thereof.

5. The nucleotide encoding human ALDH2 according to claim 1, characterized in that The nucleic acid is a linear RNA, which includes 5'UTR, any one of the nucleotide sequences in SEQ ID NOs: 7-13, 3'UTR and poly(A).

6. The nucleotide encoding human ALDH2 according to claim 5, characterized in that The 5'UTR is selected from the nucleotide sequence of SEQ ID NO: 3, and the 3'UTR is selected from the nucleotide sequence of SEQ ID NO:

4.

7. The nucleotide encoding human ALDH2 according to claim 5, characterized in that The nucleotide is a nucleotide sequence selected from any one of SEQ ID NOs: 14-16 or a combination thereof.

8. The nucleotide encoding human ALDH2 according to any one of claims 5 to 7, wherein the RNA further comprises a 5'CAP, wherein the 5'CAP is preferably one or more selected from 7-methyl-guanosine-5'-triphosphate-5'-adenosine, guanosine-5'-triphosphate-5'-adenosine, 7-methyl-guanosine-5'-triphosphate-5'-guanosine, guanosine-5'-triphosphate-5'-guanosine, and m7G(5')(2'-OMeA)pG. 9 . The nucleotide encoding human ALDH2 according to claim 5 , wherein the number of base A in the poly(A) sequence is 60-120.

10. The nucleotide encoding human ALDH2 according to any one of claims 1 to 9, characterized in that Further comprising a Flag tag nucleotide sequence; preferably, the Flag tag nucleotide sequence is selected from the nucleotide sequence of SEQ ID NO: 6; more preferably, the nucleotide is selected from the linear nucleotide sequence of SEQ ID NO: 20 or the circularized nucleotide sequence of SEQ ID NO:

21.

11. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the nucleotide encoding human ALDH2 according to any one of claims 1 to 10 and a pharmaceutically acceptable excipient.

12. The pharmaceutical composition according to claim 11, characterized in that The composition is lipid nanoparticles (LNPs); preferably, the lipid nanoparticles (LNPs) contain SM-102 amino cationic lipids.

13. Use of the nucleotide encoding human ALDH2 according to any one of claims 1 to 10 or the pharmaceutical composition according to any one of claims 11 to 12 in the preparation of a medicament for a disease treated by supplementing ALDH2 protein.

14. The use according to claim 13, wherein the disease treated by supplementing ALDH2 protein is selected from the group consisting of: metabolic diseases, cardiovascular diseases, nervous system diseases, and cancer.

15. The use of claim 13, wherein the disease treated by supplementing ALDH2 protein is selected from the group consisting of alcoholic liver disease, non-alcoholic fatty liver disease, hepatitis, myocardial ischemia-reperfusion injury, acute ischemic stroke, arrhythmia, Parkinson's disease, Alzheimer's disease, liver cancer, colorectal cancer, gastric cancer, esophageal cancer, lung cancer, bladder cancer, pulmonary hypertension, Fanconi anemia, or osteoporosis.

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