CN114891808B

CN114891808B - mRNA molecule encoding ALDH2 polypeptide, application and mRNA medicament

Info

Publication number: CN114891808B
Application number: CN202210705211.XA
Authority: CN
Inventors: 彭育才; 张振东; 方子辉; 刘隽; 刘琪; 罗丽平; 雷奕欣; 李爽
Original assignee: Zhuhai Lifanda Biotechnology Co ltd
Current assignee: Zhuhai Lifanda Biotechnology Co ltd
Priority date: 2022-06-21
Filing date: 2022-06-21
Publication date: 2023-10-27
Anticipated expiration: 2042-06-21
Also published as: WO2023246354A1; CN114891808A

Abstract

The invention relates to the technical field of pharmacy, in particular to an mRNA molecule for encoding ALDH2 polypeptide, application and an mRNA medicament. The survival experiment of mice shows that the mRNA medicine prepared by adopting the mRNA molecule for encoding the ALDH2 polypeptide provided by the invention can improve the survival rate of white spirit gastric lavage mice, and the survival rate is improved along with the improvement of the medicine dosage. Meanwhile, various detection results of Western Blot detection, ALDH2 polypeptide activity detection, ethanol metabolite content detection and liver pathological section detection show that the mRNA drug can successfully express the ALDH2 polypeptide in a mouse body, remarkably improve the activity of the ALDH2 polypeptide and play a role in promoting acetaldehyde metabolism in the liver, thereby improving the survival rate of white spirit gastric lavage mice.

Description

mRNA molecule encoding ALDH2 polypeptide, application and mRNA medicament

Technical Field

The invention relates to the technical field of pharmacy, in particular to an mRNA molecule for encoding ALDH2 polypeptide, application and an mRNA medicament.

Background

Human ALDH2 (acetaldehyde dehydrogenase 2,aldehyde dehydrogenase-2) is a 517 amino acid polypeptide encoded by a nuclear gene located on chromosome 12q 24. Like most members of the ALDH family, ALDH2 is a tetrameric enzyme that is ubiquitously expressed in all tissues, but is most abundant in the liver and also in organs that require high mitochondrial oxidative phosphorylation (e.g., heart and brain). ALDH2 is well known for its key role in ethanol metabolism, and ALDH2 may be the only ALDH enzyme in humans that contributes significantly to acetaldehyde metabolism. The ethanol detoxification pathway of the human body mainly occurs in the liver, by two enzymatic steps. The first step is catalyzed by Alcohol Dehydrogenase (ADH) and the second step is catalyzed primarily by ALDH 2.

ALDH2 x 2 alleles are the most widespread human ALDH2 variants, which are found in up to one third of the east asian and 8% of the world population. The individual heterozygotes (ALDH 2 x 1/. Times.2) had ALDH2 enzyme activity less than 50% of the wild-type, whereas the homozygotes ALDH 2/. Times.2 had ALDH2 enzyme activity less than 1-4% of the wild-type. ALDH2 enzyme activity was lower in ALDH2 carriers and exhibited facial flushing, headache, nausea, dizziness and palpitations, which are characteristic of drinking.

Studies have shown that acetaldehyde has a clear role in poisoning associated with the ingestion of ethanol. Ingested ethanol is rapidly metabolized to acetaldehyde (by alcohol dehydrogenase) and then to acetate by the mitochondrial enzyme ALDH 2. In subjects lacking ALDH2, the concentration of acetaldehyde in the blood increases significantly even after an appropriate amount of ethanol. Acute exposure to acetaldehyde can lead to a number of unpleasant effects associated with alcoholism, including nausea, palpitations, vomiting, dizziness, and headache. The risk of overdrinking ALDH2 x 1/x2 subjects with esophageal cancer and other cancers of the upper respiratory digestive tract is significant, which may be the result of DNA damaging effects of acetaldehyde. However, after the existing method is aimed at alcohol (alcohol) intake, no effective way for promoting the decomposition of alcohol or acetaldehyde in the body exists, such as naloxone, diuretics and the like, wherein the former is an excessive antidote of opioid drugs, can also relieve central inhibition of alcohol, shortens the coma time and only enables people to become awake; the latter is by strengthening alcohol and its metabolite acetaldehyde and ketone body excreted through urine, can't accelerate the "detoxication" process of acetaldehyde, can't protect liver from being damaged by alcohol and its metabolite.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a nucleic acid molecule for encoding ALDH2 polypeptide, which comprises an mRNA molecule, and an mRNA medicament prepared from the mRNA molecule, so as to accelerate the acetaldehyde metabolic process and make up for the blank of the current medicament for treating the alcohol metabolism-induced diseases.

In order to solve the technical problems and achieve the purposes, the invention provides the following technical scheme:

in a first aspect, the invention provides a nucleic acid molecule encoding a ALDH2 polypeptide comprising any one of (a) to (c):

(a) An mRNA molecule having a nucleotide sequence encoding a ALDH2 polypeptide as set forth in any one of SEQ ID Nos. 15 to 18;

(b) An mRNA molecule derived from (a) having the function of encoding ALDH2 polypeptide and the same GC base pair percentage content as the original nucleotide sequence, by substitution, deletion or addition of one or several nucleotides in the nucleotide sequence defined by (a);

(c) A nucleic acid molecule that hybridizes under stringent conditions to an mRNA molecule defined in (a) or (b), and which is a ALDH2 polypeptide.

In a second aspect, the invention provides a construct comprising a nucleic acid molecule according to the preceding embodiment, the nucleotide sequence of the construct further comprising a UTR sequence, a 5' cap and a poly (a) sequence.

Preferably, the 5' cap comprises 7-methyl-guanosine-5 ' -triphosphate-5 ' -adenosine, 7-methyl-guanosine-5 ' -triphosphate-5 ' -guanosine, guanosine-5 ' -triphosphate-5 ' -guanosine and m ⁷ One or more of G (5 ') (2' -OMeA) pG; preferably m ⁷ G(5’)(2’-OMeA) pG。

Preferably, the number of bases A in the poly (A) sequence is 60 to 120, more preferably 100.

Preferably, the UTR sequences include a 5'UTR sequence and a 3' UTR sequence.

Further preferably, the 5' UTR sequence has the nucleotide sequence shown as SEQ ID No.1 or SEQ ID No. 13.

Further preferably, the 3' UTR sequence has the nucleotide sequence shown as SEQ ID No.3 or SEQ ID No. 14.

In a third aspect, the invention provides the use of a nucleic acid molecule according to the preceding embodiment or a construct according to the preceding embodiment for the preparation of an mRNA drug.

In an alternative embodiment, the use of the mRNA drug comprises (i) or (ii):

supplementing ALDH2 enzyme;

(ii) preventing and/or treating alcohol metabolic diseases.

Preferably, the alcohol metabolic disease comprises alcohol-induced liver injury or alcoholic hepatitis.

In a fourth aspect, the invention provides an mRNA drug comprising a nucleic acid molecule according to the previous embodiment or a construct according to the previous embodiment.

In an alternative embodiment, the mRNA drug further comprises a lipid nanoparticle comprising Dlin-MC3-DMA, DSCP, cholesterol, and PEG-DMG encapsulating the nucleic acid molecule of the previous embodiment or the construct of the previous embodiment.

In an alternative embodiment, the lipid nanoparticle comprises, in mole parts, 20 to 50 parts Dlin-MC3-DMA, 5 to 20 parts DSCP, 20 to 50 parts cholesterol, and 1 to 5 parts PEG-DMG.

In an alternative embodiment, the lipid nanoparticle comprises 50 parts Dlin-MC3-DMA, 10 parts DSCP, 38.5 parts cholesterol, and 1.5 parts PEG-DMG in mole parts.

In an alternative embodiment, the method of preparation comprises:

(A) Dissolving RNA encoding ALDH2 polypeptide in buffer solution, and regulating the concentration to be 0.05 mg/mL-0.5 mg/mL to obtain water phase;

(B) Dissolving Dlin-MC3-DMA, DOPG, cholesterol and PEG-DMG in absolute ethyl alcohol, and adjusting the concentration of lipid components in an organic phase to 5 mg/mL-7 mg/mL to obtain an organic phase;

(C) The aqueous phase of step (A) and the organic phase of step (B) are combined according to 1:3, removing ethanol, and concentrating until the concentration of mRNA in the system is 50-1000 mug/mL, thereby obtaining the lipid nanoparticle containing RNA encoding ALDH2 polypeptide.

In an alternative embodiment, the method of preparation comprises:

(A) Dissolving RNA encoding ALDH2 polypeptide in citrate buffer with pH of 4, and adjusting concentration to 0.1mg/ml to obtain water phase;

(B) Dissolving Dlin-MC3-DMA, DOPG, cholesterol and PEG-DMG in absolute ethyl alcohol, and adjusting the concentration of lipid component in the organic phase to 6mg/mL to obtain an organic phase;

(C) The aqueous phase of step (a) and the organic phase of step (b) are combined according to 1:3, mixing at a flow rate of 12mL/min using a microfluidic device, immediately diluting the mixture 100-fold with PBS solution ph7.4, removing ethanol from the solution using tangential flow filtration, and concentrating to a mRNA concentration of 550 μg/mL in the system to obtain lipid nanoparticles comprising RNA encoding ALDH2 polypeptide.

The survival experiment of mice shows that the mRNA medicine prepared by adopting the mRNA molecule for encoding the ALDH2 polypeptide provided by the invention can improve the survival rate of white spirit gastric lavage mice, and the survival rate is improved along with the improvement of the medicine dosage. Meanwhile, various detection results of Western Blot detection, ALDH2 polypeptide activity detection, ethanol metabolite content detection and liver pathological section detection show that the mRNA drug can successfully express the ALDH2 polypeptide in a mouse body, remarkably improve the activity of the ALDH2 polypeptide and play a role in promoting acetaldehyde metabolism in the liver, thereby improving the survival rate of white spirit gastric lavage mice.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the Western Blot detection result in experimental example 1 of the present invention;

FIG. 2 shows the results of the test performed on the construction of ALDH2-KO mice in Experimental example 2 of the present invention;

FIG. 3 shows the results of expression of hADH-2 and mALDH2 proteins in experimental example 3 of the present invention;

FIG. 4 is a graph showing the GC base pair percentage content of SEQ ID No.4 in experimental example 4 of the present invention;

FIG. 5 is a GC base pair percentage of SEQ ID No.15 of Experimental example 4 of the present invention;

FIG. 6 shows the results of the expression of ALDH2 protein by each mRNA sequence in experimental example 3 of the present invention;

FIG. 7 is a GC base pair percentage of SEQ ID No.16 of the present invention;

FIG. 8 is a GC base pair percentage of SEQ ID No.17 of the present invention;

FIG. 9 is a GC base pair percentage of SEQ ID No.18 of the present invention;

FIG. 10 is a GC base pair percentage of SEQ ID No.20 of the present invention;

FIG. 11 is a GC base pair percentage of SEQ ID No.21 of the present invention;

FIG. 12 shows the results of detecting the expression level of ALDH2 polypeptide in experimental example 6 of the present invention;

FIG. 13 shows the results of detecting ALDH2 polypeptide activity in experimental example 6 of the present invention;

FIG. 14 shows the results of the ethanol metabolism test in Experimental example 6 of the present invention;

FIG. 15 is a graph showing the comparison between the low dose group and the control group in the liver pathological section of the mouse in experimental example 6 of the present invention;

FIG. 16 is a graph showing the comparison of the low dose group and the high dose group in the liver pathological section of the mouse in experimental example 6 of the present invention;

FIG. 17 shows the results of high dose death group microscopy in liver pathological sections of mice in Experimental example 6 of the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In a particular embodiment, the invention provides, in a first aspect, a nucleic acid molecule encoding a ALDH2 polypeptide comprising any one of (a) to (c):

The partial GC base pair percentage content in the nucleotide sequence of the coding ALDH2 polypeptide shown in SEQ ID No. 13-15 is not less than 40%, and the total GC base pair percentage content is 55% -65%. Wherein "local GC base pair percentage content" refers to the GC base pair percentage content in a local sequence having 60bp as window size from the 3 'end to the 5' end of the ORF sequence.

The "GC base pair percentage content" in the above nucleic acid molecule (b) includes a local GC base pair percentage content and an overall GC base pair percentage content.

It is understood that the term "hybridization of the nucleic acid molecule (c) with the mRNA of (a) or (b) under stringent conditions" means a nucleic acid molecule (c), such as a single-stranded DNA or other RNA molecule, obtained by hybridization using the mRNA molecule (a) or (b) as a template, based on the principle of base complementary pairing. For specific "stringent conditions" of the reaction conditions, the person skilled in the art will be able to make routine selections based on the desired product, with reference to the prior art and common general knowledge.

In a second aspect, the invention provides a construct comprising an mRNA molecule according to the previous embodiments, the nucleotide sequence of the construct further comprising a UTR sequence, a 5' cap and a poly (a) sequence.

Preferably, the UTR sequences include a 5'UTR sequence and a 3' UTR sequence.

In a third aspect, the invention provides the use of an mRNA molecule according to the previous embodiment or a construct according to the previous embodiment for the preparation of an mRNA drug.

In an alternative embodiment, the use of the mRNA drug comprises (i) or (ii):

supplementing ALDH2 enzyme;

(ii) preventing and/or treating alcohol metabolic diseases.

In a fourth aspect, the invention provides an mRNA drug comprising an mRNA molecule according to the previous embodiment or a construct according to the previous embodiment.

In an alternative embodiment, the mRNA drug further comprises a lipid nanoparticle encapsulating the mRNA molecule of claim 1 or the construct of claim 2, the lipid nanoparticle composition comprising Dlin-MC3-DMA, DSCP, cholesterol, and PEG-DMG.

In an alternative embodiment, the method of preparation comprises:

Some embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following embodiments and features of the embodiments may be combined with each other without conflict.

Example 1

The present example provides four mRNAs encoding ALDH2 polypeptides, the specific nucleotide sequences of which are shown in SEQ ID Nos. 15 to 18, respectively.

Example 2

This example provides 8 sets of mRNAs encoding ALDH2 polypeptides comprising, in addition to an ORF encoding the ALDH2 polypeptide, a 5 'cap, UTR and 3' tail, and in which the uracil is replaced by 1-methyl pseudouridine, with the following specific sequence numbers and compositions:

example 3

This example provides 8 sets of mRNA drugs, comprising mRNA encoding ALDH2 polypeptides and lipid nanoparticles encapsulating the mRNA, wherein the lipid nanoparticles comprise Dlin-MC3-DMA 50%, DSCP 10%, cholesterol 38.5% and PEG-DMG 1.5% in mole percent.

The preparation method of the mRNA medicament comprises the following steps:

(a) 8 groups of mRNA (sequences shown as SEQ ID Nos. 5 to 12) encoding ALDH2 polypeptide synthesized artificially are dissolved in a citrate buffer solution with pH of 4, and the concentration is adjusted to 0.1mg/ml, so as to obtain an aqueous phase.

(b) Dlin-MC3-DMA, DOPG, cholesterol and PEG-DMG were dissolved in absolute ethanol in the amounts of the formulation, and the concentration of the lipid component in the organic phase was adjusted to 6mg/mL to give an organic phase.

(c) Mixing the aqueous phase of step (a) and the organic phase of step (b) at a volume ratio of 1:3 using a microfluidic device at a flow rate of 12mL/min, immediately diluting the mixture 100-fold with a PBS solution at ph7.4, removing the ethanol component from the solution using Tangential Flow Filtration (TFF), and concentrating to a mRNA concentration of 550 μg/mL in the system to obtain lipid nanoparticles comprising RNA encoding ALDH2 polypeptides.

Example 4

This example provides an additional 8 sets of mRNA drugs, differing from example 3 in that the lipid nanoparticle comprises, in mole parts, 20 parts Dlin-MC3-DMA, 5 parts DSCP, 20 parts cholesterol, and 1 part PEG-DMG.

Example 5

This example provides an additional 8 sets of mRNA drugs, differing from example 3 in that the lipid nanoparticle comprises 50 parts Dlin-MC3-DMA, 20 parts DSCP, 50 parts cholesterol and 5 parts PEG-DMG in mole parts.

Experimental example 1

The experimental example detects the expression condition of the ALDH2 polypeptide provided in the example 1 at the in vitro cell level, and the specific method is as follows:

(1) Transfection

Lip2000 reagent 10. Mu.L and 40. Mu.L opti-MEM were mixed uniformly and allowed to stand, to which was added 46. Mu.L opti-MEM, then 2. Mu.L DNA plasmid and 2. Mu.L mRNA (. Mu.g/. Mu.L) were added, respectively, allowed to stand, added to HEK293 cells, and incubated in a cell incubator.

(2) Detection of intracellular ALDH2 protein expression Using Western Blot

The cell supernatant was removed, the cells were washed 2 times with PBS, and 250. Mu.L of the cell lysate (containing PMSF) was added to the tube, followed by centrifugation, and the supernatant was dispensed into 1.5mL tubes. The BCA kit was used to determine the protein concentration of the cells. Then, electrophoresis was performed using a pre-prepared gel, and a recombinant Anti-ALDH2 antibody and a Goat Anti-Rabbit IgG H & L (HRP) were added to chemiluminescent and develop a PVDF membrane. The results are shown in FIG. 1, where Control is the empty lipid group; 1 is a circular plasmid; 2 is the mRNA provided in example 1, it can be seen that the DNA plasmid and mRNA group ALDH2 protein expression levels were significantly higher than the control group, and that the ALDH2 protein DNA and mRNA were able to be stably expressed in HEK293 cells.

Experimental example 2

The experimental example constructs an ALDH2-KO mouse, and the specific method is as follows:

f0 generation ALDH2-KO mice were obtained by CRISPR/Cas9 technology by the Hainan model biotechnology Co., ltd. The expression of ALDH2 protein in mouse liver was detected using Western Blot technique. As a result, as shown in FIG. 2, the expression of ALDH2 in the liver of the ALDH2-KO mouse was much lower than that of the wild-type mouse, and the KO mouse model was established.

Experimental example 3

The experimental example examines the expression conditions of human ALDH2 mRNA and murine ALDH2 mRNA in KO mice, and the specific method is as follows:

number of ALDH2-KO mice ("ALDH 2-KO mice" refers to "mice homozygous for ALDH2 x 2/x2"): 9; the control group is PBS group, the administration group is human ALDH2 mRNA (hADH 2) group, and the murine ALDH2 mRNA (mALDH 2) group; a total of 3 groups of 3 mice each.

Experimental protocol: (1) control group: mu.L of PBS solution ALDH2-KO mice were injected by tail vein injection; drug administration group: KO mice were administered with human-derived ALDH2 mRNA (hADH 2) (the ORF sequence of the mRNA is SEQ ID No. 15) or murine-derived ALDH2 mRNA (mALDH 2) (the mRNA sequence is SEQ ID No.19 and the 5' -cap is m7G (5 ') (2 ' -OMeA) pG) at a dose of 2.5mg/kg by tail intravenous injection according to the body weight of the mice, wherein the mRNA was prepared as LNP according to the method of example 1.

(2) After 4 hours of administration, 9 ALDH2-KO mice were given 52 degree distilled spirit by gavage at a dose of 13. Mu.g/g, based on the body weight of the mice.

Liver sampling time points of mice: and 2h and 6h after the white spirit is filled into the stomach. And (5) after sample collection, placing the sample into liquid nitrogen for quick freezing, and then placing the sample into a-80 refrigerator for preservation.

The expression of ALDH-2 protein in mouse liver was detected by Western Blot: the liver tissue block is placed in a centrifuge tube for shearing, 250 mu L of tissue lysate (containing PMSF) is added into the centrifuge tube, homogenization and centrifugation are carried out, and the supernatant is taken and split-packed into 1.5mL centrifuge tubes. Protein concentration of the samples was determined using BCA kit. Then, electrophoresis was performed using a pre-prepared gel, and a recombinant Anti-ALDH2 antibody and a Goat Anti-Mouse IgG H & L (HRP) were added to chemiluminescent and develop a PVDF membrane.

The experimental results are shown in fig. 3: (1) All mice in the control group die after alcohol lavage, while 1 mice in the mALDH-2 group survived and 2 mice in the hADH-2 group survived; (2) Both hADH-2 and mALDH2 proteins in the hADH-2 and mALDH-2 groups were expressed in mouse livers.

Experimental example 4

The GC base pair percentages of SEQ ID No.4 and SEQ ID No.15 described above were examined, as shown in FIGS. 4 and 5. The total GC% content of SEQ ID No.4 is 54.03%, wherein the base A% content is 24.24%, the base C% content is 25.21%, the base G% content is 28.82%, and the base U% content is 21.73%. From FIG. 4 it can be seen that the percentage of GC base pairs localized to SEQ ID No.4 is between 40% and 65%.

The total GC% content of SEQ ID No.15 was 58.75, with a base A% content of 21.24%, a base C% content of 27.8%, a base G% content of 30.95% and a base U% content of 20.01%. From FIG. 5 it can be seen that the partial GC base pair percentage content of SEQ ID No. is 41% -83%.

The total GC% content of SEQ ID No.16 was 59.72%, with a base A% content of 21.75%, a base C% content of 28.19%, a base G% content of 31.53% and a base U% content of 18.53%. As can be seen from FIG. 7, the partial GC base pair percentage content of SEQ ID No. is 41% -83%

The total GC% content of SEQ ID No.17 was 59.38%, with a base A% content of 23.02%, a base C% content of 29.08%, a base G% content of 30.3%, and a base U% content of 17.6%. From FIG. 8 it can be seen that the partial GC base pair percentage content of SEQ ID No. is 41% to 78%.

The total GC% content of SEQ ID No.18 was 59.42% with a base A% content of 23.02%, a base C% content of 29.21%, a base G% content of 30.11% and a base U% content of 17.67%. From FIG. 9 it can be seen that the partial GC base pair percentage content of SEQ ID No. is 42% -77%.

The total GC% content of SEQ ID No.20 was 52.49%, with a base A% content of 25.66%, a base C% content of 23.15%, a base G% content of 29.34%, and a base U% content of 21.86%. From FIG. 10 it can be seen that the partial GC base pair percentage content of SEQ ID No. is 40% -63%.

The total GC% content of SEQ ID No.21 was 66.73%, with a base A% content of 18.57%, a base C% content of 35.01%, a base G% content of 31.72% and a base U% content of 14.7%. From FIG. 11 it can be seen that the partial GC base pair percentage content of SEQ ID No. is 52% -87%.

Experimental example 5

The result of detecting ALDH2 protein expressed by each mRNA sequence in experimental example 3 by referring to the Western blot method is shown in FIG. 6, and the experimental conclusion is that: (1) The expression level of the ORF sequence of SEQ ID No.15 in the mRNA of the edited ALDH2 polypeptide is far higher than that of the ORF sequence of SEQ ID No. 4; (2) When the 5'UTR is SEQ ID No.1 and the 3' UTR is SEQ ID No.2, the expression level of the ALDH2 polypeptide is higher than that of other combinations of the 5'UTR and the 3' UTR.

Further screening and editing the ORF sequence of the ALDH2 polypeptide, and finding that when the total GC% content of the ORF sequence is 30-70%, the local GC% content is not lower than 40% (as shown in SEQ ID No. 15-18), and the expression level of the ALDH2 polypeptide is higher; furthermore, when the local GC% content of the ORF sequence is not less than 40%, the expression level of the ALDH2 polypeptide with the total GC% content of 55% -60% (SEQ ID No. 15-18) is slightly higher than that of the sequence with the total GC% content of 30% -55% and 60% -70% (SEQ ID No.4, 20, 21).

Experimental example 6

The experimental example examines the condition that the ALDH2 mRNA drug protects KO mice from alcohol damage, and the specific method is as follows:

the number of 15 ALDH2-KO mice was randomly divided into 3 groups (PBS group, high dose group and low dose group, respectively) of 5.

Experimental protocol: (1) mu.L of PBS solution was injected into the control group ALDH2-KO mice by tail vein injection; according to the body weight of mice, the mRNA medicine encoding mALDH2 polypeptide provided in example 3 is injected into the high dose group and the low dose group ALDH2-KO mice by tail vein injection according to the dosages of 2.5mg/kg and 1.25mg/kg respectively, wherein the nucleotide sequence of the mRNA is shown as SEQ ID No. 10.

(2) After 4 hours, 15 ALDH2-KO mice delivered 52 degree white spirit in a gavage manner at a dose of 13. Mu.g/g according to the body weight of the mice.

Time point of KO mouse serum (50 μl) sampling: before mRNA administration, before and after gastric lavage for 1, 6, 10, 24 and 48 hours, collecting, quick freezing in liquid nitrogen, and storing in a refrigerator at-80 ℃.

Experimental results:

1. survival situation

24 hours after the white spirit is filled into the stomach, 5 mice in the PBS group die completely; mice in the low dose group (1.25 mg/kg) died 2 and survived 3; the high dose group (2.5 mg/kg) died 1, and survived 4.

2. Liver expression of ALDH2 polypeptide

Western Blot was used to detect expression of ALDH-2 protein in mouse livers, respectively. The liver tissue block is placed in a centrifuge tube for shearing, 250 mu L of tissue lysate is added into the centrifuge tube for splitting (containing PMSF), homogenization and centrifugation are carried out, and the supernatant is taken and split-packed into a 1.5mL centrifuge tube. Protein concentration of the samples was determined using BCA kit. Then, electrophoresis was performed using a pre-prepared gel, and a recombinant Anti-ALDH2 antibody and a Goat Anti-Mouse IgG H & L (HRP) were added to chemiluminescent and develop a PVDF membrane. The results are shown in FIG. 12, where ALDH2 protein was expressed in the liver of mice in both the high and low dose groups.

3. Activity of ALDH2 polypeptide

The enzymatic activity of ALDH2 in mouse liver tissue was detected using ELISA. About 10mg of mouse liver tissue is taken, added into Buffer, cracked and evenly mixed on ice, and centrifuged by using a centrifuge. The supernatant was diluted 20 times in 96-well plates. After preparing a standard curve, the substrate is hydrolyzed, 20 mu L of hydrolyzed substrate is added into a 96-well plate for incubation, and finally 200 mu L of stop solution is used for stopping the reaction, and the reaction is put into a multifunctional enzyme-labeled instrument for detection. The results are shown in FIG. 13, which shows that the ALDH2 enzyme activity in the high and low dose groups was significantly increased as compared with the PBS group, and the enzyme activity was also significantly increased as the mRNA administration dose was increased.

4. Variation of ethanol, acetaldehyde and acetic acid content in serum

Detecting headspace sample injection conditions by adopting a gas chromatography method: a constant mode; the furnace temperature is 65 ℃; the balancing time is 10min; the quantitative temperature is 105 ℃; the temperature of the transmission line is 110 ℃; the needle temperature is 90 ℃; the sample pressurization time is 0.1min; the filling time of the quantitative ring is 0.1min; quantitative ring equilibration time was 0.05min. Taking 0.2. 0.2 mL to-be-detected mouse serum, placing the to-be-detected mouse serum in a 2mL sample bottle, pressing a bottle cap, and placing the to-be-detected mouse serum on a headspace sampler sample rack for measurement.

(1) The contents of ethanol, acetaldehyde and acetic acid in the blood of the mice before and after the white spirit is infused with the stomach are shown in fig. 12, and the results show that: 24 hours after the white spirit is infused, the following steps can be seen:

(a) The ethanol, acetaldehyde and acetic acid contents (24 h) in the blood of the mice in the PBS group, the high-dose group and the low-dose group are obviously increased compared with those before the white spirit gastric lavage administration (0 h).

(b) The ethanol content in the blood of mice in PBS group is obviously higher than that in the blood of mice in high-dose group and low-dose group (P < 0.05) 24 hours after the white spirit is subjected to gastric lavage administration, and the ALDH2-KO mice are supplemented with the hADH 2 polypeptide in the form of injecting mRNA encoding the hADH 2 polypeptide, so that the ethanol metabolism can be accelerated.

(c) The acetaldehyde content in the blood of the mice in the PBS group is significantly higher than that in the blood of the mice in the high-dose group and the low-dose group (P < 0.05), the acetic acid content in the blood of the mice in the PBS group is significantly lower than that in the blood of the mice in the high-dose group and the low-dose group (P < 0.05), the acetaldehyde content in the blood of the mice in the low-dose group is significantly higher than that in the blood of the mice in the high-dose group (P < 0.05), and the acetic acid content in the blood of the mice in the low-dose group is significantly lower than that in the blood of the mice in the high-dose group (P < 0.05). Demonstrating that supplementation of ALDH2-KO mice with an hdh 2 polypeptide in the form of injection of mRNA encoding the hdh 2 polypeptide can accelerate acetaldehyde metabolism to acetic acid; and exhibits dose dependence as shown in figure 14.

5. Pathological section of mouse liver

The method comprises the steps of dehydrating and slicing the liver tissue of a mouse, and firstly, dehydrating the liver tissue through a series of alcohols, embedding paraffin and slicing; paraffin sections were then hematoxylin-eosin stained and finally sections mounted. The pathological condition of the liver tissue of the mice was observed under a microscope.

As shown in FIGS. 15-17, liver of PBS group mice showed liver cell swelling, lipid droplets diffused, inflammatory cell infiltration, round or quasi-round lipid droplet vacuoles, individual cell swelling was balloon-like, and cell morphology was severely destroyed. The mice in the mRNA dosage group have reduced liver cell swelling, reduced lipid drop dispersion and slightly intact nuclear morphology. The morphology of the liver cells of the mRNA high-dose group is recovered to be normal, the outlines of the liver cells are clear, lipid drops are obviously reduced, and no obvious inflammatory cell infiltration exists.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

SEQUENCE LISTING

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<213> Artificial Sequence

<220>

<223> 3' UTR sequence one

<400> 3

ggacuaguua uaagacugac uagcccgaug ggccucccaa cgggcccucc uccccuccuu 60

gcaccgagau uaau 74

<210> 4

<211> 1551

<212> RNA

<213> Artificial Sequence

<220>

<223> ORF sequence one

<400> 4

augcuuagag ccgcugcgag auuuggaccu agacugggua ggagacugcu cagcgcugca 60

gcuacacagg ccgucccugc uccuaaccaa cagccagaag uguuuugcaa ucagaucuuu 120

auuaacaacg aguggcauga cgcgguuuca cgcaagaccu uucccaccgu gaaucccuca 180

acuggggagg ucauuuguca gguggccgaa ggggauaaag aggacguuga uaaggcggua 240

aaagcugcac gggccgccuu ccaguugggu aguccaugga ggagaaugga cgcgucucac 300

agggggagac uccugaauag guuggccgac cugauugaac gggaucgaac cuaucuggcu 360

gcccuggaaa cgcucgauaa cggcaaacca uauguuauau ccuaucuugu cgaccuugau 420

augguccuga agugccuccg cuacuaugcc ggcugggccg auaaguacca cgggaagaca 480

aucccuauug acggggacuu cuucucauau acucggcaug aaccugucgg agucuguggc 540

cagaucaucc cuuggaacuu cccucugcug augcaggccu ggaaacuggg accagccuug 600

gccaccggca acguugucgu gaugaaaguc gcugagcaga caccccugac ugcccucuac 660

guggcgaauc ucaucaaaga ggcgggguuu ccccccggug uugugaauau cgugccagga 720

uuuggcccca cagccggagc ugcuauugcc ucucacgaag auguggacaa gguggcuuuu 780

accggcucca ccgagauugg aagggugauu cagguggcgg cagguuccag uaaccucaaa 840

cgggucacac uggagcuggg aggaaaaucc cccaauauca ucauguccga cgccgauaug 900

gacugggcag ucgaacaggc gcacuucgcc uuguuuuuua accaggggca guguuguugu 960

gccggaucua gaacuuuugu gcaggaggau auauacgacg aauucgucga gaggucuguc 1020

gcgcgggcua aaagucgggu cgugggaaac cccuucgauu ccaagaccga gcagggccca 1080

cagguggaug agacacaguu caagaagauc uuggguuaca uuaacaccgg aaaacaggag 1140

ggugcuaagc uucugugcgg aggagggauc gcugcugaca gaggauacuu uauucagccg 1200

acaguguucg gcgacgugca ggaugguaug accaucgcua aggaggagau cuucggaccg 1260

guaaugcaga uucugaaauu caagacaauu gaagagguug uggggcgcgc caauaacucc 1320

accuauggcc ucgcagcagc cguuuucaca aaagaccugg acaaggcaaa cuaccuuagc 1380

caggcgcuuc aggcuggcac ugucugggug aacugcuacg acguguucgg agcccagucc 1440

cccuuuggag gcuacaagau gagcggcucu ggaagagaac ugggagaaua cggccuccag 1500

gcauacacag aaguuaaaac cgugaccguc aaggucccac agaaaaauuc u 1551

<210> 5

<211> 1743

<212> RNA

<213> Artificial Sequence

<220>

<223> example 3 provides mRNA coding sequence one

<400> 5

gggagaaagc uuaccauguu gcgcgcugcc gcccgcuucg ggccccgccu gggccgccgc 60

cucuugucag ccgccgccac ccaggccgug ccugccccca accagcagcc cgaggucuuc 120

ugcaaccaga uuuucauaaa caaugaaugg cacgaugccg ucagcaggaa aacauucccc 180

accgucaauc cguccacugg agaggucauc ugucagguag cugaagggga caaggaagau 240

guggacaagg cagugaaggc cgcccgggcc gccuuccagc ugggcucacc uuggcgccgc 300

auggacgcau cacacagggg ccggcugcug aaccgccugg ccgaucugau cgagcgggac 360

cggaccuacc uggcggccuu ggagacccug gacaauggca agcccuaugu caucuccuac 420

cugguggauu uggacauggu ccucaaaugu cuccgguauu augccggcug ggcugauaag 480

uaccacggga aaaccauccc cauugacgga gacuucuuca gcuacacacg ccaugaaccu 540

gugggggugu gcgggcagau cauuccgugg aauuucccgc uccugaugca agcauggaag 600

cugggcccag ccuuggcaac uggaaacgug guugugauga agguagcuga gcagacaccc 660

cucaccgccc ucuauguggc caaccugauc aaggaggcug gcuuuccccc uggugugguc 720

aacauugugc cuggauuugg ccccacggcu ggggccgcca uugccuccca ugaggaugug 780

gacaaagugg cauucacagg cuccacugag auuggccgcg uaauccaggu ugcugcuggg 840

agcagcaacc ucaagagagu gaccuuggag cuggggggga agagccccaa caucaucaug 900

ucagaugccg auauggauug ggccguggaa caggcccacu ucgcccuguu cuucaaccag 960

ggccagugcu gcugugccgg cucccggacc uucgugcagg aggacaucua ugaugaguuu 1020

guggagcgga gcguugcccg ggccaagucu cggguggucg ggaaccccuu ugauagcaag 1080

accgagcagg ggccgcaggu ggaugaaacu caguuuaaga agauccucgg cuacaucaac 1140

acggggaagc aagagggggc gaagcugcug uguggugggg gcauugcugc ugaccguggu 1200

uacuucaucc agcccacugu guuuggagau gugcaggaug gcaugaccau cgccaaggag 1260

gagaucuucg ggccagugau gcagauccug aaguucaaga ccauagagga gguuguuggg 1320

agagccaaca auuccacgua cgggcuggcc gcagcugucu ucacaaagga uuuggacaag 1380

gccaauuacc ugucccaggc ccuccaggcg ggcacugugu gggucaacug cuaugaugug 1440

uuuggagccc agucacccuu ugguggcuac aagaugucgg ggaguggccg ggaguugggc 1500

gaguacgggc ugcaggcaua cacugaagug aaaacuguca cagucaaagu gccucagaag 1560

aacucauaag gacuaguuau aagacugacu agcccgaugg gccucccaac gggcccuccu 1620

ccccuccuug caccgagauu aauaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740

aaa 1743

<210> 6

<211> 1743

<212> RNA

<213> Artificial Sequence

<220>

<223> example 3 provides mRNA sequence two

<400> 6

gggagaaagc uuaccauguu gcgcgcugcc gcccgcuucg ggccccgccu gggccgccgc 60

cucuugucag ccgccgccac ccaggccgug ccugccccca accagcagcc cgaggucuuc 120

ugcaaccaga uuuucauaaa caaugaaugg cacgaugccg ucagcaggaa aacauucccc 180

accgucaauc cguccacugg agaggucauc ugucagguag cugaagggga caaggaagau 240

guggacaagg cagugaaggc cgcccgggcc gccuuccagc ugggcucacc uuggcgccgc 300

auggacgcau cacacagggg ccggcugcug aaccgccugg ccgaucugau cgagcgggac 360

cggaccuacc uggcggccuu ggagacccug gacaauggca agcccuaugu caucuccuac 420

cugguggauu uggacauggu ccucaaaugu cuccgguauu augccggcug ggcugauaag 480

uaccacggga aaaccauccc cauugacgga gacuucuuca gcuacacacg ccaugaaccu 540

gugggggugu gcgggcagau cauuccgugg aauuucccgc uccugaugca agcauggaag 600

cugggcccag ccuuggcaac uggaaacgug guugugauga agguagcuga gcagacaccc 660

cucaccgccc ucuauguggc caaccugauc aaggaggcug gcuuuccccc uggugugguc 720

aacauugugc cuggauuugg ccccacggcu ggggccgcca uugccuccca ugaggaugug 780

gacaaagugg cauucacagg cuccacugag auuggccgcg uaauccaggu ugcugcuggg 840

agcagcaacc ucaagagagu gaccuuggag cuggggggga agagccccaa caucaucaug 900

ucagaugccg auauggauug ggccguggaa caggcccacu ucgcccuguu cuucaaccag 960

ggccagugcu gcugugccgg cucccggacc uucgugcagg aggacaucua ugaugaguuu 1020

guggagcgga gcguugcccg ggccaagucu cggguggucg ggaaccccuu ugauagcaag 1080

accgagcagg ggccgcaggu ggaugaaacu caguuuaaga agauccucgg cuacaucaac 1140

acggggaagc aagagggggc gaagcugcug uguggugggg gcauugcugc ugaccguggu 1200

uacuucaucc agcccacugu guuuggagau gugcaggaug gcaugaccau cgccaaggag 1260

gagaucuucg ggccagugau gcagauccug aaguucaaga ccauagagga gguuguuggg 1320

agagccaaca auuccacgua cgggcuggcc gcagcugucu ucacaaagga uuuggacaag 1380

gccaauuacc ugucccaggc ccuccaggcg ggcacugugu gggucaacug cuaugaugug 1440

uuuggagccc agucacccuu ugguggcuac aagaugucgg ggaguggccg ggaguugggc 1500

gaguacgggc ugcaggcaua cacugaagug aaaacuguca cagucaaagu gccucagaag 1560

aacucauaag gacuaguuau aagacugacu agcccgaugg gccucccaac gggcccuccu 1620

ccccuccuug caccgagauu aauaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740

aaa 1743

<210> 7

<211> 1762

<212> RNA

<213> Artificial Sequence

<220>

<223> example 3 provides mRNA sequence III

<400> 7

gagacccaag cuggcuagcg ggagaaagcu uaccauguug cgcgcugccg cccgcuucgg 60

gccccgccug ggccgccgcc ucuugucagc cgccgccacc caggccgugc cugcccccaa 120

ccagcagccc gaggucuucu gcaaccagau uuucauaaac aaugaauggc acgaugccgu 180

cagcaggaaa acauucccca ccgucaaucc guccacugga gaggucaucu gucagguagc 240

ugaaggggac aaggaagaug uggacaaggc agugaaggcc gcccgggccg ccuuccagcu 300

gggcucaccu uggcgccgca uggacgcauc acacaggggc cggcugcuga accgccuggc 360

cgaucugauc gagcgggacc ggaccuaccu ggcggccuug gagacccugg acaauggcaa 420

gcccuauguc aucuccuacc ugguggauuu ggacaugguc cucaaauguc uccgguauua 480

ugccggcugg gcugauaagu accacgggaa aaccaucccc auugacggag acuucuucag 540

cuacacacgc caugaaccug ugggggugug cgggcagauc auuccgugga auuucccgcu 600

ccugaugcaa gcauggaagc ugggcccagc cuuggcaacu ggaaacgugg uugugaugaa 660

gguagcugag cagacacccc ucaccgcccu cuauguggcc aaccugauca aggaggcugg 720

cuuucccccu ggugugguca acauugugcc uggauuuggc cccacggcug gggccgccau 780

ugccucccau gaggaugugg acaaaguggc auucacaggc uccacugaga uuggccgcgu 840

aauccagguu gcugcuggga gcagcaaccu caagagagug accuuggagc ugggggggaa 900

gagccccaac aucaucaugu cagaugccga uauggauugg gccguggaac aggcccacuu 960

cgcccuguuc uucaaccagg gccagugcug cugugccggc ucccggaccu ucgugcagga 1020

ggacaucuau gaugaguuug uggagcggag cguugcccgg gccaagucuc ggguggucgg 1080

gaaccccuuu gauagcaaga ccgagcaggg gccgcaggug gaugaaacuc aguuuaagaa 1140

gauccucggc uacaucaaca cggggaagca agagggggcg aagcugcugu gugguggggg 1200

cauugcugcu gaccgugguu acuucaucca gcccacugug uuuggagaug ugcaggaugg 1260

caugaccauc gccaaggagg agaucuucgg gccagugaug cagauccuga aguucaagac 1320

cauagaggag guuguuggga gagccaacaa uuccacguac gggcuggccg cagcugucuu 1380

cacaaaggau uuggacaagg ccaauuaccu gucccaggcc cuccaggcgg gcacugugug 1440

ggucaacugc uaugaugugu uuggagccca gucacccuuu gguggcuaca agaugucggg 1500

gaguggccgg gaguugggcg aguacgggcu gcaggcauac acugaaguga aaacugucac 1560

agucaaagug ccucagaaga acucauaagg acuaguuaua agacugacua gcccgauggg 1620

ccucccaacg ggcccuccuc cccuccuugc accgagauua auaaaaaaaa aaaaaaaaaa 1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740

aaaaaaaaaa aaaaaaaaaa aa 1762

<210> 8

<211> 1762

<212> RNA

<213> Artificial Sequence

<220>

<223> example 3 provides mRNA sequence four

<400> 8

gagacccaag cuggcuagcg ggagaaagcu uaccauguug cgcgcugccg cccgcuucgg 60

gccccgccug ggccgccgcc ucuugucagc cgccgccacc caggccgugc cugcccccaa 120

ccagcagccc gaggucuucu gcaaccagau uuucauaaac aaugaauggc acgaugccgu 180

cagcaggaaa acauucccca ccgucaaucc guccacugga gaggucaucu gucagguagc 240

ugaaggggac aaggaagaug uggacaaggc agugaaggcc gcccgggccg ccuuccagcu 300

gggcucaccu uggcgccgca uggacgcauc acacaggggc cggcugcuga accgccuggc 360

cgaucugauc gagcgggacc ggaccuaccu ggcggccuug gagacccugg acaauggcaa 420

gcccuauguc aucuccuacc ugguggauuu ggacaugguc cucaaauguc uccgguauua 480

ugccggcugg gcugauaagu accacgggaa aaccaucccc auugacggag acuucuucag 540

cuacacacgc caugaaccug ugggggugug cgggcagauc auuccgugga auuucccgcu 600

ccugaugcaa gcauggaagc ugggcccagc cuuggcaacu ggaaacgugg uugugaugaa 660

gguagcugag cagacacccc ucaccgcccu cuauguggcc aaccugauca aggaggcugg 720

cuuucccccu ggugugguca acauugugcc uggauuuggc cccacggcug gggccgccau 780

ugccucccau gaggaugugg acaaaguggc auucacaggc uccacugaga uuggccgcgu 840

aauccagguu gcugcuggga gcagcaaccu caagagagug accuuggagc ugggggggaa 900

gagccccaac aucaucaugu cagaugccga uauggauugg gccguggaac aggcccacuu 960

cgcccuguuc uucaaccagg gccagugcug cugugccggc ucccggaccu ucgugcagga 1020

ggacaucuau gaugaguuug uggagcggag cguugcccgg gccaagucuc ggguggucgg 1080

gaaccccuuu gauagcaaga ccgagcaggg gccgcaggug gaugaaacuc aguuuaagaa 1140

gauccucggc uacaucaaca cggggaagca agagggggcg aagcugcugu gugguggggg 1200

cauugcugcu gaccgugguu acuucaucca gcccacugug uuuggagaug ugcaggaugg 1260

caugaccauc gccaaggagg agaucuucgg gccagugaug cagauccuga aguucaagac 1320

cauagaggag guuguuggga gagccaacaa uuccacguac gggcuggccg cagcugucuu 1380

cacaaaggau uuggacaagg ccaauuaccu gucccaggcc cuccaggcgg gcacugugug 1440

ggucaacugc uaugaugugu uuggagccca gucacccuuu gguggcuaca agaugucggg 1500

gaguggccgg gaguugggcg aguacgggcu gcaggcauac acugaaguga aaacugucac 1560

agucaaagug ccucagaaga acucauaagg acuaguuaua agacugacua gcccgauggg 1620

ccucccaacg ggcccuccuc cccuccuugc accgagauua auaaaaaaaa aaaaaaaaaa 1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740

aaaaaaaaaa aaaaaaaaaa aa 1762

<210> 9

<211> 1740

<212> RNA

<213> Artificial Sequence

<220>

<223> example 3 provides mRNA sequence five

<400> 9

gggagaaagc uuaccaugcu uagagccgcu gcgagauuug gaccuagacu ggguaggaga 60

cugcucagcg cugcagcuac acaggccguc ccugcuccua accaacagcc agaaguguuu 120

ugcaaucaga ucuuuauuaa caacgagugg caugacgcgg uuucacgcaa gaccuuuccc 180

accgugaauc ccucaacugg ggaggucauu ugucaggugg ccgaagggga uaaagaggac 240

guugauaagg cgguaaaagc ugcacgggcc gccuuccagu uggguagucc auggaggaga 300

auggacgcgu cucacagggg gagacuccug aauagguugg ccgaccugau ugaacgggau 360

cgaaccuauc uggcugcccu ggaaacgcuc gauaacggca aaccauaugu uauauccuau 420

cuugucgacc uugauauggu ccugaagugc cuccgcuacu augccggcug ggccgauaag 480

uaccacggga agacaauccc uauugacggg gacuucuucu cauauacucg gcaugaaccu 540

gucggagucu guggccagau caucccuugg aacuucccuc ugcugaugca ggccuggaaa 600

cugggaccag ccuuggccac cggcaacguu gucgugauga aagucgcuga gcagacaccc 660

cugacugccc ucuacguggc gaaucucauc aaagaggcgg gguuuccccc cgguguugug 720

aauaucgugc caggauuugg ccccacagcc ggagcugcua uugccucuca cgaagaugug 780

gacaaggugg cuuuuaccgg cuccaccgag auuggaaggg ugauucaggu ggcggcaggu 840

uccaguaacc ucaaacgggu cacacuggag cugggaggaa aaucccccaa uaucaucaug 900

uccgacgccg auauggacug ggcagucgaa caggcgcacu ucgccuuguu uuuuaaccag 960

gggcaguguu guugugccgg aucuagaacu uuugugcagg aggauauaua cgacgaauuc 1020

gucgagaggu cugucgcgcg ggcuaaaagu cgggucgugg gaaaccccuu cgauuccaag 1080

accgagcagg gcccacaggu ggaugagaca caguucaaga agaucuuggg uuacauuaac 1140

accggaaaac aggagggugc uaagcuucug ugcggaggag ggaucgcugc ugacagagga 1200

uacuuuauuc agccgacagu guucggcgac gugcaggaug guaugaccau cgcuaaggag 1260

gagaucuucg gaccgguaau gcagauucug aaauucaaga caauugaaga gguugugggg 1320

cgcgccaaua acuccaccua uggccucgca gcagccguuu ucacaaaaga ccuggacaag 1380

gcaaacuacc uuagccaggc gcuucaggcu ggcacugucu gggugaacug cuacgacgug 1440

uucggagccc agucccccuu uggaggcuac aagaugagcg gcucuggaag agaacuggga 1500

gaauacggcc uccaggcaua cacagaaguu aaaaccguga ccgucaaggu cccacagaaa 1560

aauucuggac uaguuauaag acugacuagc ccgaugggcc ucccaacggg cccuccuccc 1620

cuccuugcac cgagauuaau aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740

<210> 10

<211> 1740

<212> RNA

<213> Artificial Sequence

<220>

<223> example 3 provides mRNA sequence six

<400> 10

gggagaaagc uuaccaugcu uagagccgcu gcgagauuug gaccuagacu ggguaggaga 60

cugcucagcg cugcagcuac acaggccguc ccugcuccua accaacagcc agaaguguuu 120

ugcaaucaga ucuuuauuaa caacgagugg caugacgcgg uuucacgcaa gaccuuuccc 180

accgugaauc ccucaacugg ggaggucauu ugucaggugg ccgaagggga uaaagaggac 240

guugauaagg cgguaaaagc ugcacgggcc gccuuccagu uggguagucc auggaggaga 300

auggacgcgu cucacagggg gagacuccug aauagguugg ccgaccugau ugaacgggau 360

cgaaccuauc uggcugcccu ggaaacgcuc gauaacggca aaccauaugu uauauccuau 420

cuugucgacc uugauauggu ccugaagugc cuccgcuacu augccggcug ggccgauaag 480

uaccacggga agacaauccc uauugacggg gacuucuucu cauauacucg gcaugaaccu 540

gucggagucu guggccagau caucccuugg aacuucccuc ugcugaugca ggccuggaaa 600

cugggaccag ccuuggccac cggcaacguu gucgugauga aagucgcuga gcagacaccc 660

cugacugccc ucuacguggc gaaucucauc aaagaggcgg gguuuccccc cgguguugug 720

aauaucgugc caggauuugg ccccacagcc ggagcugcua uugccucuca cgaagaugug 780

gacaaggugg cuuuuaccgg cuccaccgag auuggaaggg ugauucaggu ggcggcaggu 840

uccaguaacc ucaaacgggu cacacuggag cugggaggaa aaucccccaa uaucaucaug 900

uccgacgccg auauggacug ggcagucgaa caggcgcacu ucgccuuguu uuuuaaccag 960

gggcaguguu guugugccgg aucuagaacu uuugugcagg aggauauaua cgacgaauuc 1020

gucgagaggu cugucgcgcg ggcuaaaagu cgggucgugg gaaaccccuu cgauuccaag 1080

accgagcagg gcccacaggu ggaugagaca caguucaaga agaucuuggg uuacauuaac 1140

accggaaaac aggagggugc uaagcuucug ugcggaggag ggaucgcugc ugacagagga 1200

uacuuuauuc agccgacagu guucggcgac gugcaggaug guaugaccau cgcuaaggag 1260

gagaucuucg gaccgguaau gcagauucug aaauucaaga caauugaaga gguugugggg 1320

cgcgccaaua acuccaccua uggccucgca gcagccguuu ucacaaaaga ccuggacaag 1380

gcaaacuacc uuagccaggc gcuucaggcu ggcacugucu gggugaacug cuacgacgug 1440

uucggagccc agucccccuu uggaggcuac aagaugagcg gcucuggaag agaacuggga 1500

gaauacggcc uccaggcaua cacagaaguu aaaaccguga ccgucaaggu cccacagaaa 1560

aauucuggac uaguuauaag acugacuagc ccgaugggcc ucccaacggg cccuccuccc 1620

cuccuugcac cgagauuaau aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740

<210> 11

<211> 1759

<212> RNA

<213> Artificial Sequence

<220>

<223> example 3 provides mRNA sequence seven

<400> 11

gagacccaag cuggcuagcg ggagaaagcu uaccaugcuu agagccgcug cgagauuugg 60

accuagacug gguaggagac ugcucagcgc ugcagcuaca caggccgucc cugcuccuaa 120

ccaacagcca gaaguguuuu gcaaucagau cuuuauuaac aacgaguggc augacgcggu 180

uucacgcaag accuuuccca ccgugaaucc cucaacuggg gaggucauuu gucagguggc 240

cgaaggggau aaagaggacg uugauaaggc gguaaaagcu gcacgggccg ccuuccaguu 300

ggguagucca uggaggagaa uggacgcguc ucacaggggg agacuccuga auagguuggc 360

cgaccugauu gaacgggauc gaaccuaucu ggcugcccug gaaacgcucg auaacggcaa 420

accauauguu auauccuauc uugucgaccu ugauaugguc cugaagugcc uccgcuacua 480

ugccggcugg gccgauaagu accacgggaa gacaaucccu auugacgggg acuucuucuc 540

auauacucgg caugaaccug ucggagucug uggccagauc aucccuugga acuucccucu 600

gcugaugcag gccuggaaac ugggaccagc cuuggccacc ggcaacguug ucgugaugaa 660

agucgcugag cagacacccc ugacugcccu cuacguggcg aaucucauca aagaggcggg 720

guuucccccc gguguuguga auaucgugcc aggauuuggc cccacagccg gagcugcuau 780

ugccucucac gaagaugugg acaagguggc uuuuaccggc uccaccgaga uuggaagggu 840

gauucaggug gcggcagguu ccaguaaccu caaacggguc acacuggagc ugggaggaaa 900

aucccccaau aucaucaugu ccgacgccga uauggacugg gcagucgaac aggcgcacuu 960

cgccuuguuu uuuaaccagg ggcaguguug uugugccgga ucuagaacuu uugugcagga 1020

ggauauauac gacgaauucg ucgagagguc ugucgcgcgg gcuaaaaguc gggucguggg 1080

aaaccccuuc gauuccaaga ccgagcaggg cccacaggug gaugagacac aguucaagaa 1140

gaucuugggu uacauuaaca ccggaaaaca ggagggugcu aagcuucugu gcggaggagg 1200

gaucgcugcu gacagaggau acuuuauuca gccgacagug uucggcgacg ugcaggaugg 1260

uaugaccauc gcuaaggagg agaucuucgg accgguaaug cagauucuga aauucaagac 1320

aauugaagag guuguggggc gcgccaauaa cuccaccuau ggccucgcag cagccguuuu 1380

cacaaaagac cuggacaagg caaacuaccu uagccaggcg cuucaggcug gcacugucug 1440

ggugaacugc uacgacgugu ucggagccca gucccccuuu ggaggcuaca agaugagcgg 1500

cucuggaaga gaacugggag aauacggccu ccaggcauac acagaaguua aaaccgugac 1560

cgucaagguc ccacagaaaa auucuggacu aguuauaaga cugacuagcc cgaugggccu 1620

cccaacgggc ccuccucccc uccuugcacc gagauuaaua aaaaaaaaaa aaaaaaaaaa 1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740

aaaaaaaaaa aaaaaaaaa 1759

<210> 12

<211> 1759

<212> RNA

<213> Artificial Sequence

<220>

<223> example 3 provides mRNA sequence eight

<400> 12

gagacccaag cuggcuagcg ggagaaagcu uaccaugcuu agagccgcug cgagauuugg 60

accuagacug gguaggagac ugcucagcgc ugcagcuaca caggccgucc cugcuccuaa 120

ccaacagcca gaaguguuuu gcaaucagau cuuuauuaac aacgaguggc augacgcggu 180

uucacgcaag accuuuccca ccgugaaucc cucaacuggg gaggucauuu gucagguggc 240

cgaaggggau aaagaggacg uugauaaggc gguaaaagcu gcacgggccg ccuuccaguu 300

ggguagucca uggaggagaa uggacgcguc ucacaggggg agacuccuga auagguuggc 360

cgaccugauu gaacgggauc gaaccuaucu ggcugcccug gaaacgcucg auaacggcaa 420

accauauguu auauccuauc uugucgaccu ugauaugguc cugaagugcc uccgcuacua 480

ugccggcugg gccgauaagu accacgggaa gacaaucccu auugacgggg acuucuucuc 540

auauacucgg caugaaccug ucggagucug uggccagauc aucccuugga acuucccucu 600

gcugaugcag gccuggaaac ugggaccagc cuuggccacc ggcaacguug ucgugaugaa 660

agucgcugag cagacacccc ugacugcccu cuacguggcg aaucucauca aagaggcggg 720

guuucccccc gguguuguga auaucgugcc aggauuuggc cccacagccg gagcugcuau 780

ugccucucac gaagaugugg acaagguggc uuuuaccggc uccaccgaga uuggaagggu 840

gauucaggug gcggcagguu ccaguaaccu caaacggguc acacuggagc ugggaggaaa 900

aucccccaau aucaucaugu ccgacgccga uauggacugg gcagucgaac aggcgcacuu 960

cgccuuguuu uuuaaccagg ggcaguguug uugugccgga ucuagaacuu uugugcagga 1020

ggauauauac gacgaauucg ucgagagguc ugucgcgcgg gcuaaaaguc gggucguggg 1080

aaaccccuuc gauuccaaga ccgagcaggg cccacaggug gaugagacac aguucaagaa 1140

gaucuugggu uacauuaaca ccggaaaaca ggagggugcu aagcuucugu gcggaggagg 1200

gaucgcugcu gacagaggau acuuuauuca gccgacagug uucggcgacg ugcaggaugg 1260

uaugaccauc gcuaaggagg agaucuucgg accgguaaug cagauucuga aauucaagac 1320

aauugaagag guuguggggc gcgccaauaa cuccaccuau ggccucgcag cagccguuuu 1380

cacaaaagac cuggacaagg caaacuaccu uagccaggcg cuucaggcug gcacugucug 1440

ggugaacugc uacgacgugu ucggagccca gucccccuuu ggaggcuaca agaugagcgg 1500

cucuggaaga gaacugggag aauacggccu ccaggcauac acagaaguua aaaccgugac 1560

cgucaagguc ccacagaaaa auucuggacu aguuauaaga cugacuagcc cgaugggccu 1620

cccaacgggc ccuccucccc uccuugcacc gagauuaaua aaaaaaaaaa aaaaaaaaaa 1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740

aaaaaaaaaa aaaaaaaaa 1759

<210> 13

<211> 34

<212> RNA

<213> Artificial Sequence

<220>

<223> sequence two of 5' UTR

<400> 13

gagacccaag cuggcuagcg ggagaaagcu uacc 34

<210> 14

<211> 109

<212> RNA

<213> Artificial Sequence

<220>

<223> 3' UTR sequence two

<400> 14

gcuggagccu cgguagccgu uccuccugcc cgcugggccu cccaacgggc ccuccucccc 60

uccuugcacc ggcccuuccu ggucuuugaa uaaagucuga gugggcagc 109

<210> 15

<211> 1554

<212> RNA

<213> Artificial Sequence

<220>

<223> ORF sequence two

<400> 15

auguugcgcg cugccgcccg cuucgggccc cgccugggcc gccgccucuu gucagccgcc 60

gccacccagg ccgugccugc ccccaaccag cagcccgagg ucuucugcaa ccagauuuuc 120

auaaacaaug aauggcacga ugccgucagc aggaaaacau uccccaccgu caauccgucc 180

acuggagagg ucaucuguca gguagcugaa ggggacaagg aagaugugga caaggcagug 240

aaggccgccc gggccgccuu ccagcugggc ucaccuuggc gccgcaugga cgcaucacac 300

aggggccggc ugcugaaccg ccuggccgau cugaucgagc gggaccggac cuaccuggcg 360

gccuuggaga cccuggacaa uggcaagccc uaugucaucu ccuaccuggu ggauuuggac 420

augguccuca aaugucuccg guauuaugcc ggcugggcug auaaguacca cgggaaaacc 480

auccccauug acggagacuu cuucagcuac acacgccaug aaccuguggg ggugugcggg 540

cagaucauuc cguggaauuu cccgcuccug augcaagcau ggaagcuggg cccagccuug 600

gcaacuggaa acgugguugu gaugaaggua gcugagcaga caccccucac cgcccucuau 660

guggccaacc ugaucaagga ggcuggcuuu cccccuggug uggucaacau ugugccugga 720

uuuggcccca cggcuggggc cgccauugcc ucccaugagg auguggacaa aguggcauuc 780

acaggcucca cugagauugg ccgcguaauc cagguugcug cugggagcag caaccucaag 840

agagugaccu uggagcuggg ggggaagagc cccaacauca ucaugucaga ugccgauaug 900

gauugggccg uggaacaggc ccacuucgcc cuguucuuca accagggcca gugcugcugu 960

gccggcuccc ggaccuucgu gcaggaggac aucuaugaug aguuugugga gcggagcguu 1020

gcccgggcca agucucgggu ggucgggaac cccuuugaua gcaagaccga gcaggggccg 1080

cagguggaug aaacucaguu uaagaagauc cucggcuaca ucaacacggg gaagcaagag 1140

ggggcgaagc ugcugugugg ugggggcauu gcugcugacc gugguuacuu cauccagccc 1200

acuguguuug gagaugugca ggauggcaug accaucgcca aggaggagau cuucgggcca 1260

gugaugcaga uccugaaguu caagaccaua gaggagguug uugggagagc caacaauucc 1320

acguacgggc uggccgcagc ugucuucaca aaggauuugg acaaggccaa uuaccugucc 1380

caggcccucc aggcgggcac uguguggguc aacugcuaug auguguuugg agcccaguca 1440

cccuuuggug gcuacaagau gucggggagu ggccgggagu ugggcgagua cgggcugcag 1500

gcauacacug aagugaaaac ugucacaguc aaagugccuc agaagaacuc auaa 1554

<210> 16

<211> 1554

<212> RNA

<213> Artificial Sequence

<220>

<223> ORF sequence three

<400> 16

augcugcgcg cugccgcucg cuuuggcccc cggcuggggc ggagacugcu guccgccgcc 60

gccacccagg ccgugcccgc ccccaaccag cagcccgagg uguucuguaa ccagaucuuu 120

aucaacaacg aguggcacga cgccgugagc cggaagaccu uuccuacugu gaaccccucc 180

accggcgagg ugaucuguca gguggccgag ggcgacaagg aggacgugga caaggccgug 240

aaagcagcca gggccgccuu ccagcugggc ucacccuggc ggagaaugga cgccagccau 300

agaggcaggc ugcugaacag acuggccgac cucaucgaga gggauaggac uuaucuggca 360

gcccuggaga cacuggacaa uggcaagccc uacgugaucu cuuaucuggu ggaucucgac 420

auggugcuga agugucugcg cuauuaugcc ggcugggccg auaaauauca cggcaagacc 480

aucccaaucg auggagauuu uuucuccuau accaggcaug aaccaguggg agugugcggc 540

cagaucauuc cauggaacuu uccccugcua augcaggccu ggaaguuagg cccugcucug 600

gccacuggaa acgugguggu gaugaaggug gccgagcaga caccccucac ugcucuguac 660

guggccaacc ugaucaaaga ggccggcuuu ccuccuggag ucgucaacau ugugcccggg 720

uucggaccca cagccggugc cgcuaucgcc ucccacgagg acguggacaa gguggcuuuc 780

acaggcagca ccgagaucgg ccgggugauc cagguggccg ccggcagcuc caaccugaag 840

cgggugacau uggagcuggg cggcaagucc cccaauauca ucaugagcga cgccgacaug 900

gacugggccg uggagcaggc ccauuuugcc cuguucuuua aucaggggca gugcuguugu 960

gccgggucca ggaccuucgu gcaggaggau aucuaugacg aguuuguuga gagaagcgug 1020

gccagggcca agagcagggu ggugggcaac cccuucgaua gcaagaccga gcagggcccu 1080

cagguggacg aaacccaguu caagaaaauc cugggguaca ucaacaccgg aaaacaggag 1140

ggcgcuaagc ugcugugcgg gggcggcauc gccgccgaua gaggauauuu uauccagccc 1200

accgucuucg gggacgucca agacggaaug accauugcca aggaggagau cuuuggccca 1260

gugaugcaga uccugaaguu caagaccauc gaggaagugg ugggcagagc caauaauagc 1320

accuacgggc uggcugccgc cguguuuacc aaggaucugg acaaggccaa cuaccuguca 1380

caggcccugc aggccggcac cgugugggug aacugcuacg acgucuuugg cgcucagucu 1440

cccuucgggg guuacaagau gucugggagc gggagagagc ugggcgagua cggccugcag 1500

gcuuacaccg aggugaaaac cgugaccgug aaagugccac agaagaauag cuga 1554

<210> 17

<211> 1551

<212> RNA

<213> Artificial Sequence

<220>

<223> ORF sequence four

<400> 17

augcugcggg ccgcugccag auucggcccc agacugggaa ggcggcugcu guccgccgcc 60

gccacccagg ccguuccugc uccuaaucaa cagccugagg uuuucugcaa ccaaaucuuc 120

aucaacaacg aguggcacga cgccgucagc agaaagacau uccccaccgu caauccuagc 180

acaggcgagg ugaucugcca ggucgccgag ggcgacaaag aggacgugga caaggccgug 240

aaagccgccc gcgccgccuu ucagcugggc ucuccuuggc ggagaaugga ugccagccac 300

agaggcaggc ugcucaacag acuggccgau cugaucgaga gagauagaac auaccuggcu 360

gcucuggaaa cacuggacaa cggcaagccc uacgugauca gcuaccuggu ggaccuggau 420

auggugcuga agugccugag auacuacgcc ggaugggcug auaaguacca cgggaaaaca 480

aucccuaucg acggcgauuu cuucagcuac accagacacg agccuguggg cgugugcgga 540

cagaucaucc ccuggaacuu cccauugcug augcaagcuu ggaagcuggg cccugcccug 600

gccacaggaa acgugguggu caugaaggug gcugaacaga ccccucugac cgcccuguac 660

guggccaauc ugaucaagga agccggauuu ccuccuggag uggugaacau cgugcccggc 720

uuuggcccca cagccggcgc cgccaucgcc ucucaugagg acguggauaa gguggccuuc 780

accggcagca ccgagauagg acgggugauc cagguggcag cuggcagcag caaccugaag 840

cgggugacac uggagcuggg aggcaagagc cccaacauca ucaugagcga cgccgacaug 900

gacugggccg uggaacaggc ucacuucgcc cuguucuuca accagggcca gugcuguugu 960

gccggcagca gaaccuucgu gcaggaggac aucuacgaug aguucgugga aagaagcgug 1020

gccagagcca aguccagagu ggugggaaau cccuucgacu ccaagaccga gcagggccca 1080

cagguggaug agacacaguu caaaaagauc cugggauaua ucaauaccgg caagcaggag 1140

ggcgccaagc ugcuuugugg cggcggcauc gcugccgacc ggggcuacuu cauccagccu 1200

acaguguucg gcgacguuca ggacggcaug accaucgcua aggaagaaau cuuuggccca 1260

gugaugcaga uucugaaauu caagaccauc gaggaagugg ugggccgggc caacaacagc 1320

acuuacggcc uggccgcugc cguguucacc aaggaccugg acaaggccaa cuaccugucc 1380

caggcccugc aggccggcac cgucugggug aacugcuacg acguguuugg agcccagucu 1440

ccuuucggcg gcuauaagau gagcggcucu ggccgggaac ugggcgaaua cggccugcaa 1500

gccuacaccg aggugaagac cgugaccgug aaggugccuc agaaaaacuc u 1551

<210> 18

<211> 1551

<212> RNA

<213> Artificial Sequence

<220>

<223> ORF sequence five

<400> 18

augcugaggg cugcugccag auucggcccg cggcucggaa gaagacugcu cuccgccgcu 60

gcuacacagg ccguccccgc cccuaaucag cagccugagg uguucugcaa ccagauuuuc 120

aucaacaacg aguggcacga ugccguuucc agaaagaccu uuccaaccgu aaaucccagc 180

accggcgagg ugaucugcca gguggccgag ggcgauaaag aggacgugga caaggccgug 240

aaagccgccc gggccgccuu ucagcugggc agcccuuggc ggagaaugga cgcuucucau 300

agaggaaggc ugcugaacag acuggcugac cugaucgaac gggacagaac uuaccuggcc 360

gcccuggaaa cacuggacaa cggcaagccu uacgugauca gcuaccuggu ggaccuggau 420

auggugcuga agugccugcg guacuacgcc ggcugggccg acaaauacca cggcaagaca 480

auccccaucg acggcgacuu cuucagcuac accagacacg agccuguggg cgugugcggc 540

cagaucaucc cuuggaacuu cccucugcug augcaggccu ggaaacuggg accugcccug 600

gccacaggca augugguggu caugaaggug gccgagcaaa caccucugac agcccuguac 660

guggccaacc ugaucaagga ggccggauuu ccccccggcg uggugaacau cgugccuggc 720

uucggcccua ccgccggcgc cgccaucgcc agccacgagg augucgauaa gguggcuuuu 780

acaggaagca ccgagauugg ccgggugauc caagucgcgg ccgggucuag caaccugaaa 840

agagugaccc uggaacuggg agguaaaagc ccuaacauca ucaugagcga cgccgauaug 900

gauugggccg uggaacaggc ccacuucgcc uuguuuuuca accagggaca guguuguugc 960

gccggcagcc ggaccuucgu ucaggaggac aucuacgacg aauucgugga aagaagcgug 1020

gcuagagcca agagcagagu ggugggaaac cccuucgaca gcaagaccga gcagggccca 1080

cagguggacg aaacacaauu uaagaagauc cugggcuaca ucaacaccgg caagcaggag 1140

ggcgccaagc ugcugugugg cggcggcauc gccgcugaua gaggcuacuu cauccagcca 1200

accguguucg gcgacgugca ggauggcaug accaucgcca aggaagaaau cuucggaccu 1260

gugaugcaga uccugaaguu caagaccauc gaggaagugg uuggccgcgc uaacaacucc 1320

accuacggcc uggccgccgc cguguucacc aaggaccugg acaaggccaa cuaccugucu 1380

caggcccugc aagcugggac cguguggguc aauugcuaug acguguucgg cgcucagucu 1440

ccuuucggcg gcuauaagau guccggcucu ggcagagaac ugggcgagua cggccuccag 1500

gccuacacag aggucaagac cgugaccgug aaagugcccc agaagaacag c 1551

<210> 19

<211> 1416

<212> RNA

<213> Artificial Sequence

<220>

<223> murine ORF sequence

<400> 19

augcugcgag cagcucuuac cacuguccgc agggguccca gauuaagccg gcuacugucu 60

gcagccgcaa cgagugcagu gcccgccccc aaccaccagc cagaaguuuu cugcaaucag 120

aucuucauca auaaugagug gcaugaugcu guuucucgua agacuuuccc gacaguuaac 180

cccuccacag gggaggucau cugccagguc gcugagggga acaaggaaga cguagauaaa 240

gccgucaaag cugcgagggc cgcauuccag cugggaaguc cauggagacg aauggaugca 300

ucagacagag gucgcuuauu guaucgucua gcagauuuga uugaacggga cagaaccuac 360

cuggccgcgc uugaaacccu ugacaacggg aaaccuuaug ugaucuccua ccuggucgac 420

cuggauaugg ugcugaagug uuugagguac uaugccggcu gggcagauaa guaccacggg 480

aagaccauuc caaucgaugg agacuucuuu ucauacacuc gccacgagcc uguuggagug 540

uguggucaaa ucauucccug gaacuuccca cuucucaugc aagccuggaa gcucgggccu 600

gccuuggcaa cgggcaaugu gguuguuaug aagguggcag agcaaacacc acuaacagcu 660

cuguacguag ccaaccugau caaagaagcc ggcuuccccc cgggaguagu caauauugug 720

cccggauuug gaccaacagc gggugccgcu auugccucuc augaaggcgu ggacaaaguu 780

gccuuuacug guagcacaga ggugggccac cucauccagg uagcagcugg cagcagcaau 840

cucaagcggg ucacacugga gcuggguggg aagucuccua acauaaucau guccgaugcc 900

gacauggacu gggcugugga gcaagcucau uuugcccucu ucuucaacca ggggcagugc 960

ugcugugcag gcucacgcac auuuguccaa gagaaugucu augaugaguu cguggaaaga 1020

agcguagcuc gggccaaauc gaggguugug ggaaacccau uugacagcag gaccgagcag 1080

ggcccucagg uggaugagac ccaguucaag aagauacuug gauacaucaa gagcggccag 1140

caggaaggag ccaagcugcu cuguggcggg ggagcugcug cugacagggg cuacuucaua 1200

cagccuaccg uguuuggaga ugugaaggac ggcaugacua uagcuaagga ggagauuuuu 1260

ggcccuguga ugcagauuuu aaaauuuaaa acuaucgagg aagugguggg gagagcaaau 1320

gacuccaagu auggccuggc cgcggccgug uuuacuaaag aucuggacaa agcuaacuau 1380

cuuucucagg cccugcaggc agggaccguc ugguga 1416

<210> 20

<211> 1551

<212> RNA

<213> Artificial Sequence

<220>

<223> ORF sequence six

<400> 20

augcucagag cugccgcaag auuugggccu agauugggaa gaagacugcu gucugcagca 60

gcuacacaag cugucccggc accuaaccag cagcccgaag uauuuuguaa ucagaucuuc 120

aucaauaacg aguggcauga cgccgugagu cggaaaacau uccccacugu gaauccgagu 180

acaggcgaag ugauaugcca ggucgcugaa ggagauaagg aggaugugga caaggcuguc 240

aaggcugcca gggcugcauu ucaguugggc aguccuugga gaaggaugga ugccucucac 300

aggggaagac ugcugaaucg gcuggccgac cugaucgaga gagauaggac auacuuggcc 360

gcccuggaga cacuggacaa cggaaaacca uacgugaucu cauaccuugu ugaucuggac 420

auggugcuga agugccugag guauuaugca gggugggcug acaaguauca ugggaaaacc 480

auccccauag auggggauuu cuuuagcuac acuaggcaug aaccgguugg gguguguggc 540

cagaucaucc ccuggaacuu cccccugcuc augcaagcuu ggaagcucgg accugcccuu 600

gcaaccggca acgucguugu gaugaaagug gccgagcaga caccucugac agcacuguac 660

guggcaaacc ugaucaagga ggcagguuuc ccuccaggcg uuguuaauau cguucccggu 720

uuuggaccaa cugcaggcgc agcaaucgca ucacaugaag auguagauaa agucgcuuuu 780

acaggcucaa ccgaaaucgg caggguuauu caaguggccg ccgguucuag uaaucuuaaa 840

cgggucacac uggagcucgg cgguaagagu cccaacauua ucaugagcga ugcugauaug 900

gacugggcag uggagcaggc acacuucgca cuguuuuuca accaggggca guguuguugu 960

gccgggucca gaacuuuugu ucaagaggac auuuaugaug aguuugugga gcgcucugug 1020

gcucgggcaa aauccagagu uguggggaac cccuuugacu caaagacuga acaaggcccc 1080

cagguggaug aaacgcaauu caaaaagauu cuggguuaca ucaauacggg caagcaggaa 1140

ggggcuaaac uucugugugg cggcgggauu gcugcugaua gaggguacuu uauccagccu 1200

acagucuucg gcgacgucca ggacgggaug acgaucgcaa aggaggaaau cuucgggccc 1260

gugaugcaaa uacugaaguu caagaccauc gaagaaguag ucggaagagc caacaauagu 1320

acauacggcu uggccgcugc cguguucacc aaggaccucg auaaggccaa uuaccugagc 1380

caggcacugc aggcuggaac agugugggug aacugcuacg acguuuucgg cgcgcaaagc 1440

cccuuugggg gauacaaaau gagcgguagu gggcgggagc uuggugagua cgguuugcag 1500

gcuuauaccg aagugaaaac ugugacugug aaagugcccc aaaagaacuc c 1551

<210> 21

<211> 1551

<212> RNA

<213> Artificial Sequence

<220>

<223> ORF sequence seven

<400> 21

augcugcgcg ccgccgcccg cuucggcccc cggcuggggc ggcgccugcu guccgccgcc 60

gccacccagg ccgugcccgc ccccaaccag cagcccgagg uguucugcaa ucagaucuuc 120

aucaacaacg aguggcacga cgccgugagc cggaagaccu uccccacugu gaaccccagc 180

accggcgagg ugaucugcca gguggccgag ggcgacaagg aggacgugga caaggccgug 240

aaggccgccc gggccgccuu ccagcugggc ucucccuggc ggcggaugga cgccucucac 300

cgcggccgcc ugcugaaccg gcuggccgac cugaucgagc gcgaccggac cuaccuggcc 360

gcccucgaga cccuggacaa cggcaagccc uacgugauca gcuaccuggu ggaccuggac 420

auggugcuga agugccugcg guacuacgcc ggcugggccg acaaguacca cggcaagacc 480

auccccaucg acggcgacuu cuucagcuac acccgccacg agcccguggg cgugugcggc 540

cagaucaucc ccuggaacuu cccccugcug augcaggccu ggaagcuggg acccgcccug 600

gccaccggca augugguggu caugaaggug gccgagcaaa ccccccugac cgcccuguac 660

guggccaacc ugaucaagga ggccggauuc ccccccggcg uggugaacau cgugcccggc 720

uucggcccca ccgccggcgc cgccaucgcc agccacgagg acguggacaa gguggccuuc 780

accggcagca ccgagaucgg ccgggugauc cagguggccg ccggcagcuc caaccugaag 840

cgggugaccu uggagcuggg cggcaagucc cccaauauca ucaugagcga cgccgacaug 900

gacugggccg uggagcaggc ccacuucgcc uuguucuuca accagggaca guguuguugc 960

gccggcagcc ggaccuucgu ccaggaggac aucuacgacg aguucgugga gcgcagcgug 1020

gcccgcgcca agagccgcgu ggugggaaac cccuucgaca gcaagaccga gcagggcccc 1080

cagguggacg agacccaauu caagaagauc cugggcuaca ucaacaccgg caagcaggag 1140

ggcgccaagc ugcugugugg cggcggcauc gccgccgacc gcggcuacuu cauccagccc 1200

accguguucg gcgacgugca ggacggcaug accaucgcca aggaggagau cuucggaccc 1260

gugaugcaga uccugaaguu caagaccauc gaggaggugg ucggccgcgc caacaacucc 1320

accuacggcc uggccgccgc cguguucacc aaggaccugg acaaggccaa cuaccugucu 1380

caggcccugc aagccgggac cguguggguc aauugcuaug acguguucgg cgcccagucu 1440

cccuucggcg gcuauaagau guccggcucu ggccgcgagc ugggcgagua cggccuccag 1500

gccuacaccg aggucaagac cgugaccgug aaggugcccc agaagaacag c 1551

Claims

1. mRNA comprising an ORF encoding an ALDH2 polypeptide, a 5' UTR sequence, a 3' UTR sequence, a 5' cap and a poly (A) sequence; the ORF for encoding the ALDH2 polypeptide is shown as SEQ ID No. 15; the 5'UTR sequence is shown as SEQ ID No.1, and the 3' UTR sequence is shown as SEQ ID No. 3.

2. The mRNA of claim 1, wherein the 5' cap comprises 7-methyl-guanosine-5 ' -triphosphate-5 ' -adenosine, 7-methyl-guanosine-5 ' -triphosphate-5 ' -guanosine, and m ⁷ G (5 ') (2' -OMeA) pG.

3. The mRNA of claim 2, wherein the 5' cap is m ⁷ G（5’）（2’-OMeA）pG。

4. The mRNA according to claim 1, wherein the number of base A in the poly (A) sequence is 60 to 120.

5. The mRNA according to claim 4, wherein the number of the base A in the poly (A) sequence is 100.

6. Use of an mRNA according to any one of claims 1 to 5 for the preparation of an mRNA drug, the use of said mRNA drug comprising (i) or (ii):

supplementing ALDH2 enzyme;

(ii) treating liver injury or alcoholic hepatitis caused by the absence of ALDH2 enzyme combined with alcohol.

7. An mRNA drug, characterized in that the mRNA drug comprises the mRNA of any one of claims 1 to 5, and the use of the mRNA drug comprises (i) or (ii):

supplementing ALDH2 enzyme;

8. The mRNA drug of claim 7, further comprising lipid nanoparticles encapsulating the mRNA of any one of claims 1-5, the lipid nanoparticle composition comprising Dlin-MC3-DMA, DSCP, cholesterol, and PEG-DMG.

9. The mRNA drug of claim 8, wherein the lipid nanoparticle comprises, in parts by mole, 20-50 parts Dlin-MC3-DMA, 5-20 parts DSCP, 20-50 parts cholesterol, and 1-5 parts PEG-DMG.

10. The mRNA drug of claim 9, wherein the lipid nanoparticle comprises 50 parts Dlin-MC3-DMA, 10 parts DSCP, 38.5 parts cholesterol, and 1.5 parts PEG-DMG in mole parts.

11. The method for preparing the mRNA drug according to any one of claims 7 to 10, characterized in that the method comprises:

(A) Dissolving mRNA encoding ALDH2 polypeptide in a buffer solution, and adjusting the concentration to be 0.05 mg/mL-0.5 mg/mL to obtain a water phase;

(C) The aqueous phase of step (A) and the organic phase of step (B) are combined according to 1:3, removing ethanol, and concentrating until the concentration of mRNA in the system is 50-1000 mug/mL to obtain the lipid nanoparticle containing the mRNA for encoding the ALDH2 polypeptide.

12. The method of manufacturing according to claim 11, characterized in that the method of manufacturing comprises:

(A) Dissolving mRNA encoding ALDH2 polypeptide in citrate buffer solution with pH of 4, and adjusting the concentration to 0.1mg/ml to obtain water phase;

(C) The aqueous phase of step (a) and the organic phase of step (b) are combined according to 1:3, mixing the mixed solution at a flow rate of 12mL/min by using a microfluidic device, immediately diluting the mixed solution 100 times by using a PBS solution with pH7.4, removing ethanol in the solution by using tangential flow filtration, and concentrating the solution until the concentration of mRNA in the system is 550 mug/mL to obtain lipid nanoparticles containing mRNA encoding ALDH2 polypeptide.