CN114891808B - 编码ALDH2多肽的mRNA分子、应用及mRNA药物 - Google Patents

编码ALDH2多肽的mRNA分子、应用及mRNA药物 Download PDF

Info

Publication number
CN114891808B
CN114891808B CN202210705211.XA CN202210705211A CN114891808B CN 114891808 B CN114891808 B CN 114891808B CN 202210705211 A CN202210705211 A CN 202210705211A CN 114891808 B CN114891808 B CN 114891808B
Authority
CN
China
Prior art keywords
mrna
aldh2
parts
encoding
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210705211.XA
Other languages
English (en)
Other versions
CN114891808A (zh
Inventor
彭育才
张振东
方子辉
刘隽
刘琪
罗丽平
雷奕欣
李爽
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhuhai Lifanda Biotechnology Co ltd
Original Assignee
Zhuhai Lifanda Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhuhai Lifanda Biotechnology Co ltd filed Critical Zhuhai Lifanda Biotechnology Co ltd
Priority to CN202210705211.XA priority Critical patent/CN114891808B/zh
Publication of CN114891808A publication Critical patent/CN114891808A/zh
Priority to PCT/CN2023/093362 priority patent/WO2023246354A1/zh
Application granted granted Critical
Publication of CN114891808B publication Critical patent/CN114891808B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/0101—Acetaldehyde dehydrogenase (acetylating) (1.2.1.10)

Abstract

本发明涉及制药技术领域，尤其是涉及编码ALDH2多肽的mRNA分子、应用及mRNA药物。经过小鼠生存实验验证表明，采用本发明提供的编码ALDH2多肽的mRNA分子制备得到的mRNA药物，能够提高白酒灌胃小鼠的生存率，并且生存率随着药物剂量的提高而提高。同时Western Blot检测、ALDH2多肽活性检测、乙醇代谢产物含量检测和肝脏病理切片检测的多种检测结果表明，该mRNA药物能够在小鼠体内成功表达ALDH2多肽，显著提高ALDH2多肽活性，并在肝脏发挥促进乙醛代谢的作用，从而提高白酒灌胃小鼠生存率。

Description

编码ALDH2多肽的mRNA分子、应用及mRNA药物
技术领域
本发明涉及制药技术领域，尤其是涉及编码ALDH2多肽的mRNA分子、应用及mRNA药物。
背景技术
人类ALDH2(乙醛脱氢酶2，aldehyde dehydrogenase-2)是一种517个氨基酸的多肽，由位于染色体12q24的核基因编码。与ALDH家族的大多数成员一样，ALDH2是一种四聚体酶，在所有组织中普遍表达，但在肝脏中含量最高，并且在需要高线粒体氧化磷酸化的器官(如心脏和大脑)中也有大量表达。ALDH2因其在乙醇代谢中的关键作用而广为人知，且ALDH2可能是人体内唯一对乙醛代谢有显着贡献的ALDH酶。人体的乙醇解毒途径主要发生在肝脏中，由两个酶促步骤进行。第一步由乙醇脱氢酶(ADH)催化，第二步主要由ALDH2催化。
ALDH2*2等位基因是人类最广泛的ALDH2变体，它存在于多达三分之一的东亚人和8％世界人口中。杂合子个体(ALDH2*1/*2)的ALDH2酶活性不到野生型的50％，而ALDH2*2/*2纯合子的ALDH2酶活性不到野生型的 1-4％。ALDH2*2携带者的ALDH2酶活性较低，表现为饮酒后特征性的面部潮红、头痛、恶心、头晕和心悸。
研究表明乙醛在与摄入乙醇相关的中毒中具有明确的作用。摄入的乙醇会迅速代谢为乙醛(通过乙醇脱氢酶)，然后通过线粒体酶ALDH2代谢为乙酸盐。在ALDH2缺乏的受试者中，即使在适量的乙醇之后，血液中的乙醛浓度也会显着升高。急性暴露于乙醛会导致许多与乙醇中毒相关的不愉快影响，包括恶心、心悸、呕吐、头晕和头痛。过度饮酒的ALDH2*1/*2受试者患食道癌和其他上呼吸消化道癌症的风险很大，这可能是乙醛的DNA破坏作用的结果。但是现有针对乙醇(酒精)摄入后，并没有有效促进体内乙醇或乙醛分解的方式，如纳络酮、利尿剂等，前者是阿片类药物过量的解毒剂，也能解除酒精的中枢抑制、缩短昏迷时间，只能让人变清醒；后者是通过加强乙醇及其代谢产物乙醛和酮体经尿液***，都无法加速乙醛的“解毒”过程，更无法保护肝脏被乙醇及其代谢产物损伤。
鉴于此，特提出本发明。
发明内容
本发明的目的在于，提供一种编码ALDH2多肽的核酸分子，包括mRNA分子，采用该mRNA分子制备得到的 mRNA药物，以期能够加速乙醛代谢过程，弥补目前酒精代谢引发疾病治疗药物的空白。
为了解决上述技术问题，实现上述目的，本发明提供以下技术方案：
第一方面，本发明提供编码ALDH2多肽的核酸分子，包括(a)～(c)任一种：
(a)具有SEQ ID No.15～18任一条所示的编码ALDH2多肽的核苷酸序列的mRNA分子；
(b)在(a)限定的核苷酸序列中经过取代、缺失或添加一个或几个核苷酸，具有编码ALDH2多肽功能且与原核苷酸序列具有相同GC碱基对百分比含量的由(a)衍生的mRNA分子；
(c)在严格条件下与(a)或(b)限定的mRNA分子杂交，且ALDH2多肽的核酸分子。
第二方面，本发明提供构建体，所述构建体包括前述实施方式所述的核酸分子，所述构建体的核苷酸序列还包括 UTR序列、5’帽子和poly(A)序列。
优选地，所述5’帽子包括7-甲基-鸟苷-5’-三磷酸-5’-腺苷、鸟苷-5’-三磷酸-5’-腺苷、7-甲基-鸟苷-5’-三磷酸-5’-鸟苷、鸟苷-5’-三磷酸-5’-鸟苷和m7G(5’)(2’-OMeA)pG中的一种或几种；优选为m7G(5’)(2’-OMeA) pG。
优选地，所述poly(A)序列中碱基A的数量为60～120，进一步优选为100。
优选地，所述UTR序列包括5’UTR序列和3’UTR序列。
进一步优选地，所述5’UTR序列具有SEQ ID No.1或SEQ ID No.13所示的核苷酸序列。
进一步优选地，所述3’UTR序列具有SEQ ID No.3或SEQ ID No.14所示的核苷酸序列。
第三方面，本发明提供前述实施方式所核酸分子或前述实施方式所述构建体在制备mRNA药物中的应用。
在可选的实施方式中，所述mRNA药物的用途包括(ⅰ)或(ⅱ)：
(ⅰ)补充ALDH2酶；
(ⅱ)预防和/或治疗酒精代谢性疾病。
优选地，所述酒精代谢性疾病包括酒精引发的肝损伤或酒精性肝炎。
第四方面，本发明提供mRNA药物，所述mRNA药物包括前述实施方式所述的核酸分子或前述实施方式所述的构建体。
在可选的实施方式中，所述mRNA药物还包括包裹前述实施方式所述核酸分子或前述实施方式所述构建体的脂质纳米颗粒，所述脂质纳米颗粒组成包括Dlin-MC3-DMA、DSCP、胆固醇和PEG-DMG。
在可选的实施方式中，所述脂质纳米颗粒按照摩尔份数计包括，20～50份的Dlin-MC3-DMA、5～20份的DSCP、 20～50份的胆固醇和1～5份的PEG-DMG。
在可选的实施方式中，所述脂质纳米颗粒按照摩尔份数计包括50份的Dlin-MC3-DMA、10份的DSCP、38.5份的胆固醇和1.5份PEG-DMG。
在可选的实施方式中，所述制备方法包括：
(A)将编码ALDH2多肽的RNA溶解于缓冲液，将浓度调整为0.05mg/mL～0.5mg/mL，得到水相；
(B)将Dlin-MC3-DMA、DOPG、胆固醇和PEG-DMG溶解于无水乙醇，将有机相中的脂质成分的浓度调整至 5mg/mL～7mg/mL，得到有机相；
(C)将步骤(A)的水相和步骤(B)的有机相按照1：3的体积比混合，除去乙醇后浓缩至体系中的mRNA的浓度为50μg/mL～1000μg/mL，得到包含编码ALDH2多肽的RNA的脂质纳米颗粒。
在可选的实施方式中，所述制备方法包括：
(A)将编码ALDH2多肽的RNA溶解于pH4的柠檬酸盐缓冲液，将浓度调整为0.1mg/ml，得到水相；
(B)将Dlin-MC3-DMA、DOPG、胆固醇和PEG-DMG溶解于无水乙醇，将有机相中的脂质成分的浓度调整至 6mg/mL，得到有机相；
(C)将步骤(a)的水相和步骤(b)的有机相按照1：3的体积比，使用微流控设备，按照12mL/min的流速混合，混合液立刻用pH7.4的PBS溶液稀释100倍，并使用切向流过滤除去溶液中乙醇，然后浓缩至体系中的mRNA 的浓度为550μg/ml，得到包含编码ALDH2多肽的RNA的脂质纳米颗粒。
经过小鼠生存实验验证表明，采用本发明提供的编码ALDH2多肽的mRNA分子制备得到的mRNA药物，能够提高白酒灌胃小鼠的生存率，并且生存率随着药物剂量的提高而提高。同时Western Blot检测、ALDH2多肽活性检测、乙醇代谢产物含量检测和肝脏病理切片检测的多种检测结果表明，该mRNA药物能够在小鼠体内成功表达 ALDH2多肽，显著提高ALDH2多肽活性，并在肝脏发挥促进乙醛代谢的作用，从而提高白酒灌胃小鼠生存率。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实验例1中Western Blot检测结果；
图2为本发明实验例2中ALDH2-KO小鼠构建检测结果；
图3为本发明实验例3中hALDH-2和mALDH2蛋白表达结果；
图4为本发明实验例4中SEQ ID No.4的GC碱基对百分含量；
图5为本发明实验例4中SEQ ID No.15的GC碱基对百分含量；
图6为本发明实验例3中各mRNA序列表达的ALDH2蛋白的结果；
图7为本发明SEQ ID No.16的GC碱基对百分含量；
图8为本发明SEQ ID No.17的GC碱基对百分含量；
图9为本发明SEQ ID No.18的GC碱基对百分含量；
图10为本发明SEQ ID No.20的GC碱基对百分含量；
图11为本发明SEQ ID No.21的GC碱基对百分含量；
图12为本发明实验例6中ALDH2多肽表达量检测结果；
图13为本发明实验例6中ALDH2多肽活性检测结果；
图14为本发明实验例6中乙醇代谢检测结果；
图15为本发明实验例6中小鼠肝脏病理切片中低剂量组和对照组对比结果；
图16为本发明实验例6中小鼠肝脏病理切片中低剂量组和高剂量组对比结果；
图17为本发明实验例6中小鼠肝脏病理切片中高剂量死亡组镜检结果。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。通常在此处附图中描述和示出的本发明实施例的组件可以以各种不同的配置来布置和设计。
因此，以下对在附图中提供的本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
在某次具体实施方式中，第一方面，本发明提供编码ALDH2多肽的核酸分子，包括(a)～(c)任一种：
(a)具有SEQ ID No.15～18任一条所示的编码ALDH2多肽的核苷酸序列的mRNA分子；
(b)在(a)限定的核苷酸序列中经过取代、缺失或添加一个或几个核苷酸，具有编码ALDH2多肽功能且与原核苷酸序列具有相同GC碱基对百分比含量的由(a)衍生的mRNA分子；
(c)在严格条件下与(a)或(b)限定的mRNA分子杂交，且ALDH2多肽的核酸分子。
本发明提供的SEQ ID No.13～15所示的编码ALDH2多肽的核苷酸序列中局部的GC碱基对百分比含量不低于 40％，总体的GC碱基对百分比含量为55％～65％。其中“局部的GC碱基对百分比含量”是指从ORF序列的3’端到 5’端，以60bp作为window size的局部序列中GC碱基对百分比含量。
上述核酸分子(b)中“GC碱基对百分比含量”包括局部的GC碱基对百分比含量和总体的GC碱基对百分比含量。
可以理解的是，上述核酸分子(c)在“严格条件”与(a)或(b)所述的mRNA杂交，是指严格依据碱基互补配对原则，以mRNA分子(a)或(b)为模板杂交获得的核酸分子(c)，例如单链DNA或其他RNA分子。对于具体的“严格条件”的反应条件，本领域技术人员能够参考现有技术和公知常识，根据目的产物进行常规选择。
第二方面，本发明提供构建体，所述构建体包括前述实施方式所述的mRNA分子，所述构建体的核苷酸序列还包括UTR序列、5’帽子和poly(A)序列。
优选地，所述5’帽子包括7-甲基-鸟苷-5’-三磷酸-5’-腺苷、鸟苷-5’-三磷酸-5’-腺苷、7-甲基-鸟苷-5’-三磷酸-5’-鸟苷、鸟苷-5’-三磷酸-5’-鸟苷和m7G(5’)(2’-OMeA)pG中的一种或几种；优选为m7G(5’)(2’-OMeA) pG。
优选地，所述poly(A)序列中碱基A的数量为60～120，进一步优选为100。
优选地，所述UTR序列包括5’UTR序列和3’UTR序列。
进一步优选地，所述5’UTR序列具有SEQ ID No.1或SEQ ID No.13所示的核苷酸序列。
进一步优选地，所述3’UTR序列具有SEQ ID No.3或SEQ ID No.14所示的核苷酸序列。
第三方面，本发明提供前述实施方式所述mRNA分子或前述实施方式所述构建体在制备mRNA药物中的应用。
在可选的实施方式中，所述mRNA药物的用途包括(ⅰ)或(ⅱ)：
(ⅰ)补充ALDH2酶；
(ⅱ)预防和/或治疗酒精代谢性疾病。
优选地，所述酒精代谢性疾病包括酒精引发的肝损伤或酒精性肝炎。
第四方面，本发明提供mRNA药物，所述mRNA药物包括前述实施方式所述的mRNA分子或前述实施方式所述的构建体。
在可选的实施方式中，所述mRNA药物还包括包裹权利要求1所述mRNA分子或权利要求2所述构建体的脂质纳米颗粒，所述脂质纳米颗粒组成包括Dlin-MC3-DMA、DSCP、胆固醇和PEG-DMG。
在可选的实施方式中，所述脂质纳米颗粒按照摩尔份数计包括，20～50份的Dlin-MC3-DMA、5～20份的DSCP、 20～50份的胆固醇和1～5份的PEG-DMG。
在可选的实施方式中，所述脂质纳米颗粒按照摩尔份数计包括50份的Dlin-MC3-DMA、10份的DSCP、38.5份的胆固醇和1.5份PEG-DMG。
在可选的实施方式中，所述制备方法包括：
(A)将编码ALDH2多肽的RNA溶解于缓冲液，将浓度调整为0.05mg/mL～0.5mg/mL，得到水相；
(B)将Dlin-MC3-DMA、DOPG、胆固醇和PEG-DMG溶解于无水乙醇，将有机相中的脂质成分的浓度调整至 5mg/mL～7mg/mL，得到有机相；
(C)将步骤(A)的水相和步骤(B)的有机相按照1：3的体积比混合，除去乙醇后浓缩至体系中的mRNA的浓度为50μg/mL～1000μg/mL，得到包含编码ALDH2多肽的RNA的脂质纳米颗粒。
在可选的实施方式中，所述制备方法包括：
(A)将编码ALDH2多肽的RNA溶解于pH4的柠檬酸盐缓冲液，将浓度调整为0.1mg/ml，得到水相；
(B)将Dlin-MC3-DMA、DOPG、胆固醇和PEG-DMG溶解于无水乙醇，将有机相中的脂质成分的浓度调整至 6mg/mL，得到有机相；
(C)将步骤(a)的水相和步骤(b)的有机相按照1：3的体积比，使用微流控设备，按照12mL/min的流速混合，混合液立刻用pH7.4的PBS溶液稀释100倍，并使用切向流过滤除去溶液中乙醇，然后浓缩至体系中的mRNA 的浓度为550μg/ml，得到包含编码ALDH2多肽的RNA的脂质纳米颗粒。
下面结合附图，对本发明的一些实施方式作详细说明。在不冲突的情况下，下述的实施例及实施例中的特征可以相互组合。
实施例1
本实施例提供了四条编码ALDH2多肽的mRNA，具体的核苷酸序列分别如SEQ IDNo.15～18所示。
实施例2
本实施例提供了8组编码ALDH2多肽的mRNA，所述mRNA除了包含编码ALDH2多肽的ORF外，还包含5’端帽子、UTR和3’尾巴，并且mRNA中尿嘧啶均被替换为1-甲基假尿苷，具体序列编号及组成如下：
实施例3
本实施例提供8组mRNA药物，包括编码ALDH2多肽的mRNA和包裹该mRNA的脂质纳米颗粒，其中脂质纳米颗粒按照摩尔百分比计包括Dlin-MC3-DMA 50％、DSCP 10％、胆固醇38.5％和PEG-DMG 1.5％。
该mRNA药物的制备方法如下：
(a)将人工合成的编码ALDH2多肽的8组mRNA(序列分别如SEQ ID No.5～12所示)溶解于pH4的柠檬酸盐缓冲液，将浓度调整为0.1mg/ml，得到水相。
(b)按照配方量将Dlin-MC3-DMA、DOPG、胆固醇和PEG-DMG按照配方量溶解于无水乙醇，将有机相中的脂质成分的浓度调整至6mg/mL，得到有机相。
(c)将步骤(a)的水相和步骤(b)的有机相按照1:3的体积比，使用微流控设备，按照12mL/min的流速混合，混合液立刻用pH7.4的PBS溶液稀释100倍，并使用切向流过滤(TFF)除去溶液中乙醇成分，然后浓缩至体系中的 mRNA的浓度为550μg/ml，得到包含编码ALDH2多肽的RNA的脂质纳米颗粒。
实施例4
本实施例提供另外8组mRNA药物，与实施例3的区别在于，脂质纳米颗粒按照摩尔份数计包括20份的Dlin- MC3-DMA、5份的DSCP、20份胆固醇和1份PEG-DMG。
实施例5
本实施例提供另外8组mRNA药物，与实施例3的区别在于，脂质纳米颗粒按照摩尔份数计包括50份的Dlin- MC3-DMA、20份的DSCP、50份胆固醇和5份PEG-DMG。
实验例1
本实验例检测实施例1提供的ALDH2多肽在体外细胞水平的表达情况，具体方法如下：
(1)转染
将Lip2000试剂10μL与40μL opti-MEM混合均匀静置，向其中再加入46μL opti-MEM，然后分别加入2μL DNA质粒和2μL mRNA(μg/μL)，静置，将其加入HEK293细胞中，并在细胞培养箱中孵育。
(2)使用Western Blot法检测细胞内ALDH2蛋白的表达
将细胞上清液去除，使用PBS清洗细胞2次，加250μL细胞裂解液裂(含PMSF)于离心管中，进行离心，取上清分装于1.5mL离心管中。使用BCA试剂盒对细胞的蛋白浓度进行测定。接着使用预制胶进行电泳，加入重组 Anti-ALDH2抗体和Goat Anti-Rabbit IgG H&L(HRP)，对PVDF膜进行化学发光，显色。结果如图1所示，其中Control 为空脂质组；1为环状质粒；2为实施例1提供的mRNA，可以看出，DNA质粒和mRNA组ALDH2蛋白表达量显著高于对照组，ALDH2蛋白的DNA和mRNA能够在HEK293细胞中稳定表达。
实验例2
本实验例构建了ALDH2-KO小鼠，具体方法如下：
委托上海南方模式生物科技股份有限公司通过CRISPR/Cas9技术获得F0代ALDH2-KO小鼠。使用Western Blot 技术检测小鼠肝脏中ALDH2蛋白的表达。结果如图2显示，ALDH2-KO小鼠肝脏中的ALDH2表达远低于野生型小鼠，KO小鼠模型成立。
实验例3
本实验例考察人源ALDH2 mRNA和鼠源ALDH2 mRNA药物在KO小鼠中的表达情况，具体方法如下：
ALDH2-KO小鼠数量(“ALDH2-KO小鼠”是指“ALDH2*2/*2纯合子的小鼠”)：9只；对照组为PBS组，给药组为人源ALDH2 mRNA(hALDH2)组，鼠源ALDH2 mRNA(mALDH2)组；共3组，每组3只小鼠。
实验方案：(1)对照组：以尾静脉注射方式注射100μL PBS溶液ALDH2-KO小鼠；给药组：根据小鼠的体重，以尾静脉注射方式2.5mg/kg的剂量给予KO小鼠人源ALDH2 mRNA(hALDH2)(mRNA的ORF序列为SEQ ID No.15)或鼠源ALDH2 mRNA(mALDH2)(mRNA序列为SEQID No.19，5’帽子为：m7G(5’)(2’-OMeA) pG)，其中mRNA按照实施例1的方法制备为LNP形式。
(2)给药4小时后，根据小鼠的体重，9只ALDH2-KO小鼠按照13μg/g的剂量以灌胃方式给与52度白酒。
小鼠的肝脏采样时间点：白酒灌胃后2h和6h。样品采集后放入液氮中速冻，然后放入-80冰箱保存。
采用Western Blot检测小鼠肝脏中ALDH-2蛋白的表达：将肝脏组织块置于离心管剪碎，加250μL组织裂解液 (含PMSF)于离心管中，进行匀浆离心，取上清分装于1.5mL离心管中。使用BCA试剂盒对样品的蛋白浓度进行测定。接着使用预制胶进行电泳，加入重组Anti-ALDH2抗体和Goat Anti-Mouse IgG H&L(HRP)，对PVDF膜进行化学发光，显色。
实验结果如图3所示：(1)对照组小鼠酒精灌胃后全部死亡，而mALDH-2组小鼠存活1只，hALDH-2组小鼠存活2只；(2)hALDH-2组和mALDH-2组中hALDH-2和mALDH2蛋白均在小鼠肝脏中表达。
实验例4
对上述SEQ ID No.4和SEQ ID No.15的GC碱基对百分含量进行考察，如图4和图5所示。SEQ ID No.4的总 GC％含量为54.03％，其中碱基A％含量为24.24％，碱基C％含量为25.21％，碱基G％含量为28.82％，碱基U％含量为21.73％。由图4可以看出SEQ ID No.4局部的GC碱基对百分比含量在40％～65％。
SEQ ID No.15的总GC％含量为58.75，其中碱基A％含量为21.24％，碱基C％含量为27.8％，碱基G％含量为 30.95％，碱基U％含量为20.01％。由图5可以看出SEQ ID No.局部的GC碱基对百分比含量在41％～83％。
SEQ ID No.16的总GC％含量为59.72％，其中碱基A％含量为21.75％，碱基C％含量为28.19％，碱基G％含量为31.53％，碱基U％含量为18.53％。由图7可以看出SEQ ID No.局部的GC碱基对百分比含量在41％～83％
SEQ ID No.17的总GC％含量为59.38％，其中碱基A％含量为23.02％，碱基C％含量为29.08％，碱基G％含量为30.3％，碱基U％含量为17.6％。由图8可以看出SEQ ID No.局部的GC碱基对百分比含量在41％～78％。
SEQ ID No.18的总GC％含量为59.42％，其中碱基A％含量为23.02％，碱基C％含量为29.21％，碱基G％含量为30.11％，碱基U％含量为17.67％。由图9可以看出SEQ ID No.局部的GC碱基对百分比含量在42％～77％。
SEQ ID No.20的总GC％含量为52.49％，其中碱基A％含量为25.66％，碱基C％含量为23.15％，碱基G％含量为29.34％，碱基U％含量为21.86％。由图10可以看出SEQ IDNo.局部的GC碱基对百分比含量在40％～63％。
SEQ ID No.21的总GC％含量为66.73％，其中碱基A％含量为18.57％，碱基C％含量为35.01％，碱基G％含量为31.72％，碱基U％含量为14.7％。由图11可以看出SEQ ID No.局部的GC碱基对百分比含量在52％～87％。
实验例5
参考前述Western blot法检测实验例3中各mRNA序列表达的ALDH2蛋白，结果如图6所示，实验结论：(1) 编辑ALDH2多肽的mRNA中ORF序列为SEQ ID No.15的表达量远远高于ORF序列为SEQ ID No.4的表达量； (2)5’UTR为SEQ ID No.1和3’UTR为SEQ ID No.2时，ALDH2多肽的表达量高于其他5’UTR和3’UTR的组合方式。
经过进一步的筛选编辑ALDH2多肽的ORF序列，发现当ORF序列总体的GC％含量为30～70％时，局部的GC％含量不低于40％时(如SEQ ID No.15～18)，ALDH2多肽的表达量较高；更进一步的，ORF序列局部的GC％含量不低于40％时，总体的GC％含量为55％～60％的序列(如SEQ ID No.15～18)ALDH2多肽的表达量略高于总体的GC％含量30％～55％以及60％～70％的序列(如SEQ ID No.4，20、21)。
实验例6
本实验例考察ALDH2 mRNA药物保护KO小鼠免受酒精损伤情况，具体方法如下：
随机将15只ALDH2-KO小鼠数量分为3组(分别为PBS组、高剂量组和低剂量组)，每组5只。
实验方案：(1)给对照组ALDH2-KO小鼠用尾静脉注射方式注射100μL PBS溶液；根据小鼠的体重，分别按照2.5mg/kg和1.25mg/kg的剂量给高剂量组和低剂量组ALDH2-KO小鼠用尾静脉注射方式注射实施例3提供的编码mALDH2多肽的mRNA药物，其中mRNA的核苷酸序列如SEQ ID No.10所示。
(2)4小时后，根据小鼠的体重，15只ALDH2-KO小鼠按照13μg/g的剂量以灌胃方式递送52度白酒。
KO小鼠血清(50μL)采样时间点：mRNA给药前、灌胃前、灌胃后1、6、10、24、48h，采集后放入液氮中速冻，然后放入-80℃冰箱保存。
实验结果：
1、生存情况
白酒灌胃后24h，PBS组小鼠5只全部死亡；低剂量给药组(1.25mg/kg)小鼠死亡2只，存活3只；高剂量给药组(2.5mg/kg)小鼠死亡1只，存活4只。
2、ALDH2多肽的肝脏表达情况
采用Western Blot分别检测小鼠肝脏中ALDH-2蛋白的表达情况。将肝脏组织块置于离心管剪碎，加250μL组织裂解液裂(含PMSF)于离心管中，进行匀浆离心，取上清分装于1.5mL离心管中。使用BCA试剂盒对样品的蛋白浓度进行测定。接着使用预制胶进行电泳，加入重组Anti-ALDH2抗体和Goat Anti-Mouse IgG H&L(HRP)，对PVDF 膜进行化学发光，显色。结果如图12显示，在高、低剂量组中ALDH2蛋白均在小鼠肝脏中表达。
3、ALDH2多肽的活性
使用ELISA对小鼠肝脏组织中的ALDH2酶活性进行检测。取约10mg小鼠肝脏组织，加入Buffer于冰上裂解混匀，使用离心机离心。取上清，稀释20倍于96孔板中。制备标准曲线后，将底物进行水解，将20μL水解底物加入96孔板中孵育，最后使用200μL终止液来终止反应，放入多功能酶标仪中进行检测。结果如图13所示，表明与 PBS组相比，高、低剂量组中ALDH2酶活性均显著提高，并且随着mRNA给药剂量的提高，其酶活性也显著提高。
4、血清中乙醇、乙醛和乙酸的含量变化
采用气相色谱法检测顶空进样条件：恒定模式；炉温为65℃；平衡时间为10min；定量温度为105℃；传输线温度为110℃；针温为90℃；样品加压时间为0.1min；定量环充满时间为0.1min；定量环平衡时间:0.05min。取0.2 mL待检小鼠血清，将其置于2mL样品瓶中，压紧瓶盖，置于顶空进样器样品架上进行测定。
(1)白酒灌胃前后小鼠血液中的乙醇、乙醛和乙酸含量，检测结果如图12所示，结果说明：白酒灌胃给药后 24h，可以看到：
(a)PBS组、高剂量组和低剂量组的小鼠血液中的乙醇、乙醛和乙酸含量(24h)比白酒灌胃给药前(0h)都明显升高。
(b)白酒灌胃给药后24h，PBS组小鼠血液中的乙醇含量显著高于高剂量组和低剂量组的小鼠血液中的乙醇含量(P<0.05)，以注射编码hALDH2多肽的mRNA的形式对ALDH2-KO小鼠补充hALDH2多肽，可以加速乙醇代谢。
(c)PBS组小鼠血液中的乙醛含量显著高于高剂量组和低剂量组的小鼠血液中的乙醛含量(P<0.05)，PBS组小鼠血液中的乙酸含量显著低于高剂量组和低剂量组的小鼠血液中的乙酸(P<0.05)，低剂量组小鼠血液中的乙醛含量显著高于高剂量组的小鼠血液中的乙醛含量(P<0.05)，低剂量组小鼠血液中的乙酸含量显著低于高剂量组的小鼠血液中的乙酸含量(P<0.05)。说明以注射编码hALDH2多肽的mRNA的形式对ALDH2-KO小鼠补充hALDH2多肽，可以加速乙醛代谢为乙酸；并呈现剂量依赖，如图14所示。
5、小鼠肝脏病理切片
将小鼠肝脏组织进行脱水切片，首先将肝脏组织经过一系列的酒精脱水以及石蜡包埋与切片；然后将石蜡切片进行苏木素-伊红染色，最后切片封固。在显微镜下观察鼠肝脏组织的病理情况。
结果如图15～17所示，PBS组小鼠肝脏出现肝细胞肿胀，脂滴弥漫，炎细胞浸润，见圆形或类圆形脂滴空泡，个别细胞肿大呈气球样变，细胞形态严重破坏。mRNA中剂量组小鼠肝脏细胞肿胀程度减少，脂滴弥撒程度减轻，细胞核形态稍微完整规则。mRNA高剂量组肝细胞形态恢复正常，肝细胞轮廓清晰可见，脂滴显著减少，无显著炎细胞浸润。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
SEQUENCE LISTING
<110> 珠海丽凡达生物技术有限公司
<120> 编码ALDH2多肽的mRNA分子、应用及mRNA药物
<160> 21
<170> PatentIn version 3.5
<210> 1
<211> 15
<212> RNA
<213> Artificial Sequence
<220>
<223> 5'UTR序列一
<400> 1
gggagaaagc uuacc 15
<210> 2
<211> 100
<212> RNA
<213> Artificial Sequence
<220>
<223> 3'polyA尾
<400> 2
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 100
<210> 3
<211> 74
<212> RNA
<213> Artificial Sequence
<220>
<223> 3'UTR序列一
<400> 3
ggacuaguua uaagacugac uagcccgaug ggccucccaa cgggcccucc uccccuccuu 60
gcaccgagau uaau 74
<210> 4
<211> 1551
<212> RNA
<213> Artificial Sequence
<220>
<223> ORF序列一
<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> 实施例3提供mRNA编码序列一
<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> 实施例3提供mRNA序列二
<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> 实施例3提供mRNA序列三
<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> 实施例3提供mRNA序列四
<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> 实施例3提供mRNA序列五
<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> 实施例3提供mRNA序列六
<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> 实施例3提供mRNA序列七
<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> 实施例3提供mRNA序列八
<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> 5'UTR的序列二
<400> 13
gagacccaag cuggcuagcg ggagaaagcu uacc 34
<210> 14
<211> 109
<212> RNA
<213> Artificial Sequence
<220>
<223> 3'UTR序列二
<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序列二
<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序列三
<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序列四
<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序列五
<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> 鼠源ORF序列
<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序列六
<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序列七
<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 (12)

1. mRNA，其特征在于，包括编码ALDH2多肽的ORF、5’UTR序列、3’UTR序列、5’帽子和poly（A）序列；所述编码ALDH2多肽的ORF如SEQ ID No.15所示；所述5’UTR序列如SEQ IDNo.1所示，所述3’UTR序列如SEQ ID No.3所示。
2.根据权利要求1所述的mRNA，其特征在于，所述5’帽子包括7-甲基-鸟苷-5’-三磷酸-5’-腺苷、鸟苷-5’-三磷酸-5’-腺苷、7-甲基-鸟苷-5’-三磷酸-5’-鸟苷、鸟苷-5’-三磷酸-5’-鸟苷和m7G（5’）（2’-OMeA）pG中的一种或几种。
3.根据权利要求2所述的mRNA，其特征在于，所述5’帽子为m7G（5’）（2’-OMeA）pG。
4.根据权利要求1所述的mRNA，其特征在于，所述poly（A）序列中碱基A的数量为60~120。
5.根据权利要求4所述的mRNA，其特征在于，所述poly（A）序列中碱基A的数量为100。
6.权利要求1~5任一项所述mRNA在制备mRNA药物中的应用，所述mRNA药物的用途包括（ⅰ）或（ⅱ）：
（ⅰ）补充ALDH2酶；
（ⅱ）治疗缺乏ALDH2酶合并酒精引发的肝损伤或酒精性肝炎。
7. mRNA药物，其特征在于，所述mRNA药物包括权利要求1~5任一项所述mRNA，所述mRNA药物的用途包括（ⅰ）或（ⅱ）：
（ⅰ）补充ALDH2酶；
（ⅱ）治疗缺乏ALDH2酶合并酒精引发的肝损伤或酒精性肝炎。
8.根据权利要求7所述的mRNA药物，其特征在于，所述mRNA药物还包括包裹权利要求1~5任一项所述mRNA的脂质纳米颗粒，所述脂质纳米颗粒组成包括Dlin-MC3-DMA、DSCP、胆固醇和PEG-DMG。
9. 根据权利要求8所述的mRNA药物，其特征在于，所述脂质纳米颗粒按照摩尔份数计包括，20~50份的Dlin-MC3-DMA 、5~20份的DSCP、20~50份的胆固醇和1~5份的PEG-DMG。
10.根据权利要求9所述的mRNA药物，其特征在于，所述脂质纳米颗粒按照摩尔份数计包括50份的Dlin-MC3-DMA、10份的DSCP、38.5份的胆固醇和1.5份PEG-DMG。
11.权利要求7~10任一项所述mRNA药物的制备方法，其特征在于，所述制备方法包括：
（A）将编码ALDH2多肽的mRNA溶解于缓冲液，将浓度调整为 0.05mg/mL~0.5mg/mL，得到水相；
（B）将Dlin-MC3-DMA、DOPG、胆固醇和PEG-DMG溶解于无水乙醇，将有机相中的脂质成分的浓度调整至5mg/mL~7mg/mL，得到有机相；
（C）将步骤（A）的水相和步骤（B）的有机相按照1：3的体积比混合，除去乙醇后浓缩至体系中的mRNA的浓度为50μg/mL~1000μg/mL，得到包含编码ALDH2多肽的mRNA的脂质纳米颗粒。
12.根据权利要求11所述的制备方法，其特征在于，所述制备方法包括：
（A）将编码ALDH2多肽的mRNA溶解于pH4的柠檬酸盐缓冲液，将浓度调整为 0.1mg/ml，得到水相；
（B）将Dlin-MC3-DMA、DOPG、胆固醇和PEG-DMG溶解于无水乙醇，将有机相中的脂质成分的浓度调整至6mg/mL，得到有机相；
（C）将步骤（a）的水相和步骤（b）的有机相按照1：3的体积比，使用微流控设备，按照12mL/min的流速混合，混合液立刻用pH7.4的PBS 溶液稀释100倍，并使用切向流过滤除去溶液中乙醇，然后浓缩至体系中的mRNA的浓度为550μg/ml，得到包含编码ALDH2多肽的mRNA的脂质纳米颗粒。
CN202210705211.XA 2022-06-21 2022-06-21 编码ALDH2多肽的mRNA分子、应用及mRNA药物 Active CN114891808B (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202210705211.XA CN114891808B (zh) 2022-06-21 2022-06-21 编码ALDH2多肽的mRNA分子、应用及mRNA药物
PCT/CN2023/093362 WO2023246354A1 (zh) 2022-06-21 2023-05-11 编码ALDH2多肽的mRNA分子、应用及mRNA药物

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210705211.XA CN114891808B (zh) 2022-06-21 2022-06-21 编码ALDH2多肽的mRNA分子、应用及mRNA药物

Publications (2)

Publication Number Publication Date
CN114891808A CN114891808A (zh) 2022-08-12
CN114891808B true CN114891808B (zh) 2023-10-27

Family

ID=82727220

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210705211.XA Active CN114891808B (zh) 2022-06-21 2022-06-21 编码ALDH2多肽的mRNA分子、应用及mRNA药物

Country Status (2)

Country Link
CN (1) CN114891808B (zh)
WO (1) WO2023246354A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114891808B (zh) * 2022-06-21 2023-10-27 珠海丽凡达生物技术有限公司 编码ALDH2多肽的mRNA分子、应用及mRNA药物

Citations (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007016953A1 (en) * 2005-07-29 2007-02-15 Matuschka-Greiffenclau Markus Composition for reducing alcohol induced liver cancer risk
AU2006278823A1 (en) * 2005-07-29 2007-02-15 Tima Foundation Composition for moderating alcohol metabolism and for reducing the risk of alcohol induced diseases
WO2012095509A1 (en) * 2011-01-14 2012-07-19 Tima Foundation Composition for accelerating alcohol metabolism and for reducing the risk of alcohol induced diseases
CN104120180A (zh) * 2014-07-15 2014-10-29 江苏伟禾生物科技有限公司 一种人类aldh2基因型检测试剂盒
CN105400871A (zh) * 2015-11-16 2016-03-16 北京晋祺生物科技有限公司 Aldh2基因的检测引物组、其构成的反应体系及应用
CN107050148A (zh) * 2017-06-13 2017-08-18 广州中天康顺生物医药有限公司 一种解酒保肝中药组合物及其制备方法
CN107789370A (zh) * 2017-11-29 2018-03-13 温州医科大学附属第医院 预防和治疗酒精性肝病的药剂
CN109952114A (zh) * 2016-07-26 2019-06-28 康奈尔大学 用于治疗醛脱氢酶缺乏的基因疗法
CN110452951A (zh) * 2019-08-16 2019-11-15 珠海丽凡达生物技术有限公司 监测mRNA Ploy(A)尾长度的方法及应用
CN110638759A (zh) * 2019-10-29 2020-01-03 珠海丽凡达生物技术有限公司 一种用于体外转染和体内递送mRNA的制剂
CN110812366A (zh) * 2019-11-18 2020-02-21 珠海丽凡达生物技术有限公司 一种可用于激素补充的mRNA药物及其制备方法
CN111565733A (zh) * 2017-09-13 2020-08-21 Z生命科学公司 用于益生微生物的基因表达***
CN111601891A (zh) * 2018-01-16 2020-08-28 迪克纳制药公司 用于抑制aldh2表达的组合物和方法
CN112210519A (zh) * 2019-07-09 2021-01-12 深伦生物科技（深圳）有限公司 一种以食用菌分泌乙醛脱氢酶的基因工程菌
CN112930198A (zh) * 2018-09-04 2021-06-08 德克萨斯大学***董事会 用于核酸的器官特异性递送的组合物和方法
CN112996519A (zh) * 2018-09-04 2021-06-18 德克萨斯大学***董事会 用于核酸的器官特异性递送的组合物和方法
WO2022092310A1 (ja) * 2020-11-02 2022-05-05 株式会社アークメディスン 化合物、アルデヒドデヒドロゲナーゼ２活性化剤、医薬組成物、並びに治療及び／又は予防薬
WO2023056914A1 (en) * 2021-10-08 2023-04-13 Suzhou Abogen Biosciences Co., Ltd. Lipid compounds and lipid nanoparticle compositions

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019195592A1 (en) * 2018-04-04 2019-10-10 The University Of Chicago Genetically-engineered microbes and compositions thereof
WO2021167356A1 (ko) * 2020-02-18 2021-08-26 주식회사 피코엔텍 글루타치온과 알데히드탈수소효소를 함유하는 숙취해소제
CN114891808B (zh) * 2022-06-21 2023-10-27 珠海丽凡达生物技术有限公司 编码ALDH2多肽的mRNA分子、应用及mRNA药物

Patent Citations (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2006278823A1 (en) * 2005-07-29 2007-02-15 Tima Foundation Composition for moderating alcohol metabolism and for reducing the risk of alcohol induced diseases
WO2007016953A1 (en) * 2005-07-29 2007-02-15 Matuschka-Greiffenclau Markus Composition for reducing alcohol induced liver cancer risk
WO2012095509A1 (en) * 2011-01-14 2012-07-19 Tima Foundation Composition for accelerating alcohol metabolism and for reducing the risk of alcohol induced diseases
CN104120180A (zh) * 2014-07-15 2014-10-29 江苏伟禾生物科技有限公司 一种人类aldh2基因型检测试剂盒
CN105400871A (zh) * 2015-11-16 2016-03-16 北京晋祺生物科技有限公司 Aldh2基因的检测引物组、其构成的反应体系及应用
CN109952114A (zh) * 2016-07-26 2019-06-28 康奈尔大学 用于治疗醛脱氢酶缺乏的基因疗法
CN107050148A (zh) * 2017-06-13 2017-08-18 广州中天康顺生物医药有限公司 一种解酒保肝中药组合物及其制备方法
CN111565733A (zh) * 2017-09-13 2020-08-21 Z生命科学公司 用于益生微生物的基因表达***
CN107789370A (zh) * 2017-11-29 2018-03-13 温州医科大学附属第医院 预防和治疗酒精性肝病的药剂
CN111601891A (zh) * 2018-01-16 2020-08-28 迪克纳制药公司 用于抑制aldh2表达的组合物和方法
CN112930198A (zh) * 2018-09-04 2021-06-08 德克萨斯大学***董事会 用于核酸的器官特异性递送的组合物和方法
CN112996519A (zh) * 2018-09-04 2021-06-18 德克萨斯大学***董事会 用于核酸的器官特异性递送的组合物和方法
CN112210519A (zh) * 2019-07-09 2021-01-12 深伦生物科技（深圳）有限公司 一种以食用菌分泌乙醛脱氢酶的基因工程菌
CN110452951A (zh) * 2019-08-16 2019-11-15 珠海丽凡达生物技术有限公司 监测mRNA Ploy(A)尾长度的方法及应用
CN110638759A (zh) * 2019-10-29 2020-01-03 珠海丽凡达生物技术有限公司 一种用于体外转染和体内递送mRNA的制剂
CN110812366A (zh) * 2019-11-18 2020-02-21 珠海丽凡达生物技术有限公司 一种可用于激素补充的mRNA药物及其制备方法
WO2022092310A1 (ja) * 2020-11-02 2022-05-05 株式会社アークメディスン 化合物、アルデヒドデヒドロゲナーゼ２活性化剤、医薬組成物、並びに治療及び／又は予防薬
WO2023056914A1 (en) * 2021-10-08 2023-04-13 Suzhou Abogen Biosciences Co., Ltd. Lipid compounds and lipid nanoparticle compositions

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
乙醛脱氢酶2基因多态性与慢性乙型肝炎和酒精性肝病之间的关系;熊志娇 等;《热带医学杂志》;全文 *
王维 等主编.《消化疾病护理与专科实践》.吉林科学技术出版社,2019,第179-180页. *

Also Published As

Publication number Publication date
CN114891808A (zh) 2022-08-12
WO2023246354A1 (zh) 2023-12-28

Similar Documents

Publication Publication Date Title
CA3024625A1 (en) Polynucleotides encoding citrin for the treatment of citrullinemia type 2
TW201839136A (zh) 治療血色素異常症之組合物及方法
CA3024507A1 (en) Polynucleotides encoding .alpha.-galactosidase a for the treatment of fabry disease
WO2011147086A1 (zh) 载有干扰核糖核酸的细胞微粒子、其制备方法及其应用
CN114891808B (zh) 编码ALDH2多肽的mRNA分子、应用及mRNA药物
CA2527109A1 (en) Oligo double-stranded rna inhibiting the expression of bcl-2 and medicinal composition containing the same
WO2012131365A1 (en) Therapeutic molecules for use in the suppression of parkinson&#39;s disease
JPH06508622A (ja) ｂｃｒ−ａｂ１アンチセンスオリゴヌクレオチドによる白血球増殖の選択的抑制
Grobner et al. Effect of allopurinol and its metabolic derivatives on the configuration of human orotate phosphoribosyltransferase and orotidine 5'-phosphate decarboxylase
Schapira et al. An improved technique for preparation of skeletal muscle cell plasma membrane
CN112996913A (zh) 寡聚核酸分子及其应用
WO2022105903A1 (zh) 一种治疗肝纤维化的siRNA及其递送制剂
Bister et al. Biological and biochemical studies on the inactivation of avian oncoviruses by ultraviolet irradiation
Bossart et al. Enucleation of cells by density gradient centrifugation
AU2022256039A1 (en) Specific oligonucleotide-programmed readthrough of nonsense codons
CN116162620A (zh) 一种小干扰核酸和制剂及其制药应用
CN116510001B (zh) 一种水产养殖用mRNA疫苗及其制备方法
Rothgangl et al. Treatment of a genetic liver disease in mice through transient prime editor expression
CN114836379B (zh) 抗血液肿瘤药物活性成分的获得方法及其用途
CN113209301B (zh) γCamkⅡ表达促进剂或稳定剂在制备减缓衰老和抗神经退行性药物中的应用
CN114457045B (zh) 抑制Slc2a1的RNAi腺相关病毒及其制备和应用
Ali et al. Characterization of Circulating Exosomal MicroRNAs and Its Role as Biomarkers for Metabolic Syndrome
CN116478893B (zh) 一种产丙酸的工程益生菌的构建与应用
CN117137936A (zh) 以NOX1为靶点的小干扰siRNA对心肌缺血再灌注损伤的应用
Pouwels et al. A transcriptional barrier in the regulatory region of the tryptophan operon of Escherichia coli: Its role in the regulation of repressor-independent RNA synthesis

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant