vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/vjgb-25-81vavilov-4801Research ArticleМОЛЕКУЛЯРНАЯ И КЛЕТОЧНАЯ БИОЛОГИЯMOLECULAR AND CELL BIOLOGYНовая комбинация 5’- и 3’-нетранслируемых областей способствует повышению экспрессии мРНК in vitro и in vivoA new combination of 5’- and 3’-untranslated regions increases the expression of mRNAs in vitro and in vivoАнтроповД. Н.AntropovD. N.

 Новосибирск 

 Novosibirsk 

МарковО. В.MarkovO. V.

 Новосибирск 

 Novosibirsk 

ДомеА. С.DomeA. S.

 Новосибирск 

 Novosibirsk 

ПучковП. А.PuchkovP. A.

 Москва 

 Moscow 

ШмендельЕ. В.ShmendelE. V.

 Шмендель

 Moscow 

ГладкихД. В.GladkikhD. V.

 Новосибирск 

 Novosibirsk 

ГолышевВ. М.GolyshevV. M.

 Новосибирск 

 Novosibirsk 

МатвееваА. М.MatveevaA. M.

 Новосибирск 

 Novosibirsk 

МасловМ. А.MaslovM. A.

 Москва 

 Moscow 

СтепановГ. А.StepanovG. A.

 Новосибирск 

 Novosibirsk 

stepanovga@niboch.nsc.ruИнститут химической биологии и фундаментальной медицины Сибирского отделения Российской академии наукРоссияInstitute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of SciencesRussian FederationИнститут тонких химических технологий им. М.В. Ломоносова, МИРЭА – Российский технологический университетРоссияLomonosov Institute of Fine Chemical Technologies, MIREA – Russian Technological UniversityRussian Federation202509102025296737743Copyright © Антропов Д.Н., Марков О.В., Доме А.С., Пучков П.А., Шмендель Е.В., Гладких Д.В., Голышев В.М., Матвеева А.М., Маслов М.А., Степанов Г.А., 20252025Антропов Д.Н., Марков О.В., Доме А.С., Пучков П.А., Шмендель Е.В., Гладких Д.В., Голышев В.М., Матвеева А.М., Маслов М.А., Степанов Г.А.Antropov D.N., Markov O.V., Dome A.S., Puchkov P.A., Shmendel E.V., Gladkikh D.V., Golyshev V.M., Matveeva A.M., Maslov M.A., Stepanov G.A.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/4801

Технология мРНК-вакцин начала активно развиваться в начале XXI в. и получила хороший стимул за счет расширения знаний о функционировании иммунной системы человека и успехов в синтезе вариантов молекул-доставщиков. Иммунизация с помощью мРНК-вакцин является более эффективной, чем иммунизация с помощью ДНК, благодаря более быстрой разработке, гибкости технологии и отсутствию интеграции в геном. В наши дни искусственные мРНК используют в различных биотехнологических и медицинских целях, включая разработку противовирусных и противораковых мРНК-вакцин. Для их эффективной экспрессии необходимо правильно подобрать структурные элементы мРНК. Помимо добавления в структуру мРНК 5’-кэпа, достаточного уровня полиаденилирования и оптимизации последовательности кодонов, 5’- и 3’-нетранслируемые области (НТО) играют важную роль в трансляционной эффективности терапевтических мРНК. В настоящем исследовании для получения искусственных мРНК были сконструированы плазмидные конструкции, содержащие в своем составе новую комбинацию нетранслируемых областей – 5’-UTR-4 и 3’-UTR AES-mtRNR1. Для новой комбинации НТО, впервые описанной в данной работе, было показано значительное увеличение уровня трансляции кодон-оптимизированных последовательностей репортерных мРНК, кодирующих GFP (зеленый флюоресцентный белок) и FLuc (люцифераза светлячка), содержащих в своем составе псевдоуридин и поли(А)-последовательность. В ходе работы были сформированы комплексы вышеупомянутых репортерных мРНК с липосомами, состоящими из катионного липида 2Х3 (1,26-бис(холест- 5-ен-3β-илоксикарбониламино)-7,11,16,20-тетраазогексакозан тетрагидрохлорид) и липида-хелпера DOPE (1,2-диолеил-sn-глицеро-3-фосфоэтаноламин). Для экспериментов in vivo в состав липосомальной композиции добавляли 2 % 1,2-дистеароил-sn-глицеро-3-фосфоэтаноламин-N-[амино(полиэтиленгликоль)-2000] (DSPE-PEG2000). Новая комбинация НТО продемонстрировала более высокую эффективность трансляции мРНК в сравнении с β-глобиновыми НТО как in vitro, так и in vivo. При внутримышечном введении мРНК предложенная комбинация НТО обеспечивает длительную экспрессию более четырех суток. Результаты исследования показали высокую эффективность новой комбинации НТО для повышения уровня трансляции искусственных мРНК, что может быть использовано для снижения терапевтической дозы мРНК в составе вакцин. 

mRNA vaccine technologies have been actively developing since the beginning of the 21st century and have received a major boost from new findings about the functioning of the immune system and the development of efficient vehicles for nucleic acid delivery. The mRNA vaccine demonstrates superior properties compared to the DNA vaccine, primarily due to accelerated mRNA vaccine development, enhanced flexibility, and the absence of integration into the genome. Artificial mRNAs have biotechnological and medical applications, including the development of antiviral and anticancer mRNA therapeutics. The effective expression of therapeutic mRNA depends upon the appropriate selection of structural elements. Along with the addition of the 5’-cap, appropriate polyadenylation, and sequence codon optimization, 5’- and 3’-untranslated regions (UTRs) play an important role in the translation efficiency of therapeutic mRNAs. In this study, new plasmids containing a novel combination of UTR pairs, namely 5’-UTR-4 and 3’-UTR AES-mtRNR1, were constructed to obtain artificial mRNAs encoding green fluorescent protein (GFP) and firefly luciferase (FLuc) with new structural elements and properties. The novel combination of the UTRs, which is described in this article for the first time, in addition to sufficient polyadenylation and pseudouridinilation of mRNA, was demonstrated to strongly increase the translation of codon-optimized sequences of reporter mRNAs. We generated lipoplexes containing the aforementioned reporter mRNAs and liposomes composed of cationic lipid 2X3 (1,26-bis(cholest-5-en-3beta-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride) and helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). For in vivo experiments, the liposomes were decorated with 2 % of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000). The translation efficiency of mRNAs was found to be superior for the novel UTR combination compared with HBB gene UTRs, both in vitro and in vivo. When mRNA is administered intramuscularly, the proposed combination of UTRs provides lasting expression for more than 4 days. The results demonstrated that the novel UTR pair combination could be useful in the development of artificial mRNAs with enhanced translation efficiency, potentially reducing the dose for mRNA-based therapeutics.

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The authors declare that there are no conflicts of interest present.