mRNA-based therapeutics represent a highly promising platform for the prevention and treatment of diseases. However, further optimization is required to improve mRNA stability and translational efficiency. The 5' untranslated region (5' UTR) is a critical regulatory element that governs pre-initiation complex assembly and directly influences protein expression levels, making it an important target for rational design. The diverse challenges facing mRNA therapeutics necessitate the development of improved 5' UTR elements adaptable to specific applications. In this study, we evaluated the effects of five different 5' UTR variants, selected based on published data, on firefly luciferase (FLuc) production in cell culture and in an in vivo bioluminescence imaging model in animals. Subsequently, all variants were tested as components of mRNA vaccines against SARS-CoV-2. Our findings demonstrate that the H1.2-TISU and Synth 5' UTRs with mRNA constructs produce increased levels of reporter protein expression in mice, whereas mRNA constructs incorporating the H1.2-TISU 5' UTR demonstrated enhanced humoral immunogenicity compared to the 5' UTR derived from human alpha-globin (HBA) mRNA. Thus, the identified 5' UTRs (H1.2-TISU and Synth) represent promising, in vivo-validated candidates for incorporation into mRNA-based therapeutics and may substantially enhance their efficacy.

Capping efficiency is a critical quality attribute of mRNA therapeutics, however its accurate assessment remains a nontrivial analytical challenge. The primary difficulty arises from the minimal contribution of cap structure to the overall molecular mass, since for mRNAs spanning several hundred to thousands of nucleotides. As a result, distinguishing between capped and uncapped species requires multistep sample preparation and highly sensitive analytical instrumentation. Additional complexity is introduced by heterogeneity at the 5′ end, which may arise during transcription, capping, and subsequent analytical procedures.In this work, we provide a comprehensive overview of current analytical strategies for the quantitative assessment of mRNA capping efficiency and cap composition characterization. We also discuss sources of 5′-end heterogeneity identified and characterized in methodological studies. This review will enable researchers to identify the most effective strategy for evaluating RNA capping efficiency based on their objectives and the phase of mRNA therapeutic development.

mRNA therapeutics represent a promising frontier in the development of vaccines, anti-cancer treatments, and gene replacement therapies. The mRNA platform facilitates the rapid design of effective and safe candidates. However, native mRNA molecules are inherently characterized by limited stability and high reactogenicity, which may compromise their in vivo efficacy. The incorporation of modified nucleoside analogues can address these challenges by extending the mRNA half-life and subsequently enhancing protein expression. Despite the discovery and widespread adoption of pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ) in mRNA therapeutics, these modifications have not fully addressed all inherent limitations of mRNA-based platforms. Consequently, identifying novel nucleoside analogues, their combinations, and alternative therapeutic strategies remains a critical area of research. This review provides a comprehensive overview of how commonly used nucleoside analogues influence mRNA stability, translation, and immunogenicity, while also discussing their natural distribution patterns in native mRNA. Furthermore, we examine experimental in vitro and in vivo data regarding the application of non-canonical nucleosides in cell and animal models and evaluate the future prospects of modified mRNA-based therapies. In conclusion, this review provides an extensive analysis of the properties of modified nucleosides employed in mRNA formulations and characterizes their impact on the overall efficacy of mRNA-based therapeutics in detail.

The worldwide number of deaths from complications caused by severe influenza and COVID-19 is about 1 million cases annually. Development of the effective antiviral therapy strategies for the disease treatment is one of the most important tasks. Use of the CRISPR/Cas13 system, which specifically degrades viral RNA and significantly reduces titer of the virus, could be a solution of this problem. Despite the fact that Cas13 nucleases have been discovered only recently, they already have shown high efficiency in suppressing viral transcripts in cell cultures. The recent advances in mRNA technology and improvements in non-viral delivery systems have made it possible to effectively use CRISPR/Cas13 in animal models as well. In this review, we analyzed experimental in vitro and in vivo studies on the use of CRISPR/Cas13 systems as an antiviral agent in cell cultures and animal models and discussed main directions for improving the CRISPR/Cas13 system. These data allow us to understand prospects and limitations of the further use of CRISPR/Cas13 in the treatment of viral diseases.

Tuberculosis is a leading cause of death from a bacterial infection agent. The development of new tuberculosis vaccines can reduce the number of new cases and tuberculosis-related deaths. One of the most promising areas in vaccination is development of mRNA vaccines, which have already proven their high effectiveness against COVID-19 and other viral infections. Using modern immunoinformatic methods, we developed four new antituberculosis multiepitope mRNA vaccines differing in the encoded adjuvants and codon composition and tested their immunogenicity and protectivity in mice. Most of the developed mRNA vaccines induced the formation of both cellular and humoral immunity. The adaptive response was stronger for the vaccines with the RpfE adjuvant; however, the best protective response was elicited by the mRNA-mEp21-FL-IDT vaccine with the FL adjuvant. This vaccine reduced the mycobacterial load in the lungs of mice infected with Mycobacterium tuberculosis and increased their survival rate. Altogether, our results indicate that the mRNA-mEp21-FL-IDT vaccine ensures effective protection against tuberculosis comparable to that provided by the BCG vaccine.

mRNA vaccines turned out to be highly effective in combating the COVID-19 pandemic and other viral infections. Despite extensive study of mRNA vaccines in the last five years, the issue of safety of their use is still relevant. The study aimed to assess immunogenicity of two anti-tuberculosis mRNA vaccine doses in female and male rats 2 and 4 weeks after vaccination. Hematological and biochemical parameters of blood were determined within the same timeframe. The dose-dependent nature of mRNA vaccine immunogenicity was confirmed in both females and males. Vaccination led to moderate lymphopenia and neutrophilia in male rats, as well as to apparent dose-dependent and sex-related changes in blood biochemistry parameters at various time points. The findings suggest moderate toxicity of the anti-tuberculosis mRNA vaccine and the importance of assessing its toxic effects at various time points in animals of both sexes.

IntroductionTo comprehensively identify and provide an overview of in vivo or clinical studies of nucleic acids (NA)-based vaccines against TB we included human or animal studies of NA vaccines for the prevention or treatment of TB and excluded in vitro or in silico research, studies of microorganisms other than M. tuberculosis, reviews, letters, and low-yield reports.MethodsWe searched PubMed, Scopus, Embase, selected Web of Science and ProQuest databases, Google Scholar, eLIBRARY.RU, PROSPERO, OSF Registries, Cochrane CENTRAL, EU Clinical Trials Register, clinicaltrials.gov, and others through WHO International Clinical Trials Registry Platform Search Portal, AVMA and CABI databases, bioRxiv, medRxiv, and others through OSF Preprint Archive Search. We searched the same sources and Google for vaccine names (GX-70) and scanned reviews for references. Data on antigenic composition, delivery systems, adjuvants, and vaccine efficacy were charted and summarized descriptively.ResultsA total of 18,157 records were identified, of which 968 were assessed for eligibility. No clinical studies were identified. 365 reports of 345 animal studies were included in the review. 342 (99.1%) studies involved DNA vaccines, and the remaining three focused on mRNA vaccines. 285 (82.6%) studies used single-antigen vaccines, while 48 (13.9%) used multiple antigens or combinations with adjuvants. Only 12 (3.5%) studies involved multiepitope vaccines. The most frequently used antigens were immunodominant secretory antigens (Ag85A, Ag85B, ESAT6), heat shock proteins, and cell wall proteins. Most studies delivered naked plasmid DNA intramuscularly without additional adjuvants. Only 4 of 17 studies comparing NA vaccines to BCG after M. tuberculosis challenge demonstrated superior protection in terms of bacterial load reduction. Some vaccine variants showed better efficacy compared to BCG.Systematic review registrationhttps://osf.io/, identifier F7P9G.

Background/Objectives. Tuberculosis is a deadly bacterial disease and the second most common cause of death from monoinfectious diseases worldwide. Comprehensive measures taken by health authorities in various countries in recent decades have saved tens of millions of lives, but the number of new cases of this infection has been steadily increasing in the last few years and already exceeds 10 million new cases annually. The development of new vaccines against tuberculosis is a priority area in the prevention of new cases of the disease. mRNA vaccines have already shown high efficacy against COVID-19 and other viral infections and can currently be considered a promising field of antituberculosis vaccination. In our previous study, we assessed the immunogenicity and protective activity of several types of antituberculosis mRNA vaccines with different 5′ untranslated regions, but the efficacy of these vaccines was either comparable with or lower than that of BCG. Methods. Here, we conducted a comprehensive experiment to investigate the effects of cotranscriptional capping conditions and of cap structure on the magnitude of the mRNAs’ translation in HEK293T and DC2.4 cells. The most effective cap version was used to create an antituberculosis mRNA vaccine called mEpitope-ESAT6. Results and Conclusions. We compared immunogenicity and protective activity between mEpitope-ESAT6 and BCG and found that the vaccine with the new cap type is more immunogenic than BCG. Nonetheless, the increased immunogenicity did not enhance vaccine-induced protection. Thus, the incorporation of different cap analogs into mRNA allows to modulate the efficacy of mRNA vaccines.

mRNA vaccines have been shown to be effective in combating the COVID-19 pandemic. The amount of research on the use of mRNAs as preventive and therapeutic modalities has undergone explosive growth in the last few years. Nonetheless, the issue of the stability of mRNA molecules and their translation efficiency remains incompletely resolved. These characteristics of mRNA directly affect the expression level of a desired protein. Regulatory elements of RNA—5′ and 3′ untranslated regions (UTRs)—are responsible for translation efficiency. An optimal combination of the regulatory sequences allows mRNA to significantly increase the target protein’s expression. We assessed the translation efficiency of mRNA encoding of firefly luciferase with various 5′ and 3′UTRs in vitro on cell lines DC2.4 and THP1. We found that mRNAs containing 5′UTR sequences from eukaryotic genes HBB, HSPA1A, Rabb, or H4C2, or from the adenoviral leader sequence TPL, resulted in higher levels of luciferase bioluminescence 4 h after transfection of DC2.4 cells as compared with 5′UTR sequences used in vaccines mRNA-1273 and BNT162b2 from Moderna and BioNTech. mRNA containing TPL as the 5′UTR also showed higher efficiency (as compared with the 5′UTR from Moderna) at generating a T-cell response in mice immunized with mRNA vaccines encoding a multiepitope antigen. By contrast, no effects of various 5′UTRs and 3′UTRs were detectable in THP1 cells, suggesting that the observed effects are cell type specific. Further analyses enabled us to identify potential cell type-specific RNA-binding proteins that differ in landing sites within mRNAs with various 5′UTRs and 3′UTRs. Taken together, our data indicate high translation efficiency of TPL as a 5′UTR, according to experiments on DC2.4 cells and C57BL/6 mice.

Today, lipid nanoparticles (LNPs) are some of the main delivery systems for mRNA-based therapeutics. The scope of LNP applications in terms of RNA is not limited to antiviral vaccines but encompasses anticancer drugs and therapeutics for genetic (including rare) diseases. Such widespread use implies high customizability of targeted delivery of LNPs to specific organs and tissues. This review addresses vector-free options for targeted delivery of LNPs, namely the influence of lipid composition of these nanoparticles on their biodistribution. In the review, experimental studies are examined that are focused on the biodistribution of mRNA or of the encoded protein after mRNA administration via LNPs in mammals. We also performed a comprehensive analysis of individual lipids’ functional groups that ensure biodistribution to desired organs. These data will allow us to outline prospects for further optimization of lipid compositions of nanoparticles for targeted delivery of mRNA therapeutics.

Bacterial infections have accompanied humanity for centuries. The discovery of the first antibiotics and the subsequent golden era of their discovery temporarily shifted the balance in this confrontation to the side of humans. Nevertheless, the excessive and improper use of antibacterial drugs and the evolution of bacteria has gotten the better of humans again. Therefore, today, the search for new antibacterial drugs or the development of alternative approaches to the prevention and treatment of bacterial infections is relevant and topical again. Vaccination is one of the most effective strategies for the prevention of bacterial infections. The success of new-generation vaccines, such as mRNA vaccines, in the fight against viral infections has prompted many researchers to design mRNA vaccines against bacterial infections. Nevertheless, the biology of bacteria and their interactions with the host’s immunity are much more complex compared to viruses. In this review, we discuss structural features and key mechanisms of evasion of an immune response for nine species of bacterial pathogens against which mRNA vaccines have been developed and tested in animals. We focus on the results of experiments involving the application of mRNA vaccines against various bacterial pathogens in animal models and discuss possible options for improving the vaccines’ effectiveness. This is one of the first comprehensive reviews of the use of mRNA vaccines against bacterial infections in vivo to improve our knowledge.

mRNK-vakciny pokazali vysokuyu effektivnost' v bor'be s pandemiej COVID-19 i drugimi virusnymi infekciyami. Nesmotrya na intensivnoe izuchenie mRNK-vakcin v poslednie pyat' let, vopros o bezopasnosti ih primeneniya vse eshche ostaetsya aktual'nym. Cel'yu raboty bylo ocenit' immunogennost' protivotuberkuleznoj mRNK vakciny v dvuh dozah u samok i samcov krys cherez 2 i 4 nedeli posle vakcinacii. V eti zhe sroki opredelyali gematologicheskie i biohimicheskie pokazateli krovi. Podtverzhdena dozozavisimost' immunogennosti mRNK vakcin kak u samok, tak i u samcov. Vakcinaciya privela k umerennoj limfocitopenii i nejtrofilii u samcov krys, a takzhe k vyrazhennym dozo- i genderzavisimym izmeneniyam v biohimicheskih parametrah krovi v razlichnyh vremennyh tochkah. Poluchennye rezul'taty svidetel'stvuyut ob umerennoj toksichnosti protivotuberkuleznoj mRNK vakciny i vazhnosti issledovaniya ee toksicheskih dejstvij v razlichnyh vremennyh tochkah u zhivotnyh oboih polov.

Neuroinflammation in the early postnatal period can disturb trajectories of the completion of normal brain development and can lead to mental illnesses, such as depression, anxiety disorders, and personality disorders later in life. In our study, we focused on evaluating short- and long-term effects of neonatal inflammation induced by lipopolysaccharide, poly(I:C), or their combination in female and male C57BL/6 and BTBR mice. We chose the BTBR strain as potentially more susceptible to neonatal inflammation because these mice have behavioral, neuroanatomical, and physiological features of autism spectrum disorders, an abnormal immune response, and several structural aberrations in the brain. Our results indicated that BTBR mice are more sensitive to the influence of the neonatal immune activation (NIA) on the formation of neonatal reflexes than C57BL/6 mice are. In these experiments, the injection of lipopolysaccharide had an effect on the formation of the cliff aversion reflex in female BTBR mice. Nonetheless, NIA had no delayed effects on either social behavior or anxiety-like behavior in juvenile and adolescent BTBR and C57BL/6 mice. Altogether, our data show that NIA has mimetic-, age-, and strain-dependent effects on the development of neonatal reflexes and on exploratory activity in BTBR and C57BL/6 mice.

Animal models of psychopathologies are of exceptional interest for neurobiologists because these models allow us to clarify molecular mechanisms underlying the pathologies. One such model is the inbred BTBR strain of mice, which is characterized by behavioral, neuroanatomical, and physiological hallmarks of schizophrenia (SCZ) and autism spectrum disorders (ASDs). Despite the active use of BTBR mice as a model object, the understanding of the molecular features of this strain that cause the observed behavioral phenotype remains insufficient. Here, we analyzed recently published data from independent transcriptomic and proteomic studies on hippocampal and corticostriatal samples from BTBR mice to search for the most consistent aberrations in gene or protein expression. Next, we compared reproducible molecular signatures of BTBR mice with data on postmortem samples from ASD and SCZ patients. Taken together, these data helped us to elucidate brain-region-specific molecular abnormalities in BTBR mice as well as their relevance to the anomalies seen in ASDs or SCZ in humans.

Razrabotka novyh vakcin protiv tuberkuleza, effektivnyh v tom chisle i u vzroslyh, yavlyaetsya aktual'noj zadachej, poskol'ku ezhegodnaya smertnost' ot etogo zabolevaniya vo vsem mire prevyshaet 1,5 mln sluchaev. Nesmotrya na mnozhestvo issledovanij v poslednie desyatiletiya, effektivnoj vakciny vse eshche ne polucheno. Soprotivlyaemost' tuberkulezu sostoit iz mnogih faktorov, v etom issledovanii sdelan akcent na T-kletochnom otvete — mekhanizme, pozvolyayushchem eliminirovat' vnutrikletochnye patogeny, takie kak M. tuberculosis. Cel'yu issledovaniya bylo razrabotat' mRNK-vakcinu, sposobnuyu formirovat' vyrazhennyj T-kletochnyj otvet na antigeny M. tuberculosis. S pomoshch'yu analiza in silico byli vybrany epitopy sekretornogo belka ESAT6 (Rv3875) M. tuberculosis dlya dizajna mul'tiepitopnoj mRNK-vakciny. Provedena ocenka effektivnosti T-kletochnogo otveta u myshej, immunizirovannyh mRNK-vakcinami, kotorye kodiruyut polnorazmernyj ili mul'tiepitopnyj antigen. Rezul'taty pokazali, chto pri immunizacii mul'tiepitopnoj mRNK-vakcinoj kolichestvo IFNγ-sekretiruyushchih splenocitov v otvet na specifichnuyu stimulyaciyu v dva raza vyshe, v sravnenii s kolichestvom IFNγ-sekretiruyushchih kletok u myshej, immunizirovannyh mRNK-vakcinoj, kodiruyushchej polnorazmernyj belok. Takim obrazom, razrabotannaya mul'tiepitopnaya mRNK-vakcina mozhet stat' effektivnym preparatom dlya profilaktiki M. tuberculosis posredstvom formirovaniya vyrazhennogo T-kletochnogo otveta.

Vaccination is among the most effective measures to reduce tuberculosis morbidity and mortality. In 1974, BCG vaccination was included in the Expanded Program on Immunization. Today, it covers 80% of all children around the globe. Unfortunately, BCG vaccine provides no protection against pulmonary tuberculosis, the most prevalent form of tuberculosis. It is necessary to urgently develop new vaccination strategies to stop large-scale dissemination of infection caused by the multidrugresistant pathogen. The study was aimed to compare the capabilities of three variants of mRNA vaccines encoding Esat6 epitopes of stimulating adaptive immune response formation in C57BL/6 mice (ELISpot, delayed hypersensitivity, IgG titers), as well as of protecting I/St mice against M. tuberculosis infection. Efficacy of mRNA vaccines comprising different untranslated regions packaged in lipid nanoparticles was compared with that of BCG vaccine. The 5'-TPL-Esat6-3'-Mod vaccine demonstrated the highest efficacy in our experimental model. Thus, the 5'-TPL-Esat6-3'-Mod mRNA vaccine can be considered as a candidate vaccine for further optimization, improving efficacy and subsequent use for prevention of tuberculosis.

Development of the new tuberculosis vaccines that would be effective in adults is an urgent task: worldwide, the annual death toll of this disease exceeds 1.5 million. In the recent decades, the matter has been addressed in numerous studies, but none has yielded an effective vaccine so far. There are many factors to resistance against tuberculosis; this study focuses on the T-cell response, a mechanism that enables elimination of intracellular pathogens, such as M. tuberculosis. We aimed to develop an mRNA vaccine capable of triggering a pronounced T-cell response to the M. tuberculosis antigens. The in silico analysis allowed us to select epitopes of the M. tuberculosis secreted protein ESAT6 (Rv3875) and design a multi-epitope mRNA vaccine thereon. We assessed the intensity of T-cell response in mice immunized with mRNA vaccines that encode a full-length or multi-epitope antigen. The results of this study in mice show that immunization with a multi-epitope mRNA vaccine produces twice as many IFNγ-secreting splenocytes in response to specific stimulation than immunization with an mRNA vaccine encoding the full-length protein. Thus, the developed multi-epitope mRNA vaccine can be an effective M. tuberculosis prevention agent the mode of action of which involves formation of a pronounced T-cell response.

Explosive developments in mRNA vaccine technology in the last decade have made it possible to achieve great success in clinical trials of mRNA vaccines to prevent infectious diseases and develop cancer treatments and mRNA-based gene therapy products. The approval of the mRNA-1273 and BNT162b2 mRNA vaccines against SARS-CoV-2 by the U.S. Food and Drug Administration has led to mass vaccination (with mRNA vaccines) of several hundred million people around the world, including children. Despite its effectiveness in the fight against COVID-19, rare adverse effects of the vaccination have been shown in some studies, including vascular microcirculation disorders and autoimmune and allergic reactions. The biodistribution of mRNA vaccines remains one of the most poorly investigated topics. This mini-review discussed the results of recent experimental studies on humans and rodents regarding the biodistribution of mRNA vaccines, their constituents (mRNA and lipid nanoparticles), and their encoded antigens. We focused on the dynamics of the biodistribution of mRNA vaccine products and on the possibility of crossing the blood–brain and blood–placental barriers as well as transmission to infants through breast milk. In addition, we critically assessed the strengths and weaknesses of the detection methods that have been applied in these articles, whose results’ reliability is becoming a subject of debate.

mRNA-based therapeutics have been found to be a promising treatment strategy in immunotherapy, gene therapy, and cancer treatments. Effectiveness of mRNA therapeutics depends on the level and duration of a desired protein’s expression, which is determined by various cis- and trans-regulatory elements of the mRNA. Sequences of 5′ and 3′ untranslated regions (UTRs) are responsible for translational efficiency and stability of mRNA. An optimal combination of the regulatory sequences allows researchers to significantly increase the target protein’s expression. Using both literature data and previously obtained experimental data, we chose six sequences of 5′UTRs (adenoviral tripartite leader [TPL], HBB, rabbit β-globin [Rabb], H4C2, Moderna, and Neo2) and five sequences of 3′UTRs (mtRNR-EMCV, mtRNR-AES, mtRNR-mtRNR, BioNTech, and Moderna). By combining them, we constructed 30 in vitro transcribed RNAs encoding firefly luciferase with various combinations of 5′- and 3′UTRs, and the resultant bioluminescence was assessed in the DC2.4 cell line at 4, 8, 24, and 72 h after transfection. The cellular data enabled us to identify the best seven combinations of 5′- and 3′UTRs, whose translational efficiency was then assessed in BALB/c mice. Two combinations of 5′- and 3′UTRs (5′Rabb-3′mtRNR-EMCV and 5′TPL-3′Biontech) led to the most pronounced increase in the luciferase amount in the in vivo experiment in mice. Subsequent analysis of the stability of the mRNA indicated that the increase in luciferase expression is explained primarily by the efficiency of translation, not by the number of RNA molecules. Altogether, these findings suggest that 5′UTR-and-3′UTR combinations 5′Rabb-3′mtRNR- EMCV and 5′TPL-3′Biontech lead to high expression of target proteins and may be considered for use in preventive and therapeutic modalities based on mRNA.

Vakcinaciya yavlyaetsya odnim iz naibolee uspeshnyh medicinskih meropriyatij po snizheniyu zabolevaemosti i smertnosti ot tuberkuleza. V 1974 g. vakcinaciya BCZH byla vklyuchena v Rasshirennuyu programmu vakcinacii, i na segodnya ohvatyvaet 80% vsekh detej na zemnom share. K sozhaleniyu, vakcina BCZH ne zashchishchaet ot naibolee rasprostranennoj formy tuberkuleza — tuberkuleza legkih. Trebuetsya srochno razrabotat' novye strategii vakcinacii, chtoby ostanovit' shirokomasshtabnoe rasprostranenie infekcii s mnozhestvennoj lekarstvennoj ustojchivost'yu vozbuditelya. Cel'yu issledovaniya bylo sravnit' sposobnost' trekh variantov mRNK-vakcin, kodiruyushchih epitopy Esat6, stimulirovat' formirovanie adaptivnogo immuniteta u myshej C57BL/6 (ELISpot, GZT, titry IgG), a takzhe zashchishchat' myshej I/St ot zarazheniya M. tuberculosis. Effektivnost' upakovannyh v nanolipidnye chasticy mRNK-vakcin, razlichayushchihsya posledovatel'nostyami netransliruemyh regionov, sravnivali s effektivnost'yu BCZH. V poluchennoj nami eksperimental'noj modeli maksimal'nuyu effektivnost' po bol'shinstvu pokazatelej prodemonstrirovala vakcina 5'-TPL-Esat6- 3'-Mod. Takim obrazom, mRNK-vakcina 5'-TPL-Esat6-3'-Mod mozhet byt' rassmotrena v kachestve kandidatnoj dlya dal'nejshej optimizacii, povysheniya ee effektivnosti i posleduyushchego primeneniya dlya profilaktiki tuberkuleza.