mRNA Research Articles

CAPZB mRNA is a novel biomarker for cervical high-grade squamous lesions

This study aimed to evaluate the potential of capping protein (actin filament) muscle Z-line subunit β (CAPZB) messenger ribonucleic acid (mRNA) levels as a biomarker for distinguishing low-grade squamous intraepithelial lesions of the cervix (LSIL) from high-grade squamous intraepithelial lesions of the cervix (HSIL). We collected a total of 166 cervical exfoliated cells and divided them into five groups based on histopathological results. Each sample was divided into two portions, one for fluorescence in situ hybridization (FISH) detection and the other for bisulfite sequencing polymerase chain reaction (BSP) detection. We found that FISH detection of CAPZB mRNA mean fluorescence intensity (MFI) and BSP detection of CAPZB deoxyribonucleic acid (DNA) percentage of methylation rate (PMR) performed as biomarkers for distinguishing HSIL from LSIL, with an area under the receiver operating characteristic curve (AUC), sensitivity, specificity and cut-off value of 0.893, 81.25%, 80.39% and 0.616, 0.794, 64.06%, 81.37% and 0.454, respectively. Furthermore, FISH detection of CAPZB mRNA exhibited a greater AUC (0.893) for the detection of HSIL than the CAPZB DNA methylation method (0.794), indicating the CAPZB mRNA levels can be used as a biomarker for assessing cervical lesions.

Integrative in silico approaches to analyse microRNA-mediated responses in human diseases.

Advancements in sequencing technologies have facilitated omics level information generation for various diseases in human. High-throughput technologies have become a powerful tool to understand differential expression studies and transcriptional network analysis. An understanding of complex transcriptional networks in human diseases requires integration of datasets representing different RNA species including microRNA (miRNA) and messenger RNA (mRNA). This review emphasises on conceptual explanation of generalized workflow and methodologies to the miRNA mediated responses in human diseases by using different in silico analysis. Although,there have been many prior explorations in miRNA-mediated responses in human diseases, the advantages, limitations and overcoming the limitation through different statistical techniques have not yet been discussed. This review focuses on miRNAs as important gene regulators in human diseases, methodologies for miRNA-target gene prediction and data driven methods for enrichment and network analysis for miRnome-targetome interactions. Additionally, it proposes an integrative workflow to analyse structural components of networks obtained from high-throughput data. This review explains how to apply the existing methods to analyse miRNA-mediated responses in human diseases. It addresses unique characteristics of different analysis, its limitations and its statistical solutions influencing the choice of methods for the analysis through a workflow. Moreover, it provides an overview of promising common integrative approaches to comprehend miRNA-mediated gene regulatory events in biological processes in humans. The proposed methodologies and workflow shall help in the analysis of multi-source data to identify molecular signatures of various human diseases.

N-acetyltransferase 10 mediates cognitive dysfunction through the acetylation of GABABR1 mRNA in sepsis-associated encephalopathy

Sepsis-associated encephalopathy (SAE) is a critical neurological complication of sepsis and represents a crucial factor contributing to high mortality and adverse prognosis in septic patients. This study explored the contribution of NAT10-mediated messenger RNA (mRNA) acetylation in cognitive dysfunction associated with SAE, utilizing a cecal ligation and puncture (CLP)-induced SAE mouse model. Our findings demonstrate that CLP significantly upregulates NAT10 expression and mRNA acetylation in the excitatory neurons of the hippocampal dentate gyrus (DG). Notably, neuronal-specific Nat10 knockdown improved cognitive function in septic mice, highlighting its critical role in SAE. Proteomic analysis, RNA immunoprecipitation, and real-time qPCR identified GABABR1 as a key downstream target of NAT10. Nat10 deletion reduced GABABR1 expression, and subsequently weakened inhibitory postsynaptic currents in hippocampal DG neurons. Further analysis revealed that microglia activation and the release of inflammatory mediators lead to the increased NAT10 expression in neurons. Microglia depletion with PLX3397 effectively reduced NAT10 and GABABR1 expression in neurons, and ameliorated cognitive dysfunction induced by SAE. In summary, our findings revealed that after CLP, NAT10 in hippocampal DG neurons promotes GABABR1 expression through mRNA acetylation, leading to cognitive dysfunction.

Differential microRNA expression in adolescent anxiety proneness.

Biological mechanisms underlying anxiety proneness (AP), the tendency to react fearfully to stressors due to the belief that experiencing anxiety has harmful consequences, remain unclear. Epigenetic mechanisms, such as microRNAs (small, non-coding RNAs 19-20 nucleotides long), may be contributory. This study investigated AP-associated differences in microRNA expression among South African adolescents with variable exposure to childhood trauma (CT). AP was assessed using a composite score reflecting trait anxiety and anxiety sensitivity, while CT exposure was assessed with the Childhood Trauma Questionnaire. High-quality total RNA (n = 88) extracted from whole blood underwent microRNA-sequencing. Differential microRNA expression analysis was conducted with DESeq2 in R, messenger RNA target prediction analysis was performed using TargetScan and DIANA-microT, and the DIANA mirPATH tool was used for KEGG pathway analysis. The majority of participants were female (75.86%) with an average age of 15 (±1.19) years. MicroRNA expression analysis identified upregulation of hsa-miR-28-5p and downregulation of hsa-miR-502-3p and hsa-miR-500a-3p in high-AP individuals, irrespective of CT. Four KEGG pathways, each with ≥10% of their constituent genes predicted to be targets of the differentially expressed microRNAs, were identified and were enriched for genes involved in calcineurin and glutamate signalling. These findings suggest that epigenetically mediated effects on neuronal function contribute to the molecular aetiology of AP.

Probing the higher order structure of oligonucleotides through anion exchange chromatography

Large synthetic oligonucleotides such as guide ribonucleic acid (gRNA), a critical reagent in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing, have complex higher order structures (HOS) inherent in their design. In this study, we first developed a generic anion exchange chromatography (AEX) method for the comprehensive analysis of a 100mer single guide ribonucleic acid (sgRNA) impurity profiling. AEX demonstrated superior resolution compared to other common chromatographic methods employed for sgRNA analysis, such as Ion-Pairing Reversed Phase Liquid Chromatography (IP-RPLC) and Hydrophilic Interaction Chromatography (HILIC). Moreover, we discovered AEX's potential in probing the HOS of RNAs by adjusting the temperature and using organic additives. Our study also highlighted that sgRNA possesses a unique HOS distinctly different from other therapeutic nucleic acids, such as antisense oligonucleotides and messenger RNAs.

Effects of Continuous Prenatal Low Dose Rate Irradiation on Neurobehavior, Hippocampal Cellularity, Messenger RNA and MicroRNA Expression on B6C3F1 Mice.

Epidemiological, experimental, and ecological data have indicated the controversial effect of in utero chronic low dose rate (<6 mGy/h) with accumulative low (≤100 mGy) or high (>100 mGy) dose radiation exposure. Our main goal of this study was to examine if different low dose rates of chronic pre- and/or post-natal radiation exposure with accumulative high doses could induce hippocampal cellular, mRNA, and miRNA changes leading to neuropsychiatric disorders. The comprehensive mouse phenotypic traits, organ weight, pathological, and blood mRNA and miRNA changes were also studied. Using different approaches including SmithKline, Harwell, Imperial College, Royal Hospital, Phenotype Assessment (SHIRPA), neurobehavioral tests, pathological examination, immunohistochemistry, mRNA and miRNA sequencing, and real-time quantitative polymerase chain reaction (qRT-PCR) validation, we found that in prenatally irradiated (100 mGy/d for 18 days with an accumulative dose of 1.8 Gy) 1-year-old mice, no cellular changes, including immature neurons in the subgranular zone, mature neurons and glial cells in the hilus of the dentate gyrus and development of cognitive impairment, neuropsychiatric disorders, occurred. However, a significant reduction in body weight and mass index (BMI) was indicated by the SHIRPA test. A reduced exploratory behavior was shown by an open field test. Organ weights showed significant reductions in the testes, kidneys, heart, liver and epididymides with no abnormal pathology. mRNA and miRNA sequencing and qRT-PCR validation revealed the upregulation of Rubcnl and Abhd14b, and downregulation of Hspa1b, P4ha1, and Banp genes in both the hippocampus and blood of mice prenatally irradiated with 100 mGy/d. Meanwhile, downregulation of miR-448-3p and miR1298-5p in the hippocampus, miR-320-3p, miR-423-5p, miR-486b-5p, miR-486b-3p, miR-423-3p, miR-652-3p, miR-324-3p, miR-181b-5p, miR-let-7b, and miR-6904-5p in the blood was induced. The target scan revealed that Rubcnl is one of the miR-181b-5p targets in the blood. We, therefore, concluded that prenatal chronic irradiation with a low dose rate of 100 mGy/d and accumulative dose of 1.8 Gy or below might not induce significant adverse health effects on the offspring. Further study of different low dose rate radiation exposures with accumulative high doses may provide threshold doses for authorities or regulators to set new radiation safety guidelines to replace those extrapolated from acute high dose/dose rate irradiation to reduce unnecessary emergency evacuation or spending once a nuclear accident or leakage occurs.

Brain lncRNA-mRNA co-expression regulatory networks and alcohol use disorder

Prolonged alcohol consumption can disturb the expression of both coding and noncoding genes in the brain. These dysregulated genes may co-express in modules and interact within networks, consequently influencing the susceptibility to developing alcohol use disorder (AUD). In the present study, we performed an RNA-seq analysis of the expression of both long noncoding RNAs (lncRNAs) and messenger RNAs (mRNAs) in 192 postmortem tissue samples collected from eight brain regions (amygdala, caudate nucleus, cerebellum, hippocampus, nucleus accumbens, prefrontal cortex, putamen, and ventral tegmental area) of 12 AUD and 12 control subjects of European ancestry. Applying the limma-voom method, we detected a total of 57 lncRNAs and 51 mRNAs exhibiting significant differential expression (Padj < 0.05 and fold-change ≥2) across at least one of the eight brain regions investigated. Machine learning analysis further confirmed the potential of these top genes in predicting AUD. Through Weighted Gene Co-expression Network Analysis (WGCNA), we identified distinct lncRNA-mRNA co-expression modules associated with AUD in each of the eight brain regions. Additionally, lncRNA-mRNA co-expression networks were constructed for each brain region using Cytoscape to reveal gene regulatory interactions implicated in AUD. Hub genes within these networks were found to be enriched in several key KEGG pathways, including Axon Guidance, MAPK Signaling, p53 Signaling, Adherens Junction, and Neurodegeneration. Our results underscore the significance of networks involving AUD-associated lncRNAs and mRNAs in modulating neuroplasticity in response to alcohol exposure. Further elucidating these molecular mechanisms holds promise for the development of targeted therapeutic interventions for AUD.

Strategic deactivation of mRNA COVID-19 vaccines: New applications for siRNA therapy and RIBOTACs.

The rapid development and authorization of messenger ribonucleic acid (mRNA) vaccines by Pfizer-BioNTech (BNT162b2) and Moderna (mRNA-1273) in 2020 marked a significant milestone in human mRNA product application, overcoming previous obstacles such as mRNA instability and immunogenicity. This paper reviews the strategic modifications incorporated into these vaccines to enhance mRNA stability and translation efficiency, such as the inclusion of nucleoside modifications and optimized mRNA design elements including the 5' cap and poly(A) tail. We highlight emerging concerns regarding the wide systemic biodistribution of these mRNA vaccines leading to prolonged inflammatory responses and other safety concerns. The regulatory framework guiding the biodistribution studies is pivotal in assessing the safety profiles of new mRNA formulations in use today. The stability of mRNA vaccines, their pervasive distribution, and the longevity of the encapsulated mRNA along with unlimited production of the damaging and potentially lethal spike (S) protein call for strategies to mitigate potential adverse effects. Here, we explore the potential of small interfering RNA (siRNA) and ribonuclease targeting chimeras (RIBOTACs) as promising solutions to target, inactivate, and degrade residual and persistent vaccine mRNA, thereby potentially preventing uncontrolled S protein production and reducing toxicity. The targeted nature of siRNA and RIBOTACs allows for precise intervention, offering a path to prevent and mitigate adverse events of mRNA-based therapies. This review calls for further research into siRNA and RIBOTAC applications as antidotes and detoxication products for mRNA vaccine technology.

APOBEC3C-mediated NF-κB activation enhances clear cell renal cell carcinoma progression.

Renowned as the predominant form of kidney cancer, clear cell renal cell carcinoma (ccRCC) exhibits susceptibility to immunotherapies due to its specific expression profile as well as notable immune cell infiltration. Despite this, effectively treating metastatic ccRCC remains a significant challenge, necessitating a more profound comprehension of the underlying molecular mechanisms governing its progression. Here, we unveil that the enhanced expression of the RNA-binding protein DNA dC → dU-editing enzyme APOBEC-3C (APOBEC3C; also known as A3C) in ccRCC tissue and ccRCC-derived cell lines serves as a catalyst for tumor growth by amplifying nuclear factor-kappa B (NF-κB) activity. By employing RNA-sequencing and cell-based assays in ccRCC-derived cell lines, we determined that A3C is a stress-responsive factor and crucial for cell survival. Furthermore, we identified that A3C binds and potentially stabilizes messenger RNAs (mRNAs) encoding positive regulators of the NF-κB pathway. Upon A3C depletion, essential subunits of the NF-κB family are abnormally restrained in the cytoplasm, leading to deregulation of NF-κB target genes. Our study illuminates the pivotal role of A3C in promoting ccRCC tumor development, positioning it as a prospective target for future therapeutic strategies.

Propyl acetate protects intestinal barrier during parenteral nutrition in mice and Caco-2 cells.

Gut microbiota dysbiosis induces intestinal barrier damage during parenteral nutrition (PN). However, the underlying mechanisms remain unclear. This study aimed to investigate gut microbiota dysbiosis, luminal short-chain fatty acids, and autophagy in a mouse model and how these short-chain fatty acids regulate autophagy. Eight-week-old male specific-pathogen-free mice were randomly divided into a Chow group (standard diet and intravenous normal saline infusion) and a PN group (continuous infusion of PN nutrient solution) for 7 days. Caco-2 cells were also treated with intestinal rinse solutions from Chow and PN mouse models. Compared with the Chow group, the PN group exhibited increased Proteobacteria and decreased Firmicutes, correlating with decreased propyl acetate. In the PN group, intestinal tissue exhibited elevated adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, LC3II protein levels, and Atg3 and Atg7 messenger RNA levels. P62 protein levels were decreased, indicating an increase of autophagy flux in the PN group. In the Caco-2 cell model, cells treated with PN solution plus propyl acetate exhibited increased Claudin-1 and occluding along with decreased interleukin-6 and tumor necrosis factor α compared with those treated with PN solution alone. Propyl acetate addition inhibited the AMPK-mammalian target of rapamycin (mTOR) pathway, mitigating the excessive autophagy induced by the PN intestinal rinse solution in Caco-2 cells. PN led to a significant reduction in propyl acetate levels in the intestine, excessive activation of autophagy, and barrier dysfunction. Propyl acetate inhibited excessive autophagy via the AMPK/mTOR signaling pathway and protected the intestinal barrier during PN.

DRpred: A Novel Deep Learning-Based Predictor for Multi-Label mRNA Subcellular Localization Prediction by Incorporating Bayesian Inferred Prior Label Relationships.

The subcellular localization of messenger RNA (mRNA) not only helps us to understand the localization regulation of gene expression but also helps to understand the relationship between RNA localization pattern and human disease mechanism, which has profound biological and medical significance. Several predictors have been proposed for predicting the subcellular localization of mRNA. However, there is still considerable room for improvement in their predictive performance, especially regarding multi-label prediction. This study proposes a novel multi-label predictor, DRpred, for mRNA subcellular localization prediction. This predictor first utilizes Bayesian networks to capture the dependencies among labels. Subsequently, it combines these dependencies with features extracted from mRNA sequences using Word2vec, forming the input for the predictor. Finally, it employs a neural network combining BiLSTM and an attention mechanism to capture the internal relationships of the input features for mRNA subcellular localization. The experimental validation on an independent test set demonstrated that DRpred obtained a competitive predictive performance in multi-label prediction and outperformed state-of-the-art predictors in predicting single subcellular localizations, obtaining accuracies of 82.14%, 93.02%, 80.37%, 94.00%, 90.58%, 84.53%, 82.01%, 79.71%, and 85.67% for the chromatin, cytoplasm, cytosol, exosome, membrane, nucleolus, nucleoplasm, nucleus, and ribosome, respectively. It is anticipated to offer profound insights for biological and medical research.

Identification of differentially expressed genes and screening for key genes involved in ovarian cancer prognosis: An integrated bioinformatics and network analysis approach

Objectives: Ovaries are important and essential organs of animals in producing and releasing eggs. Ovarian cancer (OvCa) is one of the most prevalent lethal gynecological malignancies with a lack of distinct biomarkers. Advances in high-throughput genomic data and the continued refinement of bioinformatics tools enable the identification of potential biomarkers. Leveraging these insights, we can employ systems biology approaches to enhance the accuracy of diagnosis and prognosis. Material and Methods: A comparative analysis was conducted between normal and tumor samples, employing bioinformatics software and tools. Differential expression analysis utilized fold-change statistics, while DAVID 6.8 software was used to perform gene ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. The protein-protein interaction (PPI) network was constructed differentially expressed genes (DEGs) using Search Tool for the Retrieval of Interacting Genes database, and Cytoscape 3.9.1, along with its Molecular Complex Detection and CytoHubba plugins, facilitated network visualization, analysis, and module detection. Hub gene expression and overall survival were explored through the Kaplan–Meier plotter, while Gene Expression Profiling Interactive Analysis 2 analyzed the tumor stage of OvCa patients. Hub genes protein expression was analyzed using the human protein atlas database through immunostaining results. The NetworkAnalyst program and Cytoscape were employed to analyze and visualize the transcription factor-hub gene associations. Subsequently, single-nucleotide variation, methylation, and pathway activity of hub genes were examined. Validation of hub genes messenger RNA expression was done using quantitative real-time polymerase chain reaction analysis. Results: 607 DEGs, including 248 upregulated and 359 downregulated genes, were identified. The top 20 candidate genes were screened out through PPI network analysis. We discovered that the genes BUB1 Mitotic Checkpoint Serine/Threonine Kinase B (BUB1B), Cyclin A2 (CCNA2), Mitotic Arrest Deficient 2 Like 1 (MAD2L1), Protein Regulator of Cytokinesis 1 (PRC1), Thyroid Hormone Receptor Interactor 13 (TRIP13), and ZW10 Interacting Kinetochore Protein (ZWINT) exhibited significant importance in OvCa prognosis. Conclusion: Six genes, BUB1B, CCNA2, MAD2L1, PRC1, TRIP13, and ZWINT (identified as functional hub genes), are probably playing tumor-promotive roles, except TRIP13. All genes product is functionally related to the cell cycle. These can be targeted in quest of potential therapeutics for OvCa treatment.

Benchmarking the negatives: Effect of negative data generation on the classification of miRNA-mRNA interactions.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally. In animals, this regulation is achieved via base-pairing with partially complementary sequences on mainly 3' UTR region of messenger RNAs (mRNAs). Computational approaches that predict miRNA target interactions (MTIs) facilitate the process of narrowing down potential targets for experimental validation. The availability of new datasets of high-throughput, direct MTIs has led to the development of machine learning (ML) based methods for MTI prediction. To train an ML algorithm, it is beneficial to provide entries from all class labels (i.e., positive and negative). Currently, no high-throughput assays exist for capturing negative examples. Therefore, current ML approaches must rely on either artificially generated or inferred negative examples deduced from experimentally identified positive miRNA-target datasets. Moreover, the lack of uniform standards for generating such data leads to biased results and hampers comparisons between studies. In this comprehensive study, we collected methods for generating negative data for animal miRNA-target interactions and investigated their impact on the classification of true human MTIs. Our study relies on training ML models on a fixed positive dataset in combination with different negative datasets and evaluating their intra- and cross-dataset performance. As a result, we were able to examine each method independently and evaluate ML models' sensitivity to the methodologies utilized in negative data generation. To achieve a deep understanding of the performance results, we analyzed unique features that distinguish between datasets. In addition, we examined whether one-class classification models that utilize solely positive interactions for training are suitable for the task of MTI classification. We demonstrate the importance of negative data in MTI classification, analyze specific methodological characteristics that differentiate negative datasets, and highlight the challenge of ML models generalizing interaction rules from training to testing sets derived from different approaches. This study provides valuable insights into the computational prediction of MTIs that can be further used to establish standards in the field.

Methyltransferase ATMETTL5 writes m6A on 18S ribosomal RNA to regulate translation in Arabidopsis.

Aberrant RNA modifications can lead to dysregulated gene expression and impeded growth in plants. Ribosomal RNA (rRNA) constitutes a substantial portion of total RNA, while the precise functions and molecular mechanisms underlying rRNA modifications in plants remain largely elusive. Here, we elucidated the exclusive occurrence of the canonical RNA modification N6-methyladenosine (m6A) solely 18S rRNA, but not 25S rRNA. We identified a completely uncharacterized protein, ATMETTL5, as an Arabidopsis m6A methyltransferase responsible for installing m6A methylation at the 1771 site of the 18S rRNA. ATMETTL5 is ubiquitously expressed and localized in both nucleus and cytoplasm, mediating rRNA m6A methylation. Mechanistically, the loss of ATMETTL5-mediated methylation results in attenuated translation. Furthermore, we uncovered the role of ATMETTL5-mediated methylation in coordinating blue light-mediated hypocotyl growth by regulating the translation of blue light-related messenger RNAs (mRNAs), specifically HYH and PRR9. Our findings provide mechanistic insights into how rRNA modification regulates ribosome function in mRNA translation and the response to blue light, thereby advancing our understanding of the role of epigenetic modifications in precisely regulating mRNA translation in plants.

Synthesizing Lipid Nanoparticles by Turbulent Flow in Confined Impinging Jet Mixers.

Lipid nanoparticles (LNPs) have demonstrated their enormous potential as therapeutic delivery vehicles, as evidenced by the approval and global usage of two COVID-19 messenger RNA (mRNA) vaccines. On a small scale, LNPs are often made using microfluidics; however, the limitations of these devices preclude their use on a large scale. The COVID-19 vaccines are manufactured in large quantities using confined impinging jet (CIJ) turbulent mixers. CIJ technology enables production at a laboratory scale with the confidence that it can be scaled to production volumes. The key concepts in CIJ mixing are that the mixing length and time scale are determined by the turbulence intensity in the mixing cavity and that the nanoparticle formation occurs away from walls, eliminating the problem of deposition on surfaces and fouling. This work demonstrates the process of making LNPs using confined impinging jet mixer technology with two geometries: the two-jet CIJ and the four-jet multi-inlet vortex mixer (MIVM). The advantages and disadvantages of each mixing geometry are discussed. In these geometries, LNPs are formed by rapid mixing of an organic solvent stream (usually ethanol containing the ionizable lipids, co-lipids, and stabilizing PEG-lipids) with an aqueous anti-solvent stream (aqueous buffer containing RNA or DNA). The operating parameters for the CIJ and MIVM mixers are presented to prepare reproducible LNPs with controlled size, zeta potential, stability, and transfection effectiveness. The differences between LNPs made with poor mixing (pipetting solutions) compared to CIJ mixing are also presented.

Therapeutic Antisense Oligonucleotides in Oncology: From Bench to Bedside.

Advancements in our comprehension of tumor biology and chemoresistance have spurred the development of treatments that precisely target specific molecules within the body. Despite the expanding landscape of therapeutic options, there persists a demand for innovative approaches to address unmet clinical needs. RNA therapeutics have emerged as a promising frontier in this realm, offering novel avenues for intervention such as RNA interference and the utilization of antisense oligonucleotides (ASOs). ASOs represent a versatile class of therapeutics capable of selectively targeting messenger RNAs (mRNAs) and silencing disease-associated proteins, thereby disrupting pathogenic processes at the molecular level. Recent advancements in chemical modification and carrier molecule design have significantly enhanced the stability, biodistribution, and intracellular uptake of ASOs, thereby bolstering their therapeutic potential. While ASO therapy holds promise across various disease domains, including oncology, coronary angioplasty, neurological disorders, viral, and parasitic diseases, our review manuscript focuses specifically on the application of ASOs in targeted cancer therapies. Through a comprehensive examination of the latest research findings and clinical developments, we delve into the intricacies of ASO-based approaches to cancer treatment, shedding light on their mechanisms of action, therapeutic efficacy, and prospects.

Supramolecular assembly of polycation/mRNA nanoparticles and in vivo monocyte programming

Size-dependent phagocytosis is a well-characterized phenomenon in monocytes and macrophages. However, this size effect for preferential gene delivery to these important cell targets has not been fully exploited because commonly adopted stabilization methods for electrostatically complexed nucleic acid nanoparticles, such as PEGylation and charge repulsion, typically arrest the vehicle size below 200 nm. Here, we bridge the technical gap in scalable synthesis of larger submicron gene delivery vehicles by electrostatic self-assembly of charged nanoparticles, facilitated by a polymer structurally designed to modulate internanoparticle Coulombic and van der Waals forces. Specifically, our strategy permits controlled assembly of small poly(β-amino ester)/messenger ribonucleic acid (mRNA) nanoparticles into particles with a size that is kinetically tunable between 200 and 1,000 nm with high colloidal stability in physiological media. We found that assembled particles with an average size of 400 nm safely and most efficiently transfect monocytes following intravenous administration and mediate their differentiation into macrophages in the periphery. When a CpG adjuvant is co-loaded into the particles with an antigen mRNA, the monocytes differentiate into inflammatory dendritic cells and prime adaptive anticancer immunity in the tumor-draining lymph node. This platform technology offers a unique ligand-independent, particle-size-mediated strategy for preferential mRNA delivery and enables therapeutic paradigms via monocyte programming.

Advances in the study of LNPs for mRNA delivery and clinical applications.

Messenger ribonucleic acid (mRNA) was discovered in 1961 as an intermediary for transferring genetic information from DNA to ribosomes for protein synthesis. The COVID-19 pandemic brought worldwide attention to mRNA vaccines. The emergency use authorization of two COVID-19 mRNA vaccines, BNT162b2 and mRNA-1273, were major achievements in the history of vaccine development. Lipid nanoparticles (LNPs), one of the most superior non-viral delivery vectors available, have made many exciting advances in clinical translation as part of the COVID-19 vaccine and therefore has the potential to accelerate the clinical translation of many gene drugs. In addition, due to these small size, biocompatibility and excellent biodegradability, LNPs can efficiently deliver nucleic acids into cells, which is particularly important for current mRNA therapeutic regimens. LNPs are composed cationic or pH-dependent ionizable lipid bilayer, polyethylene glycol (PEG), phospholipids, and cholesterol, represents an advanced system for the delivery of mRNA vaccines. Furthermore, optimization of these four components constituting the LNPs have demonstrated enhanced vaccine efficacy and diminished adverse effects. The incorporation of biodegradable lipids enhance the biocompatibility of LNPs, thereby improving its potential as an efficacious therapeutic approach for a wide range of challenging and intricate diseases, encompassing infectious diseases, liver disorders, cancer, cardiovascular diseases, cerebrovascular conditions, among others. Consequently, this review aims to furnish the scientific community with the most up-to-date information regarding mRNA vaccines and LNP delivery systems.

Unraveling RNA contribution to the molecular origins of bacterial surface-enhanced Raman spectroscopy (SERS) signals

Surface-enhanced Raman spectroscopy (SERS) is widely utilized in bacterial analyses, with the dominant SERS peaks attributed to purine metabolites released during sample preparation. Although adenosine triphosphate (ATP) and nucleic acids are potential molecular origins of these metabolites, research on their exact contributions remains limited. This study explored purine metabolite release from E. coli and RNA integrity following various sample preparation methods. Standard water washing generated dominant SERS signals within 10 s, a duration shorter than the anticipated RNA half-lives under starvation. Evaluating RNA integrity indicated that the most abundant ribosomal RNA species remained intact for hours post-washing, whereas messenger RNA and transfer RNA species degraded gradually. This suggests that bacterial SERS signatures observed after the typical washing step could originate from only a small fraction of endogenous purine-containing molecules. In contrast, acid depurination led to degradation of most RNA species, releasing about 40 times more purine derivatives than water washing. Mild heating also instigated the RNA degradation and released more purine derivatives than water washing. Notably, differences were also evident in the dominant SERS signals following these treatments. This work provides insights into SERS-based studies of purine metabolites released by bacteria and future development of methodologies.

Synthesis of a novel adapter lipid using Fc-region mediated antibody modification for post-insert preparation of transferrin receptor targeted messenger RNA-loaded lipid nanoparticles

Lipid nanoparticles (LNPs) are promising delivery systems with the ability to deliver small interfering RNA (siRNA) and messenger RNA (mRNA) in diseased tissues and intracellular sites of action. However, delivery to non-hepatic tissues via systemic administration remains challenging. Antibody modification of LNPs is a hopeful approach for improving their selectivity to target tissues. The conventional method of antibody modification via thiol–maleimide linkage is concerned with reduced recognition efficiency of the disease-related target molecules owing to variations in antibody orientation on the surface of the LNPs. In this study, we developed a novel adapter lipopeptide for antibody modification of LNPs via the Fc-region. Here, we selected RI7-217, an anti-transferrin receptor antibody, as the ligand. Through optimization of spacer peptides, we found a FcBP-EKGG-lipid exhibits high water-dispersibility for post-insertion method to LNPs. We prepared RI7-217-modified LNPs by modifying LNPs with FcBP-EKGG-lipids and mixing the antibodies. We found that the luciferase protein expression of RI7-217-modified LNPs was significantly enhanced in an antibody-specific manner against transferrin receptor-expressing U-87 MG cells. This information would be valuable in the development of antibody-modified LNPs for cell-selective targeting.