Search Results (317)

Radon (Rn) exposure has a strong association with lung cancer risk and is influenced by epigenetic modifications. To investigate the characterization of DNA methylation (DNAm) episignatures for radon-induced lung cancer, we detected the specific changes in DNAm in blood and lung tissues using reduced representation bisulfite sequencing (RRBS). We identified the differentially methylated regions (DMRs) induced by radon exposure. The bioinformatics analysis of the DMR-mapped genes revealed that pathways in cancer were affected by radon exposure. Among them, the DNAm episignatures of MAPK10, PLCG1, PLCβ3 and PIK3R2 were repeated between lung tissue and blood, and validated by the MassArray. In addition, radon exposure promoted lung cancer development in the genetic engineering mouse model (GEMM), accompanied by decreased MAPK10 and increased PLCG1, PLCβ3, and PIK3R2 with mRNA and protein levels. Conclusively, radon exposure significantly changes the genomic DNAm patterns in lung tissue and blood. The DNAm episignatures of MAPK10, PLCG1, PLCβ3 and PIK3R2 have a significant influence on radon-induced lung cancer. This brings a new perspective to understanding the pathways involved in radon-induced lung cancer and offers potential targets for developing blood-based biomarkers and epigenetic therapeutics. Full article

Chemical modifications are the standard for small interfering RNAs (siRNAs) in therapeutic applications, but predicting their off-target effects remains a significant challenge. Current approaches often rely on sequence-based encodings, which fail to fully capture the structural and protein–RNA interaction details critical for off-target prediction. In this study, we developed a framework to generate reproducible structure-based chemical features, incorporating both molecular fingerprints and computationally derived siRNA–hAgo2 complex structures. Using an RNA-Seq off-target study, we generated over 30,000 siRNA–gene data points and systematically compared nine distinct types of feature representation strategies. Among the datasets, the highest predictive performance was achieved by Dataset 3, which used extended connectivity fingerprints (ECFPs) to encode siRNA and mRNA features. An energy-minimized dataset (7R), representing siRNA–hAgo2 structural alignments, was the second-best performer, underscoring the value of incorporating reproducible structural information into feature engineering. Our findings demonstrate that combining detailed structural representations with sequence-based features enables the generation of robust, reproducible chemical features for machine learning models, offering a promising path forward for off-target prediction and siRNA therapeutic design that can be seamlessly extended to include any modification, such as clinically relevant 2′-F or 2′-OMe. Full article

Pediatric high-grade gliomas (pHGGs), particularly diffuse midline gliomas (DMGs), are among the most lethal brain tumors due to poor survival and resistance to therapies. DMGs possess a distinct genetic profile, primarily driven by hallmark mutations such as H3K27M, ACVR1, and PDGFRA mutations/amplifications and TP53 inactivation, all of which contribute to tumor biology and therapeutic resistance. Developing physiologically relevant preclinical models that replicate both tumor biology and the tumor microenvironment (TME) is critical for advancing effective treatments. This review highlights recent progress in in vitro, ex vivo, and in vivo models, including patient-derived brain organoids, genetically engineered mouse models (GEMMs), and region-specific midline organoids incorporating SHH, BMP, and FGF2/8/19 signaling to model pontine gliomas. Key genetic alterations can now be introduced using lipofectamine-mediated transfection, PiggyBac plasmid systems, and CRISPR-Cas9, allowing the precise study of tumor initiation, progression, and therapy resistance. These models enable the investigation of TME interactions, including immune responses, neuronal infiltration, and therapeutic vulnerabilities. Future advancements involve developing immune-competent organoids, integrating vascularized networks, and applying multi-omics platforms like single-cell RNA sequencing and spatial transcriptomics to dissect tumor heterogeneity and lineage-specific vulnerabilities. These innovative approaches aim to enhance drug screening, identify new therapeutic targets, and accelerate personalized treatments for pediatric gliomas. Full article

HIV-1 Tat acts as a central molecular switch governing the transition between viral latency and active replication, making it a pivotal target for HIV-1 functional cure strategies. By binding to the viral long terminal repeat (LTR) and hijacking host transcriptional machinery, Tat dynamically regulates RNA polymerase II processivity to alter viral transcription states. Recent studies reveal its context-dependent variability: while Tat recruits chromatin modifiers and scaffolds non-coding RNAs to stabilize epigenetic silencing in latently infected cells, it also triggers rapid transcriptional amplification upon cellular activation. This review systematically analyzes the bistable regulatory mechanism of Tat and investigates advanced technologies for reprogramming this switch to eliminateviral reservoirs and achieve functional cures. Conventional approaches targeting Tat are limited by compensatory viral evolution and poor bioavailability. Next-generation interventions will employ precision-engineered tools, such as AI-optimized small molecules blocking Tat-P-TEFb interfaces and CRISPR-dCas9/Tat chimeric systems, for locus-specific LTR silencing or reactivation (“block and lock” or “shock and kill”). Advanced delivery platforms, including brain-penetrant lipid nanoparticles (LNPs), enable the targeted delivery of Tat-editing mRNA or base editors to microglial reservoirs. Single-cell multiomics elucidates Tat-mediated clonal heterogeneity, identifying “switchable” subpopulations for timed interventions. By integrating systems-level Tat interactomics, epigenetic engineering, and spatiotemporally controlled delivery, this review proposes a roadmap to disrupt HIV-1 persistence by hijacking the Tat switch, ultimately bridging mechanistic insights to clinical applications. Full article

The therapeutic potential of exosomes (Exos), a subpopulation of extracellular vesicles (EVs) secreted by various cell types, has been broadly emphasized. Exos are endosome-derived membrane-bound vesicles 50–150 nm in size. Exos can be general or cell type-specific. Their contents enable them to function as multi-signaling and vectorized vehicles. Exos are important for maintaining cellular homeostasis. They are released into extracellular spaces, leading to uptake by neighboring or distant cells and delivering their contents to modulate cell signaling. Exos influence tissue responses to injury, infection, and disease by fusion with the target cells and transferring their cargo, including cytokines, growth and angiogenic factors, signaling molecules, lipids, DNA, mRNAs, and non-coding RNAs. They are implicated in various physiological and pathological conditions, including ocular surface events, such as corneal scarring, wound healing, and inflammation. Their biocompatibility, stability, low immunogenicity, and easy detectability in bodily fluids (blood, tears, saliva, and urine) make them promising tools for diagnosing and treating ocular diseases. The potential to engineer specific Exo cargos makes them outstanding therapeutic delivery vehicles. The objective of this review is to provide novel insights into the functions of Exo cargos and their applications as biomarkers and therapeutics, or targets in the cornea. Full article

Therapeutic cancer vaccines are a new growth point of biomedicine with broad industrial prospects in the post-COVID-19 era. Many large international pharmaceutical companies and emerging biotechnology companies are deploying different tumor therapeutic cancer vaccine projects, focusing on promoting their clinical transformation, and the vaccine industry has strong momentum for development. Such vaccines are also the core engine and pilot site for the development of new vaccine targets, new vectors, new adjuvants, and new technologies, which play a key role in promoting the innovation and development of vaccines. Various therapeutic cancer vaccines, such as viral vector vaccines, bacterial vector vaccines, cell vector vaccines, peptide vaccines, and nucleic acid vaccines, have all been applied in clinical research. With the continuous development of technology, therapeutic cancer vaccines are evolving towards the trends of precise antigens, efficient carriers, diversified adjuvants, and combined applications. For instance, the rapidly advancing mRNA-4157 vaccine is a typical representative that combines personalized antigens with efficient delivery vectors (lipid nanoparticles, LNPs), and it also shows synergistic advantages in melanoma patients treated in combination with immune checkpoint inhibitors. In this article, we will systematically discuss the current research and development status and clinical research progress of various therapeutic cancer vaccines. Full article

mRNA-based drug development is revolutionizing tumor therapies by enabling precise cancer immunotherapy, tumor suppressor gene restoration, and genome editing. However, the success of mRNA therapies hinges on efficient delivery systems that can protect mRNA from degradation and facilitate its release into the cytoplasm for translation. Despite the emergence of lipid nanoparticles (LNPs) as a clinically advanced platform for mRNA delivery, the efficiency of endo/lysosomal escape still represents a substantial bottleneck. Here, we summarize the intracellular fate of mRNA-loaded LNPs, focusing on their internalization pathways and processing within the endo-lysosomal system. We also discuss the impact of endo-lysosomal processes on mRNA delivery and explore potential strategies to improve mRNA escape from endo-lysosomal compartments. This review focuses on molecular engineering strategies to enhance LNP-mediated endo/lysosomal escape by optimizing lipid composition, including ionizable lipids, helper lipids, cholesterol, and PEGylated lipids. Additionally, ancillary enhancement strategies such as surface coating and shape management are discussed. By comprehensively integrating mechanistic insights into the journey of LNPs within the endo-lysosome system and recent advances in lipid chemistry, this review offers valuable inspiration for advancing mRNA-based cancer therapies by enabling robust protein expression. Full article

(1) Background: Vascular mural cells reside in the media and outer layers of the vessel wall. Their ability to proliferate and migrate or to change phenotype in response to external cues is a central feature of the vascular response to injury. Genetically engineered mice are used for loss- or gain-of-function analyses or lineage tracing in vivo, their primary cells for mechanistic studies in vitro. Whether and how cultivation conditions affect their phenotype and function is often overlooked. (2) Methods: Here, we systematically studied how the cultivation of primary mural cells isolated from the aorta of adult wild-type mice in either basal medium (DMEM) or special media formulated for the cultivation of fibroblasts or pericytes affects their phenotype and function. (3) Results: Medium composition did not alter cell viability, but the mRNA levels of differentiated smooth muscle cell markers were highest in vascular mural cells expanded in DMEM. Conversely, significantly higher numbers of proliferating and migrating cells were observed in cells expanded in Pericyte medium, and cytoskeletal rearrangements supported increased migratory capacities. Significantly reduced telomere lengths and metabolic reprogramming was observed in aortic mural cells cultured in Fibroblast medium. (4) Conclusions: Our findings underline the plasticity of primary aortic mural cells and highlight the importance of the culture media composition during their expansion, which could be exploited to interrogate their responsiveness to external stimuli or conditions observed in vivo or in patients. Full article

Background/Objectives: Developing next-generation mRNA-based seasonal influenza vaccines remains challenging, primarily because of the relatively low immunogenicity of influenza B hemagglutinin (HA) antigens. We describe a systematic vaccine development strategy that combined vector and antigen design optimization. Methods: Novel untranslated region (UTR) sequences and a hybrid poly(A) tail were used to increase plasmid stability and mRNA expression. Fusion proteins containing HA antigens linked by T4 foldon domains were engineered to enhance the immune responses against influenza B HA antigens and to permit the expression of multiple HA ectodomains from a single mRNA species. The vaccine performance was verified in a traditional encapsulated lipid nanoparticle (LNP) formulation that requires long-term storage at temperatures below −15 °C as well as in a proprietary thermo-stable LNP formulation developed for the long-term storage of the mRNA vaccine at 2–8 °C. Results: In preclinical studies, our next-generation seasonal influenza vaccine tested alone or as a combination influenza/COVID-19 mRNA vaccine elicited hemagglutination inhibition (HAI) titers significantly higher than Fluzone HD, a commercial inactivated influenza vaccine, across all 2024/2025 seasonal influenza strains, including the B/Victoria lineage strain. At the same time, the combination mRNA vaccine demonstrated superior neutralizing antibody titers to 2023/2024 Spikevax, a commercial COVID-19 comparator mRNA vaccine. Conclusions: Our data demonstrate that the multimerization of antigens expressed as complex fusion proteins is a powerful antigen design approach that may be broadly applied toward mRNA vaccine development. Full article

The emergence of complex and rapidly evolving pathogens necessitates innovative vaccine platforms that move beyond traditional methods. This review explores the transformative potential of next-generation vaccine technologies, focusing on the combined use of synthetic biology, nanotechnology, and systems immunology. Synthetic biology provides modular tools for designing antigenic components with improved immunogenicity, as seen in mRNA, DNA, and peptide-based platforms featuring codon optimization and self-amplifying constructs. At the same time, nanotechnology enables precise antigen delivery and controlled immune activation through engineered nanoparticles such as lipid-based carriers, virus-like particles, and polymeric systems to improve stability, targeting, and dose efficiency. Systems immunology aids these advancements by analyzing immune responses through multi-omics data and computational modeling, which assists in antigen selection, immune profiling, and adjuvant optimization. This approach enhances both humoral and cellular immunity, solving challenges like antigen presentation, response durability, and vaccine personalization. Case studies on SARS-CoV-2, Epstein–Barr virus, and Mycobacterium tuberculosis highlight the practical application of these platforms. Despite promising progress, challenges include scalability, safety evaluation, and ethical concerns with data-driven vaccine designs. Ongoing interdisciplinary collaboration is crucial to fully develop these technologies for strong, adaptable, globally accessible vaccines. This review emphasizes next-generation vaccines as foundational for future immunoprophylaxis, especially against emerging infectious diseases and cancer immunotherapy. Full article

The human coronavirus 229E (HCoV-229E) is a member of the human coronavirus family that includes SARS-CoV-2, the causative agent of COVID-19. Developing antiviral strategies and compounds is crucial to treat and prevent HCoV-229E infections and the associated diseases. Ribozymes derived from ribonuclease P (RNase P) catalytic RNA represent a novel class of promising gene-targeting agents by cleaving their target mRNA and knocking down the expression of the target mRNA. However, it has not been reported whether RNase P ribozymes block the infection and replication of HCoV-229E. We report here the engineering of an anti-HCoV-229E RNase P ribozyme to target an overlapping region of viral genomic RNA and the mRNA encoding the nucleocapsid (N) protein, which is vital for viral replication and growth. The engineered ribozyme actively hydrolyzed the viral RNA target in vitro. HCoV-229E-infected cells expressing the engineered, catalytically active ribozyme exhibited a reduction of about 85% in viral RNA levels and N protein expression, and a reduction of about 750-fold in infectious particle production, compared to cells expressing no ribozymes or a control, catalytically inactive ribozyme. Our study provides the first direct evidence of the therapeutic potential of RNase P ribozymes against human coronaviruses such as HCoV-229E. Full article

Background: The global burden of obesity and type 2 diabetes mellitus is a significant contributor to mortality and disability in the modern world. In this regard, the modification of adipocyte metabolism has been identified as a promising approach to develop new genetic and cellular engineering therapeutics. In this study, we activate the expression of creatine kinase B (CKB), a key enzyme of a non-canonical futile cycle and the regulator of energy storage, to promote catabolic processes in mature adipocytes. Methods: The protein-coding sequence of CKB was amplified by PCR from Mus musculus brain mRNA. Lentiviral transduction was used to transfer the CKB sequence into mature adipocytes. Adipocyte metabolism was analyzed by radioisotope monitoring of labeled [3H]-2-deoxyglucose and [14C]-glucose. Confocal microscopy was applied to estimate lipid droplets morphology (BODIPY493/503 dye), mitochondrial membrane potential (JC-1 dye), and thermogenesis (ERthermAC dye). Results: After lentiviral delivery of the CKB-coding sequence, CKB mRNA level increased 75-fold and protein expression fivefold. CKB overexpression does not cause significant changes in lipid droplet morphology. Despite this, enhanced glucose uptake and reduced lipid synthesis under adrenergic stimulation are detected during CKB overexpression. CKB causes an increase in mitochondrial potential with no effect on thermogenesis in adipocytes. Conclusions: In this study, we have shown that CKB overexpression in mature adipocytes allows us to obtain adipocytes with high glucose uptake, potency of ATP synthesis, and suppressed lipogenesis. These genetically modified cells may potentially exhibit a favorable metabolic effect in the context of excessive nutrient utilization. Full article

cAMP (cyclic adenosine-3′,5′-monophosphate) has extensive physiological functions and nutritional value for living organisms, and it regulates cellular metabolism mainly by modulating PKA (protein kinase A) activity. The current yields of cAMP synthesized by microbial fermentation are still low, which is arousing interest in developing high-yield cAMP strains. In this work, two baker’s yeasts with high cAMP content were constructed by knocking out BCY1, TPK3, and TPK2 genes, and truncating the promoter of the TPK1 gene. The content of cAMP in BN5-126 and BN5-310 (with the TPK1 gene promoter truncated by 126 and 310 bp in BN5) was improved by 30- and 9-fold, respectively, relative to the wild strain. The TPK1 gene mRNA levels of BN5-126 and BN5-310 were decreased by 18% and 40%, respectively, without significant changes in growth performance. The results of heat shock tolerance of engineered strains also reflected the enhanced PKA activity. This work demonstrates a novel strategy for regulating gene expression to boost cAMP biosynthesis in yeast, providing a promising platform for producing nutritionally enriched and functional fermented products. Full article

Self-amplifying RNA is synthetic nucleic acid engineered to replicate within cells without generating viral particles. Derived from alphavirus genomes, saRNA retains the non-structural elements essential for replication while replacing the structural elements with an antigen of interest. By enabling efficient intracellular amplification, saRNA offers a promising alternative to conventional mRNA vaccines, enhancing antigen expression while requiring lower doses. However, this advantage comes with challenges. In this review, we highlight the key limitations of saRNA technology and explore potential strategies to overcome them. By identifying these challenges, we aim to provide insights that can guide the future design of saRNA-based therapeutics, extending their potential beyond vaccine applications. Full article

Background/Objectives: The AXL receptor tyrosine kinase is a promising therapeutic target in solid tumors, yet conventional viral vector-engineered CAR-T cells face critical limitations, including risks of insertional mutagenesis and immunogenicity from murine-derived single-chain variable fragments (scFvs). This study aimed to develop and evaluate mRNA-engineered fully human AXL CAR-T (^mfhAXL CAR-T) cells as a safer, scalable alternative for solid tumor immunotherapy. Methods:^mfhAXL CAR-T cells were generated via electroporation-mediated delivery of in vitro transcribed mRNA encoding a fully human AXL-specific CAR. CAR expression kinetics and T-cell viability were quantified by flow cytometry. Antitumor activity was assessed through in vitro co-cultures with AXL-positive lung and pancreatic cancer cells, measuring cytotoxicity, cytokine secretion, and specificity. In vivo efficacy was evaluated in a lung cancer xenograft mouse model, with tumor volume and body weight monitored over 14 days. Results: Flow cytometry confirmed transient but high CAR expression (>90% at 24 h) with preserved T-cell viability (>90%). In vitro, ^mfhAXL CAR-T cells exhibited dose-dependent cytotoxicity and antigen-specific cytokine secretion. In vivo, four administrations of ^mfhAXL CAR-T cells suppressed tumor growth without body weight loss. Conclusions: The mRNA-electroporated ^mfhAXL CAR-T platform enables cost-effective, large-scale production, offering a safer alternative to viral vector-based approaches by eliminating risks of insertional mutagenesis and immunogenicity. Full article