Search Results (190)

Background: Transfection is vital for gene therapy, mRNA treatments, CAR-T cell therapy, and regenerative medicine. While viral vectors are effective, non-viral systems like lipid nanoparticles (LNPs) offer safer, more flexible alternatives. This work explores emerging non-viral transfection technologies to improve delivery efficiency and therapeutic outcomes. Methods: This review synthesizes the current literature and recent advancements in non-viral transfection technologies. It focuses on the mechanisms, advantages, and limitations of various delivery systems, including lipid nanoparticles, biodegradable polymers, electroporation, peptide-based carriers, and microfluidic platforms. Comparative analysis was conducted to evaluate their performance in terms of transfection efficiency, cellular uptake, biocompatibility, and potential for clinical translation. Several academic search engines and online resources were utilized for data collection, including Science Direct, PubMed, Google Scholar Scopus, the National Cancer Institute’s online portal, and other reputable online databases. Results: Non-viral systems demonstrated superior performance in delivering mRNA, siRNA, and antisense oligonucleotides, particularly in clinical applications. Biodegradable polymers and peptide-based systems showed promise in enhancing biocompatibility and targeted delivery. Electroporation and microfluidic systems offered precise control over transfection parameters, improving reproducibility and scalability. Collectively, these innovations address key challenges in gene delivery, such as stability, immune response, and cell-type specificity. Conclusions: The continuous evolution of transfection technologies is pivotal for advancing gene and cell-based therapies. Non-viral delivery systems, particularly LNPs and emerging platforms like microfluidics and biodegradable polymers, offer safer and more adaptable alternatives to viral vectors. These innovations are critical for optimizing therapeutic efficacy and enabling personalized medicine, immunotherapy, and regenerative treatments. Future research should focus on integrating these technologies to develop next-generation transfection platforms with enhanced precision and clinical applicability. Full article

Background/Objectives: SARS-CoV-2 vaccine candidates comprising the receptor binding domain (RBD) of the spike protein have been shown to confer protection against infection. Previous research evaluating vaccine candidates with SARS-CoV-2 RBD fused to ferritin (RBD-ferritin) and other scaffolds suggested that multimeric assemblies of RBD can enhance antigen presentation to improve the potency and breadth of immune responses. Though RBDs directly fused to a self-assembling scaffold can be delivered as messenger RNA (mRNA) formulated with lipid nanoparticles (LNPs), reports of SARS-CoV-2 vaccine candidates that combine these approaches remain scarce. Methods: Here, we designed RBD fused to AP205 or TIP60 self-assembling nanoparticles following a search of available structures focused on several scaffold properties. RBD-AP205 and RBD-TIP60 were tested for antigenicity following transfection and for immunogenicity and neutralization potency when delivered as mRNA in mice, with RBD-ferritin as a direct comparator. Results: All scaffolded RBD constructs were readily secreted to transfection supernatant and showed antigenicity in ELISA, though clear heterogeneity in assembly was observed. RBD-AP205 and RBD-TIP60 also exhibited robust antibody binding and neutralization titers in mice that were comparable to those elicited by RBD-ferritin or a full-length membrane-bound spike. Conclusions: These data suggest that AP205 and TIP60 can present RBD as effectively as ferritin and induce similar immune responses. By describing additional scaffolds for multimeric display that accommodate mRNA delivery platforms, this work can provide new tools for future vaccine design efforts. Full article

Background: Lipid nanoparticles (LNPs) represent one of the most effective non-viral vectors for nucleic acid delivery and have demonstrated clinical success in siRNA therapies and mRNA vaccines. While considerable research has focused on optimizing ionizable lipids and helper lipids, the impact of PEGylated lipid content on LNP-mediated mRNA delivery, especially in terms of in vitro transfection efficiency and in vivo performance, remains insufficiently understood. Methods: In this study, LNPs were formulated using a self-synthesized ionizable lipid and varying molar ratios of DMG-PEG2000. Nanoparticles were prepared via nanoprecipitation, and their physicochemical properties, mRNA encapsulation efficiency, cellular uptake, and transfection efficiency were evaluated in HeLa and DC2.4 cells. In vivo delivery efficiency and organ distribution were assessed in mice following intravenous administration. Results: The PEGylated lipid content exerted a significant influence on both the in vitro and in vivo performance of LNPs. A bell-shaped relationship between PEG content and transfection efficiency was observed: 1.5% DMG-PEG2000 yielded optimal mRNA transfection in vitro, while 5% DMG-PEG2000 resulted in the highest transgene expression in vivo. This discrepancy in optimal PEG content may be attributed to the trade-off between cellular uptake and systemic circulation: lower PEG levels enhance cellular internalization, whereas higher PEG levels improve stability and in vivo bioavailability at the expense of cellular entry. Furthermore, varying the PEG-lipid content enabled the partial modulation of organ distribution, offering a formulation-based strategy to influence biodistribution without altering the ionizable lipid structure. Conclusions: This study highlights the critical role of PEGylated lipid content in balancing nanoparticle stability, cellular uptake, and in vivo delivery performance. Our findings provide valuable mechanistic insights and suggest a straightforward formulation-based strategy to optimize LNP/mRNA systems for therapeutic applications. Full article

Background: Acinetobacter baumannii (A. baumannii) has emerged as a critical human pathogen, causing high mortality rates among hospitalized patients and frequently triggering nosocomial outbreaks. The increasing prevalence of multidrug-resistant (MDR) A. baumannii poses a pressing threat to public health. To date, no commercially available vaccine against A. baumannii has been developed for clinical use. messenger RNA (mRNA)–lipid nanoparticle (LNP) vaccines have emerged as a promising vaccination strategy. Methods: In this work, we developed two mRNA vaccines targeting SmpA-PLD and the fusion protein of outer membrane proteins OmpK and Omp22. The mRNA was encapsulated in LNP and administered to BALB/c mice. We evaluated humoral and cellular immune responses, bacterial burden, inflammation, and protective efficacy against A. baumannii infection in a sepsis model. Results: These mRNA vaccines triggered robust humoral and cellular immune responses in BALB/c mice, reduced bacterial burden and inflammation in sepsis models, and provided significant protection against A. baumannii infection. Notably, the OmpK-Omp22 vaccine exhibited superior protective efficacy, reducing bacterial loads in various organs and improving survival rates in the sepsis model compared to the SmpA-PLD vaccine. Conclusions: Our findings demonstrate mRNA-LNP vaccine technology as a versatile and promising platform for the development of innovative therapeutics against A. baumannii, with the potential to mitigate acute disease and promote bacterial decolonization. These findings pave the way for the development of urgently needed and effective antibacterial vaccines. Full article

Background: Many new mRNA-based vaccine candidates in liquid mRNA-LNP formulations are under development; however, their stability limitations necessitate frozen storage, posing a significant challenge for long-term storage and transportation. Methods: In this study, an mRNA-LNP rabies vaccine, ABO1005, was prepared, freeze-dried and stored at 2–8 °C for 12-month storage stability evaluation. The immunogenicity, vaccine potency (the NIH method), and protective efficacy of ABO1005 were assessed in mice or dogs and compared to a commercialized inactivated vaccine. Results: Research conducted in mice indicated that the lyophilized vaccine exhibited comparable immunogenicity to its liquid form counterpart. Furthermore, the vaccine candidate elicited a robust humoral response lasting at least 175 days, and the specific antibody titers were not affected by the pre-administration of hyperimmune serum. In comparison to the commercialized inactivated vaccine (HDCV or PVRV), ABO1005 elicited significantly higher levels of humoral and cellular immunity. Vaccine potency testing (NIH) revealed that the potency of ABO1005 at 15 μg/dose was 8.85 IU/dose, which is substantially higher than the standard required for the lot release of rabies vaccines for current human use. In a post-exposure prophylaxis (PEP) study in Beagle dogs, the lyophilized vaccine provided 100% protection for dogs following a two-dose regimen (D0-D7), whereas commercially approved inactivated vaccine offered 83% protection. After storage at 2–8 °C for 12 months, no notable changes were observed in the particle size, encapsulation efficiency, and integrity of mRNA or in the immunogenicity of the lyophilized vaccine. Conclusions: This study successfully developed a formulation and process of freeze-drying for a rabies mRNA vaccine, paving the way for future lyophilized mRNA vaccine development. Full article

Nipah and Hendra viruses are lethal zoonotic pathogens with no approved vaccines or therapeutics. mRNA produced via in vitro transcription enables endogenous protein expression and cost reduction. Here, we systematically screened natural and artificial untranslated regions (UTRs) and identified an optimal combination for expressing henipavirus-neutralizing antibody 1E5. We generated mRNA-1E5 encapsulated in lipid nanoparticles (mRNA-1E5-LNPs). In vitro, mRNA-1E5-LNPs achieved functional antibody expression levels of >1500 ng/mL. In BALB/c mice, intravenous administration of mRNA-1E5-LNPs induced rapid antibody elevation (peak at day 3), without hepatic toxicity or tissue inflammation. We established two Hendra pseudovirus models in biosafety level 2 facilities to evaluate the efficacy of mRNA-1E5-LNPs. Low-dose prophylactic administration effectively blocked entry of the Hendra pseudovirus. Notably, a single 0.5 mg/kg dose of mRNA-1E5-LNPs, stored at 4 °C for two months and administered 7 days prior, provided good protection. Our findings provide a therapeutic strategy for henipaviral infections and a blueprint for the development of mRNA-based antibodies against emerging viruses. Full article

Background: While mRNA vaccines effectively limit hospitalization and severe COVID-19 disease, the precise early innate immune mechanisms associated with their efficacy and reactogenicity remain underexplored. The identification of innate immune correlates prior to vaccination could provide mechanistic insights and potentially predict responses. Methods: We developed an in vitro model to study the innate immune activation of pre-vaccination peripheral blood mononuclear cells (PBMCs) collected from participants enrolled in a well-characterized COVID-19 BioNTech/Pfizer BNT162b2 vaccine (BNT162b2 vaccine) cohort. Pre-vaccination PBMCs were stimulated with empty lipid nanoparticle (LNP), mRNA-LNP, or Toll-like receptor (TLR) agonists. Using multiparameter spectral flow cytometry, we analyzed the baseline immune state, innate responsiveness to stimuli, and cytokine profiles of study participants. These pre-vaccination in vitro results were analyzed for correlations with post-vaccination symptoms and spike-specific IgG responses. Results: Baseline dendritic cell (DC) states inversely correlated with the magnitude of symptoms following BNT162b2 vaccination. Heightened conventional (cDC) and weaker plasmacytoid DC (pDC) responses to RNA stimuli correlated with the magnitude of an acute IgG response. IgG durability modestly correlated with a lower pDC state but higher cDC2 and monocyte baseline states and inversely correlated with TLR3 agonist responsiveness. Conclusions: The pre-vaccination assessment of innate immune function and resting states can be used to fit models potentially predictive of immunogenicity and reactogenicity to BNT162b2 vaccination. Pre-vaccination DC states may influence reactogenicity, while the response to RNA may impact antibody responses. Our data suggest that pre-vaccination assessment offers insights into the innate mechanisms driving mRNA vaccine responses and has predictive potential. 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

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

The mRNA vaccine has protected humans from the Coronavirus disease 2019 (COVID-19) and has taken the lead in reversing the epidemic efficiently. However, the Centre of Disease Control (CDC) reported and raised the alarm of allergic or acute inflammatory adverse reactions after vaccination with mRNA-LNP vaccines. Meanwhile, the US Food and Drug Administration (FDA) has added four black-box warnings in the instructions for mRNA-LNP vaccines. Numerous studies have proven that the observance of side effects after vaccination is indeed positively correlated to the level of anti-PEG antibodies (IgM or IgG), which are enhanced by PEGylated preparations like LNP vaccine and environmental exposure. After literature research and review in the past two decades, it was found that the many clinical trial failures (BIND-014, RB006 fell in phase II) of PEG modified delivery system or PEGylated drug were related to the high expression of anti-PEG IgM and IgG. In the background of shooting multiple mRNA-LNP vaccines in billions of people around the world in the past three years, the level of anti-PEG antibodies in the population may have significantly increased, which brings potential risks for PEG-modified drug development and clinical safety. This review summarizes the experience of using mRNA-LNP vaccines from the mechanism of the anti-PEG antibodies generation, detection methods, clinical failure cases of PEG-containing products, harm analysis of abuse of PEGylation, and alternatives. In light of the increasing prevalence of anti-PEG antibodies in the population and the need to avoid secondary injuries, this review article holds greater significance by offering insights for drug developers. It suggests avoiding the use of PEG excipients when designing PEGylated drugs or PEG-modified nano-formulations and provides references for strategies such as utilizing PEG-free or alternative excipients. Full article

Adiponectin (APN) is a secreted adipokine that plays a key role in modulating energy and bone metabolism, as well as regulating inflammatory responses. The overexpression of APN has been proposed as a potential therapeutic strategy for treating obesity and related disorders. Lipid nanoparticles (LNPs) are promising vectors for transporting messenger ribonucleic acid (mRNA) molecules. This study tested whether delivering a stabilized version of adiponectin mRNA (APN mRNA) using lipid nanoparticles could reduce fat formation and promote bone repair in vitro and in vivo. We demonstrated that transfection with APN-LNP upregulated the mRNA and protein expression of APN, while inhibiting adipogenesis in 3T3-L1 adipocytes. APN-LNP enhanced osteogenic gene expression in MC3T3-E1 cells in a dose-dependent manner. It also reduced matrix metalloproteinase 9 expression in receptor activator of nuclear factor-kappaB ligand (RANKL)-stimulated RAW264.7 cells, suggesting an anti-resorptive effect. In vivo, a femoral fracture model was established to explore the application of APN-LNP in promoting bone healing in diet-induced obese mice. Micro-computed tomography and histology analysis indicated that intravenous injection with APN-LNP promoted bone healing. Fasting blood glucose and body weight were decreased in the APN-LNP group. Moreover, APN-LNP increased bone sialoprotein and runt-related transcription factor 2 expression in contralateral femurs, as well as interleukin-10 expression in white adipose tissues. Thus, our study provides promising preclinical data on the potential use of APN-LNP for treating bone disorders in obesity. 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

Background: The transmembrane fusion (F) protein of RSV plays important roles in RSV pathogenesis as it mediates the fusion between the virus and the target cell membrane. During the fusion process, the F protein transits from a metastable state (prefusion, preF) to a stable state (postfusion, postF) after the merging of the virus and cell membranes. The majority of highly neutralizing antibodies induced by natural infection or immunization target the preF form, which makes it the preferred antigen for vaccine development. Methods: Here, we designed an effective RSV mRNA vaccine, STR-V003, consisting of mRNA encoding preF protein in lipid nanoparticles (LNPs). The immunogenicity, protection efficacy and toxicity were measured in multiple animal models. Results: STR-V003 demonstrated robust immunogenicity in both mice and cotton rats, inducing high levels of neutralizing antibodies and RSV preF-specific IgG antibodies and significantly reducing the RSV viral loads in the lung and nose tissue of challenged animals. In addition, STR-V003 did not show significant enhancement of lung pathology without causing vaccine-enhanced disease (VED). The repeated dose general toxicology studies and local tolerance studies of STR-V003 were evaluated in rats and non-human primate (NHP). Conclusions: STR-V003 demonstrates a favorable safety profile and induces robust protective immunity against RSV. Full article

Self-amplifying RNA-based (saRNA) technology represents the last frontier in using synthetic RNA in vaccinology. Typically, saRNA consists of positive-strand RNA molecules of viral origin (almost exclusively from alphaviruses) where the sequences of structural proteins are replaced with the open reading frame coding the antigen of interest. For in vivo delivery, they are complexed with lipid nanoparticles (LNPs), just like current COVID-19 vaccines based on synthetic messenger RNA (mRNA). Given their ability to amplify themselves inside the cell, optimal intracellular levels of the immunogenic antigen can be achieved by delivering lower amounts of saRNA molecules compared to mRNA-based vaccines. However, the excessive intracellular accumulation of saRNA may represent a relevant drawback since, as already described in alphavirus-infected cells, the recipient cell may react by incorporating excessive RNA molecules into extracellular vesicles (EVs). These EVs can shed and enter neighboring as well as distant cells, where the EV-associated saRNA can start a new replication cycle. This mechanism could lead to an unwanted and unnecessary spread of saRNA throughout the body, posing relevant safety issues. This perspective article discusses the molecular mechanisms through which saRNAs can be transmitted among different cells/tissues. In addition, a simple way to control the possible excessive saRNA intercellular propagation through the co-expression of an EV-anchored protein inhibiting the saRNA replication is proposed. Based on current knowledge, a safety improvement of saRNA-based vaccines appears to be mandatory for their usage in healthy humans. Full article