A large body of evidence has highlighted the role of non-coding RNAs in neurodevelopment and neuroinflammation. This evidence has led to increasing speculation that non-coding RNAs may be involved in the pathophysiological mechanisms underlying hydrocephalus, one of the most common neurological conditions worldwide. In this review, we first outline the basic concepts and incidence of hydrocephalus along with the limitations of existing treatments for this condition. Then, we outline the definition, classification, and biological role of non-coding RNAs. Subsequently, we analyze the roles of non-coding RNAs in the formation of hydrocephalus in detail. Specifically, we have focused on the potential significance of non-coding RNAs in the pathophysiology of hydrocephalus, including glymphatic pathways, neuroinflammatory processes, and neurological dysplasia, on the basis of the existing evidence. Lastly, we review the potential of non-coding RNAs as biomarkers of hydrocephalus and for the creation of innovative treatments.

The growing interest in RNA-based therapeutics has positioned small interfering RNA (siRNA) as a promising tool for gene silencing with high specificity and efficacy. However, the successful clinical application of siRNA therapies requires efficient delivery systems to overcome extracellular and intracellular barriers. Chitosan, a naturally derived polysaccharide, has gained significant attention as a non-viral vector due to its biodegradability, biocompatibility, mucoadhesive properties, and capacity to enhance cellular uptake. These attributes make chitosan an attractive alternative to lipid-based nanoparticles, which currently dominate siRNA delivery platforms. Recent advancements in chitosan-based nanoformulations, including chemical modifications and functionalization strategies, have improved siRNA stability, targeting efficiency, and transfection potential, addressing key limitations such as low bioavailability and immunogenicity. Despite these advances, challenges remain in achieving optimal release kinetics, scalability, and consistent therapeutic efficacy. Future research efforts will focus on engineering chitosan derivatives with enhanced physicochemical properties, integrating multifunctional nanocarriers, and refining formulation strategies to bridge the gap between preclinical research and clinical translation. The continued development of chitosan-based siRNA therapeutics holds significant potential for advancing precision medicine and expanding treatment options for a variety of diseases, including cancer, metabolic disorders, and inflammatory conditions.

With the rise of small interfering RNA (siRNA) as a therapeutic tool, effective siRNA design is crucial. Current methods often emphasize sequence-related features, overlooking structural information. To address this, we introduce ENsiRNA, a multimodal approach utilizing a geometric graph neural network to predict the efficacy of both standard and modified siRNA. ENsiRNA integrates sequence features from a pretrained RNA language model, structural characteristics, and thermodynamic data or chemical modifications to enhance prediction accuracy. Our results indicate that ENsiRNA outperforms existing methods, achieving over a 13% improvement in Pearson Correlation Coefficient (PCC) for standard siRNA across various tests. For modified siRNA, despite challenges associated with RNA folding methods, ENsiRNA still demonstrates competitive performance in different datasets. This novel method highlights the significance of structural information and multimodal strategies in siRNA prediction, advancing the field of therapeutic design.

AKT3, a key component of the PI3K-AKT-MTOR pathway, is highly expressed in the brain, and its activating variants cause megalencephaly and cortical malformations. In this study, we functionally verified a novel missense AKT3 variant (p.Q78R) identified in a patient with extreme megalencephaly and intractable epilepsy. We transiently transfected HEK-293T cells with the AKTWT or AKT3Q78R and observed a significant increase of phospho-S6, a marker of mTOR complex 1 (mTORC1) activity, in AKT3Q78R transfected cells. Furthermore, considering its application in epilepsy treatment research, we identified a small interfering RNA (siRNA) capable of reducing the mRNA levels of AKTQ78R without affecting the expression levels of AKT3WT. Finally, the siRNA we identified specifically suppressed the AKT3Q78R-mediated mTORC1 activity, suggesting that this allele-specific siRNA approach holds promise for ameliorating the pathological condition.

Gain-of-function variants in the KCNT1 gene, which encodes a sodium-activated potassium ion channel, drive severe early onset developmental epileptic encephalopathies including epilepsy of infancy with migrating focal seizures and sleep-related hypermotor epilepsy. No therapy provides more than sporadic or incremental improvement. Here, we report suppression of seizures in a genetic mouse model of KCNT1 epilepsy by reducing Kcnt1 transcript with divalent small interfering RNA (siRNA), an emerging variant of oligonucleotide technology developed for the central nervous system.

The ATL-201 molecule is two identical synthetic double-stranded siRNAs, covalently linked, with 100% nucleotide base pair match to sequence present in both human KCNT1 and mouse Kcnt1 that does not contain any known pathogenic variant. ATL-201 activity was tested in cortical neurons cultured from wild-type mice and in mice homozygous for Kcnt1-Y777H, the mouse ortholog to the human pathogenic KCNT1-Y796H missense variant. Seizures and nest-building behavior were measured in freely behaving Kcnt1-Y777H mice. The number and duration of seizures were measured by electrocorticography in mice dosed with ATL-201 or phosphate-buffered saline in a 6-month durability study and in a 2-month dose-efficacy study.

In vitro, ATL-201 reduced KCNT1 transcript from whole-cell lysate and eliminated potassium currents from KCNT1 channels in heterologous expression. ATL-201 also eliminated sodium-activated potassium currents recorded from individual cortical neurons. In vivo, ATL-201 suppressed seizures in Kcnt1-Y777H homozygous mice in a dose-dependent manner with near-complete suppression from 2 weeks to at least 4 months. Kcnt1-Y777H mice had defects in nest building, whereas in ATL-201-treated mice nest building was equivalent to wild-type mice.

Patients with KCNT1-driven epilepsy experience up to hundreds of seizures per day and have severe impairment in cognitive, motor, and language development and high mortality. The dose-dependent efficacy and long durability of ATL-201 in mice show promise for ATL-201 as a disease-modifying treatment of KCNT1 epilepsy.

Severe fever with thrombocytopenia syndrome virus (SFTSV) has a high mortality rate, particularly in regions such as South Korea. Although vaccines and antiviral drugs are available, their effectiveness is limited, and they often have significant side effects. This study presents a novel therapeutic strategy aimed at effectively inhibiting SFTSV gene expression through the use of biomimetic nanospheres designed for siRNA delivery. In this study, we developed a biomimetic nanosphere for siRNA delivery by combining Gn-modified Vero cell membrane with siRNA-cationic peptide nanocomplexes. The biomimetic nanospheres exhibited an average size of ∼175 nm, a ζ-potential of +20 mV, and an IC50 of 18.44 nM, demonstrating high antiviral efficacy against SFTSV.

In vivo degradation of bone scaffolds is significantly influenced by osteoclast (OC) activity, which is orchestrated by the interplay between receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG). The ratio of RANKL/OPG is a crucial determinant of OC-mediated bone resorption, which plays an integral role in bone remodeling and scaffold degradation. Elevated levels of RANKL relative to OPG enhance osteoclastogenesis, thereby accelerating the degradation process essential for integrating bone scaffolds into the host tissue.

To elucidate the effects of OPG gene silencing on osteoclastogenesis within rat bone marrow-derived mesenchymal stem cells (BMSCs). By investigating these effects, the study aimed to provide deeper insights into the regulatory mechanisms that influence bone scaffold degradation, potentially leading to improved bone repair and regeneration strategies.

We employed recombinant lentiviral plasmids to silence the OPG gene in rat BMSCs to achieve the aims. The efficacy of gene silencing was assessed using quantitative reverse transcription polymerase chain reaction and western blot analysis to measure the expression levels of OPG and RANKL. Tartrate-resistant acid phosphatase staining was utilized to evaluate the formation of OCs. Additionally, co-immunoprecipitation assays were conducted to explore the interactions between RANKL and OPG proteins, further assessing the biochemical pathways involved in osteoclastogenesis.

The silencing of the OPG gene in BMSCs resulted in a significant increase in the RANKL/OPG ratio, evidenced by decreased expression levels of OPG and increased levels of RANKL. Enhanced osteoclastogenesis was observed through tartrate-resistant acid phosphatase staining, which indicated a substantial rise in OC formation in response to the altered RANKL/OPG balance. The co-immunoprecipitation assays provided concrete evidence of the direct interaction between RANKL and OPG proteins, substantiating their pivotal roles in regulating OC activity.

The findings from this study underscore the critical role of the RANKL/OPG axis in osteoclastogenesis. Silencing of the OPG gene in BMSCs effectively increases the RANKL/OPG ratio, promoting OC activity and potentially enhancing bone scaffold degradation. This regulatory mechanism offers a promising avenue for modulating bone remodeling processes, which is essential for effective bone repair and the successful integration of bone scaffolds into damaged sites. Future research might focus on optimizing the control of this axis to better facilitate bone tissue engineering and regenerative therapies.

Increasing evidence revealed that restoring the correct expression of lncRNAs could have implications in the management of melanoma patients. In this context, here, we aim to dissect the main characteristics of lncRNAs altered in melanoma and their crosstalk with the signaling pathways involved in the progression of this disease. We also highlight the role of nucleic acid-based techniques and natural compounds (i.e., phytochemicals) as a therapeutic tool to increase or silence their expression in cancer cells. Finally, we explore the advances in nanotechnologies as delivery systems to efficiently carry these chemicals into cancer cells, thus limiting their potential off-target effects. The analysis of the literature showed that HOTAIR, MALAT1, and H19 are the oncogenic lncRNAs most studied in melanoma, while MEG3 is an important tumor suppressor decreased in this cancer. The aberrant expression of these lncRNAs affects several hallmarks of cancer, e.g., proliferation, motility, and epithelial to mesenchymal transition, promoting the melanoma plasticity and drug resistance. In this frame, siRNA, antisense oligonucleotide, and CRISPR-Cas9 genome editing appear to be the most effective nucleic acid strategies to restore the physiologic expression of lncRNA, while curcumin, resveratrol, and quercetin are the main phytochemicals able to target and influence the expression of lncRNAs altered in cancer. Overall, this study provides a comprehensive overview regarding the role of lncRNAs in the phenotype plasticity of melanoma cells and their potential targeting using RNA-based therapy and natural products.

Small interfering RNA (siRNA) has been deemed a promising therapeutic method for treating diverse diseases. siRNA-based therapeutics provide a distinct mechanism of action by selectively targeting and silencing disease-causing genes at the post-transcriptional level. This paper provides an overview of the present state of siRNA-based therapeutics, highlighting their potential in different therapeutic areas. The first section of this review introduces the basic principles of siRNA technology, including its mechanism of action and delivery methods. Subsequently, we discuss the impediments associated with siRNA delivery and manufacturing development and the strategies for overcoming these obstacles. The clinical advancement of siRNA therapeutics in various disease areas, including cancer, genetic disorders, viral infections, and inflammatory diseases, is summarized. Lastly, we summarize the successes, failures, and lessons learned from the development of siRNAs. With advancements in delivery systems and improvements in target selection, the field of medicine can be revolutionized, and siRNA therapeutics can offer new treatment options for patients.

Anticoagulation is indicated for treatment and prevention of arterial and venous thrombosis. Targeting different steps of the coagulation process, currently available anticoagulants entail an increased risk of bleeding, which detrimentally impacts on prognosis and hinders the administration of an effective antithrombotic regimen. Factor XI (FXI) inhibition has emerged as a strategy to uncouple prevention of thrombosis from bleeding. Indeed, while FXI is crucial for the amplification phase in pathological thrombosis, it is ancillary in physiological hemostasis. A comprehensive search in several scientific databases has been performed to identify relevant studies in the field. In addition, ongoing trials have been searched for in proper datasets to provide an updated and comprehensive assessment of the current state of investigations on FXI inhibition. Many compounds have been tested to inhibit FXI at different stages (i.e., synthesis, activation, or interactions with target molecules and coagulation factors). These include antisense oligonucleotides, monoclonal antibodies, small molecules, natural peptides and aptamers. In phase 2 studies, FXI inhibitors reduced thrombotic complications without any corresponding increase in bleeding. FXI inhibitors were noninferior and potentially superior to low-molecular-weight heparin in orthopedic surgery and reduced bleeding compared to apixaban in patients with atrial fibrillation. FXI inhibition is also under testing in other conditions, including end-stage renal disease, cancer, or noncardioembolic stroke. FXI inhibition represents a promising and rapidly emerging approach for a number of clinical indications. This article reviews the rationale, evidence, pharmacology, and future applications of FXI inhibition.

Delivery of exogenous nucleic acids (NAs) for gene regulation in bacteria, bypassing the barrier of the cell wall, is essential for advancing fundamental microbiology and genetic engineering, and the treatment of bacterial diseases. However, current methods that rely on electrical or chemical interventions are limited by their complexity, specialized expertise, and laboratory-specific instrumentation. This study explores the capability of gold nanoclusters (AuNCs) as carriers for delivering small-interfering RNA and antisense oligonucleotides into bacteria for targeted gene regulation while shielding them from degradation during transport. By enhancing the cytoplasmic membrane permeability, the AuNCs enable efficient internalization of NAs into both Gram-positive and Gram-negative bacteria while exerting negligible influence on bacterial activity. It is demonstrated that the rationally designed NAs can be released from the AuNCs within bacteria, enabling ~70% knockdown of mecA in Methicillin-resistant Staphylococcus aureus (MRSA). This significantly reduces MRSA's antibiotic resistance and enhances oxacillin treatment efficacy. Furthermore, the successful silencing of ligA in Escherichia coli and pilQ in Pseudomonas aeruginosa highlights the broad adaptability of the approach across diverse bacterial species. The AuNCs-based next-generation NA delivery system has the potential to transform bacterial gene regulation-previously restricted to laboratory settings-into a versatile and scalable solution for real-world application.

Gastric cancer (GC) is an exceedingly aggressive disease and ranks as the third leading cause of cancer-related deaths, which poses a huge health burden globally. Chemotherapy is commonly employed during the middle to advanced stages of cancer, although it faces frequent treatment failures attributed to drug resistance. Thus, it is imperative for researchers to identify potential targets for overcoming therapeutic resistance, thereby facilitating the development of novel anti-cancer agents for GC patients with advanced stages. Long noncoding RNAs (lncRNAs) are a diverse group of transcripts with limited protein-coding capacity, which have been recognized for functional molecules for regulating cancer progression including cell proliferation, metastasis, and drug resistance in GC. In this review, we examine the intricate molecular networks on the role of lncRNAs in drug resistance of GC. LncRNAs conferred cancer cell resistance to anti-cancer drug through various molecular mechanisms, therefore functioning as promising therapeutic targets for GC patients. Additionally, we discuss current advancements of strategies targeting lncRNAs in cancer therapy, which may pave the way for lncRNA-mediated precision medicine for this malignant disease.

This study aimed to identify key molecular targets that drive osteoclasts (OCs) influenced progression of osteoarthritis (OA) and to explore their mechanisms influencing OCs differentiation and OA progression. We conducted weighted gene co-expression network analysis (WGCNA) and differential expression analysis using OA datasets from the GEO database, cross-referencing these findings with OCs differentiation datasets, ultimately identifying COL6A1 as a hub gene. Validation results indicated that COL6A1 expression was increased during both OA progression and OCs differentiation. Immune-related analysis indicates that the expression level of COL6A1 can influence the immune microenvironment in the subchondral bone of OA. Subsequent in vitro perturbation and rescue experiments demonstrated that COL6A1 enhances OCs differentiation and formation by activating the EPAC/RAP1 signaling axis. In vivo experiments further confirmed that COL6A1 knockdown reduced OC-mediated subchondral bone remodeling and slowed OA progression in DMM mouse models. Additionally, the molecular docking results suggest that ingenol-mebutate is a potential functional inhibitor of COL6A1. In summary, this study indicates that COL6A1 promotes the differentiation and formation of OCs by activating the EPAC/RAP1 signaling axis. Targeted blockade of COL6A1 can alleviate subchondral bone remodeling and OA progression in DMM model mice. Ingenol mebutate is a potential therapeutic drug.

Non-coding RNAs (ncRNAs) are a class of RNAs that largely lack the capacity to encode proteins. They have garnered significant attention due to their central regulatory functions across numerous cellular and physiological processes at transcriptional, post-transcriptional, and translational levels. Over the past decade, ncRNA-based therapies have gained considerable attention in the diagnosis, treatment, and prevention of diseases, and many studies have revealed a significant relationship between ncRNAs and diseases. At the same time, due to their tissue specificity, an increasing number of projects have focused on the application of ncRNAs as biomarkers in diseases, as well as the design and development of novel ncRNA-based vaccines and therapies for clinical use. These ncRNAs may also drive research into the potential molecular mechanisms and complex pathogenesis of related diseases. However, new biomarkers need to be validated for their clinical effectiveness. Additionally, to produce safe and stable RNA products, factors such as purity, precise dosage, and effective delivery methods must be ensured to achieve optimal bioactivity. These challenges remain key issues in the clinical application of ncRNAs. This review summarizes the prospects of ncRNAs as potential biomarkers, as well as the current research status and clinical applications of ncRNAs in therapies and vaccines, and discusses the challenges and expectations of ncRNAs in disease diagnosis and drug therapy.

Prior studies have established that myometrial hypoxia during labor is pivotal in intensifying contractions, the alterations in gene expression and histone modifications in myometrial cells under hypoxia have yet to be documented. Here, hypoxia's enhancement of cellular contractility was confirmed, and RNA-seq identified 2,262 differentially expressed genes in human myometrial smooth muscle cells (hMSMCs) under hypoxia. Chromatin immunoprecipitation (ChIP), high-throughput chromosome conformation capture followed by ChIP (Hi-ChIP) were employed to investigate the epigenetic changes, specifically histone modifications (H3K27ac, H3K4me1, H3K27me3, and H3K4me3), in hMSMCs under hypoxia. We identified the enhancer and super-enhancer regions in hMSMCs and found HIF1A as the key mediator of these H3K27ac changes under hypoxia. Labor-associated genes regulated by HIF1A have been identified. Validation experiments on these genes such as CXCL8, RUNX1, IL-6, and PTGES3 demonstrated that HIF1A knockdown reduces their expression and associated H3K27ac modifications in peak regions of their promoters or enhancers. These findings indicate that HIF1A probably mediate changes in histone H3K27ac modifications to regulate myometrial cell contractions under hypoxia, providing potential therapeutic and intervention targets for disorders related to parturition.

Studies have shown that in the pathogenesis of head and neck squamous cell carcinoma, immune circulation obstruction caused by various factors including metabolic abnormalities, gene mutations, and matrix barrier, is a critical factor for the induction of tumor development and progression. Therefore, the immunotherapy strategy of killing head and neck squamous cell carcinoma cells by an enhanced immune circulation mechanism has attracted much attention. In addition, the rapid development of new nucleic acid drug therapy, such as mRNA, oligonucleotide and small guide RNA (sgRNA), has taken immunotherapy of head and neck squamous cell carcinoma (immune checkpoint inhibitors, tumor vaccines, cellular immunotherapy, cytokines and adjuvants, etc.) to a new level. The combination of nucleic acid therapy with immunotherapy developed for its therapeutic properties has brought a new direction for the diagnosis and treatment of head and neck squamous cell carcinoma, and the combination of the two has had considerable curative effect to patients with refractory/recurrent head and neck squamous cell carcinoma. In this review, we summarized the latest progress of nucleic acid therapy applied to conventional immunotherapy for head and neck squamous cell carcinoma, discussed its mechanism of action and efficacy, and looked into the future development trend.

Oligonucleotide (ON) is one of the rapidly developing fields in biotherapeutics. Ultra high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) has been widely used for the identification of metabolites due to its high sensitivity, high resolution, and ability to provide structural information. The identification of ON metabolites in matrix has been reported by UHPLC-HRMS, however, manual data processing is time-consuming. In this study, an analytical strategy based on UHPLC-QTOF-MS/MS and molecule profiler software was established and employed for the automatic identification of metabolites of ONs. Fomivirsen (FMVS), a 21-mer antisense oligonucleotide with 20 phosphorothioate linkages, was selected as proof of concept. Firstly, the sample preparation and UHPLC-QTOF-MS/MS condition were optimized. Secondly, the feasibility of the automatic identification of ON metabolites by this strategy was verified using enzymatic digests of FMVS. Finally, in vitro and in vivo metabolites of FMVS were identified. As a result, the enzymatic digests of FMVS were successfully identified by the established strategy, and a total of 17 metabolites were identified from serum, plasma and tissues. FMVS was mainly metabolized by 3'-exonuclease in plasma, liver and kidney. This is the first study on metabolites identification of FMVS, and the proposed strategy would simplify the identification of ON metabolites, thus could be used for other ON metabolites.

The TOR signaling pathway regulator-like (TIPRL) gene plays a multifaceted role in cancer, yet its pan-cancer profile remains underexplored. This study investigates TIPRL expression across multiple cancers and its associations with survival, genetic alterations, immune infiltration, and functional pathways, providing insights into TIPRL's role as a potential prognostic and therapeutic target.

TIPRL expression and prognostic significance across tumor types were analyzed using TCGA_GTEx and CPTAC data in R software and platforms like GEPIA2 and UALCAN. Genetic alterations and 3D structures were evaluated through cBioPortal. Associations with RNA modifications, immune checkpoints, immune cell infiltration, TMB, MSI, HRD, and enriched pathways were assessed via R and STRING databases, employing survival analysis, ssGSEA, and enrichment analyses.

TIPRL expression was elevated in most cancers, with significant stage-specific associations observed in KICH, KIRP, and LUSC. High TIPRL expression correlated with worse overall survival in ACC, BRCA, HNSC, KICH, LIHC, and MESO, suggesting its role in prognosis. Genetic analysis identified amplifications as the main alteration, with varied clinical relevance across cancers. RNA modifications in TIPRL, particularly m1A, m5C, and m6A, suggested potential regulatory mechanisms. Immune infiltration analysis revealed TIPRL's varied correlations with immune cell types and immune scores, differing by cancer type. TIPRL also positively correlated with TMB, MSI, and HRD in several cancers, indicating its association with genomic instability. Enrichment analyses highlighted TIPRL's involvement in processes like oxidative phosphorylation and autophagy, underscoring its influence in tumorigenesis.

These findings establish TIPRL as a significant biomarker in cancer progression and immune regulation, warranting further exploration into its therapeutic implications across diverse tumor types.

Small interfering RNA (siRNA) drugs were first proposed in 1999. They have reached the market for administration to patients after more than 20 years of development. The US Food and Drug Administration has approved six siRNA drugs in recent years: patisiran, givosiran, lumasiran, vutrisiran, inclisiran, and nedosiran. siRNA drugs are based on the post-transcriptional gene regulation mechanism of RNA interference. These drugs have gained widespread attention for their effectiveness, low dosage, and low frequency of administration. Theoretically, siRNA drugs have great potential due to their ability to silence almost any target gene. However, drug delivery, especially the extrahepatic one, remains a major challenge. Currently, all approved drugs target the liver. The high blood flow, natural filtration function, and drug delivery methods of the liver overall ensure high efficacy and stability of the drugs themselves. This review summarizes the history of siRNA drug development and the mechanisms of action, with a focus on the drug targets, indications, and key clinical trial results to introduce the status of both marketed drugs and those currently in clinical trials. Additionally, this review provides a brief analysis of several key stages of the commercialization process of siRNA drugs.

Peptide-based self-assembled nanosystems show great promise as non-viral gene and siRNA delivery vectors. In the current study, we designed and functionalized nanofibers for the delivery of siRNA, targeting and silencing EGFR gene overexpressed in triple-negative breast cancer. The nanofiber-mediated siRNA delivery was characterized in terms of zeta potential, morphology, and structural stability by circular dichroism spectroscopy. In cytotoxicity studies, nanofibers presented high biocompatibility showing a negligible effect on cell viability both on healthy and cancer cell lines. The binding between nanofibers and EGFR-siRNA was investigated and ascertained by performing different biophysical studies. The complex siRNA:NF was stable over time, under fetal bovine serum, temperature and ionic strength effects. Moreover, nanofibers effectiveness in stabilizing and delivering an ad hoc selected siRNA for EGFR gene expression silencing was verified in a preclinical model of triple-negative breast cancer. Specifically, a significant gene knockdown was obtained with the complex siRNA:NF, that is comparable with the effect obtained by lipofectamine/siRNA transfection. This effective gene silencing derived from the successful internalization of nanofibers by cancer cells as observed by confocal microscopy. These results suggested that this peptide-based nanofiber could be an effective and safe systemic siRNA delivery system for application in biomedical areas.

Nanotechnology involves the utilization of materials with exceptional properties at the nanoscale. Over the past few years, nanotechnologies have demonstrated significant potential in improving human health, particularly in medical treatments. The self-assembly characteristic of RNA is a highly effective method for designing and constructing nanostructures using a combination of biological, chemical, and physical techniques from different fields. There is great potential for the application of RNA nanotechnology in therapeutics. This review explores various nano-based drug delivery systems and their unique features through the impressive progress of the RNA field and their significant therapeutic promises due to their unique performance in the COVID-19 pandemic. However, a significant hurdle in fully harnessing the power of RNA drugs lies in effectively delivering RNA to precise organs and tissues, a critical factor for achieving therapeutic effectiveness, minimizing side effects, and optimizing treatment outcomes. There have been many efforts to pursue targeting, but the clinical translation of RNA drugs has been hindered by the lack of clear guidelines and shared understanding. A comprehensive understanding of various principles is essential to develop vaccines using nucleic acids and nanomedicine successfully. These include mechanisms of immune responses, functions of nucleic acids, nanotechnology, and vaccinations. Regarding this matter, the aim of this review is to revisit the fundamental principles of the immune system's function, vaccination, nanotechnology, and drug delivery in relation to the creation and manufacturing of vaccines utilizing nanotechnology and nucleic acids. RNA drugs have demonstrated significant potential in treating a wide range of diseases in both clinical and preclinical research. One of the reasons is their capacity to regulate gene expression and manage protein production efficiently. Different methods, like modifying chemicals, connecting ligands, and utilizing nanotechnology, have been essential in enabling the effective use of RNA-based treatments in medical environments. The article reviews stimuli-responsive nanotechnologies for RNA delivery and their potential in RNA medicines. It emphasizes the notable benefits of these technologies in improving the effectiveness of RNA and targeting specific cells and organs. This review offers a comprehensive analysis of different RNA drugs and how they work to produce therapeutic benefits. Recent progress in using RNA-based drugs, especially mRNA treatments, has shown that targeted delivery methods work well in medical treatments.

Nucleic acid (NA) therapy holds tremendous potential for treating a wide range of genetic diseases by the delivery of therapeutic genes into target cells. However, significant challenges exist in safely and effectively delivering these genes to their intended locations. Viral vectors, though efficient, pose risks such as immunogenicity and mutagenesis. This has resulted in growing interest in non-viral, nanoparticle-based NA delivery systems. This review article describes various physiological barriers to NA delivery and explores nanoparticle-based NA delivery systems, including bioengineered nanoparticles, peptides, lipid nanoparticles, and polymeric nanoparticles, highlighting their unique features to overcome in vivo barriers for NA delivery. While these nanoparticle-based NA delivery systems offer a promising alternative to viral vectors, challenges related to cytotoxicity, reproducible synthesis, and cost need to be addressed. The current clinical landscape of NA delivery is also discussed, emphasizing the need for safer, scalable, and cost-effective solutions. Nanoparticles represent a promising future in NA therapy, with the possibility of developing clinically relevant, non-toxic, stable, and non-immunogenic delivery vehicles, paving the way for broader therapeutic applications and improved clinical outcomes.

Platelet-rich plasma (PRP) is a promising regenerative therapy due to its simplicity, clinical application, safety, and ability to promote angiogenesis. It utilizes various angiogenic growth factors in platelets, including platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF), which are integral to the tissue repair. Critical limb ischemia (CLI) is a major symptom of peripheral arterial disease (PAD), and PRP therapy aims to improve blood circulation to the distal limb through the development of blood vessels. This review focuses on the extensive research on the molecular mechanisms of PRPs in treating CLI. A comprehensive search was conducted on Web of Science, PubMed, Google Scholar, and Scopus to find studies published during PRP therapy in critical limb ischemia up to June 2024. Current studies reveal that PRP composition varies by case, affecting preparation methods, storage duration, storage methods, and interaction with other materials. PRP-derived growth factors have shown promising results in treating CLI, but well-controlled human research is scarce despite positive animal studies.

Lipid nanoparticle (LNP)-mediated RNA delivery holds significant potential for the treatment of various liver diseases. Ionizable lipids play a crucial role in the formulation of LNPs and directly influence their delivery efficiency. In this study, we introduced an innovative concept by incorporating an ether bond into the hydrophobic tail of ionizable lipids for the first time. Three ionizable lipids, namely, ND-O1, ND-O2, and ND-O3, were synthesized based on 1-octylnonyl 8-[(2-hydroxyethyl)-[8-(nonyloxy)-8-oxooctyl] amino] octanoate (Lipid M). The efficacy of lipids-based LNPs for the delivery of the heat shock protein 47 (HSP47)-targeted siRNA to the liver was investigated. Compared to Lipid M-based LNP (LNP-M), it was observed that ND-O1 based LNP (LNP-O1) exhibited enhanced siRNA transfection efficiency in activated fibroblasts. In the fibrosis mice, LNP-O1 effectively suppressed HSP47 expression by approximately 84%, which was three times more effective than LNP-M, resulting in a significant decrease of collagen deposition and an amelioration of liver fibrosis. These findings highlighted the potential application of ND-O1 as an ionizable lipid for enhancing the efficient delivery of LNPs-delivered siRNA to the liver. Furthermore, this ionizable lipid design strategy offers a promising avenue for the improvement of the LNP delivery system.

Metal-organic frameworks (MOFs) are a class of inorganic-organic hybrid nanoparticles formed by the coordination of metal ions/clusters and organic ligands. Due to their high porosities, large surface areas, adjustable structures, and responsiveness to light/sound, etc., MOFs have shown great clinical potential in the field of tumor therapy. Tumor immunotherapy exerts antitumor effects through reshaping tumor immune microenvironment, showing significant preclinical and clinical advantages. Based on the mechanisms of immunity activation, the tumor immunotherapy agents can be divided into chemotherapeutic agents, immunomodulators, enzymes, tumor vaccines and oligonucleotide drugs, etc. Herein, we review the MOFs-based drug delivery systems for tumor immunotherapy. The classification of MOFs, followed by their antitumor immunity activation mechanisms, are first introduced. Drug delivery systems based on MOFs with different immunotherapy agents are also summarized, especially the synergetic immunity activation mechanisms triggered by MOFs and their loadings. Furthermore, the merits and drawbacks of MOFs and the potential strategies for MOFs to promote their clinical applications are discussed.

We are on the brink of a paradigm shift in both theoretical and clinical oncology. Genomic and transcriptomic profiling, alongside personalized approaches that account for individual patient variability, are increasingly shaping discourse. Discussions on the future of personalized cancer medicine are mainly dominated by the potential of non-coding RNAs (ncRNAs), which play a prominent role in cancer progression and metastasis formation by regulating the expression of oncogenic or tumor suppressor proteins at transcriptional and post-transcriptional levels; furthermore, their cell-free counterparts might be involved in intercellular communication. Non-coding RNAs are considered to be promising biomarker candidates for early diagnosis of cancer as well as potential therapeutic agents. This review aims to provide clarity amidst the vast body of literature by focusing on diverse species of ncRNAs, exploring the structure, origin, function, and potential clinical applications of miRNAs, siRNAs, lncRNAs, circRNAs, snRNAs, snoRNAs, eRNAs, paRNAs, YRNAs, vtRNAs, and piRNAs. We discuss molecular methods used for their detection or functional studies both in vitro and in vivo. We also address the challenges that must be overcome to enter a new era of cancer diagnosis and therapy that will reshape the future of oncology.

Elevated blood pressure poses a significant global health challenge, affecting over 1.28 billion adults worldwide, with a staggering 46% unaware of their condition. Despite its pervasive impact and association with cardiovascular disease, hypertension remains inadequately controlled, highlighting the urgent need for innovative treatment approaches. This review explores the potential of small interfering RNA (siRNA) therapeutics, focusing on zilebesiran, as a promising strategy for hypertension management. SiRNA therapy represents a groundbreaking approach to selectively modulate protein production, offering targeted intervention in the pathophysiological mechanisms underlying hypertension. Zilebesiran, a siRNA, targets hepatic angiotensinogen (AGT) synthesis through interaction with the asialoglycoprotein receptor, ultimately reducing angiotensin II levels and reducing blood pressure. Zilebesiran demonstrates remarkable pharmacokinetic properties, with sustained efficacy observed after single-dose administration. Clinical trials evaluating zilebesiran have shown significant reductions in blood pressure, with effects lasting up to 24 weeks post-administration. Moreover, combination therapy with angiotensin receptor blockers has demonstrated enhanced efficacy, highlighting the potential for synergistic effects in hypertension management. Importantly, zilebesiran exhibits a favorable safety profile, with manageable adverse events, primarily injection site reactions. Zilebesiran represents a transformative therapy in hypertension management, offering targeted and potent blood pressure reduction with favorable safety and dosing characteristics. Its emergence highlights the ongoing evolution of cardiovascular pharmacology and underscores the importance of innovative approaches to address the global burden of hypertension. Moving forward, concerted efforts in research and clinical practice are necessary to realize the benefits of zilebesiran into hypertension management protocols, ultimately advancing cardiovascular health worldwide.

This study examines the potential of small interfering RNA (siRNA) as a therapeutic agent for cancer targeting long non-coding RNAs (lncRNAs). The article begins with an analysis of the structure and biogenesis of lncRNA. It explains the diverse functions of lncRNAs in cancer, establishing a foundation for assessing approaches to inhibit these molecules. The analysis focuses on the consequences of lncRNA suppression through siRNA on signaling pathways associated with cancer, connecting theoretical understanding to practical applications. An evaluation of ongoing clinical trials and applications contributes to the discourse by revealing the potential for siRNA-mediated interventions to be practiced. Furthermore, an evaluation of the advantages and disadvantages of this therapeutic approach offers a nuanced viewpoint. In conclusion, the paper synthesizes significant discoveries and outlines potential avenues for future research, contributing to the dialogue surrounding personalized cancer therapeutics and precision medicine. Future challenges in using siRNA to target lncRNAs in oncology include optimizing delivery systems for efficient tumor cell uptake, minimizing off-target effects, enhancing RNA stability for a longer therapeutic window, and overcoming barriers in the tumor microenvironment. Addressing these factors is essential for the practical application of siRNA-based cancer therapies.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a complex multifactorial disease and becoming the leading cause of liver-related morbidity and mortality. MASLD spans from isolated steatosis to metabolic dysfunction-associated steatohepatitis (MASH), that may progress to cirrhosis and hepatocellular carcinoma (HCC). Genetic, metabolic, and environmental factors strongly contribute to the heterogeneity of MASLD. Lifestyle intervention and weight loss represent a viable treatment for MASLD. Moreover, Resmetirom, a thyroid hormone beta receptor agonist, has recently been approved for MASLD treatment. However, most individuals treated did not respond to this therapeutic, suggesting the need for a more tailored approach to treat MASLD. Oligonucleotide-based therapies, namely small-interfering RNA (siRNA) and antisense oligonucleotide (ASO), have been recently developed to tackle MASLD by reducing the expression of genes influencing MASH progression, such as PNPLA3 and HSD17B13. Here, we review the latest progress made in the synthesis and development of oligonucleotide-based agents targeting genetic determinants of MASH.

Molecular therapy uses nucleic acid-based therapeutics agents and becomes a promising alternative for disease conditions unresponsive to traditional pharmaceutical approaches. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are two well-known strategies used to modulate gene expression. RNA-targeted therapy can precisely modulate the function of target RNA with minimal off-target effects and can be rationally designed based on sequence data. ASOs and siRNA-based drugs have unique capabilities for using in target groups of patients or can be tailored as patient-customized N-of-1 therapeutic approach. Antisense therapy can be utilized not only for the treatment of monogenic diseases but also holds significant promise for addressing polygenic and complex diseases by targeting key genes and molecular pathways involved in disease pathogenesis. In the context of endocrine disorders, molecular therapy is particularly effective in modulating pathogenic mechanisms such as defective insulin signaling, beta-cell dysfunction and hormonal imbalances. Furthermore, siRNA and ASOs have the ability to downregulate overactive signaling pathways that contribute to complex, non-monogenic endocrine disorders, thereby addressing these conditions at their molecular origin. ASOs are also being studied worldwide as unique candidates for developing therapies for N-of-1 therapies. The sequence-specific ASOs binding provides exceptional accuracy in N-of-1 approaches, when the oligonucleotide can be targeted to a patient's exact mutant sequence. In this review we focus on diseases of the endocrine system and discuss potential RNA-targeted therapeutic opportunities in diabetes mellitus, including monogenic beta cell diabetes, and obesity, including syndrome obesity and monogenic obesity, as well as in non-monogenic or complex endocrine disorders. We also provide an overview of currently developed and available antisense molecules, and describe potentials of antisense-based therapeutics for the treatment of rare and «ultrarare» endocrine diseases.

Pharmaceutical advancements and an improved understanding of pathophysiology have enabled innovative therapies for chronic conditions like dyslipidemia. This condition is marked by abnormalities in lipid homeostasis. Nucleic acid therapeutics, including antisense oligonucleotides and small interfering RNAs, are novel management strategies that silence genes by targeting mRNA. Antisense oligonucleotides modify mRNA to inhibit protein production, whereas small interfering RNAs induce mRNA degradation via the RNA-induced silencing complex (RISC), thus offering promising treatments for dyslipidemia and atherosclerotic cardiovascular disease. Chemical modifications improve their stability and mRNA targeting. RNA-based therapies targeting PCSK9, Lp(a), ApoC-III, and ANGPTL3 hold transformative potential for treating dyslipidemia effectively. This article discusses the latest data from completed and ongoing trials on RNA therapies for dyslipidemia, including inclisiran, pelacarsen, olpasiran, zerlasiran, lepodisiran, volanesorsen, olezarsen, plozasiran, zodasiran, and solbinsiran. Each therapy targets specific molecules while also significantly impacting other lipid parameters. The promising results of these trials indicate potential improvements in lipid therapy and cardiovascular risk reduction, with ongoing studies expected to further refine the role of the novel RNA-based agents in effective lipid management.

Cancer immunotherapy has shown significant potential in treating several malignancies by stimulating the host immune system to recognize and attack cancer cells. Immunogenic cell death (ICD) can amplify the antitumor immune responses and reverse the immunosuppressive tumor microenvironment, thus increasing the sensitivity of cancer immunotherapy. In recent years, noncoding RNAs (ncRNAs) have emerged as key regulatory factors in ICD and oncologic immunity. Accordingly, ICD-related ncRNAs hold promise as novel therapeutic targets for optimizing the efficacy of cancer immunotherapy. However, the immunomodulatory properties of ICD-related ncRNAs have not yet been comprehensively summarized. Hence, we summarize the current knowledge on ncRNAs involved in ICD and their potential roles in cancer immunotherapy in this review. It deepens our understanding of ncRNAs associated with ICD and provides a new strategy to enhance cancer immunotherapy by specifically targeting the ICD-related ncRNAs.

Small interfering RNAs (siRNAs) have been successfully used as therapeutics to silence disease-causing genes when conjugated to ligands or formulated in lipid nanoparticles to target relevant cell types for efficacy while sparing other cells for safety. To support the development of new methods for delivery of siRNA therapeutics, we developed and characterized a panel of antibodies generated against chemically modified nucleotides used in therapeutic siRNA molecules, identifying a monoclonal antibody that detects a broad range of siRNA representing distinct sequences and modification patterns. By integrating this anti-siRNA antibody with additional reagents, we created a multiplex siRNA immunoassay that simultaneously quantifies siRNA uptake, trafficking, and silencing activity. Using immunohistochemistry (IHC), we applied our method on tissues from mice treated with unconjugated, GalNAc-conjugated, or cholesterol-conjugated siRNAs and quantitatively assessed the biodistribution and activity of siRNAs in various organs. In addition, we used high-content imaging (HCI) and applied our multiplex siRNA immunoassay in tissue culture to enable simultaneous quantification of siRNA uptake, activity, and intracellular colocalization with endosome markers. These methods provide a robust platform for testing nucleic acid delivery methods in vitro and in vivo, allowing precise analysis and visualization of the pharmacokinetics and pharmacodynamics of siRNA therapeutics with cellular and subcellular resolution.

Mutations in the KRAS gene are well-known tumourigenic drivers of colorectal, pancreatic and lung cancers. Mechanistically, these mutations promote uncontrolled cell proliferation and alter the tumour microenvironment during early carcinoma stages. Given their critical carcinogenic functions, significant progress has been made in developing KRAS inhibitors for cancer treatment. However, clinical applications of these KRAS inhibitor compounds are limited to specific cancer types which carry the relevant KRAS mutations. Additionally, clinical findings have shown that these compounds can induce moderate to serious side effects. Therefore, new approaches have emerged focusing on the development of universal therapeutics capable of targeting a wider range of KRAS mutations, minimising toxicity and enhancing the therapeutic efficacy. This review aims to examine these therapeutic strategies in the context of cancer treatment. It firstly provides an overview of fundamental KRAS biology within the cell signalling landscape and how KRAS mutations are associated with cancer pathogenesis. Subsequently, it introduces the development of current KRAS inhibitors which target certain KRAS mutants in different types of cancer. It then explores the potential of gene therapy approaches, including siRNA, miRNA and CRISPR methodologies. Furthermore, it discusses the use of lipid-based nanocarriers to deliver gene cargos for targeting KRAS gene mutants. Finally, it provides the insights into the future prospects for combatting KRAS mutation-associated cancers.

Plaque rupture in atherosclerosis (AS) is a major cause of acute cardiovascular events. Macrophage-induced inflammatory responses and accumulation of excess reactive oxygen species (ROS) primarily induce unstable plaques. Therefore, targeting ROS clearance and functional modulation of macrophages are clinically crucial for improving plaque stability and inhibiting AS progression. Here, we constructed a bionic nano-delivery platform, PBP@siR@PM, using platelet membranes (PM) coated with black phosphorus nanosheets (BPNSs) to target macrophages in atherosclerotic plaques. Meanwhile, PM-coated BPNSs (PBP@siR@PM) were used to deliver small interfering RNA silencing Ca2+/calmodulin-dependent protein kinase γ (CaMKIIγ) into macrophages. Furthermore, macrophage efferocytosis was restored by inhibiting CaMKIIγ and increasing the expression of MerTK, a cytosolic receptor, thus promoting the clearance of apoptotic cells from plaques. This study demonstrated that intraplaque macrophage-targeted therapy using the bionic nano-delivery platform PBP@siR@PM effectively removed excess ROS from macrophages, promoted efferocytosis, cleared apoptotic cells in plaques, improved plaque stability, and largely inhibited AS progression in ApoE-/- mice after high fat diet. In summary, this study proposes a therapeutic strategy for AS and highlights the outstanding therapeutic potential of biomimetic nanomaterials in this type of chronic inflammatory disease. STATEMENT OF SIGNIFICANCE: Rupture of atherosclerotic unstable plaques is a major cause of acute cardiovascular events. Macrophage-induced chronic inflammation and oxidative stress due to overloaded ROS are major contributors to plaque rupture. In this study, we focused on the improvement of macrophage efferocytosis within the plaque for the effective treatment of atherosclerosis. A bionic nano-delivery platform was constructed using platelet membranes (PM) coated black phosphorus nanosheets (BPNSs) to target macrophages in atherosclerotic plaques. In conclusion, intraplaque macrophage-targeted therapy based on the bionic nano-delivery platform PBP@siR@PM effectively scavenges overloaded ROS in macrophages, promotes efferocytosis, removes apoptotic cells from plaques, and improves plaque stability, which significantly inhibits the progression of atherosclerosis in ApoE-/- mice after a high-fat diet.

Nucleic acid (NA)-based drugs are promising therapeutics agents. Beyond efficacy, addressing safety concerns-particularly those specific to this class of drugs-is crucial. Here, we propose an in vitro approach to screen for potential adverse off-target effects of NA-based drugs. Human peripheral blood mononuclear cells (PBMCs), purified from buffy coats of healthy donors, were used to investigate the ability of NA-drugs to trigger toxicity pathways and inappropriate immune stimulation. PBMCs were selected for their ability to represent potential human responses, given their likelihood of interacting with administered drugs. As proof of concept, a small interfering RNA (siRNA) targeting Ryanodine Receptor mRNA (RyR2) identified by the Italian National Center for Gene Therapy and Drugs based on RNA Technology as a potential therapeutic target for dominant catecholaminergic polymorphic ventricular tachycardia, was selected. This compound and its scramble were formulated within a calcium phosphate nanoparticle-based delivery system. Positive controls for four toxicity pathways were identified through literature review, each associated with a specific type of cellular stress: oxidative stress (tert-butyl hydroperoxide), mitochondrial stress (rotenone), endoplasmic reticulum stress (thapsigargin), and autophagy (rapamycin). These controls were used to define specific mRNA signatures triggered in PBMCs, which were subsequently used as indicators of off-target effects. To assess immune activation, the release of pro-inflammatory cytokines (interleukin-6, interleukin-8, tumor necrosis factor-α, and interferon-γ) was measured 24 h after exposure. The proposed approach provides a rapid and effective screening method for identifying potential unintended effects in a relevant human model, which also allows to address gender effects and variability in responses.

Current antiviral therapy for the chronic hepatitis B virus (HBV) has a low clinical cure rate, high administration frequency, and limited efficacy in reducing HBsAg levels, leading to poor patient compliance. Novel agents are required to achieve HBV functional cure, and reduction of HBV antigenemia may enhance the activation of effective and long-lasting host immune control. HT-101 is a siRNA currently in phase I clinical trials with promising prospects for future applications. By designing and synthesizing siRNA targeting the conserved HBV S region, we evaluated its inhibitory effect on HBV biomarkers across four different genotypes (A-D). Additionally, potential cytotoxic effects were investigated. The in vivo effects and duration of inhibition were assessed using a HBV/adeno-associated virus mouse model. The EC50 values for HBV DNA, HBsAg, HBeAg, and HBV RNA in the supernatant of HepG2.2.15 cells were determined to be 0.3348 0.1696, 4.329, and 2.831 nM, respectively, while the CC50 of HT-101 against the viability of Hep2, H1 HeLa, MRC-5, HEK293, and Huh7 cell lines all exceeded 1 μM significantly. Compared with the vehicle group from days 7 to 70 postdosing, especially in the high-dose group (9 mpk), plasma levels of HBsAg, HBeAg, and HBV DNA were significantly reduced with mean reduction values ranging from 1.72 to 3.38 log10 copy/mL due to long-lasting suppression of HBsAg below the lower limit of quantitation (LLOQ), ultimately leading to induction of anti-HBs. In summary, the preclinical data demonstrate that HT-101 represents a significant breakthrough in reducing antigens and provides a promising strategy for functional cure of HBV.

Monogenic disorders are a group of human diseases caused by mutations in single genes. While some disease-altering treatments offer relief and slow the progression of certain conditions, the majority of monogenic disorders still lack effective therapies. In recent years, gene therapy has appeared as a promising approach for addressing genetic disorders. However, despite advancements in gene manipulation tools and delivery systems, several challenges remain unresolved, including inefficient delivery, lack of sustained expression, immunogenicity, toxicity, capacity limitations, genomic integration risks, and limited tissue specificity. This review provides an overview of the plasmid-based gene therapy techniques and delivery methods currently employed for monogenic diseases, highlighting the challenges they face and exploring potential strategies to overcome these barriers.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and devastating lung disorder. In response to transforming growth factor-β (TGF-β), normal lung cells proliferate and differentiate into myofibroblasts, which are instrumental in promoting disease progression. Small interfering RNA (siRNA) targeting heat shock protein 47 (HSP47) has been demonstrated to alleviate IPF by blocking collagen synthesis and secretion. Exosomes (EXOs) have been investigated for drug delivery due to their superior carrier properties. However, their loading efficiency has been a limiting factor in widely application as drug carriers. In this study, an ultrasonic microfluidic method was employed to enhance the loading efficiency of siHSP47 into EXOs, achieving 31.1% efficiency rate. EXOs were isolated from human embryonic kidney cells (293F) and loaded with siHSP47 (EXO-siHSP47). The findings indicated that EXO-siHSP47 penetrated the collagen barrier and effectively silenced HSP47 expression in activated fibroblasts in vitro. Western blotting and immunofluorescence analyses confirmed that EXO-siHSP47 significantly reduced the secretion and deposition of extracellular matrix (ECM) proteins. Wound healing and Transwell migration assays demonstrated that EXO-siHSP47 inhibited fibroblast differentiation and migration. In conclusion, 293F-derived EXOs loaded with siHSP47 present a promising therapeutic strategy for IPF.