Small interfering RNA (siRNA) therapeutics provide a targeted approach to silence disease-related genes, with notable success in liver-targeting applications. However, the quantitative effects of siRNA properties, such as stability and affinity, as well as biological factors like cell proliferation, mRNA turnover, and abundance, on gene silencing, particularly for extrahepatic targets, remain poorly understood. To identify determinants influencing gene knockdown extent and duration, we developed a mechanistic intracellular pharmacokinetic/pharmacodynamic (PK/PD) model for RNAiMAX-delivered siRNA, based on cytoplasmic siRNA disposition, RISC-loaded siRNA exposure, and mRNA knockdown across different targets in MCF7 and BT474 cells. The model highlighted the critical roles of cell proliferation in silencing duration and mRNA turnover rates on knockdown extent. In rapid-dividing cells, mRNA half-life drives knockdown profiles, whereas chemical siRNA stabilization extends silencing in slow-dividing cells. Targets with extremely low or high mRNA abundance pose silencing challenges. While sufficient RISC occupancy is essential, increasing RISC exposure has minimal impact on silencing extent; enhancing siRNA-mRNA target engagement is more effective. The model also defined a quantitative relationship for maximal mRNA knockdown, governed by cell proliferation, mRNA half-life, and RISC-mediated cleavage rates. This mechanistic PK/PD modeling provides insights into optimizing siRNA design and target selection in therapeutic development.

RNA interference (RNAi)-based pesticides are emerging as the next generation of pest control solutions. We have previously identified Pseudomonas chlororaphis strain R3-3, which exhibits toxicity towards Plagiodera versicolora.

We engineered a mutant strain derived from R3-3, named P. chlororaphis BM, through knocking out the RNase III gene and incorporating a T7 RNA polymerase expression system to boost dsRNA production. We revealed that P. chlororaphis BM produced comparable amounts of dsRNA to the strain E. coli HT115 (DE3) while maintaining its insecticidal activity. Importantly, insect feeding bioassays demonstrated that P. chlororaphis BM expressing dsRNA targeted β-Actin (encoding β-actin protein) of P. versicolora and Srp54K (encoding signal recognition particle protein 54 k) of Henosepilachna vigintioctopunctata exhibited enhanced insecticidal efficacy compared to E. coli HT115 (DE3).

The development of P. chlororaphis BM, a novel dsRNA-expressing bacterium, holds promise for pest management due to its robust dsRNA production and sustained insecticidal activity. This research paves the way for leveraging biocontrol bacteria in RNAi-based pest management strategies. © 2025 Society of Chemical Industry.

RNA silencing regulates diverse cellular processes in plants. Argonaute (AGO), Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) proteins are core components of RNA interference (RNAi). Despite their functional significance, the systematic identification and characterization of these families have remained largely unexplored in Siraitia grosvenorii. Using HMMER and Pfam analyses, we identified six SgAGO, four SgDCL, and six SgRDR genes. Phylogenetic analysis classified SgAGOs, SgDCLs, and SgRDRs into five, four, and four clades, respectively, all of which clustered closely with homologs from other Cucurbitaceae species, demonstrating lineage-specific evolutionary conservation. Promoter cis-element analysis revealed the significant enrichment of hormonal (methyl jasmonate, abscisic acid) and stress-responsive (light, hypoxia) elements, indicating their roles in environmental adaptation. Tissue-specific expression profiling showed that most SgAGO, SgDCL, and SgRDR genes were highly expressed in flowers and mid-stage fruits (35 days after pollination), while SgAGO10.1 exhibited stem-specific expression. By contrast, SgRDR1.2 displayed no tissue specificity. Notably, sex-biased expression patterns in dioecious flowers suggested the RNAi-mediated regulation of gametophyte development and their potential roles in reproductive and secondary metabolic processes. This study lays the foundation for further exploration of RNAi machinery's role in coordinating mogroside biosynthesis and stress resilience in S. grosvenorii while providing potential targets for genetic improvement.

We previously developed a modified ethanol injection (MEI) method to construct small interfering RNA (siRNA) lipoplexes by mixing a lipid-ethanol solution with an siRNA-containing phosphate-buffered saline solution. Here, we constructed siRNA lipoplexes with 11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and poly(ethylene glycol) (PEG)-lipid using our MEI method. The siRNA lipoplexes were PEGylated with 1, 3, 5, and 10 mol% PEG cholesteryl ether (PEG-Chol), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (mPEG-DMG), or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy[polyethylene glycol]) (mPEG-DSPE). PEGylation of siRNA lipoplexes with PEG-Chol did not attenuate the inhibitory effects of Luc and polo-like kinase 1 (PLK1) siRNA lipoplexes on the luciferase (Luc) activity and proliferation of human cervical carcinoma HeLa-Luc, human ovarian cancer SK-OV-3-Luc, and human breast cancer MCF-7-Luc cells stably expressing Luc. In contrast, PEGylated lipoplexes with 10 mol% mPEG-DMG inhibited Luc activity by Luc siRNA but considerably attenuated the PLK1 siRNA-mediated cytotoxic effects. For PEGylated siRNA lipoplexes with mPEG-DSPE, inhibitory effect of Luc siRNA on Luc activity decreased with increasing amounts of PEG modification, and PLK1 siRNA-mediated cytotoxic effects disappeared at more than 3 mol% PEGylation. Erythrocyte aggregation and hemolysis induction by the siRNA lipoplexes were effectively inhibited by 10 mol% PEGylation, irrespective of the PEG-lipid. Compared to those with 1 mol% PEG-Chol, PEGylated siRNA lipoplexes with 10 mol% PEG-Chol potently reduced siRNA accumulation in mouse lungs post-intravenous administration. Overall, TC-1-12-based siRNA lipoplexes with 10 mol% PEG-Chol exerted PLK1 siRNA-mediated cytotoxic effects, without inducing hemolysis and erythrocyte aggregation.

RNA switches regulated by specific inducer molecules have become a powerful synthetic biology tool for precise gene regulation in mammalian systems. The engineered RNA switches can be integrated with natural RNA-mediated gene regulatory functions as a modular and customizable approach to probe and control cellular behavior. RNA switches have been used to advance synthetic biology applications, including gene therapy, bio-production, and cellular reprogramming. This review explores recent progress in the design and functional implementation of synthetic riboswitches in mammalian cells based on diverse RNA regulation mechanisms by highlighting recent studies and emerging technologies. We also discuss challenges such as off-target effects, system stability, and ligand delivery in complex biological environments. In conclusion, this review emphasizes the potential of synthetic riboswitches as a platform for customizable gene regulation in diverse biomedical applications.

Organisms that overwinter in temperate climates may experience freezing and freezing-induced oxidative stress during winter. While many insect species can survive freezing, reverse genetics techniques such as RNA interference (RNAi) have not been used to understand the physiological mechanisms underlying freeze tolerance. The spring field cricket Gryllus veletis can survive freezing following a 6-week fall-like acclimation. We used RNAi to knock down expression of an antioxidant enzyme in G. veletis to test the hypothesis that minimizing oxidative stress is important for freeze tolerance. In fat body tissue, Catalase mRNA abundance and enzyme activity increased during the fall-like acclimation that induces freeze tolerance. Other tissues such as midgut and Malpighian tubules had more stable or lower Catalase expression and activity during this acclimation. In summer-acclimated (freeze-intolerant) crickets, RNA interference (RNAi) effectively knocked down production of the Catalase mRNA and protein in fat body and midgut, but not Malpighian tubules. In fall-acclimated (freeze-tolerant) crickets, RNAi efficacy was temperature-dependent, functioning well at warm (c. 22 °C) but not cool (15 °C or lower) temperatures. This highlights a challenge of using RNAi in organisms acclimated to low temperatures, as they may need to be warmed up for RNAi to work, potentially affecting their stress physiology. Knockdown of Catalase via RNAi in fall-acclimated crickets also had no effect on the ability of the crickets to survive a mild freeze treatment, suggesting that Catalase may not be necessary for freeze tolerance. Our study is the first to demonstrate that RNAi is possible in a freeze-tolerant insect, but further research is needed to examine whether other genes and antioxidants are needed for G. veletis freeze tolerance.

The role of Argonaute (AGO) proteins and the RNA interference (RNAi) machinery in mammalian antiviral response has been debated. Therefore, we set out to investigate how mammalian RNAi impacts influenza A virus (IAV) infection. We reveal that IAV infection triggers nuclear accumulation of AGO2, which is directly facilitated by p53 activation. Mechanistically, we show that IAV induces nuclear AGO2 targeting of TRIM71and type-I interferon-pathway genes for silencing. Accordingly, Tp53-/- mice do not accumulate nuclear AGO2 and demonstrate decreased susceptibility to IAV infection. Hence, the RNAi machinery is highjacked by the virus to evade the immune system and support viral replication. Furthermore, the FDA-approved drug, arsenic trioxide, prevents p53 nuclear translocation, increases interferon response and decreases viral replication in vitro and in a mouse model in vivo. Our data indicate that targeting the AGO2:p53-mediated silencing of innate immunity may offer a promising strategy to mitigate viral infections.

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.

Fibrotic diseases, contributing to a significant portion of global mortality, highlight the need for innovative therapies. This study explores a novel approach to disrupt the expression of collagen by using adenine base editing to target Col1a1, a key gene driving both fibrosis and cancer metastasis. Editing Col1a1 in fibroblasts demonstrated 18% editing efficiency. An analysis of a specific clone harboring a CCAAT-to-CCGGA mutation in the Col1a1 promoter revealed reduced collagen production. Notably, when wild-type fibroblasts were cultured on the Col1a1-edited matrix, no compensatory collagen upregulation was detected, suggesting a lack of feedback mechanism in fibroblasts. Furthermore, the matrix derived from edited fibroblasts did not support the growth of MCF-7 cancer cells. These findings suggest that Col1a1 gene editing holds promise as a potential therapeutic strategy for fibrotic diseases. Further investigation is warranted to fully elucidate the implications of these findings for fibrosis and cancer.

The recent global pandemic illustrates the importance of understanding the host cellular infection processes of emerging zoonotic viruses. Nipah virus (NiV) is a deadly zoonotic biosafety level 4 encephalitic and respiratory paramyxovirus. Our knowledge of the molecular cell biology of NiV infection is extremely limited. This study identified changes in cellular components during NiV infection of human cells using a multi-platform, high-throughput transcriptomics, proteomics, lipidomics, and metabolomics approach. Remarkably, validation via multi-disciplinary approaches implicated viral glycoproteins in enriching mitochondria-associated proteins despite an overall decrease in protein translation. Our approach also allowed the mapping of significant fluctuations in the metabolism of glucose, lipids, and several amino acids, suggesting periodic changes in glycolysis and a transition to fatty acid oxidation and glutamine anaplerosis to support mitochondrial ATP synthesis. Notably, these analyses provide an atlas of cellular changes during NiV infections, which is helpful in designing therapeutics against the rapidly growing Henipavirus genus and related viral infections.

Lipid nanoparticles (LNPs) are often liver tropic, presenting challenges for LNP-delivered mRNA therapeutics intended for other tissues, as off-target expression in the liver may increase side effects and modulate immune responses. To avoid off-target expression in the liver, miR-122 binding sites have been used by others in viral and non-viral therapeutics. Here, we use a luciferase reporter system to compare different copy numbers and insertion locations of miR-122 binding sequences to restrict liver expression. We inserted one to five miR-122 binding sites into the 5' or 3' untranslated regions (UTRs) of luciferase mRNAs and tested them in LNPs in vitro and in vivo via systemic intravenous and local intramuscular injections in mice. Our results showed no significant differences in de-targeting efficacy between mRNAs harboring one or multiple miR-122 binding sites or between those with 5' or 3' UTR placements. To test the impact of miR-122 binding sites on antibody response to a mRNA vaccine, Ebola virus matrix protein VP40 mRNAs were modified with or without miR-122 binding sites and injected in mice intramuscularly. This work reinforces the utility of miR-122 binding sites while providing a comparison of these sites to aid the future development of LNP-mRNA therapies for non-hepatic tissues.

RNA interference (RNAi)-based control technologies are gaining popularity as potential alternatives to synthetic fungicides in the ongoing effort to manage plant pathogenic fungi. Among these methods, spray-induced gene silencing (SIGS) emerges as particularly promising due to its convenience and feasibility for development. This approach is a new technology for plant disease management, in which double-stranded RNAs (dsRNAs) targeting essential or virulence genes are applied to plants or plant products and subsequently absorbed by plant pathogens, triggering a gene silencing effect and the inhibition of the infection process. Spray-induced gene silencing has demonstrated efficacy in laboratory settings against various fungal pathogens. However, as research progressed from the laboratory to the greenhouse and field environments, novel challenges arose, such as ensuring the stability of dsRNAs and their effective delivery to fungal targets. Here, we provide a practical guide to SIGS for the control of plant pathogenic fungi. This guide outlines the essential steps and considerations needed for designing and assessing dsRNA molecules. It also addresses key challenges inherent to SIGS, including delivery and stability of dsRNA molecules, and how nanoencapsulation of dsRNAs can aid in overcoming these obstacles. Additionally, the guide underscores existing knowledge gaps that warrant further research and aims to provide assistance to researchers, especially those new to the field, encouraging the advancement of SIGS for the control of a broad range of fungal pathogens.

In Drosophila melanogaster, the siRNA-directed RNAi pathway provides crucial antiviral defenses. Cell-autonomously, Dicer-2 (Dcr-2) recognizes and cleaves viral dsRNA into siRNAs, which are incorporated into the RNA-induced silencing complex (RISC). Argonaute 2 (Ago2) then targets and cleaves viral RNA, preventing replication. Non-cell-autonomously, infected hemocytes secrete exosomes containing viral siRNAs, spreading antiviral signals to other cells. Additionally, tunneling nanotubes can transfer RNAi components between neighboring cells, further enhancing systemic immunity. These findings highlight the sophisticated antiviral strategies in Drosophila, offering insights for broader antiviral research.

Facioscapulohumeral muscular dystrophy (FSHD) is a common genetic disorder characterized by progressive muscle weakness, especially in the face, shoulders, and upper limbs. Despite extensive research, the underlying pathogenesis and clinical variability remain incompletely understood. This review aims to summarize recent advances in FSHD research, focusing on genetic and epigenetic factors and the potential for precision medicine.

A comprehensive review of recent literature was conducted, examining molecular mechanisms such as mutations in the D4Z4 region, DUX4 expression, RNA interference (RNAi) and antisense oligonucleotides (AOs). Clinical variability was analyzed to assess different disease phenotypes. Clinical trials investigating potential treatments, especially those targeting DUX4, were also reviewed.

FSHD shows significant clinical variability, with different progression rates across phenotypes. The 4qA allele is linked to more typical forms of the disease, but epigenetic factors, including DNA methylation and miRNA expression, also influence disease severity. Despite progress, the exact molecular mechanisms driving disease expression remain unclear. Clinical trials, such as Losmapimod, show promise in slowing muscle degeneration, though results remain inconsistent.

FSHD presents significant challenges for therapy development due to its genetic complexity and clinical variability. Ongoing research is needed to clarify pathogenesis and identify reliable biomarkers. Future therapeutic strategies should focus on precision medicine, integrating genetic, clinical, and imaging data to optimize patient stratification and treatment efficacy.

MicroRNAs (miRNAs) have emerged as intricate players in rheumatoid arthritis (RA), holding promise as discerning biomarkers for diagnostic and prognostic purposes. The lack of sensitivity and specificity in current diagnostic techniques, such as rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA), causes diagnosis delays in RA. The miR-146a and miR-155 act in inflammatory cascades and reduce joint deterioration, and miR-223 is paradoxical, acting differently in different illness scenarios. The microenvironment of RA is shaped by the complex modulation of gene expression and cytokine dynamics by miR-126 and miR-24. miRNAs serve as a promising candidate for precision medicine in the management of RA. There are obstacles encountered in validation, delivery optimization, and off-target effect mitigation before miRNA-based biomarkers may be applied in clinical settings. Machine learning (ML) and artificial intelligence (AI) have been used to integrate miRNA expression patterns with clinical data to greatly advance the treatment of RA. Because of the disease's inherent complexity and variability, these state-of-the-art models provide accurate predictions regarding the onset, development, and response to treatment of RA. By using clinical information and miRNA expression data, ML algorithms are revolutionizing the treatment of RA by predicting the onset and course of the disease with remarkably high accuracy. The development of therapeutic modalities and miRNA profiling has great potential to transform the diagnosis, prognosis, and treatment of RA, providing fresh hope for better patient outcomes.

Mitochondrial gene expression needs to be balanced with cytosolic translation to produce oxidative phosphorylation complexes. In yeast, translational feedback loops involving lowly expressed proteins called translational activators help to achieve this balance. Synthesis of cytochrome b (Cytb or COB), a core subunit of complex III in the respiratory chain, is controlled by three translational activators and the assembly factor Cbp3-Cbp6. However, the molecular interface between the COB translational feedback loop and complex III assembly is yet unknown. Here, using protein-proximity mapping combined with selective mitoribosome profiling, we reveal the components and dynamics of the molecular switch controlling COB translation. Specifically, we demonstrate that Mrx4, a previously uncharacterized ligand of the mitoribosomal polypeptide tunnel exit, interacts with either the assembly factor Cbp3-Cbp6 or with the translational activator Cbs2. These reciprocal interactions determine whether the translational activator complex with bound COB mRNA can interact with the mRNA channel exit on the small ribosomal subunit for translation initiation. Organization of the feedback loop at the tunnel exit therefore orchestrates mitochondrial translation with respiratory chain biogenesis.

The mite Varroa destructor is the most serious pest of the western honey bee (Apis mellifera) and a major factor in the global decline of colonies. Traditional control methods, such as chemical pesticides, although quick and temporarily effective, leave residues in hive products, harming bees and operators' health, while promoting pathogen resistance and spread. As a sustainable alternative, RNA interference (RNAi) technology has shown great potential for honey bee pest control in laboratory assays, but evidence of effectiveness in the field has been lacking.

We investigated the efficacy and feasibility of a RNAi treatment to improve bee health under natural beekeeping conditions by integrating a honey bee diet with a mixture of dsRNA targeting V. destructor acetyl-CoA carboxylase, Na+/K+ ATPase and endochitinase genes.

In treated hives, we observed that the average infestation rate of phoretic Varroa mite was reduced by 33% and 42% relative to control bees fed with sucrose and GFP-dsRNA, respectively. The dsRNA treatment did not affect bee survival, and the beekeepers involved in the project found the method manageable in the apiary and non-intrusive to production activities.

Our findings demonstrate the feasibility and effectiveness of RNAi technology in reducing Varroa mite infestations under natural rearing conditions. This study supports the potential of RNAi as a promising alternative to chemical pesticides, offering a targeted, efficient and sustainable solution for managing V. destructor in honey bee populations.

In this chapter, we provide a method for silencing target genes in epidermal cells via RNA interference. Specifically, we describe a protocol for transfection-mediated delivery of small interfering RNA oligonucleotides (siRNA). Functional assays are indispensable to characterize the biological consequences of gene knockdowns, and we also provide a method to analyze alterations in cell adhesion properties, consequent to knockdown of genes involved in this process.

Current treatments for retinal disorders are anti-angiogenic agents, laser photocoagulation, and photodynamic therapies. These conventional treatments focus on reducing abnormal blood vessel formation in the retina, which, in a low-oxygen environment, can lead to harmful proliferation of endothelial cells. This results in dysfunctional, leaky blood vessels that cause retinal edema, hemorrhage, and vision loss. Age-related Macular Degeneration is a primary cause of vision loss and blindness in the elderly, impacting around 20% of those over 50 years old. This complex disease is also closely related to oxidative stress in retina. In this review, we explore the challenge of treating retinal diseases, alternatives and possibilities of enhancing the effectiveness of therapies using co-delivery systems containing both antiangiogenic and antioxidant therapeutic agents. Despite recent proposals potential, the lack of extensive clinical studies on the long-term outcomes and optimal combinations of therapies means that the full risk profile and effectiveness of combined therapy are not yet completely understood. These factors must be carefully considered and managed by healthcare providers to optimize treatment outcomes and ensure patient safety.

Although omics technologies allow us to identify genes involved in various biological processes, we still rely on the analysis of the function of individual genes. Studies of the function of a specific gene include technologies such as gene silencing by RNAi or genome editing by CRISPR-cas9. However, most of them depend on the availability of transformation methods, so they are not established for all plants. In this chapter, we report a protocol that involves the exogenous application of miRNAs for the specific silencing of genes of interest. The advantage of this protocol is that it does not require a transformation event and can be applied to a certain tissue or developmental stage. This novel technology facilitates the analysis of specific gene functions in crops of economic interest.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality globally. HCC is highly heterogenous with diverse etiologies leading to different driver mutations potentiating unique tumor immune microenvironments. Current therapeutic options, including immune checkpoint inhibitors and combinations, have achieved limited objective response rates for the majority of patients. Thus, a precision medicine approach is needed to tailor specific treatment options for molecular subsets of HCC patients. Lipid nanovesicle platforms, either liposome- (synthetic) or extracellular vesicle (natural)-derived present are improved drug delivery vehicles which may be modified to contain specific cargos for targeting specific tumor sites, with a natural affinity for liver with limited toxicity. This mini-review provides updates on the applications of novel lipid nanovesicle-based therapeutics for HCC precision medicine and the challenges associated with translating this therapeutic subclass from preclinical models to the clinic.

Double-stranded RNA (dsRNA) has emerged as key player in gene silencing for the past two decades. Tailor-made dsRNA is now recognized a versatile raw material, suitable for a wide range of applications in biopesticide formulations, including insect control to pesticide resistance management. The mechanism of RNA interference (RNAi) acts at the messenger RNA (mRNA) level, utilizing a sequence-dependent approach that makes it unique in term of effectiveness and specificity compared to conventional agrochemicals. Two primary categories of small RNAs, known as short interfering RNAs (siRNAs) and microRNAs (miRNAs), function in both somatic and germline lineages in a broad range of eukaryotic species to regulate endogenous genes and to defend the genome from invasive nucleic acids. Furthermore, the application of RNAi in crop protection can be achieved by employing plant-incorporated protectants through plant transformation, but also by non-transformative strategies such as the use of formulations of sprayable RNAs as direct control agents, resistance factor repressors or developmental disruptors. This review explores the agricultural applications of RNAi, delving into its successes in pest-insect control and considering its broader potential for managing plant pathogens, nematodes, and pests. Additionally, the use of RNAi as a tool for addressing pesticide-resistant weeds and insects is reviewed, along with an evaluation of production costs and environmental implications.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is a desirable gene modification tool covering a wide area in various sectors of medicine, agriculture, and microbial biotechnology. The role of this incredible genetic engineering technology has been extensively investigated; however, it remains formidable with cargo choices, nonspecific delivery, and insertional mutagenesis. Various nanomaterials including lipid, polymeric, and inorganic are being used to deliver the CRISPR-Cas system. Progress in nanomaterials could potentially address these challenges by accelerating precision targeting, cost-effectiveness, and one-step delivery. In this review, we highlighted the advances in nanotechnology and nanomaterials as smart delivery systems for CRISPR-Cas so as to ameliorate applications for environmental remediation including biomedical research and healthcare, strategies for mitigating antimicrobial resistance, and to be used as nanofertilizers for enhancing crop growth, and reducing the environmental impact of traditional fertilizers. The timely co-evolution of nanotechnology and CRISPR technologies has contributed to smart novel nanostructure hybrids for improving the onerous tasks of environmental remediation and biological sustainability.

To develop a novel approach for increasing radiosensitivity in glioblastoma (GBM) by using targeted nanoparticles to deliver siRNA aimed at silencing the EGFR and RELA/P65 genes, which are implicated in radioresistance.

We engineered biodegradable, tumor-targeted, self-assembled, and stimuli-responsive peptide nanoparticles for efficient siRNA delivery. We evaluated the nanoparticles' ability to induce gene silencing and enhance DNA damage under radiation in vitro and in vivo. The nanoparticles were designed to exhibit pH-responsive endosomal escape and αvβ3 integrin targeting, allowing for preferential accumulation at tumor sites and traversal of the blood-brain tumor barrier.

The application of these nanoparticles resulted in significant gene silencing, increased apoptosis, and decreased cell viability. The treatment impaired DNA repair mechanisms, thereby enhancing radiosensitivity in GBM cells. In a GBM mouse model, the combination of nanoparticle treatment with radiotherapy notably prolonged survival without apparent toxicity.

Our findings suggest that nanoparticle-mediated dual gene silencing can effectively overcome GBM radioresistance. This strategy has the potential to improve clinical outcomes in GBM treatment, proposing a promising therapeutic avenue for this challenging malignancy.

Comparative ecophysiologists strive to understand physiological problems in non-model organisms, but molecular tools such as RNA interference (RNAi) are under-used in our field. Here, we provide a framework for invertebrate ecophysiologists to use RNAi to answer questions focused on physiological processes, rather than as a tool to investigate gene function. We specifically focus on non-model invertebrates, in which the use of other genetic tools (e.g., genetic knockout lines) is less likely. We argue that because RNAi elicits a temporary manipulation of gene expression, and resources to carry out RNAi are technically and financially accessible, it is an effective tool for invertebrate ecophysiologists. We cover the terminology and basic mechanisms of RNA interference as an accessible introduction for "non-molecular" physiologists, include a suggested workflow for identifying RNAi gene targets and validating biologically relevant gene knockdowns, and present a hypothesis-testing framework for using RNAi to answer common questions in the realm of invertebrate ecophysiology. This review encourages invertebrate ecophysiologists to use these tools and workflows to explore physiological processes and bridge genotypes to phenotypes in their animal(s) of interest.

The development of precise RNA-editing tools is essential for the advancement of RNA therapeutics. CRISPR (clustered regularly interspaced short palindromic repeats) PspCas13b is a programmable RNA nuclease predicted to offer superior specificity because of its 30-nucleotide spacer sequence. However, its design principles and its on-target, off-target and collateral activities remain poorly characterized. Here, we present single-base tiled screening and computational analyses that identify key design principles for potent and highly selective RNA recognition and cleavage in human cells. We show that the de novo design of spacers containing guanosine bases at precise positions can greatly enhance the catalytic activity of inefficient CRISPR RNAs (crRNAs). These validated design principles (integrated into an online tool, https://cas13target.azurewebsites.net/ ) can predict highly effective crRNAs with ~90% accuracy. Furthermore, the comprehensive spacer-target mutagenesis revealed that PspCas13b can tolerate only up to four mismatches and requires ~26-nucleotide base pairing with the target to activate its nuclease domains, highlighting its superior specificity compared to other RNA or DNA interference tools. On the basis of this targeting resolution, we predict an extremely low probability of PspCas13b having off-target effects on other cellular transcripts. Proteomic analysis validated this prediction and showed that, unlike other Cas13 orthologs, PspCas13b exhibits potent on-target activity and lacks collateral effects.

Despite an existing safe and effective vaccine for hepatitis B virus (HBV), it is still a major public health concern. Nowadays, several drugs are used to treat chronic hepatitis B; however, full healing remains controversial. The viral covalently closed circular DNA (cccDNA) formed by HBV forms a major challenge in its treatment, as does the ability of HBV to integrate itself into the host genome, which enables infection reactivation. Interfering RNA (RNAi) is a gene-silencing post-transcriptional mechanism which forms as a promising alternative to treat chronic hepatitis B. The aim of the present review is to assess the evolution of hepatitis B treatment approaches based on using RNA interference.

Data published between 2016 and 2023 in scientific databases (PubMed, PMC, LILACS, and Bireme) were assessed.

In total, 76,949 articles were initially identified and quality-checked, and 226 eligible reports were analyzed in depth. The main genomic targets, delivery systems, and major HBV therapy innovations are discussed in this review. This review reinforces the therapeutic potential of RNAi and identifies the need for conducting further studies to fill the remaining gaps between bench and clinical practice.

Euchromatic histone lysine methyltransferase 2 (EHMT2, also known as G9a) is a mammalian histone methyltransferase that catalyzes the dimethylation of histone 3 lysine 9 (H3K9). On human chromosome 15, the parental-specific expression of Prader-Willi Syndrome (PWS)-related genes, such as SNRPN and SNORD116, are regulated through the genetic imprinting of the PWS imprinting center (PWS-IC). On the paternal allele, PWS genes are expressed whereas the epigenetic maternal silencing of PWS genes is controlled by the EHMT2-mediated methylation of H3K9 in PWS-IC. Here, we measured the effects of RNA interference of EHMT2 on the maternal expression of genes deficient in PWS in mouse model and patient iPSC-derived cells.

We used small interfering RNA (siRNA) oligonucleotides and lentiviral short harpin RNA (shRNA) to reduce Ehtm2/EHMT2 expression in mouse Snord116 deletion primary neurons, PWS patient-derived induced pluripotent stem cell (iPSC) line and PWS iPSC-derived neurons. We then measured the expression of transcript or protein (if relevant) of PWS genes normally silenced on the maternal allele.

With an approximate reduction of 90% in EHMT2 mRNA and more than 80% of the EHMT2 protein, we demonstrated close to a 2-fold increase in the expression of maternal transcripts for SNRPN and SNORD116 in PWS iPSCs treated with siEHMT2 compared to PWS iPSC siControl. A similar increase in SNORD116 and SNRPN RNA expression was observed in PWS iPSC-derived neurons treated with shEHMT2.

RNAi reduction in EHMT2 activates maternally silenced PWS genes. Further studies are needed to determine whether the increase is therapeutically relevant. This study confirms the role of EHMT2 in the epigenetic regulation of PWS genes.

The Dicer protein is an indispensable player in such fundamental cell pathways as miRNA biogenesis and regulation of protein expression in a cell. Most recently, both germline and somatic mutations in DICER1 have been identified in diverse types of cancers, which suggests Dicer mutations can lead to cancer progression. In addition to well-known hotspot mutations in RNAase III domains, DICER1 is characterized by a wide spectrum of variants in all the functional domains; most are of uncertain significance and unstated clinical effects. Moreover, various new somatic DICER1 mutations continuously appear in cancer genome sequencing. The latest contemporary methods of variant effect prediction utilize machine learning algorithms on bulk data, yielding suboptimal correlation with biological data. Consequently, such analysis should be conducted based on the functional and structural characteristics of each protein, using a well-grounded targeted dataset rather than relying on large amounts of unsupervised data. Domains are the functional and evolutionary units of a protein; the analysis of the whole protein should be based on separate and independent examinations of each domain by their evolutionary reconstruction. Dicer represents a hallmark example of a multidomain protein, and we confirmed the phylogenetic multidomain approach being beneficial for the clinical effect prediction of Dicer variants. Because Dicer was suggested to have a putative role in hematological malignancies, we examined variants of DICER1 occurring outside the well-known hotspots of the RNase III domain in this type of cancer using phylogenetic reconstruction of individual domain history. Examined substitutions might disrupt the Dicer function, which was demonstrated by molecular dynamic simulation, where distinct structural alterations were observed for each mutation. Our approach can be utilized to study other multidomain proteins and to improve clinical effect evaluation.

RNA interference (RNAi) has become a widely used experimental approach for post-transcriptional regulation and is increasingly showing its potential as future targeted drugs. However, the prediction of highly efficient siRNAs (small interfering RNAs) is still hindered by dataset biases, the inadequacy of prediction methods, and the presence of off-target effects. To overcome these limitations, we propose an accurate and robust prediction method, OligoFormer, for siRNA design.

OligoFormer comprises three different modules including thermodynamic calculation, RNA-FM module, and Oligo encoder. Oligo encoder is the core module based on the transformer encoder. Taking siRNA and mRNA sequences as input, OligoFormer can obtain thermodynamic parameters, RNA-FM embedding, and Oligo embedding through these three modules, respectively. We carefully benchmarked OligoFormer against six comparable methods on siRNA efficacy datasets. OligoFormer outperforms all the other methods, with an average improvement of 9% in AUC, 6.6% in PRC, 9.8% in F1 score, and 5.1% in PCC compared to the best method among them in our inter-dataset validation. We also provide a comprehensive pipeline with prediction of siRNA efficacy and off-target effects using PITA score and TargetScan score. The ablation study shows RNA-FM module and thermodynamic parameters improved the performance and accelerated convergence of OligoFormer. The saliency maps by gradient backpropagation and base preference maps show certain base preferences in initial and terminal region of siRNAs.

The source code of OligoFormer is freely available on GitHub at: https://github.com/lulab/OligoFormer. Docker image of OligoFormer is freely available on the docker hub at https://hub.docker.com/r/yilanbai/oligoformer.

Achieving ideal plant architecture is of utmost importance for plant improvement to meet the demands of ever-increasing population. The wish list of ideal plant architecture traits varies with respect to its utilization and environmental conditions. Late seed development in woody plants poses difficulties for their propagation, and an increase in regeneration capacity can overcome this problem. The transition of a plant through sequential developmental stages e.g., embryonic, juvenile, and maturity is a well-orchestrated molecular and physiological process. The manipulation in the timing of phase transition to achieve ideal plant traits and regulation of metabolic partitioning will unlock new plant potential. Previous studies demonstrate that micro RNA156 (miR156) impairs the expression of its downstream genes to resist the juvenile-adult-reproductive phase transition to prolonged juvenility. The phenomenon behind prolonged juvenility is the maintenance of stem cell integrity and regeneration is an outcome of re-establishment of the stem cell niche. The previously reported vital and diverse functions of miR156 make it a more important case of study to explore its functions and possible ways to use it in molecular breeding. In this review, we proposed how genetic manipulation of miR156 can be used to reshape plant development phase transition and achieve ideal plant architecture. We have summarized recent studies on miR156 to describe its functional pattern and networking with up and down-stream molecular factors at each stage of the plant developmental life cycle. In addition, we have highlighted unaddressed questions, provided insights and devised molecular pathways that will help researchers to design their future studies.

Mycotoxin contamination of food and feed is a worldwide problem that needs to be addressed with highly efficient and biologically safe techniques. RNA interference (RNAi) is a natural mechanism playing an important role in different processes in eukaryotes, including the regulation of gene expression, maintenance of genome stability, protection against viruses and others. Recently, RNAi-based techniques have been widely applied for the purposes of food safety and management of plant diseases, including those caused by mycotoxin-producing fungi. In this review, we summarize the current state-of-the-art RNAi-based approaches for reducing the aggressiveness of key toxigenic fungal pathogens and mycotoxin contamination of grain and its products. The ways of improving RNAi efficiency for plant protection and future perspectives of this technique, including progress in methods of double-stranded RNA production and its delivery to the target cells, are also discussed.

DNA demethylases TET2 and TET3 play a fundamental role in thymic invariant natural killer T (iNKT) cell differentiation by mediating DNA demethylation of genes encoding for lineage specifying factors. Paradoxically, differential gene expression analysis revealed that significant number of genes were upregulated upon TET2 and TET3 loss in iNKT cells. This unexpected finding could be potentially explained if loss of TET proteins was reducing the expression of proteins that suppress gene expression. In this study, we discover that TET2 and TET3 synergistically regulate Drosha expression, by generating 5hmC across the gene body and by impacting chromatin accessibility. As DROSHA is involved in microRNA biogenesis, we proceed to investigate the impact of TET2/3 loss on microRNAs in iNKT cells. We report that among the downregulated microRNAs are members of the Let-7 family that downregulate in vivo the expression of the iNKT cell lineage specifying factor PLZF. Our data link TET proteins with microRNA expression and reveal an additional layer of TET mediated regulation of gene expression.

The foundation of most food production systems underpinning global food security is the careful management of soil resources. Embedded in the concept of soil health is the impact of diverse soil-borne pests and pathogens, and phytoparasitic nematodes represent a particular challenge. Root-knot nematodes and cyst nematodes are severe threats to agriculture, accounting for annual yield losses of US$157 billion. The control of soil-borne phytoparasitic nematodes conventionally relies on the use of chemical nematicides, which can have adverse effects on the environment and human health due to their persistence in soil, plants, and water. Nematode-resistant plants offer a promising alternative, but genetic resistance is species-dependent, limited to a few crops, and breeding and deploying resistant cultivars often takes years. Novel approaches for the control of phytoparasitic nematodes are therefore required, those that specifically target these parasites in the ground whilst minimizing the impact on the environment, agricultural ecosystems, and human health. In addition to the development of next-generation, environmentally safer nematicides, promising biochemical strategies include the combination of RNA interference (RNAi) with nanomaterials that ensure the targeted delivery and controlled release of double-stranded RNA. Genome sequencing has identified more than 75 genes in root knot and cyst nematodes that have been targeted with RNAi so far. But despite encouraging results, the delivery of dsRNA to nematodes in the soil remains inefficient. In this review article, we describe the state-of-the-art RNAi approaches targeting phytoparasitic nematodes and consider the potential benefits of nanotechnology to improve dsRNA delivery.

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy, characterized by progressive and asymmetric muscle atrophy, primarily affecting muscles of the face, shoulder girdle, and upper arms before affecting muscles of the lower extremities with age and greater disease severity. FSHD is a disabling condition, and patients may also present with various extramuscular symptoms. FSHD is caused by the aberrant expression of double homeobox 4 (DUX4) in skeletal muscle, arising from compromised epigenetic repression of the D4Z4 array. DUX4 encodes the DUX4 protein, a transcription factor that activates myotoxic gene programs to produce the FSHD pathology. Therefore, sequence-specific oligonucleotides aimed at reducing DUX4 levels in patients is a compelling therapeutic approach, and one that has received considerable research interest over the last decade. This review aims to describe the current preclinical landscape of oligonucleotide therapies for FSHD. This includes outlining the mechanism of action of each therapy and summarizing the preclinical results obtained regarding their efficacy in cellular and/or murine disease models. The scope of this review is limited to oligonucleotide-based therapies that inhibit the DUX4 gene, mRNA, or protein in a way that does not involve gene editing.

It is well acknowledged that tobacco-derived lung carcinogens can induce lung injury and even lung cancer through a complex mechanism. MicroRNAs (MiRNAs) are differentially expressed in tobacco-derived carcinogen nicotine-derived nitrosamine ketone (NNK)-treated A/J mice.

RNA sequencing was used to detect the level of long non-coding RNAs (lncRNAs). Murine and human lung normal and cancer cells were used to evaluate the function of lncRNA XIST and miR-328-3p in vitro, and NNK-treated A/J mice were used to test their function in vivo. In vivo levels of miR-328-3p and lncRNA XIST were analysed, using in situ hybridization. miR-328-3p agomir and lncRNA XIST-specific siRNA were used to manipulate in vivo levels of miR-328-3p and lncRNA XIST in A/J mice.

LncRNA XIST was up-regulated in NNK-induced lung injury and dominated the NNK-induced ectopic miRNA expression in NNK-induced lung injury both in vitro and in vivo. Either lncRNA XIST silencing or miR-328-3p overexpression exerted opposing effects in lung normal and cancer cells regarding cell migration. LncRNA XIST down-regulated miR-328-3p levels as a miRNA sponge, and miR-328-3p targeted the 3'-UTR of FZD7 mRNA, which is ectopically overexpressed in lung cancer patients. Both in vivo lncRNA XIST silencing and miR-328 overexpression could rescue NNK-induced lung injury and aberrant overexpression of the lung cancer biomarker CK19 in NNK-treated A/J mice.

Our results highlight the promotive effect of lncRNA XIST in NNK-induced lung injury and elucidate its post-transcriptional mechanisms, indicating that targeting lncRNA XIST/miR-328-3p could be a potential therapeutic strategy to prevent tobacco carcinogen-induced lung injury in vivo.

DNA demethylases TET2 and TET3 play a fundamental role in thymic invariant natural killer T (iNKT) cell differentiation by mediating DNA demethylation of genes encoding for lineage specifying factors. Paradoxically, differential gene expression analysis revealed that significant number of genes were upregulated upon TET2 and TET3 loss in iNKT cells. This unexpected finding could be potentially explained if loss of TET proteins was reducing the expression of proteins that suppress gene expression. In this study, we discover that TET2 and TET3 synergistically regulate Drosha expression, by generating 5hmC across the gene body and by impacting chromatin accessibility. As DROSHA is involved in microRNA biogenesis, we proceed to investigate the impact of TET2/3 loss on microRNAs in iNKT cells. We report that among the downregulated microRNAs are members of the Let-7 family that downregulate in vivo the expression of the iNKT cell lineage specifying factor PLZF. Our data link TET proteins with microRNA expression and reveal an additional layer of TET mediated regulation of gene expression.

MIR125B, particularly its 5p strand, is apparently involved in multiple cellular processes, including osteoblastogenesis and osteoclastogenesis. Given that MIR125B is transcribed from the loci Mir125b1 and Mir125b2, three mature transcripts (MIR125B-5p, MIR125B1-3p, and MIR125B2-3p) are generated (MIR125B-5p is common to both); however, their expression profiles and roles in the bones remain poorly understood. Both primary and mature MIR125B transcripts were differentially expressed in various organs, tissues, and cells, and their expression patterns did not necessarily correlate in wild-type (WT) mice. We generated Mir125b2 knockout (KO) mice to examine the contribution of Mir125b2 to MIR125B expression profiles and bone phenotypes. Mir125b2 KO mice were born and grew normally without any changes in bone parameters. Interestingly, in WT and Mir125b2 KO, MIR125B-5p was abundant in the calvaria and bone marrow stromal cells. These results indicate that the genetic ablation of Mir125b2 does not impinge on the bones of mice, attracting greater attention to MIR125B-5p derived from Mir125b1. Future studies should investigate the conditional deletion of Mir125b1 and both Mir125b1 and Mir125b2 in mice.

MicroRNAs (miRNAs) are small RNA molecules that regulate genes and are involved in various biological processes, including cancer development. Researchers have been exploring the potential of miRNAs as therapeutic agents in cancer treatment. Specifically, targeting the mammalian target of the rapamycin (mTOR) pathway with miRNAs has shown promise in improving the effectiveness of radiotherapy (RT), a common cancer treatment. This review provides an overview of the current understanding of miRNAs targeting mTOR as therapeutic agents to enhance RT outcomes in cancer patients. It emphasizes the importance of understanding the specific miRNAs that target mTOR and their impact on radiosensitivity for personalized cancer treatment approaches. The review also discusses the role of mTOR in cell homeostasis, cell proliferation, and immune response, as well as its association with oncogenesis. It highlights the different ways in which miRNAs can potentially affect the mTOR pathway and their implications in immune-related diseases. Preclinical findings suggest that combining mTOR modulators with RT can inhibit tumor growth through anti-angiogenic and anti-vascular effects, but further research and clinical trials are needed to validate the efficacy and safety of using miRNAs targeting mTOR as therapeutic agents in combination with RT. Overall, this review provides a comprehensive understanding of the potential of miRNAs targeting mTOR to enhance RT efficacy in cancer treatment and emphasizes the need for further research to translate these findings into improved clinical outcomes.

In Drosophila melanogaster, Dcr-2:R2D2 heterodimer binds to the 21 nucleotide siRNA duplex to form the R2D2/Dcr-2 Initiator (RDI) complex, which is critical for the initiation of siRNA-induced silencing complex (RISC) assembly. During RDI complex formation, R2D2, a protein that contains three dsRNA binding domains (dsRBD), senses two aspects of the siRNA: thermodynamically more stable end (asymmetry sensing) and the 5'-phosphate (5'-P) recognition. Despite several detailed studies to date, the molecular determinants arising from R2D2 for performing these two tasks remain elusive. In this study, we have performed structural, biophysical, and biochemical characterization of R2D2 dsRBDs. We found that the solution NMR-derived structure of R2D2 dsRBD1 yielded a canonical α1-β1-β2-β3-α2 fold, wherein two arginine salt bridges provide additional stability to the R2D2 dsRBD1. Furthermore, we show that R2D2 dsRBD1 interacts with thermodynamically asymmetric siRNA duplex independent of its 5'-phosphorylation state, whereas R2D2 dsRBD2 prefers to interact with 5'-P siRNA duplex. The mutation of key arginine residues, R53 and R101, in concatenated dsRBDs of R2D2 results in a significant loss of siRNA duplex recognition. Our study deciphers the active roles of R2D2 dsRBDs by showing that dsRBD1 initiates siRNA recognition, whereas dsRBD2 senses 5'-phosphate as an authentic mark on functional siRNA.