Suppression of Thrips palmi population by spray-on application of dsRNA targeting V-ATPase-B.

The RNA interference (RNAi)-based gene silencing technique has enormous potential as a non-chemical and eco-friendly alternative to hazardous pesticides. This study reports a spray-induced gene silencing (SIGS) approach for managing Thrips palmi by lowering survival and offspring development. Vacuolar ATP synthases (V-ATPases) are responsible for survival, egg-laying, and viability of eggs in insects. In the current study, T. palmi V-ATPase-B was targeted to suppress the pest population by spray-on application of double-stranded RNA (dsRNA). Silencing of V-ATPase-B was first validated by oral administration of dsV-ATPase-B. The expression of V-ATPase-B was reduced by 5.40-fold post-dsRNA feeding leading to increased mortality (57.03 %) and reduced reproductive fitness (67.73 %). Spray-on application of naked dsV-ATPase-B at concentrations of 3.0 µg/mL and 5.0 µg/mL effectively suppressed the population by 30.00 % and 43.33 %, respectively. The expression of the target gene was downregulated by up to 4.24-fold. Two consecutive sprays at a concentration of 5.0 µg/mL provided substantial protection against the fresh release of T. palmi for up to 10 days. The spray-on application of dsV-ATPase-B would be an eco-friendly alternative for managing T. palmi populations thereby reducing crop damage and limiting the spread of orthotospoviruses.

miRNA-mediated gene silencing in Drosophila larval development involves GW182-dependent and independent mechanisms.

MicroRNAs (miRNAs) regulate a wide variety of biological processes by silencing their target genes. Argonaute (AGO) proteins load miRNAs to form an RNA-induced silencing complex (RISC), which mediates translational repression and/or mRNA decay of the targets. A scaffold protein called GW182 directly binds AGO and the CCR4-NOT deadenylase complex, initiating the mRNA decay reaction. Although previous studies have demonstrated the critical role of GW182 in cultured cells as well as in cell-free systems, its biological significance in living organisms remains poorly explored, especially in Drosophila melanogaster. Here, we generated gw182-null flies using the CRISPR/Cas9 system and found that, unexpectedly, they can survive until an early second-instar larval stage. Moreover, in vivo miRNA reporters can be effectively repressed in gw182-null first-instar larvae. Nevertheless, gw182-null flies have defects in the expression of chitin-related genes and the formation of the larval trachea system, preventing them from completing larval development. Our results highlight the importance of both GW182-dependent and -independent silencing mechanisms in vivo.

Identification of the ribosomal protein L18 (RPL18) gene family reveals that TaRPL18-1 positively regulates powdery mildew resistance in wheat.

The Ribosomal protein L18 (RPL18) protein gene family plays an important role in plant growth, development and stress response. Although the RPL18 genes have been identified in several plant species, the RPL18 gene family in wheat (Triticum aestivum) is still unexplored. This study found 8 TaRPL18 genes, each of which has a significantly different gene sequence length and is evenly distributed on the chromosome; Additionally, these proteins have similar physicochemical characteristics as well as secondary and tertiary structures. 17 RPL18 genes in 4 species (wheat, Arabidopsis, rice, and maize) were classified into 5 groups, and the TaRPL18 genes within the same group showed similar structures and conserved motifs. Analysis of the cis-acting elements in the TaRPL18 genes promoter regions revealed the presence of developmental and stress-responsive elements in the majority of the genes. Through yeast two-hybrid (Y2H) experiments, it was confirmed that the powdery mildew resistance protein TaPm46 physically interacts with the Class IV TaRPL18-1. Functional analysis indicated that TaRPL18-1-silenced wheat plants show reduced resistance to powdery mildew compared to the wild type (WT), with decreased expression levels of PAL and PPO genes, and increased expression levels of the PR gene. The findings of this study provide a basis for clarifying the function of the TaRPL18 genes and will be useful for the selection of disease-resistant varieties of wheat.

Hydrogen Sulfide-Triggered Artificial DNAzyme Switches for Precise Manipulation of Cellular Functions.

The development of synthetic molecular tools responsive to biological cues is crucial for advancing targeted cellular regulation. A significant challenge is the regulation of cellular processes in response to gaseous signaling molecules such as hydrogen sulfide (H2S). To address this, we present the design of Gas signaling molecule-Responsive Artificial DNAzyme-based Switches (GRAS) to manipulate cellular functions via H2S-sensitive synthetic DNAzymes. By incorporating stimuli-responsive moieties to the phosphorothioate backbone, DNAzymes are strategically designed with H2S-responsive azide groups at cofactor binding locations within the catalytic core region. These modifications enable their activation through H2S-reducing decaging, thereby initiating substrate cleavage activity. Our approach allows for the flexible customization of various DNAzymes to regulate distinct cellular processes in diverse scenarios. Intracellularly, the enzymatic activity of GRAS promotes H2S-induced cleavage of specific mRNA sequences, enabling targeted gene silencing and inducing apoptosis in cancer cells. Moreover, integrating GRAS with dynamic DNA assembly allows for grafting these functional switches onto cell surface receptors, facilitating H2S-triggered receptor dimerization. This extracellular activation transmits signals intracellularly to regulate cellular behaviors such as migration and proliferation. Collectively, synthetic switches are capable of rewiring cellular functions in response to gaseous cues, offering a promising avenue for advanced targeted cellular engineering.

Omics approaches to unravel insecticide resistance mechanism in Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae).

Bemisia tabaci (Gennadius) whitefly (BtWf) is an invasive pest that has already spread worldwide and caused major crop losses. Numerous strategies have been implemented to control their infestation, including the use of insecticides. However, prolonged insecticide exposures have evolved BtWf to resist these chemicals. Such resistance mechanism is known to be regulated at the molecular level and systems biology omics approaches could shed some light on understanding this regulation wholistically. In this review, we discuss the use of various omics techniques (genomics, transcriptomics, proteomics, and metabolomics) to unravel the mechanism of insecticide resistance in BtWf. We summarize key genes, enzymes, and metabolic regulation that are associated with the resistance mechanism and review their impact on BtWf resistance. Evidently, key enzymes involved in the detoxification system such as cytochrome P450 (CYP), glutathione S-transferases (GST), carboxylesterases (COE), UDP-glucuronosyltransferases (UGT), and ATP binding cassette transporters (ABC) family played key roles in the resistance. These genes/proteins can then serve as the foundation for other targeted techniques, such as gene silencing techniques using RNA interference and CRISPR. In the future, such techniques will be useful to knock down detoxifying genes and crucial neutralizing enzymes involved in the resistance mechanism, which could lead to solutions for coping against BtWf infestation.

Profiling of Phakopsora pachyrhizi transcriptome revealed co-expressed virulence effectors as prospective RNA interference targets for soybean rust management.

Soybean rust (SBR), caused by an obligate biotrophic pathogen Phakopsora pachyrhizi, is a devastating disease of soybean worldwide. However, the mechanisms underlying plant invasion by P. pachyrhizi are poorly understood, which hinders the development of effective control strategies for SBR. Here we performed detailed histological characterization on the infection cycle of P. pachyrhizi in soybean and conducted a high-resolution transcriptional dissection of P. pachyrhizi during infection. This revealed P. pachyrhizi infection leads to significant changes in gene expression with 10 co-expressed gene modules, representing dramatic transcriptional shifts in metabolism and signal transduction during different stages throughout the infection cycle. Numerous genes encoding secreted protein are biphasic expressed, and are capable of inhibiting programmed cell death triggered by microbial effectors. Notably, three co-expressed P. pachyrhizi apoplastic effectors (PpAE1, PpAE2, and PpAE3) were found to suppress plant immune responses and were essential for P. pachyrhizi infection. Double-stranded RNA coupled with nanomaterials significantly inhibited SBR infection by targeting PpAE1, PpAE2, and PpAE3, and provided long-lasting protection to soybean against P. pachyrhizi. Together, this study revealed prominent changes in gene expression associated with SBR and identified P. pachyrhizi virulence effectors as promising targets of RNA interference-based soybean protection strategy against SBR.

Molecular approaches for the management of papaya ringspot virus infecting papaya: a comprehensive review.

Papaya ringspot virus (PRSV) is a catastrophic disease that causes huge yield losses in papaya cultivation around the world. Yield losses in severely infected plants can be upto 100%. Because of this disease, papaya cultivation has been shifted to other crops in some areas of the world. Many conventional methods and breeding approaches are used against this disease, which turns out to be less effective. Considering the yield loss caused by PRSV in papaya, it is high time to focus on alternative control methods. To implement effective management strategies, molecular approaches such as Marker Assisted Breeding (MAS) or transgenic methods involving post-transcriptional gene silencing targeting the genome viz., coat protein, replicase gene, or HC Pro can be pursued. However, the public's reluctance to widely accept the transgenic approach due to health and environmental concerns necessitates a consideration of non-transgenic alternatives. Prioritizing safety and ensuring efficient virus control, non-transgenic approaches which encompass cross-protection, genome editing, and topical applications of dsRNA to induce gene silencing within the host, can be adopted. This review aims to provide comprehensive insights of various molecular tools used in managing PRSV which in turn will help in sustainable agriculture.

The transcription factor TabZIP156 acts as a positive regulator in response to drought tolerance in Arabidopsis and wheat (Triticum aestivum L.).

Drought stress strongly restricts the growth, development, and yield of wheat worldwide. Among the various transcription factors (TFs) involved in the wheat drought response, the specific functions of many basic leucine zipper (bZIP) TFs related to drought tolerance are still not well understood. In this study, we focused on the bZIP TF TabZIP156 in wheat. Our analysis showed that TabZIP156 was highly expressed in both roots and leaves, and it responded to drought and abscisic acid (ABA) stress. Through subcellular localization and transactivation assays, we confirmed that TabZIP156 was located to the nucleus and functioned as a transcriptional activator. Overexpression of TabZIP156 in Arabidopsis enhanced drought tolerance, as evidenced by higher germination rate, longer root length, lower water loss rate, reduced ion leakage, increased proline accumulation, decreased levels of H2O2, O2- and MDA, and improved activities of POD, SOD, and CAT enzymes. Additionally, the expression of drought- and antioxidant-related genes were significantly upregulated in TabZIP156 transgenic Arabidopsis under drought stress. However, silencing TabZIP156 in wheat led to decreased proline content, increased accumulation of H2O2, O2- and MDA, reduced activities of antioxidant enzymes, and downregulation of many drought- and antioxidant-related genes under drought stress. Furthermore, the dual-luciferase assay demonstrated that TabZIP156 could activate the expression of TaP5CS, TaDREB1A, and TaPOD by binding to their promoters. Taken together, this study highlights the significant role of TabZIP156 in drought stress and provides valuable insights for its potential application in breeding drought-resistant wheat.

Impacts of phosphoenolpyruvate carboxylase gene silencing on photosynthetic efficiency and carbon fixation in Skeletonema costatum.

Diatoms play a crucial role in marine primary productivity through carbon fixation, which is essential for understanding the operation of marine biological pumps and carbon sinks. This study focuses on the phosphoenolpyruvate carboxylase (PEPC) gene, a key enzyme in the carbon assimilation pathway of diatoms, by investigating the consequences of its silencing in Skeletonemacostatum. Through this approach, we aimed to clarify the distinct contributions of PEPC to the overall carbon fixation process. The mutant strains of S. costatum were subjected to thorough analysis to identify any shifts in physiological behavior, alterations in the gene expression of key carbon-fixing enzymes, and changes in the associated enzyme activities. Notably, the inhibition of the PEPC gene did not significantly affect the growth rate of S. costatum; however, it did have a notable impact on the photosynthetic apparatus, as evidenced by a reduction in the maximal electron transport rate and a decline in light utilization efficiency. A significant decrease was observed in both the enzymatic activity and gene expression of PEPCase. This down-regulation also affected other enzymes integral to the carbon fixation pathway, such as phosphoenolpyruvate carboxykinase and pyruvate-phosphate dikinase, indicating a wider metabolic perturbation. In contrast, the expression and activity of the Rubisco enzyme suggested that some facets of carbon fixation remained resilient. Furthermore, the substantial upregulation of carbonic anhydrase expression and activity probably represented an adaptive mechanism to sustain the inorganic carbon supply necessary for the carboxylation process of Rubisco. This research not only underscores the pivotal role of the PEPC gene in the carbon fixation of S. costatum but also expands our comprehension of carbon fixation mechanisms in diatoms.

Biotechnological frontiers in harnessing allelopathy for sustainable crop production.

Allelopathy, the phenomenon in which plants release biochemical compounds that influence the growth and development of neighbouring plants, presents promising opportunities for revolutionizing agriculture towards sustainability. This abstract explores the role of biotechnological advancements in unlocking the potential of allelopathy for sustainable crop production and its applications in agriculture, ecology, and natural resource management. By combining molecular, genetic, biochemical, and bioinformatic tools, researchers can unravel the complexities of allelopathic interactions and their potential for sustainable crop production and environmental stewardship. The development of novel management methods for weed control is getting a lot of attention with the introduction of new genetic technologies such as Gene drive, Transgene technologies, Gene silencing, Marker-assisted selection (MAS), and Clustered regularly interspaced short palindromic repeats (CRISPR-Cas9). By strengthening competitive characteristics these tools hold great promise for boosting crops' ability to compete with weeds. Considering recent literature, this review highlights the genetic, transcriptomics, and metabolomics approaches to allelopathy. Employing allelopathic properties in agriculture offer sustainable benefits like natural weed management, pest management, and reduced chemical pollution, but challenges include environmental factors, toxicity, regulatory hurdles, and limited resources. Effective integration requires continued research, regulatory support, and farmer education​. Also, we aimed to identify the biotechnological domains requiring more investigation and to provide the basis for future advances through this assessment.

Metabolite and Transcriptome Profiling Analysis Provides New Insights into the Distinctive Effects of Exogenous Melatonin on Flavonoids Biosynthesis in Rosa rugosa.

Although the petals of Rosa rugosa are rich in flavonoids and their bioactivity has a significant impact on human health, the flavonoid content decreases during flower development. In this study, R. rugosa 'Feng hua' was used to investigate the effects of the melatonin foliar spray on enhancing the quality of rose by focusing on major flavonoids. The results showed that the contents of total flavonoids in rose petals at the full bloom stage induced by melatonin obeyed a bell-shaped curve, with a maximum at 0.3 mM, indicating the concentration-dependent up-regulation of flavonoid biosynthesis. In the treatment with 0.3 mM melatonin, metabolomic analyses showed that the concentrations of ten main flavonoids were identified to be increased by melatonin induction, with high levels and increases observed in three flavonols and two anthocyanins. KEGG enrichment of transcriptomic analysis revealed a remarkable enrichment of DEGs in flavonoid and flavonol biosynthesis, such as Rr4CL, RrF3H, and RrANS. Furthermore, functional validation using virus-induced gene silencing technology demonstrated that Rr4CL3 is the crucial gene regulating flavonoid biosynthesis in response to the stimulant of melatonin. This study provides insights into the exogenous melatonin regulation mechanism of biosynthesis of flavonoids, thereby offering potential industrial applications.

Expression Pattern and Functional Analysis of MebHLH149 Gene in Response to Cassava Bacterial Blight.

The significant reduction in cassava (Manihot esculenta Crantz) yields attributed to cassava bacterial blight (CBB) constitutes an urgent matter demanding prompt attention. The current study centered on the MebHLH149 transcription factor, which is acknowledged to be reactive to CBB and exhibits augmented expression levels, as indicated by laboratory transcriptome data. Our exploration, encompassing Xanthomonas phaseoli pv. manihotis strain CHN01 (Xpm CHN01) and hormone stress, disclosed that the MebHLH149 gene interacts with the pathogen at the early stage of infection. Furthermore, the MebHLH149 gene has been discovered to be responsive to the plant hormones abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid (SA), intimating a potential role in the signaling pathways mediated by these hormones. An analysis of the protein's subcellular localization suggested that MebHLH149 is predominantly located within the nucleus. Through virus-induced gene silencing (VIGS) in cassava, we discovered that MebHLH149-silenced plants manifested higher disease susceptibility, less ROS accumulation, and significantly larger leaf spot areas compared to control plants. The proteins MePRE5 and MePRE6, which are predicted to interact with MebHLH149, demonstrated complementary downregulation and upregulation patterns in response to silencing and overexpression of the MebHLH149 gene. This implies a potential interaction between MebHLH149 and these proteins. Both MePRE5 and MePRE6 genes are involved in the initial immune response to CBB. Notably, MebHLH149 was identified as a protein that physically interacts with MePRE5 and MePRE6. Based on these findings, it is hypothesized that the MebHLH149 gene likely functions as a positive regulator in the defense mechanisms of cassava against CBB.

Regulation of Anthocyanin Accumulation in Tomato Solanum lycopersicum L. by Exogenous Synthetic dsRNA Targeting Different Regions of SlTRY Gene.

RNA interference (RNAi) is a regulatory and protective mechanism that plays a crucial role in the growth, development, and control of plant responses to pathogens and abiotic stresses. In spray-induced gene silencing (SIGS), exogenous double-stranded RNAs (dsRNA) are used to efficiently regulate target genes via plant surface treatment. In this study, we aimed to evaluate the effect of specific exogenous dsRNAs on silencing different regions (promoter, protein-coding and intron) of the target SlTRY tomato gene, encoding an R3-type MYB repressor of anthocyanin biosynthesis. We also assessed the impact of targeting different SlTRY regions on the expression of genes involved in anthocyanin and flavonoid biosynthesis. This study demonstrated the critical importance of selecting the appropriate gene target region for dsRNA action. The highest inhibition of the SlTRY gene expression and activation of anthocyanin biosynthesis was achieved by dsRNA complementary to the protein-coding region of SlTRY gene, compared with dsRNAs targeting the SlTRY promoter or intron regions. Silencing the SlTRY gene increased the content of anthocyanins and boosted levels of other substances in the phenylpropanoid pathway, such as caffeoyl putrescine, chlorogenic acid, ferulic acid glucoside, feruloyl quinic acid, and rutin. This study is the first to examine the effects of four different dsRNAs targeting various regions of the SlTRY gene, an important negative regulator of anthocyanin biosynthesis.

Advancements in surgical treatments for Huntington disease: From pallidotomy to experimental therapies.

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder characterized by choreic movements, behavioral changes, and cognitive impairment. The pathogenesis of this process is a consequence of mutant protein toxicity in striatal and cortical neurons. Thus far, neurosurgical management of HD has largely been limited to symptomatic relief of motor symptoms using ablative and stimulation techniques. These interventions, however, do not modify the progressive course of the disease. More recently, disease-modifying experimental therapeutic strategies have emerged targeting intrastriatal infusion of neurotrophic factors, cell transplantation, HTT gene silencing, and delivery of intrabodies. Herein we review therapies requiring neurosurgical intervention, including those targeting symptom management and more recent disease-modifying agents, with a focus on safety, efficacy, and surgical considerations.

LinQURE: A novel AAV gene silencing platform that supports multi-transcript targeting for complex disorders.

Given that numerous genetic disorders, driven by diverse pathogenic mechanisms, may be amenable to recombinant adeno-associated virus (rAAV)-delivered gene therapies, the sustained innovation of rAAV-based therapeutic modalities is crucial. The progression and severity of genetic diseases can be reduced by targeting the toxic transcripts of a defective gene using microRNA (miRNA)-based miQURE technology delivered within an AAV vector. By adapting the delivered cassette, it may be possible to simultaneously regulate the expression profile of multiple genes involved in the pathogenesis of complex genetic diseases. The established miQURE gene silencing strategy was expanded by concatenating several miQURE molecules in a single construct, resulting in the novel linQURE platform. Here, a proof of mechanism is established by demonstrating that linQURE technology enables the concomitant expression of two synthetic miRNAs in vitro and in vivo, allowing more efficient downregulation of their disease-causing mRNA targets. This approach supports the development of multi-targeting therapeutic strategies, enabling gene therapy products to adapt to more complex multigenic indications, thus expanding the toolbox of readily available gene therapies.

Sustained intra-cellular siRNA release from poly(L-arginine) multilayered nanoparticles for prolonged gene silencing.

BACKGROUND: Sustained siRNA release from nanocarriers is difficult to achieve inside the cell after entry: typically, all nanocarriers exhibit burst release of the cargo into the cytoplasm. RESEARCH DESIGN AND METHODS: Layer-by-layer (LbL) nanoparticles (NPs) can be constructed so that they escape endosomes intact, and subsequently exhibit sustained release of the cargo. Our work quantifies intra-cellular siRNA release from multilayered NPs, evaluates mechanism behind the sustained release, and optimizes the duration of release. RESULTS: Intra-cellular studies showed that NPs developed with four layers of poly-L-arginine, alternated with three layers of siRNA layers, were able to elicit effective and prolonged SPARC knockdown activity over 21 days with a single-dose treatment. For the first time, we have quantified the amounts of released siRNA in the cytoplasm and the amount of siRNA remaining inside the NPs at each timepoint. Furthermore, we have correlated the amount of released siRNA within cells by LbL NPs to the cellular knockdown efficiency of multilayered delivery system. CONCLUSIONS: This methodology may provide an excellent screening tool for assessing the duration of gene silencing by various nanocarrier formulations.

N6-Methyladenosine RNA Modification Regulates Maize Resistance to Maize Chlorotic Mottle Virus Infection.

Maize chlorotic mottle virus (MCMV) is one of the main viruses causing significant losses in maize. N6-methyladenosine (m6A) RNA modification has been proven to play important regulatory roles in plant development and stress response. In this study, we found that MCMV infection significantly up-regulated the m6A level in maize, and methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were performed to investigate the distribution of m6A modified peaks and gene expression patterns in MCMV-infected maize plants. The results showed that 1325 differentially methylated genes (DMGs) and 47 differentially methylated and expressed genes (DMEGs) were identified and analyzed. Moreover, the results of virus-induced gene silencing (VIGS) assays showed that ZmECT18 and ZmGST31 were required for MCMV infection, while silencing of ZmMTC, ZmSCI1 or ZmTIP1 significantly promoted MCMV infection in maize. Our findings provided novel insights into the regulatory roles of m6A modification in maize response to MCMV infection.

Virus-Induced galactinol-sucrose galactosyltransferase 2 Silencing Delays Tomato Fruit Ripening.

Tomato fruit ripening is an elaborate genetic trait correlating with significant changes at physiological and biochemical levels. Sugar metabolism plays an important role in this highly orchestrated process and ultimately determines the quality and nutritional value of fruit. However, the mode of molecular regulation is not well understood. Galactinoal-sucrose galactosyltransferase (GSGT), a key enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs), can transfer the galactose unit from 1-α-D-galactosyl-myo-inositol to sucrose and yield raffinose, or catalyze the reverse reaction. In the present study, the expression of SlGSGT2 was decreased by Potato Virus X (PVX)-mediated gene silencing, which led to an unripe phenotype in tomato fruit. The physiological and biochemical changes induced by SlGSGT2 silencing suggested that the process of fruit ripening was delayed as well. SlGSGT2 silencing also led to significant changes in gene expression levels associated with ethylene production, pigment accumulation, and ripening-associated transcription factors (TFs). In addition, the interaction between SlGSGT2 and SlSPL-CNR indicated a possible regulatory mechanism via ripening-related TFs. These findings would contribute to illustrating the biological functions of GSGT2 in tomato fruit ripening and quality forming.

Therapeutic targeting of siRNA/anti-cancer drug delivery system for non-melanoma skin cancer. Part I: Development and gene silencing of JAK1siRNA/5-FU loaded liposome nanocomplexes.

Non-melanoma skin cancer (NMSC) is one of the most prevalent cancers, leading to significant mortality rates due to limited treatment options and a lack of effective therapeutics. Janus kinase (JAK1), a non-receptor tyrosine kinase family member, is involved in various cellular processes, including differentiation, cell proliferation and survival, playing a crucial role in cancer progression. This study aims to provide a more effective treatment for NMSC by concurrently silencing the JAK1 gene and administering 5-Fluorouracil (5-FU) using liposome nanocomplexes as delivery vehicles. Utilizing RNA interference (RNAi) technology, liposome nanocomplexes modified with polyethylene imine (PEI) were conjugated with siRNA molecule targeting JAK1 and loaded with 5-FU. The prepared formulations (NL-PEI) were characterized in terms of their physicochemical properties, morphology, encapsulation efficiency, in vitro drug release, and stability. Cell cytotoxicity, cell uptake and knockdown efficiency were evaluated in human-derived non-melanoma epidermoid carcinoma cells (A-431). High contrast transmission electron microscopy (CTEM) images and dynamic light scattering (DLS) measurements revealed that the nanocomplexes formed spherical morphology with uniform sizes ranging from 80-120 nm. The cationic NL-PEI nanocomplexes successfully internalized within the cytoplasm of A-431, delivering siRNA for specific sequence binding and JAK1 gene silencing. The encapsulation of 5-FU in the nanocomplexes was achieved at 0.2 drug/lipid ratio. Post-treatment with NL-PEI for 24, 48 and 72 h showed cell viability above 80 % at concentrations up to 8.5 × 101 µg/mL. Notably, 5-FU delivery via nanoliposome formulations significantly reduced cell viability at 5-FU concentration of 5 µM and above (p < 0.05) after 24 h of incubation. The NL-PEI nanocomplexes effectively silenced the JAK1 gene in vitro, reducing its expression by 50 %. Correspondingly, JAK1 protein level decreased after transfection with JAK1 siRNA-conjugated liposome nanocomplexes, leading to a 37 % reduction in pERK (phosphor extracellular signal-regulated kinase) protein expression. These findings suggest that the combined delivery of JAK1 siRNA and 5-FU via liposomal formulations offers a promising and novel treatment strategy for targeting genes and other identified targets in NMSC therapy.