Silencing Efficiency Research Articles

Nanocarrier-mediated transdermal delivery of Lmidgf4 dsRNA expedites biological control of locusts by Beauveria bassiana

Locusts have been a major global agricultural pest that poses a serious threat to crop and livestock production. Entomopathogenic fungi (EPF) provide an eco-friendly control method; however, their efficacy usually takes slow and is unstable. To achieve an enhancement of the biocontrol efficacy of Beauveria bassiana (B. bassiana) against locusts, we developed a new strategy by which B. bassiana and nanocarrier-mediated dsRNA are co-applied across the locust cuticle. The nanocarrier star polycation (SPc) effectively delivers Lmidgf4 dsRNA (dsLmidgf4) into the locust, which targets Locusta migratoria imaginal disc growth factor 4 (Lmidgf4). SPc protects dsLmidgf4 from degradation by the hemolymph and enables efficient gene silencing. Furthermore, SPc has no adverse effects on B. bassiana spore germination and growth. Lmidgf4 interference leads to a thinner layer of endocuticle, thus facilitates infection of B. bassiana, and finally reduces the median lethal time of locusts infected with B. bassiana. In conclusion, the combination of B. bassiana and dsRNA/SPc complex overcomes the slow action of fungi, providing a novel strategy for field control of locusts.

Neoadjuvant Chemotherapy With Cisplatin Up-Regulates GSDMD to Enhance Oral Squamous Cell Carcinoma Metastasis Through MMP14-Mediated EMT Activation.

Neoadjuvant chemotherapy has been widely used for the treatment of solid tumors. However, clinical observations have shown that patients with oral squamous cell carcinoma (OSCC) who are receiving neoadjuvant chemotherapy with cisplatin still face issues such as a poor lymph node response and even lymph node progression, but the underlying mechanisms remain unidentified. In this work, it is found that low-dose cisplatin promoted oral squamous cell carcinoma migration, invasion and lymph node metastasis, and gasdermin D (GSDMD) is identified as a potential regulator. GSDMD interacted with MMP14, promoting its expression and epithelial‒mesenchymal transition (EMT) activation without activating pyroptosis. Moreover, pH-responsive nanoparticles (NPs) for the systemic delivery of a GSDMD siRNA (siGSDMD) is developed and showed that this NP-delivered siGSDMD can effectively inhibit OSCC tumor growth and metastasis via the efficient silencing of GSDMD expression in vivo. This findings indicate that GSDMD can be a biomarker to predict the prognosis of OSCC patients receiving neoadjuvant chemotherapy and that NP-mediated GSDMD silencing can be a promising strategy for the treatment of patients with advanced OSCC receiving neoadjuvant chemotherapy with cisplatin.

NIR-II photo-accelerated polymer nanoparticles boost tumor immunotherapy via PD-L1 silencing and immunogenic cell death.

NIR-II photo-accelerated polymer nanoparticles boost tumor immunotherapy via PD-L1 silencing and immunogenic cell death.

Evolution of Lipo-Xenopeptide Carriers for siRNA Delivery: Interplay of Stabilizing Subunits.

Although small interfering RNA (siRNA) holds immense promise for treating genetic diseases and cancers, its clinical application is constrained by instability, cellular uptake barriers, and inefficient cytosolic delivery, underscoring the need for effective delivery systems. Therefore, this study focuses on screening novel T-shaped lipo-xenopeptide (XP) nanocarriers for siRNA polyplex formulation, integrating two single succinoyl-tetraethylene pentamine (Stp) units for electrostatic interaction and tyrosine tripeptides (Y3) for aromatic stabilization, along with structural modifications such as the addition of histidine (H) with or without terminal cysteines (C), and the incorporation of various fatty acids (FAs). A systematic evaluation of siRNA binding, nanoparticle stability, and gene silencing efficiency in multiple cell lines illustrated that the novel Stp1-HC lipo-XPs carriers outperform their Stp2-HC analogs, despite having fewer cationizable Stp units. This advantage stems from increased fatty acid, Y3, and C density, which compensates for reduced electrostatic interactions. The presence of H in combination with unsaturated FAs significantly improved the functional siRNA delivery. Our findings highlight the complex interplay of electrostatic, hydrophobic, covalent, hydrogen-bonded, and aromatic interactions to achieve efficient siRNA delivery, which is best-balanced in the oleic acid-containing Stp1-HC/OleA lipo-XP, exceeding the previously best standard carrier Stp2-HC/OleA in efficiency.

Targeted melanoma therapy: High-efficiency siRNA delivery with R8-PEI dissolvable microneedles.

Targeted melanoma therapy: High-efficiency siRNA delivery with R8-PEI dissolvable microneedles.

Ionic Coating of siRNA Polyplexes with cRGD-PEG-Hyaluronic Acid To Modulate Serum Stability and In Vivo Performance.

Efficient delivery of siRNA-based polyplexes to tumors remains a major challenge. Nonspecific interactions in the bloodstream, limited circulation time, and nontargeted biodistribution hamper sufficient tumor accumulation. To address these challenges, we developed an ionic hyaluronic acid (HA) coating to shield sequence-defined oligoaminoamide-based polyplexes. This coating should shield the positive polyplex surface charge, thus reducing nonspecific interactions and enhancing serum stability. Additionally, we modified the HA coating with the cyclic RGDfK (cRGD) peptide to specifically target tumor endothelial cells (TECs). Optionally, a polyethylene glycol (PEG) spacer was also introduced to improve ligand presentation on the polyplex surface. The HA-coated polyplexes exhibited favorable physicochemical properties, including a negative zeta potential and effective siRNA retention within the polyplex, which was not adversely affected by PEG or cRGD modification. In vitro analyses revealed that these polyplexes not only enhanced tumor cell association and preserved the high transfection efficiency of plain cationic polyplexes but also exhibited coating-dependent cellular internalization, as evidenced by a competitive inhibition experiment. Even in the presence of serum, the HA-coated polyplexes encapsulated siRNA effectively, exhibited suitable particle sizes, and maintained a high gene silencing efficiency. In vivo studies involving intravenous administration into Neuro2a tumor-bearing mice showed that the HA coating, particularly when modified with PEG and cRGD, significantly increased the tumor accumulation of polyplexes. HA-PEG-cRGD-shielded polyplexes exhibited significantly enhanced in vivo gene silencing in tumors compared with plain polyplexes. Collectively, our results indicate a superior performance of HA-coated polyplexes in terms of stability and cellular uptake, both in vitro and in vivo.

Coat protein I genes are essential for the morphogenesis of the intestinal tract in Locusta migratoria.

Coat protein I genes are essential for the morphogenesis of the intestinal tract in Locusta migratoria.

ATP-responsive tumor targeted lipid nanoparticle for enhanced siRNA delivery and improved treatment efficacy in melanoma.

ATP-responsive tumor targeted lipid nanoparticle for enhanced siRNA delivery and improved treatment efficacy in melanoma.

Chemosensory protein 8 confers thiamethoxam resistance in Diaphorina citri.

Chemosensory protein 8 confers thiamethoxam resistance in Diaphorina citri.

CRISPR/Cas12a-mediated gene silencing across diverse functional genes demonstrates single gene-specific spacer efficacy in Mycobacterium smegmatis

BackgroundTuberculosis, a persistent global health threat, necessitates a comprehensive understanding of the genes and pathways crucial for the survival and virulence of the causative pathogen, Mycobacterium tuberculosis. Working with M. tuberculosis (M.tb) presents significant challenges; therefore, the use of M. smegmatis as a surrogate system for conducting genetic studies of M.tb has proven to be highly valuable. Development of novel genetic tools to probe cellular processes accelerates the progress in the field of drug development and also helps in understanding the basic physiology of the bacterium.ResultsThis study reports the successful implementation and evaluation of the CRISPR-Cas12a system for gene repression in Mycobacterium smegmatis, a surrogate for M. tuberculosis. We engineered a Cas12a-based CRISPR interference (CRISPRi) system and assessed its functionality. Targeting 45 genes with a single sgRNA per gene, we achieved efficient gene repression, leading to marked phenotypic changes. Each knockdown strain was evaluated individually for growth phenotypes, and a comparison of the results with the reported essential gene library probed with dCas9 demonstrated congruous results across diverse gene categories. The study shows that CRISPR/Cas12a system can be effectively utilised with a single gene specific target for efficient silencing of the gene and highlights the importance of subsequent growth assays required to evaluate the vulnerability of targeted gene silencing.ConclusionOur findings reveal the robustness and versatility of the dCas12a-CRISPRi system in M. smegmatis, providing a valuable tool for functional genomics research. This work showcases the potential of the dCas12a-CRISPRi system in investigating essential genes, enabling a deeper understanding of the biology and potential therapeutic targets in mycobacterium species.

Research progress in the engineering strains for producing double-stranded RNA

Ensuring food security requires new green pesticides. Double-stranded RNA (dsRNA) pesticides trigger RNA interference by exogenous dsRNA specifically targeting pests and diseases. They can inhibit the expression of key genes in pathogens or pests, thereby achieving effective control of specific pests and diseases. DsRNA pesticides are environmentally friendly, with strong specificity and efficient gene silencing ability, while they have problems such as high production costs. Using engineering strains to produce dsRNA is a feasible strategy, whereas currently there is no cost-effective engineering strain for producing dsRNA. This article reviews the research progress and production strategies of using microorganisms to produce dsRNA, hoping to provide reference for dsRNA production.

ADAMTS5-specific gapmer release from an albumin biomolecular assembly and cartilage internalization triggered by ultrasound

Objective Antisense oligonucleotides (ASOs) have reached the clinic; however, they lack tissue specificity. Albumin is a plasma-abundant macromolecule that has been shown to accumulate in inflamed tissues. In this work, we have designed a recombinant human albumin (rHA)-based biomolecular assembly incorporating a DNase-resistant phosphorothioate-based complementary oligonucleotide (cODN) and an anti-ADAMTS5 ASO for potential delivery to inflamed sites. Ultrasound (US) was used to trigger ASO release from the assembly and enhance internalization into articular cartilage. Methods A phosphorothioate cODN was conjugated to rHA through a maleimide cross-linker after which, a therapeutic ADAMTS5-specific gapmer ASO was annealed to the cODN. ASO release was assessed after exposing the biomolecular assembly to different US conditions using an US-actuated medical needle operating at 32.2 kHz. Gene silencing efficiency of US-treated anti-ADAMTS5 ASO was assessed in human primary chondrocytes isolated from osteoarthritic patients. US-mediated ASO penetration into articular cartilage was assessed on ex vivo bovine articular cartilage. Results ASO release was observed after exposure to US waves in continuous mode conditions that did not compromise ASO gene silencing efficiency in human chondrocytes. Furthermore, US increased ASO internalization into bovine articular cartilage after 30 min of application without detrimental effects on chondrocyte viability. Conclusion A medical needle driven by continuous US waves at 32.2 kHz has the capability of disassembling a duplex oligonucleotide and enhancing released ASOs internalization into articular cartilage. This work offers the potential delivery and the local triggered release of ASOs at the surface of articular cartilage providing potential benefits for the treatment of diverse cartilage pathologies.

Beyond species and spatial boundaries: Enabling long-distance gene silencing in plants via guanidinium-siRNA nanoparticles.

RNA interference (RNAi) has been widely used in agriculture. However, it is well accepted that common methods of plant RNAi are species-dependent and lack systematic efficiency. This study designed a thiolated siRNA nanoparticle, guanidinium (Gu+)-containing disulfide assembled siRNA (Gu+-siRNA), demonstrating remarkable species independence and efficient systemic gene silencing across different plant species. Our results indicate that this approach effectively utilizes the plant vascular system to deliver siRNA, enabling long-distance gene silencing across both monocot and dicot plants, such as rice and Arabidopsis. By applying this method, we successfully targeted and silenced key genes like STM, WER, MYB23, GD1, EIL1, and EIL2, which regulate plant development and enhance salt tolerance. This delivery system significantly expands the application of RNAi technology across different plants, serving as a valuable tool for advancing agricultural biotechnology, enhancing crop resistance, and improving agricultural productivity, while aligning with global goals for sustainable food production and crop improvement.

SIL-204 siRNA encapsulated in extended release microparticles for the treatment of localized cancer that harbors a KRAS G12x or G13D mutation.

745 Background: In a Phase 2 trial, treatment of non-resectable locally advanced pancreatic cancer (LAPC) patients harboring the KRAS G12D or G12V mutations, with SiG12DLoder combined with chemotherapy resulted in superior median overall survival (OS) of 9.3 months over chemotherapy (not statistically significant) and Objective Response Rate of 55% (35% over the chemotherapy control), when administered once every 3 months (Clinical trial # NCT01676259, ESMO 2023, abstract FPN: 1626). SIL-204 which is a modified version of siRNA siG12D is composed of a 21-base sense and 23-base antisense strands, both chemically modified and linked to a lipid. SIL-204 is encapsulated in biodegradable PLGA microparticles (MP), for direct release and injection into KRAS mutated solid tumors using an endoscopic ultrasound procedure. The strategy of preventing the synthesis of KRAS mutated protein may have an advantage over other approaches targeting KRAS function using small molecules. Methods: Methods are presented in results section. Results: SIL-204 has an increase stability over siG12D.To enhance stability, base modifications and phosphorothiate bonds at cleavage sites were implemented. SIL-204 exhibited a half-life of more than 48 hours in human serum. SIL-204 has robust KRAS silencing . When tested in Hepa1-6 cells transfected with individual human KRAS mutations, SIL-204 effectively silenced the various G12x mutations found in pancreatic cancer (D,V,R,C) at sub-nanomolar concentration (IC50 range=0.19-0.59) as well as G13D (IC50 =0.37). Addition of a hydrophobic tail to a prototype of SIL-204, SIL-101, increased the silencing of KRAS. Using PANC-1 cells (KRAS G12D mutation), lipid conjugation of the siRNA doubled the siRNA’s silencing efficiency in these cells. In addition, release of SIL-204 from the MP was tested in rats and indicates a prolong and sustained release over our intended treatment regimen SIL-204-MP was administered to tumors from human pancreatic tumor cell lines Capan-1 (KRAS G12V mutation and labelled with luciferase) grown in NSG mice and Panc-1 (grown in Athymic Nude mice). Bioluminescence imaging of tumor growth showed that SIL-204-MP significantly reduced Capan-1 tumor growth compared to vehicle controls, p<0.0005. Furthermore, histopathological analyses of tumor center slices from Capan-1 and Panc-1 models showed induction of significant tumor necrosis with SIL-204 compared to vehicle controls. Conclusions: The preclinical results of SIL-204-MP are promising and as the potential to further enhance the anti-tumor effect demonstrated by siG12DLoder in pancreatic patients. SIL-204 is now in toxicology studies as preparation for clinical trials in non-resectable pancreatic cancer.

Vacuolar (H+)-ATPase Genes Are Essential for Cuticle and Wing Development in Locusta migratoria.

Vacuolar (H+)-ATPases (V-ATPases) are crucial in several significant biological processes, including intracellular transport, endocytosis, autophagy and protein degradation. However, their role in the growth and development of insects remains largely unknown. This study aimed to explore the molecular and functional properties of V-ATPases in Locusta migratoria. LmV-ATPase genes were identified based on the locust transcriptome database and bioinformatics analysis. Quantitative reverse-transcription polymerase chain reaction was used to assess the relative expression of LmV-ATPases in different tissues and developmental stages. RNA interference combined with hematoxylin-eosin staining and transmission electron microscopy was used to explore the functions of LmV-ATPases. Ten V-ATPase genes were identified in L. migratoria and were named LmV-ATPase A, B, C, D, E, F, G, c″, d and e, respectively. These genes were highly expressed in the head, integument, gastric caecum, midgut, hindgut, fat body, trachea and ovary. The transcripts of LmV-ATPases were expressed in the developmental stages examined (from the 3rd to 5th instar nymphs). The injection of double-stranded RNA (dsRNA) against each LmV-ATPase induced high silencing efficiency in the 3rd instar nymphs. Knockdown of LmV-ATPases resulted in lethal phenotypes, with visible defects of the wing and cuticle. We further demonstrated that the deformation was caused by the defects of epidermal cells and fewer new cuticles. These findings suggest that LmV-ATPases are required for the wing and cuticle development of L. migratoria, which could be potential targets for the control of locusts.

SiRNA Knocking Down in HepG2 Cells Identifies PFKFB4 and HNF4α as Key Genes Important for Cancer Cell Survival.

Liposomes are versatile delivery systems for encapsulating small interfering RNAs (siRNAs) because they enhance cellular uptake and gene silencing. This study compares the new liposome formula to commercial lipofectamine in delivering siRNAs targeting hepatic carcinoma genes, focusing on HNF4-α and PFKFB4. Flow cytometry and confocal microscopy revealed efficient internalization of PE-Rhod- B labeled lipoplexes in HepG2 cells, while cytotoxicity assays demonstrated significant reductions in cell viability, particularly with siHNF4-α and siPFKFB4. The newly formulated liposomes showed superior efficacy, achieving nearly 93% cytotoxicity at 100 nM, compared to just 50% with lipofectamine at the same concentration. Furthermore, real-time PCR confirmed that the liposome-encapsulated siHNF4-α reduced HNF4-α mRNA expression by tenfold at 100 nM, compared to a twofold reduction with lipofectamine at 200 nM. Similarly, siPFKFB4 delivered via liposomes showed a dose-dependent 35-fold reduction in PFKFB4 mRNA expression at 100 nM, outperforming the maximum reduction achieved by lipofectamine. The IC50 values for all siRNA treatment groups were significantly lower when using the liposome formula, reflecting improved delivery efficiency. These results demonstrate the potential of liposome formulations for therapeutic siRNA delivery. The encapsulation enhances cellular uptake and gene silencing efficiency, making the liposome formula a promising candidate for targeted gene therapy in hepatic carcinoma. Further research should explore it's in vivo biodistribution and potential combination therapies.

Diving into RNAi Therapy: An Inhalable Formulation Based on Lipid-Polymer Hybrid Systems for Pulmonary Delivery of siRNA.

Here, a pulmonary formulation based on lipid-polymer hybrid nanoparticles carrying small interfering RNA (siRNA) was developed to realize a RNA interference-based therapy to treat respiratory diseases. Toward this aim, a new copolymer was synthesized, by functionalization of the α,β-poly(N-2-hydroxyethyl)-d,l-aspartamide with 35 mol % of 1,2-bis(3-aminopropylamino)ethane, 0.4 mol % of fluorescent dye, and 4.5 mol % of poly(lactic-co-glycolic acid). This was used to encapsulate siRNA targeting the green fluorescent protein (siGFP), within a lipid shell made from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-distearoyl-sn-glycero-phosphoethanolamine-N-(polyethylene glycol)2000. siGFP-loaded lipid-polymer hybrid nanoparticles (LPHFNPs@siGFP) exhibited colloidal size (∼164 nm), positive ζ potential, high siRNA encapsulation efficiency (∼99%), and a core-shell morphology. They showed high cellular uptake and a gene silencing efficiency of ∼50% in human lung cancer cells expressing GFP. To address aerodynamic challenges, LPHFNPs@siGFP were spray-dried with trehalose, yielding spherical particles (∼3 μm) with 80% siRNA encapsulation efficiency, excellent aerosolization properties, and a gene silencing efficiency comparable to the fresh LPHFNPs@siGFP sample.

Establishment of a Virus-Induced Gene Silencing System in Abelmoschus manihot L.

Abelmoschus manihot L. (Jinhuakui, JHK) is widely cultivated for its pharmacological properties owing to its high flavonoid content and is commonly used as a garden landscape plant. However, the absence of an efficient genetic transformation system poses significant challenges for functional gene studies in this species. Virus-induced gene silencing (VIGS) is a well-established technique for exploring plant gene functions; however, this technique has not been applied to JHK. Here, a tobacco rattle virus (TRV)-VIGS system was successfully developed for the first time in JHK using the gene encoding phytoene desaturase (AmPDS) as a marker gene. This study investigated the impact of various Agrobacterium infection methods on the efficiency of AmPDS silencing. The results demonstrated that administering two injections-the first on the day of complete cotyledon expansion and the second 14 days later-using pTRV1 and pTRV2-AmPDS cultures resuspended to an OD600 of 1.0 and via the backside of the blade-led to significant photobleaching in the cotyledons 2 days after the second injection. Subsequent analyses revealed a marked reduction in both chlorophyll content and AmPDS expression. These findings suggest that a VIGS system was successfully developed in JHK, thus providing a rapid and effective method for studying gene function in this species and facilitating future research in JHK genetics.

Development of a robust and efficient virus-induced gene silencing system for reverse genetics in recalcitrant Camellia drupifera capsules

BackgroundVirus-induced gene silencing (VIGS) is a rapid and powerful method for gene functional analysis in plants that pose challenges in stable transformation. Numerous VIGS systems based on Agrobacterium infiltration has been widely developed for tender tissues of various plant species, yet none is available for recalcitrant perennial woody plants with firmly lignified capsules, such as tea oil camellia. Therefore, there is an urgent need for an efficient, robust, and cost-effective VIGS system for recalcitrant tissues.ResultsHerein, we initiated the Tobacco rattle virus (TRV)-elicited VIGS in Camellia drupifera capsules with an orthogonal analysis including three factors: silencing target, virus inoculation approach, and capsule developmental stage. To facilitate observation and statistical analysis, two genes predominantly involved in pericarp pigmentation were selected for silencing efficiency: CdCRY1 (coding for a key photoreceptor affecting light-responsive perceivable anthocyanin accumulation in exocarps) and CdLAC15 (coding for an oxidase catalyzing the oxidative polymerization of proanthocyanidins in mesocarps, resulting in unperceivable red-hued mesocarps). The infiltration efficiency achieved for each gene was ~ 93.94% by pericarp cutting immersion. The optimal VIGS effect for each gene was observed at early (~ 69.80% for CdCRY1) and mid stages (~ 90.91% for CdLAC15) of capsule development.ConclusionsUsing our optimized VIGS system, CdCRY1 and CdLAC15 expression was successfully knocked down in Camellia drupifera pericarps, leading to fading phenotypes in exocarps and mesocarps, respectively. The established VIGS system may facilitate functional genomic studies in tea oil camellia and other recalcitrant fruits of woody plants.

A Family of Transglutaminases Is Essential for the Development of Appressorium-Like Structures and Phytophthora infestans Virulence in Potato.

Transglutaminases (TGases) are enzymes highly conserved among prokaryotic and eukaryotic organisms, where their role is to catalyze protein cross-linking. One of the putative TGases of Phytophthora infestans has previously been shown to be localized to the cell wall. Based on sequence similarity we were able to identify six more genes annotated as putative TGases and show that these seven genes group together in phylogenetic analysis. These seven proteins are predicted to contain both a TGase domain and a MANSC domain, the latter of which was previously shown to play a role in protein stability. Chemical inhibition of transglutaminase activity and silencing of the entire family of the putative cell wall TGases are both lethal to P. infestans indicating the importance of these proteins in cell wall formation and stability. The intermediate phenotype obtained with lower drug concentrations and less efficient silencing displays a number of deformations to germ tubes and appressoria. Both chemically treated and silenced lines show lower virulence than the wild type in leaf infection assays. Finally, we show that appressoria of P. infestans possess the ability to build up turgor pressure and that this ability is decreased by chemical inhibition of TGases.