Delivery Of Double-stranded RNA Research Articles

Cationic Polystyrene Latex Nanocarriers for Immunostimulatory Long Double-Stranded RNA Delivery to Ovarian Cancer Cells.

Long double-stranded (ds)RNA, a potent stimulator of type I interferon and the innate immune response. In the present study, we demonstrated, for the first time, the efficacy of cationic polystyrene latex nanostructures (clNPs) as a dsRNA carrier, improving cellular delivery and robustly potentiating the immunostimulatory capacity of dsRNA in the ovarian cancer cell line SKOV3. The clNPs complexed with an in vitro transcribed dsRNA molecule, were bound by SKOV3 cells, and had increased cellular association compared to uncomplexed clNPs. clNPs complexed with dsRNA induced a more robust innate immune response compared to dsRNA alone. Transcript expression of two interferon-stimulated genes, were increased 47- and 108-fold over dsRNA and induced a significant antiviral state against vesicular-stomatitis virus, resulting in a 3.3-fold improvement on the efficacy of dsRNA. These data highlight the potential of polystyrene latex nanostructures as dsRNA carriers for anticancer immunotherapies, improving the uptake and efficacy of the nucleic acid.

RNAi-Based Biopesticides Against 28-Spotted Ladybeetle Henosepilachna vigintioctopunctata Does Not Harm the Insect Predator Propylea japonica.

RNA interference (RNAi)-mediated control of the notorious pest Henosepilachna vigintioctopunctata is an emerging environment friendly research area. However, the characterization of key target genes in H. vigintioctopunctata is crucial for this. Additionally, assessing the risk of RNAi to nontarget organisms (NTOs) is necessary for environmental safety. In this study, the potential of RNAi technology in controlling H. vigintioctopunctata infestation has been investigated by the oral delivery of double-stranded RNA (dsRNA). The results revealed that the silencing of six genes, including HvABCH1, HvHel25E, HvProsbeta5, HvProsalpha6, HvProsbeta6, and HvSrp54k, was highly lethal to H. vigintioctopunctata. The LC50 values of the dsRNAs used to silence these six genes were found to be less than 13 ng/μL. Moreover, the use of the bacterially expressed dsRNAs caused high mortality in the lab and field populations of H. vigintioctopunctata. Further, administration of HvHel25E and HvSrp54k dsRNAs in the predatory lady beetle Propylea japonica confirmed no transcriptional or organismal levels effects. This risk-assessment result ensured no off-target RNAi effects on the NTOs. Overall, the findings of the study suggested that HvABCH1, HvHel25E, HvProsbeta5, HvProsalpha6, HvProsbeta6, and HvSrp54k can be novel promising molecular targets with high specificity for H. vigintioctopunctata management with negligible effects on the NTOs.

Silencing of juvenile hormone epoxide hydrolase gene in Spodoptera litura (Lepidoptera: Noctuidae) by oral delivery of double-stranded RNA

Silencing of juvenile hormone epoxide hydrolase gene in Spodoptera litura (Lepidoptera: Noctuidae) by oral delivery of double-stranded RNA

Molecular characterization of an arginine kinase from Lasioderma serricorne and the effect of gene silencing on larval survival

Arginine kinase (AK) is a phosphagen kinase that is critical for energy metabolism in insects, such as the cigarette beetle, Lasioderma serricorne, which is an important pest species of stored products. Nevertheless, the AK gene has not been identified in L. serricorne. Accordingly, this study describes the identification and characterization of the AK gene in L. serricorne (designated LsAK). The cDNA sequence of LsAK contains an open reading frame of 1068-bp and encodes the LsAK protein consisting of 355 amino-acid residues. LsAK possesses features typical of other insect AKs and shares a high amino-acid identity and a close phylogenetic relationship with orthologs from other coleopteran species. The transcripts of LsAK mRNA can be detected at all developmental stages, and the highest levels of LsAK transcription are observed at the fourth-instar larval stages. Recombinant LsAK protein expressed in Escherichia coli displayed activity to convert L-arginine to phosphoarginine. Oral delivery of double-stranded RNA significantly reduces the LsAK mRNA level and AK activity, resulting in a noticeably decreased survival rate in L. serricorne larvae. The findings of this study indicate that the LsAK gene is essential for larval survival of L. serricorne and could be a viable target for RNA-interference-based pest management.

Cationic Polystyrene Latex Nanocarriers for Immunostimulatory Long Double-Stranded Rna Delivery to Ovarian Cancer Cells

Cationic Polystyrene Latex Nanocarriers for Immunostimulatory Long Double-Stranded Rna Delivery to Ovarian Cancer Cells

Defined diets for freshwater planarians.

Planarian flatworms are popular invertebrate models for basic research on stem cell biology and regeneration. These animals are commonly maintained on a diet of homogenized calf liver or boiled egg yolk in the laboratory, introducing a source of uncontrolled experimental variability. Here, we report the development of defined diets, prepared entirely from standardized, commercially sourced ingredients, for the freshwater species Schmidtea mediterranea, Dugesia japonica, and Girardia dorotocephala. These food sources provide an opportunity to test the effects of specific nutritional variables on biological phenomena of interest. Defined diet consumption was not sufficient for growth and only partially induced the increase in stem cell division that normally accompanies feeding, suggesting these responses are not solely determined by caloric intake. Our defined diet formulations enable delivery of double-stranded RNA for gene knockdown in a manner that provides unique advantages in some experimental contexts. We also present a new approach for preserving tissue integrity during hydrogen peroxide bleaching of liver-fed animals. These tools will empower research on the connections between diet, metabolism, and stem cell biology in the experimentally tractable planarian system.

Plant Functional Genomics in A Few Days: Laser-Assisted Delivery of Double-Stranded RNA to Higher Plants.

The technology of transgenic plants is challenging and time consuming, especially for higher plants and trees such as citrus. Double-stranded RNA (dsRNA) delivery via a plant virus is an alternative method to create transgenic plants by suppressing the expression of plant endogenous genes. Citrus tristeza virus-based vector has been constructed specifically for use in citrus trees. However, this is time-consuming, as it can take up to nine months to produce the desired phenotype. Here we describe a much faster method for the study of gene function in citrus trees. In the current study, we used laser light for the delivery of dsRNA to citrus leaves. We targeted the endogenous reporter gene phytoene desaturase (PDS) and obtained the classical phenotype (leaf bleaching) in only three days after the laser-assisted delivery. Interestingly, the phenotype response was systemic, which indicates the movement of dsRNA and/or ssRNA within the plants. In addition, dsRNAs were taken up by phloem cells and the bleaching phenotype was clear around the main veins. In conclusion, the delivery of dsRNA to plants through laser treatment may provide a fast and more specific tool to study the gene function in higher plants and trees.

DsRNA Uptake in Plant Pests and Pathogens: Insights into RNAi-Based Insect and Fungal Control Technology.

Efforts to develop more environmentally friendly alternatives to traditional broad-spectrum pesticides in agriculture have recently turned to RNA interference (RNAi) technology. With the built-in, sequence-specific knockdown of gene targets following delivery of double-stranded RNA (dsRNA), RNAi offers the promise of controlling pests and pathogens without adversely affecting non-target species. Significant advances in the efficacy of this technology have been observed in a wide range of species, including many insect pests and fungal pathogens. Two different dsRNA application methods are being developed. First, host induced gene silencing (HIGS) harnesses dsRNA production through the thoughtful and precise engineering of transgenic plants and second, spray induced gene silencing (SIGS) that uses surface applications of a topically applied dsRNA molecule. Regardless of the dsRNA delivery method, one aspect that is critical to the success of RNAi is the ability of the target organism to internalize the dsRNA and take advantage of the host RNAi cellular machinery. The efficiency of dsRNA uptake mechanisms varies across species, and in some uptake is negligible, rendering them effectively resistant to this new generation of control technologies. If RNAi-based methods of control are to be used widely, it is critically important to understand the mechanisms underpinning dsRNA uptake. Understanding dsRNA uptake mechanisms will also provide insight into the design and formulation of dsRNAs for improved delivery and provide clues into the development of potential host resistance to these technologies.

RNAi-mediated mortality in southern green stinkbug Nezara viridula by oral delivery of dsRNA.

The southern green stinkbug, Nezara viridula (Hemiptera: Pentatomidae), is an important emerging polyphagous pest infesting soybean in the United States, Brazil and Argentina. The indiscriminate use of synthetic insecticides to control stinkbugs has limited the effectiveness of current management strategies. Alternatively, RNA interference (RNAi) has emerged as a novel mode of action to control pests in an eco-friendly manner. Here, we assessed the potential of RNAi technology by oral delivery of double-stranded RNA (dsRNA) for the control of N. viridula. Initially, ten candidate genes were tested by microinjection assay to select the best target genes for oral delivery. Seven genes resulted in more than 90% mortality after microinjection. To evaluate RNAi efficacy by oral delivery of dsRNA, five genes were tested by feeding the insects on gene-specific dsRNA mixed with an artificial diet. Significant mortality of 43% and 45% was observed after 14 days of treatment with dsαCop and dsvATPase A, respectively. To elucidate the lower RNAi efficacy via oral delivery of dsRNA, ex vivo dsRNA degradation in the saliva and the midgut juice was performed, which indicated that the reduced RNAi efficacy is accompanied by a rapid degradation of dsRNA by digestive secretions. This study proves that RNAi can be triggered by orally delivered dsRNA in N. viridula and can be exploited to control this economically important pest. The reduced stability of dsRNA in saliva and midgut that was observed indicates a need to further improve RNAi efficacy, for example by use of specific formulations.

Polymer-Coated Hydroxyapatite Nanocarrier for Double-Stranded RNA Delivery.

Conventional synthetic insecticides have limited success due to insect resistance and negative effects on off-target biota and the environment. Although RNA interference (RNAi) is a tool that is becoming more widely utilized in pest control products, naked dsRNA has limited success in most taxa. Nanocarriers have shown promising results in enhancing the efficacy of this tool. In this study, we used a layer-by-layer electrostatic assembly where we synthesized poly(acrylic acid) (PAA)-coated hydroxyapatite (HA) nanoparticles (PAA-HA NPs) as inorganic nanocarriers, which were then coated with a layer of a cationic poly(amino acid), 10 kDa poly-l-arginine (PLR10), to allow for binding of a layer of negatively charged dsRNA. Binding of PLR10-PAA-HA NPs to dsRNA was found to increase as the mass ratio of NPs to dsRNA increased. In vitro studies with transgenic SF9 cells (from Spodoptera frugiperda) expressing the firefly luciferase gene showed a significant gene silencing (35% decrease) at a 5:1 NP-to-dsRNA ratio, while naked dsRNA was ineffective at gene silencing. There was a significant concentration-response relationship in knockdown; however, cytotoxicity was observed at higher concentrations. Confocal microscopy studies showed that dsRNA from PLR10-PAA-HA NPs was not localized within endosomes, while naked dsRNA appeared to be entrapped within the endosomes. Overall, polymer-functionalized HA nanocarriers enabled dsRNA to elicit gene knockdown in cells, whereas naked dsRNA was not effective in causing gene knockdown.

Double-stranded RNAs targeting HvRPS18 and HvRPL13 reveal potential targets for pest management of the 28-spotted ladybeetle, Henosepilachna vigintioctopunctata.

RNA interference (RNAi) is a potential tool for plant protection against insect pests. The great challenge for effective pest control using RNAi in the field is the development of efficient and reliable methods for the production and delivery of double-stranded RNA (dsRNA). In the present study, we investigated the potential of feeding in vitro synthesized or bacterially expressed dsRNA to populations of the 28-spotted ladybeetle Henosepilachna vigintioctopunctata as a method of biological pest control. Ingestion of in vitro synthesized dsHvRPS18 or dsHvRPL13 led to significant down-regulation of the ribosomal protein-encoding genes HvRPS18 and HvRPL13, respectively, and significantly decreased the survival of H. vigintioctopunctata. Such silencing of HvRPS18 or HvRPL13 expression appeared to be partially dose-dependent and also inhibited the growth of H. vigintioctopunctata and significantly suppressed the expression of digestive enzyme-related genes. Finally, ingestion of bacterially expressed dsHvRPS18 or dsHvRPL13 induced significant mortality in the first and third instars, and in adults. The effectiveness of RNAi-based gene silencing in H. vigintioctopunctata provides a powerful reverse genetic tool for the functional annotation of its genes. This study demonstrates that HvRPS18 and HvRPL13 represent candidate genes for RNAi-based biological control of H. vigintioctopunctata. © 2020 Society of Chemical Industry.

Bacteria-Mediated RNA Interference for Management of Plagiodera versicolora (Coleoptera: Chrysomelidae).

RNA interference (RNAi) has emerged as a novel and feasible strategy for pest management. Methods for cost-effective production and stable delivery of double-stranded RNA (dsRNA) to the target insects are crucial for the wide application of RNAi for pest control. In this study, we tested the expression of dsRNA in RNaseIII-deficient Escherichia coli HT115 which was then fed to Plagiodera versicolora larvae, an insect pest of Salicaceae plants worldwide. By targeting six potential genes, including actin (ACT), signal recognition particle protein 54k (SRP54), heat shock protein 70 (HSC70), shibire (SHI), cactus (CACT), and soluble N-ethylmaleimide-sensitive fusion attachment proteins (SNAP), we found that feeding bacteria-expressed dsRNA successfully triggered the silencing of the five target genes tested and the suppression of ACT and SRP54 genes caused significant mortality. Our results suggest that the oral delivery of bacteria-expressed dsRNA is a potential alternative for the control of P. versicolora, and that ACT and SRP54 genes are the potent targets.

Gene silencing by double-stranded RNA from C. elegans neurons reveals functional mosaicism of RNA interference.

Delivery of double-stranded RNA (dsRNA) into animals can silence genes of matching sequence in diverse cell types through mechanisms that have been collectively called RNA interference. In the nematode Caenorhabditis elegans, dsRNA from multiple sources can trigger the amplification of silencing signals. Amplification occurs through the production of small RNAs by two RNA-dependent RNA polymerases (RdRPs) that are thought to be tissue-specific - EGO-1 in the germline and RRF-1 in somatic cells. Here we demonstrate that EGO-1 can compensate for the lack of RRF-1 when dsRNA from neurons is used to silence genes in intestinal cells. However, the lineal origins of cells that can use EGO-1 varies. This variability could be because random sets of cells can either receive different amounts of dsRNA from the same source or use different RdRPs to perform the same function. Variability is masked in wild-type animals, which show extensive silencing by neuronal dsRNA. As a result, cells appear similarly functional despite underlying differences that vary from animal to animal. This functional mosaicism cautions against inferring uniformity of mechanism based on uniformity of outcome. We speculate that functional mosaicism could contribute to escape from targeted therapies and could allow developmental systems to drift over evolutionary time.

RNAi mediated knockdown of RpL11, RpS2, and tra-2 led to reduced reproduction of Phytoseiulus persimilis.

Phytoseiulus persimilis is one of the most important biological control agents of spider mites. Multiple studies have been conducted on factors affecting its reproduction, but limited research on related molecular mechanisms has been carried out. In this study, RNA interference of three genes, ribosomal protein L11 (RpL11), ribosomal protein S2 (RpS2), and transformer-2 (tra-2), to newly emerged females were performed through oral delivery of double-stranded RNA, and knockdown of target genes was verified using qRT-PCR analysis. When RpL11 or RpS2 was interfered, 42 and 30% P. persimilis individuals either laid no eggor had no egg hatched, whereas the remaining females had their oviposition duration reduced by 31.8 and 49.9%, fecundity reduced by 48.1 and 67.8%, and egg hatching rate reduced by 20.4 and 22.4%, respectively. In addition, offspring sex ratios were significantly male biased especially at low fecundities. When tra-2 was interfered, no significant difference in fecundity was detected, but egg hatching rate reduced by 30.6%. This study verified the possibility of RNA interference in Phytoseiidae through oral delivery, and indicated that RpL11 and RpS2 are involved in egg formation, whereas tra-2 is involved in embryo development in P. persimilis. Phytoseiid mites have different sex determination pathways compared to insects. The present study provides data and evidence at molecular biological level for future research on reproduction and sex determination of phytoseiid mites.

Nano-mediated delivery of double-stranded RNA for gene therapy of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common type of malignant gliomas, characterized by genetic instability, intratumoral histopathological variability and unpredictable clinical behavior. Disappointing results in the treatment of gliomas with surgery, radiation and chemotherapy have fueled a search for new therapeutic targets and treatment modalities. Here we report new approach towards RNA interference therapy of glioblastoma multiforme based on the magnetic nanoparticles delivery of the double-stranded RNA (dsRNA) with homological sequences to mRNA of tenascin-C (TN-C), named ATN-RNA. The obtained nanocomposite consisted of polyethyleneimine (PEI) coated magnetic nanoparticles conjugated to the dsRNA show high efficiency in ATN-RNA delivery, resulting not only in significant TN-C expression level suppressesion, but also impairing the tumor cells migration. Moreover, synthesized nanomaterials show high contrast properties in magnetic resonance imaging (MRI) and low cytotoxicity combining with lack of induction of interferon response. We believe that the present work is a successful combination of effective, functional, non-immunostimulatory dsRNA delivery system based on magnetic nanoparticles with high potential for further application in GBM therapy.

Methods to Determine the Role of Autophagy Proteins in C. elegans Aging.

This chapter describes methods for the analysis of autophagy proteins in C. elegans aging. We discuss the strains to be considered, the methods for the delivery of double-stranded RNA, and the methods to measure autophagy levels, autophagic flux, and degradation by autophagy.

Gene silencing by RNA interference in the ectoparasitic mite, Psoroptes ovis

The presence of components of the RNA interference (RNAi) pathway in Psoroptes ovis, an ectoparasitic mite responsible for psoroptic mange, was investigated through interrogation of the P. ovis genome. Homologues of transcripts representing critical elements for achieving effective RNAi in the mite, Tetranychus urticae and the model organisms Caenorhabditis elegans and Drosophila melanogaster were identified and, following the development of a non-invasive immersion method of double stranded RNA delivery, gene silencing by RNAi was successfully demonstrated in P. ovis. Significant reductions in transcript levels were achieved for three target genes which encode the Group 2 allergen (Pso o 2), mu-class glutathione S-transferase (PoGST-mu1) and beta-tubulin (Poβtub). This is the first demonstration of RNAi in P. ovis and provides a mechanism for mining transcriptomic and genomic datasets for novel control targets against this economically important ectoparasite.

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches.

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

Host-mediated RNA interference targeting acuticular protein gene impaired fecundity inthe green peach aphid Myzus persicae.

The green peach aphid (Myzus persicae) is a devastating sap-sucking insect pest that damages many host plants worldwide and causes billions of dollars of crop losses. Induction of RNA interference (RNAi) through oral feeding of small interfering RNA (siRNA) has been demonstrated in aphids. Therefore, host-mediated delivery of double-stranded RNA (dsRNA) specific to vital structural genes of aphids has been envisaged as a tool for the development of resistance against this aphid species. Cuticular protein (CP) senses seasonal photoperiodism and drives a shift from clonal to sexual generation in aphids. Thus, attenuation of CP gene expression is likely to result in a different reproductive orientation in aphids and thereby affect their fecundity. A gene encoding CP in M. persicae has been targeted for RNAi-mediated knockdown. Transgenic Arabidopsis expressing dsRNA homologous to the MyCP gene was developed. The dsRNA-transgenics produced gene-specific siRNAs fed by aphids infesting the transgenics. A reverse transcription-quantitative polymerase chain reaction (RT-qPCR) study revealed an attenuated level of transcripts of the CP gene in aphid nymphs reared on the transgenic plants. Decreased expression of the CP gene resulted in a noticeable decline in aphid fecundity on the transgenic Arabidopsis plants. Increasing genetic resistance is the only sustainable way of minimizing the use of toxic agrochemicals to protect plants. Host-mediated RNAi of important insect genes has been proposed as a potential avenue for developing crop resistance against insect pests. This study demonstrated the potential of MyCP dsRNA in developing RNAi-based resistance to M. persicae. RNAi-mediated resistance is expected to be more durable compared with other transgenic strategies. © 2018 Society of Chemical Industry.