Application Of RNA Interference Research Articles

CRISPR/Cas9-Mediated Hitchhike Expression of Functional shRNAs at the Porcine miR-17-92 Cluster.

Successful RNAi applications depend on strategies allowing stable and persistent expression of minimal gene silencing triggers without perturbing endogenous gene expression. In this study, we proposed an endogenous microRNA (miRNA) cluster as a novel integration site for small hairpin RNAs (shRNAs). We successfully integrated exogenous shRNAs at the porcine miRNA-17-92 (pmiR-17-92) cluster via a CRISPR/Cas9-mediated knock-in strategy. The anti-EGFP or anti-CSFV shRNAs could be stably and effectively expressed at the control of the endogenous promoter of the pmiR-17-92 cluster. Importantly, we confirmed that hitchhike expression of anti- classical swine fever (CSFV) shRNA had no effect on cell growth, blastocyst development and endogenous pmiR-17-92 expression in selected transgene (TG) porcine fetal fibroblasts (PFFs) clones. Moreover, these TG PFFs could inhibit the replication of CSFV by half and could be further used for generation of transgenic pigs. Taken together, these results show that our RNA interference (RNAi) expression strategy benefits numerous applications, from miRNA, genome and transgenic research, to gene therapy.

Applications of RNA Interference in insect pest management

RNA interference (RNAi) is a technique which is being used for analyzing the functions of individual genes by employing loss-of-function analyses. It has been widely used in entomological research to study the functions of genes in variety of insects. This technology has great potential in insect pest management due to its high specificity. In this paper, we focus on the approaches of introducing dsRNA (double strand RNA) into insect like microinjection, oral feeding, topical, plant mediated and nanoparticles. The significant effects of silencing various genes involved in morphogenesis, reproduction and detoxification of insecticides in insect pests are also discussed.

The combined application of long term hepatic co-culture and RNA interference for reaction phenotyping: lessons learned

The combined application of long term hepatic co-culture and RNA interference for reaction phenotyping: lessons learned

VEGFA Gene Silencing in CXCR4-Expressing Cells via siRNA Delivery by Means of Targeted Peptide Carrier.

Discovery of small interfering RNA as a tool for specific gene inhibition led to the development of new therapeutic strategy for the treatment of cancers. The efficacious delivery of therapeutic siRNAs into the cells is a crucial step in RNA interference (RNAi) application, but it remains challenging. Non-viral vectors can provide specific cellular uptake, stable siRNA complex formation, and intracellular siRNA release. Recently, we evaluated modular peptide carrier L1 bearing CXCR4 targeting ligand for its ability to condense siRNA and facilitate endosomal escape and VEGFA gene silencing in CXCR4-expressing endothelial and glioblastoma cells. The present chapter showcases the ability of L1 targeted peptide carrier to form complexes with siRNA and provide efficient VEGFA gene knockdown. We showed that siRNA delivery by means of L1 peptide carrier can result in significant decrease of VEGFA gene expression in A172 glioblastoma cells and in EA.hy 926 endothelial cells. Also, delivery of anti-VEGFA siRNA/peptide complexes led to significant inhibition of endothelial cell migration. Our results showed that L1 peptide carrier modified with CXCR4 ligand is a promising tool for targeted siRNA delivery into CXCR4-expressing cancer and endothelial cells.

Gene Silencing With siRNA (RNA Interference): A New Therapeutic Option During Ex Vivo Machine Liver Perfusion Preservation

RNA interference (RNAi) is a natural process of posttranscriptional gene regulation that has raised a lot of attention culminating with the Nobel Prize in Medicine in 2006. RNAi-based therapeutics have been tested in experimental transplantation to reduce ischemia/reperfusion injury (IRI) with success. Modulation of genes of the innate immune system, as well as apoptotic genes, and those involved in the nuclear factor kappa B pathways can reduce liver injury in rodent liver pedicle clamping and transplantation models of IRI. However, in vivo use of RNAi faces limitations regarding the method of administration, uptake, selectivity, and stability. Machine perfusion preservation, a more recent alternative approach for liver preservation showing superior results to static cold preservation, could be used as a platform for gene interference therapeutics. Our group was the first to demonstrate uptake of small interfering RNA (siRNA) during liver machine preservation under both normothermic and hypothermic perfusion. Administering siRNA in the perfusion solution during ex vivo machine preservation has several advantages, including more efficient delivery, lower doses and cost-saving, and none/fewer side effects to other organs. Recently, the first RNAi drug was approved by the US Food and Drug Administration for clinical use, opening a new avenue for new drugs with different clinical applications. RNAi has the potential to have transformational therapeutic applications in several areas of medicine including transplantation. We believe that machine preservation offers great potential to be the ideal delivery method of siRNA to the liver graft, and future studies should be initiated to improve the clinical applicability of RNAi in solid organ transplantation.

Microbial-Based Double-Stranded RNA Production to Develop Cost-Effective RNA Interference Application for Insect Pest Management.

RNA interference (RNAi) is a convenient tool to identify and characterize biological functions in organisms. Recently, it has become an alternative to chemical insecticides as a biologically based control agent. This promising technology has the potential to avoid many problems associated with conventional chemical insecticides. In order for RNAi application to be practical for field use, a major hurdle is the development of a cost-effective system of double-stranded RNA (dsRNA) production for a large quantity of dsRNA. A handful of research reports has demonstrated microbial-based dsRNA production using L4440 vector and HT115 (DE3) Escherichia coli for application to vertebrate and invertebrate systems. However, the dsRNA yield, production efficiency, and biological purity from this in vitro system is still unclear. Thus, our study detailed biochemical and molecular tools for large-scale dsRNA production using the microbial system and investigated the production efficiency and yield of crude and purified dsRNAs. An unrelated insect gene, green fluorescent protein (GFP), and an insect neuropeptide gene, pyrokinin (PK) identified from Drosophila suzukii, were used to construct the recombinant L4440 to be expressed in the HT115 (DE3) cell. A considerable amount of dsRNA, 19.5 µg/mL of liquid culture, was isolated using ultrasonic disruption followed by phenol extraction. The sonication method was further evaluated to extract crude dsRNA without the additional phenol extraction and nuclease treatments and also to reduce potential bacterial viability. The results suggest that the ultrasonic method saved time and costs to isolate crude dsRNA directly from large volumes of cell culture without E coli contamination. We investigated whether the injection of PK dsRNA into flies resulted in increased adult mortality, but it was not statistically significant at 95% confidence level. In this study, the microbial-based dsRNA production has potential for applied RNAi technology to complement current insect pest management practices.

High resolution fingerprinting of single and double-stranded RNA using ion-pair reverse-phase chromatography

The emergence of new sustainable approaches for insect management using RNA interference (RNAi) based insecticides has created the demand for high throughput analytical techniques to fully characterise and accurately quantify double stranded RNA (dsRNA) prior to downstream RNAi applications. In this study we have developed a method for the rapid characterisation of single stranded and double stranded RNA using high resolution RNase mapping in conjunction with ion-pair reverse-phase chromatography utilising a column with superficially porous particles. The high resolution oligoribonucleotide map provides an important ‘fingerprint’ for identity testing and bioprocess monitoring. Reproducible RNA mapping chromatograms were generated from replicate analyses. Moreover, this approach was used to provide a method to rapidly distinguish different RNA sequences of the same size, based on differences in the resulting chromatograms. Principal components analysis of the high resolution RNA mapping data enabled us to rapidly compare multiple HPLC chromatograms and distinguish two dsRNA sequences of different size which share 72% sequence homology. We used the high resolution RNase mapping method to rapidly fingerprint biomanufactured dsRNA across a number of different batches. The resulting chromatograms in conjunction with principal components analysis demonstrated high similarity in the dsRNA produced across the different batches highlighting the potential ability of this method to provide information for batch release in a high throughput manner.

RNA-mediated silencing of PKS1 gene in Colletotrichum falcatum causing red rot in sugarcane

Red rot is one of the major threatening diseases of sugarcane. Due to frequent breakdown of resistance by simultaneous evolution of pathogen variants with the introduction of newer varieties red rot remains as a challenge for successful breeding programme in India. Earlier studies established that the pathogen has limited genotypic variation with enormous phenotypic variation, which could be mainly due to adaptability of pathogen to the newly released varieties and expression of pathogenicity factors including secondary metabolites viz., toxins, enzymes and pigments like melanin. It is also proved that a positive correlation remains between the production of melanin and disease expression and hence the melanin biosynthesizing genes viz., Polyketide synthase1 (PKS1), Scytalone dehydratase1 (SCD1) and Trihydroxynapthalene reductase1 (THR1) were characterized. In this study, we have utilized RNA interference (RNAi) approach using pSilent-1 vector which facilitates the generation of hair pin constructs that suppress the expression of target gene through Agrobacterium mediated transformation (ATMT) to functionally analyze the major gene PKS1 involved in the production of dihydroxy naphthalene (DHN) melanin to determine its role in virulence in C. falcatum. Functionally active sites of the PKS1 gene i.e., acyl transferase (AT) domain and the region responsible for conidial pigment (CP) polyketide synthase were chosen for knockdown approach. The above said regions were amplified in the virulent C. falcatum isolate (Cf671) and both the sense and antisense fragments were separately cloned into pSilent-1 vector. The expression cassette was then cloned into the binary vector and introduced into Agrobacterium tumefaciens LBA4404 which was later co-cultivated with the fungal spore suspension. Results indicated that the knockdown AT mutant failed to produce spores whereas the CP mutants produced spores as that of the parental strains. Further pathogenicity testing revealed that the disease symptoms of both AT and CP mutants were restricted within the inoculated internode until 15th and 5th dpi respectively while the symptoms of wild type were prominent and crossed beyond 3rd internode at 15 dpi during the host-pathogen interaction. Molecular confirmation of the knockdown mutants with the expression of hygromycin gene and absence of functional domains under RT-PCR clearly indicated that the PKS1 gene has a possible role in C. falcatum pathogenesis. However expression analysis by qRT-PCR indicated regaining of virulence in mutants during later part of host pathogen interaction. The results confirm the applicability of RNAi mediated silencing for functional analysis of pathogenicity gene homologs in C. falcatum.

RNA Interference Mechanisms and Applications in Plant Pathology.

The origin of RNA interference (RNAi), the cell sentinel system widely shared among eukaryotes that recognizes RNAs and specifically degrades or prevents their translation in cells, is suggested to predate the last eukaryote common ancestor ( 138 ). Of particular relevance to plant pathology is that in plants, but also in some fungi, insects, and lower eukaryotes, RNAi is a primary and effective antiviral defense, and recent studies have revealed that small RNAs (sRNAs) involved in RNAi play important roles in other plant diseases, including those caused by cellular plant pathogens. Because of this, and because RNAi can be manipulated to interfere with the expression of endogenous genes in an intra- or interspecific manner, RNAi has been used as a tool in studies of gene function but also for plant protection. Here, we review the discovery of RNAi, canonical mechanisms, experimental and translational applications, and new RNA-based technologies of importance to plant pathology.

Technology of RNA Interference in Advanced Medicine.

RNA interference (RNAi) and related pathways involving small interfering RNAs (siRNAs), microRNAs (miRNAs), and PIWI-interacting RNAs (piRNAs) regulate processes such as antiviral defense, genome surveillance, heterochromatin formation, and gene expression in animals, plants, and fungi. Studies on RNAi have revealed a two-step mechanism: (i) Degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity. (ii) The siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the mRNA and degrades it. Molecular structures of Dicer, Argonaute proteins, and RNA-bound complexes have offered insights into the underlying mechanisms of RNA-silencing pathways. Sequence specific gene silencing using small interfering RNA (siRNA) is now being evaluated as a novel therapeutic strategy. Recently, promising data have been obtained from clinical trials for the treatment of respiratory syncytial virus and age-related macular degeneration. The exact mechanism of the RNAi pathways is still unclear. Our review summarizes the RNAi pathways and the known functions of siRNAs, miRNAs, and piRNAs in lower and higher organisms (mostly focusing on mammals) and discusses the potential applications of RNAi.

Quantification of dsRNA using stable isotope labeling dilution liquid chromatography/mass spectrometry.

Recent developments in RNA interference (RNAi) have created a need for cost-effective and large-scale synthesis of double-stranded RNA (dsRNA), in conjunction with high-throughput analytical techniques to fully characterise and accurately quantify dsRNA prior to downstream RNAi applications. Stable isotope labeled dsRNA was synthesised both in vivo (15 N) and in vitro (13 C,15 N-guanosine-containing dsRNA) prior to purification and quantification. The stable isotope labeled dsRNA standards were subsequently spiked into total RNA extracted from E. coli engineered to express dsRNA. RNase mass mapping approaches were subsequently performed using liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) for both the identification and absolute quantification of the dsRNA using the ratios of the light and heavy oligonucleotide pairs. Absolute quantification was performed based on the resulting light and heavy oligoribonucleotides identified using MS. Using this approach we determined that 624.6ng/μL and 466.5ng/μL of dsRNA was present in 80μL total RNA extracted from 108 E. coli cells expressing 765bp and 401bp dsRNAs, respectively. Stable isotope labeling of dsRNA in conjunction with MS enabled the characterisation and quantification of dsRNA in complex total RNA mixtures.

A Single Amide Linkage in the Passenger Strand Suppresses Its Activity and Enhances Guide Strand Targeting of siRNAs.

Potential in vivo applications of RNA interference (RNAi) require suppression of various off-target activities. Herein, we report that replacement of a single phosphate linkage between the first and second nucleosides of the passenger strand with an amide linkage almost completely abolished its undesired activity and restored the desired activity of guide strands that had been compromised by unfavorable amide modifications. Molecular modeling suggested that the observed effect was most likely due to suppressed loading of the amide-modified strand into Ago2 caused by inability of amide to adopt the conformation required for the backbone twist that docks the first nucleotide of the guide strand in the MID domain of Ago2. Eliminating off-target activity of the passenger strand will be important for improving therapeutic potential of RNAi.

RNA interference-based therapy and its delivery systems.

RNA interference (RNAi) is considered a highly specific approach for gene silencing and holds tremendous potential for treatment of various pathologic conditions such as cardiovascular diseases, viral infections, and cancer. Although gene silencing approaches such as RNAi are widely used in preclinical models, the clinical application of RNAi is challenging primarily because of the difficulty in achieving successful systemic delivery. Effective delivery systems are essential to enable the full therapeutic potential of RNAi. An ideal nanocarrier not only addresses the challenges of delivering naked siRNA/miRNA, including its chemically unstable features, extracellular and intracellular barriers, and innate immune stimulation, but also offers "smart" targeted delivery. Over the past decade, great efforts have been undertaken to develop RNAi delivery systems that overcome these obstacles. This review presents an update on current progress in the therapeutic application of RNAi with a focus on cancer therapy and strategies for optimizing delivery systems, such as lipid-based nanoparticles.

Research on the effect of RNA interference on mast cells in respiratory allergic diseases

Respiratory allergic disease has become a major health problem in the 21st century.Mast cells are key effector cells of the respiratory anaphylaxis, and can form a variety of features of allergic inflammation by producting large numbers of inflammatory mediators.RNAi can affect the disease process by silencing gene expression, and has made significant process in the study of many diseases, but the research of targeting mast cells for the treatment of respiratory allergic diseases is rare.This review focuses on recent advances of the application of RNA interference on mast cell in respiratory allergic diseases, aming to further understand its application in the respiratory allergic diseases and research status, and broaden new ideas of treating respiratory allergies.

Polyethylenimine Nanoparticle-Mediated siRNA Delivery to Reduce α-Synuclein Expression in a Model of Parkinson's Disease.

RNA interference (RNAi)-based strategies that mediate the specific knockdown of target genes by administration of small interfering RNAs (siRNAs) could be applied for treatment of presently incurable neurodegenerative diseases such as Parkinson’s disease. However, inefficient delivery of siRNA into neurons hampers in vivo application of RNAi. We have previously established the 4–12 kDa branched polyethylenimine (PEI) F25-LMW with superior transfection efficacy for delivery of siRNA in vivo. Here, we present that siRNA complexed with this PEI extensively distributes across the CNS down to the lumbar spinal cord after a single intracerebroventricular infusion. siRNA against α-synuclein (SNCA), a pre-synaptic protein that aggregates in Parkinson’s disease, was complexed with PEI F25-LMW and injected into the lateral ventricle of mice overexpressing human wild-type SNCA (Thy1-aSyn mice). Five days after the single injection of 0.75 μg PEI/siRNA, SNCA mRNA expression in the striatum was reduced by 65%, accompanied by reduction of SNCA protein by ∼50%. Mice did not show signs of toxicity or adverse effects. Moreover, ependymocytes and brain parenchyma were completely preserved and free of immune cell invasion, astrogliosis, or microglial activation. Our results support the efficacy and safety of PEI nanoparticle-mediated delivery of siRNA to the brain for therapeutic intervention.

Inhibiting PHD2 in bone marrow mesenchymal stem cells via lentiviral vector-mediated RNA interference facilitates the repair of periodontal tissue defects in SD rats.

Hypoxia-inducible factors (HIFs) play an important role in angiogenesis, and they can activate the expression of several downstream angiogenic factors. HIF-1 is a major transcriptor of HIFs, composed of α and β subunits. Prolyl hydroxylase domain-containing protein 2 (PHD2) is the main catabolic enzyme for HIF-1α, and it can accelerate its degradation under normoxic conditions. PHD2 expression in bone marrow mesenchymal stem cells (BMMSCs) of SD rats was down-regulated under normoxic conditions in this study by utilizing lentiviral vector-mediated RNA interference to promote HIF-1α accumulation, thus enhancing the expression of angiogenic factors. A tissue-engineered compound was constructed using the composite collagen membrane of BMMSCs after PHD2 gene silencing to repair periodontal fenestration defects in SD rats. The results of this study indicated that, after PHD2 gene silencing, the osteogenic differentiation of BMMSCs was enhanced in vitro, the resistance of cells to oxidative stress was also validated in vitro, thereby illustrating the promotion of the repair of artificially constructed periodontal tissue defects in rats. The results of this study provide a reference and guidance for future applications of RNA interference in periodontal tissue engineering and serve as a basis for improving the survival of seed cells in recipient tissues.

A double-stranded RNA degrading enzyme reduces the efficiency of oral RNA interference in migratory locust

Application of RNA interference (RNAi) for insect pest management is limited by variable efficiency of RNAi in different insect species. In Locusta migratoria, RNAi is highly efficient through injection of dsRNA, but oral delivery of dsRNA is much less effective. Efforts to understand this phenomenon have shown that dsRNA is more rapidly degraded in midgut fluid than in hemolymph due to nuclease enzyme activity. In the present study, we identified and characterized two full-length cDNAs of double-stranded RNA degrading enzymes (dsRNase) from midgut of L. migratoria, which were named LmdsRNase2 and LmdsRNase3. Gene expression analysis revealed that LmdsRNase2 and LmdsRNase3 were predominantly expressed in the midgut, relatively lower expression in gastric caeca, and trace expression in other tested tissues. Incubation of dsRNA in midgut fluid from LmdsRNase3-suppressed larvae or control larvae injected with dsGFP resulted in high levels of degradation; however, dsRNA incubated in midgut fluid from LmdsRNase2-suppressed larvae was more stable, indicating LmdsRNase2 is responsible for dsRNA degradation in the midgut. To verify the biological function of LmdsRNase2 in vivo, nymphs were injected with dsGFP, dsLmdsRNase2 or dsLmdsRNase3 and chitinase 10 (LmCht10) or chitin synthase 1 (LmCHS1) dsRNA were orally delivered. Mortality associated with reporter gene knockdown was observed only in locusts injected with dsLmdsRNase2 (48% and 22%, for dsLmCht10 and dsLmCHS1, respectively), implicating LmdsRNase2 in reducing RNAi efficiency. Furthermore, recombinantly expressed LmdsRNase2 fusion proteins degraded dsRNA rapidly, whereas LmdsRNase3 did not. These results suggest that rapid degradation of dsRNA by dsRNase2 in the midgut is an important factor causing low RNAi efficiency when dsRNA is orally delivered in the locust.

English

Rift Valley fever (RVF) is a mosquito-borne disease that affects humans and livestock. It results in economically disastrous livestock deaths in Africa. The causative agent is RVF virus (RVFV), which possesses a tripartite negative-sense, single-stranded RNA genome, with large (L), medium (M) and small (S) segments. The lack of effective vaccines and anti-RVFV therapies motivates concerted efforts in search for safe and effective remedies. Recent advances in the RNA interference (RNAi) may provide promising tools to control RVFV. This study aimed to use the Baculovirus Expression Vector System to demonstrate that the RVFV S-genome segment is susceptible to RNAi. A multiplex short hairpin RNA (shRNA) transcription cassette was constructed for generating small interfering RNA (siRNA) that target both the RVFV genomic RNA transcripts and the Green Fluorescent Protein (GFP) which was used as a reporter gene. Two expression vectors were constructed; one for expressing the RVFV S-genome alone and the other one for simultaneous expression of both the RVFV S-genome and the shRNA cassette. A separate transiently expressed GFP only vector was included as an internal positive control to be expressed simultaneously when co-transfected into Sf21 insect cells with each RVFV-S construct to monitor the effectiveness of the shRNA to trigger RNAi. By design, the RNAi effect on both the RVFV-S and the GFP transcripts was driven from the same shRNA. A statistically significant reduction in the relative GFP fluorescence (P< 0.05) was demonstrated and a drop in GFP and the RVFV S transcript levels observed. Collectively, these results suggest the potential application of RNAi as an antiviral strategy against RVFV.   Key words: Baculovirus Expression, rift valley fever, RNA interference, green fluorescent protein.

Bivalent aptamer-dual siRNA chimera is emerging as a new combination therapy

The selective delivery of siRNAs in a cell type-specific manner represents the major challenge for the application of RNA interference for disease treatment. Aptamers have great potentials as carriers for tumor specific siRNA delivery. With the nature of nucleic acid, aptamers can be ease of modification and editing. Novel bivalent aptamer-dual siRNA chimera (PSMA aptamer- survivin siRNA -EGFR siRNA -PSMA aptamer, PSEP) was developed by fusing two siRNAs (specific to EGFR and survivin) between two PSMA aptamers. Bivalent aptamer offers increased siRNA internalization compared with monovalent counterpart. PSEP chimera is able to inhibit EGFR and survivin simultaneously in a cell type-specific manner. In PSMA expressing tumor xenografts, PSEP significantly inhibits tumor growth and angiogenesis. Our results highlight that co-delivery of multiple siRNAs with bivalent aptamer represents a novel approach for targeted combination therapy.

Multifunctional Envelope-Type siRNA Delivery Nanoparticle Platform for Prostate Cancer Therapy.

With the capability of specific silencing of target gene expression, RNA interference (RNAi) technology is emerging as a promising therapeutic modality for the treatment of cancer and other diseases. One key challenge for the clinical applications of RNAi is the safe and effective delivery of RNAi agents such as small interfering RNA (siRNA) to a particular nonliver diseased tissue (e.g., tumor) and cell type with sufficient cytosolic transport. In this work, we proposed a multifunctional envelope-type nanoparticle (NP) platform for prostate cancer (PCa)-specific in vivo siRNA delivery. A library of oligoarginine-functionalized and sharp pH-responsive polymers was synthesized and used for self-assembly with siRNA into NPs with the features of long blood circulation and pH-triggered oligoarginine-mediated endosomal membrane penetration. By further modification with ACUPA, a small molecular ligand specifically recognizing prostate-specific membrane antigen (PSMA) receptor, this envelope-type nanoplatform with multifunctional properties can efficiently target PSMA-expressing PCa cells and silence target gene expression. Systemic delivery of the siRNA NPs can efficiently silence the expression of prohibitin 1 (PHB1), which is upregulated in PCa and other cancers, and significantly inhibit PCa tumor growth. These results suggest that this multifunctional envelope-type nanoplatform could become an effective tool for PCa-specific therapy.