GFP Silencing Research Articles

Mutations in the WG and GW motifs of the three RNA silencing suppressors of grapevine fanleaf virus alter their systemic suppression ability and affect virus infectivity.

Viral suppressors of RNA silencing (VSRs) encoded by grapevine fanleaf virus (GFLV), one of the most economically consequential viruses of grapevine (Vitis spp.), were recently identified. GFLV VSRs include the RNA1-encoded protein 1A and the putative helicase protein 1BHel, as well as their fused form (1ABHel). Key characteristics underlying the suppression function of the GFLV VSRs are unknown. In this study, we explored the role of the conserved tryptophan-glycine (WG) motif in protein 1A and glycine-tryptophan (GW) motif in protein 1BHel in their systemic RNA silencing suppression ability by co-infiltrating Nicotiana benthamiana 16c line plants with a GFP silencing construct and a wildtype or a mutant GFLV VSR. We analyzed and compared wildtype and mutant GFLV VSRs for their (i) efficiency at suppressing RNA silencing, (ii) ability to limit siRNA accumulation, (iii) modulation of the expression of six host genes involved in RNA silencing, (iv) impact on virus infectivity in planta, and (v) variations in predicted protein structures using molecular and biochemical assays, as well as bioinformatics tools such as AlphaFold2. Mutating W to alanine (A) in WG of proteins 1A and 1ABHel abolished their ability to induce systemic RNA silencing suppression, limit siRNA accumulation, and downregulate NbAGO2 expression by 1ABHel. This mutation in the GFLV genome resulted in a non-infectious virus. Mutating W to A in GW of proteins 1BHel and 1ABHel reduced their ability to suppress systemic RNA silencing and abolished the downregulation of NbDCL2, NbDCL4,, and NbRDR6 expression by 1BHel. This mutation in the GFLV genome delayed infection at the local level and inhibited systemic infection in planta. Double mutations of W to A in WG and GW of protein 1ABHel abolished its ability to induce RNA silencing suppression, limit siRNA accumulation, and downregulate NbDCL2 and NbRDR6 expression. Finally, in silico protein structure prediction indicated that a W to A substitution potentially modifies the structure and physicochemical properties of the three GFLV VSRs. Together, this study provided insights into the specific roles of WG/GW not only in GFLV VSR functions but also in GFLV biology.

Distinct tomato yellow leaf curl Chuxiong virus isolated from whiteflies and plants in China and its symptom determinant and suppressor of post-transcriptional gene silencing

A begomovirus isolated from whiteflies (Bemisia tabaci) and tomato, sweet potato in China was found to be representative of a distinct begomovirus species, for which the name tomato yellow leaf curl Chuxiong virus (TYLCCxV) is proposed. The results of genomic identification and sequence comparison showed that TYLCCxV shares the highest complete nucleotide sequence identity (88.3%) with croton yellow vein mosaic virus (CroYVMV), and may have originated from the recombination between synedrella leaf curl virus (SyLCV) and squash leaf curl Yunnan virus (SLCuYV). Agrobacterium-mediated inoculation showed that TYLCCxV is highly infectious for a range of plant species, producing upward leaf curling, leaf crumpling, chlorosis, distortion, and stunt symptoms in Solanum lycopersicum plants. The results of Southern blot indicated that TYLCCxV is capable of efficiently replicating two heterologous betasatellites. The inoculation of PVX::C4 on Nicotiana benthamiana induced upward leaf curling and stem elongation symptoms, suggesting that TYLCCxV C4 functions as a symptom determinant. TYLCCxV V2 is an important virulence factor that induces downward leaf curling symptoms, elicits systemic necrosis, and suppresses local and systemic GFP silencing in co-agroinfiltrated N. benthamiana and transgenic 16c plants. Considering the multifunctional virulence proteins V2 and C4, the possibility of TYLCCxV causing devastating epidemics on tomato in China is discussed.

Polycondensed Peptide-Based Polymers for Targeted Delivery of Anti-Angiogenic siRNA to Treat Endometriosis.

Endometriosis (EM) is a prevalent gynecological disease characterized by the abnormal growth of tissue similar to the endometrium outside of the uterus. This condition is accompanied by the development of new blood vessels in endometriotic lesions. While surgical intervention is effective in removing endometriotic lesions, some patients require multiple surgeries. Therefore, finding non-surgical treatments for EM is of great interest. One of the promising approaches is anti-angiogenic therapy using siRNA-therapeutics to target the expression of the VEGFA gene. Peptide-based polymers have shown promise as siRNA delivery systems due to their biocompatibility and ease of modification. We conducted a study to evaluate the effectiveness of the R6p-cRGD peptide carrier as a non-viral vehicle for delivering siRNA to endothelial cells in vitro and endometrial implants in vivo. We investigated the physicochemical properties of the siRNA-complexes, assessed cellular toxicity, and examined the efficiency of GFP and VEGFA genes silencing. Furthermore, we tested the anti-angiogenic effects of these complexes in cellular and animal models. The transfection with siRNA complexes led to a significant increase in VEGFA gene knockdown efficiency and a decrease in the migration of endothelial cells. For the animal model, we induced endometriosis in rats by transplanting endometrial tissue subcutaneously. We evaluated the efficiency of anti-angiogenic therapy for EM in vivo using anti-VEGF siRNA/R6p-RGD complexes. During this assessment, we measured the volume of the implants, analyzed VEGFA gene expression, and conducted CD34 immunohistochemical staining. The results showed a significant decrease in the growth of endometriotic implants and in VEGFA gene expression. Overall, our findings demonstrate the potential of the R6p-cRGD peptide carrier as a delivery system for anti-angiogenic therapy of EM.

Microbe-induced gene silencing boosts crop protection against soil-borne fungal pathogens.

Small RNA (sRNA)-mediated trans-kingdom RNA interference (RNAi) between host and pathogen has been demonstrated and utilized. However, interspecies RNAi in rhizospheric microorganisms remains elusive. In this study, we developed a microbe-induced gene silencing (MIGS) technology by using a rhizospheric beneficial fungus, Trichoderma harzianum, to exploit an RNAi engineering microbe and two soil-borne pathogenic fungi, Verticillium dahliae and Fusarium oxysporum, as RNAi recipients. We first detected the feasibility of MIGS in inducing GFP silencing in V. dahliae. Then by targeting a fungal essential gene, we further demonstrated the effectiveness of MIGS in inhibiting fungal growth and protecting dicotyledon cotton and monocotyledon rice plants against V. dahliae and F. oxysporum. We also showed steerable MIGS specificity based on a selected target sequence. Our data verify interspecies RNAi in rhizospheric fungi and the potential application of MIGS in crop protection. In addition, the in situ propagation of a rhizospheric beneficial microbe would be optimal in ensuring the stability and sustainability of sRNAs, avoiding the use of nanomaterials to carry chemically synthetic sRNAs. Our finding reveals that exploiting MIGS-based biofungicides would offer straightforward design and implementation, without the need of host genetic modification, in crop protection against phytopathogens.

IRGD-Targeted Peptide Nanoparticles for Anti-Angiogenic RNAi-Based Therapy of Endometriosis.

Anti-angiogenic RNAi-based therapy can be considered as a possible strategy for the treatment of endometriosis (EM), which is the most common gynecological disease. Targeted delivery of siRNA therapeutics is a prerequisite for successful treatment without adverse effects. Here we evaluated the RGD1-R6 peptide carrier as a non-viral vehicle for targeted siRNA delivery to endothelial cells in vitro and endometrial implants in vivo. The physicochemical properties of the siRNA complexes, cellular toxicity, and GFP and VEGFA gene silencing efficiency were studied, and anti-angiogenic effects were proved in cellular and animal models. The modification of siRNA complexes with iRGD ligand resulted in a two-fold increase in gene knockdown efficiency and three-fold decrease in endothelial cells' migration in vitro. Modeling of EM in rats with the autotransplantation of endometrial tissue subcutaneously was carried out. Efficiency of anti-angiogenic EM therapy in vivo by anti-VEGF siRNA/RGD1-R6 complexes was evaluated by the implants' volume measurement, CD34 immunohistochemical staining, and VEGFA gene expression analysis. We observed a two-fold decrease in endometriotic implants growth and a two-fold decrease in VEGFA gene expression in comparison with saline-treated implants. RNAi-mediated therapeutic effects were comparable with Dienogest treatment efficiency in a rat EM model. Taken together, these findings demonstrate the advantages of RGD1-R6 peptide carrier as a delivery system for RNAi-based therapy of EM.

A GFP splicing reporter in a coilin mutant background reveals links between alternative splicing, siRNAs, and coilin function in Arabidopsis thaliana.

Coilin is a scaffold protein essential for the structure of Cajal bodies, which are nucleolar-associated, nonmembranous organelles that coordinate the assembly of nuclear ribonucleoproteins (RNPs) including spliceosomal snRNPs. To study coilin function in plants, we conducted a genetic suppressor screen using a coilin (coi1) mutant in Arabidopsis thaliana and performed an immunoprecipitation-mass spectrometry analysis on coilin protein. The coi1 mutations modify alternative splicing of a GFP reporter gene, resulting in a hyper-GFP phenotype in young coi1 seedlings relative to the intermediate wild-type level. As shown here, this hyper-GFP phenotype is extinguished in older coi1 seedlings by posttranscriptional gene silencing triggered by siRNAs derived from aberrant splice variants of GFP pre-mRNA. In the coi1 suppressor screen, we identified suppressor mutations in WRAP53, a putative coilin-interacting protein; SMU2, a predicted splicing factor; and ZCH1, an incompletely characterized zinc finger protein. These suppressor mutations return the hyper-GFP fluorescence of young coi1 seedlings to the intermediate wild-type level. Additionally, coi1 zch1 mutants display more extensive GFP silencing and elevated levels of GFP siRNAs, suggesting the involvement of wild-type ZCH1 in siRNA biogenesis or stability. The immunoprecipitation-mass spectrometry analysis reinforced the roles of coilin in pre-mRNA splicing, nucleolar chromatin structure, and rRNA processing. The participation of coilin in these processes, at least some of which incorporate small RNAs, supports the hypothesis that coilin provides a chaperone for small RNA trafficking. Our study demonstrates the usefulness of the GFP splicing reporter for investigating alternative splicing, ribosome biogenesis, and siRNA-mediated silencing in the context of coilin function.

AC5 protein encoded by squash leaf curl China virus is an RNA silencing suppressor and a virulence determinant.

Squash leaf curl China virus (SLCCNV) is a bipartite Begomovirus. The function of the protein AC5, which is encoded by SLCCNV, is unknown. Here, we confirmed that the 172-amino acids (aa) long AC5 protein of SLCCNV could suppress single-stranded RNA but not double-stranded RNA-induced post-transcriptional gene silencing (PTGS). Furthermore, we determined that the C-terminal domain (96–172 aa) of the AC5 protein was responsible for RNA silencing suppressor (RSS) activity via deletion mutant analysis. The AC5 protein can reverse GFP silencing and inhibit systemic silencing of GFP by interfering with the systemic spread of the GFP silencing signal. The SLCCNV AC5 protein was localized to both the nucleus and cytoplasm of Nicotiana benthamiana cells. Furthermore, deletion analysis showed that the putative nuclear localization signal (NLS, 102–155 aa) was crucial for the RNA silencing suppression activity of AC5. In addition, the AC5 protein elicited a hypersensitive response and enhanced potoao virus X (PVX) RNA accumulation in infected N. benthamiana plants. Using the infectious clones of the SLCCNV and SLCCNV-AC5 null mutants, mutational analysis confirmed that knockout of the AC5 gene abolished SLCCNV-induced leaf curl symptoms, showing SLCCNV AC5 is also a virulence determinant.

The novel C5 protein from tomato yellow leaf curl virus is a virulence factor and suppressor of gene silencing

Tomato yellow leaf curl virus (TYLCV) is known to encode 6 canonical viral proteins. Our recent study revealed that TYLCV also encodes some additional small proteins with potential virulence functions. The fifth ORF of TYLCV in the complementary sense, which we name C5, is evolutionarily conserved, but little is known about its expression and function during viral infection. Here, we confirmed the expression of the TYLCV C5 by analyzing the promoter activity of its upstream sequences and by detecting the C5 protein in infected cells by using a specific custom-made antibody. Ectopic expression of C5 using a potato virus X (PVX) vector resulted in severe mosaic symptoms and higher virus accumulation levels followed by a burst of reactive oxygen species (ROS) in Nicotiana benthamiana plants. C5 was able to effectively suppress local and systemic post-transcriptional gene silencing (PTGS) induced by single-stranded GFP but not double-stranded GFP, and reversed the transcriptional gene silencing (TGS) of GFP. Furthermore, the mutation of C5 in TYLCV inhibited viral replication and the development of disease symptoms in infected plants. Transgenic overexpression of C5 could complement the virulence of a TYLCV infectious clone encoding a dysfunctional C5. Collectively, this study reveals that TYLCV C5 is a pathogenicity determinant and RNA silencing suppressor, hence expanding our knowledge of the functional repertoire of the TYLCV proteome.

Exploring the Effectiveness and Durability of Trans-Kingdom Silencing of Fungal Genes in the Vascular Pathogen Verticillium dahliae.

Host-induced gene silencing (HIGS) based on trans-kingdom RNA interference (RNAi) has been successfully exploited to engineer host resistance to pests and pathogens, including fungi and oomycetes. However, revealing the mechanisms underlying trans-kingdom RNAi between hosts and pathogens lags behind applications. The effectiveness and durability of trans-kingdom silencing of pathogenic genes are uncharacterized. In this study, using our transgenic 35S-VdH1i cotton plants in which dsVdH1-derived small RNAs (siVdH1) accumulated, small RNA sequencing analysis revealed that siVdH1s exclusively occur within the double-stranded (ds)VdH1 region, and no transitive siRNAs were produced beyond this region in recovered hyphae of Verticillium dahliae (V. dahliae). Accordingly, we found that VdH1 silencing was reduced over time in recovered hyphae cultured in vitro, inferring that once the fungus got rid of the 35S-VdH1i cotton plants would gradually regain their pathogenicity. To explore whether continually exporting dsRNAs/siRNAs from transgenic plants into recipient fungal cells guaranteed the effectiveness and stability of HIGS, we created GFP/RFP double-labeled V. dahliae and transgenic Arabidopsis expressing dsGFP (35S-GFPi plants). Confocal images visually demonstrate the efficient silencing of GFP in V. dahliae that colonized host vascular tissues. Taken together, our results demonstrate that HIGS effectively triggers long-lasting trans-kingdom RNAi during plant vasculature V. dahliae interactions, despite no amplification or transitivity of RNAi being noted in this soil-borne fungal pathogen.

Progressive DNA demethylation in epigenetic hybrids between parental plants with and without methylation of the transgene promoter.

Crosses of parents that differ in their DNA methylation states leads to progressive demethylation in the F1 hybrids. In plant breeding research, hybrid vigor in F1 hybrids is known to be a very important phenomenon. Hybrid vigor, or heterosis, refers to the fact that F1 hybrids from crosses with a certain combination of parents have traits that are superior to those of the parents. In addition, DNA methylation is an important factor that affects gene expression in plant genomes and contributes to hybrid vigor. We introduced the 35S promoter sequence into the cucumber mosaic virus (CMV)-based vector and inoculated the GFP-expressing transgenic Nicotiana benthamiana line 16c with the recombinant virus specifically to induce DNA methylation on the 35S promoter. For plants that had transcriptional gene silencing (TGS) of GFP established by methylation of the 35S promoter (35S-TGS), TGS was fully maintained in their later self-pollinated generations. When the 35S-TGS plants were crossed with 16c, which does not contain DNA methylation in the 35S promoter, the F1 hybrids unexpectedly became progressively DNA demethylated as the plants grew. We hypothesis that in F1 hybrids that are produced by a cross between parents with extremely different gene methylation states, the methylation state of the genes in question may shift more and more to hypomethylation as the plants grow. This progressive demethylation phenomenon observed in this study may be important in plant breeding to reactivate the genes which were silenced by DNA methylation.

Fluorescence Lifetime Imaging Microscopy (FLIM) of Intracellular Transport by Means of Doubly Labelled siRNA Architectures.

For monitoring the intracellular pathway of small interfering RNA (siRNA), both strands were labelled at internal positions by two ATTO dyes as an interstrand Förster resonance energy transfer pair. siRNA double strands show red emission and a short donor lifetime as readout, whereas siRNA antisense single strands show green emission and a long donor lifetime. This readout signals if GFP silencing can be expected (green) or not (red). We attached both dyes to three structurally different alkyne anchors by postsynthetic modifications. There is only a slight preference for the ribofuranoside anchors with the dyes at their 2’‐positions. For the first time, the delivery and fate of siRNA in live HeLa cells was tracked by fluorescence lifetime imaging microscopy (FLIM), which revealed a clear relationship between intracellular transport using different transfection methods and knockdown of GFP expression, which demonstrates the potential of our siRNA architectures as a tool for future development of effective siRNA.

Impact of crystalline and amorphous matrices on successful spray drying of siRNA polyplexes for inhalation of nano-in-microparticles.

To develop stable and inhalable dry powder formulations with long shelf life, we spray dried polyplexes consisting of siRNA and a polyethylenimine based block copolymer in presence of mannitol or trehalose. We investigated the effect of inlet (T-In) and outlet (T-Out) temperature on the recovery of siRNA as well as adsorption effects within the tubing material. Choosing a low abrasion silicon tubing prevented siRNA loss due to adsorption. Mannitol and trehalose formulations preserved siRNA integrity regardless of excipient concentration and temperature at T-Out below the siRNA melting temperature. Trehalose formulations allowed full siRNA recovery whereas mannitol formulations resulted in spray drying induced losses of ~20 % siRNA and of 50-60 % polymer. Mannitol formulations showed optimal aerodynamic characteristics as confirmed by next generation impaction analysis based upon siRNA content. All spray dried formulations resulted in GFP silencing comparable or better than freshly prepared polyplexes. To test if the observed results could be transferred, formulations of siRNA and transferrin-PEI conjugates were spray dried, characterized and used to transfect primary human T cells ex vivo. Results confirmed successful silencing of the Th2 transcription factor GATA3 in primary CD4+ T cells with spray dried formulations as a potential treatment for severe asthma.

Hydrogel microspheres for spatiotemporally controlled delivery of RNA and silencing gene expression within scaffold-free tissue engineered constructs

Delivery systems for controlled release of RNA interference (RNAi) molecules, including small interfering (siRNA) and microRNA (miRNA), have the potential to direct stem cell differentiation for regenerative musculoskeletal applications. To date, localized RNA delivery platforms in this area have focused predominantly on bulk scaffold-based approaches, which can interfere with cell-cell interactions important for recapitulating some native musculoskeletal developmental and healing processes in tissue regeneration strategies. In contrast, scaffold-free, high density human mesenchymal stem cell (hMSC) aggregates may provide an avenue for creating a more biomimetic microenvironment. Here, photocrosslinkable dextran microspheres (MS) encapsulating siRNA-micelles were prepared via an aqueous emulsion method and incorporated within hMSC aggregates for localized and sustained delivery of bioactive siRNA. siRNA-micelles released from MS in a sustained fashion over the course of 28 days, and the released siRNA retained its ability to transfect cells for gene silencing. Incorporation of fluorescently labeled siRNA (siGLO)-laden MS within hMSC aggregates exhibited tunable siGLO delivery and uptake by stem cells. Incorporation of MS loaded with siRNA targeting green fluorescent protein (siGFP) within GFP-hMSC aggregates provided sustained presentation of siGFP within the constructs and prolonged GFP silencing for up to 15 days. This platform system enables sustained gene silencing within stem cell aggregates and thus shows great potential in tissue regeneration applications. Statement of SignificanceThis work presents a new strategy to deliver RNA-nanocomplexes from photocrosslinked dextran microspheres for tunable presentation of bioactive RNA. These microspheres were embedded within scaffold-free, human mesenchymal stem cell (hMSC) aggregates for sustained gene silencing within three-dimensional cell constructs while maintaining cell viability. Unlike exogenous delivery of RNA within culture medium that suffers from diffusion limitations and potential need for repeated transfections, this strategy provides local and sustained RNA presentation from the microspheres to cells in the constructs. This system has the potential to inhibit translation of hMSC differentiation antagonists and drive hMSC differentiation toward desired specific lineages, and is an important step in the engineering of high-density stem cell systems with incorporated instructive genetic cues for application in tissue regeneration.

Efficiency of Chitosan-Coated PLGA Nanocarriers for Cellular Delivery of siRNA and CRISPR/Cas9 Complex

Most studies based on siRNA or CRISPR/Cas9 systems rely on viral vectors for their transfection. However, these viral vectors are immunogenic, which limits their application in gene therapy. Thus, identification of novel vectors with higher safety and improved targeting is desirable. Hence, we have explored the potential of biodegradable, chitosan coated PLGA nanocarriers for intracellular delivery of CRISPR/Cas9, and siRNA. In this investigation, we have compared the efficiency of chitosan-coated PLGA NPs (CS-PLGA NPs) with that of the PLGA NPs for the delivery of CRISPR and siRNA. Presented here is the preparation and evaluation of specifically surface-modified CS-PLGA NPs and PLGA NPs on their efficacy for binding and delivery of siRNA and CRISPR/Cas9 complex. Cy3 siRNA loaded on PLGA NPs showed an internalization of 4.6% and mean fluorescent intensity (MFI) of 13.76%, while that of CS PLGA resulted in 89% internalization and MFI of 67.95%. The in vitro GFP silencing assessed by anti-GFP siRNA and NPs resulted in 10–15% silencing by PLGA NPs and 50–55% silencing by CS-PLGA NPs. The GFP silencing by CRISPR-Cas9 plasmid pX459 with CS PLGA was 80–83%, while that of PLGA was 11% and of commercial Lipofectamine agent was 13%. The biodegradable CS-PLGA NPs exhibited successful loading and high binding efficiency for siRNA as well as CRISPRCas9 and resulted in effective silencing. Our studies report that the CS-PLGA NPs can be a novel suitable candidate for the effective delivery of siRNA and CRISPR/Cas9 complex.

Evaluating the strength of RNA silencing suppressor proteins encoded by two geminiviruses using assay based on reversal of GFP silencing

Virus Induced Gene Silencing (VIGS) is conserved and dynamic gene regulation machinery, which plays an active role in shielding plants against viral infections. In counter-defense, viruses encode proteins that can suppress RNA silencing to ensure successful systemic invasion. The AC2 protein of Tomato Leaf Curl New Delhi Virus (ToLCV-AC2) and Mungbean Yellow Mosaic India Virus (MYMIV-AC2) are well-characterized suppressors of RNA silencing. In this study we have compared the structure and suppressor strength of ToLCV-AC2 and MYMIV-AC2, to gain insight on the conservation of the RNA silencing suppression activities. The relative suppressor strength was measured using an assay based on the reversal of GFP silencing and quantitated in terms of the amount of reversal of GFP expression and the sustenance of the reversal activity. The phenotypic observations in the reversal of silencing assay were further corroborated with molecular analysis. Our results indicate that ToLCV-AC2 exhibited weaker RNA silencing suppression than MYMIV-AC2. The suppression activity of ToLCV-AC2 is supplemented by related, though weak, activities encoded by ToLCV-AC4 and ToLCV-AV2 proteins. Collectively the three ToLCV proteins showed suppressor strength comparable to MYMIV-AC2. These results indicate that in all probability the ToLCV deploys more than one RNA silencing suppressor protein to synergistically counteract the host RNA silencing mechanism.

Hydrogel Microspheres for Spatiotemporally Controlled Delivery of Rna and Silencing Gene Expression within Scaffold-Free Tissue Engineered Constructs

Delivery systems for controlled release of RNA interference (RNAi) molecules, including small interfering (siRNA) and microRNA (miRNA), have the potential to direct stem cell differentiation for regenerative musculoskeletal applications. To date, localized RNA delivery platforms in this area have focused predominantly on bulk scaffold-based approaches, which can interfere with cell-cell interactions important for recapitulating some native musculoskeletal developmental and healing processes in tissue regeneration strategies. In contrast, scaffold-free, high density human mesenchymal stem cell (hMSC) condensations may provide an avenue for creating a more biomimetic microenvironment. Here, photocrosslinkable dextran microspheres (MS) encapsulating siRNA-micelles were prepared via an aqueous emulsion method and incorporated within hMSC aggregates for localized and sustained delivery of bioactive siRNA. siRNA-micelles released from MS in a sustained fashion over the course of 28 days, and the released siRNA retained its ability to transfect cells for gene silencing. Incorporation of fluorescently labeled siRNA (siGLO)-laden MS within hMSC aggregates exhibited tunable siGLO delivery and uptake by stem cells. Incorporation of MS loaded with siRNA targeting green fluorescent protein (siGFP) within GFP-hMSC aggregates provided sustained presentation of siGFP within the constructs and prolonged GFP silencing for up to 15 days. This platform system enables sustained gene silencing within stem cell condensations and thus shows great potential in tissue regeneration applications.

Silencing of GFP expression in human mesenchymal stem cells using quaternary polyplexes of siRNA-PEI with glycosaminoglycans and albumin

In recent years evidence has been mounting for a role for mesenchymal stem cells (MSCs) in immunomodulation, anti-inflammatory processes, and paracrine signaling via secreted extracellular vesicles. In order to exploit these biological functions, systems to efficiently deliver genetic material into MSCs would therefore be highly desirable. In this study, efficient silencing of GFP expression by combining high N/P ratio siRNA and branched PEI (bPEI) polyplexes (siRNA-bPEI) polyplexes with glycosaminoglycans (GAGs), namely hyaluronic acid (HA), chondroitin sulfate (CS) and heparin sulfate (HS), and human serum albumin (HSA) is reported. These quaternary systems were characterized using surface charge, size and morphology and applied to MSCs, which represent a challenge due to their typically low transfection efficiency. The quaternary polyplexes promoted efficient charge shielding and release of siRNA in the cytoplasm with reduced toxicity. A high silencing efficiency of >90% (i.e., less than 10% remaining GFP expression) was achieved with noticeably reduced cellular toxicity, especially with siRNA-bPEI polyplexes modified with HA and HA + HSA. In general addition of GAGs led to more compact polyplexes. Endocytosis studies point to improved endosomal escape at high N/P ratios as a reason for high transfection efficiency and a role for hyaluronic acid in the uptake mechanism likely via CD44 interactions. Co-localization studies showed the polyplexes are stable in the cytosol over time, which correlates with a proper disassembly and subsequent silencing of GFP. Furthermore, GAG containing polyplexes were frequently co-localized with the nucleus. These findings in sum suggest that PEI/HSA/GAG based quaternary polyplexes are promising as transfection agents for MSCs. Statement of SignificanceSince mesenchymal stem cells (MSCs) are recruited to the site of tissue repair and play a role in immunomodulation, anti-inflammatory processes, and paracrine signaling, they present an excellent target for genetic engineering. However, delivery of genetic material into MSCs is challenging. In this study, >97% silencing of constitutive green fluorescent protein expression in human MSCs (hMSCs) using high N/P ratio polyplexes of branched-PEI-siRNA incorporating glycosaminoglycan as a charge neutralizer and human serum albumin as co-complexing agent is demonstrated. In addition to possessing good cytocompatibility and excellent cytosolic stability; polyplexes incorporating GAGs also showed altered endocytic uptake, with incorporation of hyaluronic acid promoting caveolae-mediated entry. Our system highlights the importance of physiologically derived macromolecules in delivery of genetic material into hMSCs.

Systemic Delivery of Sirna-Based Therapeutics Using Single-Walled Carbon Nanotubes

Background: Carbon nanotubes (CNTs) are potential candidates for drug, antigen and nucleic acid delivery vehicle in nanomedicine. The large surface of CNTs provides structural advantages and allows loading of functional groups or therapeutics such as nucleic acid, drugs and proteins. Aim: Our study is to deliver siRNA to cells using single walled carbon nanotube (SWNT) to achieve gene silencing effect. Methods: SWNT was functionalized by dissolving 1 mg of HiPCo SWNTs and 5 mg of PL-PEG-NH or PL-PEG-maleimide in 5 mL of water, sonicated for 60 min at room temperature and centrifuged for 6 h. The supernatant were collected and measured their concentration at 808 nm by ultraviolet-VIS-NIR spectrometer. The resulted noncovalent functionalized SWNTs were further conjugated with a 5′-thiolated siRNA against GFP (siGFP) and RFP (siRFP). In vitro silencing of GFP and RFP expression by SWNT-siRNA were evaluated in stable expression cell lines by fluorescence spectroscopy. Results: A range of 50%–80% GFP expression knocked down was observed in H1299, HeLa, MCF-7 and 293T cells by SWNT-siGFP. SWNT conjugated with both siGFP and siRFP were shown knocking down both GFP and RFP simultaneously in H1299 stable coexpression cell line. Also, gene silencing was observed despite incubation with inhibitors on different cellular internalization pathways. They are chlorpromazine for clathrin-mediated endocytosis inhibitor, genistein for caveolae-mediated endocytosis inhibitor and sodium azide for energy depletion agent. We also found that the internalization of SWNT by the nonphagocytic cells (H1299) did not solely depend on single cellular entry pathway to achieve the gene silencing effect. Conclusion: The successful knockdown of GFP expression in different cell lines indicated that siRNA were released from the conjugated SWNT-siRNA in the cytoplasm and silent the gene expression. It is also indicated that 2 different types of siRNA targets could be conjugated with SWNT and achieved 2 different gene silencing effects simultaneously.

Sugarcane Mosaic Virus-Based Gene Silencing in Nicotiana benthamiana

Background: Potyvirus-based virus-induced gene silencing (VIGS) is used for knocking down the expression of a target gene in numerous plant species. Sugarcane mosaic virus (SCMV) is a monopartite, positive single strand RNA virus. Objectives: pBINTRA6 vector was modifi ed by inserting a gene segment of SCMV in place of Tobacco rattle virus (TRV) genome part 1 (TRV1 or RNA1) and the two nonstructural proteins of TRV2(RNA2). Materials and Methods: SCMV construct was inoculated into 3-4 weeks Nicotiana benthamiana plant leaves either by using a needleless syringe or applying pricking with a toothpick. Results: The construct (SCMV-RNA2) successfully induced post-transcriptional gene silencing (PTGS) of the target genes GFP and ChlI through agroinoculation proving that SCMV is a substitute of the RNA1, which plays a pivotal role in the systemic gene silencing. 2-3-weeks of post inoculation, target genes' silencing was observed in the newly developed noninoculated leaves. Conclusions: The newly developed construct expresses the knocked down of the endogenous as well as exogenous genes and only four weeks are required for the transient expression of the gene silencing based on SCMV-VIGS system.

Circular siRNAs for Reducing Off-Target Effects and Enhancing Long-Term Gene Silencing in Cells and Mice.

Circular non-coding RNAs are found to play important roles in biology but are still relatively unexplored as a structural motif for chemically regulating gene function. Here, we investigated whether small interfering RNA (siRNA) with a circular structure can circumvent off-target gene silencing, a problem often observed with standard linear duplex siRNA. In the present work, we, for the first time, synthesized a series of circular siRNAs by cyclizing two ends of a single-stranded RNA (sense or antisense strand) to construct circular siRNAs that were more resistant to enzymatic degradation. Gene silencing of GFP and luciferase was successfully achieved using these circular siRNAs with circular sense strand RNAs and their complementary linear antisense strand RNAs. The off-target effect of sense strand RNAs was evaluated and no cross off-target effects were observed. In addition, we successfully achieved longer gene-silencing efficiency in mice with circular siRNAs than with linear siRNAs. These results indicate the promise of circular siRNAs for overcoming off-target effects of siRNAs and enhancing the possible long-term effect of siRNA gene silencing in basic research and drug development.