siRNA based drug design, quality, delivery and clinical translation.

Gene silencing by RNA interference represents a promising therapeutic approach. The development of carriers, e.g., polymers, lipids, peptides, antibodies, aptamers, small molecules, exosome and red blood cells, is crucial for the systemic delivery of siRNA. Cell-specific targeting ligands in the nano-carriers can improve the pharmacokinetics, biodistribution, and selectivity of siRNA therapeutics. The safety, effectiveness, quality and prosperity of production and manufacturing are important considerations for selecting the appropriate siRNA carriers. Efficacy of systemic delivery of siRNA requires considerations of trafficking through the blood, off-target effects, innate immune response and endosomal escape avoiding lysosomal degradation for entering into RNAi process. Multifunctional nanocarriers with stimuli-responsive properties such as pH, magnetic and photo-sensitive segments can enhance the efficacy of siRNA delivery. The improved preclinical characterization of suitable siRNA drugs, good laboratory practice, that reduce the differences between in vitro and in vivo results may increase the success of siRNA drugs in clinical settings.

Key factors determining variations in RNA interference efficacy mediated by different double-stranded RNA lengths in Tribolium castaneum.

Double-stranded RNA (dsRNA) length may affect RNA interference (RNAi) efficacy. Herein, variation in RNAi efficacy associated with dsRNA molecular length was confirmed via comparison of knockdown results following dsRNA injection into Tribolium castaneum. Through in vitro experiments with T. castaneum midgut, dsRNA accumulation in the midgut, degradation by midgut homogenates and persistence in haemolymph after injection were tested to determine the causes of RNAi efficacy variation. The comparative efficacies of dsRNAs were 480 bp ≈ 240 bp > 120 bp > 60 bp >> 21 bp. The combined midgut dsRNA accumulation and midgut homogenate-induced degradation analyses suggested cellular uptake to be the key barrier for 21 bp dsRNA functioning, but was likely not the main determinant of the variation in longer dsRNAs' (≥60 bp) bioactivity. In vitro RNAi experiment with T. castaneum midgut showed that long dsRNAs all significantly depleted the expression of corresponding genes, suggesting little variation in intracellular RNAi machinery's affinity for different dsRNA lengths. In vivo haemolymph content dynamics of different dsRNAs following injection indicated higher persistence of longer dsRNAs. In addition, comparison of the in vivo and in vitro RNAi efficacy also indicated the importance of haemolymph degradation. Thus, the varied efficacy of long dsRNAs resulted from their degradation by nucleases, which varied with dsRNA length.

Colorado potato beetle (Coleoptera) gut transcriptome analysis: expression of RNA interference-related genes.

In the search for new methods of pest control, the potential of RNA interference (RNAi) is being explored. Because the gut is the first barrier for the uptake of double-stranded (ds)RNA, pyrosequencing of the gut transcriptome is a powerful tool for obtaining the necessary sequences for specific dsRNA-mediated pest control. In the present study, a dataset representing the gut transcriptome of the Colorado potato beetle (CPB; Leptinotarsa decemlineata) was generated and analysed for the presence of RNAi-related genes. Almost all selected genes that were implicated in silencing efficiency at different levels in the RNAi pathway (core machinery, associated intracellular factors, dsRNA uptake, antiviral RNAi, nucleases), which uses different types of small RNA (small interfering RNA, microRNA and piwi-RNA), were expressed in the CPB gut. Although the database is of lower quality, the majority of the RNAi genes are also found to be present in the gut transcriptome of the tobacco hornworm [TH; Manduca sexta (19 out of 35 genes analysed)]. The high quality of the CPB transcriptome database will lay the foundation for future gene expression and functional studies regarding the gut and RNAi.

Trichoderma atroviride hyphal regeneration and conidiation depend on cell-signaling processes regulated by a microRNA-like RNA.

The ability to respond to injury is essential for the survival of an organism and involves analogous mechanisms in animals and plants. Such mechanisms integrate coordinated genetic and metabolic reprogramming events requiring regulation by small RNAs for adequate healing of the wounded area. We have previously reported that the response to injury of the filamentous fungus Trichoderma atroviride involves molecular mechanisms closely resembling those of plants and animals that lead to the formation of new hyphae (regeneration) and the development of asexual reproduction structures (conidiophores). However, the involvement of microRNAs in this process has not been investigated in fungi. In this work, we explore the participation of microRNA-like RNAs (milRNAs) molecules by sequencing messenger and small RNAs during the injury response of the WT strain and RNAi mutants. We found that Dcr2 appears to play an important role in hyphal regeneration and is required to produce the majority of sRNAs in T. atroviride. We also determined that the three main milRNAs produced via Dcr2 are induced during the damage-triggered developmental process. Importantly, elimination of a single milRNA phenocopied the main defects observed in the dcr2 mutant. Our results demonstrate the essential role of milRNAs in hyphal regeneration and asexual development by post-transcriptionally regulating cellular signalling processes involving phosphorylation events. These observations allow us to conclude that fungi, like plants and animals, in response to damage activate fine-tuning regulatory mechanisms.

Small RNAs - Big Players in Plant-Microbe Interactions.

Eukaryotic small RNAs (sRNAs) are short non-coding regulatory molecules that induce RNA interference (RNAi). During microbial infection, host RNAi machinery is highly regulated and contributes to reprogramming gene expression and balancing plant immunity and growth. While most sRNAs function endogenously, some can travel across organismal boundaries between hosts and microbes and silence genes in trans in interacting organisms, a mechanism called "cross-kingdom RNAi." During the co-evolutionary arms race between fungi and plants, some fungi developed a novel virulence mechanism, sending sRNAs as effector molecules into plant cells to silence plant immunity genes, whereas plants also transport sRNAs, mainly using extracellular vesicles, into the pathogens to suppress virulence-related genes. In this Review, we highlight recent discoveries on these key roles of sRNAs and RNAi machinery. Understanding the molecular mechanisms of sRNA biogenesis, trafficking, and RNAi machinery will help us develop innovative strategies for crop protection.