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Background Small interfering RNA (siRNA) is a powerful tool for gene silencing, but its clinical application is limited by instability and potential immunogenicity. While chemical modification is essential to overcome these hurdles, data on chemically modified siRNAs are currently scattered, hindering rational drug design and development. Results We developed CMsiRNAdb, a comprehensive database integrating data resources, analytical tools, and efficacy prediction for chemically modified siRNAs. We consolidated 43,153 experimentally validated sequences and silencing efficiency data derived from 90 patents, covering 36 modification types and 13 therapeutic target genes. The database offers multi-dimensional retrieval, visualization, and batch download functions. Furthermore, we developed ModMapper, a Trie tree-based tool for precise identification of modification sites, and integrated the Cm-siRPred model for efficacy evaluation. CMsiRNAdb is freely accessible at https://cellknowledge.com.cn/CMsiRNAdb/ . Conclusion CMsiRNAdb provides critical data support and analytical tools for the rational design and rapid optimization of siRNA drugs. By standardizing data and offering predictive capabilities, it significantly advances the development of nucleic acid therapeutics.

Advances in RNA interference technology have established it as a powerful therapeutic tool with important future potential. The design and the chemical modifications of the siRNA nucleotide backbone have greatly enhanced stability, durability, and pharmacokinetics while minimizing tolerability risks. The optimal combination of these modifications depends on the target gene, tissue, and RNA sequence, necessitating an iterative, experimental approach that currently relies heavily on animal models. To reduce the reliance and number of (humanized) animals required, we developed a human long‐term liver 3D spheroid model designed for screening GalNAc‐conjugated siRNAs which captures the process of uptake, potency, and durability for early in vitro screening. These liver spheroids remain viable in culture for at least 5 weeks while maintaining expression of the asialoglycoprotein receptor to facilitate GalNAc mediated uptake. siRNA was efficiently internalized by the spheroids without the need for transfection reagents, and its durable silencing efficiency was assessed by monitoring AHSA1 target gene expression over time. Target gene silencing in the spheroid model persisted up to 5 weeks post‐treatment. Fluorescently labeled siRNA enabled visualization of uptake and distribution within the spheroid, revealing somewhat reduced siRNA accumulation in pericentral CYP3A4+ hepatocytes accompanied with somewhat reduced ASGR1 expression. No signs of hepatotoxicity were observed under the conditions used. By varying the number of phosphorothioate modifications in the siRNA backbone, distinct differences in silencing efficiency and durability were observed which were principally similar as obtained in vivo in mice. We propose that this long‐term human liver spheroid model provides a valuable preclinical platform for evaluating siRNA‐based therapeutics with respect to uptake, durability, and silencing efficiency, and could refine early in vitro screening and accelerate drug development. Study Highlights What is the current knowledge on the topic? ○Chemical modifications to the siRNA backbone are essential for enhancing molecular stability, reducing immunogenicity, increasing resistance to enzymatic degradation, and minimizing toxicity. ○Such modifications are pivotal for optimizing siRNA performance in therapeutic settings. ○However, despite growing interest in siRNA‐based therapeutics, no long‐term in vitro model exists to evaluate silencing efficiency and durability. ○As a result, preclinical development still relies heavily on humanized mouse models. What question did this study address? ○Can the human liver spheroids be used as an in vitro model to evaluate the effect of backbone modifications of the GalNAc‐conjugated siRNA on the silencing efficiency and durability? What does this study add to our knowledge? ○We present a new human spheroid model for assignment of the properties of structurally different GalNAc‐conjugated siRNA molecules. ○The spheroids were maintained in culture for up to 5 weeks while retaining expression of the asialoglycoprotein receptor. ○These spheroids can be directly treated with GalNAc‐conjugated siRNA without the need for transfection reagents, enabling sustained target gene silencing to be monitored over the entire five‐week period. ○Using fluorescently labeled siRNA, intracellular trafficking was visualized, revealing heterogeneity in hepatocyte uptake capacity. ○Moreover, the model enabled clear discrimination of silencing performance across five distinct siRNA backbones. How might this change clinical pharmacology or translational science? ○This study shows that human liver spheroids provide a robust, scalable platform for evaluating GalNAc‐conjugated siRNA therapeutics. ○The model offers a valuable tool for early‐stage compound selection, potentially accelerating screening workflows and reducing reliance on animal models in preclinical development.

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) regulate gene expression in a sequence-specific manner. Genes with partial complementarity to siRNA/miRNA sequences in their 3′-untranslated regions (UTRs) are suppressed by a mechanism referred to as the siRNA off-target effect or miRNA-mediated RNA silencing. However, the determinants of such RNA silencing efficiency are poorly understood. Previously, I and co-workers reported that the efficiency of RNA silencing is strongly correlated with the thermodynamic stability of base pairing in the duplex formed within an siRNA/miRNA and between the seed region and its target mRNA. In this review, I first summarize our previous studies that identified the thermodynamic parameter to estimate the silencing efficiency using the calculated base pairing stability: siRNAs downregulate the expression of off-target genes depending on the stability of binding between the siRNA seed region (nucleotides 2–8) and off-target mRNAs, and miRNAs downregulate target mRNA expression depending on the stability of the duplex formed between the 5′ terminus of the miRNA and its target mRNA. I further discuss the possibility that such thermodynamic features of silencing efficiency may have arisen during evolution with increasing body temperature in various organisms.

Background A challenge in gene therapy is the efficient delivery of DNA/siRNA to the diseased cells. The physicochemical characteristics of siRNA, such as high molecular weight, negative charges and hydrophilic nature—prevent passive diffusion across the plasma membrane for most cells. A therapeutically feasible carrier for intra-cellular delivery of gene materials should accomplish a series of tasks such as: condensing nucleic acid, protecting nucleic acid from leaking in vivo , facilitating endosome escape and releasing DNA/siRNA to the target site. To meet these requirements, an efficient gene vector based on polycation synthesis for siRNA delivery both in vitro and in vivo was developed. Results The polymer was synthesized by 1, 4-butanediol bis (chloroformate) and PEI 800 Da to form PEI-Bu which could condense siRNA at the N/P ratio of 38.35 or above. The size of the nanoparticles was 100–300 nm and zeta potential was in the range of 10–30 mV at different N/P ratios. The nanoparticles can achieve the ability of cellular uptake and the silencing efficiency was about 46.63% in SMMC-7721 cell line which was generated to stably express GL3 luciferase gene. The cytotoxicity of the polyplex nanoparticles was almost negligible on SMMC-7721 cells by MTT assay, indicating that the reduced luciferase expression was the effect of RNAi, not the influence of cytotoxicity of polyplexes. The polyplex nanoparticle formulated by PEI-Bu and siRNA at N/P ratio of 115.05 was injected into the SMMC-7721 tumor bearing mice locally and the expression of luciferase can reduce to 63.17% compared with control group. Conclusions Results in this study suggested that PEI-Bu polycation might provide a promising solution for siRNA delivery and had the potential in anti-tumor gene therapy.

Small interfering RNAs (siRNAs) are a powerful tool for specific suppression of protein synthesis in the cell, and this determines the attractiveness of siRNAs as a drug. Low resistance of siRNA to nucleases and inability to enter into target cells are the most crucial issues in developing siRNA-based therapy. To face this challenge, we designed multilayer nanoconstruct (MLNC) with AuNP core bearing chemically modified siRNAs. We applied chemical modifications 2′-OMe and 2′-F substitutions as well as their combinations with phosphoryl guanidine group in the internucleotide phosphate. The effect of modification on the efficiency of siRNA loading into nanocarriers was examined. The introduction of the internucleotide modifications into at least one of the strands raised the efficiency of siRNA adsorption on the surface of gold core. We also tested the stability of modified siRNA adsorbed on gold core in the presence of serum. Based on loading efficiency and stability, MLNCs with the most siRNA effective cargo were selected, and they showed an increase in biological activity compared to control MLNCs. Our study demonstrated the effect of chemical modifications of siRNA on its binding to the AuNP-based carrier, which directly affects the efficiency of target protein expression inhibition.

MicroRNAs are small, non-coding RNAs that inhibit gene expression posttranscriptionally through interaction with the 3′ untranslated region (3′ UTR) of target mRNAs. The most important factor for downregulation of target genes by miRNA is the “seed region,” which encompasses nucleotides 2–7 at the 5′ end of the miRNA. In this study, sequence determinants for efficient downregulation of target genes were investigated by employing growth-suppressive miR-608 and miR-4651, which shares the seed sequence with miR-608. Cell growth experiments revealed that miR-4651 and miR-608-scram in which the seed sequences were mutated did not inhibit the growth of A549 cells in contrast to wild-type miR-608, which significantly inhibited cell growth. When similarity to miR-608 was increased by replacing sequences of miR-4651 with that of miR-608, cell growth was gradually more inhibited. Similar results were obtained from a luciferase reporter assay using a reporter plasmid containing the 3′ UTR of BCL2L1 and from western blot analysis of BCL2L1, CCND3, and phosphoinositide 3-kinase regulatory subunit 2. Moreover, microarray analyses revealed that overexpression of miR-4651 and miR-608-scram resulted in inefficient downregulation of target genes, and the number of downregulated genes was increased when transfected with MT-3 mimic, which differs from miR-608 by four nucleotides located in the central region. Together, our findings provide a basis for understanding the mechanism underlying target recognition and/or downregulation of target genes by miR-608 and indicate that in addition to seed sequence, central and 3′ parts of miR-608 play an important role in mediating efficient downregulation of target genes.

In 2014, we performed a nationwide survey in Korean radish fields to investigate the distribution and variability of Turnip mosaic virus (TuMV). Brassica rapa ssp. pekinensis sap-inoculated with three isolates of TuMV from infected radish tissue showed different symptom severities, whereas symptoms in Raphanus sativus were similar for each isolate. The helper component-protease (HC-Pro) genes of each isolate were sequenced, and phylogenetic analysis showed that the three Korean isolates were clustered into the basal-BR group. The HC-Pro proteins of these isolates were tested for their RNA silencing suppressor (VSR) activity and subcellular localization in Nicotiana benthamiana . A VSR assay by co-agroinfiltration of HC-Pro with soluble-modified GFP (smGFP) showed that HC-Pro of isolate R007 and R041 showed stronger VSR activity than R065. The HC-Pros showed 98.25 % amino acid identity, and weak VSR isolate (R065) has a single variant residue in the C-terminal domain associated with protease activity and self-interaction compared to isolates with strong VSR activity. Formation of large subcellular aggregates of GFP:HC-Pro fusion proteins in N. benthamiana was only observed for HC-Pro from isolates with strong VSR activity, suggesting that R065 ‘weak’ HC-Pro may have diminished self-association; substitution of the variant C-terminal residue largely reversed the HC-Pro aggregation and silencing suppressor characteristics. The lack of correlation between VSR efficiency and induction of systemic necrosis (SN) suggests that differences in viral accumulation due to HC-Pro are not responsible for SN.

Variable antigens are large proteins located on the outer membrane of parasitic but also of free-living protists. Multigene families encoding surface antigens demonstrate an exclusive expression of proteins. The resulting presence of just one protein species on the cell surface is required for surface antigen function; therefore, the molecular mechanism of exclusive expression is of main interest. Regulation of gene expression and mechanisms establishing switching of antigens are hardly understood in any organism. Here we report on the reaction of Paramecium to the artificial knock down of surface antigen 51A expression by bacteria-mediated RNAi. This technique involves the feeding of dsRNA-producing bacteria. We analyzed different fragments of the target gene for dsRNA template regarding their specific knock down efficiency and found great differences. Treatment of Paramecia with RNAi against the 51A antigen demonstrated that although a massive amount of mRNA was present, the protein was not detected on the cell surface. Moreover, a minor abundance of 51D transcripts resulted in an exclusive presence of 51D proteins on the cell surface. This posttranscriptional regulation was confirmed by the transcript ratio (51A/51D) determined by real-time (RT) PCR of single cells. Because we were able to document unexclusive transcription also in wild-type cells our results indicate that this posttranscriptional regulation is a main factor of enabling exclusive gene expression. The comparison of serotype shifts, caused by efficient and inefficient knock down, indicates an involvement of full-length transcripts in regulation of gene expression. Thus, our study gives new insights into the mechanism of exclusive expression on the molecular level: (i) exclusive transcription does not occur, (ii) posttranscriptional regulation is a powerful factor enabling exclusive antigen expression, and (iii) surface antigen mRNA is shown to be involved in this mechanism in a regulating way.

Exploiting the RNA interference (RNAi) gene mechanism to silence essential genes in pest insects, leading to toxic effects, has surfaced as a promising new control strategy in the past decade. While the first commercial RNAi-based products are currently coming to market, the application against a wide range of insect species is still hindered by a number of challenges. In this review, we discuss the current status of these RNAi-based products and the different delivery strategies by which insects can be targeted by the RNAi-triggering double-stranded RNA (dsRNA) molecules. Furthermore, this review also addresses a number of physiological and cellular barriers, which can lead to decreased RNAi efficacy in insects. Finally, novel non-transgenic delivery technologies, such as polymer or liposomic nanoparticles, peptide-based delivery vehicles and viral-like particles, are also discussed, as these could overcome these barriers and lead to effective RNAi-based pest control.

Summary Recent discoveries show that fungi can take up environmental RNA, which can then silence fungal genes through environmental RNA interference. This discovery prompted the development of Spray‐Induced Gene Silencing (SIGS) for plant disease management. In this study, we aimed to determine the efficacy of SIGS across a variety of eukaryotic microbes. We first examined the efficiency of RNA uptake in multiple pathogenic and non‐pathogenic fungi, and an oomycete pathogen. We observed efficient double‐stranded RNA (dsRNA) uptake in the fungal plant pathogens Botrytis cinerea, Sclerotinia sclerotiorum, Rhizoctonia solani, Aspergillus niger and Verticillium dahliae, but no uptake in Colletotrichum gloeosporioides, and weak uptake in a beneficial fungus, Trichoderma virens. For the oomycete plant pathogen, Phytophthora infestans, RNA uptake was limited and varied across different cell types and developmental stages. Topical application of dsRNA targeting virulence‐related genes in pathogens with high RNA uptake efficiency significantly inhibited plant disease symptoms, whereas the application of dsRNA in pathogens with low RNA uptake efficiency did not suppress infection. Our results have revealed that dsRNA uptake efficiencies vary across eukaryotic microbe species and cell types. The success of SIGS for plant disease management can largely be determined by the pathogen’s RNA uptake efficiency.