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Colorectal cancer (CRC) involves various genetic alterations, with liver metastasis posing a significant clinical challenge. Furthermore, CRC cells mostly show an increase in resistance to traditional treatments like chemotherapy. It is essential to investigate more advanced and effective therapies to prevent medication resistance and metastases and extend patient life. As a result, it is anticipated that small interfering RNAs (siRNAs) would be exceptional instruments that can control gene expression by RNA interference (RNAi). In eukaryotes, RNAi is a biological mechanism that destroys specific messenger RNA (mRNA) molecules, thereby inhibiting gene expression. In the management of CRC, this method of treatment represents a potential therapeutic agent. However, it is important to acknowledge that siRNA therapies have significant issues, such as low serum stability and nonspecific absorption into biological systems. Delivery mechanisms are thus being created to address these issues. In the current work, we address the potential benefits of siRNA therapy and outline the difficulties in treating CRCby focusing on the primary signaling pathways linked to metastasis as well as genes implicated in the multi-drug resistance (MDR) process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Anxiety and depression are central nervous system illnesses that are among the most prevalent medical concerns of the twenty-first century. Patients with this condition and their families bear psychological, financial, and societal hardship. There are currently restrictions when utilizing the conventional course of treatment. RNA interference is expected to become an essential approach in anxiety and depression due to its potent and targeted gene silencing. Silencing of genes by post-transcriptional modification is the mechanism of action of small interfering RNA (siRNA). The suppression of genes linked to disease is typically accomplished by siRNA molecules in an efficient and targeted manner. Unfavourable immune responses, off-target effects, naked siRNA instability, nuclease vulnerability, and the requirement to create an appropriate delivery method are some of the challenges facing the clinical application of siRNA. This review focuses on the use of siRNA in the treatment of anxiety and depression., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Traumatic brain injury (TBI) activates the NF-κB pathway in microglia and astrocytes, which secrete pro-inflammatory cytokines that disrupt the blood-brain barrier (BBB). Curdlan derivatives are promising carriers for the delivery of siRNA drugs. Herein, we evaluated the glial cell specificity, siRNA delivery efficiency, and the subsequent phenotypic regulation of glial cells by the Curdlan derivatives in the TBI mouse model. Our in vitro and in vivo studies confirmed that the (1) pAVC4 or CuMAN polymer encapsulating siRNA were internalized by astrocytes and microglia in a receptor-dependent manner; (2) systemic administration of the pAVC4 or CuMAN polymer encapsulating siRNA resulted in significant gene silencing efficiency, altered the phenotypic polarization of glial cells, and regulated the secretion of inflammatory cytokines; (3) this lessened neuroinflammation, ameliorated BBB destruction, and improved vascular recovery. These data suggested that pAVC4 and CuMAN polymers are promising RNA delivery vehicles that can efficiently deliver siRNA to the target cells.

In this study, we report the synthesis of 2'-formamidonucleoside phosphoramidite derivatives and their incorporation into siRNA strands to reduce seed-based off-target effects of small interfering RNAs (siRNAs). Formamido derivatives of all four nucleosides (A, G, C and U) were synthesized in 5-11 steps from commercial compounds. Introducing these derivatives into double-stranded RNA slightly reduced its thermodynamic stability, but X-ray crystallography and CD spectrum analysis confirmed that the RNA maintained its natural A-form structure. Although the introduction of the 2'-formamidonucleoside derivative at the 2nd position in the guide strand of the siRNA led to a slight decrease in the on-target RNAi activity, the siRNAs with different sequences incorporating 2'-formamidonucleoside with four kinds of nucleobases into any position other than 2nd position in the seed region revealed a significant suppression of off-target activity while maintaining on-target RNAi activity. This indicates that 2'-formamidonucleosides represent a promising approach for mitigating off-target effects in siRNA therapeutics., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Recognition of viral infection often relies on the detection of double-stranded RNA (dsRNA), a process that is conserved in many different organisms. In mammals, proteins such as MDA5, RIG-I, OAS, and PKR detect viral dsRNA, but struggle to differentiate between viral and endogenous dsRNA. This study investigates an shRNA targeting DDX54's potential to activate PKR, a key player in the immune response to dsRNA. Knockdown of DDX54 by a specific shRNA induced robust PKR activation in human cells, even when DDX54 is overexpressed, suggesting an off-target mechanism. Activation of PKR by the shRNA was enhanced by knockdown of ADAR1, a dsRNA binding protein that suppresses PKR activation, indicating a dsRNA-mediated mechanism. In vitro assays confirmed direct PKR activation by the shRNA. These findings emphasize the need for rigorous controls and alternative methods to validate gene function and minimize unintended immune pathway activation., (© 2024. The Author(s).)

Temporomandibular joint osteoarthritis (TMJOA) is the most common and severe subtype of temporomandibular disease characterized by inflammation and cartilage matrix degradation. Compared with traditional conservative treatment, small interfering RNAs (siRNAs) have emerged as a more efficient gene-targeted therapeutic tool for TMJOA treatment. Nuclear factor kappaB (NF-κB) is a transcription factor orchestrating the inflammatory processes in the pathogenesis of TMJOA. Employing siRNA-NF-κB could theoretically control the development of TMJOA. However, the clinical applications of siRNA-NF-κB are limited by its structural instability, poor cellular uptake, and short TMJ retention. To overcome these shortcomings, we developed a tetrahedral framework nucleic acid (tFNA) system carrying siRNA-NF-κB, named Tsi. The results indicated that Tsi exhibited excellent structural stability and excellent cellular uptake efficiency. It also demonstrated a superior NF-κB silencing effect over siRNA alone, attenuating the activation of NF-κB and upregulating the NRF2/HO-1 pathway. This system effectively reduced the release of inflammatory factors and reactive oxygen species (ROS), inhibiting cellular oxidative stress and apoptosis. In vivo , Tsi displayed enhanced TMJ retention capacity in comparison to siRNA alone and offered significant protective effects on both the cartilage matrix and subchondral bone, presenting a promising approach for TMJOA treatment.

In this study, we developed an oleoyl-siRNA conjugate in which oleic acid was conjugated at the 5'-end of the sense strand of the siRNA. Furthermore, we examined the effects of RNAi in a mouse model of pancreatic cancer with liver metastasis. The mouse model of pancreatic cancer with liver metastasis was developed by implanting Sui67Luc human pancreatic cancer cells into the portal veins of mice. Sui67Luc cells have high expression of tumor-related genes such as β -catenin , vascular endothelial growth factor , and programmed cell death ligand-1 . All genes were knocked down using siRNA, among which siRNA targeting β -catenin exhibited the most suitable RNAi effect. Therefore, we investigated the in vitro RNAi effect of oleoyl-siRNA (Ole-siRNA) targeting the β -catenin gene in Sui67Luc cells and found that it was stronger than that of unmodified siRNA. For in vivo experiments, we investigated the biodistribution, antitumor effect, and change in life expectancy of mice upon systemic administration of Ole-siRNA complexed with Invivofectamine 3.0 (IVF). In terms of biodistribution, the Ole-siRNA/IVF complex likely accumulates in the liver of mice. The antitumor effect of Ole-siRNA in a portal vein infusion liver-metastatic Sui67Luc tumor mouse model was evaluated using an in vivo imaging system. Ole-siRNA had a significant antitumor effect compared with nonmodified siRNA. In addition, mice with metastatic liver Sui67Luc tumors treated with Ole-siRNA showed increased survival. These results suggest that Ole-siRNAs are useful novel RNAi molecules for treating pancreatic cancer and liver metastasis.

Motivation: RNA interference (RNAi) has become a widely used experimental approach for post-transcriptional regulation and is increasingly showing its potential as future targeted drugs. However, the prediction of highly efficient siRNAs (small interfering RNAs) is still hindered by dataset biases, the inadequacy of prediction methods, and the presence of off-target effects. To overcome these limitations, we propose an accurate and robust prediction method, OligoFormer, for siRNA design., Results: OligoFormer comprises three different modules including thermodynamic calculation, RNA-FM module, and Oligo encoder. Oligo encoder is the core module based on the transformer encoder. Taking siRNA and mRNA sequences as input, OligoFormer can obtain thermodynamic parameters, RNA-FM embedding, and Oligo embedding through these three modules, respectively. We carefully benchmarked OligoFormer against six comparable methods on siRNA efficacy datasets. OligoFormer outperforms all the other methods, with an average improvement of 9% in AUC, 6.6% in PRC, 9.8% in F1 score, and 5.1% in PCC compared to the best method among them in our inter-dataset validation. We also provide a comprehensive pipeline with prediction of siRNA efficacy and off-target effects using PITA score and TargetScan score. The ablation study shows RNA-FM module and thermodynamic parameters improved the performance and accelerated convergence of OligoFormer. The saliency maps by gradient backpropagation and base preference maps show certain base preferences in initial and terminal region of siRNAs., Availability and Implementation: The source code of OligoFormer is freely available on GitHub at: https://github.com/lulab/OligoFormer. Docker image of OligoFormer is freely available on the docker hub at https://hub.docker.com/r/yilanbai/oligoformer., (© The Author(s) 2024. Published by Oxford University Press.)

Non-melanoma skin cancer (NMSC) is one of the most prevalent cancers, leading to significant mortality rates due to limited treatment options and a lack of effective therapeutics. Janus kinase (JAK1), a non-receptor tyrosine kinase family member, is involved in various cellular processes, including differentiation, cell proliferation and survival, playing a crucial role in cancer progression. This study aims to provide a more effective treatment for NMSC by concurrently silencing the JAK1 gene and administering 5-Fluorouracil (5-FU) using liposome nanocomplexes as delivery vehicles. Utilizing RNA interference (RNAi) technology, liposome nanocomplexes modified with polyethylene imine (PEI) were conjugated with siRNA molecule targeting JAK1 and loaded with 5-FU. The prepared formulations (NL-PEI) were characterized in terms of their physicochemical properties, morphology, encapsulation efficiency, in vitro drug release, and stability. Cell cytotoxicity, cell uptake and knockdown efficiency were evaluated in human-derived non-melanoma epidermoid carcinoma cells (A-431). High contrast transmission electron microscopy (CTEM) images and dynamic light scattering (DLS) measurements revealed that the nanocomplexes formed spherical morphology with uniform sizes ranging from 80-120 nm. The cationic NL-PEI nanocomplexes successfully internalized within the cytoplasm of A-431, delivering siRNA for specific sequence binding and JAK1 gene silencing. The encapsulation of 5-FU in the nanocomplexes was achieved at 0.2 drug/lipid ratio. Post-treatment with NL-PEI for 24, 48 and 72 h showed cell viability above 80 % at concentrations up to 8.5 × 10 1 µg/mL. Notably, 5-FU delivery via nanoliposome formulations significantly reduced cell viability at 5-FU concentration of 5 µM and above (p < 0.05) after 24 h of incubation. The NL-PEI nanocomplexes effectively silenced the JAK1 gene in vitro, reducing its expression by 50 %. Correspondingly, JAK1 protein level decreased after transfection with JAK1 siRNA-conjugated liposome nanocomplexes, leading to a 37 % reduction in pERK (phosphor extracellular signal-regulated kinase) protein expression. These findings suggest that the combined delivery of JAK1 siRNA and 5-FU via liposomal formulations offers a promising and novel treatment strategy for targeting genes and other identified targets in NMSC therapy., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Small interfering RNA (siRNA) has emerged as a valuable tool to address RNA interference (RNAi) to modulate gene expression also in therapy. However, challenges such as inefficient cell targeting and rapid degradation in biological systems have limited its success. To address these issues, the development of a receptor-specific shuttle system represents a promising solution. [F 7 ,P 34 ]-NPY analogues were modified by solid-phase peptide synthesis, enabling non-covalent conjugation with siRNA. This modification yielded an efficient siRNA vehicle capable of binding and transporting its cargo into target cells without adversely affecting receptor activation or cell viability. Mass spectrometry and gel shift assays confirmed successful and stable siRNA binding under various conditions. Microscopy experiments further demonstrated the co-internalization of labeled peptides and siRNA in Hepa1c1 cells, highlighting the stability of the complex. In vitro quantitative RT-PCR experiments, targeting the TSC22D4 gene to normalize systemic glucose homeostasis and insulin resistance, revealed a functional peptide-based siRNA shuttle system with the ability to decrease mRNA expression to approximately 40%. These findings strengthen the potential of receptor-specific siRNA shuttle systems as efficient tools for gene therapy that offer a possibility for reducing side effects., (© 2024 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

VEGF-A or vascular endothelial growth factor-A is an important factor in enabling neovascularization and angiogenesis. VEGF-A is regulated transcriptionally as well as post transcriptionally. Human antigen R (HuR) belonging to the embryonic lethal abnormal vision (ELAV) family is a key regulator promoting stabilization of VEGF-A mRNA. In this research we investigate, whether HuR targeted RNA interference would enable the reduction of the VEGF-A protein in human retinal pigment epithelial cells (ARPE-19) in-vitro, in normoxic conditions. Three siRNA molecules with sequences complementary to three regions of the HuR mRNA were designed. The three designed siRNA molecules were individually transfected in ARPE-19 cells using Lipofectamine™2000 reagent. Post-transfection (24 h, 48 h, 72 h), downregulation of HuR mRNA was estimated by real-time polymerase reaction, while HuR protein and VEGF-A protein levels were semi-quantitatively determined by western blotting techniques. VEGF-A protein levels were additionally quantified using ELISA techniques. All experiments were done in triplicate. The designed siRNA could successfully downregulate HuR mRNA with concomitant decreases in HuR and VEGF-A protein. The study reveals that HuR downregulation can prominently downregulate VEGF-A, making the protein a target for therapy against pathological angiogenesis conditions such as diabetic retinopathy., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Treatments for cancer that incorporate small interfering RNA (siRNA) to target iron-dependent ferroptosis are thought to be highly promising. However, creating a reliable and clinically feasible siRNA delivery system continues to be a major obstacle in the field of cancer treatment. Here, three imidazole-based ionizable lipid nanoparticles (LNPs) with pH-sensitive effects are rationally designed and synthesized for siRNA delivery. LNPs formulated with the top-performing lipid (O12-D3-I3) encapsulating FVII siRNA (FVII@O-LNP) elicited greater gene silencing than those with the benchmark Onpattro lipid DLin-MC3-DMA (MC3) due to its stronger endosomal escape. Moreover, Fc-siRNA@O-LNPs encapsulated with ferrocene (Fc) and SLC7A11/Nrf2-targeted siRNA is formulated. The outcomes demonstrate optimal safety profiles and a significant anti-tumor effect by inducing long-lasting and efficient ferroptosis through a synergistic action in vivo. In summary, this work shows that imidazolyl lipid-prepared LNPs are efficient delivery vehicles for cancer therapy and ferroptosis-targeting siRNA administration, both of which have extensive clinical application potential., (© 2024 Wiley‐VCH GmbH.)

Small interfering RNA (siRNA) holds promise as a therapeutic approach for various diseases, yet challenges persist in achieving efficient delivery, biodistribution, and minimizing off-target effects. Lipidic nanoformulations are being developed to address these hurdles, but the optimal dose for preclinical investigations remains unclear. This systematic review and meta-analysis aims to determine the optimal dose of nanoformulated siRNA and explore factors influencing dose and biodistribution, informing future research in this field. A comprehensive search across four electronic databases identified 25 potential studies, with 15 selected for meta-analysis after screening. Quality assessment was conducted using SYRCLE's risk of bias tool modified for animal studies based on research question. Study found an average siRNA dose of 1.513 ± 0.377 mg/kg with mean downregulation of 65.79 % achieved, with siRNA-LNPs mainly accumulating in the liver. While individual factors showed no significant correlation, a positive association between dose and downregulation was observed, alongside other influencing factors. Extrapolating intravenous doses to potential oral doses, we suggest an initial oral dose range of 1.5 to 8 mg/kg, considering siRNA-LNPs bioavailability. These findings contribute to advancing RNA interference research and encourage further exploration of siRNA-based treatments in personalized medicine., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

RNA interference (RNAi) is a process in which small RNA molecules (such as small interfering RNAs or siRNAs) bind to specific messenger RNAs (mRNAs), leading to its degradation and inhibition of protein synthesis. Our studies have shown that RNAi can effectively silence genes involved in the replication of the Chikungunya virus (CHIKV) in cells. However, these investigations were performed only in laboratory settings and have yet to be tested in human clinical trials. Researchers need to conduct more research to determine the safety and efficacy of RNAi-based therapies as a therapeutic agent to treat viral infections. In this review, the history of evolution of siRNA as an inhibitor of protein synthesis, along with its current developments, is discussed based on our experience. Moreover, this review examines the hurdles and future implications associated with siRNA based therapeutic approaches.

Small RNAs (sRNAs) are essential for normal plant development and range in size classes of 21-24 nucleotides. The 22nt small interfering RNAs (siRNAs) and miRNAs are processed by Dicer-like 2 (DCL2) and DCL1 respectively and can initiate secondary siRNA production from the target transcript. 22nt siRNAs are under-represented due to competition between DCL2 and DCL4, while only a small number of 22nt miRNAs exist. Here we produce abundant 22nt siRNAs and other siRNA size classes using long hairpin RNA (hpRNA) transgenes. By introducing asymmetric bulges into the antisense strand of hpRNA, we shifted the dominant siRNA size class from 21nt of the traditional hpRNA to 22, 23 and 24nt of the asymmetric hpRNAs. The asymmetric hpRNAs effectively silenced a β-glucuronidase (GUS) reporter transgene and the endogenous ethylene insensitive-2 (EIN2) and chalcone synthase (CHS) genes. Furthermore, plants containing the asymmetric hpRNA transgenes showed increased amounts of 21nt siRNAs downstream of the hpRNA target site compared to plants with the traditional hpRNA transgenes. This indicates that these asymmetric hpRNAs are more effective at inducing secondary siRNA production to amplify silencing signals. The 22nt asymmetric hpRNA constructs enhanced virus resistance in plants compared to the traditional hpRNA constructs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Coordination of gene regulatory networks is necessary for proper execution of cellular programs throughout development. RNA interference (RNAi) is an essential regulatory mechanism in all metazoans. Proper RNAi-mediated gene regulation requires coordination of several RNAi branches to ensure homeostasis. For example, in Caenorhabditis elegans, the Argonautes, ALG-3 and ALG-4, are expressed specifically during spermatogenesis (L4 stage) and bind small interfering RNAs (siRNAs) complementary to sperm-enriched genes. We find that alg-3 and alg-4 are regulated by siRNAs. Our work shows that gene switches are operated via these siRNAs to regulate the Argonautes' expression in a temporal manner. This RNAi-to-RNAi regulatory cascade is essential for coordinating ALG-3/4 pathway function, particularly during heat stress, to provide thermotolerant sperm-based fertility. This work provides insight into one regulatory motif used to maintain RNAi homeostasis, across developmental stages, despite environmental stressors. As RNAi pathways are evolutionarily conserved, other species likely use similar regulatory architectures to maintain RNAi homeostasis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Competing Interests: Conflict of interest: Professor Webb’s employer, the University of Edinburgh, has been paid by Alnylam for his contributions to the zilebesiran programme in hypertension. He has undertaken recent consultancy roles with Idorsia, Janssen, and Silence pharmaceuticals in the field of cardiovascular research.

Cell culture has long been essential for preclinical modeling of human development and disease. However, conventional two-dimensional (2D) cell culture fails to faithfully model the complexity found in vivo, and novel drug candidates that show promising results in 2D models often do not translate to the clinic. More recently, three-dimensional (3D) cell culture models have gained popularity owing to their greater physiological relevance to in vivo biology. In particular, 3D spheroid models are becoming widely used due to their ability to mimic solid tumors, both in architecture and gradation of nutrients distributed from the outer, proliferative layers into the inner, quiescent layers of cells. Similar to in vivo tumors, cell lines grown in 3D spheroid models tend to be more resistant to antitumor drug treatments than their 2D cultured counterparts, though distinct signaling pathways and gene targets conferring this resistance have yet to be fully explored. RNA interference (RNAi) is an effective tool to elucidate gene function and discover novel druggable targets in 2D models; however, only a few studies have successfully performed RNAi in complex 3D models to date. Here, we demonstrate efficient RNAi-mediated knockdown using "transfection-free" Dharmacon Accell siRNAs in three spheroid culture models, in the presence or absence of the extracellular matrix. This methodology has the potential to be scaled up for complex arrayed screening experiments, which may aid in the identification of novel druggable targets with greater clinical relevance than those identified in 2D experiments. © 2024 Dharmacon, Inc. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of 3D spheroids in matrix-free ULA plates Alternate Protocol 1: Generation of Matrigel matrix-embedded 3D spheroids Alternate Protocol 2: Generation of GrowDex hydrogel-embedded 3D spheroids Basic Protocol 2: Delivery of siRNA and collection of matrix-free 3D spheroids Alternate Protocol 3: Delivery of siRNA and collection of matrix-embedded spheroids Basic Protocol 3: RNA and protein extraction from spheroids for characterization of gene knockdown., (© 2024 Dharmacon, Inc. Current Protocols published by Wiley Periodicals LLC.)

Even though RNA treatments were first proposed as a way to change aberrant signaling in cancer, research in this field is currently ongoing. The term "RNAi" refers to the use of several RNAi technologies, including ribozymes, riboswitches, Aptamers, small interfering RNA (siRNA), antisense oligonucleotides (ASOs), and CRISPR/Cas9 technology. The siRNA therapy has already achieved a remarkable feat by revolutionizing the treatment arena of cancers. Unlike small molecules and antibodies, which need administration every three months or even every two years, RNAi may be given every quarter to attain therapeutic results. In order to overcome complex challenges, delivering siRNAs to the targeted tissues and cells effectively and safely and improving the effectiveness of siRNAs in terms of their action, stability, specificity, and potential adverse consequences are required. In this context, the three primary techniques of siRNA therapies for hepatocellular carcinoma (HCC) are accomplished for inhibiting angiogenesis, decreasing cell proliferation, and promoting apoptosis, are discussed in this review. We also deliberate targeting issues, immunogenic reactions to siRNA therapy, and the difficulties with their intrinsic chemistry and transportation., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Lentiviral vector (LVV) has been used as one of the common carriers for gene therapy in clinical trials. LVV-mediated clinical trials have being reported in successfully treating hundreds of β-thalassemia cases. These LVVs bear an inversely placed β-hemoglobin (HBB) gene expression cassette for preserving introns during the viral RNA packaging. Consequently, these LVVs often produce a small amount of negatively orientated transcript driven by its internal gene promoter and would lower the viral titer by the minus-strand complemented with the viral backbone. To overcome this problem, we designed shRNAs specifically target the minus-strand RNA driven by the LVV internal promoter that resulted in a notable increase in the viral titer. This report demonstrates a simple and positive mean for increasing the effectiveness for gene therapy with the LVV system., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Hepatitis E virus (HEV) poses a significant global health threat, with an estimated 20 million infections occurring annually. Despite being a self-limiting illness, in most cases, HEV infection can lead to severe outcomes, particularly in pregnant women and individuals with pre-existing liver disease. In the absence of specific antiviral treatments, the exploration of RNAi interference (RNAi) as a targeted strategy provides valuable insights for urgently needed therapeutic interventions against Hepatitis E. We designed small interfering RNAs (siRNAs) against HEV, which target the helicase domain and the open reading frame 3 (ORF3). These target regions will reduce the risk of viral escape through mutations, as they belong to the most conserved regions in the HEV genome. The siRNAs targeting the ORF3 efficiently inhibited viral replication in A549 cells after HEV infection. Importantly, the siRNA was also highly effective at inhibiting HEV in the persistently infected A549 cell line, which provides a suitable model for chronic infection in patients. Furthermore, we showed that a 5' triphosphate modification on the siRNA sense strand activates the RIG-I receptor, a cytoplasmic pattern recognition receptor that recognizes viral RNA. Upon activation, RIG-I triggers a signaling cascade, effectively suppressing HEV replication. This dual-action strategy, combining the activation of the adaptive immune response and the inherent RNAi pathway, inhibits HEV replication successfully and may lead to the development of new therapies.

Background: Olpasiran, a small interfering RNA (siRNA), blocks lipoprotein(a) (Lp(a)) production by preventing translation of apolipoprotein(a) mRNA. In phase 2, higher doses of olpasiran every 12 weeks (Q12W) reduced circulating Lp(a) by >95%., Objectives: This study sought to assess the timing of return of Lp(a) to baseline after discontinuation of olpasiran, as well as longer-term safety., Methods: OCEAN(a)-DOSE (Olpasiran Trials of Cardiovascular Events And LipoproteiN[a] Reduction-DOSE Finding Study) was a phase 2, dose-finding trial that enrolled 281 participants with atherosclerotic cardiovascular disease and Lp(a) >150 nmol/L to 1 of 4 active doses of olpasiran vs placebo (10 mg, 75 mg, 225 mg Q12W, or an exploratory dose of 225 mg Q24W given subcutaneously). The last dose of olpasiran was administered at week 36; after week 48, there was an extended off-treatment follow-up period for a minimum of 24 weeks., Results: A total of 276 (98.2%) participants entered the off-treatment follow-up period. The median study exposure (treatment combined with off-treatment phases) was 86 weeks (Q1-Q3: 79-99 weeks). For the 75 mg Q12W dose, the off-treatment placebo-adjusted mean percent change from baseline in Lp(a) was -76.2%, -53.0%, -44.0%, and -27.9% at 60, 72, 84, and 96 weeks, respectively (all P < 0.001). The respective off-treatment changes in Lp(a) for the 225 mg Q12W dose were -84.4%, -61.6%, -52.2%, and -36.4% (all P < 0.001). During the extension follow-up phase, no new safety concerns were identified., Conclusions: Olpasiran is a potent siRNA with prolonged effects on Lp(a) lowering. Participants receiving doses ≥75 mg Q12W sustained a ∼40% to 50% reduction in Lp(a) levels close to 1 year after the last dose. (Olpasiran Trials of Cardiovascular Events And LipoproteiN[a] Reduction-DOSE Finding Study [OCEAN(a)-DOSE]; NCT04270760)., Competing Interests: Funding Support and Author Disclosures The OCEAN(a)-DOSE study was funded by Amgen. Dr O’Donoghue has received grant funding through Brigham and Women’s Hospital from Novartis, Amgen, AstraZeneca, and Janssen; and has received honoraria from Novartis, Amgen, AstraZeneca, and Janssen. Dr Rosenson has received institutional grant funding from Amgen, Arrowhead, Lilly, Novartis, and Regeneron; has received consulting fees from Amgen, Arrowhead, CRISPER Therapeutics, Lilly, Novartis, Precision Biosciences, Regeneron, and Ultragenyx; has received nonpromotional speaker fees from Amgen and Kowa; and has held stock in MediMergent. Dr López is an employee of Amgen; and/or has owned Amgen stock or stock options. Dr Lepor has received grant support from Amgen, Novartis, Regeneron, New Amsterdam, Merck, Arrowhead, and AstraZeneca; and has received speaker and/or consulting fees from Amgen, Novartis, Regeneron, Merck, AstraZeneca, and Boehringer Ingelheim. Dr Baum has received consulting fees from Altimmune, Amgen, Axcella, Boehringer Ingelheim, Lilly, Esperion, Ionis, Madrigal, Merck, Novartis, and Regeneron; and has received speaker fees from Amgen, Lilly, and Regeneron. Dr Gaudet has received grants and personal fees from Amgen during the conduct of the study; has received grants from Acasti, Kowa, Uniqure, Sanofi, Aegerion, Applied Therapeutics, AstraZeneca, Boehringer Ingelheim, Ceapro, Dalcor, Esperion, Arrowhead, Regeneron, Akcea, Allergan, Amryt, Ionis, Novartis, Novo Nordisk, Pfizer, Lilly, and The Medicine Company, outside the submitted work; and has received personal fees from Arrowhead, Regeneron, Akcea, Allergan, Amryt, Ionis, Novartis, Novo Nordisk, Pfizer, Lilly, CRISPR Therapeutics, Saliogen, and Ceapro, outside the submitted work. Dr Knusel is an employee of Amgen; and/or has owned Amgen stock or stock options. Ms Kuder has served as a member of the TIMI Study Group, which has received institutional research grant support through Brigham and Women’s Hospital from Abbott, Amgen, Anthos Therapeutics, AstraZeneca, Bayer HealthCare Pharmaceuticals, Daiichi-Sankyo, Eisai, Intarcia, MedImmune, Merck, Novartis, Pfizer, Quark Pharmaceuticals, Regeneron, Roche, Siemens Healthcare Diagnostics, The Medicines Company, and Zora Biosciences. Ms Murphy has served as a member of the TIMI Study Group, which has received institutional research grant support through Brigham and Women’s Hospital from Abbott, Amgen, Anthos Therapeutics, AstraZeneca, Bayer HealthCare Pharmaceuticals, Daiichi-Sankyo, Eisai, Intarcia, MedImmune, Merck, Novartis, Pfizer, Quark Pharmaceuticals, Regeneron, Roche, Siemens Healthcare Diagnostics, The Medicines Company, and Zora Biosciences. Dr H. Wang is an employee of Amgen; and/or has owned Amgen stock or stock options. Dr Wu is an employee of Amgen; and/or has owned Amgen stock or stock options. Dr Shah is an employee of Amgen; and has owned Amgen stock. Dr J. Wang is an employee of Amgen; and has owned Amgen stock. Dr Wilmanski is an employee of Amgen; and has owned Amgen stock. Dr Sohn is an employee of Amgen; and/or has owned Amgen stock or stock options; and has existing or pending patents related to olpasiran. Dr Kassahun is an employee of Amgen; and/or has owned Amgen stock or stock options; and has existing or pending patents related to olpasiran. Dr Sabatine has received research grant support through Brigham and Women’s Hospital from Abbott, Amgen, Anthos Therapeutics, Inc, AstraZeneca, Boehringer Ingelheim, Daiichi-Sankyo, Ionis, Merck, Novartis, Pfizer, Saghmos Therapeutics, and Verve Therapeutics; has served as a consultant for Amgen, AMPEL BioSolutions, Anthos Therapeutics, Inc, AstraZeneca, Beren Therapeutics, Boehringer Ingelheim, Dr Reddy’s Laboratories, Fibrogen, Merck, Moderna, Novo Nordisk, Precision BioSciences, and Silence Therapeutics; and has served as a member of the TIMI Study Group, which has also received institutional research grant support through Brigham and Women’s Hospital from ARCA Biopharma, Janssen Research and Development, Siemens Healthcare Diagnostics, Softcell Medical Limited, Regeneron, Roche, and Zora Biosciences. Dr Stout has reported that he has no relationships relevant to the contents of this paper to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A series of successes in RNA interference (RNAi) therapies for liver diseases using lipid nanoparticles and N -acetylgalactosamine have heralded a current era of RNA therapeutics. However, alternative delivery strategies are required to take RNAi out of the comfort zone of hepatocytes. Here we report SIRPα IgV/anti-CD47 siRNA (vS-siCD47) conjugates that selectively and persistently disrupt the antiphagocytic CD47/SIRPα axis in solid tumors. Conjugation of the SIRPα IgV domain protein to siRNAs enables tumor dash through CD47-mediated erythrocyte piggyback, primarily blocking the physical interaction between CD47 on cancer cells and SIRPα on phagocytes. After internalization of the vS-siCD47 conjugates within cancer cells, the detached free-standing anti-CD47 siRNAs subsequently attack CD47 through the RNAi mechanism. The dual-action approach of the vS-siCD47 conjugate effectively overcomes the "don't eat me" barrier and stimulates phagocyte-mediated tumor destruction, demonstrating a highly selective and potent CD47-blocking immunotherapy. This delivery strategy, employing IgV domain protein-siRNA conjugates with a dual mode of target suppression, holds promise for expanding RNAi applications beyond hepatocytes and advancing RNAi-based cancer immunotherapies for solid tumors.

Rising global populations have amplified food scarcity and ushered in the development of genetically modified (GM) crops containing small interference RNAs (siRNAs) that control gene expression to overcome these challenges. The use of RNA interference (RNAi) in agriculture remains controversial due to uncertainty regarding the unintended release of genetic material and downstream nontarget effects, which have not been assessed in environmental bacteria to date. To evaluate the impacts of siRNAs used in agriculture on environmental bacteria, this study assessed microbial growth and viability as well as transcription activity with and without the presence of environmental stressors. Results showed a statistically significant reduction in growth capacity and maximum biomass achieved when bacteria are exposed to siRNAs alone and with additional external stress ( p < 0.05). Further transcriptomic analysis demonstrated that nutrient cycling gene activities were found to be consistently and significantly altered following siRNA exposure, particularly among carbon ( xylA, FBPase , limEH, Chitinase, rgl, rgh, rgaE, mannanase, ara ) and nitrogen ( ureC, nasA, narB, narG, nirK ) cycling genes ( p < 0.05). Decreases in carbon cycling gene transcription profiles were generally significantly enhanced when siRNA exposure was coupled with nutrient or antimicrobial stress. Collectively, findings suggest that certain conditions facilitate the uptake of siRNAs from their surrounding environments that can negatively affect bacterial growth and gene expression activity, with uncertain downstream impacts on ecosystem homeostasis.

In recent years, therapeutic siRNA projects are booming in the biotech and pharmaceutical industries. As these drugs act by silencing the target gene expression, a critical step is the binding of antisense strands of siRNA to RNA-induced silencing complex (RISC) and then degrading their target mRNA. However, data that we recently obtained suggest that double-stranded siRNA can also load to RISC. This brings a new understanding of the mechanism of RISC loading which may have a potential impact on how quantification of RISC loaded siRNA should be performed. By combining RNA immune precipitation and probe-based hybridization LC-fluorescence approach, we have developed a novel assay that can accurately quantify the RISC-bound antisense strand, irrespective of which form (double-stranded or single-stranded) is loaded on RISC. In addition, this novel assay can discriminate between the 5'-phosphorylated antisense (5'p-AS) and the nonphosphorylated forms, therefore specifically quantifying the RISC bound 5'p-AS. In comparison, stem-loop qPCR assay does not provide discrimination and accurate quantification when the oligonucleotide analyte exists as a mixture of double and single-stranded forms. Taking together, RISC loading assay with probe-hybridization LC-fluorescence technique would be a more accurate and specific quantitative approach for RISC-associated pharmacokinetic assessment.

Multidrug resistance (MDR) is a major obstacle limiting the effectiveness of chemotherapy against cancer. The combination strategy of chemotherapeutic agents and siRNA targeting drug efflux has emerged as an effective cancer treatment to overcome MDR. Herein, stimuli-responsive programmable tetrahedral DNA-RNA nanocages (TDRN) have been rationally designed and developed for dynamic co-delivery of the chemotherapeutic drug doxorubicin and P-glycoprotein (P-gp) siRNA. Specifically, the sense and antisense strand sequences of the P-gp siRNA, which are programmable bricks with terminal disulfide bond conjugation, are precisely embedded in one edge of the DNA tetrahedron. TDRN provides a stimuli-responsive release element for dynamic control of functional cargo P-gp siRNA that is significantly more stable than the "tail-like" TDN nanostructures. The stable and highly rigid 3D nanostructure of the siRNA-organized TDRN nanocages demonstrated a notable improvement in the stability of RNase A and mouse serum, as well as long-term storage stability for up to 4 weeks, as evidenced by this study. These biocompatible and multifunctional TDRN nanocarriers with gold nanocluster-assisted delivery (TDRN@Dox@AuNC p ) are successfully used to achieve synergistic RNAi/Chemo-therapy in vitro and in vivo. This programmable TDRN drug delivery system, which integrates RNAi therapy and chemotherapy, offers a promising approach for treating multidrug-resistant tumors., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

RNA interference (RNAi) has not been tested in the pandemic amphibian pathogen, Batrachochytrium dendrobatidis, but developing this technology could be useful to elucidate virulence mechanisms, identify therapeutic targets, and may present a novel antifungal treatment option for chytridiomycosis. To manipulate and decipher gene function, rationally designed small interfering RNA (siRNA) can initiate the destruction of homologous messenger RNA (mRNA), resulting in the "knockdown" of target gene expression. Here, we investigate whether siRNA can be used to manipulate gene expression in B. dendrobatidis via RNAi using differing siRNA strategies to target genes involved in glutathione and ornithine synthesis. To determine the extent and duration of mRNA knockdown, target mRNA levels were monitored for 24-48 h after delivery of siRNA targeting glutamate-cysteine ligase, with a maximum of ~56% reduction in target transcripts occurring at 36 h. A second siRNA design targeting glutamate-cysteine ligase also resulted in ~53% knockdown at this time point. siRNA directed toward a different gene target, ornithine decarboxylase, achieved 17% reduction in target transcripts. Although no phenotypic effects were observed, these results suggest that RNAi is possible in B. dendrobatidis, and that gene expression can be manipulated in this pathogen. We outline ideas for further optimization steps to increase knockdown efficiency to better harness RNAi techniques for control of B. dendrobatidis., (© 2024 The Author(s). Journal of Basic Microbiology published by Wiley‐VCH GmbH.)

Herpes simplex virus-1 (HSV-1) infection can cause various diseases and the current therapeutics have limited efficacy. Small interfering RNA (siRNA) therapeutics are a promising approach against infectious diseases by targeting the viral mRNAs directly. Recently, we employed a novel tRNA scaffold to produce recombinant siRNA agents with few natural posttranscriptional modifications. In this study, we aimed to develop a specific prodrug against HSV-1 infection based on siRNA therapeutics by bioengineering technology. We screened and found that UL8 of the HSV-1 genome was an ideal antiviral target based on RNAi. Next, we used a novel bio-engineering approach to manufacture recombinant UL8-siRNA (r/si-UL8) in Escherichia coli with high purity and activity. The r/si-UL8 was selectively processed to mature si-UL8 and significantly reduced the number of infectious virions in human cells. r/si-UL8 delivered by flexible nano-liposomes significantly decreased the viral load in the skin and improved the survival rate in the preventive mouse zosteriform model. Furthermore, r/si-UL8 also effectively inhibited HSV-1 infection in a 3D human epidermal skin model. Taken together, our results highlight that the novel siRNA bioengineering technology is a unique addition to the conventional approach for siRNA therapeutics and r/si-UL8 may be a promising prodrug for curing HSV-1 infection., Competing Interests: Declaration of competing interest The authors declare no competing interests related to the present study., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In order to investigate the effects of RNAi-mediated survivin and hypoxia-inducible factor 1α (HIF-1α) gene silencing on the proliferation and apoptosis of gastric cancer BGC-823 cells, small interfering RNAs (siRNAs) targeting survivin and HIF-1α mRNAs, respectively, as well as scrambled siRNAs (SCRs) were designed and synthesized, namely siRNA-survivin group, siRNA-HIF-1α group, and SCR group. The hypoxia-sensitive gastric cancer BGC-823 cells were identified and transfected in vitro with Hifectin II under hypoxic conditions, and the expression of survivin and HIF-1α was assessed by RT-PCR and Western blotting assays, respectively. The ability of apoptosis, proliferation, invasion, and migration was measured, and the results showed that HIF-1α expression was significantly increased in BGC-823 cells under hypoxic conditions, and survival-targeted siRNA transfection decreased the expression of survivin under hypoxic conditions, while co-transfection of survivin-targeted siRNA and HIF-1α-targeted siRNA down-regulated both survivin and HIF-1α expression. Compared with the blank control group, the co-transfected siRNA group exhibited distinct characteristics, with decreased invasion and migration ability, increased apoptosis, and significantly decreased cell proliferation under hypoxic conditions. It was confirmed that the downregulation of survivin and HIF-1α in BGC-823 cells may induce anticancer effects by enhancing apoptosis and decreasing proliferation, migration, and invasion ability. The novelty lies in the application of RNAi technology to silence the expression of both survivin and HIF-1α genes in gastric cancer BGC-823 cells by single and combined interference in an established gastric cancer cell model and observed the mechanism of its effect on the proliferation and apoptosis of gastric cancer cells. Concerning the success of this highly active antiretroviral therapy of gene disruption therapies, which is the first of its kind in the world, we wonder whether we can find other better gene targets for more kinds of tumor therapy., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

RNA interference (RNAi) is a powerful and rapidly developing technology that enables precise silencing of genes of interest. However, the clinical development of RNAi is hampered by the limited cellular uptake and stability of the transferred molecules. Electroporation (EP) is an efficient and versatile technique for the transfer of both RNA and DNA. Although the mechanism of electrotransfer of small nucleic acids has been studied previously, too little is known about the potential effects of significantly larger pDNA on this process. Here we present a fundamental study of the mechanism of electrotransfer of oligonucleotides and siRNA that occur independently and simultaneously with pDNA by employing confocal fluorescence microscopy. In contrast to the conditional understanding of the mechanism, we have shown that the electrotransfer of oligonucleotides and siRNA is driven by both electrophoretic forces and diffusion after EP, followed by subsequent entry into the nucleus within 5 min after treatment. The study also revealed that the efficiency of siRNA electrotransfer decreases in response to an increase in pDNA concentration. Overall, the study provides new insights into the mechanism of electrotransfer of small nucleic acids which may have broader implications for the future application of RNAi-based strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The increasing emergence and re-emergence of RNA virus outbreaks underlines the urgent need to develop effective antivirals. RNA interference (RNAi) is a sequence-specific gene silencing mechanism that is triggered by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), which exhibits significant promise for antiviral therapy. AGO2-dependent shRNA (agshRNA) generates a single-stranded guide RNA and presents significant advantages over traditional siRNA and shRNA. In this study, we applied a logistic regression algorithm to a previously published chemically siRNA efficacy dataset and built a machine learning-based model with high predictive power. Using this model, we designed siRNA sequences targeting diverse RNA viruses, including human enterovirus A71 (EV71), Zika virus (ZIKV), dengue virus 2 (DENV2), mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and transformed them into agshRNAs. We validated the performance of our agshRNA design by evaluating antiviral efficacies of agshRNAs in cells infected with different viruses. Using the agshRNA targeting EV71 as an example, we showed that the anti-EV71 effect of agshRNA was more potent compared with the corresponding siRNA and shRNA. Moreover, the antiviral effect of agshRNA is dependent on AGO2-processed guide RNA, which can load into the RNA-induced silencing complex (RISC). We also confirmed the antiviral effect of agshRNA in vivo. Together, this work develops a novel antiviral strategy that combines machine learning-based algorithm with agshRNA design to custom design antiviral agshRNAs with high efficiency., Competing Interests: Conflict of interest Professor Xi Zhou is an editorial board member for Virologica Sinica and were not involved in the editorial review or the decision to publish this article. The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

To date, the US Food and Drug Administration (FDA) has approved six small interfering RNA (siRNA) drugs: patisiran, givosiran, lumasiran, inclisiran, vutrisiran, and nedosiran, serving as compelling evidence of the promising potential of RNA interference (RNAi) therapeutics. The successful implementation of siRNA therapeutics is improved through a combination of various chemical modifications and diverse delivery approaches. The utilization of chemically modified siRNA at specific sites on either the sense strand (SS) or antisense strand (AS) has the potential to enhance resistance to ribozyme degradation, improve stability and specificity, and prolong the efficacy of drugs. Herein, we provide comprehensive analyses concerning the correlation between chemical modifications and structure-guided siRNA design. Various modifications, such as 2'-modifications, 2',4'-dual modifications, non-canonical sugar modifications, and phosphonate mimics, are crucial for the activity of siRNA. We also emphasize the essential strategies for enhancing overhang stability, improving RISC loading efficacy and strand selection, reducing off-target effects, and discussing the future of targeted delivery., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Inhibition of disease-causing mutations using RNA interference (RNAi) has resulted in clinically approved medicines with additional candidates in late stage trials. However, targetable tissues currently in preclinical development are limited to liver following systemic intravenous (IV) administration because predictable delivery of siRNA to non-liver tissues remains an unsolved challenge. Here, evidence of durable extrahepatic gene silencing enabled by siRNA Selective ORgan Targeting lipid nanoparticles (siRNA SORT LNPs) to the kidneys, lungs, and spleen is provided. LNPs excel at dose-dependent silencing of tissue-enriched endogenous targets resulting in 60%-80% maximal knockdown after a single IV injection and up to 88% downregulation of protein expression in mouse lungs after two doses. To examine knockdown potency and unbiased organ targeting, B6.129 TdTom/EGFP mice that constitutively express the TdTomato transgene across all cell types are utilized to demonstrate 58%, 45%, and 15% reduction in TdTomato fluorescence in lungs, spleen, and kidneys, respectively. Finally, physiological relevance of siRNA SORT LNP-mediated gene silencing is established via acute suppression of endogenous Tie2 which induces lung-specific phenotypic alteration of vascular endothelial barrier. Due to plethora of extrahepatic diseases that may benefit from RNAi interventions, it is anticipated that the findings will expand preclinical landscape of therapeutic targets beyond the liver., (© 2024 The Author(s). Advanced Materials published by Wiley‐VCH GmbH.)

Endogenous banana streak virus (eBSV) integrants derived from three distinct species, present in Musa balbisiana (B) but not Musa acuminata (A) banana genomes are able to reconstitute functional episomal viruses causing banana streak disease in interspecific triploid AAB banana hybrids but not in the diploid (BB) parent line, which harbours identical eBSV loci. Here, we investigated the regulation of these eBSV. In-depth characterization of siRNAs, transcripts and methylation derived from eBSV using Illumina and bisulfite sequencing were carried out on eBSV-free Musa acuminata AAA plants and BB or AAB banana plants with eBSV. eBSV loci produce low-abundance transcripts covering most of the viral sequence and generate predominantly 24-nt siRNAs. siRNA accumulation is restricted to duplicated and inverted viral sequences present in eBSV. Both siRNA-accumulating and nonaccumulating sequences of eBSV in BB plants are heavily methylated in all three CG, CHG and CHH contexts. Our data suggest that eBSVs are controlled at the epigenetic level in BB diploids. This regulation not only prevents their awakening and systemic infection of the plant but is also probably involved in the inherent resistance of the BB plants to mealybug-transmitted viral infection. These findings are thus of relevance to other plant resources hosting integrated viruses., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

The pine wood nematode Bursaphelenchus xylophilus is a highly invasive species responsible for the widespread pine wilt disease. Double-stranded RNA (dsRNA) biopesticides represent a novel strategy for controlling plant-parasitic nematodes. The B. xylophilus arginine kinase (BxAK) features a conserved ATP-binding domain and exhibits nematode-specific divergence in the phylogenetic tree. Notably, whole-mount in situ hybridization signals are evident in the nematode head and middle sections, particularly in the juvenile stage before sex differentiation. In this study, we developed a novel dsRNA-like small interfering RNA (siRNA) assembly that specifically targets BxAK and presents highly nematicidal effects. The RNA interference (RNAi) efficiency achieved a 95.9 % reduction in second-stage juveniles. In bioassays, the median lethal concentrations of this siRNA assembly against B. xylophilus were 168.5 ng/μl for juveniles and 603.8 ng/μl for adults within 48 h. Moreover, transcriptomic results revealed significantly downregulated expression levels of genes related to metabolism and development, suggesting that the mode of action of BxAK silencing is related to disruptions in energy homeostasis and juvenile development. In conclusion, BxAK is a molecular target for controlling B. xylophilus, and our siRNA assembly significantly enhances RNAi efficiency and lowers the lethal concentration required, making it a promising candidate for future biocontrol applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In Drosophila melanogaster, Dcr-2:R2D2 heterodimer binds to the 21 nucleotide siRNA duplex to form the R2D2/Dcr-2 Initiator (RDI) complex, which is critical for the initiation of siRNA-induced silencing complex (RISC) assembly. During RDI complex formation, R2D2, a protein that contains three dsRNA binding domains (dsRBD), senses two aspects of the siRNA: thermodynamically more stable end (asymmetry sensing) and the 5'-phosphate (5'-P) recognition. Despite several detailed studies to date, the molecular determinants arising from R2D2 for performing these two tasks remain elusive. In this study, we have performed structural, biophysical, and biochemical characterization of R2D2 dsRBDs. We found that the solution NMR-derived structure of R2D2 dsRBD1 yielded a canonical α1-β1-β2-β3-α2 fold, wherein two arginine salt bridges provide additional stability to the R2D2 dsRBD1. Furthermore, we show that R2D2 dsRBD1 interacts with thermodynamically asymmetric siRNA duplex independent of its 5'-phosphorylation state, whereas R2D2 dsRBD2 prefers to interact with 5'-P siRNA duplex. The mutation of key arginine residues, R53 and R101, in concatenated dsRBDs of R2D2 results in a significant loss of siRNA duplex recognition. Our study deciphers the active roles of R2D2 dsRBDs by showing that dsRBD1 initiates siRNA recognition, whereas dsRBD2 senses 5'-phosphate as an authentic mark on functional siRNA., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Differentiation of pluripotent stem cells into desired lineages is the key aspect of regenerative medicine and cell-based therapy. Although RNA interference (RNAi) technology is exploited extensively for this, methods for long term silencing of the target genes leading to differentiation remain a challenge. Sustained knockdown of the target gene by RNAi is often inefficient as a result of low delivery efficiencies, protocol induced toxicity and safety concerns related to viral vectors. Earlier, we established octa-arginine functionalized hydroxyapatite nano vehicles (R8HNPs) for delivery of small interfering RNA (siRNA) against a pluripotency marker gene in mouse embryonic stem cells. Although we demonstrated excellent knockdown efficiency of the target gene, sustained gene silencing leading to differentiation was yet to be achieved., Methods: To establish a sustained non-viral gene silencing protocol using R8HNP, we investigated various methods of siRNA delivery: double delivery of adherent cells (Adh-D), suspension delivery followed by adherent delivery (Susp + Adh), single delivery in suspension (Susp-S) and multiple deliveries in suspension (Susp-R). Sustained knockdown of a pluripotent marker gene followed by differentiation was analysed by reverse transcriptase-PCR, fluoresence-activated cell sorting and immunofluorescence techniques. Impact on cell viability as a result of repeated exposure of the R8HNP was also tested., Results: Amongst the protocols tested, the most efficient knockdown of the target gene for a prolonged period of time was obtained by repeated suspension delivery of the R8HNP-siRNA conjugate. The long-term silencing of a pluripotency marker gene resulted in differentiation of R1 ESCs predominantly towards the extra embryonic and ectodermal lineages. Cells displayed excellent tolerance to repeated exposures of R8HNPs., Conclusions: The results demonstrate that R8HNPs are promising, biocompatible, non-viral alternatives for prolonged gene silencing and obtaining differentiated cells for therapeutics., (© 2024 The Author(s). The Journal of Gene Medicine published by John Wiley & Sons Ltd.)

Purpose of Review: RNA interference (RNAi)-based therapies that target specific gene products have impacted clinical medicine with 16 FDA approved drugs. RNAi therapy focused on reducing plasma lipoprotein(a) [Lp(a)] levels are under evaluation., Recent Findings: RNAi-based therapies have made significant progress over the past 2 decades and currently consist of antisense oligonucleotides (ASO) and small interfering RNA (siRNA). Chemical modification of the RNA backbone and conjugation of siRNA enables efficient gene silencing in hepatocytes allowing development of effective cholesterol lowering therapies. Multiple lines of evidence suggest a causative role for Lp(a) in atherosclerotic cardiovascular disease, and recent analyses indicate that Lp(a) is more atherogenic than low density lipoprotein- cholesterol (LDL-C). These findings have led to the 'Lp(a) hypothesis' that lowering Lp(a) may significantly improve cardiovascular outcomes. Four RNAi-based drugs have completed early phase clinical trials demonstrating >80% reduction in plasma Lp(a) levels. Phase 3 clinical trials examining clinical outcomes with these agents are currently underway., Summary: Currently, four RNAi-based drugs have been shown to be effective in significantly lowering plasma Lp(a) levels. Clinical outcome data from phase 3 trials will evaluate the Lp(a) hypothesis., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Canonical RNA interference (RNAi) is sequence-specific mRNA degradation guided by small interfering RNAs (siRNAs) made by RNase III Dicer from long double-stranded RNA (dsRNA). RNAi roles include gene regulation, antiviral immunity or defense against transposable elements. In mammals, RNAi is constrained by Dicer's adaptation to produce another small RNA class-microRNAs. However, a truncated Dicer isoform (ΔHEL1) supporting RNAi exists in mouse oocytes. A homozygous mutation to express only the truncated ΔHEL1 variant causes dysregulation of microRNAs and perinatal lethality in mice. Here, we report the phenotype and canonical RNAi activity in Dicer ΔHEL1/wt mice, which are viable, show minimal miRNome changes, but their endogenous siRNA levels are an order of magnitude higher. We show that siRNA production in vivo is limited by available dsRNA, but not by Protein kinase R, a dsRNA sensor of innate immunity. dsRNA expression from a transgene yields sufficient siRNA levels to induce efficient RNAi in heart and muscle. Dicer ΔHEL1/wt mice with enhanced canonical RNAi offer a platform for examining potential and limits of mammalian RNAi in vivo., (© 2024. The Author(s).)

Divalent short-interfering RNA (siRNA) holds promise as a therapeutic approach allowing for the sequence-specific modulation of a target gene within the central nervous system (CNS). However, an siRNA modality capable of simultaneously modulating gene pairs would be invaluable for treating complex neurodegenerative disorders, where more than one pathway contributes to pathogenesis. Currently, the parameters and scaffold considerations for multi-targeting nucleic acid modalities in the CNS are undefined. Here, we propose a framework for designing unimolecular 'dual-targeting' divalent siRNAs capable of co-silencing two genes in the CNS. We systematically adjusted the original CNS-active divalent siRNA and identified that connecting two sense strands 3' and 5' through an intra-strand linker enabled a functional dual-targeting scaffold, greatly simplifying the synthetic process. Our findings demonstrate that the dual-targeting siRNA supports at least two months of maximal distribution and target silencing in the mouse CNS. The dual-targeting divalent siRNA is highly programmable, enabling simultaneous modulation of two different disease-relevant gene pairs (e.g. Huntington's disease: MSH3 and HTT; Alzheimer's disease: APOE and JAK1) with similar potency to a mixture of single-targeting divalent siRNAs against each gene. This work enhances the potential for CNS modulation of disease-related gene pairs using a unimolecular siRNA., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Synthetic biology constitutes a scientific domain focused on intentional redesign of organisms to confer novel functionalities or create new products through strategic engineering of their genetic makeup. Leveraging the inherent capabilities of nature, one may address challenges across diverse sectors including medicine. Inspired by this concept, we have developed an innovative bioengineering platform, enabling high-yield and large-scale production of biological small interfering RNA (BioRNA/siRNA) agents via bacterial fermentation. Herein, we show that with the use of a new tRNA fused pre-miRNA carrier, we can produce various forms of BioRNA/siRNA agents within living host cells. We report a high-level overexpression of nine target BioRNA/siRNA molecules at 100% success rate, yielding 3-10 mg of BioRNA/siRNA per 0.25 L of bacterial culture with high purity (>98%) and low endotoxin (<5 EU/μg RNA). Furthermore, we demonstrate that three representative BioRNA/siRNAs against GFP, BCL2, and PD-L1 are biologically active and can specifically and efficiently silence their respective targets with the potential to effectively produce downstream antiproliferation effects by PD-L1-siRNA. With these promising results, we aim to advance the field of synthetic biology by offering a novel platform to bioengineer functional siRNA agents for research and drug development.

This study aimed to develop a pan-genotypic and multifunctional small interfering RNA (siRNA) against hepatitis B virus (HBV) with an efficient delivery system for treating chronic hepatitis B (CHB), and explore combined RNA interference (RNAi) and immune modulatory modalities for better viral control. Twenty synthetic siRNAs targeting consensus motifs distributed across the whole HBV genome were designed and evaluated. The lipid nanoparticle (LNP) formulation was optimized by adopting HO-PEG 2000 -DMG lipid and modifying the molar ratio of traditional polyethylene glycol (PEG) lipid in LNP prescriptions. The efficacy and safety of this formulation in delivering siHBV (tLNP/siHBV) along with the mouse IL-2 (mIL-2) mRNA (tLNP/siHBVIL2) were evaluated in the rAAV-HBV1.3 mouse model. A siRNA combination (terms "siHBV") with a genotypic coverage of 98.55% was selected, chemically modified, and encapsulated within an optimized LNP (tLNP) of high efficacy and security to fabricate a therapeutic formulation for CHB. The results revealed that tLNP/siHBV significantly reduced the expression of viral antigens and DNA (up to 3log 10 reduction; vs PBS) in dose- and time-dependent manners at single-dose or multi-dose frequencies, with satisfactory safety profiles. Further studies showed that tLNP/siHBVIL2 enables additive antigenic and immune control of the virus, via introducing potent HBsAg clearance through RNAi and triggering strong HBV-specific CD4 + and CD8 + T cell responses by expressed mIL-2 protein. By adopting tLNP as nucleic acid nanocarriers, the co-delivery of siHBV and mIL-2 mRNA enables synergistic antigenic and immune control of HBV, thus offering a promising translational therapeutic strategy for treating CHB., (© 2024. The Author(s).)

The clinical use of RNA interference (RNAi) molecular mechanisms has introduced a novel, growing class of RNA therapeutics capable of treating diseases by controlling target gene expression at the posttranscriptional level. With the newly approved nedosiran (Rivfloza), there are now six RNAi-based therapeutics approved by the United States Food and Drug Administration (FDA). Interestingly, five of the six FDA-approved small interfering RNA (siRNA) therapeutics [patisiran (Onpattro), lumasiran (Oxlumo), inclisiran (Leqvio), vutrisiran (Amvuttra), and nedosiran] were revealed to act on the 3'-untranslated regions of target mRNAs, instead of coding sequences, thereby following the common mechanistic action of genome-derived microRNAs (miRNA). Furthermore, three of the FDA-approved siRNA therapeutics [patisiran, givosiran (Givlaari), and nedosiran] induce target mRNA degradation or cleavage via near-complete rather than complete base-pair complementarity. These features along with previous findings confound the currently held characteristics to distinguish siRNAs and miRNAs or biosimilars, of which all converge in the RNAi regulatory pathway action. Herein, we discuss the RNAi mechanism of action and current criteria for distinguishing between miRNAs and siRNAs while summarizing the common and unique chemistry and molecular pharmacology of the six FDA-approved siRNA therapeutics. The term "RNAi" therapeutics, as used previously, provides a coherently unified nomenclature for broader RNAi forms as well as the growing number of therapeutic siRNAs and miRNAs or biosimilars that best aligns with current pharmacological nomenclature by mechanism of action. SIGNIFICANCE STATEMENT: The common and unique chemistry and molecular pharmacology of six FDA-approved siRNA therapeutics are summarized, in which nedosiran is newly approved. We point out rather a surprisingly mechanistic action as miRNAs for five siRNA therapeutics and discuss the differences and similarities between siRNAs and miRNAs that supports using a general and unified term "RNAi" therapeutics to align with current drug nomenclature criteria in pharmacology based on mechanism of action and embraces broader forms and growing number of novel RNAi therapeutics., (Copyright © 2024 by The Author(s).)

Oral delivery is the most widely used and convenient route of administration of medicine. However, oral administration of hydrophilic macromolecules is commonly limited by low intestinal permeability and pre-systemic degradation in the gastrointestinal (GI) tract. Overcoming some of these challenges allowed emergence of oral dosage forms of peptide-based drugs in clinical settings. Antisense oligonucleotides (ASOs) have also been investigated for oral administration but despite the recent progress, the bioavailability remains low. Given the advancement with highly potent and durable trivalent N-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) via subcutaneous (s.c.) injection, we explored their activities after oral administration. We report robust RNA interference (RNAi) activity of orally administrated GalNAc-siRNAs co-formulated with permeation enhancers (PEs) in rodents and non-human primates (NHPs). The relative bioavailability calculated from NHP liver exposure was <2.0% despite minimal enzymatic degradation in the GI. To investigate the impact of oligonucleotide size on oral delivery, highly specific GalNAc-conjugated single-stranded oligonucleotides known as REVERSIRs with different lengths were employed and their activities for reversal of RNAi effect were monitored. Our data suggests that intestinal permeability is highly influenced by the size of oligonucleotides. Further improvements in the potency of siRNA and PE could make oral delivery of GalNAc-siRNAs as a practical solution., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Malignant tumors are often associated with an immunosuppressive tumor microenvironment (TME), rendering most of them resistant to standard-of-care immune checkpoint inhibitors (CPIs). Signal transducer and activator of transcription 3 (STAT3), a ubiquitously expressed transcription factor, has well-defined immunosuppressive functions in several leukocyte populations within the TME. Since the STAT3 protein has been challenging to target using conventional pharmaceutical modalities, we investigated the feasibility of applying systemically delivered RNA interference (RNAi) agents to silence its mRNA directly in tumor-associated immune cells. In preclinical rodent tumor models, chemically stabilized acylated small interfering RNAs (siRNAs) selectively silenced Stat3 mRNA in multiple relevant cell types, reduced STAT3 protein levels, and increased cytotoxic T cell infiltration. In a murine model of CPI-resistant pancreatic cancer, RNAi-mediated Stat3 silencing resulted in tumor growth inhibition, which was further enhanced in combination with CPIs. To further exemplify the utility of RNAi for cancer immunotherapy, this technology was used to silence Cd274, the gene encoding the immune checkpoint protein programmed death-ligand 1 (PD-L1). Interestingly, silencing of Cd274 was effective in tumor models that are resistant to PD-L1 antibody therapy. These data represent the first demonstration of systemic delivery of RNAi agents to the TME and suggest applying this technology for immuno-oncology applications., Competing Interests: Declaration of interests All authors are or were employees and/or shareholders of Dicerna Pharmaceuticals, a Novo Nordisk Company. We, the authors, have patents related to this work. Patent applications PCT/US2022/018911 and PCT/US2023/064381 relate to this work., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Osteoarthritis (OA) is a common joint disorder characterized by the degeneration of cartilage and inflammation, affecting millions worldwide. The disease's complex pathogenesis involves various cell types, such as chondrocytes, synovial cells, osteoblasts, and immune cells, contributing to the intricate interplay of factors leading to tissue degradation and pain. RNA interference (RNAi) therapy, particularly through the use of small interfering RNA (siRNA), emerges as a promising avenue for OA treatment due to its capacity for specific gene silencing. siRNA molecules can modulate post-transcriptional gene expression, targeting key pathways involved in cellular proliferation, apoptosis, senescence, autophagy, biomolecule secretion, inflammation, and bone remodeling. This review delves into the mechanisms by which siRNA targets various cell populations within the OA milieu, offering a comprehensive overview of the potential therapeutic benefits and challenges in clinical application. By summarizing the current advancements in siRNA delivery systems and therapeutic targets, we provide a solid theoretical foundation for the future development of novel siRNA-based strategies for OA diagnosis and treatment, paving the way for innovative and more effective approaches to managing this debilitating disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision, (Copyright © 2024 Li, Zhao, Guo and He.)

Chronic myeloid leukemia (CML) is an oncological myeloproliferative disorder that accounts for 15 to 20% of all adult leukemia cases. The molecular basis of this disease lies in the formation of a chimeric oncogene BCR-ABL1. The protein product of this gene, p210 BCR-ABL1, exhibits abnormally high constitutive tyrosine kinase activity. Over recent decades, several targeted tyrosine kinase inhibitors (TKIs) directed against BCR-ABL1 have been developed and introduced into clinical practice. These inhibitors suppress BCR-ABL1 activity through various mechanisms. Furthermore, the advent of RNA interference technology has enabled the highly specific inhibition of BCR-ABL1 transcript expression using small interfering RNA (siRNA). This experimental evidence opens avenues for the development of a novel therapeutic strategy for CML, termed siRNA therapy. The review delves into molecular genetic mechanisms underlying the pathogenesis of CML, challenges in CML therapy, potential molecular targets for drug development, and the latest results from the application of siRNAs in in vitro and in vivo CML models.

The present study investigated the concurrent delivery of antineoplastic drug, doxorubicin, and HER2 siRNA through a targeted theranostic metallic gold nanoparticle designed using polysaccharide, PSP001. The as-synthesized HsiRNA@PGD NPs were characterized in terms of structural, functional, physicochemical, and biological properties. HsiRNA@PGD NPs exposed adequate hydrodynamic size, considerable ζ potential, and excellent drug/siRNA loading and encapsulation efficiency. Meticulous exploration of the biocompatible dual-targeted nanoconjugate exhibited an appealing biocompatibility and pH-sensitive cargo release kinetics, indicating its safety for use in clinics. HsiRNA@PGD NPs deciphered competent cancer cell internalization, enhanced cytotoxicity mediated via the induction of apoptosis, and excellent downregulation of the overexpressing target HER2 gene. Further in vivo explorations in the SKBR3 xenograft breast tumor model revealed the appealing tumor reduction properties, selective accumulation in the tumor site followed by significant suppression of the HER2 gene which contributed to the exclusive abrogation of breast tumor mass by the HsiRNA@PGD NPs. Compared to free drugs or the monotherapy constructs, the dual delivery approach produced a synergistic suppression of breast tumors both in vitro and in vivo . Hence the drawings from these findings implicate that the as-synthesized HsiRNA@PGD NPs could offer a promising platform for chemo-RNAi combinational breast cancer therapy.

Copper plays a crucial role in maintaining biological redox balance in living organisms, with elevated levels observed in cancer cells. Short interfering RNAs (siRNAs) are effective in gene silencing and find applications as both research tools and therapeutic agents. A method to regulate RNA interference using copper is especially advantageous for cancer-specific therapy. We present a chemical approach of selective siRNA activation triggered by intracellular copper ions. We designed and synthesized nucleotides containing copper-responsive moieties, which were incorporated into siRNAs. These copper-responsive siRNAs effectively silenced the target cyclin B1 mRNA in living cells. This pioneering study introduces a novel method for conditionally controlling gene silencing using biologically relevant metal ions in human cells, thereby expanding the repertoire of chemical knockdown tools., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Akimitsu Okamoto reports financial support was provided by Japan Society for the Promotion of Science. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Small interfering RNAs (siRNAs) are promising therapeutic strategies, and five siRNA drugs have been approved by the Food and Drug Administration (FDA) and the European Commission (EC). This marks a significant milestone in the development of siRNA for clinical applications. The approved siRNA agents can effectively deliver siRNAs to the liver and treat liver-related diseases. Currently, researchers have developed diverse delivery platforms for transporting siRNAs to different tissues such as the brain, lung, muscle, and others, and a large number of siRNA drugs are undergoing clinical trials. Here, these delivery technologies and the latest advancements in clinical applications are summarized, and this Review provides a concise overview of the strategies employed for siRNA delivery to both hepatic and extrahepatic tissues.