Metrics details an endogenous inhibitor of granzyme B (GzmB) has emerged as a critical factor in the resistance to immunotherapy by protecting cancer cells from GzmB-induced cytotoxicity its role in chemosensitivity remains unknown we show that gemcitabine (GEM) treatment upregulates SERPINB9 through transcription factor ATF-3 GEM also induces the expression of GzmB and knockout or knockdown of SERPINB9 results in enhanced response of tumor cells to GEM suggesting a role of GzmB/SERPINB9 axis in regulating chemosensitivity To facilitate the therapeutic translation of these findings we engineer POEM nanocarrier (consisting of lipid-derivatized polylysine (PEG-PLL-Oleic acid and GEM-conjugated polylysine (PEG-PLL-OA-GEM PPO/PPOGEM (POEM)) that is highly effective in codelivery of built-in GEM and loaded SERPINB9 short interfering RNA (siSPB9) GEM conjugation introduces an additional mechanism of carrier/siRNA interaction in addition to charge-mediated interaction and enables efficient i.v we show that co-delivery of GEM and siSPB9 significantly improves antitumor efficacy and remodels the tumor immune microenvironment in pancreatic cancer models supporting a promising therapeutic strategy selective inhibition of SPB9 activity in tumor cells and immunosuppressive immune cells while preserving its function in CTLs shall represent an attractive and effective strategy to boost ICB as well as other immunotherapies the specific role of SPB9 in modulating the response of cancer cells to chemotherapeutic agents remains poorly understood we show that GEM also induces SPB9 expression in tumor cells or tissues in vitro and in vivo suggesting another feedback mechanism that negatively regulates the antitumor immune response We study the mechanism by which GEM induces the SPB9 gene expression at transcriptional level We further evaluate the role of GzmB/SPB9 axis in regulating the chemosensitivity of PCa cells to GEM POEM (PPO/PPOGEM) nanocarrier is developed and evaluated for codelivery of GEM and siSPB9 to overcome both chemotherapy and immune resistance The broad implications of these findings in cancer chemotherapy require more studies in the future our data suggests that SPB9 KD or inhibition in tumor cells may benefit increased sensitivity to GEM-based chemotherapy in addition to its benefits in improving antitumor immune response a PPO with a PEG/OA ratio of 5:2 and without further amino acid modification formed the most effective siRNA NPs with PPOGEM in terms of the amounts of Cy5.5 signals in the tumor tissues as well as the ratios of the signals in tumors over those in other normal tissues/organs This PPO was selected for further optimization of POEM/siRNA via extensive characterizations of the biophysical properties and in vivo distribution of 18 NPs that vary in PPOGEM/PPO/siRNA ratio indicating that PPOGEM helps stabilize the system siRNA was well loaded into POEM NPs at an N/P ratio of 5/1 Increasing the amounts of PPOGEM led to a gradual decrease of zeta potential likely due to the shielding effect from PPOGEM at an N/P ratio of 5/1 and a PPOGEM/siRNA mass ratio of 50/1 the POEM/siRNA system demonstrated the most desirable biophysical properties with a size of ~45.6 nm and a zeta potential of around 9.3 mV (NPs with zeta potential in a range of −10 to +10 are considered close to neutral) the zeta potential of the NPs could not be reduced to below 10 mV at all PPO/Psugar ratios examined These data highlight the significant role of GEM in formulating the POEM NP system it is obvious that the POEM system (blue curve ~54.87 Å) exhibits lower RoG values than the control polymer (red curve This can be further explained by the representative conformation of POEM/siRNA displayed at the bottom right of the panel where siRNA effectively interacts with PPOGEM through both T-shaped π-π and hydrogen bonding interactions It is emphasized that during the simulation the whole NP system was constructed based on a molar ratio of PPOGEM (or Psugar) the simulation accurately represents the composition of the NPs NPs are composed of numerous copies of the simulation system While the RoG difference between the POEM and Psugar systems is only around 1 Å in the simulation this small difference at the unit level can lead to a significant variation in the overall size of the assembled NPs the POEM system (~40 nm) and the Psugar system (~160 nm) exhibit a substantial difference in their final dimensions The in vitro gene knockdown efficiency of POEM NPs was evaluated using KPC-C2-Luc and Panc02-Luc cell lines (Supplementary Fig. 12) POEM NPs were more effective than the commercial siRNA transfection reagent RNAiMAX in mediating the siLuc transfection and silencing luciferase transgene expression in both cell lines The long circulation time of POEM/siRNA is likely attributed to the excellent stability of the NPs in blood which contributes to the effective tumor targeting clearly demonstrating the therapeutic benefit of codelivery of gemcitabine and siSPB9 using POEM NPs These findings underscore the excellent safety profile of co-delivery of siSPB9 and GEM using POEM NPs at dosages that have shown significant therapeutic efficacy These results suggest that the combination of POEM/siSPB9 with anti-PD-1 treatment represents a promising regimen towards GEM resistant pancreatic cancer Our data suggests that KD of SPB9 can directly sensitize PCa to GEM treatment in addition to improving the antitumor immune response It remains to be studied if GzmB/SPB9 axis also affects the sensitivity of PCa as well as other cancer types to other chemotherapeutic drugs POEM represents an effective nanocarrier in codelivery of GEM and siRNA (Fig. 3) The pyrimidine structure is critical in stabilizing the siRNA NPs through π-π stacking and hydrogen bonding It is possible that the hydroxyl groups in the sugar ring are also important in promoting the interaction with siRNA albeit less important compared to pyrimidine motif The use of two individual polymers (PPO and PPOGEM) each with cationic (-NH2) and GEM motif will provide the flexibility of adjusting their ratio in formulation optimization the very similar structure of the two polymers will facilitate their mixing to form compact and stable micelles Although this study focuses on evaluating combination of GEM and siRNA this strategy can be applied to codelivery of siRNA with other nucleoside analogue-based drugs such as AZA other drugs such as cisplatin can be readily incorporated into POEM NPs following lipid-derivatization our system may be tailor-designed to develop precision medicines to suit different combination therapies may also play a role in enhancing the accumulation and penetration of POEM NPs EPR shall also contribute to the tumor targeting by POEM NPs as well especially considering their compact sizes (40–50 nm) The combination of EPR-mediated passive targeting and fibronectin-mediated active targeting contributes significantly to the effective tumor accumulation of POEM NPs in both subcutaneous and orthotopic models: around 6% of injected siRNA formulated in POEM NPs was found in the tumor tissues at 24 h and the siRNA concentrations in tumors were 3–4 folds higher than those in liver More mechanistic studies on tumor targeting may lead to further improved tumor delivery systems in the future More studies on the differential effect of different formulations and/or drug combinations on tumor cells and different immune cell subpopulations may lead to the development of an improved therapy targeting the GzmB/SPB9 axis All animals were housed under pathogen free conditions according to AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) guidelines All animal-related experiments were performed in full compliance with institutional guidelines and approved by the Animal Use and Care Administrative Advisory Committee at the University of Pittsburgh under protocol number 21099779 The maximum tumor volume permitted by the IACUC guidelines was 2000 mm3 tumor size and animal health were monitored daily and the maximum allowed tumor burden was not exceeded and KPC-C2-Luc cells were produced by following lentiviral/retroviral infection protocol as detailed in lentiviral infection section below Panc02 and PANC-1 were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (100 U/ml) and KPC-C5 were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) 1% GlutaMAX (100×) and penicillin/streptomycin (100 U/ml) MIA PaCa-2 was cultured in DMEM supplemented with 10% fetal bovine serum (FBS) Panc 02.03 and Panc 10.05 were cultured in RPMI-1640 supplemented with 15% fetal bovine serum (FBS) Gemcitabine resistant cell lines were cultured in the full medium with gemcitabine (260 ng/mL) All cells were cultured at 37 °C in a humidified atmosphere with 5% CO2 Female C57BL/6 mice aged between 4–6 and 8–10 weeks were purchased from The Jackson Laboratory (ME Mice were housed at an ambient temperature of 22 °C (range: 22–24 °C) and a humidity of 45% with a 14/10 day/night cycle (lights on at 6:00 Edit-R Mouse Serpinb9 mCMV-EGFP All-in-one Lentiviral sgRNA was purchased from Horizon Discovery Ltd psPAX2 and pMD2.G were kindly provided by Dr pLentipuro3/TO/V5-GW/EGFP-Firefly Luciferase Plasmid was purchased from Addgene (MA SERPINB9 KO cell lines were generated by using CRISPR technology Cells were infected with the lentivirus packaged by Serpinb9-All-in-one lentiviral sgRNA-CRISPR-Cas9 plasmid encoding EGFP The successfully knocked out cells were selected by cell sorting of EGFP+ population followed by single clone culture and further confirmed through Western blot analysis for the lack of SERPINB9 proteins SERPINB9 control vector cell line was generated by using a control lentiviral vector with Cas9 coding sequence but without the specific guiding sequences (Lenti CRISPR plasmid without sgRNA sequence) The luciferase-expressing cell lines were generated according to a published protocol56 Cells were infected with the lentivirus packaged from pLentipuro3/TO/V5-GW/EGFP-Firefly Luciferase plasmid encoding EGFP The luciferase-expressing cells were selected by cell sorting of the EGFP+ population and further confirmed through flow cytometry analysis for EGFP expression To examine the mRNA level of different genes from RNA-seq results murine pancreatic cancer cell lines (Panc02 and KPC-C5; WT vs GEMR) were collected and subjected to qRT-PCR as detailed below To examine the effect of GEM on the mRNA level of GranzymeB different pancreatic cancer cells were treated with various concentrations of GEM Cells were collected 24 or 48 h later and subjected to qRT-PCR as detailed below The total protein was extracted from the indicated cells by using radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific USA) through gently shaking on ice for 30 min After centrifugation at 12,500 × g for 10 min and the concentrations of proteins were determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific The protein samples were mixed with 5× SDS loading buffer loaded onto 10% SDS-polyacrylamide gel electrophoresis (PAGE) gel for electrophoresis The membranes were then blocked in 3% BSA dissolved in phosphate buffer containing 0.05% Tween-20 (PBST) for 1 h at room temperature the membranes were incubated with primary antibody in diluted buffer (3% BSA in PBST) with gentle agitation overnight at 4 °C the membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h The membranes were then washed three times with PBST before being incubated with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific Signal was visualized by films with the AX700LE film processor (Alphatek) or iBright™ FL1500 Imaging System Granzyme B activity was measured using the GranToxiLux PLUS kit Cells subjected to different treatments were incubated with the Granzyme B substrate for 1 h at 37 °C The cleaved Granzyme B substrate (GranToxiLux) exhibited an excitation peak at 488 nm and an emission peak at 520 nm GranToxiLux fluorescence was measured by flow cytometry and cell populations positive for GranToxiLux were considered to have active Granzyme B The three DNA fragments with different 5x repeated sequences were synthesized by AZENTA (MA USA) and HindIII (Thermo Fisher Scientific USA) restriction enzymes were used to digest the pGL3-Basic plasmid and the synthesized fragments The 3 DNA fragments were individually ligated with the backbone by using T4 DNA ligase (Invitrogen Single colonies were picked and expanded in the DH5α competent cells (Thermo Fisher Scientific the concentration was determined by NanoDrop and the sequence was confirmed by Sanger sequence a full length promoter sequence containing all three binding motifs was synthesized by Twist Bioscience (CA The mutated plasmid was constructed through site-directed mutagenesis a pair of primers for mutagenesis were designed and synthesized (IDT This pair of primers was designed to include the front and back sequence of each binding motif but without the motif sequence Then the PCR reaction with this pair of primers was performed using the previously generated full-length plasmid as a template The PCR product was treated with Dpn1 enzyme to remove the template the PCR product was used for the transformation in DH5α competent cells The Sanger sequencing confirmed the sequence and Panc 02.03 cells were transfected with each of the plasmids described above using Lipofectamine 3000 (Invitrogen the cells were treated with gemcitabine for an additional 48 h Cell lysis was performed using the lysis buffer from the Pierce Firefly Luciferase Glow Assay Kit (Thermo Fisher Scientific and bioluminescence was detected using a luminometer according to the manufacturer’s instructions Cytotoxicity was evaluated by MTT assay with indicated cell lines Cells were seeded in 96-well plates (attached 96 well tissue culture plate (CELLTREAT MA)) at a density of 2 × 103 cells/well with 100 μL of culture medium Cells received various treatments including GEM alone or GEM+siSPB9 combination at various GEM and siRNA concentrations for 48 h cells were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen USA) for 48 h followed by drug treatment for another 48 h MTT assay was then performed on the cultured cancer cells The absorbances of each well were measured at 590 nm The cell viability was determined via the following formula: (ODtreated- ODblank)/(ODcontrol-ODblank) × 100 % Cells were digested following different treatments for 24 h and stained with Zombie NIR and BV421 anti-mouse Annexin V or FITC anti-mouse Annexin V for flow cytometry analysis Annexin V+ population was considered apoptotic cells To synthesize PPO polymers conjugated with various amino acids tert-butyloxycarbonyl (BOC)-protected amino acids (histidine The activation of the amino acid carboxyl groups was achieved by adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) The reaction mixture was stirred for 2 h to promote conjugation the non-amino acid-decorated PPO polymer and triethylamine (TEA) were introduced and the mixture was stirred for an additional 12 h The reaction product was purified by dialysis using a dialysis membrane (MWCO 3500 Da) Dialysis was performed in a DMSO/H2O (4:1) mixture for 24 h The final product was lyophilized and stored at 4 °C The content of gemcitabine in the polymer was determined by UV absorbance The conjugation rates of cytidine and glucosamine were determined by UV absorbance To prepare the POEM/siRNA or Psugar/PPO/siRNA NPs SiRNA was dissolved in deionized water at a concentration of 25 μg/mL The DMSO solutions of PPO with PPOGEM or Psugar were prepared at different ratios followed by rapid addition of siRNA (10 μg) aqueous solution The mixture was centrifuged using an Amicon Ultra centrifugal filter device at 15,000 × g for 15 min and the centrifugation was repeated at 15,000 g for 15 min to remove residual DMSO The final nanoparticles in the remaining liquid in the tube were collected for subsequent studies To assess the resistance of POEM/siRNA NPs against nuclease-mediated degradation the NPs were incubated with RNase (50 U/mL) (NEB and the integrity of siRNA was evaluated by electrophoresis while the OL3 and OL15 force fields were used to model RNA and DNA Initial system relaxation was achieved by a 1000-step conjugated-gradient energy minimization on the entire POEM/siRNA structure constant-pressure MD simulation was performed for ~3.5 nanoseconds (ns) facilitating system shrinking and stabilization with an objective of achieving a density close to 1 a two-stage MD simulation protocol was applied to the shrunk system with a 600 ns MD simulation to continue to stabilize the system in stage one the last snapshot was solvated in a rectangular water box filled in with TIP3P water and 0.15 M Na+ and Cl− ions The whole system was neutralized and the simulation box has a dimension of 220 × 220 × 220 Å Then a 300 ns MD simulation was conducted with period boundary condition enforced in the second stage position restraints were applied to the siRNA with a force constant of 100 kcal/mol/Å2 to prevent its distortion but the other atoms were allowed to move freely The snapshot with the smallest root-mean-square deviation (RMSD) from the average structure of snapshots collected after the system reached equilibrium (the last 150 ns) was selected as the representative conformation A similar protocol was applied for the PCytidine/siRNA and Psugar/siRNA system Samples were initially examined using negative-stain electron microscopy A 3 μL aliquot was applied to a freshly glow-discharged continuous carbon copper grid and stained with 1% uranyl acetate solution The grids were then inserted into a Tecnai TF20 electron microscope (Thermo Fisher Scientific and images captured using an XF416 CMOS camera (TVIPS GmbH Germany) to assess nanoparticle uniformity and concentration 3 μL of the sample was pipetted onto a Protochips C-flat CF-2/1-3CU-T grid (Protochips USA) that had been glow discharged at 25 mA for 30 s using an Emitech KX100 glow discharger The grids were then processed in a Thermo Fisher Vitrobot Mk 4 They were blotted for 3 s with a force setting of 4 and subsequently plunged into a 40/60 mixture of liquid ethane/propane that was precooled in a liquid nitrogen bath The grids were then placed onto a Gatan 910 3-grid cryoholder (Gatan USA) and inserted into the TF20 microscope maintaining a temperature of no higher than −175 °C throughout with contrast enhanced by a 100 μm objective aperture Cryo-electron micrographs were collected at a nominal magnification of 150,000× on the TVIPS XF416 CMOS camera Low dose methods were employed to minimize electron beam damage and images were acquired using TVIPS EMplified software in movie mode to correct for drift To test the in vitro knockdown efficiency of POEM NPs KPC-C2-Luc and Panc02-Luc cells were treated with different formulations of POEM NPs with varying ratios of PPOGEM and PPO cell lysates were collected and analyzed by measuring the bioluminescent intensity Commercial Lipofectamine RNAiMAX was used as a control KPC C2 WT cells were pretreated with chlorpromazine The cells were then incubated with POEM/siRNA/DiD dye NPs for 1 h and the fluorescence intensity of DiD dye was measured KPC C2 cells were seeded into the upper chamber of a transwell and cultured for 3 days until a confluent monolayer was formed Prior to adding POEM/siRNA/DiD NPs to the upper chamber the cells were pretreated with anti-ITGA5 or IgG for 1 h the medium in the lower chamber was collected followed by measurement of its fluorescence intensity To establish the subcutaneous tumor models or KPC-C2 GEMR cells were inoculated into the right flank of C57BL/6 mice (4-6 weeks) or KPC-C2 GEMR tumor-bearing mice received different treatments when the tumor size reached the indicated volume Tumor growth in KPC-C2 SERPINB9 KO or KPC-C2 control vector tumor-bearing mice was monitored for 20 days followed by analysis of tumor-infiltrating immune cells as detailed in “Analysis of tumor-infiltrating immune cells” section anesthetized C57BL/6 mice (8–10 weeks) were shaved and the surgical area on the left flank was disinfected The pancreas was exposed through a small incision (~1 cm) on the left flank Fifty microliters Panc02-Luc or KPC-C2-Luc single cell suspension (1 × 105 per mouse) was injected cell into the tail of the pancreas using 28-gauge hypodermic needles The needle was slowly removed after the injection of cell suspension the mice were maintained at the heating pad and observed until complete recovery The development of pancreatic cancer was monitored by bioluminescence radiance intensity after the mice were injected with D-Luciferin (GoldBio mice bearing subcutaneous (~300 mm3) or orthotopic (~10 days after inoculation) pancreatic tumors were intravenously injected with free siSPB9-cy5.5 and POEM/siSPB9-cy5.5 NPs with different PPOGEM/siRNA ratios The mice were sacrificed at 24 h after injection and kidney were collected and imaged by IVIS 200 system (Perkin Elmer USA) at a constant 1 s exposure time with excitation at 679 nm and emission at 702 nm blood was collected in EDTA-containing tubes at 10 min 48 h and 72 h timepoints and plasma samples were prepared by centrifugation at 21,000 × g for 10 min and imaged by the IVIS 200 system Groups of 3 C57BL/6 mice (4–6 weeks) bearing subcutaneous KPC-C2 tumors (~300 mm3) received tail vein injection of POEM/siSPB9-cy5.5 NPs or free siSPB9-cy5.5 at a dose of 1 mg/kg for siRNA blood was collected in the tubes containing EDTA The amount of siRNA in the plasma was quantified by qRT-PCR as detailed below The PK parameters were obtained by fitting the blood siRNA concentration versus time using a one-compartment model and tumors were collected at 24 h and homogenized SiRNA in the tissues was quantified through qRT-PCR Spleen and KPC-C2 tumor tissues were harvested from C57BL/6 mice followed by the preparation of single-cell suspensions CD4+ and CD8+ T cells were isolated using CD4/CD8 MicroBeads (Miltenyi Biotec Cells were cultured in RPMI-1640 medium and stimulated by anti-CD3 (Invitrogen or Lipofectamine RNAiMAX/siSPB9-cy5.5 as a control for 4 h Cellular uptake of siRNA was examined by flow cytometry POEM NPs loaded with luciferase siRNA (siLuc) or control siRNA (siCT) were intravenously injected into KPC-C2-Luc tumor-bearing mice at a dose of 1 mg/kg The efficiency of gene knockdown was measured three times by whole body bioluminescence imaging on the 2nd day following the 1st and 3rd injection of the NPs once every 3 days The exposure time was set at 60 s for every experiment Mice were anesthetized according to protocol prior to imaging KPC-C2 tumor-bearing mice were randomly assigned to different groups (n = 5) They were intravenously administered with PBS or POEM/siSPB9 NPs three times at an interval of 5 days (on day 5 The doses for PPOGEM and siRNA were 50 mg/kg and 1 mg/kg Twenty-four hours after the final injection tumors were collected and subjected to qRT-PCR of SPB9 expression as detailed above Murine pancreatic cancer (KPC-C2 and KPC-C2 GEMR) models were established for in vivo antitumor efficacy study every 3 days for a total of 5 times (PCytidine: 50 mg/kg; PPOGEM: 50 mg/kg; siSPB9: 1 mg/kg) through intravenous injection Tumor volumes and mouse body weights were monitored every 3 days following the initiation of the treatment The tumor volumes (V) were calculated by the formula: (Length × Width2)/2 tumor tissues and major organs were harvested for histochemical staining and plasma was isolated after centrifugation at 21,000 × g for 10 min and creatinine levels in plasma were measured as indicators of hepatic and renal function the KPC-C2-Luc tumor-bearing mice were treated intravenously with PBS The treatments were conducted every 3 days for a total of five times The whole-body tumor burden was monitored and quantified by measuring the luminous intensity using Living Imaging 4.1.0 software To test the therapeutic effect of the combinational therapy of POEM/siSPB9 NPs with anti-PD-1 the treatment was started when the KPC-C2 GEMR tumors reached ~50 mm3 in size Mice were treated with anti-PD-1 (clone RMP1-14 while POEM/siSPB9 NPs were given intravenously at dosage described above Mice were followed until death or were killed if the tumor size reached 2000 mm3 the maximal tumor size permitted by the Animal Use and Care Administrative Advisory Committee at the University of Pittsburgh The images were observed under a BZ-X710 Fluorescence Microscope (Keyence tumors were dissected and transferred into RPMI-1640 medium Tumors were disrupted mechanically using scissors digested with a mixture of deoxyribonuclease I (0.3 mg/mL Sigma-Aldrich) and TL Liberase (0.25 mg/mL Roche) in serum-free RPMI-1640 at 37 °C for 30 min and dispersed through a 40 μm cell strainer (BD Biosciences) live/dead cell discrimination was performed using a Zombie NIR Fixable Viability Kit (BioLegend dilution: 1/1000) at 4 °C for 30 min in PBS Surface staining was performed at 4 °C for 30 min in FACS staining buffer (1× PBS/5% FBS/0.5% sodium azide) containing designated antibody cocktails (PerCP anti-mouse CD45 antibody Brilliant Violet 737 anti-mouse CD4 antibody Brilliant Violet 480 anti-mouse CD8 antibody Brilliant Violet 615 anti-mouse PD-1 antibody Brilliant Violet 510 anti-mouse Gr-1 antibody Brilliant Violet 737 anti-mouse CD80 antibody FITC anti-mouse CD69; dilution: 1/200 for all antibodies) Cells were fixed and permeabilized using the BD Cytofix/Cytoperm kit For intracellular cytokine staining (PE-Cyanine7 anti-mouse IFN-γ antibody and eF450 anti-mouse GzmB antibody; dilution: 1/200 for antibody) cells were stimulated with phorbol 12-myristate-13-acetate (100 ng/mL) and ionomycin (500 ng/mL) for 6 h in the presence of Monensin Cells were fixed/permeabilized using the BD Cytofix/Cytoperm kit before cell staining Statistical analysis was performed with two-tailed Student’s t-test for comparison between two groups one-way analysis of variance (ANOVA) for comparison between multiple groups and log-rank (Mantel–Cox) test for survival analysis as indicated in figure legend Results were considered statistically significant if P < 0.05 Prism 10.1.0 (GraphPad Software) was used for data analysis and graph plotting All experiments were repeated at least three times with similar results Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Immune checkpoint blockade and biomarkers of clinical response in non-small cell lung cancer Notch signaling and efficacy of PD-1/PD-L1 blockade in relapsed small cell lung cancer Molecular patterns of resistance to immune checkpoint blockade in melanoma Mechanisms of tumor resistance to immune checkpoint blockade and combination strategies to overcome resistance Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme B Intracellular versus extracellular granzyme B in immunity and disease: challenging the dogma SERPINB9 is commonly amplified and high expression in cancer cells correlates with poor immune checkpoint blockade response and partial characterization of two novel members of the ovalbumin family of serine proteinase inhibitors The biological function of Serpinb9 and Serpinb9-based therapy Serine protease inhibitor 6 protects cytotoxic T cells from self-inflicted injury by ensuring the integrity of cytotoxic granules is up-regulated during accessory cell maturation and effector cell degranulation and its overexpression enhances CTL potency is mainly expressed by dendritic cells and at immune-privileged sites Serine protease inhibitor 6-deficient mice have increased neutrophil immunity to Pseudomonas aeruginosa Blockade of the granzyme B/perforin pathway through overexpression of the serine protease inhibitor PI-9/SPI-6 constitutes a mechanism for immune escape by tumors Direct tumor killing and immunotherapy through anti-SerpinB9 therapy Signatures of T cell dysfunction and exclusion predict cancer immunotherapy response Mapping the functional interactions at the tumor-immune checkpoint interface Expression of the apoptosis inhibitor protease inhibitor 9 predicts clinical outcome in vaccinated patients with stage III and IV melanoma Low-dose gemcitabine treatment enhances immunogenicity and natural killer cell-driven tumor immunity in lung cancer Syk inhibition reprograms tumor-associated macrophages and overcomes gemcitabine-induced immunosuppression in pancreatic ductal adenocarcinoma Tumor cell-intrinsic factors underlie heterogeneity of immune cell infiltration and response to immunotherapy Integrated stress response is critical for gemcitabine resistance in pancreatic ductal adenocarcinoma Comparison of human chromosome 6p25 with mouse chromosome 13 reveals a greatly expanded ov-serpin gene repertoire in the mouse In vivo CRISPR screens reveal Serpinb9 and Adam2 as regulators of immune therapy response in lung cancer Gene expression profiling-based identification of cell-surface targets for developing multimeric ligands in pancreatic cancer The common stress responsive transcription factor ATF3 binds genomic sites enriched with p300 and H3K27ac for transcriptional regulation Master regulator activating transcription factor 3 (ATF3) in metabolic homeostasis and cancer ATF3 promotes the serine synthesis pathway and tumor growth under dietary serine restriction by tumor cells in patients with non-Hodgkin and Hodgkin lymphoma: a novel protective mechanism for tumor cells to circumvent the immune system Multifunctional oncolytic nanoparticles deliver self-replicating IL-12 RNA to eliminate established tumors and prime systemic immunity Engineered extracellular vesicles for delivery of siRNA promoting targeted repair of traumatic spinal cord injury Luo, Z., Chen, C. Y. & Li, S. Improving tumor targeting and penetration for nanoparticle-mediated cancer therapy. Small Methods e2401860 (2025). https://doi.org/10.1002/smtd.202401860 The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs RNA interference in the era of nucleic acid therapeutics active and endogenous organ-targeted lipid and polymer nanoparticles for delivery of genetic drugs Targeting Xkr8 via nanoparticle-mediated in situ co-delivery of siRNA and chemotherapy drugs for cancer immunochemotherapy Overcoming pancreatic cancer immune resistance by codelivery of CCR2 antagonist using a STING-activating gemcitabine-based nanocarrier Multifunctional cationic lipid-based nanoparticles facilitate endosomal escape and reduction-triggered cytosolic siRNA release Creatine based polymer for codelivery of bioengineered MicroRNA and chemodrugs against breast cancer lung metastasis The effect of surface charge on in vivo biodistribution of PEG-oligocholic acid based micellar nanoparticles Glutathione-mediated nanomedicines for cancer diagnosis and therapy Orthotopic pancreatic tumor mouse models of liver metastasis Radiolabeled RNA nanoparticles for highly specific targeting and efficient tumor accumulation with favorable in vivo biodistribution Protein corona of nanoparticles: isolation and analysis In situ formation of fibronectin-enriched protein corona on epigenetic nanocarrier for enhanced synthetic lethal therapy Inhibition of the SLC35B2-TPST2 axis of tyrosine sulfation attenuates the growth and metastasis of pancreatic ductal adenocarcinom Persister cell phenotypes contribute to poor patient outcomes after neoadjuvant chemotherapy in PDAC PD-1 and PD-L1: architects of immune symphony and immunotherapy breakthroughs in cancer treatment Immunogenic cell death effects induced by doxorubicin improved chemo-immunotherapy via restoration of granzyme B activity Identifying key barriers in cationic polymer gene delivery to human T cells Nanoparticles for generating antigen-specific T cells for immunotherapy Transvascular transport of nanocarriers for tumor delivery Development and characterization of gemcitabine-resistant pancreatic tumor cells Generation of luciferase-expressing tumor cell lines Transcript-level expression analysis of RNA-seq experiments with HISAT Systematic identification of non-coding pharmacogenomic landscape in cancer Targeting metabotropic glutamate receptor 4 for cancer immunotherapy Mirovascular permeability and interstitial penetration of sterically stabilized (stealth) liposomes in a human tumor xenograft Inhibition of iRhom1 by CD44-targeting nanocarrier for improved cancer immunochemotherapy Automatic atom type and bond type perception in molecular mechanical calculations Development and testing of a general amber force field A fast and high-quality charge model for the next generation general AMBER force field Routine microsecond molecular dynamics simulations with AMBER on GPUs High loading of hydrophobic and hydrophilic agents via small immunostimulatory carrier for enhanced tumor penetration and combinational therapy An immunostimulatory dual-functional nanocarrier that improves cancer immunochemotherapy A novel immunochemotherapy based on targeting of cyclooxygenase and induction of immunogenic cell death Download references This work was supported by the fund from National Institute of Health grants R01CA219399 R01GM147673 (to J.W.) and The David and Betty Brenneman Scholar Fund (to S.L.) This research used the Olympus FV3000 Confocal Microscope which is supported by 1S10OD030254-01A1 (to RBG) The Pittsburgh Center for CryoEM (RRID:SCR_025216) used for data collection in this project was supported These authors contributed equally: Haozhe Huang Department of Pharmaceutical Sciences and Computational Chemical Genomics Screening Center The authors declare no competing interests Nature Communications thanks Philippe Soubeyran reviewer(s) for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-025-59490-y Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology Metrics details AGO-clade Argonaute proteins utilize small interfering RNAs (siRNAs) as guides to recognize target with complete complementarity resulting in target RNA cleavage that is a critical step for target silencing These proteins feature a constricted nucleic acid-binding channel that limits base pairing between the guide and target beyond the seed region How the AGO–siRNA complexes overcome this structural limitation and achieve efficient target cleavage remains unclear We performed cryo-electron microscopy of human AGO–siRNA complexes bound to target RNAs of increasing lengths to examine the conformational changes associated with target recognition and cleavage conformational transition propagates from the opening of the PAZ domain and extends through a repositioning of the PIWI–L1–N domain toward the binding channel facilitating the capture of siRNA–target duplex Subsequent extension of base pairing drives the downward movement of the PIWI–L1–N domain to enable catalytic activation further base pairing toward the 3′ end of siRNA destabilizes the PAZ–N domain which might facilitate the multi-turnover action of the AGO–siRNA enzyme complex the “uni-lobed” structure of the AGO complex makes multiple contacts with the target in the central region of the siRNA–target duplex Our findings shed light on the stepwise mechanisms by which the AGO–siRNA complex executes target RNA cleavage and offer insights into the distinct operational modalities of AGO and PIWI proteins in achieving such cleavage Prices may be subject to local taxes which are calculated during checkout Argonaute proteins: Functional insights and emerging roles Emerging roles and functional mechanisms of PIWI-interacting RNAs Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway PIWI-interacting RNAs: small RNAs with big functions indicates that thousands of human genes are microRNA targets Origins and mechanisms of miRNAs and siRNAs MRNA destabilization is the dominant effect of mammalian microRNAs by the time substantial repression ensues Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish MicroRNA inhibition of translation initiation in vitro by targeting the cap-binding complex eIF4F miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay Kinetic analysis of the RNAi enzyme complex RNAi in human cells: Basic structural and functional features of small interfering RNA siRNA function in RNAi: A chemical modification analysis RNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals RNA interference is an antiviral defence mechanism in Caenorhabditis elegans RNA interference directs innate immunity against viruses in adult Drosophila A dicer-independent miRNA biogenesis pathway that requires Ago catalysis A novel miRNA processing pathway independent of dicer requires argonaute2 catalytic activity Conserved vertebrate mir-451 provides a platform for Dicer-independent Dual strategies for argonaute2-mediated biogenesis of erythroid miRNAs underlie conserved requirements for slicing in mammals The structure of human argonaute-2 in complex with miR-20a RISC is a 5′ phosphomonoester-producing RNA endonuclease Argonaute divides its RNA guide into domains with distinct functions and RNA-binding properties High-throughput analysis reveals rules for target RNA binding and cleavage by AGO2 a site-specific DNA-guided endoribonuclease provides insights into RISC-mediated mRNA cleavage Crystal structure of argonaute and its implications for RISC slicer activity Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity propagation and cleavage of target RNAs in Ago silencing complexes A biochemical framework for RNA silencing in plants A microRNA in a multiple-turnover RNAi enzyme complex Molecular basis for target RNA recognition and cleavage by human RISC Structural insights into RNA cleavage by PIWI Argonaute Water-mediated recognition of t1-adenosine anchors Argonaute2 to microRNA targets Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex Structure-based cleavage mechanism of Thermus thermophilus argonaute DNA guide strand-mediated DNA target cleavage RNAi: The nuts and bolts of the RISC machine Purified Argonaute2 and an siRNA form recombinant human RISC Highly complementary target RNAs promote release of guide RNAs from human argonaute2 A cellular function for the RNA-interference enzyme dicer in the maturation of the let-7 small temporal RNA Control of stress-dependent cardiac growth and gene expression by a microRNA Kinetic analysis reveals the fate of a microRNA following target regulation in mammalian cells The structural basis for RNA slicing by human Argonaute2 Sarkar, S., Gebert, L. F. R. & MacRae, I. J. Structural basis for gene silencing by siRNAs in humans. bioRxiv https://doi.org/10.1101/2024.12.05.627081 (2024) Structural basis for target-directed microRNA degradation The guide-RNA sequence dictates the slicing kinetics and conformational dynamics of the Argonaute silencing complex Crystal structure of silkworm PIWI-clade argonaute Siwi bound to piRNA Mammalian PIWI–piRNA–target complexes reveal features for broad and efficient target silencing GTSF1 accelerates target RNA cleavage by PIWI-clade Argonaute proteins RNA clamping by Vasa assembles a piRNA amplifier complex on transposon transcripts elegans VASA homologs control Argonaute pathway specificity and promote transgenerational silencing Rapid and specific purification of Argonaute-small RNA complexes from crude cell lysates CryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination RELION: Implementation of a Bayesian approach to cryo-EM structure determination Coot: Model-building tools for molecular graphics PHENIX: A comprehensive Python-based system for macromolecular structure solution UCSF Chimera — A visualization system for exploratory research and analysis UCSF ChimeraX: Meeting modern challenges in visualization and analysis Download references Dangsheng Li for insightful suggestions; members of Shen’s lab for discussions; Cryo-EM Facility of Westlake University for providing support on cryo-EM data collection This work was supported by Westlake Education Foundation Zhejiang Provincial Foundation of China (2021R01013) the National Natural Science Foundation of China (32070628) Westlake Education Foundation (041010140118) Zhejiang Provincial Key Laboratory Construction Project and the Westlake Laboratory of Life Sciences to E.Z.S. the National Natural Science Foundation of China (32271261) Zhejiang Provincial Natural Science Foundation of China (LR22C050003) and Institutional Startup Grant from the Westlake Education Foundation (101486021901) to J.W These authors contributed equally: Zhenzhen Li Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province Westlake Laboratory of Life Sciences and Biomedicine Key Laboratory of Structural Biology of Zhejiang Province Department of Pharmacology and Cancer Biology performed experiments and analyzed the data wrote the manuscript with help from all authors; H.G. Overview of cryo-EM density and the structural model of hAGO2D669A-siRNA-target (12-nt) Overview of cryo-EM density and the structural model of hAGO2D669A-siRNA-target (14-nt Conformational dynamics of hAGO2 ternary complexes as complementarity extended towards the siRNA 3ʹ end Conformational dynamics of MILI ternary complexes as complementarity extended towards the piRNA 3ʹ end a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law Download citation DOI: https://doi.org/10.1038/s41422-025-01114-7 Multidrug-resistant bacteria pose a major threat to human health Manipulation of bacterial genes at the transcriptional level is a potential strategy to fight antibiotic-resistant bacterial infections by silencing their resistance genes siRNAs have not been applied to regulate bacterial genes due to the lack of RNAi regulatory machinery efficient methods for delivering siRNAs to bacteria in vivo are not currently available the authors demonstrated that exosomes can serve as delivery vehicles to introduce AGO2-loaded siRNA into the cytoplasm of bacteria and in turn down-regulated gene expression of the mRNA that shares sequence complementarity to the siRNA The scientific significance of these findings is highlighted below: (1) Exosomal siRNA inhibits bacterial gene translation in an AGO2-dependent manner Despite the fact that the siRNA machinery is absent in prokaryote this study demonstrates that exosomal siRNAs can be efficiently delivered into bacteria and silence target bacterial genes The exosomal Argonaute 2 (AGO2) protein forms a complex with siRNAs which is essential for siRNA-mediated gene silencing in bacteria Even though the delivered siRNA was designed for possessing full sequence complementarity to the bacterial mRNA downregulation of the target protein was achieved at the translational level rather than on the level of mRNA stability (as would be expected in mammalian systems) (2) Exosomal siRNA converts MRSA into methicillin-sensitive bacteria Exosomal siMecA (an siRNA designed to target the mecA gene) can downregulate the mecA gene which encodes the penicillin-binding protein 2a (PBP2a) a protein at the heart of the drug-resistance phenotype in MRSA Both in vitro and in vivo (using MRSA-infected mice) data demonstrated that by reducing PBP2a levels by exosome-delivered siRNA-AGO2 complex MRSA can be converted into methicillin-sensitive bacteria (3) In vivo self-assembled siRNAs protect mice from lethal MRSA infection: potential clinical application the authors demonstrated that by intravenous injection of a plasmid encoding all relevant genes for the production of a particular siRNA into mice the mouse liver was producing siRNA-AGO2 loaded exosomes (siMecA-Exos) efficiently targeting MRSA These findings have huge potential clinical relevance since it might be possible to use this approach to also target multi-drug resistant bacterial infections in the human system (4) Exosome-mediated small RNA-AGO2 transport and bacterial gene silencing is a potential pathway by which mammalian cells regulate interacting bacteria in vivo Nanjing University School of Life Sciences Wang, C., et al. (2025). siRNA-AGO2 complex inhibits bacterial gene translation: A promising therapeutic strategy for superbug infection. Cell Reports Medicine. doi.org/10.1016/j.xcrm.2025.101997 Posted in: Cell Biology | Medical Science News | Medical Research News Cancel reply to comment Learn how experts are advancing benzodiazepine analysis and detection using insights from the lab discusses how he is addressing today’s medical challenges using the technology of the future Explore how the Radian ASAP mass spectrometer is being used to streamline and enhance seized drug screening you can trust me to find commercial scientific answers from News-Medical.net please log into your AZoProfile account first Registered members can chat with Azthena, request quotations, download pdf's, brochures and subscribe to our related newsletter content A few things you need to know before we start Read the full Terms & Conditions Metrics details Lipid nanoparticles (LNPs) are widely used for nucleic acid delivery but face challenges like limited targeting and accelerated blood clearance (ABC) effect We design three ionizable oligomers (IOs) that with polylactide-polyethylene glycol (PLA-PEG) The siRNA encapsulated IPMs escape from lysosomes upon cellular uptake A fibroblast activation protein inhibitor modified IPMs (FAPi-IPMs) show higher targeting for activated hepatic stellate cells (HSCs) compared to that for hepatocytes reducing collagen secretion and liver inflammation IPMs and FAPi-IPMs mitigate ABC effect and produce fewer PEG antibodies than LNPs and show minimal apolipoprotein adsorption in vivo compared with LNPs differentiating their targeting effects from LNPs IPMs represent a nucleic acid delivery system with alternative targeting ability and reduced ABC effect current research is still confined within the scope of LNPs unable to avoid the inherent problems of LNPs current micelle-based nucleic acid delivery system has not made satisfactory progress Three IOs with PEG-PLA were prepared by microfluidic chip to obtain IPM siHSP47 and siHMGB1 were delivered to treat hepatic fibrosis by targeting activated hepatic stellate cells with the addition of FAPi-PEG-PLA in the formulation attenuated ABC phenomenon of IPM was observed a Schematic representation of different ionizable oligomers (IO) structures c Particle size distribution of IPMs formed by different IOs with PEG5000-PLA8000 and encapsulation efficiency of IPMs at different N/P ratios (n = 3 samples) IPM achieves the smallest particle size and PDI along with higher encapsulation efficiency and zeta potential of IPMs formed by different IOs (PEG5000-PLA8000) (n = 3 samples) IPM (IOA2) exhibits the smallest particle size and zeta potential of IPMs formed by different segment molecular weights of PEG-PLA (IOA2) (n = 3 samples) IPM (PEG5000-PLA8000) shows the smallest particle size and highest encapsulation efficiency g TEM images of IPMs formed by different IOs with PEG5K-PLA8K and nucleic acid stained by thionine with IPM (IOA2) having the smallest particle size and a higher concentration of RNA with a more punctate distribution Values represent mean ± standard deviation (SD) a Confocal microscopy images of HSCs co-incubated with IPM/Cy5-siNC formed by different IOs for 2 h b Flow cytometry assay demonstrating the highest cellular uptake efficiency of IPM (IOA2 PEG5000-PLA8000) (n = 3 cell culture wells) c The confocal images of the lysosomal escape experiments revealing a decline in the co-localization signals from 4 to 6 h with LysoTracker Green labeling of lysosomes and Hochest 33342 labeling of nuclei d Statistical analysis of Pearson’s R for co-localization of Cy5 signal with lysosomal signal the Pearson’s R in the IPM (IOA2) group was <0.5 changing from moderate to low correlation (n = 3 cell culture wells) e Statistical analysis of Overlap R for co-localization of Cy5 signal with lysosomal signal (n = 3 cell culture wells) f TNS assay for the apparent pKa of the three blank carriers (n = 3 independent experiments) g In vitro RNAi using IPM encapsulating siHSP47 and siHMGB1 formed by PEG5000-PLA8000 and IOA2 with LNP serving as a positive control and LNP/siNC serving as a negative control (n = 3 cell culture wells) The qPCR results showing the effective silencing of the HSP47 (h) and HMGB1 (i) genes in vitro with LNP and Lipo2000 serving as positive controls and LNP/siNC and no siRNA serving as negative controls (n = 3 cell culture wells) Data were analyzed by One-way ANOVA followed by Tukey test a GPC molecular weight distribution of FAPi-PEG5000-PLA8000 and PEG5000-PLA8000 b qPCR results demonstrating upregulation of FAP mRNA expression in HSCs following bleomycin stimulation (n = 3 cell culture wells) c Flow cytometry analysis of the cellular uptake efficiency of IPM and FAPi-IPM by HSCs before and after bleomycin stimulation being more effectively internalized by HSCs with significant differences observed after bleomycin stimulation (n = 3 cell culture wells) d Competitive inhibition assay with varying concentrations of free FAPi showing dose-dependent cellular uptake of FAPi-IPM (n = 3 cell culture wells) e Confocal microscopy results indicating increased intracellular fluorescence intensity in FAPi-IPM/Cy5-siNC-treated cells after bleomycin stimulation with a decrease in intracellular fluorescence intensity as free FAPi concentration increases f Western blot results of IPM/siHMGB1+siHSP47 and FAPi-IPM/siHMGB1+siHSP47 after bleomycin stimulation of HSCs with LNP/siHMGB1+siHSP47 serving as a positive control h Effective downregulation of target genes HSP47 and HMGB1 relative expression by IPM and FAPi-IPM (n = 3 cell culture wells) i Schematic representation of hepatic fibrosis modeling j ROI values in the liver of mice with hepatic fibrosis (n = 3 mice) k Flow cytometry showing FAM positivity of activated hepatic stellate cells (α-SMA labeled) in the livers of mice with hepatic fibrosis (n = 3 mice) l The immunofluorescence results of liver tissues from mice with hepatic fibrosis 24 h after the tail vein injection with DAPI labeling of the nuclei and α-SMA labeling of activated hepatic stellate cells m Fluorescence imaging of the liver 24 h after injection in mice with liver fibrosis Data were analyzed by ANOVA followed by Tukey test a Schematic representation of the dosing regimen for targeting hepatic stellate cells with FAPi-IPM co-delivery of siHSP47 and siHMGB1 for the treatment of hepatic fibrosis (n = 5 mice) b H&E staining results of the liver showing histological changes c Masson’s trichrome staining results of the liver demonstrating a reduction in collagen deposition area in the FAPi-IPM group d Quantification of collagen deposition area e Immunohistochemical staining for HSP47 in the liver showing a decrease in HSP47-positive area and expression in the FAPi-IPM group g Immunohistochemical staining for HMGB1 in the liver showing a decrease in HMGB1-positive area and expression in the FAPi-IPM group The FAPi group shows a decrease in ALT and AST levels to the normal range with no significant difference compared with the negative control group l Liver appearance showing changes in the PBS group and ‌alleviation in the FAPi-IPM group m Body weight curve of mice showing weight recovery after administration in the treatment groups n Western blot results demonstrating inhibition of HSP47 and HMGB1 protein expression in the treatment groups p Significant silencing of HSP47 and HMGB1 protein expression in the FAPi-IPM group in vivo r qPCR results demonstrating significant gene silencing of HSP47 and HMGB1 in the treatment groups Data were analyzed by ANOVA followed by Tukey’s multiple comparisons test a Fluorescence imaging of healthy male C57BL/6 mice (6–8 weeks old) injected with FAPi-IPM LNP enters the liver more rapidly and is rapidly cleared while FAPi-IPM exhibits prolonged liver distribution b Immunofluorescence staining of liver tissue (F4/80 labeling KCs) at 24 h post-injection reveals differences in uptake among formulations c ROI values indicate LNP accumulates most in the liver at 4 h whereas FAPi-IPM shows delayed liver accumulation at 24 h (n = 3 mice) d Pearson’s R of FAM fluorescence and KCs in liver showing rapid phagocytosis of LNP by KCs at 2–4 h (n = 3 mice) e Cell distribution changes show LNP is captured by hepatocytes and Kupffer cells at 2 h while FAPi-IPM enters hepatocytes later at 8 h; IPM enters hepatocytes less overall (n = 3 mice) all formulations are mainly taken up by mononuclear cells with FAPi-IPM showing the highest uptake (n = 3 mice) g The 2-h FAM colocalization results in the spleen indicate that all three formulations strongly colocalize with neutrophils and are mostly taken up by neutrophils h Schematic representation of dosing regimen: PEGylated drug injections on days 0 with pharmacokinetic assays on days 1 and 10 and anti-PEG IgM levels assessed on days 1 i Pharmacokinetic curves of the three PEGylated drugs and pharmacokinetic curves rechallenged on day 10 AUC0-24 of the three PEGylated drug on day 1 (j) and day 10 (k) l Ratio of AUC0-24 rechallenged and AUC0-24 a Schematic illustration of the process for isolating the in vitro and in vivo protein coronae adsorbed on formulations b Silver staining results of protein coronae adsorbed on FAPi-IPM (F) Three times was repeated independently with similar results c–e Protein concentrations collected from the three PEGylated drugs via affinity chromatography columns f The two tubes with the highest protein concentrations collected from each formulation via affinity chromatography columns were defined as the protein coronae adsorbed on the formulations with FAPi-IPM and IPM exhibiting higher protein corona concentrations than LNP g The number of protein types adsorbed on the three formulations h Top 50 proteins adsorbed on the three formulations classified by complement j Top 20 proteins adsorbed on the three formulations Molecular function (k) and KEGG analyses (l) of the protein coronae adsorbed on IPM Data were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test we successfully designed and synthesized three IOs formulations with potential for nucleic acid delivery Through screening based on physicochemical properties we optimized the formulation and developed a potential nucleic acid delivery system we found that IOA2 with oligoalanine tails outperformed IOLA4 and IOLA8 with oligolactic acid tails to the greater stability of amide bonds compared to ester bonds in vivo along with the introduction of more nitrogen it may be due to the shorter alkyl chain between the tertiary amine head group and the hydroxyl group resulting in stronger protonation ability and enhanced capabilities for electrostatic adsorption of siRNA and escape from lysosomes To apply this potential delivery system to the treatment of hepatic fibrosis using siRNA therapy targeting activated HSCs overexpressing FAP we further introduced the targeting molecule FAPi FAPi-IPM exhibited approximately 20% higher uptake by activated hepatic stellate cells compared with IPM the RNAi efficiency targeting HSP47 and HMGB1 respectively increased to 55.39 ± 1.22% and 42.46 ± 3.49% respectively FAPi-IPM showed significant targeting to hepatic stellate cells in mice with hepatic fibrosis with reduced phagocytosis by KCs compared with IPM leading to more effective knockdown of HMGB1 and HSP47 thus effectively alleviating hepatic fibrosis we found that IPM achieved similar therapeutic effects compared with the positive control To explore the differences in the in vivo fate of IPM compared with traditional LNP we further investigated the biodistribution and pharmacokinetic performance of the three formulations The results of biodistribution showed that FAPi-IPM did not exhibit significant targeting ability which may be related to the non-activation of HSCs and low expression of FAP in healthy mice FAPi-IPM was less prone to phagocytosis by KCs and entered HSCs later than IPM IPM and FAPi-IPM were more likely to be taken up by neutrophils than LNP whereas LNP was more likely to be taken up by monocytes and DCs This cellular distribution pattern may lead to faster clearance of LNP compared with IPM and FAPi-IPM Pharmacokinetic results confirmed our hypothesis FAPi-IPM and IPM were metabolized more slowly in the liver than LNP with smaller serum clearance rate constants and longer half-lives the titer of anti-PEG IgM produced by FAPi-IPM was much lower than that of IPM and LNP IPM and LNP exhibited significant ABC effects To further analyze the reasons for the different in vivo fates we examined the differences in the composition and relative abundance of soft protein coronas adsorbed by the three formulations in vitro and in vivo The results showed that FAPi-IPM and IPM adsorbed higher concentrations and more types of proteins in vitro and in vivo than LNP The top 50 proteins adsorbed by FAPi-IPM and IPM contained fewer complement proteins and almost no apolipoproteins and most of the adsorbed proteins were functionally related to binding This explains why they are cleared more slowly in the serum possibly due to rapid adsorption of proteins in the circulation camouflaging themselves as non-“foreign” particles thereby avoiding capture and clearance by the mononuclear phagocyte system The composition of the top 50 proteins adsorbed by LNP differed significantly from the other two formulations containing apolipoprotein E and complement component 3 and the function of the adsorbed proteins was related to oxidoreductase activity with significant involvement in the complement pathway revealing the rapid distribution in hepatocytes and rapid metabolism in vivo ionizable oligomer structure modification is also significant for improving the capability of IPM All animal experiments were conducted following guidelines approved by the Ethics Committee of East China Normal University (ARXM2023088) SPF grade Male C57BL/6 mice aged 6–8 weeks were purchased from the Shanghai Laboratory Animal Center The animals are kept in the Experimental Animal Center of the Institute of Brain Functional Genomics (license number: SYXK (Shanghai) 2014-0006) and IVC independent air supply cages are used inside the barrier environment Anesthetize mice with isoflurane: Adjust the induction concentration to 3–4% wait for the anesthetic to fill the induction box then close the induction box and wait for the animal to be completely anesthetized (this process takes about 2–3 min) The induction box can be gently shaken to check if the animal is completely anesthetized If the animal is flipped over to the side and has not attempted to return to its lying position it indicates that the animal is completely anesthetized mice were anesthetized with 10% chloral hydrate (0.004 ml/g body weight) intraperitoneally and euthanized for cervical dislocation to minimize animal pain and the bodies are disinfected and placed in body bags frozen and stored for harmless incineration as soon as possible PEG-PLA and IOs were dissolved in anhydrous ethanol at a ratio of 40.2:59.8 (mol/mol) as the organic phase while siRNA (N/P = 5.45) was dissolved in pH 5.0 10 mM citrate buffer as the aqueous phase with twice the volume of the aqueous phase in pH 6.0 10 mM citrate buffer as the buffer phase The aqueous phase and organic phase were mixed at a flow rate of 3:1 using a microfluidic device (NEXSTAR NANO2 The mixture was then centrifuged in ultrafiltration centrifuge tubes (MWCO = 10 kDa) at 2000 × g for 20 min three times followed by elution with PBS and collection of the final product IPM FAPi-IPM had a composition of PEG-PLA: ionizable oligomers: FAPi-PEG-PLA = 40.2:59.8:1 (mol/mol) with the rest of the process remaining consistent ALC-0315/DSPC/Chol/PEG2000-DSPE was dissolved in ethanol at a ratio of 50:10:38.5:1.5 (mol/mol) as the organic phase siRNA (N/P = 5.67) was dissolved in pH 5.0 10 mM citrate buffer as the aqueous phase and two times the volume of the aqueous phase in pH 6.0 10 mM citrate buffer as the buffer phase C5-3 chips were selected and prepared by microfluidic instrumentation with an aqueous phase: organic phase at a flow rate of 3:1 centrifuged three times at 2000 × g for 20 minutes in ultrafiltration centrifuge tubes (MWCO = 10 kDa) eluted by the addition of PBS and the collection of final products Encapsulation efficiency was determined using Quant-iT Ribogreen RNA assay Dynamic light scattering (DLS) technique was employed with Zetasizer Nano ZS (Malvern UK) to measure the nanoparticle hydrodynamic radius Transmission electron microscopy (TEM) JEOL 100-CX (JEOL USA Inc. MA) was used to observe the morphology of nanoparticles followed by staining of RNA with thionine (0.1 mM) to observe the inner position of nucleic acid drugs Samples were prepared at a concentration of 0.5 mg/mL The prepared formulation was co-incubated with 10% (v/v) serum and the stability in serum was assessed by measuring the particle size within 7 days The pKa of the micelles was determined using the TNS method Blank nanoparticles were prepared and diluted in a series of citrate buffer solutions with pH ranging from 3 to 10 at a concentration of 6 μM TNS was dissolved in DMSO at a concentration of 300 μM and 2 μL of the TNS solution was mixed with 100 μL of blank nanoparticles Fluorescence intensity was measured under excitation at 325 nm and emission at 435 nm The fluorescence intensity readings were curve-fitted against the pH values to calculate the pKa IPMs and LNP were co-incubated with HSCs for 2 The cells were stained with Hoechst 33342 for nucleus labeling and LysoTracker Green DND-26 for lysosome labeling The changes in co-localization signals between Cy5 fluorescence and lysosomes were observed under a laser scanning confocal microscope (LEICA Pearson’s R and Overlap R were calculated and analyzed After co-incubation of micelle-encapsulated Cy5-siNC with activated HSCs for 6 h flow cytometry was performed to measure Cy5+ cells and calculate the cellular uptake rate stained with Hoechst 33342 for nucleus labeling and sealed with anti-fluorescence quenching mounting medium The Cy5 fluorescence signal in the cells was observed under a confocal microscope HSCs activation was induced by culturing cells in medium supplemented with bleomycin (1 µg/mL) for 24 h The medium was then replaced with opti-MEM and IPM/siHSP47+siHMGB1 with or without FAPi targeting moiety was added for a 6-hour co-incubation while protein was harvested after 72 h for Western blot analysis to assess RNAi efficiency at both the gene and protein levels Male C57BL/6 mice aged 6–8 weeks were intraperitoneally injected with a 20% CCl4 solution in mineral oil (2 mL/kg body weight) three times per week for 12 consecutive weeks Liver sections were evaluated for fibrosis scoring using the Metavir fibrosis scoring system and the modeling was considered successful when the pathology of the mouse liver reached stage S3 Groups of control and hepatic fibrosis model mice (n = 3) were intravenously injected with FAPi-IPM and IPM which encapsulated Cy5-siNC (1 mg/kg) and kidneys were harvested to observe the fluorescence signal intensity Groups of control and hepatic fibrosis model mice (n = 3) were intravenously injected with LNP FAPi-IPM or IPM which all encapsulated FAM-siNC (1 mg/kg) Liver tissues were further processed for histological analysis with α-SMA marking hepatic stellate cells (HSCs) CD146 marking hepatic sinusoidal endothelial cells The spleen was also examined for CD45-positive leukocytes and CD68-positive macrophages The targeting for HSCs in the liver was evaluated liver perfusion was performed to separate non-parenchymal cells Activated HSCs were labeled with α-SMA recombinant mouse mAb followed by staining with goat anti-mouse AF647 secondary antibody The uptake rate of activated hepatic stellate cells in fibrosis liver was defined as the proportion of FAM-positive cells C57BL/6 male mice with successfully established models were divided into four groups (n = 5): IPM/siHSP47+siHMGB1 with and without FAPi targeting moiety Treatment was administered twice a week for two consecutive weeks (1 mg/kg) serum levels of alanine transaminase (ALT) and tumor necrosis factor-α (TNF-α) were measured and compared with pre-treatment levels Liver and other major organs were harvested for H&E and Masson’s trichrome staining to observe collagen fiber proliferation and qPCR (Bio Rad) were employed to detect the expression levels of HMGB1 and HSP47 proteins and genes in liver tissues Male C57BL/6 mice aged 6–8 weeks were intravenously injected with LNP and kidney were harvested for fluorescence imaging using a small animal live imaging system (Bruker Corporation) Immunofluorescence staining was performed for liver and spleen tissues α-SMA was used to label hepatic stellate cells (HSCs) CD146 for liver sinusoidal endothelial cells Co-localization analysis with FAM was conducted for each marker FAPi-IPM or IPM which all encapsulated FAM-siNC RNA (1 mg/kg) Liver tissue was minced and incubated at 37 °C for 0.5 h then ground through a 70 μm cell strainer into a 50 mL centrifuge tube The sample was washed with 15 mL ice-cold HBSS-CaCl2 and centrifuged at 4 °C The supernatant was transferred to a new 50 mL tube for non-parenchymal cell isolation while the pellet was washed twice with 15 mL ice-cold HBSS-CaCl2 The final pellet was resuspended in 1 mL PBS to obtain parenchymal cells The supernatant for non-parenchymal cell isolation was further centrifuged at 4 °C The cell pellet was resuspended in 1 mL PBS and gently layered on a 25% (2.5 mL)/50% (2 mL) Percoll gradient The sample was centrifuged horizontally at 4 °C The intermediate cell layer (2 mL) was collected The pellet containing non-parenchymal cells was retained Hepatocytes were labeled with albumin antibody followed by staining with goat anti-rabbit BV421 secondary antibody Liver sinusoidal endothelial cells were labeled with APC-CD146 the spleen was transferred to an EP tube with 1000 μL PBS and ground through a 70 μm cell strainer into a six-well plate and the cell suspension was collected and centrifuged at 4 °C and 1 mL red blood cell lysis buffer was added to the cell pellet The lysis step was repeated until all red blood cells were removed cells were washed with ice-cold PBS and centrifuged at 4 °C Spleen tissues were ground over a 70 μm cell strainer and cell suspensions were collected and lysed Dendritic cells were labeled by BV421-CD11c NJ) was used to analyze the percentage of FAM+ cells Enzyme-linked immunosorbent assay (ELISA) was performed to measure the titers of anti-PEG IgM antibodies A high-binding 96-well plate was coated with mPEG 2000-DSPE at a concentration of 20 µg/mL in ethanol The plate was then blocked with 5% BSA for 1 h were added to the plate in a serial dilution manner the plate was incubated with Goat Anti-Rat IgM mu chain (HRP) secondary antibody (ab97180) diluted 1:5000 in 0.1% BSA for 1 h at 37 °C and the plate was incubated for 8 min for color development The reaction was terminated with 0.18 M sulfuric acid The optical density (OD) at 450 nm was measured using a microplate reader (Biotek) The emulsion should be disrupted using methanol after which the fluorescence intensity of FAM in the serum should be measured A standard curve should be constructed based on the fluorescence intensity of the FAM-siNC from which the concentration of siRNA can then be calculated Using a previously reported method by Zhan’s lab50 20 µL of FAM-siNC-loaded micelles and LNP (maintained at the same concentration by fluorescence quantification) were separately mixed with 60 µL of His-tagged PEG-scFv (corresponding to 400 µg of antibody for micelles and 100 µg for LNP) and incubated at room temperature for 10 min the mixtures were combined with 20 µL of C57BL/6 mouse serum and incubated in vitro at 37 °C for 30 min FAPi-IPM or IPM which all encapsulated Cy5-siNC (1 mg/kg) (maintained at the same concentration by fluorescence quantification) Blood samples (20 µL) were collected from the tail vein using EDTA anticoagulant The samples were directly mixed with 60 µL of His-tagged PEG-scFv (same as before) Ni-NTA was packed into a 3 mL gravity column and the pre-mixed solutions were slowly applied to enable a complete interaction of Ni and His-tagged PEG-scFv for 30 minut at room temperature Fractions #1-#12 were collected using PBS containing 5 mM imidazole (to wash out unbound protein components) while fraction #13 onwards was eluted using 100 µL of PEG8000 in PBS (100 mg/mL) and collected into #14 for fluorescence fraction The components collected from the three formulations were quantified using BCA assay and two tubes were combined and denatured by adding 1/4 of 5× loading buffer and heating at 100 °C for 10 min following SDS-PAGE separation with silver staining Approximately 200 ng of total peptides from each sample were separated using a nanoUPLC liquid chromatography system (nanoElute2) coupled to a mass spectrometer equipped with a nanospray ion source (timsTOF Pro2) Chromatographic separation was achieved using a 75 μm ID × 15 cm reversed-phase column (IonOpticks C18-CSI The raw data files were searched against a database using the SpectroMine (4.2.230428.52329 Biognosys AG) software application Pulsar retrieval engine for qualitative analysis label-free quantification was performed by summing the intensities of all peptides in the sample The top 50 proteins in protein corona on the three nanocarriers were functionally categorized using the DAVID database followed by Gene Ontology (GO) and Encyclopedia of Genes and Genomes (KEGG) analyses Flow cytometry data were processed using FlowJo software Laser confocal microscopy and immunofluorescence results were analyzed for colocalization using the Colocalization Finder plugin in ImageJ Immunohistochemistry results were analyzed for positivity rates using the IHC plugin in ImageJ The Split Channels function in ImageJ was used to convert images to single-color channel 8-bit grayscale images and the average grayscale values were calculated to determine the average immunofluorescence intensity The Color Threshold function in ImageJ was used to set ranges and calculate the collagen deposition area in Masson’s staining All data are presented as mean ± standard deviation Student’s t test (two-sided) was used to analyze data between two groups analysis of variance (ANOVA) was conducted followed by Tukey’s multiple comparison test Statistical significance was defined as ns (p > 0.05) All data analyses were performed using GraphPad Prism v9.5 software Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Liquid crystalline inverted lipid phases encapsulating siRNA enhance lipid nanoparticle mediated transfection various applications and unlimited future prospects Safety and efficacy of the BNT162b2 mRNA Covid-19 Vaccine Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine Multiomics analysis of naturally efficacious lipid nanoparticle coronas reveals high-density lipoprotein is necessary for their function Peptide-encoding mRNA barcodes for the high-throughput in vivo screening of libraries of lipid nanoparticles for mRNA delivery Screening for lipid nanoparticles that modulate the immune activity of helper T cells towards enhanced antitumour activity Polyethylene glycol (PEG)-associated immune responses triggered by clinically relevant lipid nanoparticles in rats Selective organ targeting (SORT) nanoparticles for tissue-specific 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Accurate pKa calculations for carboxylic acids using Complete Basis Set and Gaussian-n models combined with CPCM continuum solvation methods Download references This work was supported by the National Natural Science Foundation of China (82373813 [Yan] the National Key R&D Program of China (Grant No Shanghai Frontiers Science Center of Molecule Intelligent Syntheses These authors contributed equally: Ziyu Zhou Institute of Biomedical Engineering and Technology Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development School of Chemistry and Molecular Engineering Ministry of Education & Department of Pharmacy Department of Pharmacology School of Basic Medical Sciences & State Key Laboratory of Molecular Engineering of Polymers Eye Institute and Department of Ophthalmology and Z.Y.: Conceived and designed the experiments Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work Download citation DOI: https://doi.org/10.1038/s41467-024-55721-w Metrics details The use of combinatorial siRNAs shows great promise for drug discovery but the identification of safe and effective siRNA combinations remains challenging we develop a massively multiplexed technology for systematic screening of siRNA-based cocktail therapeutics We employ composite micro-carriers that are responsive to near infrared light and magnetic field to achieve photoporation-facilitated siRNA transfection to individual cells randomized gene silencing by different siRNA formulations can be performed with high-throughput single-cell-based analyses we test more than 1300 siRNA combinations for knocking down multiple genes related to tumor growth discovering effective 3-siRNA formulations with an emphasis on the critical role of inhibiting Cyclin D1 and survivin along with their complementary targets for synergic efficacy This approach enables orders of magnitude reduction in time and cost associated with largescale siRNA screening and resolves key insights to siRNA pharmacology that are not permissive to existing methods which may diminish the action of a single siRNA and substantially offset the silencing effects the selection of combinatorial siRNAs to form the most effective cocktail primarily relies on examining the silencing efficacy and safety of individual siRNAs which is time-consuming and lacks the information regarding the synergic or counteractive function among them it is challenging to screen different siRNA combinations and to determine the optimal cocktails and systematic high-throughput platform technology would be greatly beneficial to enhance the clinical adoption and speed up the development of siRNA-based therapeutics This work aims to address the unmet needs by developing a high-throughput technology for screening siRNA-based cocktail therapeutics in a massively multiplexed we employ a multilayer functionalization of hierarchically assembled nano-/micro composite carriers (Knock-beads) rendering them with responsiveness to near infrared light and magnetic field to facilitate photoporation-induced cellular transfection of RNAs with single cell encoding capability randomized gene silencing by different siRNA variants is performed Our technology is capable of scaling to screening hundreds or even thousands of siRNA cocktail formulations in a single well accelerating the testing of siRNA cocktails Such benefits are not just limited to reducing the time and cost associated with siRNA screening by powers of magnitude but also answer fundamental siRNA questions that are not addressable by existing methods we first perform a small screen with six cell apoptosis or cell cycle related siRNAs to test 54 siRNA combinations to seek the most effective cocktail for inhibiting cancer cell growth With a customized multiplexed encoding strategy we further demonstrate an expanded screening involving 20 siRNA target genes and test more than 1300 cocktail formulations composed of 1 or 3 siRNAs for combinatorial knockdown of genes related to tumor growth leading to the identification of the most effective 3-siRNA combinations as potential anti-cancer therapeutics with an emphasis on the critical role of inhibiting Cyclin D1 and survivin as well as their complementary gene targets for synergic co-operation We believe that this study demonstrates the path to a systematic and high-throughput platform technology that is greatly useful to accelerate further development of siRNA-based therapeutics and enhance their clinical adoption Fe3O4 beads are combined with Au nanorods and coated with PDDA The Au layer provides Knock-beads with photothermal responsiveness facilitating intracellular delivery of siRNA through photoporation by 980 nm NIR laser Knock-beads are immobilized on top of cells enabling efficient cellular transfection of siRNAs by photoporation and single-cell encoded gene silencing in a large number of cells Through only one-shot assay in a cell culture more than 1300 siRNA combinations can be simultaneously analyzed to reveal the optimal siRNA therapeutic cocktail a multiscale assembly of micro- and nano- structures along with a functionalization scheme was developed to fabricate the Knock-bead with multiple functions including magnetic controllability for tight cell-bead interfacing appropriate surface coating for siRNA loading/off-loading and responsiveness to near infrared light (NIR) for photoporation performs 3 functions: (1) a vehicle for siRNA delivery; (2) a spatially resolvable cellular interface for immobilization on the cell membrane by a magnetic field; (3) the means for fluorescence encoding of single-cell gene silencing by different siRNA combinations the total number of possible gene silencing combinations would be: a The chemical synthesis route of Knock-beads b SEM images showing the core part of Knock-beads at different scales: Fe3O4-Au (top c Fluorescence image showing Fe3O4 magnetic beads that were first labeled with Alexa-647 dye and then conjugated to Au nanorods d Fluorescence image showing the Au nanorods that were first labeled with Alexa-647 dye and then conjugated to Fe3O4 magnetic beads e Dynamic light scattering results showing the size variation of Fe3O4 Fe3O4-Au-PDDA beads and Fe3O4-Au-PDDA-siRNA conjugates (8 × 105 beads Fe3O4-Au-PDDA beads and Fe3O4-Au-PDDA-siRNA conjugates (8 × 105 beads g Fluorescent image of Knock-beads in PBS solution Three independent experiments were conducted (b Source data are provided as a Source Data file a UV absorbance spectrum of Au nanorods b Infrared imaging of the photothermal effect of Knock-beads (top) and DI water (control A 980 nm laser (16 μs pulse width) with an intensity of 0.6 W/cm2 was applied for 30 s c Time—temperature graph of Knock-beads (red) or DI water (blue) when stimulated by 980 nm laser d Impact of different NIR-irradiation times (2 s and 12 s) on cell viability 24 h after photoporation with Knock-beads e Characterization of siRNA loading efficiency of Knock-beads using prelabeled siRNAs pull-down assay f The fluorescence distribution of a siRNA-loaded Knock-bead along the yellow line in panel (g) g 3D fluorescence images of Knock-beads (blue) loaded with siRNA (red) and the top For effective gene silencing screening, cell viability is crucial for unbiased results, especially after the photoporation using the Knock-beads. 24 h following NIR irradiation, we assessed cell viability using the 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) assay (Fig. 3d) 0.6 W/cm2) with varying irradiation times (2 the cell viability was evaluated to optimize the irradiation parameters We found that more than 85% of the cells were viable after an 8 s NIR irradiation at 0.6 W/cm2; longer NIR irradiation of 10 s and 12 s reduced the cell viability to 70% and 55% NIR irradiation duration was kept brief ( < 8 s) to avoid any major damage or interference to regular cell function thus ensuring the reliability for later use for high-throughput screening a–c Intracellular delivery with a magnetic field where RNAs are labeled using cy5 (red) and Fam (green) d Cell segmentation visualized by a customize algorithm The cell outline was visualized in different colors to help cell segmentation e Number of cells categorized differently in (d): control refers cells without Knock-beads f 3D images of Knock-beads(green) and diffused siRNA(red) and the front and top view g 3D images of Knock-beads(green) and cell actin(red) and the front and top view h Cells treated with two types of Knock-beads: anti-actin (KB-A i Magnified view of (h) cells that contacts with different Knock-beads: top-left is control j Quantification of fluorescence intensity for actin and tubulin: control refers cells without Knock-beads three independent experiments were conducted These results establish the Knock-beads as a legitimate tool for high-throughput gene silencing with single-cell addressability and strongly support the development of more complex assays to enable screening of combinatorial siRNA cocktails a Effect of siRNA number on the induction of cellular apoptosis b Effect of siRNA numbers on the inhibition of cellular proliferation c Scattered plot showing the distribution of the knock-combo efficacy classified by the number of siRNAs The dotted line (f(x) = x) refers to the total anti-tumor efficacy with equal weight of cellular apoptosis and proliferation in determining the final efficacy Comparison between single or dual pathway gene silencing by different Knock-combos composed of 3 (d) or 2 (e) siRNAs The black dots indicate the measurements from individual cells f Centroid distribution illustrating the apoptosis induction and proliferation inhibition efficacy of Knock-combos containing specific siRNA g Heatmap showing the apoptosis induction and proliferation inhibition efficacy for Knock-combos containing specific siRNA The color index indicates the Z-score normalized phenotype quantification the data was analyzed using unpaired one-way ANOVA; for panel (d the data was analyzed using non-parametric Mann–Whitney test the black dots indicate an overlay of individual data points the whisker limit is 1.5 times the interquartile range due to the existence of complex signal transduction networks that are likely to be complementary or parallel and compensate for the single-gene silencing effects the use of siRNA cocktails targeting multiple disease-related pathways represents an attractive and robust approach to develop siRNA-based therapeutics emphasizing the critical role of inhibiting Cyclin D1 in the construction of effective siRNA cocktails as potential anti-cancer therapeutics a A set of 20 siRNAs targeting 6 different cellular pathways: membrane interaction (5 siRNAs) The screening space (1350) is composed of 20 1-siRNA b Phenotypic analysis of apoptosis induction for all the tested 1-siRNA (blue) 2-siRNA (red) and 3-siRNA (yellow) Knock-combos Data were collected from more than 20 cells for each of the 1315 Knock-combos c Boxplots with scattered data points showing the comparison of the efficacy on apoptosis induction for combinatorial targeting of single d K-means clustering analysis of all the Knock-combos reveals two distinct clusters in the UMAP space e Boxplots showing the comparison of apoptosis induction for the 2 identified Knock-combo clusters f Enrichment analysis of the 20 siRNA targets in each identified Knock-combo clusters g An interaction network showing the synergic co-operation in apoptosis induction for the 20 siRNAs to different gene targets where the line color and thickness indicate the phenotypic efficacy of the corresponding combination The black dots indicate an overlay of individual data points HER2 or Cyclin D1 showed the most connections to other genes suggesting the important contributions from siRNAs targeting supplementary genes uPAR gene appeared to be the best silencing partner for many leading siRNA targeting either survivin or HER2 These screening results provide valuable insights into the construction of combinatorial siRNAs therapeutics for anti-tumor purpose Since the discovery of RNAi and the demonstration of siRNA in post-transcriptional gene silencing there has been tremendous interest and effort in harnessing siRNAs for biomedical research and drug development One of the major issues is that effective gene silencing by siRNA is often hindered by the presence of complementary or parallel signal transduction pathways which may counteract the action of a single siRNA and substantially offset the therapeutic silencing effects the use of cocktail siRNAs has been suggested as a feasible and promising drug discovery route the identification of the most safe and efficient siRNA combination remains challenging and a crucial bottleneck This study aims to address the problem by developing a high-throughput technology for screening siRNA-based cocktail therapeutics we believe that the most meaningful siRNA cocktail screening should be oriented towards Knock-combos consisting of 2 to 3 siRNAs the total possible Knock-combos would reach 178,433,024 which is already a scale inaccessible to any existing methods hundreds or even thousands of siRNA cocktail formulations could be assayed in just a single well of cell culture which would be tremendously useful for accelerating the development of siRNA-based therapeutics Such benefits are not just limited to reducing the time and cost associated with siRNA screening by power of magnitude but also rely on the capability to answer fundamental siRNA questions that are not addressable by existing methods such as: (1) whether multiplexed gene silencing are more efficient than single target knockdown; (2) whether a combination of different signaling pathways related siRNAs are more efficient than multiple siRNAs targeting a single pathway; (3) which siRNAs are the most important elements in constructing an effective siRNA cocktail; (4) what is the optimal combination of siRNAs (precise and concise) to achieve the best synergic therapeutic gene-silence The answers to these fundamental questions could be extensively explored by machine learning and bioinformatic analysis of the large number of single-cell gene silencing data enabled by the scCode-fection technology as we have demonstrated in the screening of more than 1300 siRNA combinations concerning 20 genes in seeking the most effective cocktail for potential inhibition of cancer cell growth We believe this study will lead to paths to a systematic and high-throughput platform that would be greatly useful to accelerate further development of siRNA-based therapeutics and enhance their clinical adoption The fabrication is based on two processes: Connection of Fe3O4-NH2 with gold nanorods through glutaraldehyde and positive coating of PDDA 20 μL stock solution of Fe3O4-NH2 (Invitrogen Thermo Fisher 2 × 109 beads/mL) were incubated in 15% Glutaraldehyde aqueous solution (Sigma-Aldrich) at room temperature on a lab rotator (DLAB the beads were incubated with 100 μL of Au-NH2 solution (Sigma-Aldrich 1μm in length) while rotating on a lab rotator (DLAB the assemblies were collected with a magnetic field and blocked with 3% bovine serum albumin (BSA) for 2 h 1 μL NHS-Alexa-514 (Thermo Fisher) was added to Fe3O4-Au assemblies and rotated at 30 rpm (DLAB MX-RD-Pro) for 2 h followed by three rinses with deionized water Other codes were prepared in a similar way using a different mixture of the three fluorescent dyes PDDA was coated for loading negative charged RNAs Fe3O4-Au assemblies were washed twice with deionized water and then resuspended in 200 μL of 15% (v/v) PDDA solution (Sigma Aldrich) The suspension was sonicated in a bath for 10 min The Knock-beads were then washed three times with deionized water and resuspended in 100 μL of PBS for storage and Fe3O4-Au-PDDA was determined using a Particle Size Analyzer (Zeta sizer a 20 μL solution of each particle type (Fe3O4 and Fe3O4-Au-PDDA) was added to 500 μL of deionized water The Particle Size Analyzer was set to detect the size by selecting water as the detection solution and the zeta size mode with the only difference being the selection of the zeta potential mode To determine the RNA loading efficiency of Knock-beads we utilized fluorescence detection of unbound RNA-cy5 the Knock-beads were incubated with RNA-cy5 in deionized water rotating at 30 rpm at room temperature The mixture was then subjected to a magnetic field ensuring that the beads were attracted to the side of the tube The supernatant was then carefully collected The fluorescence intensity of the supernatant was measured using MD SpectraMax M5e microplate reader The RNA loading efficiency was calculated based on the fluorescence intensity of the supernatant as well as the initial concentration of RNA-cy5 This procedure was repeated three times to ensure reproducibility and accuracy of the results Scanning electron microscopy (SEM; Philips FEG SEM XL30) was employed to characterize the morphology of Fe3O4-Au the Fe3O4-Au assemblies were deposited onto a silicon wafer and allowed to dry overnight The dried samples were imaged using a scanning electron microscope at a voltage of 10 kV or 20 kV SEM images were analyzed using ImageJ software to determine the size distribution of the Fe3O4-Au assemblies The fluorescence images were captured with confocal laser scanning microscope (Leica SP8 the Knock-beads were mounted with an antifade mounting medium and imaged using a confocal laser scanning microscope and scanning speed were fixed across the microscopy process Scanning was performed with a 1 μm step size along the Z-axis spanning the whole cell 3D reconstruction was then performed to visualize the relative position of Knock-beads Confocal microscopic images were analyzed using ImageJ software to determine the fluorescence labeling and distribution of Knock-beads For data collection from a screening, imaging-based decoding was performed according to the spectrum encoding (Supplementary Table 5) The excitation of the 4 quantum dots (emission peak at 547 nm The emission window was set to be 10 nm wide we employed the navigator model of Leica Microsystems to capture over 200 tiles from a single culture dish for large area coverage The resulting large-scale tiles were merged using Leica software to ensure smooth calibration along each edge of the view arrays A549 (CRM-CCL-185 from American Type Culture Collection Duke Kunshan University) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in a humidified atmosphere containing 5% CO2 and cells were subcultured at 80–90% confluency using Trypsin-EDTA containing 1 × 107 beads/mL in 2 mL of PBS was subjected to laser irradiation using a 980 nm laser with a power density of 0.6 W/cm2 and pulse width of 16 μs Infrared photography was recorded using an infrared camera (Nikon) Cell viability was tested by adding 5 × 104 beads to A549 cells in a 24-well plate (1 × 105 cells per well) followed by irradiation with a 0.6 W/cm2 in 16 μs pulse width laser for 2 1 mg/mL) was added and incubated for further 4 h The MTT-containing medium was then removed and the formazan crystals were dissolved in dimethyl sulfoxide (DMSO) The absorbance of each well was measured at 570 nm using Molecular Devices SpectraMax M5e Microplate Reader The viability of the cells was expressed as the percentage of the absorbance relative to the control group The Knock-beads were used to deliver fluorescence RNA into A549 using photoporation under a magnetic field cells were seeded in glass bottom plates at a density of 1 × 106 cells/plate and allowed to adhere overnight The cells were then incubated with the Knock-beads at a concentration of 4 × 105 for 30 s to allow for absorption of the beads after discharged medium The cells were then irradiated with a 980-nm laser (16 μs pulse width) for 8 s at a power of 0.6 W/cm2 within a magnetic field the cells were washed 2 times with PBS within a magnetic field The efficiency of RNA delivery was determined by visualizing the cells under a confocal microscope and analyzing the uptake of the fluorescence dye with ImageJ Actin and tubulin were stained for evaluation of the single cell gene knockdown efficiency TUNEL and Edu were stained to evaluate the anti-tumor efficiency of related Knock-combo cells were washed with PBS and fixed with 4% paraformaldehyde (Thermo) at room temperature for 30 min cells were incubated in triton X-100 (0.1% in PBS) at room temperature for 10 min and then rinsed with PBS 2 μL of Actin-Tracker (Actin-tracker kit from Beyotime) were added to each dish and incubated in the dark at room temperature for 60 min 2 μL of Tubulin-tracker (Tubulin-tracker kit from Beyotime) were added to each dish and incubated in the dark for 60 min One Step TUNEL Apoptosis Assay Kit from Beyotime was used 20 h after photoporation Terminal Deoxynucleotidyl Transferase (TdT) and fluorescein-dUTP were added to each dish and cells were incubated at 37 °C for 1 h in the dark allowing for TdT-mediated dUTP Nick-End labeling BeyoClick™ EdU Cell Proliferation Kit from Beyotime was used 5-ethynyl-2’-deoxyuridine (EDU) at 10 mM was added to the cells before fixation and permeabilization and 50 μL of click additive solution were added to each dish followed by a 30-minute incubation in dark at room temperature the images were first loaded into the Python-based CellPose software The brightfield channel was used to derive an evaluation of the expected cell diameter in pixels The grayscale fluorescent channels were then selected for the segmentation processing The CellPose algorithm was then executed to generate a mask outlining the boundary of individual cells the cell outlines were exported as an ImageJ ZIP file which was imported to ImageJ using ROI Manager for further analysis For the centroid approach in Fig. 6f the centroid of a specific type of siRNA Knock-combo denoted as \({{{\boldsymbol{c}}}}_{{{\boldsymbol{s}}}}({x}_{s},\,{y}_{s})\) refers to the average position of a group of two-dimensional points For each siRNA combo \(({x}_{i},\,{y}_{i})\) where \({x}_{i}\) represents the efficacy of apoptosis induction and \({y}_{i}\) represents the efficacy of proliferation inhibition the centroid for n Knock-combos is obtained by: For clustering analysis in Fig. 7 The optimal number of clusters (k) was determined using silhouette statistics The K-Means algorithm was then applied to the preprocessed data with multiple random initializations to find the global optimum for unsupervised derivation of optimal number clusters of the Knock-combos based on the phenotypic features related to cellular apoptosis or proliferation To prioritize the enrichment of a gene targeted siRNA in the identified Knock-combo clusters (Fig. 7f) the relative enrichment ratio of each siRNA was calculated using the Z-score normalization defined as: where X is the appearance frequency of a siRNA for all the Knock-combos in a cluster μ is the mean appearance frequency of all siRNAs in that cluster and σ is the standard deviation of the appearance frequency for all siRNAs in the same cluster The appearance frequency of a siRNA in a cluster is calculated as: counts of Knock-combos containing the siRNA divided by the total number of siRNAs appeared in all Knock-combos of that cluster To analyze the interaction network and to show the synergic co-operation in apoptosis induction for the siRNAs to their 20 gene targets (Fig. 7g) The siRNAs were represented as the nodes connected by lines where the line color and thickness indicate the phenotypic efficacy of the corresponding combination siRNA silencing of the node genes The line color and thickness were indexed by calculating the mean apoptosis induction among all the Knock-combos containing the two node gene siRNAs (connection score) A hub score of each node was also derived by taking the sum of all the connections scores for the lines originated from one node At least three independent biological replicates were used for all experiments Statistical significance for comparing two experimental conditions is calculated by two-tailed unpaired t test; for comparing one control with multiple experimental conditions the statistical significance is calculated using the unpaired one-way ANOVA test unpaired nonparametric Mann–Whitney test is used to compare different groups A p value less than 0.05 is considered to be statistically significant and all the p values are indicated in the respective figures Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article The data generated in this study are provided in the Supplementary Information/Source Data file. Source data are provided with this paper 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recent advances in methodologies and regulations Pulsed electromagnetic fields stimulate osteogenic differentiation in human bone marrow and adipose tissue derived mesenchymal stem cells Highly multiplexed spatial mapping of microbial communities Approved HIV reverse transcriptase inhibitors in the past decade Download references This work was supported by National Natural Science Foundation of China (U20A20194 P.S.) from the Research Grants Council of Hong Kong SAR Shenzhen-Hong Kong-Macau Science and Technology Program (Category C by a Kunshan Shuangchuang Talent Grant (KSSC202102069 Support from Innovation and Technology Commission of Hong Kong through the Center for Cerebro-Cardiovascular Health Engineering and funds from City University of Hong Kong (7005084 These authors contributed equally: Feng Guo Hong Kong Centre for Cerebro-Cardiovascular Health Engineering Department of Chemical and Biological Engineering The Hong Kong University of Science and Technology Division of Natural and Applied Sciences & Global Health Research Center Center of Super-Diamond and Advanced Films (COSDAF) designed the experiments and carried out the investigation contributed to the validation of siRNA gene silencing and F.Gao contributed to the informatic analysis of the large-scale screening data contributed to the writing of the manuscript Download citation DOI: https://doi.org/10.1038/s41467-024-53419-7 Metrics details Therapeutic small interfering RNA (siRNA) requires sugar and backbone modifications to inhibit nuclease degradation metabolic stabilization by phosphorothioate (PS) the only backbone chemistry used clinically may be insufficient for targeting extrahepatic tissues synthesis and characterization of extended nucleic acid (exNA) consisting of a methylene insertion between the 5′-C and 5′-OH of a nucleoside exNA incorporation is compatible with common oligonucleotide synthetic protocols and the PS backbone provides stabilization against 3′ and 5′ exonucleases and is tolerated at multiple oligonucleotide positions A combined exNA–PS backbone enhances resistance to 3′ exonuclease by ~32-fold over the conventional PS backbone and by >1,000-fold over the natural phosphodiester backbone The improved efficacy and durability imparted by exNA may enable therapeutic interventions in extrahepatic tissues both with siRNA and with other oligonucleotides such as CRISPR guide RNA The data supporting the findings of this study are available from the corresponding authors upon reasonable request. The raw NMR data are included in the Supplementary Information The chemical evolution of oligonucleotide therapies of clinical utility Treating disease at the RNA level with oligonucleotides mechanism and clinical status of antisense oligonucleotides and duplex RNAs structure and function of approved oligonucleotide therapeutics Delivery of therapeutic small interfering RNA: the current patent-based landscape Re-engineering RNA molecules into therapeutic agents The chemical structure and phosphorothioate content of hydrophobically modified siRNAs impact extrahepatic distribution and efficacy Overcoming the challenges of tissue delivery for oligonucleotide therapeutics From bench to bedside: improving the clinical safety of GalNAc–siRNA conjugates using seed-pairing destabilization Analysis of RNA exonucleolytic activities in cellular extracts Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc–siRNA conjugates Advanced siRNA designs further improve in vivo performance of GalNAc–siRNA conjugates Investigating the pharmacodynamic durability of GalNAc–siRNA conjugates RNA interference using boranophosphate siRNAs: structure–activity relationship High potency silencing by single-stranded boranophosphate siRNA Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications A single amide linkage in the passenger strand suppresses its activity and enhances guide strand targeting of siRNAs Amide modifications in the seed region of the guide strand improve the on-target specificity of short interfering RNA Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity The α-(l)-threofuranosyl nucleic acid modification improves stability and Ago2 binding and mitigates off-target effects of small interfering RNAs Single-stranded siRNAs activate RNAi in animals 5′-(E)-Vinylphosphonate: a stable phosphate mimic can improve the RNAi activity of siRNA–GalNAc conjugates siRNA carrying an (E)-vinylphosphonate moiety at the 5′-end of the guide strand augments gene silencing by enhanced binding to human Argonaute-2 5′-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo Structurally constrained phosphonate internucleotide linkage impacts oligonucleotide–enzyme interaction and modulates siRNA activity and allele specificity Phosphoryl guanidines: a new type of nucleic acid analogues Impact of stereopure chimeric backbone chemistries on the potency and durability of gene silencing by RNA interference essential components of therapeutic oligonucleotides Structural analysis of human Argonaute-2 bound to a modified siRNA guide A highly durable RNAi therapeutic inhibitor of PCSK9 Metabolite profiling of the antisense oligonucleotide eluforsen using liquid chromatography–mass spectrometry Nonclinical pharmacokinetics and absorption the first approved N-acetylgalactosamine-conjugated RNA interference therapeutic Use of 5-deoxy-ribo-hexofuranose derivatives for the preparation of 5′-nucleotide phosphonates and homoribonucleosides Biomimetic modeling of the decomposition of 2′-chloro-2′-deoxynucleotides by ribonucleotide reductases to give 3(2H)-furanones which can effect mechanism-based inactivation by Michael-type alkylation hybridization and modeling studies of modified oligonucleotides Khvorova, A., Roux, L. M. & Yamada, K. Modified oligonucleotides with increased stability. US Patent 2020025017-W (2020); https://portal.unifiedpatents.com/patents/patent/WO-2020198509-A3 Khvorova, A., Roux, L. M. & Yamada, K. Synthetic oligonucleotides having regions of block and cluster modifications. US Patent 20210395739A1 (2021); https://patents.google.com/patent/US20210395739A1/en Khvorova, A., Roux, L. M. & Yamada, K. Synthesis of modified oligonucleotides with increased stability. US Patent 20220010309A1 (2022); https://patents.google.com/patent/US20220010309A1/en The chemistries and consequences of DNA and RNA methylation and demethylation Methylation across the central dogma in health and diseases: new therapeutic strategies Phospho-carboxylic anhydride of a homologated nucleoside leads to primer degradation in the presence of a polymerase Structural basis of duplex thermodynamic stability and enhanced nuclease resistance of 5′-C-methyl pyrimidine-modified oligonucleotides β-Cyanoethyl N,N-dialkylamino/N-morpholinomonochloro phosphoramidites new phosphitylating agents facilitating ease of deprotection and work-up of synthesized oligonucleotides Modified internucleoside linkages for nuclease-resistant oligonucleotides The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes siRNA carrying an (E)-vinylphosphonate moiety at the 5′ end of the guide strand augments gene silencing by enhanced binding to human Argonaute-2 Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain Hydrophobically modified siRNAs silence Huntingtin mRNA in primary neurons and mouse brain Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo Single-molecule imaging reveals that argonaute reshapes the binding properties of its nucleic acid guides Structure of yeast Argonaute with guide RNA Helix-7 in Argonaute2 shapes the microRNA seed region for rapid target recognition siRNA seed region is divided into two functionally different domains in RNA interference in response to 2′-OMe modifications Functional features defining the efficacy of cholesterol-conjugated siRNA function in RNAi: a chemical modification analysis Single modification at position 14 of siRNA strand abolishes its gene-silencing activity by decreasing both RISC loading and target degradation Target RNA-directed trimming and tailing of small silencing RNAs Highly complementary target RNAs promote release of guide RNAs from human Argonaute2 Chimeric siRNAs with chemically modified pentofuranose and hexopyranose nucleotides: altritol-nucleotide (ANA) containing GalNAc-siRNA conjugates: in vitro and in vivo RNAi activity and resistance to 5′-exonuclease Modified deoxyoligonucleotides stable to exonuclease degradation in serum 2′-O-Methyl at 20-mer guide strand 3′ termini may negatively affect target silencing activity of fully chemically modified siRNA Chemical modification of siRNAs to improve serum stability without loss of efficacy Diverse lipid conjugates for functional extra-hepatic siRNA delivery in vivo Roehl, I., Schuster, M. & Seiffert, S. Oligonucleotide detection method. US Patent 20110201006-A1 (2011); https://portal.unifiedpatents.com/patents/patent/US-20110201006-A1 A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system Selective striatal neuronal loss in a YAC128 mouse model of Huntington disease Structures of human exonuclease 1 DNA complexes suggest a unified mechanism for nuclease family Coupled 5′ nucleotide recognition and processivity in Xrn1-mediated mRNA decay Structural principles for the inhibition of the 3′–5′ exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates Overcoming cellular barriers for RNA therapeutics GalNAc-siRNA conjugates: leading the way for delivery of RNAi therapeutics Utilization of unlocked nucleic acid (UNA) to enhance siRNA performance in vitro and in vivo Allele-selective inhibition of expression of huntingtin and ataxin-3 by RNA duplexes containing unlocked nucleic acid substitutions RNA-based therapeutics: an overview and prospectus tRNA therapeutics burst onto startup scene Applications of CRISPR technologies in research and beyond Mesyl phosphoramidate backbone modified antisense oligonucleotides targeting miR-21 with enhanced in vivo therapeutic potency Synthesis and evaluation of parenchymal retention and efficacy of a metabolically stable O-phosphocholine-N-docosahexaenoyl-l-serine siRNA conjugate in mouse brain Removal of t-butyldimethylsilyl protection in RNA-synthesis 3HF) is a more reliable alternative to tetrabutylammonium fluoride (TBAF) Pharmacokinetic profiling of conjugated therapeutic oligonucleotides: a high-throughput method based upon serial blood microsampling coupled to peptide nucleic acid hybridization assay A high-throughput method for direct detection of therapeutic oligonucleotide-induced gene silencing in vivo Improved pharmacokinetic and bioavailability support of drug discovery using serial blood sampling in mice Chemical engineering of therapeutic siRNAs for allele-specific gene silencing in Huntington’s disease models Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons glmmTMB balances speed and flexibility among packages for zero-inflated generalized linear mixed modeling Download references Ali for maintaining the infrastructure for nuclear magnetic resonance (NMR) and Y Nguyen for measurement of high-resolution mass analysis University of Massachusetts Chan Medical School Harvard Medical School and Mass General Institute for Neurodegenerative Disease contributed to the experimental design for exNA studies in hepatic and extrahepatic tissues contributed to the experimental design for exNA studies in CNS contributed to the exNA thermal stability and nuclease resistance study contributed to the oligonucleotide synthesis contributed to the statistical analysis of exNA CNS studies gave inspirational intellectual contributions have filed patent applications for exNA platforms discloses ownership of stocks in RXi Pharmaceuticals and Advirna and is a founder of Atalanta Therapeutics and Comanche Biopharma is an employee of Comanche Biopharma and owns stock options The remaining authors declare no competing interests Nature Biotechnology thanks Eriks Rozners and the other Scheme 1 and Data (synthetic oligonucleotide sequence information Download citation DOI: https://doi.org/10.1038/s41587-024-02336-7 Sign up for the Nature Briefing newsletter — what matters in science Metrics details aberrant RNAs produced by endogenous genes or transgenes are normally degraded by the nuclear and cytosolic RNA quality control (RQC) pathways and aberrant RNAs are converted into siRNAs that initiate post-transcriptional gene silencing (PTGS) in the cytosol How aberrant RNAs are selected and brought to the cytoplasm is not known Here we show that the RNA-binding protein SUPPRESSOR OF GENE SILENCING (SGS)3 shuttles between the cytosol and the nucleus where it associates with the ISWI-like CHROMATIN REMODELER (CHR)11 and with RNAs transcribed from PTGS-sensitive transgene loci binding CHR11 Knocking down CHR11 and its paralog CHR17 strongly reduces transgene PTGS suggesting that SGS3 recruitment by CHR11/17 facilitates PTGS initiation CHR11 is also enriched at endogenous protein-coding genes (PCGs) producing nat-siRNAs and va-siRNAs under biotic or abiotic stresses and this production is reduced in chr11 chr17 double mutants at genome-wide level impairing CHR11 and CHR17 rescues the lethal phenotype caused by the massive production of siRNAs from PCGs in RQC-deficient mutants We propose that SGS3 recruitment by CHR11/17 allows exporting RNAs to the cytosol to initiate the production of siRNAs This suggests that viral RNAs bound to AGO1 and protected by SGS3 are transformed into dsRNA by RDR6 thus creating an amplification loop that increases the production of siRNAs and further contributes to decreasing virus titer these results indicate that many types of endogenous or exogenous genes can produce siRNAs after the transformation of abRNA into dsRNA by RDR6 major questions remain unanswered regarding the way abRNAs are selectively exported to the cytosol for transformation into dsRNA by RDR6 we show that transgenes and endogenous genes that produce siRNAs bind to the ISWI-like CHROMATIN REMODELER (CHR)11 We show that CHR11 and its paralog CHR17 interact with the RNA-binding protein SGS3 and that SGS3 shuttles between cytosol and the nucleus where it binds transgene RNAs We propose a model where CHR11/CHR17 and SGS3 are major nuclear determinants promoting the initiation of siRNA production a SGS3 and CHR11 structures. CHR11 and SGS3 domains interacting in yeast two-hybrid are shown in gray. The sequence of the CHR11 clone retrieved in the in yeast two-hybrid screen is shown in Supplementary Fig. 1 b Localization of CHR11 full-length and truncated forms (stop1 and stop2) in N benthamiana (the same results were obtained twice in N The overlay is shown for DAPI/YFP/chlorophyll fluorescences c BiFC Interaction between SGS3 and CHR11 (full-length and truncated forms) in the nucleus of N Subcellular localization of reconstructed YFP was determined in the leaf epidermis for SGS3 protein in fusion with the C- terminal part of YFP (YC) and CHR11 proteins in fusion with the N- terminal part of YFP (YN) The overlay is shown for DAPI/YFP/chlorophyll fluorescences (the same results were obtained 4 times in N d SGS3 is associated with fractions enriched in microsomal and nuclear compartments in wild-type Arabidopsis and in the chr11 chr17 double mutant complemented with the p35S:FLAG-CHR11construct (the same results were obtained twice on two different subcellular fractionations) The sgs3-1 null allele is used as a negative control suggesting a dual localization of SGS3 in the nucleus and cytosol of Arabidopsis cells c Nuclear localization of a control line expressing GFP-CHR11 under the 35S promoter (the same results were obtained more than twice in A resides in the same cytosolic siRNA bodies these results clearly indicate that only SGS3 shuttles between the nuclear and cytosolic compartments and dissociates before SGS3 re-export to the cytosol no report to date has established a link between the ISWI complex and PTGS GUS siRNA and amiRCHR11-17 accumulation at 18 days in the progeny of lines L1/ pUBQ10:amirCHR11-17 lines #125 and #135 harvested in bulk GUS activity is given below for each sample used for RNA extraction (the results are shown for the northern blot obtained from one unique extraction of each sample) indicating that impairment of CHR11/17 does not modify the level of transcription of the p35S:GUS transgene these results suggest that the reason why CHR11/17 impairment affects S-PTGS is not due to changes in the level of transcription of transgene mRNA it remains possible that transgene transcription is qualitatively affected modifying the ratio between functional mRNAs and aberrant RNAs ChIP-qPCR analyses were performed on 15-day-old seedlings of the indicated genotypes a ChIP was performed on the L1/pUBQ10:GFP-CHR11 line using GFP antibodies for IP Graphical representation shows the fold change of two biological repeats b ChIP was performed on the L1/pUBQ10:FLAG-CHR11 using Flag antibodies for IP which expressed CHR11 at a similar level than in L1/pUBQ10:FLAG-CHR11 Graphical representation shows the fold change as the mean of three biological repeats and displays every data point Error bars represent the standard deviation In addition to validating the 6b4 locus as a control this result supports the hypothesis that the 6b4 locus triggers S-PTGS in an RQC-deficient background through CHR11/SGS3 selection/recruitment these results suggest that CHR11/17 generally promotes transgene S-PTGS by binding to transgene loci thus allowing the association of transgene RNAs with SGS3 in the nucleus and their export to the cytosol to initiate S-PTGS indicating that the added NLS is functional and that the export of the SGS3-NLS-GFP protein from the nucleus to the cytosol is inhibited or strongly diminished at 38 °C RIP analysis of GUS mRNA associated with SGS3 was performed using L1/sgs3 plants complemented by pRDR6:SGS3-NLS-GFP and L1 line (without TAG) as control for normalization RT-qPCR was performed to determine the accumulation levels of 5’ terminal part qPCR Fold enrichment of the immunoprecipitated (IP) was calculated as 2exp(−ΔΔCt [RIP in SGS3-NLS-GFP line/background in L1]) Fold enrichment of the qPCR internal control PP2A was also calculated and arbitrarily set to 1 The mean values and standard deviation of three independent experiments are shown as every data point The results presented above suggest a model in which SGS3 is recruited by CHR11 and presumably its paralog CHR17 to transgene DNA allowing certain nascent transgene RNAs to bind SGS3 SGS3 shuttling between nucleus and cytosol likely promotes the export of SGS3-bound transgene RNAs to the cytosol where they can be fully converted to dsRNA by RDR6 in siRNA bodies c Variation of 21/22nt ta-siRNA accumulation between chr11 chr17 and Col-0 Values are log2 fold changes according to DESeq2 differential analysis (n = 3 biologically independent samples per genotype and 7 ta-siRNA genes) The median is represented by the horizontal line the box indicates the interquartile range (IQR and black points indicate points outside this range d Accumulation of va-siRNAs upon TCV infection of Col-0 and chr11 chr17 Values are log2 fold changes according to DESeq2 differential analysis compared to Mock treatment (n = 3 biologically independent samples per genotype and condition The number shown above indicates the p-values of the difference of log2 fold change determined by a Wilcoxon test (the value displayed “< 2.2e-16” is the smallest p-value R will return for a Wilcoxon test) e Variation of va-siRNAs upon TCV infection of Col-0 and chr11 chr17 Values of the heat map are log2 fold changes according to DESeq2 differential analysis compared to Mock treatment these results support the relevance of CHR11 in the production of siRNAs from PCGs Pictures of representative plants of the indicated genotypes grown for 27 days in short days When the genomic arrangement allows the production of sense and antisense RNAs forming dsRNA with a 5’ overhang which probably releases the CHR11-SGS3 interaction RNAs bound to SGS3 are converted to dsRNA by RDR6 followed by processing by DCL4 and DCL2 into 21-nt siRNAs that can execute PTGS and 22-nt siRNAs that allow PTGS amplification The model described for the p35S-GUS transgene locus likely applies for endogenous PCG/NAT pairs (This figure was entirely created by authors using ppt) the native SGS3 shuttles between cytosol and nucleus When nucleo-cytosolic export is not inhibited except when CHR11 level is increased in the nucleus or when SGS3 fused to a canonical NLS is forced to remain in the nucleus after heat stress suggesting that SGS3 is actively exported from the nucleus to the cytosol together with the RNA binding and protecting effect of SGS3 against degradation by RQC certainly contributes to addressing transgene RNAs to the cytosol to initiate PTGS Following cleavage at the polyadenylation site of the NPT transcript the downstream part of the read-through transcript SGS3 could then bind to the GUS/SUG duplex in the nuclei because it has a 5’ overhang the native AGO1 protein produced in the cytosol has an exposed NLS and a buried NES allowing the AGO1/miRNA complexes to be exported to the cytosol The binding of SGS3 to dsRNA and/or the dissociation of SGS3 from CHR11 may also expose its NES allowing the SGS3-dsRNA complex to be exported to the cytosol the SGS3-bound GUS/SUG duplex could be processed into 21- and 22-nt siRNAs by DCL4 and DCL2 to initiate PTGS GUS and/or SUG RNAs could also be separately transformed into dsRNA by RDR6 prior to DCL2/DCL4-mediated processing into 21- and 22-nt siRNAs after which the total aerial parts of 4 to 12 plants were harvested pRDR6:SGS3-NLS-GFP was obtained by cloning canonical NLS sequences in speI of SGS3-pDONR207 prior to recombination in the compatible destination vector pRDR6-PGWB4 and pUBC‐GFP-DEST to make pUBQ10-GFP-CHR11 pUBQ10:Flag-CHR11 was obtained by cloning in pDONR207 CHR11 cDNA amplified by attB1FlagCHR11F and attB2CHR11Rbis primers prior to recombination in the compatible destination vector pUB-DEST France) PCR‐based mutagenesis was used to introduce premature stop1 and stop2 in CHR11 cloned in pDONR207 using CHR11stop1Fbis /CHR11stop1Rbis and CHR11stop2F/CHR11stop2R pairs of primers to obtained p35S:GFP-CHR11stop1 and stop2 AmiRCHR11-17 was synthesized and cloned in pUC57 by GenScript Corporation (New Jersey 08854-3900) Primers amiRf and amiRr were used to amplified amiRCHR11-17 in its precursor backbone for cloning in the pDONR207 vector prior to recombination in the compatible destination vector pUB-Dest to obtained pUBQ10:amirCHR11-17 CDS of RDR6 without its stop codon was cloned in SalI and NotI of pENTR1A vector after amplification by the primers pairs RDR6f/RDR6r (Supplementary Table 3) prior recombination in the compatible destination vector pH7FWG2 to obtained p35S:RDR6-GFP 64 were LacZ + (2 clones issued from screen without expression A fraction of the yeast plasmid minipreparation from each His + /LacZ + clone was used to transform HB101 E coli clones was transformed again into either the L40 strain carrying LexA/SGS3 to confirm interaction or an L40 carrying an unrelated hybrid protein Only 37 His + /LacZ+ clones were specific for LexA/SGS3 and in frame with the GAL4 sequence 28 interact with the C‐terminal three coiled‐coil domains of SGS3 which included the two clones recovered on a selective medium without expression (suggesting a strong interaction between the two partners) One of these two clones corresponded to the C-terminal part of CHR11 Samples were excited with a 514 nm argon laser (50%) with an emission band of 520–550 nm for YFP detection and 640–700 nm for chlorophyll autofluorescence Arabidopsis roots were directly imaged on a Leica TCS-SP5 (Leica Microsystems) equipped with a photomultiplier tube and hybrid detectors with an HCX PL APO CS 20.0 × 0.70 IMM objective GFP was imaged with 488 nm excitation using an Argon laser For the nuclear export inhibition experiment five days-old seedlings were transferred to 100ul liquid MS-medium with Leptomycin B (Sigma LMB at 5 uM final concentration corresponding to methanol at 4.7% final) and incubated 28 h before imaging Controls were incubated 28H in 100 ul liquid MS-medium with methanol added at 4.7% final Two grams of rosette leaves of 31 days-old plants were ground in 16 ml buffer supplemented with 1.14 M sucrose (10 mM Tris–HCl pH 7.5 1 mM DTT and protease inhibitor cocktail sigma p9599) The sample was filtered through Miracloth and centrifuged at 1000 × g for 10 min at 4 °C Supernatants recovered after the first centrifugation was supplemented with 0.15% Triton X100 and was centrifuged at 100,000 × g 45 min at 4 °C to obtain the microsomal fractions (pellet) The first pellet was washed twice with 10 ml buffer (10 mM Tris–HCl pH 7.5 and protease inhibitor cocktail sigma p9599) supplemented with 0.15% Triton X100 The last pellet corresponding to the nuclear fraction was resuspend in lysis buffer (10 mM Tris–HCl pH 7 and protease inhibitor cocktail sigma p9599) and sonicate on bioruptor Protein concentration was quantified using a detergent-compatible BCA kit (Bio-Rad) and 50 μg of protein were loaded on 8% SDS PAGE Proteins were electroblotted onto nitrocellulose membranes (Amersham Hybond ECL) The membrane was blocked in 5% non-fat dry milk in 1 × TBSTT (0.25% Tween-20 and incubated with primary antibody in 5% non-fat dry milk and 1 × TBST for 1 h at room temperature The membrane was then rinsed in 1 × TBST for 20 min before incubation with HRP-coupled secondary IgGs Antigens were detected using chemiluminescence for HRP immunoblot (Amersham ECL Plus) The antibodies used were anti-SGS3 (ref B152 from Santacruz 1/200e dilution) anti-H3 (ab1791 from Abcam followed by sonication using Covaris S220 ultrasonicator 12 min with 5% duty cycle 30 μL of GFP-trap-M beads (gfm-20/500 ul chromotech) was washed twice and resuspended in 60 μL of ChIP dilution buffer 60 μL of magnetic beads G was washed twice and resuspended in 60 μL of ChIP dilution buffer 5 μL of anti-FLAG M2 clone SIGMA(F3165) were added to the beads G and incubated for at least 3 h at 4 °C with gentle rotation on a wheel After three washes the beads plus antibodies were resuspended in 100 μL of ChIP dilution buffer 1 mL of the chromatin solution was added to the antibodies plus beads (GFP-trap-M or G beads plus anti-Flag) and was incubated overnight at 4 °C with gentle rotation for GFP or Flag capture the reverse crosslinking (5 h at 65 °C) and elution were performed using an IPure kit (Diagenode) and the chromatin was stored at − 20 °C until analysis Here control is L1 for ChIP on L1/pUBQ10:GFP-CHR11 and L1/ pUBQ10:GFP-CHR11 for ChIP on L1/pUBQ10:Flag-CHR11 sample is our target and internal reference is GAPDH Two to four biological replicates were analyzed each time Results show the mean and SD of the independent biological replicates Fold enrichment in the immunoprecipitated fraction (IP) compared to input fraction was calculated as 2exp(−ΔΔCt [RIP in SGS3-NLS-GFP line/RIP background in L1 line]) which correspond to ratio between percentage of input calculated for SGS3-NLS-GFP line / L1 control line Fold enrichment was calculated for the GUS mRNA target Mean values and standard errors of three independent experiments are shown Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article and activities of endogenous silencing small RNAs in Arabidopsis 3’ fragment of miR173-programmed RISC-cleaved RNA is protected from degradation in a complex with RISC and SGS3 A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis Heat stress promotes Arabidopsis AGO1 phase separation and association with stress granule components A neomorphic sgs3 allele stabilizing miRNA cleavage products reveals that SGS3 acts as a homodimer Cytoplasmic Arabidopsis AGO7 accumulates in membrane-associated siRNA bodies and is required for ta-siRNA biogenesis SGS3 and RDR6 interact and colocalize in cytoplasmic SGS3/RDR6-bodies Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression Cytoplasmic and nuclear quality control and turnover of single-stranded RNA modulate post-transcriptional gene silencing in plants Post-transcriptional gene silencing triggered by sense transgenes involves uncapped antisense RNA and differs from silencing intentionally triggered by antisense transgenes RST1 and RIPR connect the cytosolic RNA exosome to the Ski complex in Arabidopsis A genetics screen highlights emerging roles for CPL3 RST1 and URT1 in RNA metabolism and silencing decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs The TUTase URT1 connects decapping activators and prevents the accumulation of excessively deadenylated mRNAs to avoid siRNA biogenesis Suppression of endogenous gene silencing by bidirectional cytoplasmic RNA decay in Arabidopsis Virus infection triggers widespread silencing of host genes by a distinct class of endogenous siRNAs in <em>Arabidopsis</em> Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis Genome-wide analysis of plant nat-siRNAs reveals insights into their distribution Snf2 proteins in plants: gene silencing and beyond Intersection of small RNA pathways in Arabidopsis thaliana sub-nuclear domains Ribosome stalling and SGS3 phase separation prime the epigenetic silencing of transposons Phase separation of SGS3 drives siRNA body formation and promotes endogenous gene silencing ePlant: Visualizing and exploring multiple levels of data for hypothesis generation in plant biology Overexpression of ATG8 in arabidopsis stimulates autophagic activity and increases nitrogen remobilization efficiency and grain filling a chromatin-remodeling factor essential for nuclear proliferation during female gametogenesis in Arabidopsis thaliana Imitation Switch chromatin remodeling factors and their interacting RINGLET proteins act together in controlling the plant vegetative phase in Arabidopsis A plant-specific SWR1 chromatin-remodeling complex couples histone H2A.Z deposition with nucleosome sliding DDT-RELATED PROTEIN4-IMITATION SWITCH alters nucleosome distribution to relieve transcriptional silencing in Arabidopsis Unexpected silencing effects from T-DNA tags in Arabidopsis Transcriptional silencing induced by Arabidopsis T-DNA mutants is associated with 35S promoter siRNAs and requires genes involved in siRNA-mediated chromatin silencing and XRN3 are endogenous RNA silencing suppressors The Zinc-Finger Protein SOP1 Is Required for a Subset of the Nuclear Exosome Functions in Arabidopsis The RNA helicases AtMTR4 and HEN2 target specific subsets of nuclear transcripts for degradation by the nuclear exosome in Arabidopsis thaliana Second-site mutagenesis of a hypomorphic argonaute1 allele identifies SUPERKILLER3 as an endogenous suppressor of transgene posttranscriptional gene silencing ISWI proteins participate in the genome-wide nucleosome distribution in Arabidopsis A SWI/SNF chromatin-remodeling complex acts in noncoding RNA-mediated transcriptional silencing Analysis of mRNA-derived siRNAs in mutants of mRNA maturation and surveillance pathways in Arabidopsis thaliana Hua, X. et al. Global analysis of RNA-dependent RNA polymerase-dependent small RNAs reveals new substrates and functions for these proteins and SGS3 in arabidopsis. Noncoding RNA 7, https://doi.org/10.3390/ncrna7020028 (2021) dsRNA with 5’ overhangs contributes to endogenous and antiviral RNA silencing pathways in plants Nucleo-cytosolic shuttling of ARGONAUTE1 prompts a revised model of the plant microRNA pathway A branched pathway for transgene-induced RNA silencing in plants Arabidopsis mutants impaired in cosuppression The conserved RNA trafficking proteins HPR1 and TEX1 are involved in the production of endogenous and exogenous small interfering RNA in Arabidopsis Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance AtXRN4 degrades mRNA in Arabidopsis and its substrates include selected miRNA targets Improved Gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies Highly specific gene silencing by artificial microRNAs in Arabidopsis sgs1: a neomorphic nac52 allele impairing post-transcriptional gene silencing through SGS3 downregulation Protein-protein interactions among the Aux/IAA proteins Cloning of Rac and Rho-GDI from tobacco using an heterologous two-hybrid screen Mutations in the Arabidopsis H3K4me2/3 demethylase JMJ14 suppress posttranscriptional gene silencing by decreasing transgene transcription Genome-wide identification of RETINOBLASTOMA RELATED 1 binding sites in Arabidopsis reveals novel DNA damage regulators Profiling histone modification patterns in plants using genomic tiling microarrays Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis Contrasting epigenetic control of transgenes and endogenous genes promotes post-transcriptional transgene silencing in Arabidopsis Download references We thank Hervé Ferry and Philippe Maréchal for taking care of the plants We thank François Roudier and Jerémie Bazin for the discussion and advices respectively on the ChIP protocol adapted for chromatin remodeler proteins and on the RIP protocol We also thank Martin Lacroix for fruitful discussions Research in the Vaucheret laboratory is supported by grants from the French Agence Nationale pour la Recherche (ANR-16-CE12-0032 and ANR-20-CE12-0009) The IJPB benefits from the support of the LabEx Saclay Plant Sciences-SPS (ANR-17-EUR-007) This work has benefited from the support of IJPB’s Plant Observatory technological platforms Institut Jean-Pierre Bourgin for Plant Sciences (IJPB) analyzed the data and wrote the manuscript Nature Communications thanks Yuichiro Watanabe and the other anonymous reviewer(s) for their contribution to the peer review of this work Download citation DOI: https://doi.org/10.1038/s41467-025-57394-5 Metrics details Strategies to enhance the anti-tumor immune response of stereotactic ablative radiotherapy (SABR) at primary tumors and abscopal sites are under intensive investigation Here we report a metabolizable binary supracluster (BSCgal) that combines gold nanoclusters as radiosensitizing adjuvants with small interfering RNA (siRNA) targeting the immunosuppressive mediator galectin-1 (Gal-1) BSCgal comprises reversibly crosslinked cationic gold nanoclusters and siRNA complexes in a polymer matrix that biodegrades over weeks facilitating clearance (90.3% in vivo clearance at 4 weeks) to reduce toxicity The particle size well above the renal filtration threshold facilitates passive delivery to tumors Using mouse models of head and neck cancer we show that BSCgal augments the radiodynamic and immunotherapeutic effects of SABR at the primary and metastatic tumors by promoting tumor-inhibitory leukocytes upregulating cytotoxic granzyme B and reducing immunosuppressive cell populations It outperforms SABR plus Gal-1 antagonists chemoradiation drug cisplatin or PD-1 inhibitor This work presents a translatable strategy to converge focal radiosensitization with targeted immune checkpoint silencing for personalized radioimmunotherapy Integrated MRI-guided radiotherapy—opportunities and challenges Image-guided radiotherapy: from current concept to future perspectives Immunotherapy and stereotactic ablative radiotherapy (ISABR): a curative approach Patterns of disease recurrence after stereotactic ablative radiotherapy for early stage non-small-cell lung cancer: a retrospective analysis Individualized adaptive stereotactic body radiotherapy for liver tumors in patients at high risk for liver damage: a phase 2 clinical trial and patterns of failure after single-fraction stereotactic body radiation therapy (SBRT) for oligometastases Long-term follow-up and patterns of recurrence of patients with oligometastatic NSCLC treated with pulmonary SBRT The role of stereotactic ablative body radiotherapy in renal cell carcinoma Radiotherapy and immunotherapy: a beneficial liaison Stereotactic radiotherapy and pembrolizumab for oligometastatic renal tumors: the RAPPORT trial Randomized phase II trial of nivolumab with stereotactic body radiotherapy versus nivolumab alone in metastatic head and neck squamous cell carcinoma NRG-GU012: randomized phase II stereotactic ablative radiation therapy (SABR) for patients with metastatic unresected renal cell carcinoma (RCC) receiving immunotherapy (SAMURAI) The oligometastatic spectrum in the era of improved detection and modern systemic therapy Gold nanoparticles for applications in cancer radiotherapy: mechanisms and recent advancements Low-dose X-ray radiotherapy–radiodynamic therapy via nanoscale metal–organic frameworks enhances checkpoint blockade immunotherapy Synergistic checkpoint-blockade and radiotherapy–radiodynamic therapy via an immunomodulatory nanoscale metal–organic framework First-in-human study testing a new radioenhancer using nanoparticles (NBTXR3) activated by radiation therapy in patients with locally advanced soft tissue sarcomas NBTXR3 crystalline nanoparticles and radiation therapy in treating randomized patients in two arms with soft tissue sarcoma of the extremity and trunk wall. https://clinicaltrials.gov/study/NCT02379845 Advanced multimodal nanoparticles delay tumor progression with clinical radiation therapy AGuIX® from bench to bedside—transfer of an ultrasmall theranostic gadolinium-based nanoparticle to clinical medicine Radiosensitization of multiple brain metastases using AGuIX gadolinium based nanoparticles (NANO-RAD). https://clinicaltrials.gov/study/NCT02820454 Radiosensitization by gold nanoparticles: will they ever make it to the clinic Critical parameters to translate gold nanoparticles as radiosensitizing agents into the clinic Renal clearable catalytic gold nanoclusters for in vivo disease monitoring Glutathione-mediated biotransformation in the liver modulates nanoparticle transport Proximal tubules eliminate endocytosed gold nanoparticles through an 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blockade a first-in-class radioenhancer hafnium oxide nanoparticle plus radiotherapy versus radiotherapy alone in patients with locally advanced soft-tissue sarcoma (Act.In.Sarc): a multicentre Gold-nanocluster-mediated delivery of siRNA to intact plant cells for efficient gene knockdown Influence of the size and charge of gold nanoclusters on complexation with siRNA: a molecular dynamics simulation study Target-specific gene silencing of layer-by-layer assembled gold–cysteamine/siRNA/PEI/HA nanocomplex Co-caged gold nanoclusters and methyl motifs lead to detoxification of dendrimers and allow cytosolic access for siRNA transfection Radiotherapy remodels the tumor microenvironment for enhancing immunotherapeutic sensitivity Tumor-reprogrammed resident T cells resist radiation to control tumors The paradox of radiation and T cells in tumors Fractionated radiation therapy stimulates antitumor immunity mediated by both resident and infiltrating polyclonal T-cell populations when combined with PD-1 blockade Platinum-based chemotherapy plus cetuximab in head and neck cancer Cetuximab for the treatment of colorectal cancer Download references This study was supported by the following grants: P01CA257907 (to Q.T.L. and J.L.) from the National Cancer Institute and J.L.) from the National Institute of Dental and Craniofacial Research and U54CA274511 (to Q.T.L.) from the National Cancer Institute of the National Institutes of Health Schematic illustrations were created using BioRender The anti-galectin-1 blocking antibody (aGal-1) was generously provided by Bristol Myers Squibb via a material transfer agreement Department of Pathology and Laboratory Medicine Veterans Affairs Northern California Health Care System developed supraclusters and performed in vitro characterization shared P029 cell lines and laboratory resources designed the in vivo clearance study and experiments on abscopal effect are named inventors on a patent application related to the metabolizable supraclusters and their use in cancer treatments The other authors declare no competing interests Arta Monir Monjazeb and Jianping Xie for their contribution to the peer review of this work (a) ROS production in MOC2 cells after incubation with PBS or BSCgal (50 μg/mL) followed by RT (6 Gy) ROS was indicated by green fluorescence from H2DCFDA probe 100 μm (b) Mean fluorescence intensity (MFI) of ROS production in MOC2 cells after various treatments (RT = 6 Gy; n = 5 GSC and BSCgal was determined by one-way ANOVA (P = 0.7288) The others were analyzed by two-tailed unpaired Student’s t-test not statistically significant; ns P (BSCgal + RT vs GSC + RT) = 0.7109; ****P < 0.0001 (c) Clonogenic survival assay of MOC2 cells various treatments (n = 3) P value was determined by two-tailed unpaired Student’s t-test (a) Quantification of CRT fluorescence intensity in Fig. 5b (n = 5) (b) HMGB1 concentration in the plasma of mice after various treatments (n = 5) (c) Box plot of percentage of GzmB+ cells in tumor-infiltrating CD8+ T cells from mice after various treatments (n = 5) (d) Box plot of percentage of Ki67+CD8+ T cells in tumor- infiltrating CD45+ leukocytes from mice after various treatments (n = 5) *P = 0.0319; **P (BSCgal vs GSC) = 0.0045; **P (BSCgal + SABR vs BSCgal) = 0.0082 GSC and BSCgal was determined by one-way ANOVA The other P values were determined by two-tailed unpaired Student’s t-test not statistically significant (P > 0.05) Download citation DOI: https://doi.org/10.1038/s41587-024-02448-0 Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research Metrics details Efficient cytosolic delivery is a significant hurdle when using short interfering RNA (siRNA) in therapeutic applications Here we show that cholesterol-rich exosomes are prone to entering cancer cells through membrane fusion achieving direct cytosolic delivery of siRNA Molecular dynamics simulations suggest that deformation and increased contact with the target cell membrane facilitate membrane fusion In vitro we show that cholesterol-enriched milk-derived exosomes (MEs) achieve a significantly higher gene silencing effect of siRNA inducing superior cancer cell apoptosis compared with the native and cholesterol-depleted MEs as well as conventional transfection agents When administered orally or intravenously to mice bearing orthotopic or subcutaneous tumours the cholesterol-enriched MEs/siRNA exhibit antitumour activity superior to that of lipid nanoparticles by modulating the cholesterol content of exosome membranes to facilitate cell entry via membrane fusion we provide a promising approach for siRNA-based gene therapy Thermostable ionizable lipid-like nanoparticle (iLAND) for RNAi treatment of hyperlipidemia RNA silencing: from discovery and elucidation to application and perspectives RNA interference-based therapy and its delivery systems Rekindling RNAi therapy: materials design requirements for in vivo siRNA delivery Oral delivery of nucleic acid therapeutics: challenges Sphk2 RNAi nanoparticles suppress tumor growth via downregulating cancer cell derived exosomal microRNA Tumour-derived extracellular vesicle membrane hybrid lipid nanovesicles enhance siRNA delivery by tumour-homing and intracellular freeway transportation Targeted gene silencing in vivo by platelet membrane-coated metal-organic framework nanoparticles Conformation-sensitive targeting of lipid nanoparticles for RNA therapeutics Lysosome-related organelles as mediators of metal homeostasis Targeted delivery of RNAi to cancer cells using RNA-ligand displaying exosome A tumor targeted chimeric peptide for synergistic endosomal escape and therapy by dual-stage light manipulation Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown Image-based analysis of lipid nanoparticle-mediated siRNA delivery intracellular trafficking and endosomal escape Design of synthetic materials for intracellular delivery of RNAs: from siRNA-mediated gene silencing to CRISPR/Cas gene editing Nanoescapology: progress toward understanding the endosomal escape of polymeric nanoparticles An antigen self-assembled and dendritic cell-targeted nanovaccine for enhanced immunity against cancer Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer Extracellular vesicles as a next-generation drug delivery platform Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication Routes and mechanisms of extracellular vesicle uptake Exosome uptake through clathrin-mediated endocytosis and macropinocytosis and mediating miR-21 delivery Vesicle trafficking and vesicle fusion: mechanisms and their implications for potential disease therapy Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery In situ cell membrane fusion for engineered tumor cells by worm-like nanocell mimics A plant-derived natural photosynthetic system for improving cell anabolism Membrane fusion and drug delivery with carbon nanotube porins Folate-displaying exosome mediated cytosolic delivery of siRNA avoiding endosome trapping SARS-CoV-2 requires cholesterol for viral entry and pathological syncytia formation Different regions of synaptic vesicle membrane regulate VAMP2 conformation for the SNARE assembly ABCA13 dysfunction associated with psychiatric disorders causes impaired cholesterol trafficking Lipid raft microdomains and neurotransmitter signalling Cholesterol reduction impairs exocytosis of synaptic vesicles Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles Mechanisms and kinetics of liposome-cell interactions Temporal control of membrane fusion through photolabile PEGylation of liposome membranes Exosomal lipid composition and the role of ether lipids and phosphoinositides in exosome biology a coarse-grained force field for lipid membrane simulations with implicit solvent Oral administration of bovine milk-derived extracellular vesicles induces senescence in the primary tumor but accelerates cancer metastasis Methyl-beta-cyclodextrin induces mitochondrial cholesterol depletion and alters the mitochondrial structure and bioenergetics Influence of cholesterol content on red cell membrane viscoelasticity and fluidity Nanoscale imaging and mechanical analysis of Fc receptor-mediated macrophage phagocytosis against cancer cells Hierarchical micro-/nanostructures from human hair for biomedical applications Yolk-shell cationic liposomes overcome mucus and epithelial barriers for enhanced oral drug delivery Efficient base editing in methylated regions with a human APOBEC3A-Cas9 fusion Spatial transcriptomics at subspot resolution with BayesSpace Programmably tiling rigidified DNA brick on gold nanoparticle as multi-functional shell for cancer-targeted delivery of siRNAs Integrated combination treatment using a ‘smart’ chemotherapy and microRNA delivery system improves outcomes in an orthotopic colorectal cancer model Download references We sincerely appreciate the financial support from the National Science Fund of Distinguished Young Scholars (82025032) the National Key Research and Development Program of China (2022YFA1203200) the National Natural Science Foundation of China (82073773) the Young Elite Scientists Sponsorship Program by CAST (2022QNRC001) the Key Research Program of Chinese Academy of Sciences (ZDBS-ZRKJZ-TLC005) the ‘Open Competition to Select the Best Candidates’ Key Technology Program for Nucleic Acid Drugs of NCTIB (NCTIB2022HS01006) Technology and Innovation (23HC1401200) and the Shanghai Institute of Materia Medica Chinese Academy of Sciences (SIMM0220232001) We thank the staff members of the Integrated Laser Microscopy System the Molecular Imaging System and the Large-scale Protein Preparation System at the National Facility for Protein Science in Shanghai (NFPS) The MD simulations were performed on BSCC-A6 at the Beijing Super Cloud Computing Center These authors contributed equally: Yan Zhuo State Key Laboratory of Drug Research and Center of Pharmaceutics State Key Laboratory of Fluid Power and Mechatronic Systems Shanghai University of Traditional Chinese Medicine NMPA Key Laboratory or Quality Research and Evaluation of Pharmaceutical Excipients National Institutes for Food and Drug Control analysed the data and were involved in discussions of the data All authors critically reviewed and approved the paper Nature Nanotechnology thanks the anonymous reviewers for their contribution to the peer review of this work 3D distribution of 5%Chol/MEs in HCT116 cells 3D distribution of 30%Chol/MEs in HCT116 cells Uncropped and unprocessed scans for Supplementary Fig Download citation DOI: https://doi.org/10.1038/s41565-024-01785-0 siRNAs are a new class of drugs with the potential to transform the way medicine is practiced seven siRNA-based therapeutics have received regulatory approval all targeting gene expression in the liver specifically the ability to deliver to tissues outside the liver have laid the foundation for the development of novel RNA-based therapeutics across a wide range of clinical indications We collaborate with both academic and industry partners to accelerate the clinical translation of these compounds Three of our drug candidates have now received regulatory approval to formally initiate clinical testing with many more in various stages of preclinical development Preeclampsia is a serious pregnancy complication characterized by high blood pressure and organ dysfunction extrahepatic delivery platform to selectively knock down sFLT1 targeted approach to treating preeclampsia at its molecular origin ALYS-101 – A lipophilic siRNA targeting JAK1 for the localized treatment of alopecia areata Alopecia areata is an autoimmune disorder that causes patchy or total hair loss ALYS-101 is a topically administered siRNA designed for local silencing of JAK1 enabling targeted immunomodulation without systemic side effects RNA Therapeutics Institute Privacy Statement Metrics details upregulated midkine (MDK) limits the survival benefits conferred by temozolomide (TMZ) RNA interference (RNAi) and CRISPR–Cas9 gene editing technology are attractive approaches for regulating MDK expression delivering these biologics to GBM tissue is challenging Here we demonstrate a polymer-locking fusogenic liposome (Plofsome) that can be transported across the blood–brain barrier (BBB) and deliver short interfering RNA or CRISPR–Cas9 ribonucleoprotein complexes into the cytoplasm of GBM cells Plofsome is designed by integrating a ‘lock’ into the fusogenic liposome using a traceless reactive oxygen species (ROS)-cleavable linker so that fusion occurs only after crossing the BBB and entering the GBM tissue with high ROS levels Our results showed that MDK suppression by Plofsomes significantly reduced TMZ resistance and inhibited GBM growth in orthotopic brain tumour models Plofsomes are effective only at tumour sites and not in normal tissues which improves the safety of combined RNAi and CRISPR–Cas9 therapeutics Mechanisms of immunotherapy resistance: lessons from glioblastoma Drug resistance in glioblastoma: a mini review Dual functionalized brain-targeting nanoinhibitors restrain temozolomide-resistant glioma via attenuating EGFR and MET signaling pathways Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial Interrogation of the microenvironmental landscape in brain tumors reveals disease-specific alterations of immune cells DNA damage repair alterations modulate M2 polarization of microglia to remodel the tumor microenvironment via the p53-mediated MDK expression in glioma DNA repair in personalized brain cancer therapy with temozolomide and nitrosoureas Glioblastoma cell-derived lncRNA-containing exosomes induce microglia to produce complement C5 Nanoparticle-mediated convection-enhanced delivery of a DNA intercalator to gliomas circumvents temozolomide resistance A biodegradable nanocapsule delivers a Cas9 ribonucleoprotein complex for in vivo genome editing Single siRNA nanocapsules for effective siRNA brain delivery and glioblastoma treatment Multistage delivery nanoparticle facilitates efficient CRISPR/dCas9 activation and tumor growth suppression in vivo NanoRNP overcomes tumor heterogeneity in cancer treatment Precise targeting of POLR2A as a therapeutic strategy for human triple negative breast cancer Image-based analysis of lipid nanoparticle–mediated siRNA delivery Rational design of cancer nanomedicine: nanoproperty integration and synchronization Integration of nanoassembly functions for an effective delivery cascade for cancer drugs Nanomechanical action opens endo-lysosomal compartments maintenance and disruption of the blood-brain barrier Neuroprotective nanoscavenger induces coaggregation of β-amyloid and facilitates its clearance in Alzheimer’s disease brain Self-immolative hydroxybenzylamine linkers for traceless protein modification Acid-labile traceless click linker for protein transduction Site-specific chemical modification of antibody fragments using traceless cleavable linkers Reactive oxygen species‐responsive protein modification and its intracellular delivery for targeted cancer therapy Dual‐locking nanoparticles disrupt the PD‐1/PD‐L1 pathway for efficient cancer immunotherapy Protein toxin chaperoned by LRP‐1‐targeted virus‐mimicking vesicles induces high‐efficiency glioblastoma therapy in vivo and avoiding the endosome: peptide‐targeted fusogenic porous silicon nanoparticles for delivery of siRNA Fusogenic liposomes encapsulating mitochondria as a promising delivery system for osteoarthritis therapy Tumor cell surface modification with immuno-amplified nanoparticles to enhance cancer immunotherapy Fusogenic liposomes as nanocarriers for the delivery of intracellular proteins Strategies for the intracellular delivery of nanoparticles Membrane‐fusion‐mediated multiplex engineering of tumor cell surface glycans for enhanced NK cell therapy Monensin enhanced generation of extracellular vesicles as transfersomes for promoting tumor penetration of pyropheophorbide-a from fusogenic liposome Targeted fusogenic liposomes for effective tumor delivery and penetration of lipophilic cargoes Anti-phagocytosis-blocking repolarization-resistant membrane-fusogenic liposome (ARMFUL) for adoptive cell immunotherapy A multistage cooperative nanoplatform enables intracellular co‐delivery of proteins and chemotherapeutics for cancer therapy In situ modification of the tumor cell surface with immunomodulating nanoparticles for effective suppression of tumor growth in mice Bi-specific macrophage nano-engager for cancer immunotherapy Microenvironment remodeling micelles for Alzheimer’s disease therapy by early modulation of activated microglia Biocompatible polymeric nanoparticles degrade and release cargo in response to biologically relevant levels of hydrogen peroxide Fluorescamine: a reagent for assay of amino acids Stapled liposomes enhance cross‐priming of radio‐immunotherapy Polymer‐reinforced liposomes amplify immunogenic cell death‐associated antitumor immunity for photodynamic‐immunotherapy Measurement of co‐localization of objects in dual‐colour confocal images Detection of DNA double-strand breaks by gamma-H2AX immunodetection Delivery of CRISPR-Cas tools for in vivo genome editing therapy: trends and challenges Download references This study was supported by the (1) National Natural Science Foundation of China (numbers 82372901 and 82073298 for J.C. (2) Heilongjiang Provincial Key R&D Project (number GA21C002 for J.C (3) Heilongjiang Provincial Key Project of The Educational Science 14th Five-Year Plan (number GJB1422780 for J.C.) (4) Heilongjiang Provincial Natural Science Foundation (number LH2022H022 for X.M.) (5) China Postdoctoral Science Foundation (numbers 2019M660074 and 2022T150173 for X.M.) (6) Heilongjiang Postdoctoral Science Foundation (numbers LBH-Z19029 and LBH-TZ2218 for X.M.) and (7) Harbin Medical University Marshal Initiative Funding (number HMUMIF-22009 for X.M.) These authors contributed equally: Yu Zhao The Second Affiliated Hospital of Harbin Medical University The Sixth Affiliated Hospital of Harbin Medical University Key Laboratory of Functional Polymer Materials of Ministry of Education State Key Laboratory of Medicinal Chemical Biology All authors discussed the results and commented on the paper Nature Nanotechnology thanks Pablo del Pino and the other Download citation DOI: https://doi.org/10.1038/s41565-024-01769-0 when things turn out the opposite of what you expect it's often the starting point for discovery That's what happened to a team of researchers from Memorial Sloan Kettering Cancer Center (MSK) and their collaborators at the Icahn School of Medicine at Mount Sinai Their unexpected findings in the lab point to an opportunity to improve therapies that use small RNAs to silence disease-causing genes potentially including those involved in cancer but when it doesn't turn out the way you planned it can lead you to find something else that's much more interesting." the researchers - led by Seungjae Lee a postdoctoral fellow in the Lai Lab at MSK's Sloan Kettering Institute  - were testing how a protein called ALAS1 helps to make small regulatory RNAs called microRNAs they expected to see levels of microRNAs drop we were surprised to see them increase," Dr That counterintuitive result led to the discovery of an unrecognized role for ALAS1 beyond its well-known role in the production of heme (Heme is an important player in many biological processes including in oxygen transport - giving hemoglobin its name - in energy production The team's findings were published in Science one of the world's most prestigious scientific journals Both microRNAs and the related class of small interfering RNAs (siRNAs) are small snippets of RNA - just 21 or 22 nucleotides long - that bind to specific messenger RNAs (mRNAs) and repress them There's a bucket brigade of players that together convert longer RNA molecules into the tiny active products and a key takeaway is that scientists have harnessed this knowledge to turn small RNAs into drugs that can silence genes that cause specific diseases Food and Drug Administration (FDA) in 2018 to treat a debilitating genetic disorder called hereditary transthyretin amyloidosis A handful of additional siRNA drugs have been approved since with more moving their way through clinical trials Doctors see great potential to develop siRNA medicines against both rare diseases and more common ones (siRNA drugs are sometimes called RNAi drugs meaning they work by interfering with messenger RNA accumulation) Lee had discovered that upon removing ALAS1 from cells And further experiments showed that removing any of the other enzymes in the heme biosynthesis pathway did not affect microRNA levels "This told us that ALAS1 has another job outside of helping to make heme "We can consider this a 'moonlighting' function," Dr "And here we discovered that ALAS1 has this secret role regulating microRNAs that's not connected to its normal role in heme synthesis." The discovery led the MSK researchers to partner with colleagues from the Icahn School of Medicine at Mount Sinai who specialize in heme regulation and ALAS genes -  Makiko Yasuda This allowed the MSK researchers to extend their findings from cell culture into custom animal models that the Mount Sinai group were developing removing ALAS (specifically in liver cells) led to a global increase in microRNAs "The emerging picture is that ALAS acts as a brake on the production of microRNAs," Dr. Lai says. "So we thought, now that we know how to remove this brake, maybe we can use that to improve the efficacy of siRNA drugs and their ability to silence their target genes." this knowledge might help boost the activity of siRNA drugs against any problematic gene that is overactive in disease Potentially this could include oncogenes known to drive cancer "But we're not quite there yet," he says "Therapeutic siRNA drugs don't work well enough against all targets and are currently limited in where they can be used in the body." In fact all six of the FDA-approved siRNA drugs target hepatocytes in the liver "It's straightforward to get drugs into the liver which serves as a filter for the body," Dr the team showed that not only could they deplete mouse liver cells of ALAS but doing so also enhanced the silencing activity of another model siRNA compound delivered to the mice one of the six approved siRNA drugs turns off ALAS1 to treat acute hepatic porphyrias Desnick worked on the preclinical and clinical trials for the drug Since an siRNA against ALAS1 works effectively and safely in humans this raises the possibility of combining such an agent to enhance other siRNA drugs Lai notes that this strategy could be generally applicable to any siRNA And if siRNA drugs can be made to work better this could improve their cost-effectiveness reduce side effects by making them effective at lower doses and perhaps help to target additional cell types beyond liver cells was awarded the Nobel Prize with Victor Ambros for their joint discovery of microRNA and its role in gene regulation in the early 1990s Lai did his undergraduate thesis research in Dr Ruvkun's lab at that time (on another class of gene regulator) and credits him for launching his own career "I got my first real exposure to how science was actually done and gained lifelong interests in developmental biology and small RNAs," Dr adding that his mentor's recent accolade underscores the importance of curiosity-driven research Ruvkun didn't start out looking for microRNAs," Dr he was investigating the development of nematodes And not only did this unveil an entirely new paradigm for how genes are controlled the field they started eventually resulted in a novel class of human therapies "When people ask why we're not spending all of our research dollars directly studying diseases like cancer why we're funding research into cells and processes in model organisms like fruit flies and bacteria - this is a great example of how discovery science fuels the biggest breakthroughs," he continues "And I think it is especially critical to keep this conversation active given how much uncertainty and disagreement there is in society and government about how much to publicly fund scientific research and in what areas there will be continued support to keep the engine of foundational research strong." Funding for the research includes grants from the National Institutes of Health (R01DK134783 P30-CA008748); a Cooperative Centers of Excellence in Hematology pilot grant (10040500-05S1); and a NYSTEM training award (C32559GG) The researchers have filed a patent application on their methods for enhancing the efficacy of RNAi therapy by targeting ALAS1/ALAS2 (WO2024148236A1) Yasuda and Desnick are also co-inventors of a patent for RNAi therapy of acute hepatic porphyrias They also report pharmaceutical consulting work Memorial Sloan Kettering Cancer Center Lee, S., et al. (2024). Noncanonical role of ALAS1 as a heme-independent inhibitor of small RNA–mediated silencing. Science. doi.org/10.1126/science.adp9388 Posted in: Medical Science News | Medical Research News A biochemical breakthrough using simple carbon atoms by Ken Yamada, PhD, and Anastasia Khvorova, PhD, has dramatically improved the stability and efficacy of a potential oligonucleotide therapeutic platform in mice. This discovery, published in Nature Biotechnology has the potential to improve the durability of oligonucleotide drugs and to bring these therapies to cell types outside of the liver for the first time “The innovation of our study is a modification to the chemical composition behind the oligonucleotide platform’s architecture,” said Dr the Remondi Family Chair in Biomedical Research and professor of RNA therapeutics “Using a relatively simple change—extending the RNA backbone with single carbon atoms—we’ve been able to significantly improve the duration that oligonucleotides persist in cells by effectively hiding them from the nucleases that normally break them up This type of modification is critical to the success of oligonucleotide drugs as a class.” are a new class of medicine that enables modulation of disease-causing genes for therapeutic effect There are six siRNAs approved for clinical use by the Food and Drug Administration—all in the liver—with more in late-stage clinical trials was approved in 2018 for people with hereditary transthyretin-mediated amyloidosis The enzymes responsible for breaking down oligonucleotide molecules giving the drugs a limited amount of time to achieve a therapeutic effect scientists have been challenged to design chemical scaffolds that can stabilize sustain and extend the longevity of oligonucleotide drugs necessary to achieve therapeutic success in living tissue Most oligonucleotide modifications focus on changing the phosphorothioates or sugar molecules existing in the chemical backbone homing in on the carbon molecules in the backbone’s chemical structure a single or a few methylations to nucleobases and amino acids have been shown to slightly alter the structure of DNA RNA and proteins that profoundly impacts in-cell gene expression small structural change making a big impact in the cell,” said Yamada “This process inspired us to apply an analogous approach to the oligonucleotide platform’s backbone—a small structural change making a big impact on siRNA’s durability by increased stability and enhanced efficacy” “If we could garner even a 10-fold increase in persistence we could reduce dosage or clinical treatment time from once every two weeks to every five months To have a 32-fold increase in mice models is tremendously encouraging.” Yamada and Khvorova found that an extra carbon atom inserted in the correct spot along the nucleoside effectively hid the oligonucleotide molecules from degradation by the nuclease enzyme This resulted in a more stable and efficient oligonucleotide which was dubbed extended nucleic acid or exNA that persisted 32-fold longer than its phosphorothioate-modified counterparts “It’s all about where and how you add the carbon,” said Yamada “Even a slight structural modulation can have a huge impact on efficacy if you do it in the right place and in the right way.” eye—a host of tissues and diseases that were beyond our capabilities are now potentially feasible drug targets for oligonucleotide therapies.” The next step for researchers going forward is to begin testing the safety and longevity of their oligonucleotide backbone in clinical trials Sign up Metrics details Small interfering RNAs (siRNA) technology has emerged as a promising therapeutic tool for human health conditions like cancer due to its ability to regulate gene silencing their delivery remains localized and limiting their systemic use This study used single-walled carbon nanotubes (SWNTs) functionalized with polyethylene glycolated (PEGylated) phospholipids (PL-PEG) derivatives for systemic siRNA delivery We developed an siRNA systemic delivery vehicle (SWNT-siRNA) by conjugating SWNT functionalized with PL-PEG containing either amine (PA) or maleimide (MA) The functionalized SWNT with a lower molecular weight of PA produced the SWNT-siRNA conjugate system with the highest stability and high siRNA loading quantity The system delivered siRNA to a panel of tumour cell lines of different organs (i.e H1299 and MCF-7) and a non-cancerous human embryonic kidney 293 cells (HEK293T) with high biocompatibility and low toxicity The cellular uptake of SWNT-siRNA conjugates by epithelial cells was found to be energy dependent did not inhibit SWNT-mediated siRNA delivery Mouse xenograft model further confirmed the potential of SWNT-siRNA conjugates with a significant gene knock-down without signs of acute toxicity These findings pave the way for potential gene therapy applications using SWNTs as delivery vehicles While these agents showed promising results their application is primarily limited to localized delivery to a certain target organ This limitation makes these agents less useful for systemic diseases such as cancers that can metastasise to other organs or genetic diseases that affect multiple organ systems it is pivotal to break through this challenge to design an effective siRNA agent for the treatment of systemic diseases Our team has successfully optimised the functionalization of SWNTs using different molecular weights of the PL-PEG reactive group (PA or MA) followed by a series of investigation of the efficiency of siRNA loading on these SWNT-PL-PEGylated carriers including their knock-down efficiency and the toxicity of the SWNT-siRNA conjugates on various cancer cell lines along with its cellular uptake mechanisms We also demonstrated the efficient gene knock-down in mouse xenograft model verifying the success of this siRNA system for systemic gene delivery Our work demonstrates the potential of the SWNT-PL-PEGylated vehicle as a promising delivery system of siRNA in gene therapy applications Schematic drawing processing flow of functionalization of single-walled carbon nanotubes (SWNT) with PL-PEG-NH2 and PL-PEG-maleimide (C) Aqueous suspensions of SWNT (pristine) SWNT-PL-PEG-S-S-siRNA and SWNT-PL-PEG-siRNA (Picture taken by authors with a Canon EOS 5 Fujichrome Velvia) Table 1 summarises the yield of functionalized SWNT and their subsequent conjugation with siRNA Despite a relatively low yield of functionalized SWNT from this process these functionalized SWNT were able to be conjugated with 5’-thiolated siRNA to produce SWNT-PL-PEG-S-S-siRNA and SWNT-PL-PEG-siRNA It was found that the siRNA loading capacity into these functionalized SWNT ranged from 35 to 44% for the PA functional group and 29–45% for MA functional group; with a decrease in siRNA loading capacity as the molecular size of the PEG functional group increased zeta potential measurements revealed that the SWNT-siRNA conjugates were stable in aqueous solutions with values of -38.24 ± 5.28 mV for SWNT-PL-PEG-S-S-siRNA and − 32.78 ± 3.76 mV for SWNT-PL-PEG-siRNA Particle size analyses showed average sizes of 617 ± 115 nm and 703 ± 210 nm for the respective SWNT-siRNA conjugates slightly larger than the functionalized SWNT but within the appropriate range for cellular infiltration which is a critical factor for a successful transfection These findings demonstrate an efficient loading of siRNA using functionalized SWNT Significant gene silencing efficiency using SWNT-siRNA conjugates (A) Graphical representation of SWNT-PL-PEG-S-S-siRNA (B) Graphical representation of SWNT-PL-PEG-siRNA D and E) Evaluation of gene silencing effect by SWNT-PL-PEG-S-S-siGFP and SWNT-PL-PEG-siGFP conjugates in MCF-7 Cells expressing stable green fluorescent protein (GFP) were treated with SWNT-PL-PEG-S-S-siGFP or SWNT-PL-PEG-siGFP conjugates at a concentration of 100 nM siGFP The cell nuclei were stained with DAPI (blue) Microscopic observation was captured under 100x magnification Bars represent mean ± SD of at least 3 independent experiments * represents statistical significance as compared to vehicle controls (Student’s t-test To ensure that the observed reduction of GFP signals was not due to the toxic effects of SWNT-PL-PEG-S-S-siGFP or SWNT-PL-PEG-siGFP conjugates, the cytotoxic effects of SWNT-PL-PEG-S-S-siGFP or SWNT-PL-PEG-siGFP conjugates on these cell lines were determined using the LDH release assay at 72 h after treatment. As shown in Supplement Figure S1 neither the SWNT-PL-PEG-S-S-siGFP nor SWNT-PL-PEG-siGFP conjugates significantly affected cell viability implying the non-toxic nature of SWNT-siRNA conjugates Dual-silencing efficacy of siRNA-conjugated SWNT delivery system Fluorescence microscopic images of co-expressed GFP and RFP in H1299 cells incubated with 100 nM of (A) SWNT-PL-PEG-S-S-siRNA or (B) SWNT-PL-PEG-siRNA conjugates targeting GFP (siGFP) or RFP (siRFP) or LUC (siLUC; negative control) alone or both (dual targeting; siGFP + siRFP) for 72 h The furthest right panel was the combined images Energy dependent cellular internalization mechanisms of SWNT-siRNA conjugates independent of ABCB1 Graph shows the mean relative luminescent units (RLU) of (A and B) luciferase expressing H1299 cells co-treated with SWNT only (without siLUC) SWNT-PL-PEG-S-S-siLUC or SWNT-PL-PEG-siLUC with or without sub-lethal concentrations of various endocytosis inhibitors genistein and m-βCD are inhibitors for energy-dependent cellular uptake caveolae-dependent endocytosis and macro-pinocytosis * represents statistical significance as compared to SWNT-siLUC (Student’s t-test (C) Western blot of ABCB1 and loading control GAPDH Ectopic expression of ABCB1 (multiple drug resistance) protein in H1299-LUC cells was confirmed by the immunoblotting with increased expression of ABCB1 at 141 kDA while GAPDH at 36 kDA served as the loading control Full blots available in Supplementary Figure S2-S3 (D) Expression of ABCB1 did not affect the delivery of siRNA by the SWNT * represents statistical significance as compared to controls (Student’s t-test The findings suggest that the cellular uptake of SWNT-siRNA is not impacted by ABCB1-mediated efflux indicating that SWNTs could potentially serve as effective carriers for gene therapy in multiple drug-resistant tumours Significant knock-down efficacy using SWNT-PL-PEG-S-S-siRNA conjugate in mouse xenograft model (A) In vivo bioluminescence imaging of luciferase expressing HCT-116 xenograft in BALB/C nude mice The images show intensity of luminescence as ‘heat’ maps in which red is the maximum intensity The scale shows the number of photons detected (Left) Mice were treated with saline PBS as negative controls while (right) mice were injected with single dose of SWNT-PL-PEG-S-S-siLUC at 0.8 mg/kg through tail-vein (B) Significant gene knock-down by SWNT-PL-PEG-S-S-siLUC in mouse xenograft model Quantitation of bioluminescence was determined as depicted in Methods * indicates statistical significance as compared to vehicle control (Student’s t-test (C) No significant body weight loss was observed following administration of SWNT-PL-PEG-S-S-siLUC to the HCT-116 xenograft in BALB/C nude mice While the promise of CNTs as nanocarriers is undeniable rigorous research and optimization are required to overcome these limitations PL-PEG functionalization aligns the physicochemical properties of SWNTs with the requirements of an effective systemic delivery system ensuring that the therapeutic potential of siRNA is fully utilized Our findings demonstrated a low yield of SWNT functionalization this maybe due to the dispersion of SWNT which can be improved further by optimizing sonication parameters and mildly oxidizing of the SWNT in the future studies we could further explore improving the recovery by redispersing the functionalized SWNT in a suitable solvent if necessary minimizing losses during purification and handling Future mechanistic studies using fluorescent or radiolabelled tracking as well as targeted cleavage mechanisms as compared with the commercial siRNA delivery kit the uptake of SWNT-PL-PEG-S-S-siRNA and SWNT-PL-PEG-siRNA was not affected by the presence of P-glycoprotein a membrane protein often associated with drug resistance This observation suggests that SWNTs could be applied in delivering agents that could be beneficial in treating drug-resistant tumours addressing a significant challenge in cancer treatment it is certainly warranted to investigate whether co-delivery with other synthetic compound or biological molecule may further improve its efficacy the research findings highlight the promising potential of SWNT-siRNA conjugates as gene-silencing tools Their capability to silence multiple genes simultaneously and their effectiveness in drug-resistant conditions are promising aspects for cancer treatment The transition from in vitro to in vivo efficiency is a crucial step in drug development and the success of SWNT-siRNA conjugates in animal models provides a promising lead for future investigations Cylindrical structure SWNT were functionalized as previously described66 1 mg HiPCo® SWNT with a diameter of approximately 1.0 nm (ranging between 0.8 nm and 1.2 nm) and individual SWNT length between 100 nm and 1000 nm with high aspect ratio (length to radius) is typically ranging between 8,333 and 12,500; and > 95% purity (Unidym Inc. either PL-PEG-NH2 (PA) or PL-PEG-maleimide (MA) (NOF America Corporation were dissolved in 5 mL diethylpyrocarbonate (DEPC)-treated water (nuclease-free) This mixture was sonicated for 60 min in a water bath at room temperature (approximately 22 °C) PEG derivatives and PEG chains of varying molecular weights (2000 Insoluble contaminants and bundled SWNT were eliminated through centrifugation using a Beckman Coulter Optima L-90 K Ultracentrifuge (Beckman Coulter USA) at 24,000 × g for 6 h at room temperature the supernatant was collected and further centrifuged for 10 min at 4,000 × g at room temperature using an Amicon centrifugal filter with a cut-off size of 100 kDa (Millipore The residues underwent five rounds of washing using water to ensure a thorough removal of excess PL-PEG from the SWNT solution the SWNT concentration was determined using an ultraviolet-visible spectrometer (Perkin Elmer Both the SWNT-PL-PEG-S-S-siRNA and SWNT-PL-PEG-siRNA conjugates were prepared as described above and transferred into plastic optical cuvettes Dynamic light scattering (DLS) and zeta potential measurements were performed using Malvern Zen Zetasizer Nano (Malvern Instrument Hydrodynamic dimensions of SWNT in suspension were determined by automatic software analysis of dynamic light scattering autocorrelation curves Zeta potentials were measured in Malvern Instruments Folded Capillary Cells with embedded electrodes Each measurement consisted of up to 100 runs from which the average zeta potential value was calculated human non-small cell lung carcinoma line H1299 and human cervical cancer cells HeLa were procured from the American Type Culture Collection (ATCC; Manassas They were cultured in RPMI 1640 medium (Sigma-Aldrich USA) fortified with 10% fetal bovine serum (FBS Singapore) and 1% penicillin-streptomycin (Biowest American Type Culture Collection) was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich USA) enriched with 10% fetal bovine serum and 1% penicillin-streptomycin All cell lines were kept in logarithmic growth inside a 37 °C humidified incubator with an atmosphere of 5% CO2 Stable expressions of green fluorescent protein (GFP) luciferase mRNA (LUC) and ABCB1 were established in MCF-7 HEK-293T and HeLa using lentiviral transduction Lentiviral expression plasmids were purchased from OriGene (Rockville Lentiviral stocks were produced by co-transfection with 5 µg of packaging plasmids psPAX2 (Addgene; plasmid 12260) and 5 µg envelope plasmids pMD2.G (Addgene; plasmid 12259) into HEK-293T cells as described previously using CalPhosTM Mammalian Transfection Kit (Clontech and/or LUC were seeded overnight at a density of 3 × 105 cells per 60 mm culture dish or 5 × 103 cells per well on a 96-well culture plate 100 nM of siRNA conjugated to SWNT-PL-PEG-S-S-siRNA or SWNT-PL-PEG-siRNA was introduced to the cells expressing GFP The siRNA concentration was calculated based on an absorbance at 260 nm and/or LUC post-transfection were assessed at 72 h using the Eclipse Ti-U inverted fluorescent microscope (Nikon Quantitative measurements were conducted using the Infinite F200 microplate reader (Tecan Group Statistical significance was assessed using Student’s t-tests Values of P < 0.01 were considered statistically significant (*) All data are presented as mean ± standard deviation (SD) Cell viability assay was performed using CellTiter-Glo® Luminescent Cell Viability Assay (Promega USA) as according to manufacturer’s protocol and previous studies [24,26] Quantitative measurements were conducted using a SpectraMax Plus 384 Microplate Reader (Molecular Devices To determine the cellular update efficiency H1299 cells expressing luciferase (H1299-LUC) were pre-treated with sublethal concentration of sodium azide or methyl-b-cyclodextrin (m-βCD; 5 mM) 30 min prior to incubation with SWNT-siLUC conjugates NaN3 may inhibit energy-dependent cellular uptake [27] while chlorpromazine may block clathrin-mediated endocytosis [28] Genistein and m-βCD may inhibit caveolae-dependent endocytosis and macro-pinocytosis The inhibitory effects of each pathway were verified by assessing the silencing efficiency of firefly luciferase expression by the SWNT-PL-PEG-S-S-siLUC or SWNT-PL-PEG-siLUC conjugates after 72 h incubation Luciferase expression and cell viability were measured using the ONE-Glo™ + Tox Luciferase Reporter and Cell Viability Assay (Promega Luminescence signals were read using the SpectraMax Plus 384 Microplate Reader (Molecular Devices USA) and normalised against signals from untreated cells Primary and secondary antibodies targeting ABCB1 and GAPDH (as the loading control) were sourced from Santa Cruz Biotechnology The results were viewed using ChemiDoc™ XRS + molecular imager (Bio-Rad Laboratories in vivo bioluminescence assessments were carried out daily for a week Tumour dimensions were logged bi-weekly using a calliper and volumes were calculated as: V = 0.5 a × b2 (where ‘a’ and ‘b’ represent the tumour’s long and short diameters mice were given 150 mg/kg luciferin intraperitoneally the mice were anesthetised using the mixture of oxygen and isoflurane they were placed in an imaging chamber for bioluminescence measurements using IVIS® Lumina II optical imaging system (Caliper Life Sciences Bioluminescence images from the whole body inclusive of primary and metastatic tumours The primary study endpoint was tumour growth rate (n = 5) and statistically significant differences were concluded using independent t-test when p value less than 0.05 All data generated or analysed during this study are included in this published article (and its Supplementary Information files) FDA approves patisiran to treat hereditary transthyretin amyloidosis Ongoing clinical trials of nonviral siRNA therapeutics Clinical advances of siRNA-based nanotherapeutics for cancer treatment siRNA could be a potential therapy for COVID-19 Overcoming barriers: clinical translation of siRNA nanomedicines Overcoming barriers for siRNA therapeutics: from bench to bedside Functionalized single-walled carbon nanotubes: cellular uptake biodistribution and applications in drug delivery Nucleic acid drug and delivery techniques for disease therapy: Present situation and future prospect and therapeutics in nanoparticle-based siRNA delivery systems for breast cancer Nano-formulated siRNA-based therapeutic approaches for cancer therapy Review on carbon nanotubes (CNTs) and their chemical and physical characteristics with particular emphasis on potential applications in biomedicine Advanced strategies for overcoming endosomal/lysosomal barrier in nanodrug delivery Additive manufacturing in nano drug delivery systems Insights on functionalized carbon nanotubes for cancer theranostics Functionalized carbon nanotubes: synthesis properties and applications in water purification Carbon nanotubes as an emerging nanocarrier for the delivery of doxorubicin for improved chemotherapy Synthetic strategies in construction of organic macromolecular carrier-drug conjugates Modeling prostate cancer: what does it take to build an ideal tumor model A nanoprimer to improve the systemic delivery of siRNA and mRNA Engineered nanoparticles for systemic siRNA delivery to malignant brain tumours Design of a novel PEGylated liposomal vector for systemic delivery of siRNA to solid tumors Chung, S. 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Cells 9, doi: https://doi.org/10.3390/cells9040886 (2020) Download references This research was funded by Ministry of Higher Education the Hubert Curien Partnership – Hibiscus (PHC-HIBISCUS) France-Malaysia (Grant no Technology and Innovation Nanotechnology Research Grant (NND/NA/1/TD11-002) and UCSI University Research Excellence and Innovation Grant (REIG-FPS-2023/038) Kandiah Faculty of Medicine and Health Sciences Center for Nanotechnology and Advanced Materials analyzed the results and wrote the first draft of the manuscript provided intellectual input and troubleshooting secured fundings and revised the manuscript provided intellectual inputs and troubleshooting Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Below is the link to the electronic supplementary material Download citation DOI: https://doi.org/10.1038/s41598-024-80646-1 Phase 2 trial of zerlasiran yields first demonstration of longer effect with each dose of an siRNA reduced time-averaged Lp(a) concentrations by more than 80% across 36 weeks of treatment in the randomized ALPACAR-360 trial Cleveland Clinic is a non-profit academic medical center. Advertising on our site helps support our mission. We do not endorse non-Cleveland Clinic products or services. Policy “We are still learning how the siRNA therapies affect lipoprotein(a) during extended treatment This trial shows for the first time that with each successive dose Lp(a) reduction endured longer and waned more slowly This has implications for the design of phase 3 trials of zerlasiran and potentially for optimal dosing of other drugs in this class.” Zerlasiran is among several medications in clinical trials for reduction of elevated Lp(a) a genetically determined risk factor for atherosclerotic cardiovascular disease (ASCVD) that is resistant to traditional preventive therapies No pharmacotherapies have been approved for Lp(a) reduction The investigational therapies furthest in development are four nucleic acid therapeutics consisting of the antisense oligonucleotide pelacarsen and three siRNAs “Identifying the best dosing strategy for siRNA therapies is difficult because they all have prolonged durations of action that range from weeks to months,” says Dr Chief Academic Officer of Cleveland Clinic’s Heart who is involved with clinical trials of three of the four nucleic acid therapeutics “Along with assessing the safety of zerlasiran ALPACAR-360 was designed to help identify the best dose and dosing frequency to guide a phase 3 trial.” The study involved 180 adults with stable ASCVD from 26 sites in Europe All patients had a serum Lp(a) concentration of at least 125 nmol/L a threshold that confers increased cardiovascular risk with a mean age of 63.7 years and a median baseline Lp(a) of 213 nmol/L (interquartile range Patients were randomized in a double-blind manner to one of five treatment groups: Doses were injected subcutaneously in the abdomen Patients were followed every four weeks up to 40 weeks and then at weeks 48 and 60 after randomization The primary endpoint was the time-averaged percentage change in serum Lp(a) from baseline through 36 weeks after first administration Time-averaged percentage changes from baseline to weeks 48 and 60 were secondary endpoints as were safety and tolerability through 60 weeks The Cleveland Clinic Coordinating Center for Clinical Research (C5Research) performed statistical analysis and helped manage the trial 178 received study doses and were included in results analyses mean time-averaged percentage changes in Lp(a) concentrations through 36 weeks were: Median percentage changes at week 36 were: Injection-site reactions were the most common treatment-related adverse effects; mild pain at the site was reported by 2.3% to 7.1% of participants within the first day after injection No such reactions led to study withdrawal or missed doses and no treatment-related serious adverse events occurred “This is the first trial of a nucleic acid therapy to use time-averaged Lp(a) reduction as an endpoint,” Dr noting that other trials used maximal reduction or reduction at a given time point “Our endpoint more accurately reflects the effect of treatment over time the duration of reduction was longer and the waning of Lp(a) lowering was attenuated,” he continues “In the group that received three doses of zerlasiran 300 mg the median reduction at the 60-week final follow-up visit was still 58.8% This was a full 28 weeks after the last of the three doses had been given.” He adds that any such effect hadn’t been appreciated in previous trials of nucleic acid therapies because they have involved either single doses or agents that are dosed too frequently — such as every 4 or 12 weeks — to adequately gauge the effect on Lp(a) rebound “The cumulative effect we observed with successive doses will inform the dosing interval chosen for phase 3 studies of zerlasiran,” Dr the study report includes waterfall plots that reveal responses to therapy on a patient-by-patient basis The plot for patients who received a dose every 16 weeks showed consistent responses to therapy with zerlasiran 300 mg suggesting that dosing every 16 weeks may provide more uniformly substantial reduction across all patients compared with dosing every 24 weeks “The question that naturally arises is how much Lp(a) reduction is enough,” Dr That answer is awaiting results from ongoing outcomes-based phase 3 trials of three agents — pelacarsen and the siRNAs olpasiran and lepodisiran “The collective research community is taking a lot of shots on goal to finally address the morbidity and mortality from Lp(a) elevation,” Dr “We are hopeful at least one of them will end up in the back of the net soon.” “It is exciting to have so many drugs in development to lower Lp(a),” adds Leslie Cho, MD Co-Section Head of Preventive Cardiology and Rehabilitation at Cleveland Clinic “We look forward to results from phase 3 studies to tell us whether lowering Lp(a) in the era of aggressive LDL control reduces cardiac events.” The trial was funded by Silence Therapeutics Nissen reported coordinating center funding from Silence Therapeutics Novartis and Eli Lilly during the conduct of the trial Advertisement Yet 21.4% of tested individuals had Lp(a) elevation Small interfering RNA lowered lipoprotein(a) by 94% during six-month follow-up after a single dose Resourceful approaches to the care of patients with microvascular disease and elevated Lp(a) Study authors urge reevaluation of the sweetener’s safety designation by food regulators Cleveland Clinic study finds that durable weight loss is key to health benefits How two of our surgeons are working for care equity greater representation in research and practice enhanced platelet reactivity and thrombotic potential Tech-assisted self-selection concurred with clinician-assessed eligibility in >90% of cases Metrics details Hybrid seed failure arising from wide crosses between plant species is a recurring obstacle in plant breeding This postzygotic reproductive barrier primarily occurs in the endosperm a tissue that nourishes the embryo and functions similarly to the placenta in mammals We found that incompatible seeds show a loss of DNA methylation and chromatin condensation in the endosperm similar to seeds lacking maternal RNA polymerase IV activity This similarity is linked to a decrease in small interfering RNAs in the endosperm (sirenRNAs) maternal RNA polymerase IV-dependent short interfering RNAs that regulate DNA methylation Several AGAMOUS-like MADS-box transcription factor genes (AGLs) are targeted by sirenRNAs in cis and in trans This finding aligns with the enrichment of AGL target genes among deregulated genes We propose that hybrid seed failure results from reduced maternal sirenRNAs combined with increased AGL expression leading to abnormal gene regulation in the endosperm implying the existence of a dosage-sensitive element contributing to this reproductive barrier within the endosperm the precise nature of this component remains elusive whether sirenRNAs constitute the dosage-sensitive component determining hybridization success remains an open question we demonstrate that the loss of maternal Pol IV function mirrors the loss of chromatin condensation and DNA methylation observed in Capsella hybrids This resemblance in phenotype is correlated with a shared decrease in sirenRNAs which regulate target genes in cis and trans by guiding DNA methylation Among these sirenRNA targets are several AGLs aligning with the enrichment of AGL target genes among deregulated genes we propose that the response to interspecies and interploidy hybridizations is instigated by two factors: the depletion of maternally derived sirenRNAs and the heightened expression of AGLs targeting hypomethylated regions in hybrid endosperm Cleared hybrid Capsella seeds at 4 to 6 DAP The corresponding mature seeds are shown on the right side of each panel 100 µm and 1 mm for cleared and mature seeds The colour code reflects effective ploidy as indicated in the figure Mature seed phenotypes of crosses Cr × Cbp and Cbp × Cr Mature seed phenotypes of crosses Co × Cr and 4xCo × Cr Germination data for the crosses shown in c Shown is the fraction of germinating seeds from each cross with each filled circle representing one biological replicate; there are three biological replicates per genotype The numbers at the top represent total seed numbers The asterisks represent statistical significance as calculated by one-way analysis of variance with post-hoc Tukey’s honestly significant difference test (***P < 0.001) Upset plot showing the overlap of upregulated genes between interploidy (Cr × 4xCr) and interspecies crosses of different Capsella species Heat map and dendrogram show clustering of samples based on log2 fold changes (compared with the corresponding maternal parent) of genes upregulated in the analysed hybrids Source data These data support the idea that a dosage-sensitive component generated by the maternal parent determines hybridization success these data strongly suggest that interploidy and interspecies hybridization barriers share a common molecular basis the fact that increased ploidy of the maternal parent can alleviate paternal-excess hybrid incompatibility implies the existence of a quantitative maternal factor determining hybridization success Box plots showing methylation levels of TEs in the endosperm of 6 DAP seeds of the indicated genotypes The asterisks indicate statistically significant differences as calculated by two-sided Wilcoxon tests; P values were adjusted for multiple comparisons with Benjamini and Hochberg correction (***P < 0.001) the horizontal lines show the median values the box edges show the interquartile range and the whiskers show the full range excluding outliers Source data these data reveal that interploidy and interspecies hybridization cause similar molecular defects consistent with the similar phenotypes of hybrid seeds a, DAPI-stained chromocenters from endosperm nuclei from Cr × Cr, Cr × nrpd1, nrpd1 × Cr and nrpd1 × nrpd1. Scale bars, 5 μm. b, Quantification of chromatin condensation given as mean circularity of chromocenters (Methods) The numbers below the boxes indicate the number of analysed nuclei The asterisks represent statistical significance as calculated by two-sided Wilcoxon tests; P values were adjusted for multiple comparisons with Benjamini and Hochberg correction (***P < 0.001) Metagene plots showing DNA methylation levels of TEs in the endosperm of Cr × Cr The asterisks indicate statistically significant differences as calculated by two-sided Wilcoxon tests; P values were adjusted for multiple comparisons with Benjamini and Hochberg correction (***P < 0.001; **P < 0.01) Upset plot showing the overlap of upregulated genes among Cr × 4xCr Genes were considered as upregulated on the basis of log2(fold change) > 1 and Padj < 0.05 compared with Cr × Cr Source data Box plots showing the accumulation of siRNAs (18–25 nucleotides) on siren loci in different genotypes The asterisks indicate statistically significant differences as calculated by two-sided Wilcoxon tests (***P < 0.001) siRNA accumulation and CHH methylation in the endosperm of the indicated genotypes on selected genes overlapping siren loci (marked with violet lines) The blue boxes represent genes; the purple boxes represent TEs Parental-specific accumulation of sRNAs on loci having at least 15 parental-specific reads in total The numbers above the boxes indicate the number of such genes in each genotype The asterisks indicate statistically significant differences as calculated by two-sided Wilcoxon tests (***P <0.001) Parental-specific sRNA accumulation in the endosperm of the indicated genotypes on selected genes Source data Comparison of DNA methylation levels in genes (gene body) promoters and TEs overlapping siren loci with all genes and TEs in the Cr genome Expression of genes overlapping siren loci in Cr × Cr The asterisks indicate statistically significant differences as calculated by two-sided Wilcoxon tests (***P < 0.001; **P < 0.01) Heat map and dendrogram show clustering of samples based on log2 fold changes (compared with the corresponding maternal parent) of genes overlapping siren loci Scatter plots showing Pearson’s correlation (r) (two-sided) between the loss of siRNAs and changes in genes expression (left) and CHH methylation (right) Comparison of DNA methylation in Cr × Cr versus nrpd1 × Cr (left) and Cr × Cr versus Cr × Cg (right) endosperm on upregulated genes (in the respective crosses) overlapping siren loci The asterisks indicate statistically significant differences as calculated by a two-sided Wilcoxon test (*P < 0.05) Source data making it unlikely that reduced NRPD1 expression is causally responsible for the loss of sirenRNAs in the hybrids CHG and CHH DNA methylation at sirenRNA trans targets (50-bp genomic windows with ≥1 RPM realigning sirenRNAs and ≤2 mismatches) in Cr × Cr endosperm The numbers on the plots show the number of windows in each category; the breaking points correspond to 0.7 CHG and CHH DNA methylation at sirenRNA trans targets overlapping genes (coding region +2 kb promoters; top) and TEs (bottom) The numbers on the plots show the number of windows in each category; the breaking points correspond to 0.5 Comparison of CHG and CHH DNA methylation levels in 50-bp windows differing in sirenRNA accumulation at trans targets The x axis shows the difference in DNA methylation (Cr × Cr minus Cr × Cg) and the y axis shows the difference in sirenRNA accumulation in trans (Cr × Cr minus Cr × Cg) The numbers correspond to windows in each category; the breaking points correspond to 0.6 the asterisks indicate statistically significant differences as calculated by two-sided Wilcoxon tests with Benjamini and Hochberg correction for multiple comparisons (***P < 0.001; *P < 0.05) Examples of genes and TEs with reduced sirenRNA accumulation at trans targets in Cr × Cg nrpd1 × Cr and nrpd1 × nrpd1 compared with Cr × Cr endosperm The tracks show levels of sirenRNAs and CHH methylation at the indicated genes and TEs Distance to the nearest TE upstream of the transcriptional start site of upregulated genes in Cr × Cg compared with non-upregulated genes The asterisks indicate statistically significant differences as calculated by a two-sided Wilcoxon test (***P < 0.001) Methylation levels of TEs in the promoters of upregulated genes in the Cr × Cg hybrid Statistical differences were calculated with two-sided Wilcoxon’s tests for paired samples Source data Distribution of PHE1 binding motifs in the Capsella genome in different groups of genes: all genes and genes upregulated in Cr × Cg The numbers of pericentromeric (PC) and non-pericentromeric (NPC) genes upregulated in different genotypes are compared with the numbers of all pericentromeric/non-pericentromeric genes The asterisks indicate statistically significant differences calculated by two-sided chi-square tests with Benjamini and Hochberg correction for multiple comparisons (***P < 0.001) The numbers show the number of genes in each category Heat maps and dendrograms show clustering of genes based on the expression (RPKM) of type I γ-MADS-box (b) and α*-MADS-box (c) transcription factors in different Capsella genotypes Heat map and dendrogram show clustering of genes based on log2(fold change) (in comparison to the maternal parent) of γ and α* type I MADS-box transcription factors in different Capsella hybrids Genes marked in bold overlap siren loci; genes marked in purple are targeted by trans-acting sirenRNAs Source data we propose that seed abortion in response to paternal-excess interspecies and interploidy hybridizations is triggered by two factors: the depletion of maternally derived sirenRNAs and the increased expression of AGLs targeting hypomethylated regions in hybrid endosperm This strongly implies a direct connection between the depletion of sirenRNAs and the arrest of hybrid seed development This discrepancy is probably attributed to the amplified AGL expression in the paternal parents of hybrids inducing a more pronounced response in hybrid endosperm than in the nrpd1 mutant directly testing the functional relevance of AGLs in establishing interspecies hybridization barriers in Capsella is challenging but this remains an important task of future studies we propose that maternal sirenRNAs serve as a dosage-sensitive factor that decisively influences the success of hybridization between plant species The seeds were surface sterilized with 30% bleach and 70% ethanol rinsed with distilled water and sown on agar plates containing ½ Murashige and Skoog medium and 1% sucrose The seeds were then stratified for two days in the darkness at 4 °C and moved into a growth chamber with a long-day photoperiod (16 h and 22 °C light 8 h and 19 °C darkness) with a light intensity of 110 µE Seven-day-old seedlings were transferred to pots filled with sterile soil and the plants were grown in a growth chamber with 60% humidity and daily cycles of 16 h light at 21 °C and 8 h darkness at 18 °C with a light intensity of 150 µE dry seeds were stored for 30 days to break dormancy The seeds were then surface sterilized and sown on agar plates as described above The seeds were stratified for two days at 4 °C and then moved to a growth chamber and scored after seven days for seedling establishment The siliques were opened at the side with needles and incubated overnight in fixing solution (ethanol:acetic acid (3:1)) at 4 °C The samples were washed with distilled water three times for 15 minutes and then incubated for 1 h in freshly prepared 5 N HCl the samples were washed again with distilled water three times for 15 minutes and incubated with Schiff reagent (Sigma-Aldrich) for 3–4 h The samples were washed with cold (4 °C) distilled water three times for 10 minutes and then washed in different concentrations of ethanol (10% 70% and 95%) for 10 minutes in each solution The samples were then washed with 99.5% ethanol until they remained colourless (at this point samples can be stored in this solution overnight at 4 °C) The samples were incubated in 99.5% ethanol:LR White (London Resin White + catalyst The samples were incubated in 1:1 ethanol:LR White for 1 h and then in LR White overnight mounted on a microscope slide in a drop of LR White + catalyst and baked at 60 °C for 16 h for polymerization The slides were watched under a two-photon microscope with an excitation wavelength of 800 nm and emission from 518 nm and onwards DNA was extracted from young leaf material of Cg using the CTAB method45 DNA was sized on the BluePippin system (Sage Science) to remove small fragments and then sequenced on one Oxford Nanopore PromethION flow cell and the PromethION release version was 19.05.1 corresponding to about 200× coverage of the estimated genome size of approximately 250 Mb (roughly calculated by flow cytometry analysis) Cg DNA was used to prepare an Illumina overlap library DNA was sheared using a Covaris M220 (Covaris) to 450 bp The sheared DNA was sized using a BluePippin prep system and used to prepare a PCR-free Illumina sequencing library The library was sequenced on the Illumina NovaSeq SP platform using an SP flow cell and 2 ×250 bp protocol with the default parameters) to generate a haploid representation of the heterozygous Cg assembly Seeds derived after manual pollination were dissected out of siliques at 6 DAP and collected in 20 µl of RNAlater solution (Sigma-Aldrich) RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions 500 ng of RNA was used for RNA-seq library preparation using the NEBNext Poly(A)mRNA Magnetic Isolation Module and NEBNext Ultra RNA LibraryPrep Kit for Illumina Three biological replicates were generated for each genotype The libraries were sequenced on an Illumina HiSeq X machine in 150-bp paired-end mode Manually pollinated seeds 6 DAP were removed from siliques and put on a microscopic slide covered with a piece of tissue paper soaked with a drop of RNAlater solution (Sigma-Aldrich) The seeds were gently squashed with another microscopic slide to release the endosperm and the embryos and seed coats were removed with tweezers Tissue paper with the endosperm was put in an Eppendorf tube and frozen in liquid nitrogen Approximately 600 seeds were used per replicate The samples were stored at −70 °C and then used for genomic DNA isolation using the DNeasy Plant Mini Kit (Qiagen) Biological duplicates were generated for each genotype Libraries were prepared with the Accel-NGS Methyl-Seq DNA Library Kit from Illumina and the sequencing was performed on an Illumina NovaSeq 6000 machine in 150-bp paired-end mode and cytosine methylation values calculated using the Bismark Methylation Extractor Differentially methylated regions in the CG CHG and CHH contexts were calculated using 50-bp windows across the genome as units Only hypomethylated regions (Cr × Cr > Cr × Cg Cr × Cr > Cr × 4xCr and Cr × Cr > nrpd1 × Cr) were considered Windows with differences in fractional methylation below the first decile (Fisher’s exact test P < 0.01) were selected and these were merged if they occurred within 300 bp Seeds derived after manual pollination were dissected out of siliques at 6 DAP and squeezed on facial tissue paper to release the endosperm Seed coats and embryos were removed with forceps A drop of RNAlater solution (Sigma-Aldrich) was added to the tissue containing the absorbed endosperm transferred to an Eppendorf tube and frozen in liquid nitrogen Each sample consisted of endosperm from about 600 seeds Small RNAs were extracted using the mirVana miRNA Isolation Kit (ThermoFisher Scientific) following the manufacturer’s instructions for sRNA Libraries were prepared using the NEBNext Multiplex Small RNA Library Prep Set for Illumina with 50–100 ng of input for each sample Size selection was performed on a 6% polyacrylamide gel and bands corresponding to about 150 bp were cut and purified from the gel for further analysis Sequencing was performed on a NovaSeq 6000 machine in 150-bp paired-end mode Adapters were removed from the first read of the 150-bp long read pair of each library using cutadapt and the resulting 18–25-bp long reads were selected mitochondria and structural non-coding RNAs (tRNAs rRNAs or snoRNAs) were removed using bowtie (v.1) Genes and TEs were considered as depleted of sRNAs on the basis of log2(fold change) < 0 and Padj < 0.1 in comparison to the maternal parent (for incompatible hybrids) or to Cr × Cr (for crosses with the nrpd1 mutant) we used own-built ‘pseudogenomes’ in which the above-described SNPs between these species and Cr were substituted into the Cr reference genome Endosperm nuclei spreading was performed as previously described9 Manually pollinated seeds were harvested 4 DAP and incubated overnight in a solution containing 2.5 mM 8-hydroxyquinoline The seeds were subsequently fixed in ethanol:acetic acid (3:1) for at least five hours at 4 °C the seeds were washed with 10 mM citrate buffer and incubated for five hours in an enzyme solution comprising 0.3% cytohelicase 0.3% pectolyase and 0.3% cellulase in 10 mM citrate buffer spread using 60% acetic acid and fixed on the slide with ethanol:acetic acid (3:1) The slides were mounted using Vectashield mounting medium containing DAPI (BioNordika AB) The experiment was conducted with three independent biological replicates Each nucleus was represented by the mean circularity of all chromocenters Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article The role of hybrid seed inviability in angiosperm speciation Dissection of two major components of the post-zygotic hybridization barrier in rice endosperm Parent-of-origin effects on seed development in Arabidopsis thaliana When genomes collide: aberrant seed development following maize interploidy crosses Manipulations of endosperm balance number overcome crossing barriers between diploid Solanum species Non-reciprocal interspecies hybridization barriers in the Capsella genus are established in the endosperm Differences in effective ploidy drive genome-wide endosperm expression polarization and seed failure in wild tomato hybrids Overcoming the species hybridization barrier by ploidy manipulation in the genus Oryza Hybrid seed incompatibility in Capsella is connected to chromatin condensation defects in the endosperm Endosperm evolution by duplicated and neofunctionalized type I MADS-box transcription factors Paternal easiRNAs regulate parental genome dosage in Arabidopsis Paternally acting canonical RNA-directed DNA methylation pathway genes sensitize Arabidopsis endosperm to paternal genome dosage Non-canonical RNA-directed DNA methylation A small RNA pathway mediates allelic dosage in endosperm Imprinted expression of genes and small RNA is associated with localized hypomethylation of the maternal genome in rice endosperm Abundant expression of maternal siRNAs is a conserved feature of seed development Maternal siRNAs as regulators of parental genome imbalance and gene expression in endosperm of Arabidopsis seeds RNA Pol IV induces antagonistic parent-of-origin effects on Arabidopsis endosperm Rice interspecies hybrids show precocious or delayed developmental transitions in the endosperm without change to the rate of syncytial nuclear division Endosperm-based postzygotic hybridization barriers: developmental mechanisms and evolutionary drivers Paternally expressed imprinted genes associate with hybridization barriers in Capsella Divergent mating systems and parental conflict as a barrier to hybridization in flowering plants The Capsella rubella genome and the genomic consequences of rapid mating system evolution Hybrid origins and the earliest stages of diploidization in the highly successful recent polyploid Capsella bursa-pastoris Rapid evolution of genomic imprinting in two species of the Brassicaceae Maternal components of RNA-directed DNA methylation are required for seed development in Brassica rapa ShortStack: comprehensive annotation and quantification of small RNA genes Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5′ terminal nucleotide Ovule siRNAs methylate protein-coding genes in trans Maternal small RNAs mediate spatial–temporal regulation of gene expression Endosperm and seed transcriptomes reveal possible roles for small RNA pathways in wild tomato hybrid seed failure Paternally expressed imprinted genes establish postzygotic hybridization barriers in Arabidopsis thaliana Auxin regulates endosperm cellularization in Arabidopsis Parental conflict driven regulation of endosperm cellularization by a family of Auxin Response Factors The MADS-box transcription factor PHERES1 controls imprinting in the endosperm by binding to domesticated transposons Structure-based discovery and description of plant and animal Helitrons Helitron distribution in Brassicaceae and whole genome Helitron density as a character for distinguishing plant species Polymerase IV plays a crucial role in pollen development in Capsella Nurse cell-derived small RNAs define paternal epigenetic inheritance in Arabidopsis maintaining and modifying DNA methylation patterns in plants and animals Mating system is associated with seed phenotypes upon loss of RNA-directed DNA methylation in Brassicaceae Endosperm cellularization defines an important developmental transition for embryo development Parent-dependent loss of gene silencing during interspecies hybridization Separating phases of allopolyploid evolution with resynthesized and natural Capsella bursa-pastoris Minimap2: pairwise alignment for nucleotide sequences Minimap and miniasm: fast mapping and de novo assembly for noisy long sequences FLASH: fast length adjustment of short reads to improve genome assemblies Fast and accurate short read alignment with Burrows–Wheeler transform Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement Identifying and removing haplotypic duplication in primary genome assemblies Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype featureCounts: an efficient general purpose program for assigning sequence reads to genomic features Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 Long-term balancing selection drives evolution of immunity genes in Capsella Bismark: a flexible aligner and methylation caller for bisulfite-seq applications SNPsplit: allele-specific splitting of alignments between genomes with known SNP genotypes Garrison, E. & Marth, G. Haplotype-based variant detection from short-read sequencing. Preprint at https://arxiv.org/abs/1207.3907 (2012) BEDTools: a flexible suite of utilities for comparing genomic features HTSeq—a Python framework to work with high-throughput sequencing data Improved placement of multi-mapping small RNAs Ultrafast and memory-efficient alignment of short DNA sequences to the human genome Fiji: an open-source platform for biological-image analysis Genetic dissection of morphometric traits reveals that phytochrome B affects nucleus size and heterochromatin organization in Arabidopsis thaliana Recruitment of the histone variant macroH2A1 to the pericentric region occurs upon chromatin relaxation and is responsible for major satellite transcriptional regulation Identification of the elementary structural units of the DNA damage response Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities Dziasek, K. et al. Dosage sensitive maternal siRNAs determine hybridization success in Capsella. figshare https://doi.org/10.6084/m9.figshare.27143508 (2024) Efficient test and visualization of multi-set intersections Download references Lascoux from Uppsala University for kindly sharing tetraploid Co seeds Bente for valuable suggestions about sRNA library preparation and M Incarbone and members of the Köhler group for critical reading and helpful comments on the paper This work was funded by the Knut and Alice Wallenberg Foundation (grant numbers 2018-0206 (C.K.) and 2019-0062 (C.K.)) and the Max Planck Society Open access funding provided by Max Planck Society Swedish University of Agricultural Sciences and Linnean Centre for Plant Biology Department of Plant Reproductive Biology and Epigenetics Max Planck Institute of Molecular Plant Physiology developed the methodology and provided supervision generated the genetic material used in this study acquired the funding and administered the project Nature Plants thanks the anonymous reviewers for their contribution to the peer review of this work Germination of triploid Cr seeds shown as the fraction of germinating seeds from each cross Each filled circle represents one biological replicate; there are three biological replicates per genotype Numbers on top represent total seed number Asterisks represent statistical significance as calculated by one way ANOVA with post hoc Tukey’s HSD test (*** p < 0.001) Red dots show values for three biological replicates Whiskers show the full range excluding outliers Feulgen staining of diploid and triploid Cr seeds at 6 DAP (scale bars represent 200 µm) Cr × 4xCr embryos rescued on Murashige-Skoog medium Upset plot showing the overlap of upregulated genes in comparison to the paternal parent (e) and both parents (f) between interploidy (Cr × 4xCr) and interspecies crosses of different Capsella species Source data Metagene plots show DNA methylation levels of genes in the endosperm of Cg × Cg Boxplots show methylation level of genes in the endosperm of Cg × Cg Asterisks indicate statistically significant differences as calculated by the two-sided Wilcoxon test p-values were adjusted for multiple comparisons with Benjamini and Hochberg correction (** p-value <0.01,*** p-value < 0.001 Metagene plots show DNA methylation levels of genes in the endosperm of Cg × Cr at 6 DAP in comparison to published data for Cr × Cr Boxplots show methylation level of genes in the endosperm Cg × Cr at 6 DAP in comparison to published data for Cr × Cr p-values were adjusted for multiple comparisons with Benjamini and Hochberg correction (*** p-value < 0.001) Metagene (e) and box plots (f) show DNA m2ethylation levels of genes in the endosperm of diploid and triploid 6 DAP Cr seeds Metagene (g) and box plots (h) show DNA methylation levels of TEs in the endosperm of diploid and triploid 6 DAP Cr seeds Boxplots show CHG (left panel) and CHH (right panel) methylation levels on TEs in chromosome arms and pericentromeric regions in the endosperm of Cr × Cr Cr × Cg and Cg × Cg and Cr × 4xCr at 6 DAP Asterisks indicate statistically significant differences as calculated by the two-sided Wilcoxon test (*** p-value < 0.001) Boxes show median values and the interquartile range Source data Metagene (a) and box plots (b) show DNA methylation levels of TEs in the endosperm of Cr × Co Metagene (c) and box plots (d) show DNA methylation levels of TEs in the endosperm of Cr × Co Source data Source data Barplot shows number of aborted and partially aborted seeds of three biological replicates of each genotype Numbers above the bars indicate number of analysed seeds Pictures of cleared seeds of Cr × Cr and nrpd1 × Cr at 6 DAP Inset shows enlarged regions of cellularized (Cr × Cr) and uncellularized (nrpd1 × Cr) endosperm Scale bars represents 100 µm (upper row) and 10 µm (lower row) Quantification of endosperm cellularization of indicated genotypes at 6 DAP Numbers correspond to numbers of analysed seeds Seed phenotypes of 4xCr × Cg and 4xnrpd1 × Cg seeds Barplot shows number of aborted and partially aborted and viable seeds of three biological replicates of each genotype Seed phenotypes of Cr × 4xCr and Cr × 4xnrpd1 seeds Source data Non-metric MultiDimensional Scaling (NMDS) of small RNA (sRNA) sequencing samples Correlogram showing the correlation score matrix across all sequenced sRNA libraries Barplot shows size distribution of sRNAs in all sequenced libraries Siren loci are larger than other siRNA expressing loci Genomic features overlapping with 24 nt-dominant clusters (n=28 075) and siren loci (n=1 385) in Cr The genome was subdivided into 50-bp windows (n= 2 497 626) that were assigned to either exons transposable elements and edges (50bp regions at the boundaries of gene bodies and repeats) Frequency distribution of sirenRNA clusters after dominating size class 24 nt siRNAs dominate in the majority of sirenRNA clusters defined by ShortStack 5′ nucleotide of sirenRNAs (18–24 nt) in Cr × Cr endosperm Number of siren loci normalized per size of respective genomic regions (in bp) – non-pericentromeric and pericentromeric for each of the eight Cr chromosomes Distribution of siren loci along the eight chromosomes of the Cr genome black boxes indicate positions of pericentromeric regions TE families overlapping with24 nt-dominant and siren loci compared to the genome-wide frequencies in Cr Source data siRNA accumulation and CHH methylation in the endosperm of indicated genotypes on selected genes overlapping siren loci: Carubv10025009m.g (AGL33) Boxplots show DNA methylation on γ and α* type I MADS box TFs in 6 DAP endosperm of different Capsella genotypes Asterisks indicate statistically significant differences as calculated by the two-sided Wilcoxon test for paired samples p-values were adjusted with Benjamini and Hochberg correction (*** p-value < 0.001 Barplot shows log2 fold change expression level of indicated genes in comparison to respective maternal plants Asterisks indicates statistical significance as calculated with Wald test in DESEQ2 (*** padj < 0.001 Source data Boxplot show parental-specific DNA methylation levels of genes (a) and TEs (b) in the endosperm of Cr × Cg Maternal and paternal methylation values in each genotype are compared to the respective parent in the Cr × Cg cross p-values were adjusted for multiple comparisons with Benjamini and Hochberg correction (p-value: ***< 0.001 Boxplot show parental-specific DNA methylation levels of genes (c) and TEs (d) in the endosperm of Co × Cr Maternal and paternal methylation values in each genotype are compared to the respective parent in Co × Cr cross Source data Heatmaps show expression of AGL genes (RPKM) targeted by trans-acting sirenRNAs accumulation of sirenRNAs at trans AGL targets and corresponding CHG and CHH methylation Tracks show sirenRNA accumulation at trans AGL targets and corresponding CHH and CHG methylation levels (same set of genes as on panel a) The purple line above the tracks marks cis acting siren RNAs Light green and navy-blue bars on sirenRNA tracks show reads mapping in cis at siren loci Barplots show the proportion of maternal and paternal reads in genes upregulated in Cr × Cg (c) γ-type AGLs upregulated in Cr × Cg (d) and selected orthologs of genes that have been previously reported as imprinted and involved in endosperm development or the triploid block in Arabidopsis (e) Expression data are obtained from whole seeds at 6 DAP Black horizontal line on each plot represents the expected 2:1 ratio (maternal:paternal) for biallelic genes in the endosperm Boxplot show the ratio of paternal:maternal read counts for all genes and genes upregulated in Cr × Cg Source data The table shows log2 fold changes of gene expression in the endosperm of the indicated crosses List of siren loci and overlapping genomic features The table shows the positions and normalized read counts over siren loci in the endosperm of the indicated genotypes List of potential trans targets affected in the Cr × Cg hybrid The table shows differences in sirenRNA accumulation (Diff_RPM > 0) differences in CHH and CHG methylation (diff_CHH > 0.01 OR diff_CHG > 0.05) and differences in RNA accumulation (log2 fold change > 1 p < 0.05) in Cr × Cg compared with Cr × Cr Download citation DOI: https://doi.org/10.1038/s41477-024-01844-3 Karen O’Leary is an Associate Research Analysis Editor with Nature Medicine Persistent chylomicronemia is usually caused by familial chylomicronemia syndrome People with persistent chylomicronemia have impaired clearance of chylomicrons (a type of lipoprotein) which leads to very high triglyceride levels and recurrent pancreatitis Treatment involves lifestyle modification and triglyceride-lowering therapies (such as statins) — but in trials these have failed to reduce the risk of pancreatitis doi: https://doi.org/10.1038/d41591-024-00070-w Mutation linked to thriving with little rest Native American tribe teams up with genomicists to confirm link to iconic ancient site Picuris Pueblo oral history and genomics reveal continuity in US Southwest Automated loss of pulse detection on a consumer smartwatch Clinical trial links oncolytic immunoactivation to survival in glioblastoma A COVID-19 peptide vaccine for the induction of SARS-CoV-2 T cell immunity Structure of the human DICER–pre-miRNA complex in a dicing state Sequence determinant of small RNA production by DICER poly(UG)-tailed RNAs in genome protection and epigenetic inheritance POST-DOCTORAL RESEARCH FELLOW Post Summary of the role The Characterisation & Processing of Advanced Materials HT is an interdisciplinary research institute created and supported by the Italian government whose aim is to develop innovative strategies to pr.. 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Volume 11 - 2023 | https://doi.org/10.3389/fbioe.2023.1112755 This article is part of the Research TopicNanotechnology and Nanoscience to manage SARS-CoV-2 Variants of ConcernView all 14 articles Small interfering RNA (siRNA)-mediated mRNA degradation approach have imparted its eminence against several difficult-to-treat genetic disorders and other allied diseases Viral outbreaks and resulting pandemics have repeatedly threatened public health and questioned human preparedness at the forefront of drug design and biomedical readiness During the recent pandemic caused by the SARS-CoV-2 mRNA-based vaccination strategies have paved the way for a new era of RNA therapeutics RNA Interference (RNAi) based approach using small interfering RNA may complement clinical management of the COVID-19 RNA Interference approach will primarily work by restricting the synthesis of the proteins required for viral replication thereby hampering viral cellular entry and trafficking by targeting host as well as protein factors the stability of small interfering RNA in the physiological environment is of grave concern as well as site-directed targeted delivery and evasion of the immune system require immediate attention nanotechnology offers viable solutions for these challenges The review highlights the potential of small interfering RNAs targeted toward specific regions of the viral genome and the features of nanoformulations necessary for the entrapment and delivery of small interfering RNAs In silico design of small interfering RNA for different variants of SARS-CoV-2 has been discussed Various nanoparticles as promising carriers of small interfering RNAs along with their salient properties This review will help tackle the real-world challenges encountered by the in vivo delivery of small interfering RNAs and readily available drug candidate for efficient management of SARS-CoV-2 in the future The review equivocally concludes the significance of siRNA-based nanoparticle formulation as a better alternative against SARS-CoV-2 the study did not consider any in vivo delivery approach Whether targeting viral or host targets are more effective (preferable) is an open question siRNA guide strand sequences reported in literature along with their target site FIGURE 2. Computational pipeline to predict siRNA against target segments of SARS-CoV-2. Adapted with modifications from Ref. (Chowdhury et al., 2021) Some of the potential nanocarriers are briefly discussed in this section FIGURE 3. Potential nanocarrier configurations for siRNA delivery. The carriers can be categorized into organic (i.e., derived from lipid, polymeric and polysaccharides) and inorganic types. The size and structural features of these nanocarriers are depicted. Adapted from Ref. (Zhang et al., 2022a) PEGylated lipids and cholesterol) used for lipid nanoformulations FIGURE 5. The ionization characteristics of the commercially available ILs obtained through zeta potential measurement. The LNPs showed a transition of charges-positive zeta potential at low pH and negative zeta potential at high pH, covering the range of endosomal and lysosomal pH. Adapted from Ref. (Carrasco et al., 2021) The degraded fragments of the lipids are rapidly cleared from the body allowing for multiple doses within a short duration The prepared lipidoid CS12-200 nanoparticles showed 83.8% siRNA loading when formulated with or without PEGylated helper lipids The transfection efficacy for siRNA on neural cells was also increased twice without toxicity the suitability of lipidoids for the delivery of siRNA for various purposes can be explored FIGURE 6. Folate incorporated liposome formulation for gene silencing applications. siRNA for Mcl-1 (a protein expressed in the rheumatoid joint macrophages) silencing was loaded in liposome and targeted towards folate receptor. PEGylated liposome offered stealth features against phagocytic uptake; neutral lipids exhibited low toxicity while specific targeting was observed by anchoring folate-targeted peptides. Adapted from Ref. (Nogueira et al., 2017) The γ-[32P]-ATP labeled siRNA was efficiently transfected in three different xerographs without exhibiting toxicity The knockdown efficacy in H441-luc cells due to the polymer-tyrosine-siRNA complex was also found efficient proving the capabilities of polymeric nanoparticles for siRNA delivery The schematic of an IL illustrating the main elements and how they contribute towards stability FIGURE 9. Schematic illustration of the stability of LNPs based on size. Adapted from Ref. (Sato et al., 2016) ALN-0319 (adapted from ref 113) L3 (adapted from ref 112) CL4H6 (adapted from ref 111) with cleavable ester linkage sites (highlighted in blue color) and expected hydrolysis pathway catalyzed by esterases Better understanding and control of the LNP fate in vivo is important and this has been actively explored in numerous studies Algarni et al. have explored the targeting efficiency of three ionizable cationic lipids, DLin-MC3-DMA, DLin-KC2-DMA and DODAP (1,2-dioleoyl-3-dimethylammonium-propane), for the organ-specific delivery of pDNA (Algarni et al., 2022) The intravenous administration in mice models with LNPs formulations showed that the DLin-MC3-DMA and DLin-KC2-DMA bearing LNPs more precisely and efficiently transfected the nucleic acid cargo to the spleen instead of the liver DLin-MC3-DMA and DLin-KC2-DMA with two double bonds per alkyl chains has influenced the transfection efficiency in the spleen the selection of suitable ILs may improve the targeting The LNP contained DMKE (45%) (O,O′-dimyristyl-N-lysyl glutamate) and was prepared by a methanol and chloroform mixture (2:1 FIGURE 11. (A) The structure of aptamer molecules conjugated onto LNP-RNAi complex. LNPs were first complexed with siRNAs, which were then conjugated with synthesized aptamers. (B) SELEX-based method for aptamer selection after multiple rounds of binding, separation, washing, elution and amplification. Adapted from ref. (Saify Nabiabad et al., 2022) the in vitro and in vivo targeting aspects of these aptamers are yet to be verified (A) Preparation of SORT LNPs for mRNA delivery for liver spleen and lungs by incorporation of different SORT lipids in the formulation of LNPs (B) The level of luciferase expression after administration of 5A2-SC8 SORT LNPs in mice models With the increased cationic lipid DOTAP percentage the expression of luciferase shifted from liver to spleen to lungs Effects of incorporation of SORT molecules in LNPs formulations visible from luminescence profile for tested mice models. Adapted from Ref. (Cheng et al., 2020) further bursting of endosomes releasing the siRNA in the cytoplasm following the complete disintegration of LNP structure during the process the fast mutation rate of the viral genome has to be kept in mind with potentially targeting the conserved viral domains with the help of available libraries and computational resources Approved and under trial siRNA therapeutics for different diseases Considering the ever-changing dynamic mutations in the SARS-CoV-2 genome the demand for well-targeted specific and highly effective therapeutics are needed Genomic regions of the virus conserved across the variants and sub-variants could be first targeted to inhibit viral entry and replication in the host Many in silico algorithms and web servers have been deployed to design siRNAs following the three golden rules of design The accessory parameters such as heat capacity (CP) and off-target matches are predicted computationally some validation efforts have been attempted to experimentally verify the silencing efficiency of the computationally designed siRNAs but this will require more extensive studies to assure confidence in the theoretical designs a handful of effective designs are now available that could be tested in a clinical setting should there be sufficient impetus from clinicians and industrial parties the designed siRNAs can be formulated with different nanocarriers for practical utility Lipids-based and polymeric nanoparticles offer the flexibility of conjugation with siRNAs and surface functionalization for targeted delivery siRNAs-based therapies have been approved for other diseases but in the case of COVID-19 clinical trials are yet to be undertaken Along with the efficacy of nano-formulations other inflammatory responses should be investigated through in vivo studies excessive inflammation that is ultimately cause of patient exhaustion in clinic The dosage of siRNAs should be confined to a minimum concentration preferably below 100 nM at local sites and <1 mg/kg overall for practical translation of delivery systems The siRNA-based nano-formulations appear to be promising for the therapy of contagious COVID-19 and post-COVID inflammations and LP drafted the manuscript and LP and HU edited the manuscript The authors acknowledge the Shastri Indo-Canadian Institute for the Shastri COVID-19 Pandemic Response Grant (SCPRG) 2020-21 for financial assistance for this work The studies in HU’s lab are supported by NSERC NFRF and project support by MITACS/RJH Biosciences HU has ownership interest in materials that could be used in siRNA delivery AR was supported by a MITACS fellowship sponsored by RJH Biosciences The remaining 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 All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher Transactivation response RNA-binding protein; SARS-COV 2 Severe acute respiratory syndrome coronavirus 2; LNP (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate; ALC-0315 [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate); SM-102 (1-Octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6(undecyloxy)hexyl]amino]-octanoate); DOPE 1,2-dioleoyl-3-dimethylammonium-propane; RBD Systemic Evolution of Ligands by EXponential enrichment; DMKE O,O′-dimyristyl-N-lysyl glutamate; SORT Dioleoyl-3-trimethylammonium propane; hATTR Efficacy and safety of vutrisiran for patients with hereditary transthyretin-mediated amyloidosis with polyneuropathy: A randomized clinical trial CrossRef Full Text | Google Scholar RNA-targeting and gene editing therapies for transthyretin amyloidosis PubMed Abstract | CrossRef Full Text | Google 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Google Scholar Uludag H and Pandey LM (2023) Potential of siRNA in COVID-19 therapy: Emphasis on in silico design and nanoparticles based delivery Received: 30 November 2022; Accepted: 13 January 2023;Published: 06 February 2023 Copyright © 2023 Fopase, Panda, Rajendran, Uludag and Pandey. 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Volume 5 - 2025 | https://doi.org/10.3389/finsc.2025.1574585 RNA interference (RNAi) is a promising gene-silencing technique for pest control that targets essential genes We assessed the potential of double-stranded RNA (dsRNA) and small interfering RNA (siRNA) to silence mesh or iap genes in the midguts of Spodoptera litura larvae Despite the theoretical promise of RNAi approaches our findings revealed that dsRNA did not induce significant gene silencing or impact larval growth whereas siRNA exhibited clear insecticidal effects likely by disrupting intestinal osmoregulation and impairing larval fitness Detailed analysis indicated that dsRNA could not be efficiently converted into functional siRNA in the midguts of S possibly due to the low expression levels of Dicer-2 and the rapid degradation of dsRNA within the gut environment while dsRNA demonstrated greater environmental stability than siRNA under soil conditions litura to process dsRNA effectively limits its viability as a pest control agent These findings indicate the critical role of Dicer-2 in RNAi-mediated gene silencing and highlight the challenges and limitations of employing dsRNA-based genetic pesticides in lepidopteran species the efficacy of dsRNA-based pesticides in controlling lepidopteran pests we assume that targeting mesh could reduce the integrity and normal function of larval digestive system and causing mortality Our findings indicate that the low expression of Dicer-2 coupled with the rapid degradation of dsRNA in the gut environment of S significantly impedes the conversion of dsRNA into functional siRNA thereby rendering dsRNA-based pest control strategies ineffective in this species These results highlight the importance of further research aimed at improving dsRNA stability and enhancing conversion efficiency in lepidopterans may offer a more effective approach for pest management in S Total RNA was isolated from various larval instars using TRIzol reagent (Invitrogen), following the manufacturer’s instructions. A NanoDrop OneC spectrophotometer (Thermo Fisher Scientific) was used to determine the RNA concentration. cDNA was synthesized from 500 ng of total RNA using the PrimeScript RT Reagent Kit (TaKaRa) following the manufacturer’s instructions. cDNA was diluted 10-fold for subsequent experiments (14) Double-stranded RNA (dsRNA) targeting the mesh and iap genes was synthesized using gene-specific primers designed via the Primer-BLAST tool (NCBI) The primer sequences for Mesh were 5’-CGC TTC TCA AGT GTG GGT CT-3’ (forward) and 5’-TGA CCT GAC CGT GGA TGT TG-3’ (reverse) while those for iap were 5’-CCG TGG AGA TGA AGT CCG TT-3’ (forward) and 5’-CCC AGG GTA CGT CAT GGT TC-3’ (reverse) A mesh or iap gene fragment was PCR-amplified using “Fermentas” under the following conditions: 5 min at 96°C; 40 cycles at 96°C for 30 s and 72°C for 20 s; and a final extension at 72°C for 3 min the PCR product was used as a template for the second round of PCR to add T7 promoter sequences to both ends A GenepHlow gel/PCR kit (Geneaid) was used to purify the resulting product The MEGAscript T7 Kit (Invitrogen) was used to synthesize dsRNA against the mesh or iap genes following the manufacturer’s instructions TURBO DNase digestion removed the template DNA and dsRNA was recovered using TRIzol reagent The quality and quantity of dsRNA were assessed by electrophoresis on a 1% agarose gel and spectrophotometry Taiwan conducted a mesh or iap gene sequence analysis for siRNA synthesis and further predicted and synthesized three siRNA sequences The primer of candidate genes for analysis gene expression Total protein from the dissected midgut of S litura was extracted by homogenization in 60 µL 1× RIPA solution (Bio Basic Inc. followed by 20 min of ice incubation and centrifugation at 1,000 ×g for 10 min to remove debris The samples (20 µL each) were mixed with an equal volume of 4× Laemmli Sample Buffer (Bio-Rad The samples were subsequently incubated at 99°C for 10 min and stored at −20°C until use Electrophoresis was performed using the Mini-PROTEIN Tetra System (Bio-Rad) and PowerPac Basic (Bio-Rad) Protein samples (20 µL) and 5 µL of AccuRuller RGB Pre-stained Protein Ladders (Omics Bio Taiwan) were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto an Immobilon Transfer Membrane (Millipore The membrane was blocked with 5% bovine serum albumin (UniRegion Bio-Tech Taiwan) in Tris-buffered saline with 0.1% Tween 20 (VWR Life Science AMRESCO U.S.) and subsequently incubated with rabbit polyclonal anti-Dicer-2 (1:5000; Abcam ab4732) and mouse monoclonal actin primary antibodies (1:2500; Yao-Hung Biotechnology Inc. This was followed by a 1-h incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:1000; Millipore) for the anti-Dicer-2 antibody and goat anti-mouse secondary antibody (1:1000; Millipore) for the actin antibody at 26°C The signal was detected using UVP Chemostudio (Analytik Jena) Differential RNAi-mediated silencing of the mesh gene and its impact on Spodoptera litura larval mortality (A) Expression of the mesh gene in the midgut of Spodoptera litura larvae 24 hours after feeding with dsRNA or siRNA The control group was fed with siRNA targeting the egfp gene (B) Larval mortality assessed 12 days after feeding with 0.3 μg of dsMESH or siMESH Data represent the mean ± standard deviation from three independent experiments Differences between experimental groups were analyzed using Student’s t-test Effect of dsRNA injection and feeding on Spodoptera litura (A) qPCR analysis of mesh gene expression 24 hours after injecting 0.3 μg dsRNA per individual The control group was injected with dsRNA targeting the egfp gene (B) qPCR analysis of mesh gene expression in the midgut 24 hours after feeding with 0.3 μg dsRNA per individual Expression levels in the control group were set to 100% Three biological replicates were performed Statistical differences between groups were evaluated using Student’s t-test with significance defined as p < 0.05 (*) To verify that the difference in RNAi efficiency between dsRNA and siRNA feeding is not gene-specific, we conducted an additional experiment targeting a second gene, the inhibitor of apoptosis protein (iap). Similar results were observed, showing that siRNA feeding significantly suppressed gene expression (Figure 3A) and induced higher mortality (Figure 3B) compared to dsRNA feeding Enhanced insecticidal effect of siRNA Targeting the iap Gene in Spodoptera litura larvae (A) Expression of the iap gene in the midgut of S litura larvae 24 hours after feeding with dsRNA or siRNA (B) Impact of dsIAP and siIAP feeding on larval survival over 12 days larvae were fed 0.3 μg of dsRNA or siRNA per individual per day for four consecutive days While the same mass of dsRNA and siRNA was used their molar amounts differ due to the shorter length of siRNA resulting in a higher molar concentration compared to dsRNA Statistical significance was indicated as p< 0.05 (*) Conversion Efficiency of dsRNA into siRNA and Expression Levels of RNAi-Related Genes in the Midgut of S (A) Northern blot analysis showing the conversion of dsRNA into siRNA in S Small RNA was extracted from the midgut at various time points (2 12 and 24 hours post feeding) following dsRNA ingestion C: Synthesized small RNA was directly loaded onto a gel as an untreated control (B) qRT-PCR analysis of key genes involved in the processing of long dsRNA into siRNA in the midguts of 2nd to 4th instar S Ago2-X1 and Ago2-X2 represent alternative splicing isoforms of Ago2 The scale bar indicated -ΔΔCT value subtracted by Dicer2 in each instar The number in each cell represented the average and standard deviation (n=5) This low dicer-2 expression may explain the failure of dsRNA to effectively trigger RNAi in the midgut suggesting that reduced dicer-2 expression may be an important factor in the inefficient RNAi response Reduced Dicer-2 expression in the midgut of Spodoptera litura larvae impairs the conversion of double-stranded RNA (dsRNA) into small interfering RNA (siRNA) (A) qRT-PCR analysis was performed to measure the expression levels of the dicer-2 gene in the midguts of various insect species The expression level of 18S rRNA was independently quantified for each species and standardized to 100% The relative expression levels of the dicer-2 gene were normalized against this baseline Statistical differences between experimental groups were analyzed using Student’s t-test Statistical significance was indicated as p < 0.01 (**) (B) Western blot analysis confirming Dicer-2 expression in the midgut of S Anti-actin antibodies were used as a loading control Total protein was extracted from the midgut tissue qRT-PCR analysis of dsRNA degradation in midgut fluid from S Midgut juice was extracted from a single larva per biological replicate The gut juice from one larva was mixed with PBS (1:10 dilution) before incubating with 40 μg of dsRNA Degradation was analyzed at 30 and 60 minutes using qRT-PCR The y-axis represents the relative transcript abundance A relative transcript abundance of 0 indicates undetectable dsRNA levels The experiment was conducted with four biological replicates These findings indicate that the failure of dsRNA to function in S litura is due to both low Dicer-2 expression these physiological limitations suggest that dsRNA-based pesticides are unlikely to be effective against S Impact of double-stranded RNA (dsRNA) and small interfering RNA (siRNA) on Spodoptera litura larvae whereas siRNA demonstrates notable toxicity against the larvae litura is attributed to the absence of key genes in the midgut necessary for processing dsRNA into siRNA These findings indicate the limitations of dsRNA in S litura due to inefficient RNAi processing and rapid degradation in the midgut particularly with the potential for formulation improvements that improve stability and uptake in pest species resistant to traditional RNAi approaches The raw data supporting the conclusions of this article will be made available by the authors The manuscript presents research on animals that do not require ethical approval for their study The author(s) declare that financial support was received for the research and/or publication of this article This research was funded by the grant MOST111-2313-B-002-038-MY3 to Y-LW from the Ministry of Science and Technology Alexander Barton for kindly revising the manuscript 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) declare that no Generative AI was used in the creation of this manuscript Antifeedant and larvicidal activity of bioactive compounds isolated from entomopathogenic fungi Penicillium sp for the control of agricultural and medically important insect pest (Spodoptera litura and Culex quinquefasciatus) Development of a hybrid delta-endotoxin and its expression in tobacco and cotton for control of a polyphagous pest Spodoptera litura Assessing pesticides in the atmosphere: A global study on pollution monitoring network and regulatory performance Pesticides: An alarming detrimental to health and 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thuringiensis toxin receptor RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design Biological mechanisms determining the success of RNA interference in insects doi: 10.1016/B978-0-12-800178-3.00005-1 Reduction in deformed wing virus infection in larval and adult honey bees (Apis mellifera L.) by double-stranded RNA ingestion doi: 10.1111/j.1365-2583.2012.01150.x HMGB1-like dorsal switch protein 1 of the mealworm acts as a damage-associated molecular pattern Reduced stability and intracellular transport of dsRNA contribute to poor RNAi response in lepidopteran insects A nuclease specific to lepidopteran insects suppresses RNAi Double-stranded RNA technology to control insect pests: current status and challenges Towards the elements of successful insect RNAi Lin Y-H and Wu Y-L (2025) Differential RNAi efficacy of siRNA and dsRNA targeting key genes for pest control in Spodoptera litura Received: 11 February 2025; Accepted: 01 April 2025;Published: 30 April 2025 Copyright © 2025 Lin, Lu, Liu, Su, Lin and Wu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Yu-Hsien Lin, eS5oLmxpbkB1dmEubmw=; Yueh-Lung Wu, cnVud3VAbnR1LmVkdS50dw== †These authors have contributed equally to this work Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish Metrics details small-interfering RNAs (siRNAs) mediate epigenetic silencing via the RNA-directed DNA methylation (RdDM) pathway which is particularly prominent during reproduction and seed development there is limited understanding of the origins and dynamics of reproductive siRNAs acting in different cellular and developmental contexts we used the RNaseIII-like protein RTL1 to suppress siRNA biogenesis in Arabidopsis pollen and found distinct siRNA subsets produced during pollen development We demonstrate that RTL1 expression in the late microspore and vegetative cell strongly impairs epigenetic silencing and resembles RdDM mutants in their ability to bypass interploidy hybridization barriers in the seed germline-specific RTL1 expression did not impact transgenerational inheritance of triploid seed lethality These results reveal the existence of multiple siRNA subsets accumulated in mature pollen and suggest that mobile siRNAs involved in the triploid block are produced in germline precursor cells after meiosis or in the vegetative cell during pollen mitosis but the biological significance of this mechanism remains poorly understood By using two pollen-specific promoters with very different expression dynamics we were able to identify distinct TE-derived siRNA subsets produced during pollen development and demonstrate that RTL1 expression in pollen impairs epigenetic silencing to promote triploid seed viability in the next generation a Schematic representation of RTL1 expression in mature pollen driven by the pLAT52 and pMGH3 promoters that are preferentially expressed in the vegetative cell and sperm cells b Principal component analysis after variance-stabilizing transformations shows reproducibility of small RNA sequencing experiments for two independent transgenic lines of each construct and wild-type Col-0 and highlights variance along PC1 and PC2 between all sample types c Heatmap representation of siRNA depletion in pLAT52::RTL1 and pMGH3::RTL1 pollen (vs WT Col-0) shows that distinct loci are affected in the two transgenic lines d Barplots present examples of different protein-coding genes showing reduced levels of 21/22-nt siRNA in pLAT52::RTL1 or in pMGH3::RTL1 pollen Dots and bars represent expression level of individual replicates and mean values (n = 2) a Comparative small RNA analysis between transgenic and WT Col-0 pollen shows that siRNA depletion in pLAT52::RTL1 and pMGH3::RTL1 pollen occurs primarily at TEs Among all TEs annotated in the Arabidopsis TAIR10 genome the LTR/Gypsy superfamily was found over-represented in the lists of differentially expressed siRNA loci b Different members of the LTR/Gypsy superfamily showed significantly reduced siRNA levels in pLAT52::RTL1 or pMGH3::RTL1 pollen The bar plots show the fraction of TE superfamilies in the two datasets c Many retrotransposons from the ATGP1 family lost siRNAs specifically in pLAT52::RTL1 pollen while ATGP2 and ATGP2N lost siRNAs specifically in pMGH3::RTL1 pollen The bar plots show the fraction of TE families in the two datasets d siRNAs depleted in pLAT52::RTL1 or pMGH3::RTL1 pollen are dependent on Pol IV activity c Genome browser tracks show CHH methylation (green) and RNA sequencing (black) datasets at selected loci Partial CHH hypomethylation is observed specifically in nrpd1a-3FTL and pLAT52::RTL1 pollen leading to the transcriptional activation of retrotransposons d RNA sequencing confirmed RTL1 expression in both pLAT52::RTL1 and pMGH3::RTL1 pollen but up-regulation of LTR/Gypsy retroelements from the ATHILA4A ATHILA4 and ATGP1 families occurs only in pLAT52::RTL1 and nrpd1a-3FTL pollen and normalized expression values were calculated by DESeq2 using the median of ratios method Source data are provided as a Source Data file and in Supplementary Data 2 a The triploid block response in jas-3 mutants is quantified by counting the number of collapsed seeds in six siliques of selfed plants which reflects the amount of diploid pollen produced in this mutant background (30 to 40%) Seed abortion is significantly reduced in jas-3/nrpd1a-3 double mutant plants in the F3 generation and also across five consecutive generations of three independent jas-3;pLAT52::RTL1 lines while jas-3;pMGH3::RTL1 remains similar to jas-3 controls Numbers above each box represent the number of individual plants used for each genotype and their levels of triploid seed collapse is represented by the dots on top of each box Boxes represent the interquartile range (IQR) showing the lower (Q1) and upper (Q3) quartiles surrounding the median (central line) and whiskers represent the minimum (Q1 –1.5*IQR) and maximum (Q3 + 1.5*IQR) values Statistically significant differences in the percentage of collapsed seeds were calculated by ANOVA with a post hoc Dunnett test using jas-3 as the reference group (ns is not significant b Representative images of seeds show lower levels of seed abortion in suppressor lines jas-3/nrpd1a-3 and jas-3;pLAT52::RTL1 in comparison with a non-suppressor line jas-3;pMGH3::RTL1 and jas-3 controls It is tempting to speculate that a subset of germline small RNAs is part of a surveillance mechanism that is primed to act only in situations of epigenetic instability caused by stress or hybridizations that lead to bursts of TE expression and activity These outstanding questions may now be tested by using RTL1-mediated suppression of sperm siRNAs in different plant species and developmental contexts Transgenic seeds were surface sterilized with 50% bleach followed by 70% ethanol for 2 min washed with sterile deionized water and sowed on agar plates (0.5X MS medium pH 5.7) supplemented with cefotaxime (250 mg/L Plates were placed in a growth chamber at 23 °C 120 μE m–2 light with a 16-h light/8-h dark (long days) photoperiod for two weeks and seedlings were then transferred to soil and grown in greenhouse long-day conditions to complete the life cycle (16 h light and 8 h dark) MGH3 and ACT2 were used as internal controls Approximately 500,000 pollen grains of each population were used for downstream analysis Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Vaucheret, H. & Voinnet, O. The plant siRNA landscape. Plant Cell https://doi.org/10.1093/plcell/koad253 Chow, H. T. & Mosher, R. A. Small RNA-mediated DNA methylation during plant reproduction. Plant Cell https://doi.org/10.1093/plcell/koad010 Epigenetic control of transposons during plant reproduction: From meiosis to hybrid seeds Small RNAs: big impact on plant development The expanding world of small RNAs in plants RNA-directed DNA methylation: an epigenetic pathway of increasing complexity Reinforcement of CHH methylation through RNA-directed DNA methylation ensures sexual reproduction in rice mediator of paramutation1 is required for establishment and maintenance of paramutation at multiple maize loci Life after meiosis: patterning the angiosperm male gametophyte Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes Epigenetic reprogramming and small RNA silencing of transposable elements in pollen Transposon-derived small RNAs triggered by miR845 mediate genome dosage response in Arabidopsis The miRNome function transitions from regulating developmental genes to transposable elements during pollen maturation Loss of small-RNA-directed DNA methylation in the plant cell cycle promotes germline reprogramming and somaclonal variation Silencing in sperm cells is directed by RNA movement from the surrounding nurse cell Wu, W. et al. Heterochromatic silencing is reinforced by ARID1‐mediated small RNA movement in Arabidopsis pollen. New Phytol. https://doi.org/10.1111/nph.16871 (2020) Nurse cell­–derived small RNAs define paternal epigenetic inheritance in Arabidopsis Plants encode a general siRNA suppressor that is induced and suppressed by viruses A plant germline-specific integrator of sperm specification and cell cycle progression Artificial microRNAs reveal cell-specific differences in small RNA activity in pollen Epigenetic reprogramming rewires transcription during the alternation of generations in Arabidopsis Arabidopsis male sexual lineage exhibits more robust maintenance of CG methylation than somatic tissues Pollen tetrad-based visual assay for meiotic recombination in Arabidopsis Arabidopsis RNA polymerase IV generates 21–22 nucleotide small RNAs that can participate in RNA-directed DNA methylation and may regulate genes Paternally acting canonical RNA-directed dna methylation pathway genes sensitize arabidopsis endosperm to paternal genome dosage The Arabidopsis Mutant jason produces unreduced first division restitution male gametes through a parallel/fused spindle mechanism in meiosis II An imprinted gene underlies postzygotic reproductive isolation in Arabidopsis thaliana Hypomethylated pollen bypasses the interploidy hybridization barrier in Arabidopsis Bypassing reproductive barriers in hybrid seeds using chemically induced epimutagenesis Induction of RNA-directed DNA methylation upon decondensation of constitutive heterochromatin Borg, M. et al. Targeted reprogramming of H3K27me3 resets epigenetic memory in plant paternal chromatin. Nat. Cell Biol. https://doi.org/10.1038/s41556-020-0515-y Buttress, T. et al. Histone H2B.8 compacts flowering plant sperm through chromatin phase separation. Nature https://doi.org/10.1038/s41586-022-05386-6 Non-cell-autonomous small RNA silencing in Arabidopsis female gametes Asymmetric expression of Argonautes in reproductive tissues Herridge, R. P. et al. Pseudouridine guides germline small RNA transport and epigenetic inheritance. Preprint at https://doi.org/10.1101/2023.05.27.542553 (2023) Floral dip: a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications Transgenerational effect of mutants in the RNA-directed DNA methylation pathway on the triploid block in Arabidopsis Download references This work was supported by the grant EpiHYBRIDS from the French National Agency of Research (ANR-19-CE120008 to F.B.) We thank all members of the EPIREP team for daily discussions Hervé Vaucheret for critical reading of the manuscript and sharing the p35S::RTL1-Myc plasmid and seeds and Gregory Copenhaver for the FTL1230 line The authors acknowledge technical support from Mickael Bourge and Nicolas Valentin from the cytometry facility of the I2BC The IJPB benefits from the support of Saclay Plant Sciences-SPS (ANR-17-EUR-0007) Present address: School of Biosciences and Technology analyzed the data and wrote the manuscript with contributions from K.P Download citation DOI: https://doi.org/10.1038/s41467-024-48950-6 Two start-ups have launched to develop next-generation drugs using small interfering RNA (siRNA) to silence genes is a spin-off of the venture capital firm Atlas Venture The company spent about 3 years in stealth while designing a drug candidate that combines a ligand with siRNA that should silence messenger RNA (mRNA) and reduce the number of certain solute carrier proteins that are involved in disease While siRNA is a proven therapeutic approach getting it to the right cell types is challenging The kidney is a “very complex tissue,” says Chief Scientific Officer Alfica Sehgal a former head scientist at RNA biotech firms Camp4 Therapeutics and Alnylam Pharmaceuticals The organ consists of multiple tissue types also filters out molecules by size; it excretes siRNA by default ACS’s Basic Package keeps you connected with C&EN and ACS $80 Regular Members & Society Affiliates ACS’s Standard Package lets you stay up to date with C&EN ACS’s Premium Package gives you full access to C&EN and everything the ACS Community has to offer The company spent about 3 years in stealth while designing a drug candidate that combines a ligand with siRNAthat should silence messenger RNA (mRNA) and “It’s not an easy bean to crack,” Sehgal says But Judo Bio has proposed an elegant solution The ligand acts as a hook on the siRNA so that the conjugate latches on to the kidney’s proximal tubule epithelial cells via a receptor called megalin The start-up recently closed a series A investment round that While Judo Bio was preparing for its public launch the latest project of Alnylam founding CEO John Maraganore Maraganore is partial to RNA interference (RNAi) as a drug mechanism; in 2018 Alnylam became the first company to have an RNAi drug approved by the US Food and Drug Administration So in the summer of 2023, when Maraganore came across a paper from chemist Kotaro Nakanishi at the Ohio State University, he was intrigued. Nakanishi and his colleagues had found that certain, tiny RNA molecules could activate protein argonaute-3 to slice through proteins (Proc. Natl. Acad. Sci. U.S.A. 2020, DOI: 10.1073/pnas.2015026117) They called the molecules cleavage-​inducing tiny guide RNAs A seed was planted: use those minuscule RNAs to get to tissues that large molecules—like traditional siRNA—can’t reach Maraganore sees potential for cityRNAs to help silence disease-causing genes in multiple target tissues He expects City will start testing its first drug candidate in humans by early 2026 with the help of a $135 million series A financing round led by Arch Venture Partners Maraganore envisions one to two clinical trials for new drug candidates beginning each year Sign up for C&EN's must-read weekly newsletter This article has been sent to the following recipient: Copyright © 2025 American Chemical Society Small interfering RNA (siRNA) carries great promise in the biotechnology industry As an essential component of the RNA interference (RNAi) pathway siRNA works by degrading target messenger RNA (mRNA) molecules thereby preventing them from being translated into proteins This specific mechanism has the potential to treat a wide range of diseases from genetic disorders and cancers to viral infections.  which carries genetic information for protein synthesis which regulates gene expression more broadly siRNA is designed to target and silence specific genes with precision According to Maximize Market Research the market for siRNA therapeutics is expected to grow from $12.7 billion to $39.2 billion by 2029 Here are eight companies contributing to the field of siRNA Founded in 2002 and headquartered in Cambridge, Massachusetts, Alnylam Pharmaceuticals is a leader in RNAi therapeutics The company leverages its proprietary platforms the enhanced stabilization chemistry (ESC) platform and its conjugate delivery platform.  The ESC platform incorporates chemical modifications to the siRNA molecules which enhance their stability and reduce the likelihood of degradation within the body This allows for longer-lasting therapeutic effects and less frequent dosing The conjugate delivery platform involves the use of GalNAc (N-acetylgalactosamine) conjugates By attaching GalNAc ligands to siRNA molecules Alnylam can deliver these therapeutics directly to hepatocytes (liver cells) with high precision This targeted delivery system enhances the efficacy of the siRNA while minimizing off-target effects and potential toxicity The biotech company is well-established in the siRNA field and has several commercialized products on the market:  Founded in 2017 and headquartered in Philadelphia Aro Biotherapeutics specializes in developing tissue-targeted genetic medicines The company’s proprietary Centyrin technology enables the precise delivery of therapeutic agents to specific cells Aro’s Centyrin technology involves small stable proteins that are engineered to bind specifically to disease targets This technology is particularly effective for delivering siRNA to extra-hepatic tissues Centyrin-siRNA conjugates allow for targeted delivery to tissues like muscle where traditional siRNA therapies struggle to reach This targeted approach enhances the efficacy and safety profile of siRNA therapies by minimizing off-target effects and ensuring that the therapeutic molecules reach the intended cells is a Centyrin-siRNA conjugate targeting Pompe disease inherited metabolic disorder caused by mutations in the GAA gene This gene mutation leads to a deficiency of the enzyme acid alpha-glucosidase which is crucial for breaking down glycogen into glucose causing progressive damage to muscle and nerve cells throughout the body.  This therapy is currently in phase 1 clinical trials and has received both orphan drug designation and rare pediatric disease status from the FDA ABX1100 works by targeting glycogen synthase 1 (Gys1) mRNA in muscle tissues reducing the toxic glycogen buildup that characterizes Pompe disease Aro Biotherapeutics has raised significant funding, with a recent $41.5 million series B financing round in November 2023. The company recently started its first-in-human clinical trial for ABX1100 Arrowhead Pharmaceuticals specializes in developing RNAi-based therapies Arrowhead’s Targeted RNAi Molecule (TRiM) platform is designed to enable precise delivery of siRNA molecules to specific tissues The TRiM platform optimizes siRNA stability reducing off-target effects and improving therapeutic outcomes allowing different chemical components to be swapped in and out to best suit the target and disease Arrowhead collaborates with pharmaceutical giants like Takeda and GSK to co-develop and commercialize RNAi therapies The company focuses on developing RNAi-based therapies to address genetic and rare diseases Dicerna’s GalXC RNAi technology employs a structure where siRNA molecules are chemically conjugated to a GalNAc ligand This ligand targets receptors highly expressed in liver cells ensuring the siRNA is efficiently delivered to the liver where it can bind to and degrade the target mRNA reducing the production of disease-causing proteins The two key candidates that motivated Novo Nordisk to acquire the siRNA company in addition to its technology are Rivfloza and Belcesiran.  The FDA approved Rivfloza (nedosiran) for treating primary hyperoxaluria type 1 (PH1) in children 9 years and older and adults with relatively preserved kidney function PH1 is an inherited disorder caused by a deficiency of the liver enzyme alanine-glyoxylate aminotransferase (AGT) This enzyme deficiency leads to the overproduction of oxalate a substance that combines with calcium to form insoluble calcium oxalate crystals These crystals can accumulate in the kidneys and urinary tract leading to kidney stones and progressive kidney damage Rivfloza is a once-monthly subcutaneous therapy designed to lower urinary oxalate levels by targeting the liver enzyme lactate dehydrogenase Belcesiran targets alpha-1 antitrypsin deficiency (AATD) by silencing the mutant gene responsible for the disease AATD is a genetic disorder caused by mutations in the SERPINA1 gene AATD primarily affects the liver and lungs leading to conditions such as liver cirrhosis and emphysema Prior to its acquisition, Dicerna secured a substantial total funding of $420.5 million Dicerna’s technologies and pipeline have been integrated into Novo’s broader therapeutic programs expanding the application of RNAi therapies Founded in 2017, DTx Pharma is a biotechnology company that focuses on developing RNA-based therapies using its proprietary Fatty Acid Ligand Conjugated OligoNucleotide (FALCON) platform. In July 2023, Novartis acquired the siRNA company for an upfront payment of $500 million with potential milestone payments bringing the total value up to $1 billion.  and potency of siRNA molecules by conjugating them with fatty acids This modification improves the biodistribution and cellular uptake of siRNA enabling efficient gene silencing in tissues beyond the liver such as the central nervous system and muscles This approach leverages the natural uptake mechanisms of cells for fatty acids thereby “tricking” cells into absorbing the siRNA for targeted gene repression with plans to start clinical trials in the near future Founded in 2020 and with activities in the U.K. this biotech company focuses on developing durable siRNA therapies through a combination of chemistry and artificial intelligence Eleven Therapeutics employs its proprietary TERA platform which integrates parallel combinatorial chemistry with AI algorithms to design and optimize RNA molecules The platform uses a high-throughput split-pool synthesis process to generate a vast library of chemically modified RNA molecules Each molecule is tagged with a DNA barcode enabling precise tracking and analysis of its structure-activity relationship (SAR) The process involves the following key steps: Eleven Therapeutics raised $22 million in seed funding in 2022 including a substantial contribution from the Bill & Melinda Gates Foundation The CASi platform combines properties of both single and double-stranded RNAs to enable efficient self-delivery CASi molecules are designed to be activated only under specific cellular conditions ensuring that the siRNA is delivered and activated only in target cells This specificity reduces off-target effects and enhances the therapeutic index of the siRNA therapy The siRNA company is initially focused on developing treatments for neurodegenerative diseases but also works on central nervous system diseases The biotech’s siRNA programs are still at the preclinical stage Wave Life Sciences was founded in 2012 and is headquartered in Cambridge It is a clinical-stage biotechnology company focused on developing RNA-targeting therapeutics The company’s proprietary PRISM platform integrates multiple RNA technologies The company employs siRNA constructs enhanced with GalNAc conjugation which improves targeted delivery to liver cells This method leverages the natural uptake pathways of cells enhancing the precision and effectiveness of siRNA therapies The biotech’s INHBE program targets metabolic disorders, including obesity. Utilizing Wave’s GalNAc-siRNA technology, the INHBE program has shown promising preclinical results including substantial weight loss and reduction in visceral fat in animal models The program is expected to enter clinical trials in early 2025 Wave Life Sciences has a significant partnership with GSK, focusing on siRNA and RNA editing programs. GSK has selected two siRNA programs to advance to development candidates utilizing Wave’s GalNAc-siRNA format for hepatology applications The collaboration with GSK includes the development of up to eight RNA-targeting programs with Wave leading the research up to the investigational new drug (IND) stage.  The field of siRNA therapeutics is poised for substantial growth This rapid expansion is driven by advancements in RNAi technology and the successful approval and commercialization of several siRNA-based drugs Key players like Alnylam Pharmaceuticals have paved the way with FDA-approved therapies such as ONPATTRO or GIVLAARI collaborations between biotech companies and pharmaceutical giants such as the partnership between Wave Life Sciences and GSK the field of siRNA therapeutics faces several challenges One of the primary hurdles is the effective delivery of siRNA molecules to target cells The hydrophilic and negatively charged nature of siRNA makes it difficult for these molecules to cross cell membranes and reach the cytoplasm such as lipid nanoparticles (LNPs) and GalNAc conjugates have been developed to enhance cellular uptake and target specificity Another challenge is the potential for off-target effects and immune responses Chemical modifications to the siRNA structure can help mitigate these issues enhancing stability and reducing immunogenicity Ongoing research is focused on refining these modifications and developing more sophisticated delivery systems to overcome these barriers Oncology R&D trends and breakthrough innovations As Europe’s top biotech website for over a decade Labiotech.eu offers trusted insights and updates for the global life sciences community Labiotech.eu offers trusted insights and updates for the global life sciences community.