siRNA Drugs Research Articles

SiRNAEfficacyDB: An experimentally supported small interfering RNA efficacy database.

Small interfering RNA (siRNA) has revolutionised biomedical research and drug development through precise post-transcriptional gene silencing technology. Despite its immense potential, siRNA therapy still faces technical challenges, such as delivery efficiency, targeting specificity, and molecular stability. To address these challenges and facilitate siRNA drug development, we have developed siRNAEfficacyDB, a comprehensive database that integrates experimentally validated siRNA efficacy data. This database contains 3544 siRNA records, covering 42 target genes and 5 cell lines. It provides detailed information on siRNA sequences, target genes, inhibition efficiencies, experimental techniques, cell lines, siRNA concentrations, and incubation times. siRNAEfficacyDB offers a user-friendly web interface that makes it easy to query, browse and analyse data, enabling efficient access to siRNA-related information. In summary, siRNAEfficacyDB provides a useful data foundation for siRNA drug design and optimisation, serving as a valuable resource for advancing computer-aided siRNA design research and nucleic acid drug development. siRNAEfficacyDB is freely available at https://cellknowledge.com.cn/siRNAEfficacy for non-commercial use.

Capturing Hydrophilic Chemotherapeutics Agents Into siRNA-Encapsulated Vesicle-Like Nanoparticles for Convenient ICB-Chemo Combination Therapy.

Clinical evidence has demonstrated that combining immune checkpoint blockade (ICB) therapy with chemotherapy significantly improves response rates to ICB therapy and therapeutic efficacy in various tumor types. However, a convenient method for achieving synergistic ICB therapy and chemotherapy with precise co-delivery of both agents is still highly desirable. In this study, a strategy for co-delivering small interfering RNA (siRNA) encapsulated in vesicle-like nanoparticles (VNPsiRNA) and chemotherapeutic drugs is aimed to develop. It is discovered that the hydrophilic chemotherapeutic drug mitoxantrone hydrochloride (MTO·2HCl) can be captured into VNPsiRNA. The resulting VNPsiRNACpMTO can simultaneously block immune checkpoints via RNA silencing and induce chemotherapeutic effects on tumor cells. The mechanism of MTO·2HCl is elucidates, captures, and demonstrates the superior therapeutic effect of VNPsiRNACpMTO through chemo-immunotherapy. This strategy can also be extended to deliver other hydrochloride anticancer drugs, such as doxorubicin hydrochloride (DOX·HCl), for achieving synergistic combination therapy. This study provides a facile strategy for enhancing combined ICB and chemotherapy via co-delivering siRNA and chemotherapeutic drugs, offering a promising approach to cancer treatment.

Analyte and probe melting temperature guided method development strategy for hybridization LC-MS/MS quantification of siRNAs

Small interfering RNA (siRNA) is a novel class of double-stranded oligonucleotide therapeutics rapidly growing in drug research and development. Accurate, sensitive, and reliable quantification of siRNA analytes in biological samples is required to study their pharmacokinetics, toxicokinetics, and biodistribution. Hybridization LC-MS/MS can achieve highly sensitive and specific bioanalysis of single-stranded oligonucleotides, e.g., antisense oligonucleotides (ASOs); however, its application for bioanalysis of siRNA or other double-stranded oligonucleotides is limited. The detailed rationale and principles for assay development are still not well understood. In this work, we systematically evaluated key steps and parameters of hybridization LC-MS/MS assays, including probes (five different types compared), hybridization procedure and temperature, elution temperature, and column temperature using patisiran, an approved siRNA drug, as the test siRNA. Based on the evaluation, a practical and efficient melting temperature (Tm) guided strategy was developed for fast and reliable method development of hybridization LC-MS/MS assays for siRNA bioanalysis. The strategy was successfully applied to siRNA-A, a test siRNA, in mouse plasma over the range of 1.00–1000 ng/mL and the resulting method has been used to support multiple mouse studies. This method-development strategy showed great value as a general approach for other siRNAs or double-stranded oligonucleotides.

Combination therapy with GalNAc-siRNA drugs targeting both PCSK9 and APOC3 resulted in enhanced lipid lowering in mice

Abstract Introduction Aggressive lowering of both atherogenic low-density lipoprotein cholesterol (LDL-C) and triglyceride-rich lipoproteins (TRLs) could reduce residual atherosclerotic cardiovascular disease (ASCVD) risk. Drugs targeting PCSK9 or APOC3 have shown good lipid-lowering effects in clinic. Here, we have developed GalNAc-conjugated siRNAs, RBD7022 targeting PCSK9 and RBD5044 targeting APOC3, as novel therapeutic agents to lower LDL-C and triglyceride (TG) plasma levels respectively. Each drug shows potent and durable effect in silencing its own target. However, it is unknown whether the combined therapy with siRNA drugs targeting both PCSK9 and APOC3 will generate enhanced effects in lipid lowering. Purpose To investigate the effects of fixed-dose combination of RBD7022 and RBD5044 on LDL-C, TG and total cholesterol (TC). Methods RBD7022 and RBD5044 completely match the human PCSK9 and APOC3 transcripts respectively. RBD7022 has 2 nucleotide mismatches with mouse pcsk9, but still shows silencing activity in mice, while RBD5044 has no activity due to 5 mismatches with mouse apoc3. Therefore, humanized APOC3 transgenic mice were used in this study, which had serious hypertriglyceridemia with relatively high cholesterol and TG levels. They were randomized into 8 groups with treatment of RBD7022 (1 mg/kg or 3 mg/kg), RBD5044 (3 mg/kg or 6 mg/kg), or 3 different dose combinations of RBD7022 and RBD5044, as well as PBS control. All animals were administrated once on Day 1. Plasma was collected to quantify lipid biomarkers. Results RBD7022 showed a robust effect on LDL-C reduction, with inhibition rates of 45% in 3 mg/kg and 43% in 6 mg/kg, indicating 3 mg/kg was a saturated dose for RBD7022 in LDL-C lowering. RBD5044 showed a dose-dependent effect on LDL-C reduction, with inhibition rate of 52% at 1 mg/kg and 59% at 3 mg/kg. Interestingly, significantly enhanced LDL-C reduction was observed in the combination groups, with 67% reduction in LDL-C at RBD5044 1 mg/kg plus RBD7022 3 mg/kg, 73% reduction in LDL-C at RBD5044 3 mg/kg plus RBD7022 3 mg/kg and 78% reduction in LDL-C at RBD5044 3 mg/kg plus RBD7022 6 mg/kg, showing dose-dependent effects (Figure 1A). Enhanced TC reduction was also found in combination at RBD5044 3 mg/kg plus RBD7022 6 mg/kg compared with RBD5044 or RBD7022 alone. RBD5044 showed a dose dependent effect on TG reduction with inhibition rates of 56% at 1 mg/kg and 83% at 3 mg/kg. RBD7022 treatment alone did not show significant effect on TG reduction. No added effects of RBD7022 on TG levels was found in the combination with RBD5044, indicating the TG lowering was induced by RBD5044. Conclusions The combination of RBD7022 targeting PCSK9 and RBD5044 targeting APOC3 simultaneously reduce LDL-C and TC levels. The combination of RBD7022 and RBD5044 could be a better therapeutic option to further reduce LDL-C, particularly for mixed hyperlipidemia patients and other high-risk individuals with both high LDL-C and high TG levels.Figure 1

Two firms launch for siRNA drugs

Two firms launch for siRNA drugs

Curdlan-Mediated Syngeneic RNAi against NF-κB in Glial Cells Protects Cerebral Vessels in the TBI Mouse Model.

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

Development of charged membranes for the ultrafiltration of siRNA

RNA interference (RNAi) is an exciting new therapeutic approach, using small interfering RNA (siRNA) to silence specific genes for treating various diseases. Ultrafiltration has been widely used for the concentration and purification of monoclonal antibodies and other biotherapeutics; however, its application in the downstream processing of double-stranded oligonucleotides remains underexplored. This study investigates the use of ultrafiltration in the final formulation for concentrating a siRNA drug substance. Initial experiments were conducted with Ultracel™ composite regenerated cellulose membranes over a range of molecular weight cutoffs (MWCO) at different siRNA feed concentrations. Results demonstrated significant effects of both fouling and concentration polarization, with the maximum achievable concentration falling well below the target required for subcutaneous injection. Novel surface-modified ultrafiltration membranes were made by reaction of the cellulose with bromopropanesulfonic acid. These negatively charged membranes showed improved siRNA retention, reduced fouling, and a large increase in the maximum achievable siRNA concentration (from 52 to >180 mg/mL). These findings offer critical insights into novel approaches for developing high performance ultrafiltration processes for siRNA concentration.

RNA interference therapy in cardiology: will new targets improve therapeutic goals?

The discovery of RNA interference in 1998 opened avenues for the manipulation of gene expression, leading to the development of small interfering RNA (siRNA) drugs. Patisiran, the first FDA-approved siRNA medication, targets hereditary transthyretin amyloidosis with polyneuropathy. Givosiran, lumasiran and nedosiran further expand siRNA applications in treating rare genetic diseases, demonstrating positive outcomes. In cardiology, inclisiran, approved for hypercholesterolaemia, showcases sustained reductions in LDL cholesterol levels. However, ongoing research aims to establish its impact on cardiovascular outcomes. Lipoprotein(a), an independent risk factor for atherosclerotic cardiovascular disease, has become a focus of siRNA therapies, precipitating the development of specific siRNA drugs like olpasiran, zerlasiran and lepodisiran, with promising reductions in lipoprotein(a) levels. Research to assess the effectiveness of these medications in reducing events is currently under way. Zodasiran and plozasiran address potential risk factors for cardiovascular diseases, targeting triglyceride-rich lipoproteins. Zilebesiran, which targets hepatic angiotensinogen mRNA, has demonstrated a dose-related reduction in serum angiotensinogen levels, thereby lowering blood pressure in patients with systemic arterial hypertension. The evolving siRNA methodology presents a promising future in cardiology, with ongoing studies assessing its effectiveness in various conditions. In the future, larger studies will provide insights into improvements in cardiovascular outcomes, long-term safety and broader applications in the general population. This review highlights the historical timeline of the development of siRNA-based drugs, their clinical indications, potential side-effects and future perspectives.

Advances in siRNA Drug Delivery Strategies for Targeted TNBC Therapy.

Among breast cancers, triple-negative breast cancer (TNBC) has been recognized as the most aggressive type with a poor prognosis and low survival rate. Targeted therapy for TNBC is challenging because it lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Chemotherapy, radiation therapy, and surgery are the common therapies for TNBC. Although TNBC is prone to chemotherapy, drug resistance and recurrence are commonly associated with treatment failure. Combination therapy approaches using chemotherapy, mAbs, ADC, and antibody-siRNA conjugates may be effective in TNBC. Recent advances with siRNA-based therapy approaches are promising for TNBC therapy with better prognosis and reduced mortality. This review discusses advances in nanomaterial- and nanobiomaterial-based siRNA delivery platforms for TNBC therapy exploring targeted therapy approaches for major genes, proteins, and TFs upregulated in TNBC tumors, which engage in molecular pathways associated with low TNBC prognosis. Bioengineered siRNA drugs targeting one or several genes simultaneously can downregulate desired genes, significantly reducing disease progression.

Multifunctional Biomimetic Nanocarriers for Dual-Targeted Immuno-Gene Therapy Against Hepatocellular Carcinoma.

Growing evidences have proved that tumors evade recognition and attack by the immune system through immune escape mechanisms, and PDL1/Pbrm1 genes have a strong correlation with poor response or resistance to immune checkpoint blockade (ICB) therapy. Herein, a multifunctional biomimetic nanocarrier (siRNA-CaP@PD1-NVs) is developed, which can not only enhance the cytotoxic activity of immune cells by blocking PD1/PDL1 axis, but also reduce tumor immune escape via Pbrm1/PDL1 gene silencing, leading to a significant improvement in tumor immunosuppressive microenvironment. Consequently, the nanocarrier promotes DC cell maturation, enhances the infiltration and activity of CD8+ T cells, and forms long-term immune memory, which can effectively inhibit tumor growth or even eliminate tumors, and prevent tumor recurrence and metastasis. Overall, this study presents a powerful strategy for co-delivery of siRNA drugs, immune adjuvant, and immune checkpoint inhibitors, and holds great promise for improving the effectiveness and safety of current immunotherapy regimens.

Reciprocal regulation between RACGAP1 and AR contributes to endocrine therapy resistance in prostate cancer

BackgroundEndocrine resistance driven by sustained activation of androgen receptor (AR) signaling pathway in advanced prostate cancer (PCa) is fatal. Characterization of mechanisms underlying aberrant AR pathway activation to search for potential therapeutic strategy is particularly important. Rac GTPase-activating protein 1 (RACGAP1) is one of the specific GTPase-activating proteins. As a novel tumor proto-oncogene, overexpression of RACGAP1 was related to the occurrence of various tumors.MethodsBioinformatics methods were used to analyze the relationship of expression level between RACGAP1 and AR as well as AR pathway activation. qRT-PCR and western blotting assays were performed to assess the expression of AR/AR-V7 and RACGAP1 in PCa cells. Immunoprecipitation and immunofluorescence experiments were conducted to detect the interaction and co-localization between RACGAP1 and AR/AR-V7. Gain- and loss-of-function analyses were conducted to investigate the biological roles of RACGAP1 in PCa cells, using MTS and colony formation assays. In vivo experiments were conducted to evaluate the effect of RACGAP1 inhibition on the tumor growth.ResultsRACGAP1 was a gene activated by AR, which was markedly upregulated in PCa patients with CRPC and enzalutamide resistance. AR transcriptionally activated RACGAP1 expression by binding to its promoter region. Reciprocally, nuclear RACGAP1 bound to the N-terminal domain (NTD) of both AR and AR-V7, blocking their interaction with the E3 ubiquitin ligase MDM2. Consequently, this prevented the degradation of AR/AR-V7 in a ubiquitin-proteasome-dependent pathway. Notably, the positive feedback loop between RACGAP1 and AR/AR-V7 contributed to endocrine therapy resistance of CRPC. Combination of enzalutamide and in vivo cholesterol-conjugated RIG-I siRNA drugs targeting RACGAP1 induced potent inhibition of xenograft tumor growth of PCa.ConclusionIn summary, our results reveal that reciprocal regulation between RACGAP1 and AR/AR-V7 contributes to the endocrine resistance in PCa. These findings highlight the therapeutic potential of combined RACGAP1 inhibition and enzalutamide in treatment of advanced PCa.

Pulmonary fibroblast-specific delivery of siRNA exploiting exosomes-based nanoscaffolds for IPF treatment

Idiopathic pulmonary fibrosis (IPF) is a progressive pulmonary disease that leads to interstitial inflammation, lung damage, and eventually life-threatening complications. Among various pathologic factors, Smad4 is a pivotal molecule involved in the progression and exacerbation of IPF. It mediates nuclear transfer of Smad2/Smad3 complexes and initiates the transcription of fibrosis-promoting genes. Thus, the inhibition of Smad4 expression in pulmonary fibroblasts by small interfering RNAs (siRNAs) might be a promising therapeutic strategy for IPF. Herein, we engineered exosome membranes (EM) by cationic lipid (i.e., DOTAP) to load siRNAs against Smad4 (DOTAP/siSmad4@EM), and investigated their specific delivery to pulmonary fibroblasts for treating IPF in a mouse model via pulmonary administration. As reference nanoscaffolds, undecorated DOTAP/siSmad4 complexes (lipoplexes, consisting of cationic lipid DOTAP and siRNAs) and siSmad4-loaded lipid nanoparticles (DOTAP/siSmad4@lipo, consisting of lipoplexes fused with DPPCChol liposomes) were also prepared. The results showed that DOTAP/siSmad4@EM exhibited a higher cellular uptake and gene silencing efficacies in mouse pulmonary fibroblasts (viz., MLg2908) as compared to the two reference nanoscaffolds. Furthermore, the outcomes of the in vivo experiments illustrated that DOTAP/siSmad4@EM could significantly down-regulate the Smad4 expression with augmented anti-fibrosis efficiency. Additionally, the DOTAP/siSmad4@EM conferred excellent biocompatibility with low cytokine levels in bronchoalveolar lavage fluid and proinflammatory responses in the pulmonary area. Taken together, the outcomes of our investigation imply that specific inhibition of Smad4 expression in pulmonary fibroblasts by pulmonary administrated DOTAP/siSmad4@EM is a promising therapeutic strategy for IPF, which could safely and effectively deliver siRNA drugs to the targeted site of action.

The Coming Age of Antisense Oligos for the Treatment of Hepatic Ischemia/Reperfusion (IRI) and Other Liver Disorders: Role of Oxidative Stress and Potential Antioxidant Effect.

Imbalances in the redox state of the liver arise during metabolic processes, inflammatory injuries, and proliferative liver disorders. Acute exposure to intracellular reactive oxygen species (ROS) results from high levels of oxidative stress (OxS) that occur in response to hepatic ischemia/reperfusion injury (IRI) and metabolic diseases of the liver. Antisense oligonucleotides (ASOs) are an emerging class of gene expression modulators that target RNA molecules by Watson-Crick binding specificity, leading to RNA degradation, splicing modulation, and/or translation interference. Here, we review ASO inhibitor/activator strategies to modulate transcription and translation that control the expression of enzymes, transcription factors, and intracellular sensors of DNA damage. Several small-interfering RNA (siRNA) drugs with N-acetyl galactosamine moieties for the liver have recently been approved. Preclinical studies using short-activating RNAs (saRNAs), phosphorodiamidate morpholino oligomers (PMOs), and locked nucleic acids (LNAs) are at the forefront of proof-in-concept therapeutics. Future research targeting intracellular OxS-related pathways in the liver may help realize the promise of precision medicine, revolutionizing the customary approach to caring for and treating individuals afflicted with liver-specific conditions.

Study on the molecular interactions between givosiran and theophylline by surface-enhanced Raman spectroscopy, biolayer interferometry, and visible/fluorescence spectroscopy

Givosiran is a small-interfering ribonucleic acid (siRNA) drug used to treat acute hepatic porphyria. As an exogenous double-stranded RNA, givosiran has a specific spatial conformation. When they coexist, givosiran and small-molecule drugs may interact through non-covalent bonds, which may affect their efficacies. This study aimed to evaluate the molecular interaction between givosiran and three structurally-related small-molecule drugs. We combined surface-enhanced Raman spectroscopy, biolayer interferometry, and visible/fluorescence spectroscopy with a methylene blue probe to study the molecular interaction between givosiran and theophylline, caffeine, and theobromine. Molecular interactions were observed only between theophylline and givosiran, with an affinity constant (KD) of 64.2 μM. The main binding forces were hydrogen bonds and van der Waals forces, and the equilibrium binding constant (KA) was 1.855 × 104 L·mol−1. This study focused on the 5 W (Whether, Where, Why, hoW about the tendency, hoW strong) issues of the molecular interaction between givosiran and small-molecule chemical drugs. Our findings contradict the previous perception that the probability of drug–drug interactions (DDI) between nucleic acid and small-molecular drugs is low. Our study illustrates that a combination of direct and indirect analytical methods will pave a new way for future siRNA-related unknown interactions studies.

Analysis of the pharmacokinetics and efficacy of RBD1016 – A GalNAc-siRNA targeting Hepatitis B Virus X gene using semi-mechanistic PK/PD model

Small interference RNA (siRNA) is a class of short double-stranded RNA molecules that cause mRNA degradation through an RNA interference mechanism and is a promising therapeutic modality. RBD1016 is a siRNA drug in clinical development for the treatment of chronic Hepatitis B Virus (HBV) infection, which contains a conjugated with N-acetylglucosamine moiety that can facilitate its hepatic delivery. We aimed to construct a semi-mechanistic model of RBD1016 in pre-clinical animals, to elucidate the pharmacokinetic/pharmacodynamic (PK/PD) profiles in mice and PK profiles in monkeys, which can lay the foundation for potential future translation of RBD1016 PK and PD from the pre-clinical stage to the clinic stage. The proposed semi-mechanistic PK/PD model fitted PK and PD data in HBV transgenic mice well and described plasma and liver concentrations in the monkeys well. The simulation results showed that our model has a reasonable predictive ability for Hepatitis B surface antigen (HBsAg) levels after multiple dosing in mice. Further PK and PD data for RBD1016, including clinical data, will assist in refining the model presented here. Our current effort focused on model building for RBD1016, we anticipate that the model could apply to other GalNAc-siRNA drugs.

Recent Progress of Small Interfering RNA Delivery on the Market and Clinical Stage.

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

Analysis of Self-Assembled Micelle Inhibitory RNA (SAMiRNA) Drug Using Ion-Pairing Reversed-Phase Liquid Chromatography Combined with Mass Spectrometry.

Small interfering RNA (siRNA) is known for its ability to silence the expression of specific genes, demonstrating its promising potential as a therapeutic approach. Self-assembled micelle inhibitory RNA (SAMiRNA) is an oligonucleotide duplex developed to overcome the in vivo delivery limitations of siRNA. SAMiRNA has hydrophilic and hydrophobic groups at both ends of a sense strand, forming a spherical nanostructure that enhances the in vivo delivery efficiency. Ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the most commonly used method for the analysis of oligonucleotides. Since SAMiRNA is heavily chemically modified, the behavior of SAMiRNA in IP-RPLC combined with mass spectrometry (MS) is anticipated to differ from that of the conventional siRNA drug. The current investigation using IP-RPLC-MS revealed that a distinct duplex peak along with two minor separate strands of antisense and sense was observed at column temperatures below 35 °C in the IP-RPLC system with a 100 mM ammonium bicarbonate buffer system. At column temperatures higher than 35 °C, however, two fully denatured single strands were observed. The mass spectrum from the chromatographic peak of the SAMiRNA duplex contained signals from the duplex, the antisense, and the sense, probably due to duplex denaturation during the MS ionization process. The current comprehensive analysis results will make a substantial contribution to the future application of IP-RPLC-MS in the analysis of SAMiRNA.

A First-in-Human Phase 1 Study of a Tumor-Directed RNA-Interference Drug against HIF2α in Patients with Advanced Clear Cell Renal Cell Carcinoma.

ARO-HIF2 is an siRNA drug designed to selectively target hypoxia-inducible factor-2α (HIF2α) interrupting downstream pro-oncogenic signaling in clear cell renal cell carcinoma (ccRCC). The aims of this Phase 1 study (AROHIF21001) were to evaluate safety, tolerability, pharmacokinetics, and establish a recommended Phase 2 dose. Subjects with ccRCC and progressive disease after at least 2 prior therapies that included VEGF and immune checkpoint inhibitors were progressively enrolled into dose-escalation cohorts of ARO-HIF2 administered intravenously at 225, 525, or 1,050 mg weekly. Twenty-six subjects received ARO-HIF2. The most common treatment emergent adverse events (AE) irrespective of causality were fatigue (50.0%), dizziness (26.9%), dyspnea (23.1%), and nausea (23.1%). Four subjects (15.4%) had treatment-related serious AEs. AEs of special interest included neuropathy, hypoxia, and dyspnea. ARO-HIF2 was almost completely cleared from plasma circulation within 48 hours with minimal renal clearance. Reductions in HIF2α were observed between pre- and post-dosing tumor biopsies, but the magnitude was quite variable. The objective response rate was 7.7% and the disease control rate was 38.5%. Responses were accompanied by ARO-HIF2 uptake in tumor cells, HIF2α downregulation, as well as rapid suppression of tumor produced erythropoietin (EPO) in a patient with paraneoplastic polycythemia. ARO-HIF2 downregulated HIF2α in advanced ccRCC-inhibiting tumor growth in a subset of subjects. Further development was hampered by off-target neurotoxicity and low response rate. This study provides proof of concept that siRNA can target tumors in a specific manner.

AttSiOff: a self-attention-based approach on siRNA design with inhibition and off-target effect prediction

Small interfering RNA (siRNA) is often used for function study and expression regulation of specific genes, as well as the development of small molecule drugs. Selecting siRNAs with high inhibition and low off-target effects from massive candidates is always a great challenge. Increasing experimentally-validated samples can prompt the development of machine-learning-based algorithms, including Support Vector Machine (SVM), Convolutional Neural Network (CNN), and Graph Neural Network (GNN). However, these methods still suffer from limited accuracy and poor generalization in designing potent and specific siRNAs.In this study, we propose a novel approach for siRNA inhibition and off-target effect prediction, named AttSiOff. It combines a self-attention-based siRNA inhibition predictor with an mRNA searching package and an off-target filter. The predictor gives the inhibition score via analyzing the embedding of siRNA and local mRNA sequences, generated from the pre-trained RNA-FM model, as well as other meaningful prior-knowledge-based features. Self-attention mechanism can detect potentially decisive features, which may determine the inhibition of siRNA. It captures global and local dependencies more efficiently than normal convolutions. The tenfold cross-validation results indicate that our model outperforms all existing methods, achieving PCC of 0.81, SPCC of 0.84, and AUC of 0.886. It also reaches better performance of generalization and robustness on cross-dataset validation. In addition, the mRNA searching package could find all mature mRNAs for a given gene name from the GENOMES database, and the off-target filter can calculate the amount of unwanted off-target binding sites, which affects the specificity of siRNA. Experiments on five mature siRNA drugs, as well as a new target gene (AGT), show that AttSioff has excellent convenience and operability in practical applications.Graphical

Biomimetic Nanovesicles as a Dual Gene Delivery System for the Synergistic Gene Therapy of Alzheimer's Disease.

The association between dysfunctional microglia and amyloid-β (Aβ) is a fundamental pathological event and increases the speed of Alzheimer's disease (AD). Additionally, the pathogenesis of AD is intricate and a single drug may not be enough to achieve a satisfactory therapeutic outcome. Herein, we reported a facile and effective gene therapy strategy for the modulation of microglia function and intervention of Aβ anabolism by ROS-responsive biomimetic exosome-liposome hybrid nanovesicles (designated as TSEL). The biomimetic nanovesicles codelivery β-site amyloid precursor protein cleaving enzyme-1 (BACE1) siRNA (siBACE1) and TREM2 plasmid (pTREM2) gene drug efficiently penetrate the blood-brain barrier and enhance the drug accumulation at AD lesions with the help of exosomes homing ability and angiopep-2 peptides. Specifically, an upregulation of TREM2 expression can reprogram microglia from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype while also restoring its capacity to phagocytose Aβ and its nerve repair function. In addition, siRNA reduces the production of Aβ plaques at the source by knocking out the BACE1 gene, which is expected to further enhance the therapeutic effect of AD. The in vivo study suggests that TSEL through the synergistic effect of two gene drugs can ameliorate APP/PS1 mice cognitive impairment by regulating the activated microglial phenotype, reducing the accumulation of Aβ, and preventing the retriggering of neuroinflammation. This strategy employs biomimetic nanovesicles for the delivery of dual nucleic acids, achieving synergistic gene therapy for AD, thus offering more options for the treatment of AD.