siRNA Molecules Research Articles

Analysis of RNA Interference Targeted Against Human Antigen R (HuR) to Reduce Vascular Endothelial Growth Factor (VEGF) Protein Expression in Human Retinal Pigment Epithelial Cells.

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

An In-silico Approach to Design and Validate siRNA against Monkeypox Virus.

The monkeypox virus has emerged as an uncommon zoonotic infection. The recent outbreak of MPXV in Europe and abroad in 2022 presented a major threat to individuals at risk. At present, no specific MPXV vaccinations or medications are available. In this study, we predicted the most effective siRNA against the conserved region of the MPXV and validated the activity by performing molecular docking studies. Ultimately, the most efficient siRNA molecule was shortlisted against the envelope protein gene (B6R) based on its toxicity, effectivity, thermodynamic stability, molecular interaction, and molecular dynamics simulations (MD) with the Human Argonaute 2 protein. Thus, the strategy may offer a platform for the development of potential antiviral RNA therapeutics that target MPXV at the genomic level.

RNF149 confers cisplatin resistance in esophageal squamous cell carcinoma via destabilization of PHLPP2 and activating PI3K/AKT signalling.

Chemo-resistance has been identified as a crucial factor contributing to tumor recurrence and a leading cause of worse prognosis in patients with ESCC. Therefore, unravel the critical regulators and effective strategies to overcome drug resistance will have a significant clinical impact on the disease. In our study we found that RNF149 was upregulated in ESCC and high RNF149 expression was associated with poor prognosis with ESCC patients. Functionally, we have demonstrated that overexpression of RNF149 confers CDDP resistance to ESCC; however, inhibition of RNF149 reversed this phenomenon both in vitro and in vivo. Mechanistically, we demonstrated that RNF149 interacts with PH domain and leucine rich repeat protein phosphatase 2 (PHLPP2) and induces E3 ligase-dependent protein degradation of PHLPP2, substantially activating the PI3K/AKT signalling pathway in ESCC. Additionally, we found that inhibition of PI3K/AKT signalling pathway by AKT siRNA or small molecule inhibitor significantly suppressed RNF149-induced CDDP resistance. Importantly, RNF149 locus was also found to be amplified not only in ESCC but also in various human cancer types. Our data suggest that RNF149 might function as an oncogenic gene. Targeting the RNF149/PHLPP2/PI3K/Akt axis may be a promising prognostic factor and valuable therapeutic target for malignant tumours.

N-acetylgalactosamine delivery systems for RNA therapeutics: a patent perspective

ABSTRACT Introduction SiRNA molecules with a feature of good gene-silencing are critical for drug discovery and development based on RNA interference. GalNAc-RNA therapeutics is a rapid growing area in RNA therapeutics. Areas covered This article provides patent landscape and modification feature of GalNAc-RNA therapeutics. The US-granted patents from January 2004 to April 2023 were retrieved and analyzed. Expert opinion Globally, our study is the first one to holistically depict a map of modifications and therapeutic applications for GalNAc-RNA therapeutics by patent data analysis. The results showed there were 8 major modifications and 5 new emerged modifications for GalNAc-RNA therapeutic agents. Especially, the study provides recent new emerged modifications in sugar, base, and internucleotide linkage of GalNAc-RNA therapeutic agents, e.g. morpholino-type ring, 5-methylcytosine, and phosphorodithioates. In addition, our study systematically demonstrated major therapeutic applications for GalNAc-RNA therapeutics, including liver or gallbladder disorders, anticancer, antihyperlipidemics, and disorders of the nervous system etc.

Cytocompatibility and Osteoinductive Properties of Collagen-Fibronectin Hydrogel Impregnated with siRNA Targeting Glycogen Synthase Kinase 3β: In Vitro Study.

In this study, we developed an osteoplastic material based on collagen-fibronectin hydrogel impregnated with siRNA molecules targeting glycogen synthase kinase 3β (GSK3β), which inhibits the osteogenic differentiation of mesenchymal stem cells. The hydrogel impregnated with polyplexes containing siRNA GSK3β and polyethylenimine has been shown to have no cytotoxic effect: there was no statistically significant change in the cell's viability after 7 days of incubation in its presence compared to the control group. On days 2 and 7, an increase in the level of expression of markers of osteogenic differentiation was observed, which confirms the osteoinductive qualities of the material. It has been demonstrated that the hydrogel maintains cell adhesion. Our results obtained in vitro indicate cytocompatibility and osteoinductive properties of collagen-fibronectin hydrogel impregnated with siRNA GSK3β molecules.

Proliferation-Related Features of the Human Mesenchymal Stem Cells Derived from Palatine Tonsils, Adipose Tissues, and Bone Marrow.

Mesenchymal stem cells (MSCs) are widely used in regenerative medicine and cell-based transplantations. However, an in-depth comparison of the different MSC origins is lacking. This study aimed to compare the expression of adipose-derived (AMSCs), bone marrow-derived (BMSCs), and tonsil-derived (TMSCs) and evaluate whether TMSCs are good alternatives for AMSCs or BMSCs. We analyzed the expression levels of 47,000 transcripts in AMSCs (n = 4), BMSCs (n = 4), and TMSCs (n = 4) using GeneChip. Microarray data were analyzed using the LIMMA package to compare the TMSCs, AMSCs, and BMSCs. Hub genes were analyzed using STRING and Cytoscape. To ascertain the functional roles of AURKA and AURKB, small interfering RNA (siRNA) molecules specifically targeting AURKA and AURKB mRNA were synthesized and employed to induce knockdown of AURKA and AURKB in TMSC and AMSC. We analyzed the expression level of OCT4, SOX-2, and NANOG genes in TMSC and AMSCs by cell culture and real-time PCR. We identified commonly increased 256 and decreased 160 genes in TMSCs from the differentially expressed genes (DEGs) between the TMSCs, AMSCs, and BMSCs. In the DEG-based protein-protein interaction and gene set enrichment analysis, hub genes (AURKA, AURKB, CDC20, and BUB1) highly expressed in TMSCs were enriched for development- and progression-related oocyte meiosis, the cell cycle, and ubiquitin-mediated proteolysis. In vitro analysis demonstrated that cells with downregulated expression of AURKA and AURKB exhibited a significant reduction in proliferation compared to control cells. However, silencing of the genes did not affect the differentiation capacity in TMSCs and AMSCs. Our study compared MSCs of different origins to better understand the similarities and differences among these cell types.

In silico prediction of siRNA to silence the SARS-CoV-2 omicron variant targeting BA.4, BA.5, BQ.1, BQ1.1. and XBB: an alternative to traditional therapeutics

BackgroundAfter the first infection in December 2019, the mutating strains of SARS-CoV2 have already affected a lot of healthy people around the world. But situations have not been as devastating as before the first pandemic of the omicron strains of SARS-CoV2. As of January 2023, five more Omicron offshoots, BA.4, BA.5, B.Q.1, B.Q.1.1 and XBB are now proliferating worldwide. Perhaps there are more variants already dormant that require only minor changes to resurrect. So, this study was conducted with a view to halting the infection afterwards. The spike protein found on the virus outer membrane is essential for viral attachment to host cells, thus making it an attractive target for vaccine, drug, or any other therapeutic development. Small interfering RNAs (siRNAs) are now being used as a potential treatment for various genetic conditions or as antiviral or antibacterial therapeutics. Thus, in this study, we looked at spike protein to see if any potential siRNAs could be discovered from it.ResultsIn this study, by approaching several computational assays (e.g., GC content, free energy of binding, free energy of folding, RNA–RNA binding, heat capacity, concentration plot, validation, and finally molecular docking analysis), we concluded that two siRNAs could be effective to silence the spike protein of the omicron variant. So, these siRNAs could be a potential target for therapeutic development against the SARS-CoV2 virus by silencing the spike protein of this virus.ConclusionWe believe our research lays the groundwork for the development of effective therapies at the genome level and might be used to develop chemically produced siRNA molecules as an antiviral drug against SARS-CoV2 virus infection.Graphical abstract

Impact and Advances in the Role of Bacterial Extracellular Vesicles in Neurodegenerative Disease and Its Therapeutics.

Bacterial Extracellular Vesicles (BEVs) possess the capability of intracellular interactions with other cells, and, hence, can be utilized as an efficient cargo for worldwide delivery of therapeutic substances such as monoclonal antibodies, proteins, plasmids, siRNA, and small molecules for the treatment of neurodegenerative diseases (NDs). BEVs additionally possess a remarkable capacity for delivering these therapeutics across the blood-brain barrier to treat Alzheimer's disease (AD). This review summarizes the role and advancement of BEVs for NDs, AD, and their treatment. Additionally, it investigates the critical BEV networks in the microbiome-gut-brain axis, their defensive and offensive roles in NDs, and their interaction with NDs. Furthermore, the part of BEVs in the neuroimmune system and their interference with ND, as well as the risk factors made by BEVs in the autophagy-lysosomal pathway and their potential outcomes on ND, are all discussed. To conclude, this review aims to gain a better understanding of the credentials of BEVs in NDs and possibly discover new therapeutic strategies.

Solid Lipid-Polymer Hybrid Nanoplatform for Topical Delivery of siRNA: In Vitro Biological Activity and Permeation Studies.

Small interfering RNA (siRNA) molecules have limited transfection efficiency and stability, necessitating the use of delivery systems to be effective in gene knockdown therapies. In this regard, lipid-polymeric nanocarriers have emerged as a promising class of nanoparticles for siRNA delivery, particularly for topical applications. We proposed the use of solid lipid-polymer hybrid nanoparticles (SLPHNs) as topical delivery systems for siRNA. This approach was evaluated by assessing the ability of SLPHNs-siRNA complexes to internalize siRNA molecules and both to penetrate skin layers in vitro and induce gene knocking down in a skin cell line. The SLPHNs were formed by a specific composition of solid lipids, a surfactant polymer as a dispersive agent, and a cationic polymer as a complexing agent for siRNA. The optimized nanocarriers exhibited a spherical shape with a smooth surface. The average diameter of the nanoparticles was found to be 200 nm, and the zeta potential was measured to be +20 mV. Furthermore, these nanocarriers demonstrated excellent stability when stored at 4 °C over a period of 90 days. In vitro and in vivo permeation studies showed that SLPHNs increased the cutaneous penetration of fluorescent-labeled siRNA, which reached deeper skin layers. Efficacy studies were conducted on keratinocytes and fibroblasts, showing that SLPHNs maintained cell viability and high cellular uptake. Furthermore, SLPHNs complexed with siRNA against Firefly luciferase (siLuc) reduced luciferase expression, proving the efficacy of this nanocarrier in providing adequate intracellular release of siRNA for silencing specific genes. Based on these results, the developed carriers are promising siRNA delivery systems for skin disease therapy.

SiRNA a promising tool for diabetes complications

RNA interference (RNAi) is a naturally occurring process of gene regulation that has been harnessed to silence specific genes in various cell types, including those involved in diabetes complications. Small interfering RNA (siRNA) is an RNA molecule that activates RNAi and targets specific genes for degradation. Recent research has demonstrated that siRNA holds promise as a tool for treating diabetes complications, including diabetic neuropathy, retinopathy, and nephropathy. In preclinical studies, siRNA has been shown to effectively target genes involved in these complications, resulting in improved clinical outcomes. One potential advantage of siRNA therapy is its ability to selectively target specific genes without disrupting endogenous mRNA pathways, which reduces the risk of off-target effects. Additionally, siRNA has the potential to provide long-lasting effects with a single dose, which could result in reduced treatment frequency and improved patient compliance. While promising preclinical results have been, several challenges still need to be addressed before siRNA can be used in clinical practice. These include delivery issues, as siRNA molecules rapidly degrade in the bloodstream and cannot cross cell membranes without assistance. Despite these challenges, the potential of siRNA as a tool for treating diabetes complications is exciting, and further research is needed to determine its safety and efficacy in clinical trials. With continued investigation and refinement, siRNA has the potential to become an important therapeutic tool for the treatment of diabetes complications, improving patient outcomes and quality of life.

Blockade of CD155 and Interleukin-6 Suppresses Breast Cancer Cell Growth

Background: Breast cancer is the foremost common type of cancer worldwide and the leading cause of death related to cancer. Poliovirus receptor (CD155) or nectin-like molecule-5 expression has been shown to increase in tumor cells and bind to the DNAX accessory molecule (DNAM-1)/T cell immunoreceptor with Ig and ITIM domains (TIGIT)/CD69 ligand present on the surface of T and natural killer cells, increasing inflammatory cytokines, including interleukin 6 (IL-6) in the tumor microenvironment. As a result, we intended to simultaneously target both CD155 and IL-6 as an effective therapeutic approach to prevent breast cancer growth. Methods: In this study, 4T1 cells were transfected with small interfering RNA (siRNA) molecules against CD155 and IL-6, using lipofectamine (LP). Target gene expression was analyzed using both the real-time polymerase chain reaction (RT-PCR) and an enzyme-linked immunosorbent assay (ELISA). The methyl thiazolyl tetrazolium (MTT) assay also examined the cytotoxic effect of combined therapy. Results: The results showed that the cells were effectively transfected with LP, resulting in the downregulation of IL-6 and CD155. In addition, the combined silencing of IL-6 and CD155 potently reduced the cellular viability of cancer cells. Conclusion: These findings revealed that the simultaneous targeting of CD155 and IL-6 in breast cancer cells (4T1) is an innovative and efficient therapy strategy to induce apoptosis in malignant cells.

Evaluating the Efficacy of a Small Interfering Ribonucleic Acid Molecule, Givosiran, in Treating Acute Intermittent Porphyria: A Systematic Review.

Acute intermittent porphyria (AIP) is a severe multiorgan dysfunction disorder that can be fatal if not treated promptly. The newest treatment modality involving small interfering RNA (siRNA) molecules, givosiran, is administered for AIP. Although it has very beneficial effects in treating attacks of AIP, it comes with an extensive side effect profile that is not fully understood or studied. Hence, this novel drug model treatment's risk-benefit evaluation is still necessary. For relevant medical literature, we explored medical databases such as PubMed/Medline, PubMed Central, Cochrane Library, Internet Archive Scholar, Google Scholar, and Wiley Online Library. The selected papers were screened based on eligibility criteria and filtered through quality appraisal tools, and 13 finalized research papers were included in the study. Of the 13 identified papers, three were clinical trials, and 10 were review articles. The selected papers all discussed the effectiveness and side effects of givosiran in acute and recurrent attacks of AIP. The research papers showed decreased rates of acute attacks of AIP with givosiran and terminating recurrent attacks. But there are certain non-serious side effects, like fatigue and nausea. Also, there are some severe side effects, like pain. There is limited information on renal and liver function impairment using givosiran and the use of givosiran in patients with kidney and liver disease, for which further studies are required.

Targeting BET Proteins Decreases Hyaluronidase-1 in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the presence of dense stroma that is enriched in hyaluronan (HA), with increased HA levels associated with more aggressive disease. Increased levels of the HA-degrading enzymes hyaluronidases (HYALs) are also associated with tumor progression. In this study, we evaluate the regulation of HYALs in PDAC. Using siRNA and small molecule inhibitors, we evaluated the regulation of HYALs using quantitative real-time PCR (qRT-PCR), Western blot analysis, and ELISA. The binding of BRD2 protein on the HYAL1 promoter was evaluated by chromatin immunoprecipitation (ChIP) assay. Proliferation was evaluated by WST-1 assay. Mice with xenograft tumors were treated with BET inhibitors. The expression of HYALs in tumors was analyzed by immunohistochemistry and by qRT-PCR. We show that HYAL1, HYAL2, and HYAL3 are expressed in PDAC tumors and in PDAC and pancreatic stellate cell lines. We demonstrate that inhibitors targeting bromodomain and extra-terminal domain (BET) proteins, which are readers of histone acetylation marks, primarily decrease HYAL1 expression. We show that the BET family protein BRD2 regulates HYAL1 expression by binding to its promoter region and that HYAL1 downregulation decreases proliferation and enhances apoptosis of PDAC and stellate cell lines. Notably, BET inhibitors decrease the levels of HYAL1 expression in vivo without affecting the levels of HYAL2 or HYAL3. Our results demonstrate the pro-tumorigenic role of HYAL1 and identify the role of BRD2 in the regulation of HYAL1 in PDAC. Overall, these data enhance our understanding of the role and regulation of HYAL1 and provide the rationale for targeting HYAL1 in PDAC.

GL67 lipid-based liposomal formulation for efficient siRNA delivery into human lung cancer cells

The efficient delivery of small interfering RNA (siRNA) to the targeted cells significantly affects the regulation of the overexpressed proteins involved in the progression of several genetic diseases. SiRNA molecules in naked form suffer from low internalization across the cell membrane, high susceptibility to degradation by nuclease enzyme and low stability, which hinder their efficacy. Therefore, there is an urge to develop a delivery system that can protect siRNA from degradation and facilitate their uptake across the cell membrane. In this study, the cationic lipid (GL67) was exploited, in addition to DC-Chol and DOPE lipids, to design an efficient liposomal nanocarrier for siRNA delivery. The physiochemical characterizations demonstrated that the molar ratio of 3:1 has proper particle size measurements from 144 nm to 332 nm and zeta potential of −9 mV to 47 mV that depends on the ratio of the GL67 in the liposomal formulation. Gel retardation assay exhibited that increasing the percentage of GL67 in the formulations has a good impact on the encapsulation efficiency compared to DC-Chol. The optimal formulations of the 3:1 M ratio also showed high metabolic activity against A549 cells following a 24 h cell exposure. Flow cytometry findings showed that the highest GL67 lipid ratio (100 % GL67 and 0 % DC-Chol) had the highest percentage of cellular uptake. The lipoplex nanocarriers based on GL67 lipid could potentially influence treating genetic diseases owing to the high internalization efficiency and safety profile.

Silencing Interleukin-6 and Glycoprotein 130 Suppresses Growth and Induces Apoptosis in Cancer Cells

Background: Various signaling pathways promote cancer growth and inhibit apoptosis in cancerous cells. The increased levels of interleukin (IL)-6 cytokine and its signaling by IL-6 receptors have been reported in various cancers, which were associated with poor prognosis. On the other hand, targeting IL-6 and IL-6 receptors was associated with ameliorating effects in cancers. Therefore, this study sought to inhibit both IL-6 and its receptor (glycoprotein 130, [gp130]) and to synergistically reduce cancer progression in vitro. Methods: Accordingly, 4T1 and CT26 cancer cell lines were used to analyze the efficacy of the simultaneous blockade of IL-6 and gp130. In addition, small interfering ribonucleic acid (siRNA) molecules were employed to suppress the expression of these factors. The expression of target genes was investigated using the quantitative real-time polymerase chain reaction assay. Further, an MTT assay was applied to study cell survival. Finally, cytokine was measured by enzyme-linked immunosorbent assay. Results: The transfection of cancer cells by lipofectamine led to significant downregulation of these factors in both cell lines. Moreover, the downregulation of IL-6 and gp130 caused significant cell death induction, which was associated with the reduced proliferative potential of both cells. Eventually, IL-6 silencing markedly suppressed the secretion of IL-6 in both cells. Conclusion: These findings imply that the simultaneous silencing of IL-6 and gp130 can be considered a potential anticancer therapeutic approach that should be further considered in future studies.

Research progress of miR-373 in gastrointestinal malignancies

microRNAs are a kind of higher eukaryotic genomic coding, usually non-coding small single-stranded RNA composed of 19-24 nucleotides, similar to siRNA molecules, which can be paired with the mRNA base of targeted genes, guide the MRNA to directly degrade or inhibit transcription translation, regulate the expression of targeted genes, and affect the expression of targeted proteins

Abstract 3890: TMPRSS2 serin protease is a novel biomarker for ER+ breast cancer patient prognosis and survival and mediates resistance to anti-estrogen treatment in ER+ breast cancer

Abstract Breast cancer (BC) is the most commonly diagnosed cancer in women and the second leading cause of cancer related deaths in the US. About 70-80% of patients have ER+ BC and initially respond to anti-estrogen (ie, Tamoxifen) or endocrine/homonal treatments. However, about 60% of the patients inevitably develops resistance to anti-estrogen therapies (due to intrinsic (15-20%) and acquire resistance (30-40%), representing a major clinical problem, resulting in relapses and metastasis, leading to significant patient mortality. Therefore, understanding the underlying mechanisms causing resitance to hormonal or anti-estrogen treatments is needed the for developing highly effective threapies and improving patient survival. Cell surface proteases, including serine proteases, are proteolytic modifiers of particular targets, including growth factors and protease-activated receptors, which are critical for the activation of oncogenic signaling pathways. TMPRSS2, a member of type II transmembrane serine protease (TTSP) family, has been shown to be highly expressed in prostate cancer cells and its role in carcinogenesis. We found that TMPRSS2 is highly upregulated well established tamoxifene resistant cell lines derivative of MCF7 (LCC2 and LCC9) compared to MCF7 cells. Performing Kaplan-Meier survival analyses in TCGA-BC database we also found that TMPRSS2 expression is associated with poor prognosis and significantly shorter patient survival in ER+ BC patients (p=0.015, n=221 patients). In addition, we found that TMPRSS2 is regulated by Estrogen/Estrogen Receptor-alpha (ERα) signaling as in vitro inhibition of estrogen/ERα signaling by ERα siRNA, tamoxifen or faslodex reduced TMPRSS2 protein expression ER+ MCF7 BC cells. Moreover, we found that inhibition of TMPRSS2 by 3 different siRNA molecules suppressed cell proliferation of MCF7 cells and reduced expression critical proteins for cell cycle and proliferation, including cyclin D1, c-Myc and pERK/MAPK signaling, suggesting that TMPRSS2 promotes the oncogenic signaling pathways in ER+ BC cells. Currently, we are targeting TMPRSS2 in in vitro and in vivo Tamox-resistant LCC2 and LCC9 models in mice by siRNA alone and in combination with Tamoxifen or faslodex to demostrate inhibition of TMPRSS2 sensitize tumors to anti-estrogen treatments. In conclusion, TMPRSS2 is a potential biomarker for poor patient survival and anti-estrogen resistance, and targeting TMPRSS2 alone as a monothreapy and in combination with anti- Tamoxifen or faslodex may be a promising approach for overcoming resistance to estrogen thereapies in ER+ BC patients. Citation Format: Rumeysa Ozyurt, Nermin Kahraman, Pinar Atalay Dundar, Bulent Ozpolat. TMPRSS2 serin protease is a novel biomarker for ER+ breast cancer patient prognosis and survival and mediates resistance to anti-estrogen treatment in ER+ breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3890.

Single-cell quantification and dose-response of cytosolic siRNA delivery

Endosomal escape and subsequent cytosolic delivery of small interfering RNA (siRNA) therapeutics is believed to be highly inefficient. Since it has not been possible to quantify cytosolic amounts of delivered siRNA at therapeutic doses, determining delivery bottlenecks and total efficiency has been difficult. Here, we present a confocal microscopy-based method to quantify cytosolic delivery of fluorescently labeled siRNA during lipid-mediated delivery. This method enables detection and quantification of sub-nanomolar cytosolic siRNA release amounts from individual release events with measures of quantitation confidence for each event. Single-cell kinetics of siRNA-mediated knockdown in cells expressing destabilized eGFP unveiled a dose-response relationship with respect to knockdown induction, depth and duration in the range from several hundred to thousands of cytosolic siRNA molecules. Accurate quantification of cytosolic siRNA, and the establishment of the intracellular dose-response relationships, will aid the development and characterization of novel delivery strategies for nucleic acid therapeutics.

Development of Novel siRNA Therapeutics: A Review with a Focus on Inclisiran for the Treatment of Hypercholesterolemia.

Over the past two decades, it was discovered that introducing synthetic small interfering RNAs (siRNAs) into the cytoplasm facilitates effective gene-targeted silencing. This compromises gene expression and regulation by repressing transcription or stimulating sequence-specific RNA degradation. Substantial investments in developing RNA therapeutics for disease prevention and treatment have been made. We discuss the application to proprotein convertase subtilisin/kexin type 9 (PCSK9), which binds to and degrades the low-density lipoprotein cholesterol (LDL-C) receptor, interrupting the process of LDL-C uptake into hepatocytes. PCSK9 loss-of-function modifications show significant clinical importance by causing dominant hypocholesterolemia and lessening the risk of cardiovascular disease (CVD). Monoclonal antibodies and small interfering RNA (siRNA) drugs targeting PCSK9 are a significant new option for managing lipid disorders and improving CVD outcomes. In general, monoclonal antibodies are restricted to binding with cell surface receptors or circulating proteins. Similarly, overcoming the intracellular and extracellular defenses that prevent exogenous RNA from entering cells must be achieved for the clinical application of siRNAs. N-acetylgalactosamine (GalNAc) conjugates are a simple solution to the siRNA delivery problem that is especially suitable for treating a broad spectrum of diseases involving liver-expressed genes. Inclisiran is a GalNAc-conjugated siRNA molecule that inhibits the translation of PCSK9. The administration is only required every 3 to 6 months, which is a significant improvement over monoclonal antibodies for PCSK9. This review provides an overview of siRNA therapeutics with a focus on detailed profiles of inclisiran, mainly its delivery strategies. We discuss the mechanisms of action, its status in clinical trials, and its prospects.

High-Capacity Mesoporous Silica Nanocarriers of siRNA for Applications in Retinal Delivery.

The main cause of subretinal neovascularisation in wet age-related macular degeneration (AMD) is an abnormal expression in the retinal pigment epithelium (RPE) of the vascular endothelial growth factor (VEGF). Current approaches for the treatment of AMD present considerable issues that could be overcome by encapsulating anti-VEGF drugs in suitable nanocarriers, thus providing better penetration, higher retention times, and sustained release. In this work, the ability of large pore mesoporous silica nanoparticles (LP-MSNs) to transport and protect nucleic acid molecules is exploited to develop an innovative LP-MSN-based nanosystem for the topical administration of anti-VEGF siRNA molecules to RPE cells. siRNA is loaded into LP-MSN mesopores, while the external surface of the nanodevices is functionalised with polyethylenimine (PEI) chains that allow the controlled release of siRNA and promote endosomal escape to facilitate cytosolic delivery of the cargo. The successful results obtained for VEGF silencing in ARPE-19 RPE cells demonstrate that the designed nanodevice is suitable as an siRNA transporter.