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Targeting genes using siRNA shows promise as an approach to alleviate symptoms of diabetic neuropathy. It focuses on neuropathies and distal symmetric polyneuropathy (DSPN) to explore the potential use of small interfering RNA (siRNA) as a treatment for diabetic neuropathy. Timely identification and management of neuropathy play a critical role in mitigating potential complications. RNAi success depends on understanding factors affecting small interfering RNA (siRNA) functionality and specificity. These include sequence space restrictions, structural and sequence features, mechanisms for nonspecific gene modulation, and chemical modifications. Addressing these factors enhances siRNA performance for efficient gene silencing and confidence in RNAi-mediated genomic studies. Diabetic retinopathy, particularly in South Asian, African, Latin American, and indigenous populations, is a significant concern due to its association with diabetes. Ethnicity plays a crucial role in its development and progression. Despite declining rates in the US, global trends remain concerning, and further research is needed to understand regional differences and reinforce ethnicity-based screening and treatment protocols. In this regard, siRNA emerges as a valuable instrument for early intervention strategies. While presenting promising therapeutic applications, siRNA utilization encounters challenges within insect pest control contexts, thereby providing insights into enhancing its delivery mechanisms for neuropathy treatment purposes. Recent advancements in delivery modalities, such as nanoparticles, allow for the controlled release of siRNA. More investigation is necessary to grasp the safety and efficacy of siRNA technology fully. It holds promise in transforming the treatment of diabetic neuropathy by honing in on particular genes and tackling issues such as inflammation and oxidative stress. Continuous advancements in delivery techniques have the potential to enhance patient results significantly. SiRNA targets genes in diabetic neuropathy, curbing nerve damage and pain and potentially preventing or delaying the condition. Customized treatments based on genetic variations hold promise for symptom management and enhancing quality of life., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

It is known that calcitonin gene-related peptide (CGRP) plays a key role in pain modulation in the brain. There are high expressions of CGRP and CGRP receptor in anterior cingulate cortex (ACC), an important brain structure in pain modulation. The present study explored the role and mechanisms of CGRP and CGRP receptor in nociceptive modulation in ACC in naïve rats and inflammatory rats. Administration of different does of CGRP in ACC induced significant antinociception in a dose-dependent manner in both naïve rats and rats with inflammatory pain. The CGRP-induced antinociception was attenuated by injection of the CGRP receptor antagonist CGRP8-37 in ACC. Interestingly, both CGRP-induced antinociception and CGRP receptor expression decreased in ACC in rats with inflammatory pain compared with naïve rats. Knockdown of CGRP receptor in ACC by siRNA targeting to CGRP receptor attenuated both the CGRP receptor expression and the CGRP-induced antinociception significantly in rats. These findings demonstrate that CGRP and CGRP receptor participate in nociceptive modulation in ACC in rats, inhibiting CGRP receptor expression induces decrease in CGRP-induced antinociception in ACC.

The present study aims at designing a thermosensitive gel prepared from w1/o/w2 multiple microemulsions (MMEs) for the vaginal delivery of siRNA. The w1/o/w2 MMEs were prepared by two-step emulsifications: the first step was to prepare primary emulsions (w1/o) by low energy emulsification (LEE); the second step was to obtain stable w1/o/w2 MMEs by self-emulsifying. An extensive formulation optimization process was undertaken. The final w1/o/w2 MMEs could be formed in ddH2O, phosphate buffer solution (PBS, pH 7.4) and 1640 culture media with diameter size about 166.5 ± 13.1, 271.0 ± 11.1 and 278.7 ± 12.1 nm respectively. The release rates of siRNA from solutions, MMEs and MMEs-gels were completed within 2 h, 6 h and13 h respectively. The transfection efficiency of MMEs was confirmed both in vitro and in vivo. The relative target gene expressions of MMEs were 0.07 ± 0.05% vs. 0.37 ± 0.06% in Hela cells against Lipofectamine2000® and 1.88% ± 0.00% vs. 9.65% ± 0.02% in mouse vaginal mucosa against PEI. Good biocompatibility of MMEs was verified by cytotoxicity and pathological studies. Overall, our results indicated the potential of the MMEs-gel system for the vaginal delivery of siRNA.

Methods used to control the quality of peptide products depend on the level of development of analytical and bioorganic chemistry, and the level of instrumentation. Peptide identification is a difficult task and largely depends on the complexity of its structure. There does not exist a comprehensive and simple test, except for NMR, which, however, is rather expensive and time-consuming and involves complex data interpretations. Moreover, it does not allow for unambiguous determination of the peptide purity and formula (amino acid composition, sequence, chirality of amino acid residues). For this reason, a combination of methods is often used, including amino acid analysis, TLC/HPLC and mass spectrometry, and, less frequently, sequencing. Current international practice of peptide analysis is to use HPLC in combination with mass spectrometric, mainly tandem (HPLC-MS/MS), detection. According to literature sources the amino acid sequence of linear peptides can be analysed using various enzymes and subsequent identification of proteolysis products by mass spectrometry. This article presents approaches to the development of test methods for analysis of purity and identification testing of a small interfering RNA-based novel medicinal product, which will help standardise and control the quality of the production process.

In many ways, cancer cells are different from healthy cells. A lot of tactical nano-based drug delivery systems are based on the difference between cancer and healthy cells. Currently, nanotechnology-based delivery systems are the most promising tool to deliver DNA-based products to cancer cells. This review aims to highlight the latest development in the lipids and polymeric nanocarrier for siRNA delivery to the cancer cells. It also provides the necessary information about siRNA development and its mechanism of action. Overall, this review gives us a clear picture of lipid and polymer-based drug delivery systems, which in the future could form the base to translate the basic siRNA biology into siRNA-based cancer therapies. The latest development in the lipids and polymeric nanocarrier for siRNA delivery to the cancer cells were highlighted. It also provides the necessary information about siRNA development and its mechanism of action. Image 1 [ABSTRACT FROM AUTHOR]

Hereditary genetic disorders, cancer, and infectious diseases of the liver affect millions of people around the globe and are a major public health burden. Most contemporary treatments offer limited relief as they generally aim to alleviate disease symptoms. Targeting the root cause of diseases originating in the liver by regulating malfunctioning genes with nucleic acid-based drugs holds great promise as a therapeutic approach. However, employing nucleic acid therapeutics in vivo is challenging due to their unfavorable characteristics. Lipid nanoparticle (LNP) delivery technology is a revolutionary development that has enabled clinical translation of gene therapies. LNPs can deliver siRNA, mRNA, DNA, or gene-editing complexes, providing opportunities to treat hepatic diseases by silencing pathogenic genes, expressing therapeutic proteins, or correcting genetic defects. Here we discuss the state-of-the-art LNP technology for hepatic gene therapy including formulation design parameters, production methods, preclinical development and clinical translation. Unlabelled Image [ABSTRACT FROM AUTHOR]

It is known that calcitonin gene-related peptide (CGRP) plays a key role in pain modulation in the brain. There are high expressions of CGRP and CGRP receptor in anterior cingulate cortex (ACC), an important brain structure in pain modulation. The present study explored the role and mechanisms of CGRP and CGRP receptor in nociceptive modulation in ACC in naïve rats and inflammatory rats. Administration of different does of CGRP in ACC induced significant antinociception in a dose-dependent manner in both naïve rats and rats with inflammatory pain. The CGRP-induced antinociception was attenuated by injection of the CGRP receptor antagonist CGRP8-37 in ACC. Interestingly, both CGRP-induced antinociception and CGRP receptor expression decreased in ACC in rats with inflammatory pain compared with naïve rats. Knockdown of CGRP receptor in ACC by siRNA targeting to CGRP receptor attenuated both the CGRP receptor expression and the CGRP-induced antinociception significantly in rats. These findings demonstrate that CGRP and CGRP receptor participate in nociceptive modulation in ACC in rats, inhibiting CGRP receptor expression induces decrease in CGRP-induced antinociception in ACC. [ABSTRACT FROM AUTHOR]

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Intervertebral disc degeneration (IVDD) is a pathological condition that can lead to intractable back pain or secondary neurological deficits. There is no fundamental cure for this condition, and current treatments focus on alleviating symptoms indirectly. Numerous studies have been performed to date, and the major strategy for all treatments of IVDD is to prevent cell loss due to programmed or regulated cell death. Accumulating evidence suggests that several types of cell death other than apoptosis, including necroptosis, pyroptosis, and ferroptosis, are also involved in IVDD. In this study, we discuss the molecular pathway of each type of cell death and review the literature that has identified their role in IVDD. We also summarize the recent advances in targeted therapy at the RNA level, including RNA modulations through RNA interference and regulation of non-coding RNAs, for preventing cell death and subsequent IVDD. Therefore, we review the causes and possible therapeutic targets for RNA intervention and discuss the future direction of this research field.

The siRNA-mediated inhibition of nuclear factor E2-related factor 2 (Nrf2) can be an attractive approach to overcome chemoresistance in various malignant tumors, including melanoma. This work aims at designing a new type of chitosan-shelled nanobubble for the delivery of siRNA against Nrf2 in combination with an ultrasound. A new preparation method based on a water–oil–water (W/O/W) double-emulsion was purposely developed for siRNA encapsulation in aqueous droplets within a nanobubble core. Stable, very small NB formulations were obtained, with sizes of about 100 nm and a positive surface charge. siRNA was efficiently loaded in NBs, reaching an encapsulation efficiency of about 90%. siNrf2-NBs downregulated the target gene in M14 cells, sensitizing the resistant melanoma cells to the cisplatin treatment. The combination with US favored NB cell uptake and transfection efficiency. Based on the results, nanobubbles have shown to be a promising US responsive tool for siRNA delivery, able to overcome chemoresistance in melanoma cancer cells.

Pancreatic cancer (PC) is one of the leading causes of death and is the fourth most malignant tumor in men. The epigenetic and genetic alterations appear to be responsible for development of PC. Small interfering RNA (siRNA) is a powerful genetic tool that can bind to its target and reduce expression level of a specific gene. The various critical genes involved in PC progression can be effectively targeted using diverse siRNAs. Moreover, siRNAs can enhance efficacy of chemotherapy and radiotherapy in inhibiting PC progression. However, siRNAs suffer from different off target effects and their degradation by enzymes in serum can diminish their potential in gene silencing. Loading siRNAs on nanoparticles can effectively protect them against degradation and can inhibit off target actions by facilitating targeted delivery. This can lead to enhanced efficacy of siRNAs in PC therapy. Moreover, different kinds of nanoparticles such as polymeric nanoparticles, lipid nanoparticles and metal nanostructures have been applied for optimal delivery of siRNAs that are discussed in this article. This review also reveals that how naked siRNAs and their delivery systems can be exploited in treatment of PC and as siRNAs are currently being applied in clinical trials, significant progress can be made by translating the current findings into the clinical settings.

Peptide-oligonucleotide conjugates (POCs) represent one of the increasingly successful albeit costly approaches to increasing the cellular uptake, tissue delivery, bioavailability, and, thus, overall efficiency of therapeutic nucleic acids, such as, antisense oligonucleotides and small interfering RNAs. This review puts the subject of chemical synthesis of POCs into the wider context of therapeutic oligonucleotides and the problem of nucleic acid drug delivery, cell-penetrating peptide structural types, the mechanisms of their intracellular transport, and the ways of application, which include the formation of non-covalent complexes with oligonucleotides (peptide additives) or covalent conjugation. The main strategies for the synthesis of POCs are viewed in detail, which are conceptually divided into (a) the stepwise solid-phase synthesis approach and (b) post-synthetic conjugation either in solution or on the solid phase, especially by means of various click chemistries. The relative advantages and disadvantages of both strategies are discussed and compared.

Abstract Delivery of nucleic acids into solid tumor environments remains a pressing challenge. This study examines the ability of macrophages to horizontally transfer small interfering RNA (siRNA) lipoplexes to cancer cells. Macrophages are a natural candidate for a drug carrier because of their ability to accumulate at high densities into many cancer types, including, breast, prostate, brain, and colon cancer. Here, it is demonstrated that macrophages can horizontally transfer siRNA to cancer cells during in vitro coculture. The amount of transfer can be dosed depending on the amount of siRNA loaded and total number of macrophages delivered. Macrophages loaded with calcium integrin binding protein‐1 (CIB1)‐siRNA result in decreased tumorsphere growth and decreased mRNA expression of CIB1 and KI67 in MDA‐MB‐468 human breast cancer cells. Adoptive transfer of macrophages transfected with CIB1‐siRNA localizes to the orthotopic MDA‐MB‐468 tumor. Furthermore, it is reported that macrophage activation can modulate this transfer process as well as intracellular trafficking protein Rab27a. As macrophages are heavily involved in tumor progression, understanding how to use macrophages for drug delivery can substantially benefit the treatment of tumors.

Cancer is associated with single or multiple gene defects. Recently, much research has focused on incorporating genetic materials as one of the means to treat various types of carcinomas. RNA interference (RNAi) conveys an alternative genetic approach for cancer patients, especially when conventional medications fail. RNAi involves the inhibition of expression of specific messenger RNA that signals for uncontrollable cell growth and proliferation, most notably with carcinoma cells. This molecular technology is promising as genetic materials allow us to overcome issues associated with chemotherapeutic agents including organ damage associated with severe drug toxicities. Nonetheless, vast challenges impede successful gene therapy application, including low tumor localization, low stability and rapid clearance from the blood circulation. Owing to the limited treatment opportunities for the management of cancer, the development of effective siRNA carrier systems involving nanotherapeutics has been extensively explored. Over the past years, several siRNA nanotherapeutics have undergone a period of clinical investigation, with some demonstrating promising antitumor activities and safety profiles. Extensive observation of siRNA-nanoparticles is necessary to ensure commercial success. Therefore, this review mainly focuses on the progress of siRNAs-loaded nanoparticles that have undergone clinical trials for cancer treatment. The status of the siRNA nanotherapeutics is discussed, allowing comprehensive understanding of their gene-mediated therapeutics.

Post-transcriptional gene silencing (PTGS)-mediated gene silencing exploits the cellular mechanism wherein transcripts having sequence similarity to the double-stranded RNA (dsRNA) molecules present in the cell will be subjected to degradation. PTGS is closely related to natural processes such as RNA-mediated virus resistance and cross-protection in plants. Gene silencing and the cellular machinery for affecting this phenomenon might have evolved as a natural protective measure against viral infection in plants. In PTGS, small interfering RNA (siRNA) molecules of 21–23 nucleotides length act as homology guides for triggering the systemic degradation of transcripts homologous to the siRNA molecules. PTGS phenomenon, first discovered in transgenic petunia plants harbouring chalcone synthase gene and termed co-suppression, has been subsequently exploited to target specific gene transcripts for degradation leading to manifestation of desirable traits in crop plants. Targeted gene silencing has been achieved either through the introduction of DNA constructs encoding dsRNA or antisense RNA or by deploying co-suppression constructs producing siRNAs against the transcript of interest. Understanding the mechanism of gene silencing has led to the development of several alternative strategies for inducing gene silencing in a precise and controlled way. This has paved the way for using PTGS as one of the chief functional genomics tools in plants and has helped in unraveling the mechanism of many cellular processes and identifying the focal points in pathways, besides, opening new vistas in genetic engineering of plants for human benefits. PTGS has shown great potential in silencing the deleterious genes efficiently so that value-added plant products could be obtained. Thus, PTGS has ushered in a new era in the genetic manipulation of plants for both applied and basic studies. In this review, we have outlined the basics of RNAi-mediated gene silencing and summarized the work carried out at our institute using this approach, as case studies. In particular, adopting RNAi-mediated gene silencing (a) as a method to restore fertility in transgenic male sterile lines developed based on orfH522 gene from sunflower PET1-CMS source, (b) as a tool to suppress the production of toxic proteins, ricin and RCA, in castor, and (c) as an approach to induce bud necrosis virus resistance in sunflower has been discussed. Examples from other plant systems also have been mentioned to exemplify the concept and utility of gene silencing in crop plants. [ABSTRACT FROM AUTHOR]

Radiation sensitizers that can selectively act on cancer cells hold great promise to patients who receive radiation therapy. We developed a novel targeted therapy and radiation sensitizer for non-small cell lung cancer (NSCLC) based on cetuximab conjugated nanoparticle that targets epidermal growth factor receptor (EGFR) and delivers small interfering RNA (siRNA) against polo-like kinase 1 (PLK1). EGFR is overexpressed in 50% of lung cancer patients and a mediator of DNA repair, while PLK1 is a key mitotic regulator whose inhibition enhances radiation sensitivity. The nanoparticle construct (C-siPLK1-NP) effectively targets EGFR + NSCLC cells and reduces PLK1 expression, leading to G2/M arrest and cell death. Furthermore, we show a synergistic combination between C-siPLK1-NP and radiation, which was confirmed in vivo in A549 flank tumors. We also demonstrate the translational potential of C-siPLK1-NP as a systemic therapeutic in an orthotopic lung tumor model, where administration of C-siPLK1-NP reduced tumor growth and led to prolonged survival. Our findings demonstrate that C-siPLK1-NP is effective as a targeted therapy and as a potent radiation sensitizer for NSCLC. Potential application to other EGFR + cancer types such as colorectal and breast cancer is also demonstrated. [ABSTRACT FROM AUTHOR]

The present study aims at designing a thermosensitive gel prepared from w1/o/w2 multiple microemulsions (MMEs) for the vaginal delivery of siRNA. The w1/o/w2 MMEs were prepared by two-step emulsifications: the first step was to prepare primary emulsions (w1/o) by low energy emulsification (LEE); the second step was to obtain stable w1/o/w2 MMEs by self-emulsifying. An extensive formulation optimization process was undertaken. The final w1/o/w2 MMEs could be formed in ddH2O, phosphate buffer solution (PBS, pH 7.4) and 1640 culture media with diameter size about 166.5 ± 13.1, 271.0 ± 11.1 and 278.7 ± 12.1 nm respectively. The release rates of siRNA from solutions, MMEs and MMEs-gels were completed within 2 h, 6 h and13 h respectively. The transfection efficiency of MMEs was confirmed both in vitro and in vivo. The relative target gene expressions of MMEs were 0.07 ± 0.05% vs. 0.37 ± 0.06% in Hela cells against Lipofectamine2000® and 1.88% ± 0.00% vs. 9.65% ± 0.02% in mouse vaginal mucosa against PEI. Good biocompatibility of MMEs was verified by cytotoxicity and pathological studies. Overall, our results indicated the potential of the MMEs-gel system for the vaginal delivery of siRNA. [ABSTRACT FROM AUTHOR]

In order to evaluate the in vivo effect of inhaled formulations, it is a gold standard to create a lung metastasis model by intravenously injecting cancer cells into an animal. Because the cancer grows from the blood vessel side, there is a possibility of underestimating the effect of an inhaled formulation administered to the lung epithelium side. In addition, the metastasis model has disadvantages in terms of preparation time and expense. The present study aimed to establish a new method to evaluate the effect of an inhaled small interfering RNA (siRNA) formulation that is more correct, more rapid, and less expensive. We investigated whether siRNA can suppress gene expression of plasmid DNA (pDNA) by serial pulmonary administration of siRNA and pDNA powders prepared by spray-freeze-drying. We revealed that formulations of dry siRNA powder significantly suppressed gene expression of pDNA powder compared with a control group with no siRNA. Naked siRNA inhalation powder with no vector showed the suppression of gene expression equivalent to that of an siRNA-polyethyleneimine complex without damaging tissues. These results show that the present method is suitable for evaluating the gene-silencing effect of inhaled siRNA powders. [ABSTRACT FROM AUTHOR]

Small RNAs (sRNA) are reported to play pivotal roles in the epigenetic and post-transcriptional regulation of gene expression during growth, development, and stress response in plants. Recently, the involvement of two different classes of sRNAs namely, miRNAs (microRNAs), and siRNAs (small interfering RNAs) in biotic stress response has been underlined. Notably, during virus infection, these sRNAs deploy antiviral defense by regulating the gene expression of the modulators of host defense pathways. As a counter defense, viruses have evolved strategic pathways involving the production of suppressors that interfere with the host silencing machinery. This molecular arms race between the sophisticated gene regulatory mechanism of host plants fine-tuned by sRNAs and the defense response exhibited by the virus has gained much attention among the researchers. So far, several reports have been published showing the mechanistic insights on sRNA-regulated defense mechanism in response to virus infection in several crop plants. In this context, our review enumerates the molecular mechanisms underlying host immunity against viruses mediated by sRNAs, the counter defense strategies employed by viruses to surpass this immunogenic response and the advances made in our understanding of plant-virus interactions. Altogether, the report would be insightful for the researchers working to decode the sRNA-mediated defense response in crop plants challenged with virus infection. [ABSTRACT FROM AUTHOR]

Meiotic silencing by unpaired DNA (MSUD) is a gene silencing process that occurs within meiotic cells of Neurospora crassa and other fungi. We have previously developed a high-throughput screen to identify suppressors of this silencing pathway. Here, a list of MSUD suppressor candidates from a single pass of the first 84 plates of the Neurospora knockout library is provided. [ABSTRACT FROM AUTHOR]

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is considered as the master regulator of antioxidant and cytoprotective gene expressions. Moreover, it plays a pivotal role in cancer progression. Nrf2 mediates the adaptive response which contributes to the resistance to chemotherapeutic pro-oxidant drugs, such as cisplatin (CDDP), in various tumors, including bladder cancers. For this reason, Nrf2 could be a promising target to overcome chemoresistance. There are several known Nrf2 pharmacological inhibitors; however, most of them are not specific. The use of a specific small interfering RNA (siRNA) targeting the Nrf2 gene (siNrf2) loaded into nanovehicles is an attractive alternative, since it can increase specificity. This study aimed to evaluate the biological activity of siNrf2 loaded on guanidine-terminated carbosilane dendrimers (GCDs) in overcoming CDDP resistance in bladder cancer cells with a high level of Nrf2. Parameters such as viability, proliferation, apoptosis, migration, and oxidative stress level were taken into account. Results demonstrated that siNrf2-GCD treatment sensitized CDDP-resistant cells to CDDP treatment. Moreover, data obtained by treating the non-cancerous human kidney HK-2 cell line strongly suggest a good safety profile of the carbosilane dendrimers loaded with siNrf2. In conclusion, we suggest that siNrf2-GCD is a promising drug delivery system for gene therapy to be used in vivo; and it may represent an important tool in the therapy of CDDP-resistant cancer.

Prostate cancer (PCa) accounts for a high number of deaths in males with no available curative treatments. Patients with PCa are commonly diagnosed in advanced stages due to the lack of symptoms in the early stages. Recently, the research focus was directed toward gene editing in cancer therapy. Small interfering RNA (siRNA) intervention is considered as a powerful tool for gene silencing (knockdown), enabling the suppression of oncogene factors in cancer. This strategy is applied to the treatment of various cancers including PCa. The siRNA can inhibit proliferation and invasion of PCa cells and is able to promote the anti-tumor activity of chemotherapeutic agents. However, the off-target effects of siRNA therapy remarkably reduce its efficacy in PCa therapy. To date, various carriers were designed to improve the delivery of siRNA and, among them, nanoparticles are of importance. Nanoparticles enable the targeted delivery of siRNAs and enhance their potential in the downregulation of target genes of interest. Additionally, nanoparticles can provide a platform for the co-delivery of siRNAs and anti-tumor drugs, resulting in decreased growth and migration of PCa cells. The efficacy, specificity, and delivery of siRNAs are comprehensively discussed in this review to direct further studies toward using siRNAs and their nanoscale-delivery systems in PCa therapy and perhaps other cancer types.

Polyethylene glycol (PEG)-modified (PEGylated) cationic liposomes are frequently used as delivery vehicles for small interfering RNA (siRNA)-based drugs because of their ability to encapsulate/complex with siRNA and prolong the circulation half-life in vivo. Nevertheless, we have reported that subsequent intravenous (IV) injections of siRNA complexed with PEGylated cationic liposomes (PLpx) induces the production of anti-PEG immunoglobulin M (IgM), which accelerates the blood clearance of subsequent doses of PLpx and other PEGylated products. In this study, it is interesting that splenectomy (removal of spleen) did not prevent anti-PEG IgM induction by IV injection of PLpx. This indicates that B cells other than the splenic version are involved in anti-PEG IgM production under these conditions. In vitro and in vivo studies have shown that peritoneal cells also secrete anti-PEG IgM in response to the administration of PLpx. Interleukin-6 (IL-6) is a glycoprotein that is secreted by peritoneal immune cells and has been detected in response to the in vivo administration of PLpx. These observations indicate that IV injection of PLpx stimulates the proliferation/differentiation of peritoneal PEG-specific B cells into plasma cells via IL-6 induction, which results in the production of anti-PEG IgM from the peritoneal cavity of mice. Our results suggest the mutual contribution of peritoneal B cells as a potent anti-PEG immune response against PLpx.

The prevalence of metabolic disorders is increasing exponentially and has recently reached epidemic levels. Over the decades, a large number of therapeutic options have been proposed to manage these diseases but still show several limitations. In this circumstance, RNA therapeutics have rapidly emerged as a new hope for patients with metabolic diseases. 57 years have elapsed from the discovery of mRNA, a large number of RNA-based drug candidates have been evaluated for their therapeutic effectiveness and clinical safety under clinical studies. To date, there are seven RNA drugs for treating metabolic disorders receiving official approval and entering the global market. Their targets include hereditary transthyretin-mediated amyloidosis (hATTR), familial chylomicronemia syndrome, acute hepatic porphyria, primary hyperoxaluria type 1 and hypercholesterolemia, which are all related to liver proteins. All of these seven RNA drugs are antisense oligonucleotides (ASO) and small interfering RNA (siRNA). These two types of treatment are both based on oligonucleotides complementary to target RNA through Watson-Crick base-pairing, but their mechanisms of action include different nucleases. Such treatments show greatest potential among all types of RNA therapeutics due to consecutive achievements in chemical modifications. Another method, mRNA therapeutics also promise a brighter future for patients with a handful of drug candidates currently under development., (Copyright © 2024. Published by Elsevier Inc.)

Small interfering RNAs (siRNA) technology has shown great promise as a new class of therapeutic interventions for the treatment of cancer and other diseases. It is a remarkable endogenous pathway that can regulate sequence-specific gene silencing. Despite the excitement about possible applications of this biological process for sequence-specific gene regulation, the major limitations against the use of siRNA-based therapeutics are their rapid degradation by serum nuclease, poor cellular uptake, and rapid renal clearance following systemic delivery, off-target effects and the induction of immune responses. Many researchers have tried to overcome these limitations by developing nuclease-resistant chemically-modified siRNAs and a variety of synthetic and natural biodegradable lipids and polymers to enhance the efficacy and safety profiles of siRNA delivery. Ideal siRNA-based delivery systems for cancer therapy must be clinically suitable, safe and effective. In this review, we introduce the greatest challenges in achieving efficient RNAi delivery and discuss design criteria and various delivery strategies for cancer therapy, including chemical modifications, lipid-based nanovectors, polymer-mediated delivery systems, conjugate delivery systems, and others. [ABSTRACT FROM AUTHOR]

Highlights • RNA silencing can spread from cell to cell and systemically. It is genetically determined and requires mobile RNA signals. • Genetics and mobile signals for non-cell-autonomous RNA silencing (Non-CARS) were intensively examined in past few decades. • No consensus dogma for mobile silencing can be reached in plants, yet often contradictory and sometimes controversial. • DCL2 or DCL2 -dependent network is critical for Non-CARS in both Arabidopsis and Nicotiana benthamiana. • This sheds light on genetic components and small RNA signals for mobile silencing in (across) plants and other organisms. Abstract Non-cell autonomous RNA silencing can spread from cell to cell and over long-distances in animals and plants. This process is genetically determined and requires mobile RNA signals. Genetic requirement and molecular nature of the mobile signals for non-cell-autonomous RNA silencing were intensively investigated in past few decades. No consensus dogma for mobile silencing can be reached in plants, yet published data are sometimes inconsistent and controversial. Thus, the genetic requirements and molecular signals involved in plant mobile silencing are still poorly understood. This article revisits our present understanding of intercellular and systemic non-cell autonomous RNA silencing, and summarises current debates on RNA signals for mobile silencing. In particular, we discuss new evidence on siRNA mobility, a DCL2 -dependent genetic network for mobile silencing and its potential biological relevance as well as 22 nt siRNA being a mobile signal for non-cell-autonomous silencing in both Arabidopsis and Nicotiana benthamiana. This sets up a new trend in unravelling genetic components and small RNA signal molecules for mobile silencing in (across) plants and other organisms of different kingdoms. Finally we raise several outstanding questions that need to be addressed in future plant silencing research. [ABSTRACT FROM AUTHOR]

Artemis is a key protein of NHEJ (nonhomologous end joining), which is the major pathway for the repair of IR-induced DSBs in mammalian cells. However, the expression of Artemis in tumors and the influence of silencing Artemis on tumor sensitivity to radiation have not been investigated fully. In this study, we investigated how the expression levels of Artemis may affect the treatment outcome of radiotherapy and chemotherapy in colorectal cancer cells. First, we found that the expression of Artemis is strong in some human rectal cancer samples, being higher than in adjacent normal tissues using immunohistochemical staining. We then knocked down Artemis gene in a human colorectal cancer cell line (RKO) using lentivirus-mediated siRNAs. Compared to the control RKO cells, the Artemis knockdown cells showed significantly increased sensitivity to bleomycin, etoposide, camptothecin, and IR. Induced by DNA-damaging agents, delayed DNA repair kinetics was found by the g-H2AX foci assay, and a significantly increased cell apoptosis occurred in the Artemis knockdown RKO cells through apoptosis detection methods and Western blot. We also found that the p53/p21 signaling pathway may be involved in the apoptosis process. Taken together, our study indicates that manipulating Artemis can enhance colorectal cancer cell sensitivity to DNA-damaging agents. Therefore, Artemis can serve as a therapeutic target in rectal cancer therapy. [ABSTRACT FROM AUTHOR]

Small regulatory RNAs guide gene silencing at the DNA or RNA level through repression of complementary sequences. The two main forms of small RNAs are microRNA (miRNA) and small interfering RNA (siRNAs), which are generated from the processing of different forms of double-stranded RNA (dsRNA) precursors. These two forms of small regulatory RNAs function in distinct but overlapping gene silencing pathways in plants. Gene silencing pathways in eukaryotes evolved from an ancient prokaryotic mechanism involved in genome defense against invasive genetic elements, but has since diversified to also play a crucial role in regulation of endogenous gene expression. Here, we review the biogenesis of the different forms of small RNAs in plants, including miRNAs, phased, secondary siRNAs (phasiRNAs) and heterochromatic siRNAs (hetsiRNAs), with a focus on their functions in genome defense, transcriptional and post-transcriptional gene silencing, RNA-directed DNA methylation, trans-chromosomal methylation and paramutation. We also discuss the important role that gene duplication has played in the functional diversification of gene silencing pathways in plants, and we highlight recently discovered components of gene silencing pathways in plants. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To explore the protective role of Vitamin D Receptor (VDR) in retinal ganglion cells (RGCs) after high-level glucose induction, and to investigate its underlying mechanism. MATERIALS AND METHODS: Primary RGCs were isolated from 24-hour-old Sprague Dawley (SD) rats and cultured in 50 mmol/L glucose. The expression of VDR in RGCs induced by 50 mmol/L glucose at different time points was determined by Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot, respectively. Subsequently, VDR siRNA was transfected into RGCs. Transfection efficiency was determined by qRT-PCR and Western blot, respectively. The protein expressions levels of VDR, signal transducer and activator of transcription 3 (STAT3) and p-STAT3 in RGCs after VDR knockdown were determined by Western blot. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and cell counting kit-8 (CCK-8) assay were conducted to access the viability of RGCs after high-level glucose induction. Ki-67 staining was performed to detect the proliferation of RGCs. Meanwhile, the apoptosis of RGCs was evaluated by using Annexin-V FITC/PI and TUNEL (terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling) assay, respectively. In addition, caspase-3 activity in RGCs was detected by relative commercial kit. RESULTS: After 4 days of high-level glucose induction, the viability of RGCs was remarkably decreased. VDR was highly expressed in RGCs during high-level glucose culture. The mRNA and protein expression levels of VDR were both significantly downregulated after the transfection of VDR siRNA in RGCs. Meanwhile, VDR knockdown in RGCs significantly increased the viability and proliferative ability of RGCs, whereas significantly decreased apoptotic rate and caspase-3 activity. In addition, the protein level of p-STAT3 in RGCs was remarkably downregulated after VDR knockdown. CONCLUSIONS: Inhibition of VDR exerts a protective role in high-level glucose induced RGCs damage by activating the STAT3 pathway. [ABSTRACT FROM AUTHOR]

In plants, transposable elements (TEs) represent a large fraction of the genome, with potential to alter gene expression and produce genomic rearrangements. Epigenetic control of TEs is often used to stop unrestricted movement of TEs that would result in detrimental effects due to insertion in essential genes. The current review focuses on the effects of methylation on TEs and their genomic context, and how this type of epigenetic control affects plant adaptability when plants are faced with different stresses and changes. TEs mobilize in response to stress elicitors, including biotic and abiotic cues, but also developmental transitions and 'genome shock' events like polyploidization. These events transitionally lift TE repression, allowing TEs to move to new genomic locations. When TEs fall close to genes, silencing through methylation can spread to nearby genes, resulting in lower gene expression. The presence of TEs in gene promoter regions can also confer stress inducibility modulated through alternative methylation and demethylation of the TE. Bursts of transposition triggered by events of genomic shock can increase genome size and account for differences seen during polyploidization or species divergence. Finally, TEs have evolved several mechanisms to suppress their own repression, including the use of microRNAs to control genes that promote methylation. The interplay between silencing, transient TE activation, and purifying selection allows the genome to use TEs as a reservoir of potential beneficial modifications but also keeps TEs under control to stop uncontrolled detrimental transposition. [ABSTRACT FROM AUTHOR]

Abstract: Small interfering RNAs (siRNAs) are invaluable research tools for studying gene functions in mammalian cells. siRNAs are mainly produced by chemical synthesis or by enzymatic digestion of double‐stranded RNA (dsRNA) produced in vitro. Recently, bacterial cells, engineered with ectopic plant viral siRNA binding protein p19, have enabled the production of “recombinant” siRNAs (pro‐siRNAs). Here, we describe an optimized methodology for the production of milligram amount of highly potent recombinant pro‐siRNAs from Escherichia coli cells. We first optimized bacterial culture medium and tested new designs of pro‐siRNA production plasmid. Through the exploration of multiple pro‐siRNA related factors, including the expression of p19 protein, (dsRNA) generation method, and the level of RNase III, we developed an optimal pro‐siRNA production plasmid. Together with a high–cell density fed‐batch fermentation method in a bioreactor, we have achieved a yield of ~10 mg purified pro‐siRNA per liter of bacterial culture. The pro‐siRNAs produced by the optimized method can achieve high efficiency of gene silencing when used at low nanomolar concentrations. This new method enables fast, economical, and renewable production of pure and highly potent bioengineered pro‐siRNAs at the milligram level. Our study also provides important insights into the strategies for optimizing the production of RNA products in bacteria, which is an under‐explored field. [ABSTRACT FROM AUTHOR]

In the present study, a novel dry small interfering RNA (siRNA) powder for inhalation, containing polyethyleneimine (PEI) as a delivery vector, was produced by spray freeze drying (SFD). The powder had spherical and highly porous structure of approximately 10 μm in diameter with high aerosol performance for emission and lung delivery. The reconstituted siRNA/PEI complex after dissolution of the powder had almost the same physicochemical properties and in vitro gene silencing activity as the original one constituted in the sample solution before SFD, showing that the integrity of the siRNA was maintained. In in vivo studies of intratracheal administration into lung metastasis mice and healthy mice, powder with a low dose of 3 μg siRNA exhibited strong and specific gene silencing activity against tumors metastasized to the lungs, whereas it caused no significant histological changes, lactate dehydrogenase leakage, or inflammatory cytokine induction in the lungs. These results strongly indicated that inhalable dry siRNA/PEI powders can provide effective pulmonary gene silencing without severe lung injury and that SFD can be applied to the production of such powders. [ABSTRACT FROM AUTHOR]

Therapeutic vaccine is a promising approach in cancer therapy. But tumor-associated antigen peptides have weak immunogenicity and cancer patients are often characterized by immunosuppression and tolerance, leading to less efficiency of immunotherapy. We here successfully developed indoleamine 2, 3-dioxygenase (IDO) siRNA nanoparticle-coated and tyrosinase-related protein 2 (Trp2)-displayed recombinant Saccharomyces cerevisiae (YCP). YCPs had positive charges with a diameter of approximately 5 μm, resulting in selective phagocytosis by APC cells. YCP-delivered siRNA and Trp2 successfully escaped from phagosomes, efficiently inhibited IDO expression in DCs, promoted the immune reaction of T cell against Trp2, increased the secretion of proinflammatory cytokines such as IFN-γ,TNF-α, and IL-6, and decreased the generation of regulatory T cells. Moreover, YCPs significantly inhibited melanoma tumor growth by alleviating immune tolerance and promoting Trp2-specific CD8 + T cell immune response. These results suggest that Saccharomyces cerevisiae as a combined immunotherapeutic platform to simultaneously delivery IDO-siRNA and Trp2 epitope peptide is a promising vaccine system for melanoma treatment. [ABSTRACT FROM AUTHOR]

Heat shock proteins are involved in cellular repair and protective mechanisms that counter characteristic features of neurodegenerative diseases such as protein misfolding and aggregation. The HSPA (Hsp70) multigene family includes the widely studied HSPA1A (Hsp70-1) and the little studied HSPA6 (Hsp70B’) which is present in the human genome and not in mouse and rat. The effect of knockdown of HSPA6 and HSPA1A expression was examined in relation to the ability of differentiated human SH-SY5Y neuronal cells to tolerate thermal stress. Low dose co-application of celastrol and arimoclomol, which induces Hsps, enhanced the ability of differentiated neurons to survive heat shock. Small interfering RNA (siRNA) knockdown of HSPA6 and HSPA1A resulted in loss of the protective effect of co-application of celastrol/arimoclomol. More pronounced effects on neuronal viability were apparent at 44 °C heat shock compared to 43 °C. siRNA knockdown suggests that HSPA6 and HSPA1A contribute to protection of differentiated human neuronal cells from cellular stress. [ABSTRACT FROM AUTHOR]

Small interfering RNAs (siRNA) are short nucleic acid fragments of about 20–27 nucleotides, which can inhibit the expression of specific genes. siRNA based RNAi technology has emerged as a promising method for the treatment of a variety of diseases. However, a major limitation in the therapeutic use of siRNA is its rapid degradation in plasma and cellular cytoplasm, resulting in short half-life. In addition, as siRNA molecules cannot penetrate into the cell efficiently, it is required to use a carrier system for its delivery. In this work, chemically and morphologically different calcium phosphate (CaP) nanoparticles, including spherical-like hydroxyapatite (HA-s), needle-like hydroxyapatite (HA-n) and calcium deficient hydroxyapatite (CDHA) nanoparticles were synthesized by the sol-gel technique and the effects of particle characteristics on the binding capacity of siRNA were investigated. In order to enhance the gene loading efficiency, the nanoparticles were functionalized with arginine and the morphological and their structural characteristics were analyzed. The addition of arginine did not significantly change the particle sizes; however, it provided a significantly increased binding of siRNA for all types of CaP nanoparticles, as revealed by spectrophotometric measurements analysis. Arginine functionalized HA-n nanoparticles showed the best binding behavior with siRNA among the other nanoparticles due to its high, positive zeta potential (+18.8 mV) and high surface area of Ca ++ rich “c” plane. MTT cytotoxicity assays demonstrated that all the nanoparticles tested herein were biocompatible. Our results suggest that high siRNA entrapment in each of the three modified non-toxic CaP nanoparticles make them promising candidates as a non-viral vector for delivering therapeutic siRNA molecules to treat cancer. [ABSTRACT FROM AUTHOR]

The purpose of our study was to develop and evaluate a novel integrin αvβ3-specific delivery carrier for transfection of siRNA in malignant tumors. We adopted arginine-glycine-aspartate (RGD) motif as a tissue target for specific recognition of integrin ανβ3. A chimaeric peptide was synthesized by adding nonamer arginine residues (9-arginine [9R]) at the carboxy terminus of cyclic-RGD dimer, designated as c(RGD)2-9R, to enable small interfering RNA (siRNA) binding. To test the applicability of the delivery carrier in vivo, c(RGD)2-9R was labeled with radionuclide of technetium-99m. Biodistribution and γ-camera imaging studies were performed in HepG2 xenograft-bearing nude mice. As results, an optimal 10:1 molar ratio of 99mTc-c(RGD)2-9R to siRNA was indicated by the electrophoresis on agarose gels. 99mTc-c(RGD)2-9R/siRNA remained stable under a set of conditions in vitro. For in vivo study, tumor radioactivity uptake of 99mTc-c(RGD)2-9R/siRNA in nude mice bearing HepG2 xenografts was significantly higher than that of control probe ( P < .05). The xenografts were clearly visualized at 4 hours till 6 hours noninvasively after intravenous injection of 99mTc-c(RGD)2-9R/siRNA, while the xenografts were not visualized at any time after injection of control probe. It was concluded that c(RGD)2-9R could be an effective siRNA delivery carrier. Technetium-99m radiolabeled-delivery carrier represents a potential imaging strategy for RNAi-based therapy. [ABSTRACT FROM AUTHOR]

The discovery of proprotein convertase subtilisin-kexin type 9 (PCSK9), a serine protease which binds to the low-density lipoprotein (LDL) receptors and targets the receptors for lysosomal degradation, offered an additional route through which plasma LDL-cholesterol (LDL-C) levels can be controlled. Initially, the therapeutic approaches to reduce circulating levels of PCSK9 were focused on the use of monoclonal antibodies. To that effect, evolocumab and alirocumab, two human monoclonal antibodies directed against PCSK9, given on a background of statin therapy, have been shown to markedly decrease LDL-C levels and significantly reduce cardiovascular risk. The small interfering RNA (siRNA) molecules have been used recently to target the hepatic production of PCSK9. siRNA interferes with the expression of specific genes with complementary nucleotide sequences by affecting the degradation of mRNA post-transcription, thus preventing translation. Inclisiran is a long-acting, synthetic siRNA directed against PCSK9 and it has been shown to significantly decrease hepatic production of PCSK9 and cause a marked reduction in LDL-C levels. This review aims to present and discuss the current clinical and scientific evidence pertaining to inclisiran, which is a new promising agent in the management of hypercholesterolemia.

RNA Interference (RNAi) has brought revolutionary transformations in cancer management in the past two decades. RNAi-based therapeutics including siRNA and shRNA have immense scope to silence the expression of mutant cancer genes specifically in a therapeutic context. Although tremendous progress has been made to establish catalytic RNA as a new class of biologics for cancer management, a lot of extracellular and intracellular barriers still pose a long-lasting challenge on the way to clinical approval. A series of chemically suitable, safe and effective viral and non-viral carriers have emerged to overcome physiological barriers and ensure targeted delivery of RNAi. The newly invented carriers, delivery techniques and gene editing technology made current treatment protocols stronger to fight cancer. This review has provided a platform about the chronicle of siRNA development and challenges of RNAi therapeutics for laboratory to bedside translation focusing on recent advancement in siRNA delivery vehicles with their limitations. Furthermore, an overview of several animal model studies of siRNA- or shRNA-based cancer gene therapy over the past 15 years has been presented, highlighting the roles of genes in multiple cancers, pharmacokinetic parameters and critical evaluation. The review concludes with a future direction for the development of catalytic RNA vehicles and design strategies to make RNAi-based cancer gene therapy more promising to surmount cancer gene delivery challenges.

The present study has explored the role of calcitonin gene-related peptide (CGRP) and its receptor in inflammatory pain modulation in arcuate nucleus of hypothalamus (ARC). Our study demonstrated that intra-ARC injection of CGRP induced antinociceptive effects to naïve rats and rats with inflammatory pain, the effect could be inhibited by the selective CGRP receptor antagonist CGRP8–37. Interestingly, the CGRP-induced antinociception effect was decreased in rats with inflammatory pain compared to naïve rats. Similarly, we found that calcitonin receptor like receptor (CLR), a main component of CGRP receptor, had a low decreased expression levels in the ARC regions of rats with inflammatory pain. The CGRP-induced antinociceptive effect was significantly impaired after reducing CLR expression by intra-ARC administration of CLR targeted siRNA. These findings demonstrated that CGRP might play a crucial role in nociceptive modulation in the ARC during inflammatory pain, which was mediated by CGRP receptor in the ARC. This study shed light upon CGRP and its receptor interaction might be valuable strategies for the alleviation of inflammatory pain. [ABSTRACT FROM AUTHOR]

• Ion-pair reagent-free reversed phase (RP) liquid chromatography method for siRNA separation. • Polybutylene terephthalate (PBT) RP stationary phase with mixed-mode interactions. • Impurity characterization of siRNA oligonucleotides via LC-QTOF-MS. • Retention pattern profiling indicates specific retention increments of nucleotides. Nowadays, ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the dominating generic method for the analysis of nucleic acid related compounds, such as antisense-oligonucleotides (ASO), small-interfering ribonucleic acid (siRNA) or other DNA or RNA type molecules and their conjugates. Despite of its effective performance, the usage of a high concentration of ion-pairing reagent in the eluent in IP-RPLC is unfavorable for the hyphenation with mass spectrometry (MS) which is required for a detailed structural characterization of the analytes and their structurally related impurities. In this work, we tested a polybutylene terephthalate (PBT)-bonded silica-based stationary phase for the separation of generically synthesized Patisiran as siRNA (antisense and sense single strands as well as their annealed double strand) giving some unexpected selectivity without any presence of ion-pairing reagents. Important chromatographic conditions affecting the separation have been investigated and evaluated. Furthermore, MS and tandem MS (MS/MS) characterization was possible without contamination of the MS system with ion-pair agent and related problems. [ABSTRACT FROM AUTHOR]

Background: Hepatitis C virus (HCV) is a major public health problem with 170 million chronically infected people throughout the world. Currently, the only treatment available consists of a combination of pegylated interferon (INF-a) and ribavirin, but only half of the patients treated show a sufficient antiviral response. Thus there is a great need for the development of new treatments for HCV infections. RNA interference (RNAi) represents a new promising approach to develop effective antiviral drugs and has been extremely effective against HCV gene expression in short-term cell culture. Our aim was to determine the effect of RNAi directed against the NS5B-HCV region on HCV expression in a human hepatoma cell line that expresses HCV-subgenomic replicon (Huh7 HCV replicon cells). Methods: We transfected Huh7 HCV replicon cells with different concentrations of RNAi (100-200 nM) targeting the NS5B region of the viral genome. 2-6 days post-transfection HCV-RNA was quantified by semiquantitative and real-time RT-PCR, and HCV NS5B protein levels were assayed by western blot. Cell viability was also quantified by MTT assay. Results: Our results indicate that the NS5B-siRNAs used in this study can specifically inhibit HCV-RNA replication and protein expression (more than 90%) compared to control cells. Conclusions: Synthetic siRNA against NS5B-HCV inhibited HCV replication and viral proteins levels and thereby becomes a powerful strategy to combat hepatitis C virus.

(1) Background: The great potential of RNA interference (RNAi)-based gene therapy is premised on the effective delivery of small interfering RNAs (siRNAs) to target tissues and cells. Hence, we aimed at developing and examining a novel integrin ανβ3-specific delivery carrier for targeted transfection of siRNA to malignant tumor cells; (2) Methods: Arginine-glycine-aspartate motif (RGD) was adopted as a tissue target for specific recognition of integrin ανβ3. To enable siRNA binding, a chimeric peptide was synthesized by adding nonamer arginine residues (9R) at the carboxy terminus of cyclic-RGD dimer, designated as c(RGD)2-9R. The efficiency of 9R peptide transferring siRNA was biologically evaluated in vitro by flow cytometry, confocal microscopy, and Western blot; (3) Results: An optimal 10:1 molar ratio of c(RGD)2-9R to siRNA was confirmed by the electrophoresis on agarose gels. Both the flow cytometry and confocal microscopy results testified that transfection of c(RGD)2-9R as an siRNA delivery carrier was obviously higher than the naked-siRNA group. The results of Western blot demonstrated that these 9R peptides were able to transduce siRNA to HepG2 cells in vitro, resulting in efficient gene silencing; and (4) Conclusion: The chimeric peptide of c(RGD)2-9R can be developed as an effective siRNA delivery carrier and shows potential as a new strategy for RNAi-based gene therapy. [ABSTRACT FROM AUTHOR]