Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.

CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in), by utilizing specially designed 3'-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3'-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.

A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9.

The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability should be taken into account.

Investigating the Impact of Selective Modulators on the Renin-Angiotensin-Aldosterone System: Unraveling Their Off-Target Perturbations of Transmembrane Ionic Currents.

The renin-angiotensin-aldosterone system (RAAS) plays a crucial role in maintaining various physiological processes in the body, including blood pressure regulation, electrolyte balance, and overall cardiovascular health. However, any compounds or drugs known to perturb the RAAS might have an additional impact on transmembrane ionic currents. In this retrospective review article, we aimed to present a selection of chemical compounds or medications that have long been recognized as interfering with the RAAS. It is noteworthy that these substances may also exhibit regulatory effects in different types of ionic currents. Apocynin, known to attenuate the angiotensin II-induced activation of epithelial Na+ channels, was shown to stimulate peak and late components of voltage-gated Na+ current (INa). Esaxerenone, an antagonist of the mineralocorticoid receptor, can exert an inhibitory effect on peak and late INa directly. Dexamethasone, a synthetic glucocorticoid, can directly enhance the open probability of large-conductance Ca2+-activated K+ channels. Sparsentan, a dual-acting antagonist of the angiotensin II receptor and endothelin type A receptors, was found to suppress the amplitude of peak and late INa effectively. However, telmisartan, a blocker of the angiotensin II receptor, was effective in stimulating the peak and late INa along with a slowing of the inactivation time course of the current. However, telmisartan's presence can also suppress the erg-mediated K+ current. Moreover, tolvaptan, recognized as an aquaretic agent that can block the vasopressin receptor, was noted to suppress the amplitude of the delayed-rectifier K+ current and the M-type K+ current directly. The above results indicate that these substances not only have an interference effect on the RAAS but also exert regulatory effects on different types of ionic currents. Therefore, to determine their mechanisms of action, it is necessary to gain a deeper understanding.

Off-Target Effects of P2Y12 Receptor Inhibitors: Focus on Early Myocardial Fibrosis Modulation.

Several studies have demonstrated that, beyond their antithrombotic effects, P2Y12 receptor inhibitors may provide additional off-target effects through different mechanisms. These effects range from the preservation of endothelial barrier function to the modulation of inflammation or stabilization of atherosclerotic plaques, with an impact on different cell types, including endothelial and immune cells. Many P2Y12 inhibitors have been developed, from ticlopidine, the first thienopyridine, to the more potent non-thienopyridine derivatives such as ticagrelor which may promote cardioprotective effects following myocardial infarction (MI) by inhibiting adenosine reuptake through sodium-independent equilibrative nucleoside transporter 1 (ENT1). Adenosine may affect different molecular pathways involved in cardiac fibrosis, such as the Wnt (wingless-type)/beta (ß)-catenin signaling. An early pro-fibrotic response of the epicardium and activation of cardiac fibroblasts with the involvement of Wnt1 (wingless-type family member 1)/ß-catenin, are critically required for preserving cardiac function after acute ischemic cardiac injury. This review discusses molecular signaling pathways involved in cardiac fibrosis post MI, focusing on the Wnt/ß-catenin pathway, and the off-target effect of P2Y12 receptor inhibition. A potential role of ticagrelor was speculated in the early modulation of cardiac fibrosis, thanks to its off-target effect.

Antiviral Efficacy of RNase H-Dependent Gapmer Antisense Oligonucleotides against Japanese Encephalitis Virus.

RNase H-dependent gapmer antisense oligonucleotides (ASOs) are a promising therapeutic approach via sequence-specific binding to and degrading target RNAs. However, the efficacy and mechanism of antiviral gapmer ASOs have remained unclear. Here, we investigated the inhibitory effects of gapmer ASOs containing locked nucleic acids (LNA gapmers) on proliferating a mosquito-borne flavivirus, Japanese encephalitis virus (JEV), with high mortality. We designed several LNA gapmers targeting the 3' untranslated region of JEV genomic RNAs. In vitro screening by plaque assay using Vero cells revealed that LNA gapmers targeting a stem-loop region effectively inhibit JEV proliferation. Cell-based and RNA cleavage assays using mismatched LNA gapmers exhibited an underlying mechanism where the inhibition of viral production results from JEV RNA degradation by LNA gapmers in a sequence- and modification-dependent manner. Encouragingly, LNA gapmers potently inhibited the proliferation of five JEV strains of predominant genotypes I and III in human neuroblastoma cells without apparent cytotoxicity. Database searching showed a low possibility of off-target binding of our LNA gapmers to human RNAs. The target viral RNA sequence conservation observed here highlighted their broad-spectrum antiviral potential against different JEV genotypes/strains. This work will facilitate the development of an antiviral LNA gapmer therapy for JEV and other flavivirus infections.

Genome-wide analyses of PAM-relaxed Cas9 genome editors reveal substantial off-target effects by ABE8e in rice.

PAM-relaxed Cas9 nucleases, cytosine base editors and adenine base editors are promising tools for precise genome editing in plants. However, their genome-wide off-target effects are largely unexplored. Here, we conduct whole-genome sequencing (WGS) analyses of transgenic plants edited by xCas9, Cas9-NGv1, Cas9-NG, SpRY, nCas9-NG-PmCDA1, nSpRY-PmCDA1 and nSpRY-ABE8e in rice. Our results reveal that Cas9 nuclease and base editors, when coupled with the same guide RNA (gRNA), prefer distinct gRNA-dependent off-target sites. De novo generated gRNAs by SpRY editors lead to additional, but insubstantial, off-target mutations. Strikingly, ABE8e results in ~500 genome-wide A-to-G off-target mutations at TA motif sites per transgenic plant. ABE8e's preference for the TA motif is also observed at the target sites. Finally, we investigate the timeline and mechanism of somaclonal variation due to tissue culture, which chiefly contributes to the background mutations. This study provides a comprehensive understanding on the scale and mechanisms of off-target and background mutations occurring during PAM-relaxed genome editing in plants.

A 2'-modified uridine analog, 2'-O-(methylthiomethoxy)methyl uridine, for siRNA applications.

The medicinal applications of siRNAs have been intensively examined but are still hindered by their low molecular stability under biological conditions and off-target effects, etc. The introduction of chemical modifications to the nucleoside is a promising strategy for solving these limitations. Herein, we describe the development of a new uridine analog, U*, that has a (methylthiomethoxy)methoxy group at the 2' position. The phosphoramidite reagent corresponding to U* was easily synthesized and the RNA oligonucleotides containing U* were stably prepared using a standard protocol for oligonucleotide synthesis. The introduction of U* into the siRNA resulted in positive or negative effects on the targeted gene silencing in a position-dependent manner, and the positive effects were attributed to the improved stability under biological conditions. The thermodynamic analysis of the U*-modified RNAs revealed a slight destabilization of the dsRNA, based depending on which U was strategically utilized to restrain the off-target effects of the siRNA. This study describes a rare example of nucleoside analogs with a large substitution at the 2'-position in the context of an siRNA application and is informative for the development of other analogs to further improve the molecular properties of siRNAs for medicinal applications.

Targeting cancer epigenetics with CRISPR-dCAS9: Principles and prospects.

Cancer therapeutics is an ever-evolving field due to incessant demands for effective and precise treatment options. Over the last few decades, cancer treatment strategies have shifted somewhat from surgery to targeted precision medicine. CRISPR-dCas9 is an emerging version of precision cancer therapy that has been adapted from the prokaryotic CRISPR-Cas system. Once ligated to epigenetic effectors (EE), CRISPR-dCas9 can function as an epigenetic editing tool and CRISPR-dCas9-EE complexes could be exploited to alter cancerous epigenetic features associated with different cancer hallmarks. In this article, we discuss the rationale of epigenetic editing as a therapeutic strategy against cancer. We also outline how sgRNA-dCas9 was derived from the CRISPR-Cas system. In addition, the current status of sgRNA-dCas9 use (in vivo and in vitro) in cancer is updated with a molecular illustration of CRISPR-dCas9-mediated epigenetic and transcriptional modulation. As sgRNA-dCas9 is still at the developmental phase, challenges are inherent to its use. We evaluate major challenges in targeting cancer with sgRNA-dCas9 such as off-target effects, lack of sgRNA designing rubrics, target site selection dilemmas and deficient sgRNA-dCas9 delivery systems. Finally, we appraise the sgRNA-dCas9 as a prospective cancer therapeutic by summarizing ongoing improvements of sgRNA-dCas9 methodology.

The current landscape of microRNAs (miRNAs) in bacterial pneumonia: opportunities and challenges.

MicroRNAs (miRNAs), which were initially discovered in Caenorhabditis elegans, can regulate gene expression by recognizing cognate sequences and interfering with the transcriptional or translational machinery. The application of bioinformatics tools for structural analysis and target prediction has largely driven the investigation of certain miRNAs. Notably, it has been found that certain miRNAs which are widely involved in the inflammatory response and immune regulation are closely associated with the occurrence, development, and outcome of bacterial pneumonia. It has been shown that certain miRNA techniques can be used to identify related targets and explore associated signal transduction pathways. This enhances the understanding of bacterial pneumonia, notably for "refractory" or drug-resistant bacterial pneumonia. Although these miRNA-based methods may provide a basis for the clinical diagnosis and treatment of this disease, they still face various challenges, such as low sensitivity, poor specificity, low silencing efficiency, off-target effects, and toxic reactions. The opportunities and challenges of these methods have been completely reviewed, notably in bacterial pneumonia. With the continuous improvement of the current technology, the miRNA-based methods may surmount the aforementioned limitations, providing promising support for the clinical diagnosis and treatment of "refractory" or drug-resistant bacterial pneumonia.

Improving Stability and Specificity of CRISPR/Cas9 System by Selective Modification of Guide RNAs with 2'-fluoro and Locked Nucleic Acid Nucleotides.

The genome editing approach using the components of the CRISPR/Cas system has found wide application in molecular biology, fundamental medicine and genetic engineering. A promising method is to increase the efficacy and specificity of CRISPR/Cas-based genome editing systems by modifying their components. Here, we designed and chemically synthesized guide RNAs (crRNA, tracrRNA and sgRNA) containing modified nucleotides (2'-O-methyl, 2'-fluoro, LNA-locked nucleic acid) or deoxyribonucleotides in certain positions. We compared their resistance to nuclease digestion and examined the DNA cleavage efficacy of the CRISPR/Cas9 system guided by these modified guide RNAs. The replacement of ribonucleotides with 2'-fluoro modified or LNA nucleotides increased the lifetime of the crRNAs, while other types of modification did not change their nuclease resistance. Modification of crRNA or tracrRNA preserved the efficacy of the CRISPR/Cas9 system. Otherwise, the CRISPR/Cas9 systems with modified sgRNA showed a remarkable loss of DNA cleavage efficacy. The kinetic constant of DNA cleavage was higher for the system with 2'-fluoro modified crRNA. The 2'-modification of crRNA also decreased the off-target effect upon in vitro dsDNA cleavage.

The role of cell signaling in the crosstalk between autophagy and apoptosis in the regulation of tumor cell survival in response to sorafenib and neratinib.

The molecular mechanisms by which tumor cells survive or die following therapeutic interventions are complex. There are three broadly defined categories of cell death processes: apoptosis (Type I), autophagic cell death (Type II), and necrosis (Type III). In hematopoietic tumor cells, the majority of toxic stimuli cause these cells to undergo a death process called apoptosis; apoptosis specifically involves the cleavage of DNA into large defined pieces and their subsequent localization in vesicles. Thus, 'pure' apoptosis largely lacks inflammatory potential. In carcinomas, however, the mechanisms by which tumor cells ultimately die are considerably more complex. Although the machinery of apoptosis is engaged by toxic stimuli, other processes such as autophagy ("self-eating") and replicative cell death can lead to observations that do not simplistically correspond to any of the individual Type I-III formalized death categories. The 'hybrid' forms of cell death observed in carcinoma cells result in cellular materials being released into the extracellular space without packaging, which promotes inflammation, potentially leading to the accelerated re-growth of surviving tumor cells by macrophages. Drugs as single agents or in combinations can simultaneously initiate signaling via both apoptotic and autophagic pathways. Based on the tumor type and its oncogene drivers, as well as the drug(s) being used and the duration and intensity of the autophagosome signal, apoptosis and autophagy have the potential to act in concert to kill or alternatively that the actions of either pathway can act to suppress signaling by the other pathway. And, there also is evidence that autophagic flux, by causing lysosomal protease activation, with their subsequent release into the cytosol, can directly mediate killing. This review will discuss the interactive biology between apoptosis and autophagy in carcinoma cells. Finally, the molecular actions of the FDA-approved drugs neratinib and sorafenib, and how they enhance both apoptotic and toxic autophagic processes, alone or in combination with other agents, is discussed in a bench-to-bedside manner.

Off-target effects of RNAi correlate with the mismatch rate between dsRNA and non-target mRNA.

RNAi is a potent technique for the knockdown of target genes. However, its potential off-target effects limit the widespread applications in both reverse genetic analysis and genetic manipulation. Previous efforts have uncovered rules underlying specificity of siRNA-based silencing, which has broad applications in humans, but the basis for specificity of dsRNAs, which are better suited for use as insecticides, is poorly understood. Here, we investigated the rules governing dsRNA specificity. Mutational analyses showed that dsRNAs with >80% sequence identity with target genes triggered RNAi efficiently. dsRNAs with ≥16 bp segments of perfectly matched sequence or >26 bp segments of almost perfectly matched sequence with one or two mismatches scarcely distributed (single mismatches inserted between ≥5 bp matching segments or mismatched couplets inserted between ≥8 bp matching segments) also able to trigger RNAi. Using these parameters to predict off-target risk, dsRNAs can be designed to optimize specificity and efficiency, paving the way to the widespread, rational application of RNAi in pest control.

High Concentration of an ISS-N1-Targeting Antisense Oligonucleotide Causes Massive Perturbation of the Transcriptome.

Intronic splicing silencer N1 (ISS-N1) located within Survival Motor Neuron 2 (SMN2) intron 7 is the target of a therapeutic antisense oligonucleotide (ASO), nusinersen (Spinraza), which is currently being used for the treatment of spinal muscular atrophy (SMA), a leading genetic disease associated with infant mortality. The discovery of ISS-N1 as a promising therapeutic target was enabled in part by Anti-N1, a 20-mer ASO that restored SMN2 exon 7 inclusion by annealing to ISS-N1. Here, we analyzed the transcriptome of SMA patient cells treated with 100 nM of Anti-N1 for 30 h. Such concentrations are routinely used to demonstrate the efficacy of an ASO. While 100 nM of Anti-N1 substantially stimulated SMN2 exon 7 inclusion, it also caused massive perturbations in the transcriptome and triggered widespread aberrant splicing, affecting expression of essential genes associated with multiple cellular processes such as transcription, splicing, translation, cell signaling, cell cycle, macromolecular trafficking, cytoskeletal dynamics, and innate immunity. We validated our findings with quantitative and semiquantitative PCR of 39 candidate genes associated with diverse pathways. We also showed a substantial reduction in off-target effects with shorter ISS-N1-targeting ASOs. Our findings are significant for implementing better ASO design and dosing regimens of ASO-based drugs.

N6-modified cAMP derivatives that activate protein kinase A also act as full agonists of murine HCN2 channels.

cAMP acts as a second messenger in many cellular processes. Three protein types mainly mediate cAMP-induced effects: PKA, exchange protein directly activated by cAMP (Epac), and cyclic nucleotide-modulated channels (cyclic nucleotide-gated or hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels). Discrimination among these cAMP signaling pathways requires specific targeting of only one protein. Previously, cAMP modifications at position N6 of the adenine ring (PKA) and position 2'-OH of the ribose (Epac) have been used to produce target-selective compounds. However, cyclic nucleotide-modulated ion channels were usually outside of the scope of these previous studies. These channels are widely distributed, so possible channel cross-activation by PKA- or Epac-selective agonists warrants serious consideration. Here we demonstrate the agonistic effects of three PKA-selective cAMP derivatives, N6-phenyladenosine-3',5'-cyclic monophosphate (N6-Phe-cAMP), N6-benzyladenosine-3',5'-cyclic monophosphate (N6-Bn-cAMP), and N6-benzoyl-adenosine-3',5'-cyclic monophosphate (N6-Bnz-cAMP), on murine HCN2 pacemaker channels. Electrophysiological characterization in Xenopus oocytes revealed that these derivatives differ in apparent affinities depending on the modification type but that their efficacy and effects on HCN2 activation kinetics are similar to those of cAMP. Docking experiments suggested a pivotal role of Arg-635 at the entrance of the binding pocket in HCN2, either causing stabilizing cation-π interactions with the aromatic ring in N6-Phe-cAMP or N6-Bn-cAMP or a steric clash with the aromatic ring in N6-Bnz-cAMP. A reduced apparent affinity of N6-Phe-cAMP toward the variants R635A and R635E strengthened that notion. We conclude that some PKA activators also effectively activate HCN2 channels. Hence, when studying PKA-mediated cAMP signaling with cAMP derivatives in a native environment, activation of HCN channels should be considered.

Hybrid phenotype mining method for investigating off-target protein and underlying side effects of anti-tumor immunotherapy.

BACKGROUND: It is of utmost importance to investigate novel therapies for cancer, as it is a major cause of death. In recent years, immunotherapies, especially those against immune checkpoints, have been developed and brought significant improvement in cancer management. However, on the other hand, immune checkpoints blockade (ICB) by monoclonal antiboties may cause common and severe adverse reactions (ADRs), the cause of which remains largely undetermined. We hypothesize that ICB-agents may induce adverse reactions through off-target protein interactions, similar to the ADR-causing off-target effects of small molecules. In this study, we propose a hybrid phenotype mining approach which integrates molecular level information and provides new mechanistic insights for ICB-associated ADRs. METHODS: We trained a conditional random fields model on the TAC 2017 benchmark training data, then used it to extract all drug-centric phenotypes for the five anti-PD-1/PD-L1 drugs from the drug labels of the DailyMed database. Proteins with structure similar to the drugs were obtained by using BlastP, and the gene targets of drugs were obtained from the STRING database. The target-centric phenotypes were extracted from the human phenotype ontology database. Finally, a screening module was designed to investigate off-target proteins, by making use of gene ontology analysis and pathway analysis. RESULTS: Eventually, through the cross-analysis of the drug and target gene phenotypes, the off-target effect caused by the mutation of gene BTK was found, and the candidate side-effect off-target site was analyzed. CONCLUSIONS: This research provided a hybrid method of biomedical natural language processing and bioinformatics to investigate the off-target-based mechanism of ICB treatment. The method can also be applied for the investigation of ADRs related to other large molecule drugs.

Investigation of Strand-Selective Interaction of SNA-Modified siRNA with AGO2-MID.

Small interfering RNA (siRNA) has been recognized as a powerful gene-silencing tool. For therapeutic application, chemical modification is often required to improve the properties of siRNA, including its nuclease resistance, activity, off-target effects, and tissue distribution. Careful siRNA guide strand selection in the RNA-induced silencing complex (RISC) is important to increase the RNA interference (RNAi) activity as well as to reduce off-target effects. The passenger strand-mediated off-target activity was previously reduced and on-target activity was enhanced by substitution with acyclic artificial nucleic acid, namely serinol nucleic acid (SNA). In the present study, the reduction of off-target activity caused by the passenger strand was investigated by modifying siRNAs with SNA. The interactions of SNA-substituted mononucleotides, dinucleotides, and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO)-labeled double-stranded RNA (dsRNA) with the MID domain of the Argonaute 2 (AGO2) protein, which plays a pivotal role in strand selection by accommodation of the 5'-terminus of siRNA, were comprehensively analyzed. The obtained nuclear magnetic resonance (NMR) data revealed that AGO2-MID selectively bound to the guide strand of siRNA due to the inhibitory effect of the SNA backbone located at the 5' end of the passenger strand.

Targeted Mutagenesis of the Rice FW 2.2-Like Gene Family Using the CRISPR/Cas9 System Reveals OsFWL4 as a Regulator of Tiller Number and Plant Yield in Rice.

The FW2.2-like (FWL) genes encode cysteine-rich proteins with a placenta-specific 8 domain. They play roles in cell division and organ size control, response to rhizobium infection, and metal ion homeostasis in plants. Here, we target eight rice FWL genes using the CRISPR/Cas9 system delivered by Agrobacterium-mediated transformation. We successfully generate transgenic T0 lines for 15 of the 16 targets. The targeted mutations are detected in the T0 lines of all 15 targets and the average mutation rate is found to be 81.6%. Transfer DNA (T-DNA) truncation is a major reason for the failure of mutagenesis in T0 plants. T-DNA segregation analysis reveals that the T-DNA inserts in transgenic plants can be easily eliminated in the T1 generation. Of the 30 putative off-target sites examined, unintended mutations are detected in 13 sites. Phenotypic analysis reveals that tiller number and plant yield of OsFWL4 gene mutants are significantly greater than those of the wild type. Flag leaves of OsFWL4 gene mutants are wider than those of the wild type. The increase in leaf width of the mutants is caused by an increase in cell number. Additionally, grain length of OsFWL1 gene mutants is higher than that of the wild type. Our results suggest that transgene-free rice plants with targeted mutations can be produced in the T1 generation using the Agrobacterium-mediated CRISPR/Cas9 system and that the OsFWL4 gene is a negative regulator of tiller number and plant yield.

Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice.

BACKGROUND: Application of the CRISPR/Cas9 system or its derived base editors enables targeted genome modification, thereby providing a programmable tool to exploit gene functions and to improve crop traits. RESULTS: We report that PmCDA1 is much more efficient than rAPOBEC1 when fused to CRISPR/Cas9 nickase for the conversion of cytosine (C) to thymine (T) in rice. Three high-fidelity SpCas9 variants, eSpCas9(1.1), SpCas9-HF2 and HypaCas9, were engineered to serve with PmCDA1 (pBEs) as C-to-T base editors. These three high-fidelity editors had distinct multiplex-genome editing efficiencies. To substantially improve their base-editing efficiencies, a tandemly arrayed tRNA-modified single guide RNA (sgRNA) architecture was applied. The efficiency of eSpCas9(1.1)-pBE was enhanced up to 25.5-fold with an acceptable off-target effect. Moreover, two- to five-fold improvement was observed for knock-out mutation frequency by these high-fidelity Cas9s under the direction of the tRNA-modified sgRNA architecture. CONCLUSIONS: We have engineered a diverse toolkit for efficient and precise genome engineering in rice, thus making genome editing for plant research and crop improvement more flexible.

Transduction with Lentiviral Vectors Altered the Expression Profile of Host MicroRNAs.

RNA interference (RNAi) is widely used in gene knockdown analysis and as a tool to screen host genes involved in viral infection. Owing to the limitations of transducing cells with synthetic small interfering RNAs (siRNAs), lentiviral short hairpin RNA (shRNA) vectors are more widely used. However, we found that stable transduction with lentiviral shRNA vectors inhibited hepatitis C virus (HCV) propagation in human hepatoma cells. We found by microRNA (miRNA) microarray analysis that this inhibition was induced by the alteration of host miRNA expression. In addition to one miRNA (miR-196b-5p) previously reported to be involved in HCV infection, other miRNAs (miR-216a-5p, -216b-5p, 217, and -30b-5p) were found to influence HCV infection in this study. Further studies suggested that this effect was independent of the transcription of shRNAs. The lentiviral vector itself and the integration site of the lentiviral vector might determine the change in miRNA expression. Moreover, the upregulation of JUN contributed to the dysregulation of miR-216a-5p, -216b-5p, and -217 in stably transduced cells. Although the changes in miRNA expression were beneficial for inhibiting HCV infection in our study, this off-target effect should be considered when transduction with lentiviral vectors is performed for other purposes, especially in therapy.IMPORTANCE We found that stable transduction with lentiviral shRNA was able to nonspecifically inhibit HCV infection by the dysregulation of host miRNAs. Previous studies showed that the overexpression of shRNAs oversaturated the host miRNA pathways to inhibit HCV infection. In contrast, the miRNA machinery was not affected in our study. Knockout studies suggested that the nonspecific effect was independent of the transcription of shRNAs. The lentiviral vector itself and the integration sites in the host genome determined the changes in miRNAs. Stable transduction with lentiviral vectors was able to increase the expression of JUN, which in turn upregulated miR-216a-5p, miR-216b-5p, and miR-217. miR-216a-5p and miR-216b-5p might inhibit HCV by suppressing the host autophagic machinery. Our study suggested a novel nonspecific effect of lentiviral vectors, and this side effect should be considered when transduction with lentiviral vectors is performed for other purposes, especially in therapy.

Doxycycline, salinomycin, monensin and ivermectin repositioned as cancer drugs.

Chemotherapy is one of the standard methods for the treatment of malignant tumors. It aims to cause lethal damage to cellular structures, mainly DNA. Noteworthy, in recent years discoveries of novel anticancer agents from well-known antibiotics have opened up new treatment pathways for several cancer diseases. The aim of this review article is to describe new applications for the following antibiotics: doxycycline (DOX), salinomycin (SAL), monensin (MON) and ivermectin (IVR) as they are known to show anti-tumor activity, but have not yet been introduced into standard oncological therapy. To date, these agents have been used for the treatment of a broad-spectrum of bacterial and parasitic infectious diseases and are widely available, which is why they were selected. The data presented here clearly show that the antibiotics mentioned above should be recognised in the near future as novel agents able to eradicate cancer cells and cancer stem cells (CSCs) across several cancer types.