Growth-restricting effects of siRNA transfections: a largely deterministic combination of off-target binding and hybridization-independent competition.

Perturbation of gene expression by means of synthetic small interfering RNAs (siRNAs) is a powerful way to uncover gene function. However, siRNA technology suffers from sequence-specific off-target effects and from limitations in knock-down efficiency. In this study, we assess a further problem: unintended effects of siRNA transfections on cellular fitness/proliferation. We show that the nucleotide compositions of siRNAs at specific positions have reproducible growth-restricting effects on mammalian cells in culture. This is likely distinct from hybridization-dependent off-target effects, since each nucleotide residue is seen to be acting independently and additively. The effect is robust and reproducible across different siRNA libraries and also across various cell lines, including human and mouse cells. Analyzing the growth inhibition patterns in correlation to the nucleotide sequence of the siRNAs allowed us to build a predictor that can estimate growth-restricting effects for any arbitrary siRNA sequence. Competition experiments with co-transfected siRNAs further suggest that the growth-restricting effects might be linked to an oversaturation of the cellular miRNA machinery, thus disrupting endogenous miRNA functions at large. We caution that competition between siRNA molecules could complicate the interpretation of double-knockdown or epistasis experiments, and potential interactions with endogenous miRNAs can be a factor when assaying cell growth or viability phenotypes.

Specific inhibition of diverse pathogens in human cells by synthetic microRNA-like oligonucleotides inferred from RNAi screens.

Systematic genetic perturbation screening in human cells remains technically challenging. Typically, large libraries of chemically synthesized siRNA oligonucleotides are used, each designed to degrade a specific cellular mRNA via the RNA interference (RNAi) mechanism. Here, we report on data from three genome-wide siRNA screens, conducted to uncover host factors required for infection of human cells by two bacterial and one viral pathogen. We find that the majority of phenotypic effects of siRNAs are unrelated to the intended "on-target" mechanism, defined by full complementarity of the 21-nt siRNA sequence to a target mRNA. Instead, phenotypes are largely dictated by "off-target" effects resulting from partial complementarity of siRNAs to multiple mRNAs via the "seed" region (i.e., nucleotides 2-8), reminiscent of the way specificity is determined for endogenous microRNAs. Quantitative analysis enabled the prediction of seeds that strongly and specifically block infection, independent of the intended on-target effect. This prediction was confirmed experimentally by designing oligos that do not have any on-target sequence match at all, yet can strongly reproduce the predicted phenotypes. Our results suggest that published RNAi screens have primarily, and unintentionally, screened the sequence space of microRNA seeds instead of the intended on-target space of protein-coding genes. This helps to explain why previously published RNAi screens have exhibited relatively little overlap. Our analysis suggests a possible way of identifying "seed reagents" for controlling phenotypes of interest and establishes a general strategy for extracting valuable untapped information from past and future RNAi screens.

Near surface swimming of Salmonella Typhimurium explains target-site selection and cooperative invasion.

Targeting of permissive entry sites is crucial for bacterial infection. The targeting mechanisms are incompletely understood. We have analyzed target-site selection by S. Typhimurium. This enteropathogenic bacterium employs adhesins (e.g. fim) and the type III secretion system 1 (TTSS-1) for host cell binding, the triggering of ruffles and invasion. Typically, S. Typhimurium invasion is focused on a subset of cells and multiple bacteria invade via the same ruffle. It has remained unclear how this is achieved. We have studied target-site selection in tissue culture by time lapse microscopy, movement pattern analysis and modeling. Flagellar motility (but not chemotaxis) was required for reaching the host cell surface in vitro. Subsequently, physical forces trapped the pathogen for ∼1.5-3 s in "near surface swimming". This increased the local pathogen density and facilitated "scanning" of the host surface topology. We observed transient TTSS-1 and fim-independent "stopping" and irreversible TTSS-1-mediated docking, in particular at sites of prominent topology, i.e. the base of rounded-up cells and membrane ruffles. Our data indicate that target site selection and the cooperative infection of membrane ruffles are attributable to near surface swimming. This mechanism might be of general importance for understanding infection by flagellated bacteria.

Cost-effectiveness of pembrolizumab as an adjuvant treatment for patients with resected stage IIB or IIC melanoma in Switzerland.

AIM: To assess the cost-effectiveness of adjuvant pembrolizumab versus observation for patients with resected stage IIB/IIC melanoma from a third-party payers' perspective in Switzerland over a lifetime horizon. MATERIALS AND METHODS: A Markov state transition model with four health states (recurrence-free [RF], locoregional recurrence, distant metastases [DM], and death) was developed to determine the cost-effectiveness of pembrolizumab versus observation as an adjuvant treatment in patients with stage IIB/IIC melanoma who have undergone complete resection. The model utilized data from the KEYNOTE-716 randomized controlled trial (ClinicalTrials.gov, NCT03553836). The incremental cost-effectiveness ratio (ICER) (Swiss Franc [CHF] per life year or quality-adjusted life years [QALYs] gained) was calculated. A probabilistic sensitivity analysis and deterministic sensitivity analysis were conducted to assess the robustness of the base case results. RESULTS: Model results demonstrated that pembrolizumab is highly cost-effective as an adjuvant treatment for resected stage IIB/IIC melanoma versus observation in Switzerland. Base case results showed an ICER of CHF 27,424/QALY (EUR 27,342/QALY; exchange rate: 1 CHF = 0.997 EUR) for pembrolizumab versus observation. Results were most sensitive to changes to transition probabilities from the RF state. Most sensitivity and scenario analyses resulted in ICERs below the willingness-to-pay threshold (WTP) of CHF 100,000. At this WTP, pembrolizumab had a 78.9% probability of being cost-effective versus observation. LIMITATIONS: Due to a limited follow-up period in the KEYNOTE-716 trial, data from other clinical trials in the advanced melanoma setting were synthesized in a network meta-analysis and used to inform transition probabilities from DM to death in the cost-effectiveness model, to overcome the absence of these data from the trial. CONCLUSION: The model demonstrated that pembrolizumab is highly cost-effective versus observation in patients with resected stage IIB/IIC melanoma in Switzerland. The ICER was below the WTP threshold of CHF 100,000, commonly used for cost-effectiveness models in Switzerland.

A Genome-Wide siRNA Screen Implicates Spire1/2 in SipA-Driven Salmonella Typhimurium Host Cell Invasion.

Salmonella Typhimurium (S. Tm) is a leading cause of diarrhea. The disease is triggered by pathogen invasion into the gut epithelium. Invasion is attributed to the SPI-1 type 3 secretion system (T1). T1 injects effector proteins into epithelial cells and thereby elicits rearrangements of the host cellular actin cytoskeleton and pathogen invasion. The T1 effector proteins SopE, SopB, SopE2 and SipA are contributing to this. However, the host cell factors contributing to invasion are still not completely understood. To address this question comprehensively, we used Hela tissue culture cells, a genome-wide siRNA library, a modified gentamicin protection assay and S. TmSipA, a sopBsopE2sopE mutant which strongly relies on the T1 effector protein SipA to invade host cells. We found that S. TmSipA invasion does not elicit membrane ruffles, nor promote the entry of non-invasive bacteria "in trans". However, SipA-mediated infection involved the SPIRE family of actin nucleators, besides well-established host cell factors (WRC, ARP2/3, RhoGTPases, COPI). Stage-specific follow-up assays and knockout fibroblasts indicated that SPIRE1 and SPIRE2 are involved in different steps of the S. Tm infection process. Whereas SPIRE1 interferes with bacterial binding, SPIRE2 influences intracellular replication of S. Tm. Hence, these two proteins might fulfill non-redundant functions in the pathogen-host interaction. The lack of co-localization hints to a short, direct interaction between S. Tm and SPIRE proteins or to an indirect effect.

gespeR: a statistical model for deconvoluting off-target-confounded RNA interference screens.

Small interfering RNAs (siRNAs) exhibit strong off-target effects, which confound the gene-level interpretation of RNA interference screens and thus limit their utility for functional genomics studies. Here, we present gespeR, a statistical model for reconstructing individual, gene-specific phenotypes. Using 115,878 siRNAs, single and pooled, from three companies in three pathogen infection screens, we demonstrate that deconvolution of image-based phenotypes substantially improves the reproducibility between independent siRNA sets targeting the same genes. Genes selected and prioritized by gespeR are validated and shown to constitute biologically relevant components of pathogen entry mechanisms and TGF-ß signaling. gespeR is available as a Bioconductor R-package.

BAP31 and BiP are essential for dislocation of SV40 from the endoplasmic reticulum to the cytosol.

How non-enveloped viruses overcome host cell membranes is poorly understood. Here, we show that after endocytosis and transport to the endoplasmic reticulum (ER), but before crossing the ER membrane to the cytosol, incoming simian virus 40 particles are structurally remodelled leading to exposure of the amino-terminal sequence of the minor viral protein VP2. These hydrophobic sequences anchor the virus to membranes. A negatively charged residue, Glu 17, in the α-helical, membrane-embedded peptide is essential for infection, most likely by introducing an 'irregularity' recognized by the ER-associated degradation (ERAD) system for membrane proteins. Using a siRNA-mediated screen, the lumenal chaperone BiP and the ER-membrane protein BAP31 (both involved in ERAD) were identified as being essential for infection. They co-localized with the virus in discrete foci and promoted its ER-to-cytosol dislocation. Virus-like particles devoid of VP2 failed to cross the membrane. The results demonstrated that ERAD-factors assist virus transport across the ER membrane.