We describe a statistical analysis methodology designed to minimize the impact of off-target activities upon large-scale RNA interference (RNAi) screens in mammalian cells. Application of this approach enhances reconfirmation rates and facilitates the experimental validation of new gene activities through the probability-based identification of multiple distinct and active small interfering RNAs (siRNAs) targeting the same gene. We further extend this approach to establish that the optimal redundancy for efficacious RNAi collections is between 4-6 siRNAs per gene.

Blood consists of different cell populations with distinct functions and correspondingly, distinct gene expression profiles. In this study, global miRNA expression profiling was performed across a panel of nine human immune cell subsets (neutrophils, eosinophils, monocytes, B cells, NK cells, CD4 T cells, CD8 T cells, mDCs and pDCs) to identify cell-type specific miRNAs. mRNA expression profiling was performed on the same samples to determine if miRNAs specific to certain cell types down-regulated expression levels of their target genes. Six cell-type specific miRNAs (miR-143; neutrophil specific, miR-125; T cells and neutrophil specific, miR-500; monocyte and pDC specific, miR-150; lymphoid cell specific, miR-652 and miR-223; both myeloid cell specific) were negatively correlated with expression of their predicted target genes. These results were further validated using an independent cohort where similar immune cell subsets were isolated and profiled for both miRNA and mRNA expression. miRNAs which negatively correlated with target gene expression in both cohorts were identified as candidates for miRNA/mRNA regulatory pairs and were used to construct a cell-type specific regulatory network. miRNA/mRNA pairs formed two distinct clusters in the network corresponding to myeloid (nine miRNAs) and lymphoid lineages (two miRNAs). Several myeloid specific miRNAs targeted common genes including ABL2, EIF4A2, EPC1 and INO80D; these common targets were enriched for genes involved in the regulation of gene expression (p<9.0E-7). Those miRNA might therefore have significant further effect on gene expression by repressing the expression of genes involved in transcriptional regulation. The miRNA and mRNA expression profiles reported in this study form a comprehensive transcriptome database of various human blood cells and serve as a valuable resource for elucidating the role of miRNA mediated regulation in the establishment of immune cell identity.

Aims Risdiplam (RG7916, RO7034067) is an orally administered, centrally and peripherally distributed, survival of motor neuron 2 (SMN2) mRNA splicing modifier for the treatment of spinal muscular atrophy (SMA). The objectives of this entry‐into‐human study were to assess the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics of risdiplam, and the effect of the strong CYP3A inhibitor itraconazole on the PK of risdiplam in healthy male volunteers. Methods Part 1 had a randomized, double‐blind, adaptive design with 25 subjects receiving single ascending oral doses of risdiplam (ranging from 0.6–18.0 mg, n = 18) or placebo (n = 7). A Bayesian framework was applied to estimate risdiplam's effect on SMN2 mRNA. The effect of multiple doses of itraconazole on the PK of risdiplam was also assessed using a two‐period cross‐over design (n = 8). Results Risdiplam in the fasted or fed state was well tolerated. Risdiplam exhibited linear PK over the dose range with a multi‐phasic decline with a mean terminal half‐life of 40–69 h. Food had no relevant effect, and itraconazole had only a minor effect on plasma PK indicating a low fraction of risdiplam metabolized by CYP3A. The highest tested dose of 18.0 mg risdiplam led to approximately 41% (95% confidence interval 27–55%) of the estimated maximum increase in SMN2 mRNA. Conclusions Risdiplam was well tolerated and proof of mechanism was demonstrated by the intended shift in SMN2 splicing towards full‐length SMN2 mRNA. Based on these data, Phase 2/3 studies of risdiplam in patients with SMA are now ongoing.

ObjectiveRecent advances in understanding Spinal Muscular Atrophy (SMA) etiopathogenesis prompted development of potent intervention strategies and raised need for sensitive outcome measures capable of assessing disease progression and response to treatment. Several biomarkers have been proposed; nevertheless, no general consensus has been reached on the most feasible ones. We observed a wide range of measures over 1 year to assess their ability to monitor the disease status and progression.Methods18 SMA patients and 19 healthy volunteers (HV) were followed in this 52‐weeks observational study. Quantitative‐MRI (qMRI) of both thighs and clinical evaluation of motor function was performed at baseline, 6, 9 and 12 months follow‐up. Blood samples were taken in patients for molecular characterization at screening, 9 and 12 month follow‐up. Progression, responsiveness and reliability of collected indices were quantified. Correlation analysis was performed to test for potential associations.Results QMRI indices, clinical scales and molecular measures showed high to excellent reliability. Significant differences were found between qMRI of SMA patients and HV. Significant associations were revealed between multiple qMRI measures and functional clinical scales. None of the qMRI, clinical, or molecular measures was able to detect significant disease progression over 1 year.InterpretationWe probed a variety of quantitative measures for SMA in a slowly‐progressing disease population over 1 year. The presented measures demonstrated potential to provide a closer link to underlying disease biology as compared to conventional functional scales. The proposed biomarker framework can guide implementation of more sensitive endpoints in future clinical trials and prove their utility in search for novel disease‐modifying therapies.

Primary human hepatocytes are widely used as an in vitro system for the assessment of drug metabolism and toxicity. Nevertheless a cell system with higher stability of physiological functions is required for the investigation of drugs' mode of action, pathway analyses and biomarkers evaluations. We recently discovered that the human hepatocellular carcinoma cell line, HepG2/C3A, cultured as spheroids in a 3D system can recover their main functions after trypsinisation within about 18 days. The objective of this study was to investigate whether the spheroids' metabolic functions remained stable after this recovery period. Therefore we evaluated physiological capabilities of the spheroids (cell survival, growth rate, glycogenesis, ATP, cholesterol and urea synthesis and drug metabolism) and the expression of key genes related to the main liver pathways in spheroids cultured for an additional 24 days after full recovery (day 18). Here we show that after the recovery period, the 3D spheroid culture can provide a metabolically competent homeostatic cell model which is in equilibrium with its culture environment for more than 3 weeks. Such a stable system could be used for the assessment of the drugs' mode of action, for biomarkers evaluation and for any systems biology studies which require medium-to long-term stability of metabolic functions.

Transfection of chemically synthesized short interfering RNAs (siRNAs) enables a high level of sequence-specific gene silencing. Although siRNA design algorithms have been improved in recent years, it is still necessary to prove the functionality of a given siRNA experimentally. We have functionally tested several thousand siRNAs for target genes from various gene families including kinases, phosphatases, and cancer-related genes (e.g., genes involved in apoptosis and the cell cycle). Some targets were difficult to silence above a threshold of 70% knockdown. By working with one design algorithm and a standardized validation procedure, we discovered that the level of silencing achieved was not exclusively dependent on the siRNA sequences. Here we present data showing that neither the gene expression level nor the cellular environment has a direct impact on the knockdown which can be achieved for a given target. Modifications of the experimental setting have been investigated with the aim of improving knockdown efficiencies for siRNA-target combinations that show only moderate knockdown. Use of higher siRNA concentrations did not change the overall performance of the siRNA-target combinations analyzed. Optimal knockdown at the mRNA level was usually reached 48-72 hours after transfection. Target gene-specific characteristics such as the accessibility of the corresponding target sequences to the RNAi machinery appear to have a significant influence on the knockdown observed, making certain targets easy or difficult to knock down using siRNA.

BackgroundDiscoidin domain receptor 1 (DDR1) is a collagen-activated receptor tyrosine kinase extensively implicated in diseases such as cancer, atherosclerosis and fibrosis. Multiple preclinical studies, performed using either a gene deletion or a gene silencing approaches, have shown this receptor being a major driver target of fibrosis and glomerulosclerosis.MethodsThe present study investigated the role and relevance of DDR1 in human crescentic glomerulonephritis (GN). Detailed DDR1 expression was first characterized in detail in human GN biopsies using a novel selective anti-DDR1 antibody using immunohistochemistry. Subsequently the protective role of DDR1 was investigated using a highly selective, novel, small molecule inhibitor in a nephrotoxic serum (NTS) GN model in a prophylactic regime and in the NEP25 GN mouse model using a therapeutic intervention regime.ResultsDDR1 expression was shown to be mainly limited to renal epithelium. In humans, DDR1 is highly induced in injured podocytes, in bridging cells expressing both parietal epithelial cell (PEC) and podocyte markers and in a subset of PECs forming the cellular crescents in human GN. Pharmacological inhibition of DDR1 in NTS improved both renal function and histological parameters. These results, obtained using a prophylactic regime, were confirmed in the NEP25 GN mouse model using a therapeutic intervention regime. Gene expression analysis of NTS showed that pharmacological blockade of DDR1 specifically reverted fibrotic and inflammatory gene networks and modulated expression of the glomerular cell gene signature, further validating DDR1 as a major mediator of cell fate in podocytes and PECs.ConclusionsTogether, these results suggest that DDR1 inhibition might be an attractive and promising pharmacological intervention for the treatment of GN, predominantly by targeting the renal epithelium.Electronic supplementary materialThe online version of this article (10.1186/s12967-018-1524-5) contains supplementary material, which is available to authorized users.

The identification of new biomarkers is essential in the implementation of personalized health care strategies that offer new therapeutic approaches with optimized and individualized treatment. In support of hypothesis generation and testing in the course of our biomarker research an online portal and respective function-tested reverse transcription quantitative real-time PCR assays (RT-qPCR) facilitated the selection of relevant biomarker genes. We have established workflows applicable for convenient high throughput gene expression analysis in biomarker research with cell lines (in vitro studies) and xenograft mouse models (in vivo studies) as well as formalin-fixed paraffin-embedded tissue (FFPET) sections from various human research and clinical tumor samples. Out of 92 putative biomarker candidate genes selected in silico, 35 were shown to exhibit differential expression in various tumor cell lines. These were further analysed by in vivo xenograft mouse models, which identified 13 candidate genes including potential response prediction biomarkers and a potential pharmacodynamic biomarker. Six of these candidate genes were selected for further evaluation in FFPET samples, where optimized RNA isolation, reverse transcription and qPCR assays provided reliable determination of relative expression levels as precondition for differential gene expression analysis of FFPET samples derived from projected clinical studies. Thus, we successfully applied function tested RT-qPCR assays in our biomarker research for hypothesis generation with in vitro and in vivo models as well as for hypothesis testing with human FFPET samples. Hence, appropriate function-tested RT-qPCR assays are available in biomarker research accompanying the different stages of drug development, starting from target identification up to early clinical development. The workflow presented here supports the identification and validation of new biomarkers and may lead to advances in efforts to achieve the goal of personalized health care.