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EMBO Reports (2018) e47143 Basic and translational biomedical research explores biological and pathophysiological mechanisms with the aim of developing novel therapies, preventive measures, and diagnostics to improve human health. Disappointingly, however, most new therapies fail when they are tested in clinical trials. Although the causes of this “translational attrition” are diverse and often rooted in the complexity of the underlying biology, it has also become clear that methodology is a major issue. The “translational roadblock”, along with what has been dubbed a “reproducibility crisis” [1], has fueled discussions about the reliability and reproducibility of biomedical research in general. There is strong evidence that weaknesses in planning, conducting, analyzing, and (non)reporting of research [2], as well as misidentification or contamination of reagents, biologicals, and cell lines [3], are prevalent factors. Meta‐research has shown that these problems can lead to an inflation of effect size and false positives and consequently decrease the reproducibility and predictiveness of research results. At the same time, the increasing methodological complexity combined with the immense proliferation in research outputs greatly complicates the production and evaluation of reliable evidence. Pressure to publish and hypercompetition for resources further compromise the robustness and rigor of research. Arguably, basic and translational biomedical research has a quality problem. In the 1970s, US cars had major quality problems. By comparison, Japanese cars were much more reliable. The introduction of rigorous quality management in the production process was largely credited for this competitive advantage, which helped the Japanese car industry to dominate the market for decades to come. By now, most industries, including US car manufacturers, the health and pharmaceutical industries as well as clinical medicine, have established sophisticated quality management systems (QMS) on which they spend several percent of their budget. Clearly, these investments pay off as companies …

Bioprinting is a novel technology that may help to overcome limitations associated with two-dimensional (2D) cell cultures and animal experiments, as it allows the production of three-dimensional (3D) tissue models composed of human cells. The present study describes the optimization of a bioink composed of alginate, gelatin and human extracellular matrix (hECM) to print human HepaRG liver cells with a pneumatic extrusion printer. The resulting tissue model was tested for its suitability for the study of transduction by an adeno-associated virus (AAV) vector and infection with human adenovirus 5 (hAdV5). We found supplementation of the basic alginate/gelatin bioink with 0.5 and 1 mg/mL hECM provides desirable properties for the printing process, the stability of the printed constructs, and the viability and metabolic functions of the printed HepaRG cells. The tissue models were efficiently transduced by AAV vectors of serotype 6, which successfully silenced an endogenous target (cyclophilin B) by means of RNA interference. Furthermore, the printed 3D model supported efficient adenoviral replication making it suitable to study virus biology and develop new antiviral compounds. We consider the approach described here paradigmatic for the development of 3D tissue models for studies including viral vectors and infectious viruses.

Bioprinting is a new technology, which arranges cells with high spatial resolution, but its potential to create models for viral infection studies has not yet been fully realized. The present study describes the optimization of a bioink composition for extrusion printing. The bioinks were biophysically characterized by rheological and electron micrographic measurements. Hydrogels consisting of alginate, gelatin and Matrigel were used to provide a scaffold for a 3D arrangement of human alveolar A549 cells. A blend containing 20% Matrigel provided the optimal conditions for spatial distribution and viability of the printed cells. Infection of the 3D model with a seasonal influenza A strain resulted in widespread distribution of the virus and a clustered infection pattern that is also observed in the natural lung but not in two-dimensional (2D) cell culture, which demonstrates the advantage of 3D printed constructs over conventional culture conditions. The bioink supported viral replication and proinflammatory interferon release of the infected cells. We consider our strategy to be paradigmatic for the generation of humanized 3D tissue models by bioprinting to study infections and develop new antiviral strategies.

Coxsackievirus B3 (CVB3), a single-stranded RNA virus of the picornavirus family, has been described as a novel oncolytic virus. However, the CVB3 strain used induced hepatitis and myocarditis in vivo. It was hypothesized that oncolytic activity and safety of CVB3 depends on the virus strain and its specific receptor tropism. Different laboratory strains of CVB3 (Nancy, 31-1-93, and H3), which use the coxsackievirus and adenovirus receptor (CAR), and the strain PD, which uses N- and 6-O-sulfated heparan sulfate (HS) for entry into the cells, were investigated for their potential to lyse tumor cells and for their safety profile. The investigations were carried out in colorectal carcinoma. In vitro investigations showed variable infection efficiency and lysis of colorectal carcinoma cell lines by the CVB3 strains. The most efficient strain was PD, which was the only one that could lyse all investigated colorectal carcinoma cell lines. Lytic activity of CAR-dependent CVB3 did not correlate with CAR expression on cells, whereas there was a clear correlation between lytic activity of PD and its ability to bind to HS at the cell surface of colorectal carcinoma cells. Intratumoral injection of Nancy, 31-1-93, or PD into subcutaneous colorectal DLD1 cell tumors in BALB/c nude mice resulted in strong inhibition of tumor growth. The effect was seen in the injected tumor, as well as in a non-injected, contralateral tumor. However, all animals treated with 31-1-93 and Nancy developed systemic infection and died or were moribund and sacrificed within 8 days post virus injection. In contrast, five of the six animals treated with PD showed no signs of a systemic viral infection, and PD was not detected in any organ. The data demonstrate the potential of PD as a new oncolytic virus and HS-binding of PD as a key feature of oncolytic activity and improved safety.

Adenovirus (Ad) infections are usually mild and self-limiting, but severe systemic infections and fatal diseases can occur, especially in immunosuppressed patients. Anti-adenoviral pharmacotherapy has been proven to inhibit Ad infection, but its efficiency is limited. This review addresses biological antiviral agents as a new class of therapeutics for treatment of Ad infections. One group of agents is composed of short double-stranded RNA molecules that have been developed to inhibit Ad receptor and Ad protein expression. The second group of agents includes soluble virus receptor traps which inhibit Ad uptake into cells. Anti-Ad-adoptive T-cell therapy constitutes a third approach. We also outline how the combination of biological antiviral agents and combinations of these agents with the classical antiviral drugs can increase therapeutic efficiency in anti-adenoviral treatments.

Hepatitis E virus (HEV) infection may cause acute hepatitis and lead to hepatic failure in developing and developed countries. We studied HEV seroprevalences in patients with hepatitis B virus (HBV) infection to understand the consequences of HEV superinfection in a Vietnamese population. This cross-sectional study was conducted from 2012 to 2013 and included 1318 Vietnamese patients with HBV-related liver diseases and 340 healthy controls. The case group included patients with acute (n = 26) and chronic hepatitis B (n = 744), liver cirrhosis (n = 160), hepatocellular carcinoma (n = 166) and patients with both liver cirrhosis and hepatocellular carcinoma (n = 222). Anti-HEV IgG and IgM antibodies were assessed in patients and controls by ELISA. HEV-RNA was identified by PCR assays and sequencing. Seroprevalences of anti-HEV IgG among hepatitis B patients and controls were 45% and 31%, respectively (adjusted P = 0.034). Anti-HEV IgM seroprevalences were 11.6% and 4.7% in patients and controls, respectively (adjusted P = 0.005). Seroprevalences were higher among the elder individuals. When stratifying for patient groups, those with liver cirrhosis had the highest anti-HEV IgG (52%) and anti-HEV IgM (19%) seroprevalences. Hepatitis B patients with current HEV infection had abnormal liver function tests compared to patients with past or without HEV infection. One HEV isolate was retrieved from a patient with both liver cirrhosis and hepatocellular carcinoma and identified as HEV genotype 3. This study indicates high prevalences of HEV infection in Vietnamese HBV patients and among healthy individuals and shows that HEV superinfection may influence the outcome and progression of HBV-related liver disease.

Adenoviruses (Ad) generally induce mild self-limiting respiratory or intestinal infections but can also cause serious disease with fatal outcomes in immunosuppressed patients. Antiviral drug therapy is an important treatment for adenoviral infections but its efficiency is limited. Recently, we have shown that gene silencing by RNA interference (RNAi) is a promising new approach to inhibit adenoviral infection. In the present in vitro study, we examined whether the efficiency of an RNAi-based anti-adenoviral therapy can be further increased by combination with a virus receptor trap sCAR-Fc and with the antiviral drug cidofovir. Initially, three siRNAs, siE1A_4, siIVa2_2 and Pol-si2, targeting the adenoviral E1A, IVa2 and DNA polymerase mRNAs, respectively, were used for gene silencing. Replication of the Ad was inhibited in a dose dependent manner by each siRNA, but the efficiency of inhibition differed (Pol-si2 > siIVa2_2 > siE1A_4). Double or triple combinations of the siRNAs compared with single siRNAs did not result in a measurably higher suppression of Ad replication. Combination of the siRNAs (alone or mixes of two or three siRNAs) with sCAR-Fc markedly increased the suppression of adenoviral replication compared to the same siRNA treatment without sCAR-Fc. Moreover, the triple combination of a mix of all three siRNAs, sCAR-Fc and cidofovir was about 23-fold more efficient than the combination of siRNAs mix/sCAR-Fc and about 95-fold more efficient than the siRNA mix alone. These data demonstrate that co-treatment of cells with sCAR-Fc and cidofovir is suitable to increase the efficiency of anti-adenoviral siRNAs.

Coxsackievirus B3 (CVB3), a single-stranded RNA virus of the picornavirus family, has been described as a novel oncolytic virus. However, the CVB3 strain used induced hepatitis and myocarditis in vivo. It was hypothesized that oncolytic activity and safety of CVB3 depends on the virus strain and its specific receptor tropism. Different laboratory strains of CVB3 (Nancy, 31-1-93, and H3), which use the coxsackievirus and adenovirus receptor (CAR), and the strain PD, which uses N- and 6-O-sulfated heparan sulfate (HS) for entry into the cells, were investigated for their potential to lyse tumor cells and for their safety profile. The investigations were carried out in colorectal carcinoma. In vitro investigations showed variable infection efficiency and lysis of colorectal carcinoma cell lines by the CVB3 strains. The most efficient strain was PD, which was the only one that could lyse all investigated colorectal carcinoma cell lines. Lytic activity of CAR-dependent CVB3 did not correlate with CAR expression on cells, whereas there was a clear correlation between lytic activity of PD and its ability to bind to HS at the cell surface of colorectal carcinoma cells. Intratumoral injection of Nancy, 31-1-93, or PD into subcutaneous colorectal DLD1 cell tumors in BALB/c nude mice resulted in strong inhibition of tumor growth. The effect was seen in the injected tumor, as well as in a non-injected, contralateral tumor. However, all animals treated with 31-1-93 and Nancy developed systemic infection and died or were moribund and sacrificed within 8 days post virus injection. In contrast, five of the six animals treated with PD showed no signs of a systemic viral infection, and PD was not detected in any organ. The data demonstrate the potential of PD as a new oncolytic virus and HS-binding of PD as a key feature of oncolytic activity and improved safety.

Activation of the neuronal potassium channel Kv7.2 encoded by the KCNQ2 gene has recently been shown to be an attractive mechanism to inhibit nociceptive transmission. However, potent, selective, and clinically proven activators of Kv7.2/Kv7.3 currents with analgesic properties are still lacking. An important prerequisite for the development of new drugs is a model to test the selectivity of novel agonists by abrogating Kv7.2/Kv7.3 function. Since constitutive knockout mice are not viable, we developed a model based on RNA interference-mediated silencing of KCNQ2. By delivery of a KCNQ2-specific short hairpin RNA with adeno-associated virus vectors, we completely abolished the activity of the specific Kv7.2/Kv7.3-opener ICA-27243 in rat sensory neurons. Results obtained in the silencing experiments were consistent between freshly prepared and cryopreserved dorsal root ganglion neurons, as well as in dorsal root ganglion neurons dissociated and cultured after in vivo administration of the silencing vector by intrathecal injections into rats. Interestingly, the tested associated virus serotypes substantially differed with respect to their transduction capability in cultured neuronal cell lines and primary dorsal root ganglion neurons and the in vivo transfer of transgenes by intrathecal injection of associated virus vectors. However, our study provides the proof-of-concept that RNA interference-mediated silencing of KCNQ2 is a suitable approach to create an ex vivo model for testing the specificity of novel Kv7.2/Kv7.3 agonists.

Recombinant adeno-associated virus (rAAV) vectors are promising tools in gene therapy, but accurate quantification of the vector dose remains a critical issue for their successful application. We therefore aimed at the precise determination of the titer of self-complementary AAV (scAAV) vectors to improve the reliability of RNA interference (RNAi)-mediated knockdown approaches. Vector titers were initially determined by quantitative polymerase chain reaction (qPCR) using four primer sets targeting different regions within the AAV vector genome (VG) and either coiled or linearized plasmid standards. Despite very low variability between replicates in each assay, these quantification experiments revealed up to 20-fold variation in vector titers. Therefore, we developed a novel approach for the reproducible determination of titers of scAAV vectors based on the use of purified genomic vector DNA as a standard (scAAVStd). Consistent results were obtained in qPCR assays using the four primer sets mentioned above. RNAi-mediated silencing of human cyclophilin B (hCycB) by short hairpin RNA-expressing scAAV vectors was investigated in HeLa cells using two independent vector preparations. We found that the required vector titers for efficient knockdown differed by a factor of 3.5 between both preparations. Hence, we also investigated the number of internalized scAAV vectors, termed transduction units (TUs). TUs were determined by qPCR applying the scAAVStd. Very similar values for 80% hCycB knockdown were obtained for the two AAV vector preparations. Thus, only the determination of TUs, rather than vector concentration, allows for reproducible results in functional analyses using AAV vectors.

Silencing of cardiac genes by RNA interference (RNAi) has developed into a powerful new method to treat cardiac diseases. Small interfering (si)RNAs are the inducers of RNAi, but cultured primary cardiomyocytes and heart are highly resistant to siRNA transfection. This can be overcome by delivery of small hairpin (sh)RNAs or artificial microRNA (amiRNAs) by cardiotropic adeno-associated virus (AAV) vectors. Here we describe as example of the silencing of a cardiac gene, the generation and cloning of shRNA, and amiRNAs directed against the cardiac protein phospholamban. We further describe the generation of AAV shuttle plasmids with self complementary vector genomes, the production of AAV vectors in roller bottles, and their purification via iodixanol gradient centrifugation and concentration with filter systems. Finally we describe the preparation of primary neonatal rat cardiomyocytes (PNRC), the transduction of PNRC with AAV vectors, and the maintenance of the transduced cell culture.

Silencing of cardiac genes by RNA interference (RNAi) has developed into a powerful new method to treat cardiac diseases. Small interfering (si)RNAs are the inducers of RNAi, but cultured primary cardiomyocytes and heart are highly resistant to siRNA transfection. This can be overcome by delivery of small hairpin (sh)RNAs or artificial microRNA (amiRNAs) by cardiotropic adeno-associated virus (AAV) vectors. Here we describe as example of the silencing of a cardiac gene, the generation and cloning of shRNA, and amiRNAs directed against the cardiac protein phospholamban. We further describe the generation of AAV shuttle plasmids with self complementary vector genomes, the production of AAV vectors in roller bottles, and their purification via iodixanol gradient centrifugation and concentration with filter systems. Finally we describe the preparation of primary neonatal rat cardiomyocytes (PNRC), the transduction of PNRC with AAV vectors, and the maintenance of the transduced cell culture.

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector systems. The model is obtained by repopulating the extracellular matrix of a decellularized rat liver with a human hepatocyte cell line. The model permits studies in a vascularized 3D cell system, replacing potentially harmful experiments with living animals. Another advantage is the humanized nature of the model, which is closer to human physiology than animal models. In this study, we demonstrate the transduction of this liver model with a viral vector derived from adeno-associated viruses (AAV vector). The perfusion circuit that supplies the 3D liver model with media provides an easy means to apply the vector. The system permits monitoring of the major metabolic parameters of the liver. For final analysis, tissue samples can be taken to determine the extent of recellularization by histological techniques. Distribution of the virus vector and expression of the delivered transgene can be analyzed by quantitative PCR (qPCR), Western blotting and immunohistochemistry. Numerous applications of the vector model in basic research and in the development of gene therapeutic applications can be envisioned, including the development of novel antiviral therapeutics, cancer research, and the study of viral vectors and their potential side effects.

The coxsackievirus and adenovirus receptor (CAR) is a member of the immunoglobulin superfamily (IgSF) and functions as a receptor for coxsackie B viruses (CVBs). The extracellular portion of CAR comprises two glycosylated immunoglobulin-like domains, D1 and D2. CAR-D1 binds to the virus and is essential for virus infection; however, it is not known whether D2 is also important for infection, and the role of glycosylation has not been explored. To understand the function of these structural components in CAR-mediated CVB3 infection, we generated a panel of human (h) CAR deletion and substitution mutants and analyzed their functionality as CVB receptors, examining both virus binding and replication. Lack of glycosylation of the CAR-D1 or -D2 domains did not adversely affect CVB3 binding or infection, indicating that the glycosylation of CAR is not required for its receptor functions. Deletion of the D2 domain reduced CVB3 binding, with a proportionate reduction in the efficiency of virus infection. Replacement of D2 with the homologous D2 domain from chicken CAR, or with the heterologous type C2 immunoglobulin-like domain from IgSF11, another IgSF member, fully restored receptor function; however, replacement of CAR-D2 with domains from CD155 or CD80 restored function only in part. These data indicate that glycosylation of the extracellular domain of hCAR plays no role in CVB3 receptor function and that CAR-D2 is not specifically required. The D2 domain may function largely as a spacer permitting virus access to D1; however, the data may also suggest that D2 affects virus binding by influencing the conformation of D1. IMPORTANCE An important step in virus infection is the initial interaction of the virus with its cellular receptor. Although the role in infection of the extracellular CAR-D1, cytoplasmic, and transmembrane domains have been analyzed extensively, nothing is known about the function of CAR-D2 and the extracellular glycosylation of CAR. Our data indicate that glycosylation of the extracellular CAR domain has only minor importance for the function of CAR as CVB3 receptor and that the D2 domain is not essential per se but contributes to receptor function by promoting the exposure of the D1 domain on the cell surface. These results contribute to our understanding of the coxsackievirus-receptor interactions.

Photosynthesis is one of the most fascinating fields of current interdisciplinary research. It seems miraculous that Nature, in the process of evolution, has managed to bring about a 100% quantum yield in charge separation occurring in the primary processes within the photosynthetic reaction centers. In fact, wasteful deactivation of the photoexcited electronic states into the ground state should be favoured and thus prevent charge separation. Biomimetic model compounds have been synthesized, essentially from porphyrins and quinones, resembling the redoxactive cofactors, Nature has developed. The underlying philosophy of this approach is to design and prepare suitable organic molecules allowing the specific properties of the complex electron transfer reactions to be tuned appropriately. Special emphasis is given the through comparison of structures and functions of native and artificial systems. The prominent electron transfer characteristics are discussed in particular by using time-resolved picosecond fluorescence and time-resolved EPR techniques.

RNA interference (RNAi) is an evolutionarily conserved mechanism for sequence-specific target RNA degradation in animals and plants, which plays an essential role in gene regulation. In addition, it is believed to function as a defense against viruses and transposons. In recent years, RNAi has become a widely used approach for studying gene function by targeted cleavage of a specific RNA. Moreover, the technology has been developed as a new therapeutic option that has already made its way into clinical testing. Treatment of viral infections remains a serious challenge due to the emergence of new viruses and strain variation among known virus species. RNAi holds great promise to provide a flexible approach that can rapidly be adapted to new viral target sequences. A major challenge in the development of an efficient RNAi approach still remains the design of small interfering RNAs (siRNAs) with high silencing potency. While large libraries with validated siRNAs exist for silencing of endogenously expressed genes in human or murine cells, siRNAs still have to be designed individually for new antiviral approaches. The present chapter describes strategies to design highly potent siRNAs by taking into consideration thermodynamic features of the siRNA, as well as the structural restrictions of the target RNA. Furthermore, assays for testing the siRNAs in reporter assays as well as options to improve the properties of siRNAs by the introduction of modified nucleotides will be described. Finally, experimental setups will be outlined to test the siRNAs in assays with infectious viruses.

17-18 March 2010, St Hilda's College, Oxford, United Kingdom, The Argonaute proteins constitute a highly conserved family of nucleic acid-binding proteins whose members have been implicated in RNA interference (RNAi) and related phenomena in several organisms. In particular, Argonaute 2 (Ago2) represents one of the key players in the RNA induced silencing complex (RISC). However, recently published literature describes that Ago2 itself is regulated under certain cellular conditions by post-translational modifications. In this work, we investigated the activity of human Ago2 under various conditions of cell stress. Under such conditions, the sub-cellular localization of Ago2 was altered which was coincident with its decreased function in the RNAi pathway. This work implies that specific cellular stress conditions induce an intracellular translocation of Ago2 protein excluding it from sites of action of the RNAi machinery. Details will be presented and discussed., Although microRNAs (miRNAs) have been shown to regulate genes that play roles in important biological processes such as development, differentiation and disease, identifying miRNAs of interest and characterizing their mechanism of action remains a challenge. The miR-200 family has been shown to regulate epithelial to mesenchymal transitions (EMT), a process that is critical for normal development and tumor metastasis. Furthermore, recent publications indicate that the miR-200 family regulates EMT by targeting ZEB1 and ZEB2, transcription factors that repress E-cadherin. Here, we use breast cancer cell lines as an EMT model system to characterize the miR-200 family's role in these biological processes. miRIDIAN microRNA microarray profiling was use to characterize cell lines followed by introduction of miRIDIAN miRNA mimics and hairpin inhibitors to modulate miRNA expression in these cell lines. Based on these studies we have devised a workflow for studying miRNA expression and identifying miRNA targets., Small interfering RNAs (siRNAs) are promising new active compounds in gene medicine. But one serious problem with delivering siRNAs as treatment is the well-established stimulation of innate immune reactions by some RNA duplexes and lack of effective delivery systems. Innate immune reactions towards double stranded RNAs include the 2′-5 oligoadenylate (OAS) system, the protein kinase R (PKR), RIG-I and Toll-like receptor activated pathways all resulting in activation of antiviral defence mechanism. We have previously described a high throughput animal screening model in which the level of stimulation of interferon-related anti viral effects is measured as increased resistance of siRNA-injected small fish to a pathogenic virus. Here we show how this fish model can be used to make a fast in vivo analysis of the effect of chemical base modification in the siRNAs on the level of antiviral off-target effects., Defects in DNA repair and damage response can drive tumour progression and results in genomic instability in tumour cells. Whereas these DNA repair defects can be exploited in cancer therapy where unrepaired lesions induced by anti-cancer agents increase tumour killing, these DNA repair defects can also cause resistance to anti-cancer treatments. Defects in DNA repair pathways may also render tumour cells dependent on other complementary repair pathways as seen with the synthetic lethal effect of PARP inhibitors in BRCA-defective tumours. Altogether, there is a potential use of DNA repair inhibitors in anti-cancer treatment, either in combination therapy to increase the efficacy of e.g. radiation or as mono-therapy to target essential compensatory DNA repair pathways. Homologous recombination (HR) is a repair pathway involved in repairing double-strand breaks and replication-associated lesions, the main toxic lesions induces by anti-cancer drugs. Depletion of the key protein involved in HR, the RAD51 protein, is lethal but loss of other proteins involved in HR is compatible with survival. To understand the wider network of HR proteins and with the aim to find novel proteins that can be used as targets for HR-inhibition, we have used a RNAi screen approach to create functional genetic network maps describing proteins involved in homologous recombination. Foci formation of the RAD51 protein has been studied in U2OS cells subjected to protein knock-down using a RNAi library in co-treatment with either irradiation or camptothecin. This identifies proteins involved in the HR response to different types of lesions. In addition, a GFP-reporter-based HR assay has been used to identify proteins involved in HR repair of a site-specific double-strand break. With these experiments we hope to increase our understanding of the complex network of homologous recombinational repair as well as find potential targets for anti-cancer treatments., The canonical Wnt/β-Catenin pathway controls myriad fundamental cellular processes, such as differentiation and proliferation. Aberrant regulation of this signaling cascade can result in several disease phenotypes and is a hallmark of colorectal cancer and hepatocellular carcinoma. Genetic evidence links Wnt/β-Catenin signaling to the control of bone density. The identification of new components of this pathway may lead to the development of novel therapies targeting a variety of cancers and osteoporosis. Accordingly, we have conducted genome scale RNAi screens in multiple cell contexts to discover new pathway regulators. We developed a screening process that included steps for initial hit identification, mitigation of off target effects, elimination of cell line-specific effects, and measurement of Wnt/β-Catenin signature regulation. We further validated a number of these new pathway mediators in vitro for physical association with canonical pathway members and in vivo by use of zebrafish models. These studies have characterized AGGF1, BTK, and DHFR as new modulators or the Wnt/β-Catenin pathway., SiRNA molecules have a high potential for therapeutic applications. Here we present a mechanism of cell-specific, peptide-blocked siRNA. SiRNA is bound to short peptides preventing RISC formation. Peptides contain target sequence of peptidases, exclusively active in target cells. After intracellular delivery, only in target cells, peptidases cleave peptides leading to siRNA activation. Used peptide sequence is the target sequence for caspase-4, expressed in Jeg-3 choriocarcinoma and MCF-7 mammalian cancer cells, but not in human embryonic kidney (HEK) cells. We aimed to silence Signal Transducer of Activation (STAT3) expression by such modified siRNA specifically in Jeg-3 as well as MCF-7 in contrast to HEK cells. Western blot was performed to detect STAT3 protein. Furthermore, proliferation of STAT3 silenced cells was analysed. The peptide was bound to the siRNA antisense strand via amino-C6-linker based on Fmoc chemistry. Correct binding was analysed by PAGE and Maldi-MS. In Jeg-3 and MCF-7 modified siRNA became activated and reduced STAT3 expression in contrast to HEK cells lacking caspase-4. Moreover, proliferation was significantly reduced in STAT3 silenced cells. In conclusion, STAT3, which is responsible for increased proliferation and invasive properties of various cancer cells, can be cell-specifically silenced using the presented novel technology. Preliminary experiments indicate that the presented mechanism of peptide-inhibited; peptidase-activated siRNA can be transferred to a variety of cell specific active peptidases and related disease models., Background: RNA interference (RNAi), the degradation of cognate mRNA by small interfering RNA (siRNA), has emerged as a promising therapeutic entity for viral infections, including hepatitis C virus (HCV) HCV. In plants and invertebrates, RNAi-mediated protection can spread to neighboring cells; however, such a phenomenon has not been described in mammalian cells. In this study, we investigated whether endogenous expressed liver-specific microRNA and vector-delivered siRNA can transfer between cells and whether this exchange could extend the therapeutic effect of RNAi against HCV infection. Methods: Human hepatoma cell lines Huh7 and HepG2, Huh7-ET HCV replicon cells, and renal epithelial line 293T were co-cultured with conditioned medium or cells stably transduced with integrating lentiviral vectors expressing green fluorescent protein (GFP) and small hairpin RNAs targeting the HCV NS5b (LV-shNS5b), CD81 (LV-shCD81) or non-targeted control (LV-shCon). Liver-specific microRNA, miR-122, was quantified by real-time RT-PCR. Results: MiR-122 is not only highly expressed in Huh7 cells but also detectable in Huh7-CM, suggesting release of miR-122 by the cells. Upon incubation of HepG2 or 293T cells, which are 200 and 50,000-fold lower in miR-122 expression, with Huh7-CM the miR-122 level in these cells was increased by 4-20 fold, indicating uptake of miR-122 from the medium. To further investigate whether small interfering RNA delivered by vectors can be transferred between cells, Huh7-ET was co-cultured with stably transfected Huh7 cells expressing shNS5b or shCon. A significant reduction of viral replication was observed at 1:1 ratio of shNS5b cells (52±12% p, Borane (–BH3) chemistry offers some unique chemical characteristics that make these compounds promising for enhancing the potential of three anticancer strategies; (a) RNA interference (siRNA) (b) the selection of tumor specific aptamers and (c) Boron Neutron Capture Therapy, a highly selective type of radiation therapy. Borane oligonucleotides are nuclease resistant and have increased lipophilicity compared to natural oligonucleotides, yet the modified borane nucleotide triphosphates (NTPαBs) still are efficiently recognized and utilized by RNA polymerase enzymes which enable the enzymatic synthesis of RNA (siRNA and aptamers). The novel properties of boranophosphate RNA molecules could lead to an increase in affinity and specificity of the siRNA and aptamers, as well impart stability of the nucleic acids to cellular nucleases. We hypothesize that borane-RNA molecules will interact a new diverse array of ligand sites in proteins (RISC siRNA carriers or ErbB2 therapeutic target) because of the distinct hydrophobicity, shape, and polarity properties imparted by the phosphorus-boron (P-B) chemical bond compared to the natural phosphorus-oxygen (P-O) bond. (a) A major cause of chemotherapeutic treatment failure against human cancers is the aberrant regulation of genes such as MDR1 in cancer cells. Controlling the expression of cancer genes with antisense technology is a possible cancer therapy. MDR1 codes for a p-glycoprotein (Pgp) that is overexpressed in multidrug resistant cancer cells. Specifically, using modified Small interfering RNAs (siRNAs) that target and degrade the p-glycoprotein mRNA produced by the MDR1 gene can be used to correct the overexpression of p-glycoprotein. (b) The selection of boranophosphate modified RNA aptamers by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) against ErbB2. It is likely that targeting a more specific cancer membrane target such as the ErbB2 receptor with a borane RNA aptamer may also be an effective two-prong strategy in breast cancer therapy. Like the antibody protein, Herceptin, an aptamer may assume a distinct shape and block the receptor. (c) Further, borane aptamers can be used as specific carriers of 10B in Boron Neutron Capture Therapy (BNCT). BNCT specifically destroys tumor cells near boron molecules. The selection of α-P-borano-modified RNA aptamers against receptors highly over-expressed in breast cancer should provide the opportunity of testing the efficacy of a target specific method for delivering boron to the cancer cells. Such boron molecules could be potent and unique anti-tumor surrogates for antibodies because of their ease in selection, smaller size, nuclease resistance, susceptibility to BNCT, and versatility in terms of adding chemical modifications to increase potency., The RNA silencing pathway is engaged during the cellular innate immune response to double-stranded RNA. Particularly in plants, this serves as an anti-viral defense mechanism and results in the degradation of homologous viral RNA. Several viruses have evolved mechanisms to undermine the RNA silencing pathway. Tombusviruses, such as the Carnation Italian Ringspot virus (CIRV), express a 19 kDa protein (p19) which acts as a suppressor of the RNA-silencing pathway. As a dimer, p19 binds double stranded small RNAs in a size-selective and relatively sequence-independent manner, thereby preventing their incorporation into RISC. The p19 protein binds 21 nucleotide double-stranded small RNAs with nanomolar affinity. These unique selective binding features allow p19 to be used as a molecular ruler that can be used to detect small RNAs. We have further increased the potential of the p19 protein through engineering several recombinant p19 proteins which allow us to detect protein-RNA interactions in solution. By constructing linked versions of the CIRV-p19 dimer we have improved the stability and binding affinity of the p19 dimer while still maintaining its ability to discriminate RNA according to length. A recombinant p19-CFP fusion protein allows us to use fluorescence-based techniques to detect and quantify protein-RNA interactions based on Förster Resonance Energy Transfer (FRET). In this method, when the linked recombinant p19-CFP dimer binds Cy3-labelled dsRNA, intermolecular FRET occurs between the CFP donor and the Cy3 acceptor giving a quantifiable signal. In order to extend this methodology to in vivo detection of small RNA, we have developed an intramolecular FRET probe which incorporates YFP, another GFP analog, into the 2x-p19-CFP recombinant protein. Progress towards using this ‘chameleon’ recombinant protein, CFP-2x-p19-YFP, as a FRET-based probe for live-cell imaging of small RNA production and localization will be presented., RNA interference (RNAi) is a highly evolutionary conserved RNA-dependent gene silencing process initiated by short double-stranded RNA molecules. These include micro RNA and short interfering RNA (siRNA), the latter causing sequence-specific degradation of homologous mRNA sequences. Soon after the groundbreaking discovery of siRNA-mediated regulation of gene expression, the RNA induced silencing complex (RISC) was found to be the effector complex of RNAi with argonaute proteins as the catalytic component. Here, HeLa cell cytoplasmic S100 extract was established as a minimal mechanistic in vitro model to study this process in more detail. Cleavage reactions of a radiolabeled in vitro synthesized target RNA by double-stranded siRNA were performed and cleavage products as well as turnover rates were measured as a function of Mg2+ concentration, nucleotide sequence and time. Despite a lot of advances in the understanding of RISC activity, different composition sizes and associated factors of this complex have been reported in the literature. The above described HeLa cell extract was used to isolate truly active RISCs by purification of complexes via the target mRNA component such that involved protein and nucleic acid components can be further characterized. In summary, HeLa cell extract was shown to be a valuable tool for kinetic and mechanistic studies of siRNA-mediated RNA interference., MicroRNAs (miRNAs) are small endogenous RNAs, which typically imperfectly base-pair with 3'UTRs and mediate translational repression and mRNA degradation. Dicer, an RNase III generating small RNAs in the miRNA and RNAi pathways, is essential for meiotic maturation of mouse oocytes. We found that 3'UTRs of transcripts up-regulated in Dicer1/oocytes are not enriched in miRNA binding sites implicating a weak impact of miRNAs on the maternal transcriptome. Therefore, we tested the ability of endogenous miRNAs to mediate RNA-like cleavage or translational repression of reporter mRNAs. In contrast to somatic cells, endogenous miRNAs in fully-grown GV oocytes poorly repressed reporter translation while their RNAi-like activity was much less affected. In addition, reporter mRNA carrying let-7-binding sites failed to localize to P-bodies, cytoplasmic foci containing proteins involved in RNA repression and degradation where miRNA-repressed transcripts typically localize. In fact, P-bodies are not found in fully-grown mouse oocytes but disappear early during oocyte growth. In fully-grown oocytes, several P-body components and other RNA binding proteins including DDX6, CPEB, MSY2, and the exon junction complex form transient, subcortical RNA-containing aggregates, which disperse during oocyte maturation, consistent with recruitment of maternal mRNAs that occurs during this time. In contrast, levels of P-body component DCP1A are low during oocyte growth and DCP1A does not co-localize with DDX6 in the sub-cortical aggregates. The amount of DCP1A markedly increases during meiosis, which correlates with the first wave of destabilization of maternal mRNAs. Our data suggest that normal miRNA function is down-regulated during oocyte development and this idea is further supported by normal meiotic maturation of oocytes lacking Dgcr8, which is required for miRNA but not RNAi pathway. We propose that suppression of miRNA function during oocyte growth is an early event in reprogramming gene expression during the transition of a differentiated oocyte into pluripotent blastomeres of the embryo. Furthermore, the cortex of growing oocytes serves an mRNA storage compartment, which contains a novel type of RNA granule related to P-bodies., Being a member of the Picornavirus family, Coxsackievirus B3 (CVB-3) is one of the major pathogens that may lead to dilated cardiomyopathy. It is a small, non-enveloped RNA virus. Because of the plus-stranded RNA genome it is qualified for the application of RNA interference. We developed highly efficient small interfering RNAs (siRNAs) against the viral 3D RNA dependent RNA polymerase (3Dpol). Hence we were able to inhibit CVB-3 proliferation up to 90% in a plaque reduction assay. Recent experiments revealed the potential to use siRNAs modified with locked nucleic acids (LNA) or unlocked nucleic acids (UNA) to inhibit virus replication. For in vivo applications, vector delivery of shRNA expression cassettes was found to be a suitable approach to treat mice with virus-induced myocarditis. An AAV-vector expressing two shRNAs against CVB-3 was found to improve the heart parameters in a mouse model for an enterovirus myocarditis. Finally, a synergistic and potent antiviral effect in persistently infected human myocardial fibroblasts was obtained, when the siRNA was combined with a soluble variant of the coxsackievirus-adenovirus receptor, which acts as a virus trap. Taken together, these studies demonstrate that RNA interference is a powerful tool to inhibit cardiotropic viruses., Our studies have focused on the application of imaging-capable nanoparticulate agents for the delivery of siRNA to target tissues. One example includes magnetic nanoparticles (MN), which have traditionally been utilized as contrast agents for magnetic resonance imaging. MN typically consist of a dextran-coated superparamagnetic iron oxide core (for magnetic resonance imaging), labeled with Cy5.5 dye (for near-infrared in vivo optical imaging), and conjugated to synthetic siRNA molecules targeting model or therapeutic genes. We have explored the potential of these nanoparticles as delivery modules for small interfering RNA to tumors and pancreatic islets. Furthermore, we have investigated the feasibility of combining the imaging and delivery capabilities of these nanoparticles for the tracking of siRNA bioavailability. The versatile functionalization potential of MN has allowed us to control properties of the agents, such as uptake mechanism and target organ distribution. The tumoral accumulation of MN-siRNA results in a remarkable level of target-gene down-regulation. Repeated treatment with MN-siBIRC5, targeting the tumor-specific anti-apoptotic gene, birc5, leads to the induction of apoptosis in the tumors and an overall reduction in tumor growth rate. Another application of MN-siRNA extends from the fact that these nanoparticles are also taken up by pancreatic islets following in vitro incubation. This uptake can be visualized by magnetic resonance and near-infrared fluorescence optical imaging and results in down-regulation of target genes. This approach has relevance in the context of pancreatic islet transplantation, which is a promising new treatment of type 1 diabetes. A potential application of this method would involve the selective knock-down of genes implicated in islet graft dysfunction, such as pro-apoptotic genes and genes involved in immuno-recognition. More recent studies that will be discussed briefly have focused on the delivery of knock-down LNA probes targeting specific miRNA pathways., Development of siRNA therapeutics has already demonstrated clinical benefits in more than a dozen human trials. Using siRNA cocktail to silence multiple disease genes is truly realizing the advantage of small interfering RNA (siRNA)-based drugs. We have developed a set of siRNA cocktails using our proprietary algorithm and “Tri-Blocker™” platform, as the active pharmaceutical ingredient (API). Those siRNA cocktails were further tested and validated in the disease relevant animal models using a series of polymer- and liposome-based nanoparticles. The therapeutic programs in the late preclinical development stage, STP705 for improving skin scarless wound healing, STP702 for treatment of influenza H5N1/H1N1 infections and STP601 for treatment of ocular neovascularization diseases, are all based on the local and topical siRNA delivery using the self-assembled first generation nanoparticles. We further developed the second generation nanoparticles with peptide ligands and monoclonal antibodies for targeted cancer siRNA therapeutics which were tested in mouse xenograft tumor models. In addition, we are currently developing the third generation siRNA delivery vehicles, using Infrared-activated silica-coated upconversion nanoparticle (SC-UPNP) and oral-delivered nano-microspheres (OD-NMS). When the siRNA cocktail is applied with other drug modalities, such as monoclonal antibody, the therapeutic benefit was even further improved., A UsiRNA directed against mRNA for the human survivin gene was formulated into liposomes formed with a novel Di- Alkylated Amino Acid (DiLA2) resulting in well encapsulated (>85%) small (100-130 nm) particles. UsiRNAs are novel siRNA constructs which incorporate Unlocked Nucleobase Analogs (UNAs) within the oligonucleotide sequence. The survivin UsiRNA has been demonstrated to be highly potent, with an in vitro IC50 of 10 to 30 pM, which leads to caspase induction and apoptosis in cancer cells. A murine Hep3B-based orthotopic liver cancer model was used to assess the in vivo efficacy of the survivin UsiRNA DiLA2 liposomes with systemic administration. Dosing at 2 mg/kg (q3d x 6 doses; i.v.) resulted in approximately a 50% knockdown of survivin mRNA and 60% decrease in tumor weight. This result compared favorably with Avastin™ (bevacizumab)-treated mice which served as a positive control. A similar level of survivin mRNA knockdown was noted in a xenograft model of subcutaneously implanted liver tumors and systemic administration. In an orthotopic bladder cancer model, survivin UsiRNA DiLA2 liposomes were delivered topically (intravesical dosing) at up to 1.0 mg/kg (q2d x 4 doses). Greater than 90% inhibition of the survivin mRNA was observed, and there was a dose-dependent decrease in bioluminescence of up to approximately 90% in UsiRNA-treated mice which indicates reduced tumor growth. 5'-RACE analysis confirmed an RNAi-mediated mechanism of action for mRNA inhibition in each tumor model. Data for knockdown of multiple targets in tissues of normal mice and for a liver target in non-human primates has also been demonstrated with systemic administration of DiLA2-based liposomes. Taken together these data demonstrate that DiLA2 liposomes are an effective systemic and local delivery system for UsiRNAs, in both cancer and non-cancer indications., MicroRNAs are a recently discovered class of naturally occurring short non-coding RNA molecules that regulate eukaryotic gene expression. There is emerging evidence to suggest that microRNAs are involved in the pathogenesis of many cancers including B cell lymphoma. There is however little data on the role of microRNAs in T cell lymphomas. Therefore we employed microarray analysis to investigate a possible role for microRNAs in the biology of cutaneous T-cell lymphomas; Sezary syndrome (SzS, n=21), mycosis fungoides (MF, n=26) and cutaneous anaplastic large cell lymphoma (cALCL, n=15) and relevant controls (CD4+ cells from healthy subjects (n=6), and skin biopsies from patients with inflammatory disorders (n=17). Using unsupervised cluster analysis we observed that the T-cell lymphomas have a distinct miRNA profile from their counterpart controls and have distinct profiles between diagnosis. We identified a number of miRNAs in common between the T-cell lymphomas and well as those specific for diagnosis. In summary, we have identified a number of Sz-associated microRNAs that may play a role in the pathogenesis of these diseases., The sequencing of the human genome revealed that 55% of its nucleotide sequence is composed of repetitive elements and that a large fraction of the "non-functional" DNA originated from mobile elements (1). They were considered until recently as “selfish” or "Junk DNA" (2). For example, Alu elements comprise about 10% of the nucleotides of the human genome (1), with over one million inserted copies. There is evidence showing that the presence of repetitive elements had a great influence on the human genome and it has been shown that Alu repeats are mediators of recurrent chromosomal aberrations in tumors as in the case of the chromosomal translocation of intronic Alu elements (3). Recently, it was reported that some mammalian miRNAs are derived from genomic repeats and some of them show perfect complementarity with the MIR/LINE-2 class of repetitive elements, which are present within a large number of human mRNAs and EST transcripts (4). It was hypothesized that Alu elements within the 3'-UTRs are targeted specifically by certain miRNAs (5) and it has been proposed that a dual relationship exists between miRNAs and their Alu targets that may have evolved in the same time window. One hypothesis for this dual relationship is that these miRNAs could protect against excessively high rates of duplicative transposition, which would destroy the genome (6). Here we present the computational prediction of human miRNA/transposon interactions. We performed the analysis by using a combined approach based on thermodynamics and empirical constraints and found significant matches between miRNAs and human repetitive elements such as LINE, SINE, DNA transposon and ERV1. For example, over 30 miRNAs potentially target the L180_5 element (LINE1 family; 1883 nucleotides) and over 20 miRNAs are predicted to target the RICKSHA element (Non-autonomous DNA transposon fossil; 2030 nucleotides). We considered perfect seed complementarity only (7-mers or higher), with no G:U wobbles. The obtained results suggest a potential role of miRNAs in the regulation of repetitive elements and in particular transposons.

Background— RNA interference (RNAi) has the potential to be a novel therapeutic strategy in diverse areas of medicine. Here, we report on targeted RNAi for the treatment of heart failure, an important disorder in humans that results from multiple causes. Successful treatment of heart failure is demonstrated in a rat model of transaortic banding by RNAi targeting of phospholamban, a key regulator of cardiac Ca 2+ homeostasis. Whereas gene therapy rests on recombinant protein expression as its basic principle, RNAi therapy uses regulatory RNAs to achieve its effect. Methods and Results— We describe structural requirements to obtain high RNAi activity from adenoviral and adeno-associated virus (AAV9) vectors and show that an adenoviral short hairpin RNA vector (AdV-shRNA) silenced phospholamban in cardiomyocytes (primary neonatal rat cardiomyocytes) and improved hemodynamics in heart-failure rats 1 month after aortic root injection. For simplified long-term therapy, we developed a dimeric cardiotropic adeno-associated virus vector (rAAV9-shPLB) to deliver RNAi activity to the heart via intravenous injection. Cardiac phospholamban protein was reduced to 25%, and suppression of sacroplasmic reticulum Ca 2+ ATPase in the HF groups was rescued. In contrast to traditional vectors, rAAV9 showed high affinity for myocardium but low affinity for liver and other organs. rAAV9-shPLB therapy restored diastolic (left ventricular end-diastolic pressure, dp/dt min , and τ) and systolic (fractional shortening) functional parameters to normal ranges. The massive cardiac dilation was normalized, and cardiac hypertrophy, cardiomyocyte diameter, and cardiac fibrosis were reduced significantly. Importantly, no evidence was found of microRNA deregulation or hepatotoxicity during these RNAi therapies. Conclusions— Our data show for the first time the high efficacy of an RNAi therapeutic strategy in a cardiac disease.

An efficient mechanism for the sequence‐specific inhibition of gene expression is RNA interference. In this process, double‐stranded RNA molecules induce cleavage of a selected target RNA (see picture). This technique has in recent years developed into a standard method of molecular biology. Successful applications in animal models have already led to the initiation of RNAi‐based clinical trials as a new therapeutic option.WILEY-VCH Only ten years ago Andrew Fire and Craig Mello were able to show that double‐stranded RNA molecules could inhibit the expression of homologous genes in eukaryotes. This process, termed RNA interference, has developed into a standard method of molecular biology. This Review provides an overview of the molecular processes involved, with a particular focus on the posttranscriptional inhibition of gene expression in mammalian cells, the possible applications in research, and the results of the first clinical studies., An efficient mechanism for the sequence‐specific inhibition of gene expression is RNA interference. In this process, double‐stranded RNA molecules induce cleavage of a selected target RNA (see picture). This technique has in recent years developed into a standard method of molecular biology. Successful applications in animal models have already led to the initiation of RNAi‐based clinical trials as a new therapeutic option.WILEY-VCH