Small Interfering RNAs Containing Dioxane- and Morpholino-Derived Nucleotide Analogues Show Improved Off-Target Profiles and Chirality-Dependent In Vivo Knock-Down.

To expand the applicability of recently developed dioxane- and morpholino-based nucleotide analogues, their seed region destabilizing properties in small interfering RNAs (siRNAs) were investigated in order to improve potential off-target profiles. For this purpose, the corresponding adenosine analogues were synthesized in two diastereomeric series as building blocks for the automated oligonucleotide synthesis. The obtained nucleotide precursors were integrated at position 7 of an siRNA antisense strand, targeting transthyretin messenger RNA. Evaluation of the melting temperatures revealed significant differences in the obtained duplex stabilities between the two diastereomeric series, while the influence of the central scaffold was small. All siRNAs containing these novel nucleotide structures showed improved off-target profiles in vitro compared to their parent sequence with the common 2'-OMe-modified adenosine at the same position. In contrast, in vivo potencies were highly dependent on the chirality within the six-membered nucleotide scaffolds and showed high mRNA downregulations for the (2R,6R)-configured diastereomers.

Syntheses of Morpholine-Based Nucleotide Analogs for Hepatic siRNA Targeting and Stabilization.

A morpholine-based nucleotide analog was developed as a building block for hepatic siRNA targeting and stabilization. Attachment of an asialoglycoprotein-binding GalNAc ligand at the morpholine nitrogen was realized with different linkers. The obtained morpholino GalNAc scaffolds were coupled to the sense strand of a transthyretin-targeting siRNA and tested for their knockdown potency in vitro and in vivo. A clear structure-activity relationship was developed with regard to the linker type and length as well as the attachment site of the morpholino GalNAc moieties at the siRNA sense strand. Further, simple alkylation of the morpholine nitrogen led to a nucleotide analog, which increased siRNA stability, when used as a double 3'-overhang at the sense strand sequence. Combination of the best morpholino GalNAc building blocks as targeting nucleotides with an optimized stabilizing alkyl-substituted morpholine as 3'-overhangs resulted in siRNAs without any phosphorothioate stabilization in the sense strand and clearly improved the duration of action in vivo.

Nanobody-siRNA Conjugates for Targeted Delivery of siRNA to Cancer Cells.

Targeted extrahepatic delivery of siRNA remains a challenging task in the field of nucleic acid therapeutics. An ideal delivery tool must internalize siRNA exclusively into the cells of interest without affecting the silencing activity of siRNA. Here, we report the use of anti-EGFR Nanobodies (trademark of Ablynx N.V.) as tools for targeted siRNA delivery. A straightforward procedure for site-specific conjugation of siRNA to an engineered C-terminal cysteine residue on the Nanobody (trademark of Ablynx N.V.) is described. We show that siRNA-conjugated Nanobodies (Nb-siRNA) retain their binding to EGFR and enter EGFR-positive cells via receptor-mediated endocytosis. The activity of Nb-siRNAs was assessed by measuring the knockdown of a housekeeping gene (AHSA1) in EGFR-positive and EGFR-negative cells. We demonstrate that Nb-siRNAs are active in vitro and induce mRNA cleavage in the targeted cell line. In addition, we discuss the silencing activity of siRNA conjugated to fused Nbs with various combinations of EGFR-binding building blocks. Finally, we compare the performance of Nb-siRNA joined by four different linkers and discuss the advantages and limitations of using cleavable and noncleavable linkers in the context of Nanobody-mediated siRNA delivery.

Liver-Specific Knockdown of Class IIa HDACs Has Limited Efficacy on Glucose Metabolism but Entails Severe Organ Side Effects in Mice.

Histone deacetylases (HDACs) are important regulators of epigenetic gene modification that are involved in the transcriptional control of metabolism. In particular class IIa HDACs have been shown to affect hepatic gluconeogenesis and previous approaches revealed that their inhibition reduces blood glucose in type 2 diabetic mice. In the present study, we aimed to evaluate the potential of class IIa HDAC inhibition as a therapeutic opportunity for the treatment +of metabolic diseases. For that, siRNAs selectively targeting HDAC4, 5 and 7 were selected and used to achieve a combinatorial knockdown of these three class IIa HDAC isoforms. Subsequently, the hepatocellular effects as well as the impact on glucose and lipid metabolism were analyzed in vitro and in vivo. The triple knockdown resulted in a statistically significant decrease of gluconeogenic gene expression in murine and human hepatocyte cell models. A similar HDAC-induced downregulation of hepatic gluconeogenesis genes could be achieved in mice using a liver-specific lipid nanoparticle siRNA formulation. However, the efficacy on whole body glucose metabolism assessed by pyruvate-tolerance tests were only limited and did not outweigh the safety findings observed by histopathological analysis in spleen and kidney. Mechanistically, Affymetrix gene expression studies provide evidence that class IIa HDACs directly target other key factors beyond the described forkhead box (FOXP) transcription regulators, such as hepatocyte nuclear factor 4 alpha (HNF4a). Downstream of these factors several additional pathways were regulated not merely including glucose and lipid metabolism and transport. In conclusion, the liver-directed combinatorial knockdown of HDAC4, 5 and 7 by therapeutic siRNAs affected multiple pathways in vitro, leading in vivo to the downregulation of genes involved in gluconeogenesis. However, the effects on gene expression level were not paralleled by a significant reduction of gluconeogenesis in mice. Combined knockdown of HDAC isoforms was associated with severe adverse effects in vivo, challenging this approach as a treatment option for chronic metabolic disorders like type 2 diabetes.

Utility of the RIG-I Agonist Triphosphate RNA for Melanoma Therapy.

The pattern recognition receptor RIG-I plays an important role in the recognition of nonself RNA and antiviral immunity. RIG-I's natural ligand, triphosphate RNA (ppp-RNA), is proposed to be a valuable addition to the growing arsenal of cancer immunotherapy treatment options. In this study, we present comprehensive data validating the concept and utility of treatment with synthetic RIG-I agonist ppp-RNA for the therapy of human cancer, with melanoma as potential entry indication amenable to intratumoral treatment. Using mRNA expression data of human tumors, we demonstrate that RIG-I expression is closely correlated to cellular and cytokine immune activation in a wide variety of tumor types. Furthermore, we confirm susceptibility of cancer cells to ppp-RNA treatment in different cellular models of human melanoma, revealing unexpected heterogeneity between cell lines in their susceptibility to RNA agonist features, including sequence, secondary structures, and presence of triphosphate. Cellular responses to RNA treatment (induction of type I IFN, FasR, MHC-I, and cytotoxicity) were demonstrated to be RIG-I dependent using KO cells. Following ppp-RNA treatment of a mouse melanoma model, we observed significant local and systemic antitumor effects and survival benefits. These were associated with type I IFN response, tumor cell apoptosis, and innate and adaptive immune cell activation. For the first time, we demonstrate systemic presence of tumor antigen-specific CTLs following treatment with RIG-I agonists. Despite potential challenges in the generation and formulation of potent RIG-I agonists, ppp-RNA or analogues thereof have the potential to play an important role for cancer treatment in the next wave of immunotherapy.

The role of C16:0 ceramide in the development of obesity and type 2 diabetes: CerS6 inhibition as a novel therapeutic approach.

OBJECTIVE: Ectopic fat deposition is associated with increased tissue production of ceramides. Recent genetic mouse studies suggest that specific sphingolipid C16:0 ceramide produced by ceramide synthase 6 (CerS6) plays an important role in the development of insulin resistance. However, the therapeutic potential of CerS6 inhibition not been demonstrated. Therefore, we pharmacologically investigated the selective ablation of CerS6 using antisense oligonucleotides (ASO) in obese insulin resistance animal models. METHODS: We utilized ASO as therapeutic modality, CerS6 ASO molecules designed and synthesized were initially screened for in-vitro knock-down (KD) potency and cytotoxicity. ASOs with >85% inhibition of CerS6 mRNA were selected for further investigations. Most promising ASOs verified for in-vivo KD efficacy in healthy mice. CerS6 ASO (AAGATGAGCCGCACC) was found most active with hepatic reduction of CerS6 mRNA expression. Prior to longitudinal metabolic studies, we performed a dose titration target engagement analysis with CerS6 ASO in healthy mice to select the optimal dose. Next, we utilized leptin deficiency ob/ob and high fat diet (HFD) induced obese mouse models for pharmacological efficacy study. RESULTS: CerS6 expression were significantly elevated in the liver and brown adipose, this was correlated with significantly elevated C16:0 ceramide concentrations in plasma and liver. Treatment with CerS6 ASO selectively reduced CerS6 expression by ∼90% predominantly in the liver and this CerS6 KD resulted in a significant reduction of C16:0 ceramide by about 50% in both liver and plasma. CerS6 KD resulted in lower body weight gain and accompanied by a significant reduction in whole body fat and fed/fasted blood glucose levels (1% reduction in HbA1c). Moreover, ASO-mediated CerS6 KD significantly improved oral glucose tolerance (during oGTT) and mice displayed improved insulin sensitivity. Thus, CerS6 appear to play an important role in the development of obesity and insulin resistance. CONCLUSIONS: Our investigations identified specific and selective therapeutic valid ASO for CerS6 ablation in in-vivo. CerS6 should specifically be targeted for the reduction of C16:0 ceramides, that results in amelioration of insulin resistance, hyperglycemia and obesity. CerS6 mediated C16:0 ceramide reduction could be a potentially attractive target for the treatment of insulin resistance, obesity and type 2 diabetes.

Correction: Chronic Activation of Hepatic Nrf2 Has No Major Effect on Fatty Acid and Glucose Metabolism in Adult Mice.

[This corrects the article DOI: 10.1371/journal.pone.0166110.].

Inhibition of citrate cotransporter Slc13a5/mINDY by RNAi improves hepatic insulin sensitivity and prevents diet-induced non-alcoholic fatty liver disease in mice.

OBJECTIVE: Non-alcoholic fatty liver disease is a world-wide health concern and risk factor for cardio-metabolic diseases. Citrate uptake modifies intracellular hepatic energy metabolism and is controlled by the conserved sodium-dicarboxylate cotransporter solute carrier family 13 member 5 (SLC13A5, mammalian homolog of INDY: mINDY). In Drosophila melanogaster and Caenorhabditis elegans INDY reduction decreased whole-body lipid accumulation. Genetic deletion of Slc13a5 in mice protected from diet-induced adiposity and insulin resistance. We hypothesized that inducible hepatic mINDY inhibition should prevent the development of fatty liver and hepatic insulin resistance. METHODS: Adult C57BL/6J mice were fed a Western diet (60% kcal from fat, 21% kcal from carbohydrate) ad libitum. Knockdown of mINDY was induced by weekly injection of a chemically modified, liver-selective siRNA for 8 weeks. Mice were metabolically characterized and the effect of mINDY suppression on glucose tolerance as well as insulin sensitivity was assessed with an ipGTT and a hyperinsulinemic-euglycemic clamp. Hepatic lipid accumulation was determined by biochemical measurements and histochemistry. RESULTS: Within the 8 week intervention, hepatic mINDY expression was suppressed by a liver-selective siRNA by over 60%. mINDY knockdown improved hepatic insulin sensitivity (i.e. insulin-induced suppression of endogenous glucose production) of C57BL/6J mice in the hyperinsulinemic-euglycemic clamp. Moreover, the siRNA-mediated mINDY inhibition prevented neutral lipid storage and triglyceride accumulation in the liver, while we found no effect on body weight. CONCLUSIONS: We show that inducible mINDY inhibition improved hepatic insulin sensitivity and prevented diet-induced non-alcoholic fatty liver disease in adult C57BL6/J mice. These effects did not depend on changes of body weight or body composition.

Chronic Activation of Hepatic Nrf2 Has No Major Effect on Fatty Acid and Glucose Metabolism in Adult Mice.

The transcription factor NF-E2-related factor 2 (Nrf2) induces cytoprotective genes, but has also been linked to the regulation of hepatic energy metabolism. In order to assess the pharmacological potential of hepatic Nrf2 activation in metabolic disease, Nrf2 was activated over 7 weeks in mice on Western diet using two different siRNAs against kelch-like ECH-associated protein 1 (Keap1), the inhibitory protein of Nrf2. Whole genome expression analysis followed by pathway analysis demonstrated successful knock-down of Keap1 expression and induction of Nrf2-dependent genes involved in anti-oxidative stress defense and biotransformation, proving the activation of Nrf2 by the siRNAs against Keap1. Neither the expression of fatty acid- nor carbohydrate-handling proteins was regulated by Keap1 knock-down. Metabolic profiling of the animals did also not show effects on plasma and hepatic lipids, energy expenditure or glucose tolerance. The data indicate that hepatic Keap1/Nrf2 is not a major regulator of glucose or lipid metabolism in mice.

Pau d'arco activates Nrf2-dependent gene expression via the MEK/ERK-pathway.

Pau d'arco is a plant-derived traditional medicine that acts by poorly understood molecular mechanisms. Here, we studied the effect of pau d'arco on the cytoprotective transcription factor Nrf2. An aqueous extract of pau d'arco stimulated Nrf2-dependent gene expression and led to nuclear localization of Nrf2 in vitro. Chromatographic separation and mass spectrometry of the extract identified benzene trioles or benzene tetraoles within the active fractions. The extract stimulated the mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase (MEK)/extracellular-signal-regulated kinase (ERK1/2) pathway. The pharmacological inhibition of MEK, but not of p38 mitogen-activated protein kinase, glycogen synthase kinase-3 or phosphoinositide 3-kinase was required for the activation of Nrf2-dependent gene expression by pau d'arco, but not for the nuclear translocation of Nrf2. In vivo pau d'arco increased the expression of Nrf2-target genes in the intestine. The results suggest that the activation of Nrf2 could mediate beneficial effects of pau d'arco, in particular in the intestine.

Modular activation of nuclear factor-kappaB transcriptional programs in human diabetic nephropathy.

Diabetic nephropathy (DN) is the leading cause of end-stage renal failure and a major risk factor for cardiovascular mortality in diabetic patients. To evaluate the multiple pathogenetic factors implicated in DN, unbiased mRNA expression screening of tubulointerstitial compartments of human renal biopsies was combined with hypothesis-driven pathway analysis. Expression fingerprints obtained from biopsies with histological diagnosis of DN (n = 13) and from control subjects (pretransplant kidney donors [n = 7] and minimal change disease [n = 4]) allowed us to segregate the biopsies by disease state and stage by the specific expression signatures. Functional categorization showed regulation of genes linked to inflammation in progressive DN. Pathway mapping of nuclear factor-kappaB (NF-kappaB), a master transcriptional switch in inflammation, segregated progressive from mild DN and control subjects by showing upregulation of 54 of 138 known NF-kappaB targets. The promoter regions of regulated NF-kappaB targets were analyzed using ModelInspector, and the NF-kappaB module NFKB_IRFF_01 was found to be specifically enriched in progressive disease. Using this module, the induction of eight NFKB_IRFF_01-dependant genes was correctly predicted in progressive DN (B2M, CCL5/RANTES, CXCL10/IP10, EDN1, HLA-A, HLA-B, IFNB1, and VCAM1). The identification of a specific NF-kappaB promoter module activated in the inflammatory stress response of progressive DN has helped to characterize upstream pathways as potential targets for the treatment of progressive renal diseases such as DN.

Comparative promoter analysis allows de novo identification of specialized cell junction-associated proteins.

Shared transcription factor binding sites that are conserved in distance and orientation help control the expression of gene products that act together in the same biological context. New bioinformatics approaches allow the rapid characterization of shared promoter structures and can be used to find novel interacting molecules. Here, these principles are demonstrated by using molecules linked to the unique functional unit of the glomerular slit diaphragm. An evolutionarily conserved promoter model was generated by comparative genomics in the proximal promoter regions of the slit diaphragm-associated molecule nephrin. Phylogenetic promoter fingerprints of known elements of the slit diaphragm complex identified the nephrin model in the promoter region of zonula occludens-1 (ZO-1). Genome-wide scans using this promoter model effectively predicted a previously unrecognized slit diaphragm molecule, cadherin-5. Nephrin, ZO-1, and cadherin-5 mRNA showed stringent coexpression across a diverse set of human glomerular diseases. Comparative promoter analysis can identify regulatory pathways at work in tissue homeostasis and disease processes.

Oligonucleotide-based knockdown technologies: antisense versus RNA interference.

The postgenomic era is characterized by an almost intimidating amount of information regarding the sequences and expression of previously unknown genes. In response, researchers have developed an increasing interest in functional studies. At the start of such a study, one may have little more than sequence information and bioinformatic annotation. The next step is to hypothesize a potential role in the context of a cell. Testing of the hypothesis needs to be fast, cheap, and applicable to a large number of genes. Knockdown methods that rely on binding of antisense oligonucleotides to mRNA combined with a subsequent functional assay in cell culture fulfil these requirements: sequence information is sufficient for synthesis of active inhibitors. Depending on the in vitro model chosen, knockdown of gene expression can be achieved with medium or even high throughput. The two most popular methods of knockdown in cell culture are the use of antisense oligonucleotides that rely on ribonuclease H (RNAse H)-dependent cleavage of mRNA, and RNA interference triggered by small double-stranded RNA molecules. Both methods act in a sequence-specific manner and can give efficient knockdown. In both cases, researchers struggle with nonspecific "off-target" effects and the difficulty of site selection. Studies that compare the methods differ in their judgment as to which method is superior.