REVERSIR platform for the tailored control of GalNAc-siRNA conjugate pharmacology
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ABSTRACT:
PROVIDER: PRJNA434115 | ENA |
REPOSITORIES: ENA
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Project description:Primary mouse hepatocytes were treated with 1 nM TTR-siRNA and 10 nM scrambled or TTR-REVERSIR by in vitro free uptake for 24 h. RNA extracted with the Purelink RNA kit (ThermoFisher) was used for cDNA library preparation with the TruSeq Stranded Total RNA Library Prep Kit (Illumina) and sequenced on the NextSeq500 desktop sequencer (Illumina), all according to manufacturers’ instructions. Raw RNA-seq reads were filtered with minimal mean quality scores of 25 and minimal remaining length of 36, using fastq-mcf. Filtered reads were aligned to the Mus musculus genome (NCBIM37) using STAR with default parameters. Uniquely aligned reads were counted by featureCounts. Differential gene expression (DEG) analysis was performed by the R package DESeq2. Here, we report the rapid and potent reversal of GalNAc-siRNA-mediated RNAi activity in vivo with REVERSIR technology, which is based on short, synthetic high-affinity oligonucleotide complementary to the siRNA guide strand. The modular, sequence-specific nature of the REVERSIR platform has the potential to enhance the therapeutic profile of any long-acting GalNAc-siRNA conjugate to enable fine-tuned control of RNAi pharmacology
2018-05-02 | GSE110620 | GEO
Project description:GalNAc-transferase (GalNAc-T) isoforms modify distinct subsets of the O-glycoproteome and GalNAc-type O-glycosylation is found on most proteins trafficking through the secretory pathway in metazoan cells. The O-glycoproteome is regulated by up to 20 polypeptide GalNAc-Ts and the contributions and biological functions of individual GalNAc-Ts are poorly understood. Here, we used a zinc-finger nuclease (ZFN)-directed knockout strategy to probe the contributions of the major GalNAc-Ts (GalNAc-T1 and T2) in liver cells, and explore how the GalNAc-T repertoire quantitatively affects the O-glycoproteome. We demonstrate that the majority of the O-glycoproteome is covered by redundancy, whereas distinct subsets of substrates are modified by non-redundant functions of GalNAc-T1 and T2. Differential transcriptomic analysis indicates that loss of function of a GalNAc-T induces specific transcriptional response. The non-redundant O-glycoproteome subsets for and the transcriptional responses for each isoform appeared to be related to different cellular processes, and for the GalNAc-T2 isoform supporting a role in lipid metabolism. The results demonstrate that GalNAc-Ts have non-redundant glycosylation functions, and that these may affect distinct cellular processes. The data provides a comprehensive resource for unique substrates for individual GalNAc-Ts. Our study provides a new view on the regulation of the O-glycoproteome, suggesting that the plurality of GalNAc-Ts arose to regulate distinct protein functions and cellular processes.
2015-09-30 | E-MTAB-3844 | biostudies-arrayexpress
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Project description:To development of our gene expression, we have employed whole rhesus monkey genome microarray expression profiling as a discovery platform to identify genes with the potential to induce immune response. The peripheral blood from rhesus macaques, who were immunized in groups of three with the capsular polysaccharide (CPS antigen), carrier protein tetanus toxoid (TT) or conjugate vaccine via intramuscular injection (i.m.) in the anterolateral thigh on days 0, 30 and 60 using the formulations, were obtained on days 0, 30, 60 and 90. PBMCs were collected for microarray assays. Dynamic expression variations of eight genes (KLRC1, LGALS13, LTB4DH, NUAK1, VNN2, GALNT3, LOC710050 and LOC716305) were maintained in Hib conjugate vaccine group throughout experiment comparing with the CPS antigen group and carrier protein TT group.
2016-11-24 | GSE90481 | GEO
Project description:Preclinical mechanistic studies have pointed towards RNAi-mediated off-target effects as a major driver of hepatotoxicity for GalNAc-siRNA conjugates. Here we demonstrate that a single glycol nucleic acid (GNA) modification can substantially reduce siRNA seed-mediated binding to off-target transcripts while maintaining on-target activity. In siRNAs with established off-target effects leading to hepatotoxicity, these Enhanced Stabilization Chemistry plus (ESC+) designs exhibit a substantially improved therapeutic window in rat. We utilized this strategy to improve the safety of ALN-HBV, which exhibited dose-dependent, transient, and asymptomatic alanine aminotransferase elevations in healthy volunteers.
2022-06-08 | GSE184929 | GEO
Project description: Not available
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| S-EPMC6924545 | biostudies-literature
Project description:Biological functions of nuclear proteins are regulated by post-translational modifications (PTMs) that modulate gene expression and cellular physiology. However, the role of O-linked glycosylation (O-GalNAc) as a PTM of nuclear proteins in the human cell has not been previously reported. Here, we examined the initiation of O-GalNAc glycan biosynthesis, representing a novel PTM of nuclear proteins in the nucleus of human cells, with an emphasis on HeLa cells. Using affinity chromatography and MS analyses, we identified O-GalNAc glycosylated proteins in the nucleus and present solid evidence for O-GalNAc glycan synthesis in this organelle. The demonstration of O-GalNAc glycosylation of nuclear proteins in mammalian cells reported here has important implications for cell and chemical biology.
2019-04-17 | PXD013484 | Pride
Project description:To metastasize, a tumor cell must acquire abilities such as the capacity to colonize new tissue and evade immune surveillance. Recent evidence suggests that microRNAs can promote the evolution of malignant behaviors by regulating multiple targets simultaneously. We performed a microRNA analysis of human melanoma, an aggressively invasive cancer, and found that miR-30b/30d upregulation correlates with stage, metastatic potential of primary tumors, shorter time to recurrence and reduced overall survival. Ectopic expression of miR-30b/30d promoted the metastatic behavior of melanoma cells by directly targeting the GalNAc transferase GALNT7, resulted in increased synthesis of the immunosuppressive cytokine IL-10, and reduced immune cell activation and recruitment. These data point to a key role of miR-30b/30d and GalNAc transferases in metastasis, by simultaneously promoting cellular invasion and immune suppression. MicroRNAs are emerging as key contributors to tumor metastasis because of their ability to regulate multiple targets, and thereby alter several functions, simultaneously. We found a miRNA cluster that promotes metastasis by concurrently enhancing invasive capabilities of melanoma cells and suppressing immune surveillance mechanisms, allowing the tumor cells to migrate and invade foreign tissue. Both these effects of miR-30b/30d are mediated by direct suppression of GalNAc transferases. Aberrant glycosylation has previously been connected to tumor progression, but the underlying molecular mechanisms and their impact on specific cellular pathways are poorly understood. Our work places the control of glycosylation as a novel molecular link between tumor cell migration and immune evasion, two processes that act synergistically during metastasis. 2 different melanoma cell line, 2 biological duplicates for each cell line Differentially expressed genes (mRNAs) in response to miRNA over-expression
2011-03-08 | E-GEOD-27718 | biostudies-arrayexpress
Project description:Small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand are being evaluated in investigational clinical studies for a variety of indications. The typical development candidate selection process includes evaluation of the most active compounds for toxicity in rats at pharmacologically-exaggerated doses. The subset of GalNAc-siRNAs that show rat hepatotoxicity is not advanced to clinical development. Potential mechanisms of hepatotoxicity include toxicities associated with the intracellular accumulation of oligonucleotides and their metabolites, RNA interference (RNAi)-mediated hybridization-based off-target effects, and/or perturbation of endogenous RNAi pathways. Here we show that rodent hepatotoxicity observed at supratherapeutic exposures can be largely attributed to RNAi-mediated off-target effects, but not chemical modifications or the perturbation of RNAi pathways. Furthermore, these off-target effects can be mitigated by modulating seed-pairing using a thermally destabilizing chemical modification, which significantly improves the safety profile of a GalNAc-siRNA in rat and may minimize the occurrence of hepatotoxic siRNAs across species.
2018-01-11 | GSE108823 | GEO