From Bench to Bedside and Back: Improving the Safety of GalNAc-siRNA Conjugates Using GNA-Destabilized Seed-Pairing
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA767053 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR19503300.fastq.gz | Fastqsanger.gz | |||
| SRR19503400.fastq.gz | Fastqsanger.gz | |||
| SRR19503301.fastq.gz | Fastqsanger.gz | |||
| SRR19503401.fastq.gz | Fastqsanger.gz | |||
| SRR19503302.fastq.gz | Fastqsanger.gz |
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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.
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Project description:Preclinical mechanistic studies have pointed towards RNA interference-mediated off-target effects as a major driver of hepatotoxicity for GalNAc-siRNA conjugates. Here, we demonstrate that a single glycol nucleic acid or 2'-5'-RNA modification can substantially reduce small interfering RNA (siRNA) seed-mediated binding to off-target transcripts while maintaining on-target activity. In siRNAs with established hepatotoxicity driven by off-target effects, these novel designs with seed-pairing destabilization, termed enhanced stabilization chemistry plus (ESC+), demonstrated a substantially improved therapeutic window in rats. In contrast, siRNAs thermally destabilized to a similar extent by the incorporation of multiple DNA nucleotides in the seed region showed little to no improvement in rat safety suggesting that factors in addition to global thermodynamics play a role in off-target mitigation. We utilized the ESC+ strategy to improve the safety of ALN-HBV, which exhibited dose-dependent, transient and asymptomatic alanine aminotransferase elevations in healthy volunteers. The redesigned ALN-HBV02 (VIR-2218) showed improved specificity with comparable on-target activity and the program was reintroduced into clinical development.
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Project description:Overcoming GNA/RNA Base-Pairing Limitations Using Isonucleotides Improves the Pharmacodynamic Activity of ESC+ GalNAc-siRNAs
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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.
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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
Project description:Objectives Pyrrolobenzodiazepine (PBD) dimers, tethered through inert propyldioxy or pentyldioxy linkers, possess potent bactericidal activity against a range of Gram-positive bacteria by virtue of their capacity to cross-link duplex DNA in sequence-selective fashion. Here we attempt to improve the antibacterial activity and cytotoxicity profile of PBD-containing conjugates by extension of dimer linkers and replacement of one PBD unit with phenyl-substituted or benzo-fused heterocycles that facilitate non-covalent interactions with duplex DNA. Methods DNase I footprinting was used to identify high-affinity DNA binding sites. A staphylococcal gene microarray was used to assess epidemic methicillin-resistant Staphylococcus aureus 16 phenotypes induced by PBD conjugates. Molecular dynamics simulations were employed to investigate the accommodation of compounds within the DNA helix. Results Increasing the length of the linker in PBD dimers led to a progressive reduction in antibacterial activity, but not in their cytotoxic capacity. Complex patterns of DNA binding were noted for extended PBD dimers. Modelling of DNA strand cross-linking by PBD dimers indicated distortion of the helix. A majority (26 of 43) of PBD-biaryl conjugates possessed potent antibacterial activity with little or no helical distortion and a more favourable cytotoxicity profile. Bactericidal activity of PBD-biaryl conjugates was determined by inability to excise covalently bound drug molecules from bacterial duplex DNA. Conclusions PBD-biaryl conjugates have a superior antibacterial profile compared with PBD dimers such as ELB-21. We have identified six PBD-biaryl conjugates as potential drug development candidates. [Data is also available from http://bugs.sgul.ac.uk/E-BUGS-118]
2012-04-17 | E-BUGS-118 | biostudies-arrayexpress
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.
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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
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