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Tuning siRNA Specificity through Seed Region Incorporation of Deoxyribonucleotide Stereoisomers

ABSTRACT: Precise chemical design continues to drive advances in RNA-based therapeutics. Here, we report the synthesis and site-specific incorporation of four canonical phosphoramidites (U, C, A, and G), each bearing non-natural nucleoside configurations: β-D-2′-deoxyxylonucleosides and α-L-2′-deoxyribonucleosides. These stereochemically distinct nucleoside analogs were introduced at positions 6 and 7 within siRNA seed regions. When applied to siRNAs targeting Ttr, ACTN1, and Marc1, these modifications reduced off-target gene repression in functional assays and, in several cases, in transcriptome-wide differential expression analyses, while preserving robust on-target activity. In vivo, Marc1-targeting siRNAs containing these modified nucleosides showed decreased hepatotoxicity, as evidenced by reduced serum ALT and AST levels. Collectively, these findings establish β-D-2′-deoxyxylonucleoside and α-L-2′-deoxyribonucleoside analogs as promising chemical tools for enhancing the specificity and safety of siRNA therapeutics. This work underscores the power of integrating rational nucleoside design with comprehensive functional and in vivo evaluation to advance drug development based on RNA interference (RNAi).

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE313756 | GEO | 2026/02/01

REPOSITORIES: GEO

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Project description:Non-alcoholic fatty liver disease (NAFLD) has a prevalence of ~25% worldwide, with significant public health consequences, yet few effective treatments. Human genetics can help elucidate novel biology and identify targets of new therapeutics. Genetic variants in mitochondrial amidoxime reducing component 1 (MTARC1) have been associated with NAFLD and liver-related mortality, however, its pathophysiological role and the cell type(s) mediating these effects remain unclear. We aimed to investigate how MTARC1 exerts its effects on NAFLD by integrating human genetics with in vitro and in vivo studies of mARC1 knockdown. Methods: Analyses including multi-trait colocalization and mendelian randomization were used to assess the genetic associations of MTARC1. In addition, we established an in vitro long-term primary human hepatocyte model with metabolic readouts and used the Gubra GAN-diet NASH mouse model treated with hepatocyte specific GalNAc-siRNA to understand the in vivo impacts of MTARC1. Results: We show that genetic variants within the MTARC1 locus are associated with liver enzymes, liver fat, plasma lipids and body composition and these associations are due to the same causal variant (p.A165T, rs2642438 G>A), suggesting a shared mechanism. We demonstrated that increased MTARC1 mRNA had an adverse effect on these traits using Mendelian Randomization, implying therapeutic inhibition of mARC1 could be beneficial. In vitro mARC1 knockdown decreased lipid accumulation and increased triglyceride secretion and in vivo GalNAc-siRNA mediated knockdown of mARC1 lowered hepatic, but increased plasma triglycerides. We found alterations in pathways regulating lipid metabolism and decreased secretion of 3-hydroxybutyrate upon mARC1 knockdown in vitro and in vivo. Conclusions: Collectively, our findings from human genetics, and in vitro and in vivo hepatocyte-specific mARC1 knockdown support the potential efficacy of hepatocyte-specific targeting of mARC1 for treatment of NAFLD.

Project description:Background and Aims Non-alcoholic fatty liver disease (NAFLD) has a prevalence of ~25% worldwide, with significant public health consequences yet has few effective treatments. Human genetics can help elucidate novel biology and identify targets of new therapeutics. Genetic variants in mitochondrial amidoxime reducing component 1 (MTARC1) have been associated with NAFLD and liver-related mortality, however, its pathophysiological role and the cell type(s) mediating these effects remain unclear. We aimed to investigate how MTARC1 exerts its effects on NAFLD by integrating human genetics with in vitro and in vivo studies of mARC1 knockdown. Methods Analyses including multi-trait colocalization and Mendelian randomization were used to assess the genetic associations of MTARC1. In addition, we established an in vitro long-term primary human hepatocyte model with metabolic readouts and used the Gubra GAN diet NASH mouse model treated with hepatocyte specific GalNAc-siRNA to understand the in vivo impacts of MTARC1. Results We show that genetic variants within the MTARC1 locus are associated with liver enzymes, liver fat, plasma lipids and body composition and these associations are due to the same causal variant (p.A165T, rs2642438 G>A), suggesting a shared mechanism. We demonstrated that increased MTARC1 mRNA had an adverse effect on these traits using Mendelian Randomization, implying therapeutic inhibition of mARC1 could be beneficial. In vitro mARC1 knockdown decreased lipid accumulation and increased triglyceride secretion and in vivo GalNAc-siRNA mediated knockdown of mARC1 lowered hepatic, but increased plasma triglycerides. We found alterations in pathways regulating lipid metabolism and decreased secretion of 3-hydroxybutyrate upon mARC1 knockdown in vitro and in vivo. Conclusions Collectively, our findings from human genetics, and in vitro and in vivo hepatocyte-specific mARC1 knockdown support the potential efficacy of hepatocyte-specific targeting of mARC1 for treatment of NAFLD.

Project description:IL-33 is a nuclear cytokine from the IL-1 family that plays important roles in health and disease. Under healthy conditions, IL-33 is constitutively expressed to high levels in the nucleus of producing cells in various human and mouse tissues. The extracellular function of IL-33 cytokine has been well documented, but it remains unclear whether intracellular nuclear IL-33 has additional functions in the nucleus. Here, we used a global proteomic approach based on quantification of 5000 individual proteins by high-resolution mass spectrometry to compare the extracellular and intracellular roles of IL-33 in primary human endothelial cells, a major source of IL-33 protein in human tissues. Large-scale analysis of protein expression was performed either after stimulation of the cells with the IL-33 mature form IL-3395-270 (during 6h or 24h) or after siRNA knockdown of intracellular IL-33 (two experiments, each with a different pool of distinct siRNAs, noted siRNA1 and siRNA2). In each case, proteins were fractionated by 1D SDS-PAGE in 12 gel bands, and label-free quantitative analysis was performed. The present dataset contains the files for the two experiments of knockdown of endogenous nuclear IL-33 expression: - RNA silencing strategy 1. Knockdown of endogenous nuclear IL-33 expression was performed with a pool of four distinct siRNAs (Dharmacon ON-TARGETplus SMARTpool IL-33 siRNAs) that have been specifically modified for efficient silencing of the target gene with reduced off-target effects. Cells transfected with these siRNA duplexes (si1) were compared with those transfected with the provided controls (CTsi1). Three independent biological replicates (noted _A, _B, _C) were prepared and analyzed for each condition, leading to 6 different samples. Each of them was fractionated into 12 gel bands analyzed by nanoLC-MS/MS, leading to 72 raw files. - RNA silencing strategy 2. The second knockdown strategy was based on the use of an independent pool of three siRNAs targeting IL-33, predesigned by another provider using new and critical siRNA design rules (Sigma MISSION Predesigned Il-33 siRNAs based on Rosetta siRNA design algorithm). Cells transfected with these siRNA duplexes (si2) were compared with those transfected with the provided controls (CTsi2). Three independent biological replicates (noted _A, _B, _C) were prepared and analyzed for each condition, leading to 6 different samples. Each of them was fractionated into 12 gel bands analyzed by nanoLC-MS/MS, leading to 72 raw files.

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Tuning siRNA Specificity through Seed Region Incorporation of Deoxyribonucleotide Stereoisomers

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