Year: 2025
Type: article
Abstract: Background and aim. Since increased plasma ceramide levels have recently emerged as a pro-atherogenic factor, selective modulation of hepatic ceramide biosynthesis might represent a novel therapeutic approach to reduce cardiovascular risk. Methods. The efficacy of siRNA-based treatments (siPOOLs) on ceramide level modulation was tested by targeting single enzymes or couples of enzymes playing key roles in ceramide metabolism. In vitro, the effects of siRNAs on target gene expression and lipidome changes were evaluated in Hepa1c1c7 murine hepatocytes (96 hours after treatment, 6 nM). For in vivo studies, siPOOLs were encapsulated in lipid nanoparticles (LNPs) and first tested for hepatic tropism by confocal microscopy (single intraperitoneal injection of Cy5-labelled LNPs, 2 mg/kg, in C57Bl/6 male mice). In vivo gene silencing efficacy, effects on plasma and liver lipidome, and histological analyses were assessed after four intraperitoneal injections (2 mg/kg every 72 hours) in 8-week-old C57Bl/6 male mice. Results. In vitro validation identified siRNAs effective in reducing target gene expression and lowering ceramide levels in hepatocytes. Biodistribution studies showed that siRNA-LNPs accumulated primarily in the liver and in the spleen. In vivo testing showed variable silencing efficacy of siRNA-LNPs, ranging from –17% (Sptlc1) to –80% (Sptssa), depending on the target and the combination/dose administered. Despite a promising silencing effect, lipidomic analyses of plasma and liver did not show effective modulation of ceramide levels. Treated mice displayed extramedullary hematopoiesis in the spleen and, in a few cases, hepatic necrosis. Conclusions. The siRNA-LNP approach resulted in efficient silencing but did not translate into plasma ceramide modulation. A second strategy, consisting of N-acetyl galactosamine-conjugated siRNAs, will be tested in vitro and in vivo, with the aim of achieving higher efficacy and reduced toxicity.
Source: European Atherosclerosis Journal
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: diamond
OpenAlex ID: W7123633301
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