P17-24 Investigating immunotoxicity and stress pathway activation by Morpholino ASO C9 in Human PBMC
Year: 2025
Type: article
Abstract: In recent years the identification of specific molecular targets involved in disease onset has increasingly focused pharmacological treatments on personalized therapies. The specificity and flexibility of nucleic acids have made these molecules ideal candidates for this field, with a broad spectrum of potential applications. As with traditional drugs, the potential adverse effects of these molecules must be evaluated, particularly immunotoxicity and off-target effects. The purpose of this study was to develop an experimental platform using novel methodological approaches to investigate the potential toxicity of these drugs allowing the selection of more promising drug candidates, thereby enhancing drug safety. Peripheral blood mononuclear cells (PBMCs) purified from buffy coats of healthy donors of both sexes (5 males and 5 females) were exposed for 24 hours to four reference compounds (positive controls) selected for their ability to activate the four main toxicity pathways: I) for the oxidative stress, tert-butyl hydroperoxide (tBHP); II) for the endoplasmic reticulum stress, thapsigargin (TG); III) for the mitochondrial stress, rotenone (Rot); IV) for the autophagy, rapamycin (Rapa), and the selected drug Morpholino ASO C9, along with its control. Initial experiments were conducted to determine the concentration ranges for subsequent analyses. Cell viability was assessed via lactate dehydrogenase (LDH) release and cytofluorimetry apoptosis assays. These tests identified the maximum concentrations yielding 80% cell viability for subsequent analyses: 3.125 µM for tBHP; 2 µM for TG; 50 µM for Rot and Rapa. For the drug ASO Morpholino C9 (with negative control), viability analyses showed no cytotoxicity at all the tested concentrations (tested range 1–100 µM). As indicators of an inappropriate immune stimulation, the release of pro-inflammatory cytokines such as interleukine (IL)-6, IL-8, TNF-α, and IFN-γ was measured after 24 hours of cell exposure to drugs and positive controls using ELISA assays. The results revealed different modulation of cytokine release induced by the selected chemicals tested. Specifically, TG and Rot were able to induce an increase of TNF-α release. ASO Morpholino C9 was able to induce a slight increase in TNF-α release in both genders at the highest concentration (50 µM). Furthermore, genes modulation has been evaluated in the selected toxicity pathways through Real-Time PCR after 24-hours of treatment with ASO C9. Further experiments are ongoing to better evaluate the possible immune modulation induced by ASO C9. This work is funded by the European Union – Next-Generation EU (“PNRR M4C2-Investimento 1.4-CN00000041”).
Source: Toxicology Letters
Authors: V. Bettinsoli, G. Melzi, M. Marinovich, E. Corsini
Institutions: University of Milan, University of Naples Federico II
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,270
OpenAlex ID: W4414075624