Nucleic acid (NA)-based drugs are promising therapeutics agents. Beyond efficacy, addressing safety concerns—particularly those specific to this class of drugs— is crucial. Here, we propose an in vitro approach to screen for potential adverse offtarget effects of NA-based drugs. Human peripheral blood mononuclear cells (PBMCs), purified from buffy coats of healthy donors, were used to investigate the ability of NAdrugs to trigger toxicity pathways and inappropriate immune stimulation. PBMCs were selected for their ability to represent potential human responses, given their likelihood of interacting with administered drugs. As proof of concept, a small interfering RNA (siRNA) targeting Ryanodine Receptor mRNA (RyR2) identified by the Italian National Center for Gene Therapy and Drugs based on RNA Technology as a potential therapeutic target for dominant catecholaminergic polymorphic ventricular tachycardia, was selected. This compound and its scramble were formulated within a calcium phosphate nanoparticlebased delivery system. Positive controls for four toxicity pathways were identified through literature review, each associated with a specific type of cellular stress: oxidative stress (tert-butyl hydroperoxide), mitochondrial stress (rotenone), endoplasmic reticulum stress (thapsigargin), and autophagy (rapamycin). These controls were used to define specific mRNA signatures triggered in PBMCs, which were subsequently used as indicators of off-target effects. To assess immune activation, the release of pro-inflammatory cytokines (interleukin-6, interleukin-8, tumor necrosis factor-α, and interferon-γ) was measured 24 h after exposure. The proposed approach provides a rapid and effective screening method for identifying potential unintended effects in a relevant human model, which also allows to address gender effects and variability in responses.

A Novel Approach for In Vitro Testing and Hazard Evaluation of Nanoformulated RyR2-Targeting siRNA Drugs Using Human PBMCs / V. Bettinsoli, G. Melzi, A. Crea, L. Degli Esposti, M. Iafisco, D. Catalucci, P. Ciana, E. Corsini. - In: LIFE. - ISSN 2075-1729. - 15:1(2025 Jan 14), pp. 95.1-95.15. [10.3390/life15010095]

A Novel Approach for In Vitro Testing and Hazard Evaluation of Nanoformulated RyR2-Targeting siRNA Drugs Using Human PBMCs

V. Bettinsoli
Primo
;
G. Melzi
Secondo
;
P. Ciana
Penultimo
;
E. Corsini
Ultimo
2025

Abstract

Nucleic acid (NA)-based drugs are promising therapeutics agents. Beyond efficacy, addressing safety concerns—particularly those specific to this class of drugs— is crucial. Here, we propose an in vitro approach to screen for potential adverse offtarget effects of NA-based drugs. Human peripheral blood mononuclear cells (PBMCs), purified from buffy coats of healthy donors, were used to investigate the ability of NAdrugs to trigger toxicity pathways and inappropriate immune stimulation. PBMCs were selected for their ability to represent potential human responses, given their likelihood of interacting with administered drugs. As proof of concept, a small interfering RNA (siRNA) targeting Ryanodine Receptor mRNA (RyR2) identified by the Italian National Center for Gene Therapy and Drugs based on RNA Technology as a potential therapeutic target for dominant catecholaminergic polymorphic ventricular tachycardia, was selected. This compound and its scramble were formulated within a calcium phosphate nanoparticlebased delivery system. Positive controls for four toxicity pathways were identified through literature review, each associated with a specific type of cellular stress: oxidative stress (tert-butyl hydroperoxide), mitochondrial stress (rotenone), endoplasmic reticulum stress (thapsigargin), and autophagy (rapamycin). These controls were used to define specific mRNA signatures triggered in PBMCs, which were subsequently used as indicators of off-target effects. To assess immune activation, the release of pro-inflammatory cytokines (interleukin-6, interleukin-8, tumor necrosis factor-α, and interferon-γ) was measured 24 h after exposure. The proposed approach provides a rapid and effective screening method for identifying potential unintended effects in a relevant human model, which also allows to address gender effects and variability in responses.
new approach methodologies; peripheral blood mononuclear cells; nucleic acid drugs; immunotoxicology; toxicity pathways; RyR2; nanoparticles
Settore BIOS-11/A - Farmacologia
14-gen-2025
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/1136095
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