{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,15]],"date-time":"2026-04-15T21:55:57Z","timestamp":1776290157116,"version":"3.50.1"},"reference-count":52,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,1,14]],"date-time":"2025-01-14T00:00:00Z","timestamp":1736812800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"European Union\u2014NextGeneration EU","award":["PNRR M4C2-Investimento 1.4-CN00000041-PNRR_CN3RNA_SPOKE9 and SPOKE8"],"award-info":[{"award-number":["PNRR M4C2-Investimento 1.4-CN00000041-PNRR_CN3RNA_SPOKE9 and SPOKE8"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Life"],"abstract":"<jats:p>Nucleic acid (NA)-based drugs are promising therapeutics agents. Beyond efficacy, addressing safety concerns\u2014particularly those specific to this class of drugs\u2014is crucial. Here, we propose an in vitro approach to screen for potential adverse off-target 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 NA-drugs 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 nanoparticle-based 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-\u03b1, and interferon-\u03b3) 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.<\/jats:p>","DOI":"10.3390\/life15010095","type":"journal-article","created":{"date-parts":[[2025,1,14]],"date-time":"2025-01-14T11:28:31Z","timestamp":1736854111000},"page":"95","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["A Novel Approach for In Vitro Testing and Hazard Evaluation of Nanoformulated RyR2-Targeting siRNA Drugs Using Human PBMCs"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-5511-9520","authenticated-orcid":false,"given":"Valeria","family":"Bettinsoli","sequence":"first","affiliation":[{"name":"Laboratory of Toxicology and Risk Assessment, Department of Pharmacological and Biomolecular Sciences \u201cRodolfo Paoletti\u201d, Universit\u00e0 degli Studi di Milano, 20133 Milan, Italy"},{"name":"Department of Pharmacy, Universit\u00e0 degli Studi di Napoli Federico II, 80131 Naples, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3303-6099","authenticated-orcid":false,"given":"Gloria","family":"Melzi","sequence":"additional","affiliation":[{"name":"Laboratory of Toxicology and Risk Assessment, Department of Pharmacological and Biomolecular Sciences \u201cRodolfo Paoletti\u201d, Universit\u00e0 degli Studi di Milano, 20133 Milan, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Angelica","family":"Crea","sequence":"additional","affiliation":[{"name":"Laboratory of Toxicology and Risk Assessment, Department of Pharmacological and Biomolecular Sciences \u201cRodolfo Paoletti\u201d, Universit\u00e0 degli Studi di Milano, 20133 Milan, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6596-560X","authenticated-orcid":false,"given":"Lorenzo","family":"Degli Esposti","sequence":"additional","affiliation":[{"name":"Dipartimento di Chimica e Chimica Industriale, Universit\u00e0 degli Studi di Genova, 16146 Genoa, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7813-8347","authenticated-orcid":false,"given":"Michele","family":"Iafisco","sequence":"additional","affiliation":[{"name":"Institute of Science, Technology and Sustainability for Ceramics (ISSMC), National Research Council (CNR), 48018 Faenza, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7041-6114","authenticated-orcid":false,"given":"Daniele","family":"Catalucci","sequence":"additional","affiliation":[{"name":"Institute of Genetic and Biomedical Research (IRGB), National Research Council (CNR), 20133 Milan, Italy"},{"name":"IRCCS Humanitas Research Hospital, 20089 Rozzano, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paolo","family":"Ciana","sequence":"additional","affiliation":[{"name":"Department of Health Sciences, Universit\u00e0 degli Studi di Milano, 20146 Milan, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6927-5956","authenticated-orcid":false,"given":"Emanuela","family":"Corsini","sequence":"additional","affiliation":[{"name":"Laboratory of Toxicology and Risk Assessment, Department of Pharmacological and Biomolecular Sciences \u201cRodolfo Paoletti\u201d, Universit\u00e0 degli Studi di Milano, 20133 Milan, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,1,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"673","DOI":"10.1038\/s41573-020-0075-7","article-title":"Advances in Oligonucleotide Drug Delivery","volume":"19","author":"Roberts","year":"2020","journal-title":"Nat. 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Antisense RNA Design, Delivery, and Analysis, Springer.","DOI":"10.1007\/978-1-0716-2010-6"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"226","DOI":"10.1038\/mt.2013.217","article-title":"Phase I Clinical Trial of SYL040012, a Small Interfering RNA Targeting \u03b2-Adrenergic Receptor 2, for Lowering Intraocular Pressure","volume":"22","author":"Ruz","year":"2014","journal-title":"Mol. Ther."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"6447","DOI":"10.1167\/iovs.16-20303","article-title":"Safety and Efficacy Clinical Trials for SYL1001, a Novel Short Interfering RNA for the Treatment of Dry Eye Disease","volume":"57","author":"Turman","year":"2016","journal-title":"Investig. Opthalmology Vis. 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