A Novel Approach for In Vitro Testing and Hazard Evaluation of Nanoformulated RyR2-Targeting siRNA Drugs Using Human PBMCs
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 off-target effects of NA-based drugs. Human peripheral blo...
Saved in:
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-01-01
|
| Series: | Life |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2075-1729/15/1/95 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1832588121811714048 |
|---|---|
| author | Valeria Bettinsoli Gloria Melzi Angelica Crea Lorenzo Degli Esposti Michele Iafisco Daniele Catalucci Paolo Ciana Emanuela Corsini |
| author_facet | Valeria Bettinsoli Gloria Melzi Angelica Crea Lorenzo Degli Esposti Michele Iafisco Daniele Catalucci Paolo Ciana Emanuela Corsini |
| author_sort | Valeria Bettinsoli |
| collection | DOAJ |
| description | 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 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-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. |
| format | Article |
| id | doaj-art-3ab4e2022c924a2f95d65160db16504c |
| institution | Kabale University |
| issn | 2075-1729 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Life |
| spelling | doaj-art-3ab4e2022c924a2f95d65160db16504c2025-01-24T13:38:46ZengMDPI AGLife2075-17292025-01-011519510.3390/life15010095A Novel Approach for In Vitro Testing and Hazard Evaluation of Nanoformulated RyR2-Targeting siRNA Drugs Using Human PBMCsValeria Bettinsoli0Gloria Melzi1Angelica Crea2Lorenzo Degli Esposti3Michele Iafisco4Daniele Catalucci5Paolo Ciana6Emanuela Corsini7Laboratory of Toxicology and Risk Assessment, Department of Pharmacological and Biomolecular Sciences “Rodolfo Paoletti”, Università degli Studi di Milano, 20133 Milan, ItalyLaboratory of Toxicology and Risk Assessment, Department of Pharmacological and Biomolecular Sciences “Rodolfo Paoletti”, Università degli Studi di Milano, 20133 Milan, ItalyLaboratory of Toxicology and Risk Assessment, Department of Pharmacological and Biomolecular Sciences “Rodolfo Paoletti”, Università degli Studi di Milano, 20133 Milan, ItalyDipartimento di Chimica e Chimica Industriale, Università degli Studi di Genova, 16146 Genoa, ItalyInstitute of Science, Technology and Sustainability for Ceramics (ISSMC), National Research Council (CNR), 48018 Faenza, ItalyInstitute of Genetic and Biomedical Research (IRGB), National Research Council (CNR), 20133 Milan, ItalyDepartment of Health Sciences, Università degli Studi di Milano, 20146 Milan, ItalyLaboratory of Toxicology and Risk Assessment, Department of Pharmacological and Biomolecular Sciences “Rodolfo Paoletti”, Università degli Studi di Milano, 20133 Milan, ItalyNucleic 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 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-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.https://www.mdpi.com/2075-1729/15/1/95new approach methodologiesperipheral blood mononuclear cellsnucleic acid drugsimmunotoxicologytoxicity pathwaysRyR2 |
| spellingShingle | Valeria Bettinsoli Gloria Melzi Angelica Crea Lorenzo Degli Esposti Michele Iafisco Daniele Catalucci Paolo Ciana Emanuela Corsini A Novel Approach for In Vitro Testing and Hazard Evaluation of Nanoformulated RyR2-Targeting siRNA Drugs Using Human PBMCs Life new approach methodologies peripheral blood mononuclear cells nucleic acid drugs immunotoxicology toxicity pathways RyR2 |
| title | A Novel Approach for In Vitro Testing and Hazard Evaluation of Nanoformulated RyR2-Targeting siRNA Drugs Using Human PBMCs |
| title_full | A Novel Approach for In Vitro Testing and Hazard Evaluation of Nanoformulated RyR2-Targeting siRNA Drugs Using Human PBMCs |
| title_fullStr | A Novel Approach for In Vitro Testing and Hazard Evaluation of Nanoformulated RyR2-Targeting siRNA Drugs Using Human PBMCs |
| title_full_unstemmed | A Novel Approach for In Vitro Testing and Hazard Evaluation of Nanoformulated RyR2-Targeting siRNA Drugs Using Human PBMCs |
| title_short | A Novel Approach for In Vitro Testing and Hazard Evaluation of Nanoformulated RyR2-Targeting siRNA Drugs Using Human PBMCs |
| title_sort | novel approach for in vitro testing and hazard evaluation of nanoformulated ryr2 targeting sirna drugs using human pbmcs |
| topic | new approach methodologies peripheral blood mononuclear cells nucleic acid drugs immunotoxicology toxicity pathways RyR2 |
| url | https://www.mdpi.com/2075-1729/15/1/95 |
| work_keys_str_mv | AT valeriabettinsoli anovelapproachforinvitrotestingandhazardevaluationofnanoformulatedryr2targetingsirnadrugsusinghumanpbmcs AT gloriamelzi anovelapproachforinvitrotestingandhazardevaluationofnanoformulatedryr2targetingsirnadrugsusinghumanpbmcs AT angelicacrea anovelapproachforinvitrotestingandhazardevaluationofnanoformulatedryr2targetingsirnadrugsusinghumanpbmcs AT lorenzodegliesposti anovelapproachforinvitrotestingandhazardevaluationofnanoformulatedryr2targetingsirnadrugsusinghumanpbmcs AT micheleiafisco anovelapproachforinvitrotestingandhazardevaluationofnanoformulatedryr2targetingsirnadrugsusinghumanpbmcs AT danielecatalucci anovelapproachforinvitrotestingandhazardevaluationofnanoformulatedryr2targetingsirnadrugsusinghumanpbmcs AT paolociana anovelapproachforinvitrotestingandhazardevaluationofnanoformulatedryr2targetingsirnadrugsusinghumanpbmcs AT emanuelacorsini anovelapproachforinvitrotestingandhazardevaluationofnanoformulatedryr2targetingsirnadrugsusinghumanpbmcs AT valeriabettinsoli novelapproachforinvitrotestingandhazardevaluationofnanoformulatedryr2targetingsirnadrugsusinghumanpbmcs AT gloriamelzi novelapproachforinvitrotestingandhazardevaluationofnanoformulatedryr2targetingsirnadrugsusinghumanpbmcs AT angelicacrea novelapproachforinvitrotestingandhazardevaluationofnanoformulatedryr2targetingsirnadrugsusinghumanpbmcs AT lorenzodegliesposti novelapproachforinvitrotestingandhazardevaluationofnanoformulatedryr2targetingsirnadrugsusinghumanpbmcs AT micheleiafisco novelapproachforinvitrotestingandhazardevaluationofnanoformulatedryr2targetingsirnadrugsusinghumanpbmcs AT danielecatalucci novelapproachforinvitrotestingandhazardevaluationofnanoformulatedryr2targetingsirnadrugsusinghumanpbmcs AT paolociana novelapproachforinvitrotestingandhazardevaluationofnanoformulatedryr2targetingsirnadrugsusinghumanpbmcs AT emanuelacorsini novelapproachforinvitrotestingandhazardevaluationofnanoformulatedryr2targetingsirnadrugsusinghumanpbmcs |