PhD Scientific Days 2026

Poster Session 2.B - Molecular Medicine

Developing platforms for characterization of immune activation by ASOs

Name of the presenter

Colar Zanjko, Laura

Institute/workplace of the presenter

BioTalentum Ltd, Gödöllő, Hungary

Authors

Laura Colar Zanjko^1,2, Andrea Balogh¹, Anna Meller¹, Máté Lengyel¹, Melinda Zana¹, András Dinnyés^1,3

1: BioTalentum Ltd, Gödöllő, Hungary
2: Department of Physiology and Animal Health, Institute of Physiology and Animal Nutrition,Antisense oligonucleotides (ASOs) are short, synthetic, single-stranded nucleic acids binding selectively to a target pre-mRNA or mRNA sequence consequently modulating gene expression via RNase H-mediated degradation, steric blockade of splicing elements, or inhibition of translation. ASOs can interact with immune cells like microglia or macrophages through phagocytosis or receptor-mediated uptake, potentially leading to cytotoxic or immunogenic responses. In our study we aimed to establish in vitro hiPSC-derived microglia and THP-1 derived macrophage-based models to assess immune activation upon ASO treatment. Utilizing immunocytochemistry and flow cytometry we characterized the microglia and THP-1 macrophages. We confirmed ASO uptake with miRNAScope™ using a chromogenic probe which hybridizes to the ASO sequence. Cytotoxic and immunogenic effects were assessed through ATP viability assays, cytokine profiling arrays and a machine-learning-based classifier utilizing TLR9 and p65 immune stainings to generate intensity- and morphology-derived parameters. Microglia showed higher cytotoxic effects after transfection with ASOs compared to THP-1 macrophages. All ASOs apart from nusinersen (Spinraza®), which is FDA and EMA approved for use in patients with spinal muscular atrophy, caused moderate cytotoxicity in microglia, while transfected THP-1 macrophages showed low levels of cytotoxicity. Elevated expression of pro-inflammatory cytokines such as TNF-α, emmprin, MIP-1α/MIP-1β and MIP-3α was found in THP-1 macrophages after uptake of ISIS 518477 and valeriasen, both known as immunogenic ASOs. ISIS 518477 uptake in microglia intensified TLR9 and p65 signal intensity and p65 nuclear translocation leading to immune activation. To conclude, both hiPSC-derived microglia and THP-1 derived macrophages present relevant and viable platforms to analyse immune activation upon ASO treatment. This project is funded by the Horizon Europe MSCA scheme, Medicine Made to Measure, under the grant number 101120256.
3: Department of Physiology and Animal Health, Institute of Physiology and Animal Nutrition, Hungarian University of Agriculture and Life Sciences, Gödöllő, Hungary