PhD Scientific Days 2025

Poster Session II. - B: Molecular Medicine

Utilizing miRNAscope™: A Novel Approach to Compare Transfection Efficiency of Antisense Oligonucleotides Across Cortical Cell Models

Name of the presenter

Colar Zanjko Laura

Institute/workplace of the presenter

BioTalentum Ltd, Gödöllő, Hungary

Authors

Laura Colar Zanjko^1,2, Anna Meller¹, Andrea Balogh¹, János Farkas¹, Kriengkai Chessadangkul^1,3, Seppe Hermans⁴, Tobias Pöhlmann⁵, Willeke M. C. van Roon-Mom⁶, Melinda Zana¹, András Dinnyés^1,2

1: BioTalentum Ltd, Gödöllő, Hungary
2: Department of Physiology and Animal Health, Institute of Physiology and Animal Nutrition, Hungarian University of Agriculture and Life Sciences, Gödöllő, Hungary
3: Department of Biomedical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok, Thailand
4: BianoGMP GmbH, Gera, Thüringen, Germany
5: XNApharma GmbH, Gera, Thüringen, Germany
6: Leiden University Medical Centre, Leiden, The Netherlands

Text of the abstract

Antisense oligonucleotides (ASOs) are short, synthetic, single-stranded nucleic acid oligomers that bind with high specificity to target pre-mRNA or mRNA, thereby modulating gene expression through various mechanisms. ASOs represent a promising therapeutic approach for rare and ultra-rare genetic disorders, underscoring the need for sensitive and specific methods to evaluate their cellular uptake and functional efficacy. Traditional techniques for assessing transfection efficiency, such as qPCR or western blotting, provide only indirect or cumulative information. In contrast, RNAscope™, an advanced in situ hybridisation method, enables the visualisation of RNA at single-cell resolution using sequence-specific probes.
In this study, we assessed ASO transfection efficiency using miRNAscope™, a variant of RNAscope™ (Bio-Techne) optimised for the detection of small RNAs. Using the miRNAscope™ HD Reagent Kit in combination with fluorescence microscopy, we visualised and quantified the uptake of various ASOs, including gapmer and splice-switching variants. The cell models employed were derived from human induced pluripotent stem cells (iPSCs). These were monolayers of neural progenitor cells (NPCs), differentiated neural-astroglial co-cultures, microglial cultures, and neural-astroglial-microglial tri-cultures. To mimic the complexity of the developing brain, we incorporated neurospheroids into the experimental design. ASO delivery was tested via free uptake and lipid-mediated transfection.
Our results demonstrated dose-dependent transfection efficiency across all models, with higher uptake observed in differentiated cortical cultures than in NPCs. Quantitative analysis revealed lower signal intensity for gapmer ASOs than their splice-switching counterparts when detected with the same probe, likely reflecting reduced probe binding affinity to the gapmer structure. No differences were observed in the subcellular localisation of the ASOs; nuclear and cytoplasmic compartments were positively stained.
In conclusion, miRNAscope™ provides a robust and reliable platform for visualising ASOs in complex cortical cell models. This technique offers a sensitive and versatile tool for advancing ASO research and facilitating the development of RNA-based therapeutics.

This project is funded by the Horizon Europe MSCA scheme, MMM (No.101120256) and WhyNotDry (No.101131087).