20.500.12556/RUL-183982 Optimizing in vitro efficacy assessment of the antisense oligonucleotide nusinersen in human cellular models Background/Objectives: Spinal muscular atrophy (SMA) is a rare genetic disorder caused by mutations or deletions in SMN1, resulting in the loss of SMN protein and severe neuromuscular consequences. Nusinersen, an antisense oligonucleotide that promotes full-length SMN2 transcript formation, has significantly improved SMA outcomes. However, standardized in vitro procedures for evaluating nusinersen efficacy remain limited. This study aimed to optimize in vitro efficacy assessment of nusinersen across two human cellular models. Methods: Experiments were performed using HEK293 cells and the SMA patient-derived fibroblast line GM03813. Transfection conditions were optimized for each model. In HEK293 cells, several seeding densities were evaluated for nucleofection, while in GM03813 fibroblasts, multiple transfection reagents and protocols were tested. Nusinersen activity was quantified at the transcript and protein levels, and dose–response curves were generated to determine EC50 values. Results: In HEK293 cells, a higher seeding density (1 × 106 cells) yielded the most efficient nucleofection. In GM03813 fibroblasts, Lipofectamine 3000 outperformed the other transfection reagents tested. Nusinersen exhibited dose-dependent effects in both models. The EC50 for transcript induction in HEK293 cells was 293 nM, whereas in GM03813 fibroblasts the EC50 was 10 nM, demonstrating substantial model-dependent differences in response. Conclusions: This study establishes optimized conditions for in vitro efficacy assessment of nusinersen in HEK293 and GM03813 cellular models. These protocols provide a robust and reproducible framework for evaluating nusinersen and can be readily applied to other antisense oligonucleotides designed to correct SMN2 splicing. nusinersen spinal muscular atrophy survival of motor neuron SMN antisense oligonucleotides drug validation spinalna mišična atrofija gen za preživetje motoričnih nevronov protismerni oligonukleotidi validacija zdravil true false true Angleški jezik Slovenski jezik Članek v reviji 2026-06-23 11:02:34 2026-06-23 11:02:41 2026-08-11 08:13:13 0000-00-00 00:00:00 2026 0 0 18 str. iss. 6, art. 652 Vol. 18 May 2026 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 577:615 1999-4923 10.3390/pharmaceutics18060652 280630019 RAZ_Sinreih_Masa_2026.pdf RAZ_Sinreih_Masa_2026.pdf 1 F911909FB60BA7CFA54E0AB136E44AA1 8bc5d82159e01ba4eab6774dd23ec82ee50b65bfb4cf2ea50e930cd7bb6cd7a8 9fc1100c-6ee2-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=236336 https://www.mdpi.com/1999-4923/18/6/652 1 361acd94-6ee2-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=236335 Medicinska fakulteta 0 0 0