{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T04:45:29Z","timestamp":1788583529163,"version":"build-2803163510"},"reference-count":43,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2026,5,26]],"date-time":"2026-05-26T00:00:00Z","timestamp":1779753600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Lek Pharmaceuticals, d.d."}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>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\u2013response curves were generated to determine EC50 values. Results: In HEK293 cells, a higher seeding density (1 \u00d7 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.<\/jats:p>","DOI":"10.3390\/pharmaceutics18060652","type":"journal-article","created":{"date-parts":[[2026,5,26]],"date-time":"2026-05-26T13:28:21Z","timestamp":1779802101000},"page":"652","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Optimizing In Vitro Efficacy Assessment of the Antisense Oligonucleotide Nusinersen in Human Cellular Models"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5333-9177","authenticated-orcid":false,"given":"Ma\u0161a Sinreih","family":"Tisnikar","sequence":"first","affiliation":[{"name":"Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8587-3390","authenticated-orcid":false,"given":"Alja","family":"Zottel","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0692-3573","authenticated-orcid":false,"given":"Katja","family":"Kristan","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia"},{"name":"Sandoz Development Center Slovenia, Lek Pharmaceuticals d.d., Verov\u0161kova 57, 1526 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3453-4081","authenticated-orcid":false,"given":"Tea","family":"Lani\u0161nik Ri\u017ener","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e437","DOI":"10.1542\/neo.20-8-e437","article-title":"Spinal Muscular Atrophy: Past, Present, and Future","volume":"20","author":"Ross","year":"2019","journal-title":"Neoreviews"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1016\/B978-0-444-64076-5.00038-7","article-title":"Spinal muscular atrophy","volume":"Volume 148","author":"Arnold","year":"2018","journal-title":"Handbook of Clinical Neurology"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1146\/annurev-med-041217-010829","article-title":"Therapeutic Antisense Oligonucleotides are Coming of Age","volume":"70","author":"Bennett","year":"2019","journal-title":"Annu. 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