{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T08:58:29Z","timestamp":1777539509872,"version":"3.51.4"},"reference-count":57,"publisher":"MDPI AG","issue":"20","license":[{"start":{"date-parts":[[2023,10,12]],"date-time":"2023-10-12T00:00:00Z","timestamp":1697068800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Atamyo Therapeutics and Association Fran\u00e7aise contre les Myopathies (AFM-T\u00e9l\u00e9thon)"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Limb-Girdle Muscular Dystrophy R9 (LGMDR9) is a dystroglycanopathy caused by Fukutin-related protein (FKRP) defects leading to the deficiency of \u03b1-DG glycosylation, essential to membrane integrity. Recombinant adeno-associated viral vector (rAAV) gene therapy offers great therapeutic promise for such neuromuscular disorders. Pre-clinical studies have paved the way for a phase 1\/2 clinical trial aiming to evaluate the safety and efficacy of FKRP gene therapy in LGMDR9 patients. To demonstrate product activity, quality, and consistency throughout product and clinical development, regulatory authorities request several quality controls, including a potency assay aiming to demonstrate and quantify the intended biological effect of the gene therapy product. In the present study, we generated FKRP knock-out (KO) cells fully depleted of \u03b1-DG glycosylation using CRISPR-Cas9 to assess the functional activity of a rAAV-FKRP gene therapy. We then developed a high-throughput On-Cell-Western methodology to evaluate the restoration of \u03b1-DG glycosylation in KO-FKRP cells and determine the biological activity of the FKRP transgene. The determination of the half maximal effective concentration (EC50) provides a method to compare the rAAV-FKRP batch using a reference standard. The generation of KO-FKRP muscle cells associated with the high-throughput On-Cell-Western technique may serve as a cell-based potency assay to assess rAAV-FKRP gene therapy products.<\/jats:p>","DOI":"10.3390\/cells12202444","type":"journal-article","created":{"date-parts":[[2023,10,12]],"date-time":"2023-10-12T07:28:44Z","timestamp":1697095724000},"page":"2444","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["CRISPR-Cas9 KO Cell Line Generation and Development of a Cell-Based Potency Assay for rAAV-FKRP Gene Therapy"],"prefix":"10.3390","volume":"12","author":[{"given":"Marine","family":"Geoffroy","sequence":"first","affiliation":[{"name":"G\u00e9n\u00e9thon, 91000 Evry-Courcouronnes, France"},{"name":"Universit\u00e9 Paris-Saclay\/Universit\u00e9 Evry, INSERM, G\u00e9n\u00e9thon, Integrare Research Unit, UMR_S951, 91000 Evry, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Louna","family":"Pili","sequence":"additional","affiliation":[{"name":"G\u00e9n\u00e9thon, 91000 Evry-Courcouronnes, France"},{"name":"Universit\u00e9 Paris-Saclay\/Universit\u00e9 Evry, INSERM, G\u00e9n\u00e9thon, Integrare Research Unit, UMR_S951, 91000 Evry, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Valentina","family":"Buffa","sequence":"additional","affiliation":[{"name":"G\u00e9n\u00e9thon, 91000 Evry-Courcouronnes, France"},{"name":"Universit\u00e9 Paris-Saclay\/Universit\u00e9 Evry, INSERM, G\u00e9n\u00e9thon, Integrare Research Unit, UMR_S951, 91000 Evry, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ma\u00eblle","family":"Caroff","sequence":"additional","affiliation":[{"name":"G\u00e9n\u00e9thon, 91000 Evry-Courcouronnes, France"},{"name":"Universit\u00e9 Paris-Saclay\/Universit\u00e9 Evry, INSERM, G\u00e9n\u00e9thon, Integrare Research Unit, UMR_S951, 91000 Evry, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anne","family":"Bigot","sequence":"additional","affiliation":[{"name":"Institut de Myologie, Universit\u00e9 Pierre et Marie Curie Paris 6, UM76 Univ. Paris 6\/U974 UMR7215, CNRS Piti\u00e9-Salp\u00e9tri\u00e8re-INSERM, UMRS 974, 75000 Paris, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Evelyne","family":"Gicquel","sequence":"additional","affiliation":[{"name":"G\u00e9n\u00e9thon, 91000 Evry-Courcouronnes, France"},{"name":"Universit\u00e9 Paris-Saclay\/Universit\u00e9 Evry, INSERM, G\u00e9n\u00e9thon, Integrare Research Unit, UMR_S951, 91000 Evry, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gr\u00e9gory","family":"Rouby","sequence":"additional","affiliation":[{"name":"G\u00e9n\u00e9thon, 91000 Evry-Courcouronnes, France"},{"name":"Universit\u00e9 Paris-Saclay\/Universit\u00e9 Evry, INSERM, G\u00e9n\u00e9thon, Integrare Research Unit, UMR_S951, 91000 Evry, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6505-446X","authenticated-orcid":false,"given":"Isabelle","family":"Richard","sequence":"additional","affiliation":[{"name":"G\u00e9n\u00e9thon, 91000 Evry-Courcouronnes, France"},{"name":"Universit\u00e9 Paris-Saclay\/Universit\u00e9 Evry, INSERM, G\u00e9n\u00e9thon, Integrare Research Unit, UMR_S951, 91000 Evry, France"},{"name":"Atamyo Therapeutics, 91000 Evry, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Romain","family":"Fragnoud","sequence":"additional","affiliation":[{"name":"G\u00e9n\u00e9thon, 91000 Evry-Courcouronnes, France"},{"name":"Universit\u00e9 Paris-Saclay\/Universit\u00e9 Evry, INSERM, G\u00e9n\u00e9thon, Integrare Research Unit, UMR_S951, 91000 Evry, France"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,10,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Kanagawa, M. 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