CRISPR-Cas9 KO Cell Line Generation and Development of a Cell-Based Potency Assay for rAAV-FKRP Gene Therapy

Author:

Geoffroy Marine12,Pili Louna12,Buffa Valentina12,Caroff Maëlle12,Bigot Anne3,Gicquel Evelyne12,Rouby Grégory12,Richard Isabelle124ORCID,Fragnoud Romain12

Affiliation:

1. Généthon, 91000 Evry-Courcouronnes, France

2. Université Paris-Saclay/Université Evry, INSERM, Généthon, Integrare Research Unit, UMR_S951, 91000 Evry, France

3. Institut de Myologie, Université Pierre et Marie Curie Paris 6, UM76 Univ. Paris 6/U974 UMR7215, CNRS Pitié-Salpétrière-INSERM, UMRS 974, 75000 Paris, France

4. Atamyo Therapeutics, 91000 Evry, France

Abstract

Limb-Girdle Muscular Dystrophy R9 (LGMDR9) is a dystroglycanopathy caused by Fukutin-related protein (FKRP) defects leading to the deficiency of α-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 α-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 α-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.

Funder

Atamyo Therapeutics and Association Française contre les Myopathies

Publisher

MDPI AG

Subject

General Medicine

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