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CRISPR-Cas9 KO Cell Line Generation and Development of a Cell-Based Potency Assay for rAAV-FKRP Gene Therapy
Marine Geoffroy1,2, Louna Pili1,2, Valentina Buffa1,2
1Généthon, 91000 Evry-Courcouronnes, 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.
Insights
A new cell-based assay using FKRP knock-out cells and On-Cell-Western methods effectively measures the biological activity of gene therapy for Limb-Girdle Muscular Dystrophy R9. This assay ensures gene therapy product quality and consistency.
Area of Science:
- Biochemistry
- Genetics
- Cell Biology
Background:
- Limb-Girdle Muscular Dystrophy R9 (LGMDR9) is a dystroglycanopathy caused by Fukutin-related protein (FKRP) defects, impairing α-DG glycosylation and membrane integrity.
- Recombinant adeno-associated viral vector (rAAV) gene therapy shows promise for LGMDR9, with a phase 1/2 clinical trial underway.
- Regulatory approval requires robust quality controls, including potency assays to demonstrate the biological effect of gene therapy products.
Purpose of the Study:
- To develop and validate a cell-based potency assay for rAAV-FKRP gene therapy.
- To assess the functional activity and biological effect of the FKRP transgene.
- To ensure the quality and consistency of rAAV-FKRP gene therapy products for clinical use.
Main Methods:
- Generated FKRP knock-out (KO) cells lacking α-DG glycosylation using CRISPR-Cas9.
- Developed a high-throughput On-Cell-Western methodology to evaluate α-DG glycosylation restoration.
- Determined the half maximal effective concentration (EC50) to quantify biological activity and compare batches.
Main Results:
- Successfully generated FKRP KO cells with complete α-DG glycosylation deficiency.
- Established a reliable On-Cell-Western assay to measure FKRP gene therapy functional activity.
- Demonstrated the assay's ability to quantify biological activity and serve as a potency assay for batch comparison.
Conclusions:
- FKRP KO muscle cells combined with high-throughput On-Cell-Western provide a robust cell-based potency assay for rAAV-FKRP gene therapy.
- This assay is crucial for demonstrating product activity, quality, and consistency in clinical development.
- The developed method supports regulatory requirements for gene therapy product characterization.
