CRISPR-Cas9 KO Cell Line Generation and Development of a Cell-Based Potency Assay for rAAV-FKRP Gene Therapy.
Marine GeoffroyLouna PiliValentina BuffaMaëlle CaroffAnne BigotEvelyne GicquelGrégory RoubyIsabelle RichardRomain FragnoudPublished in: Cells (2023)
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 (EC 50 ) 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.
Keyphrases
- gene therapy
- muscular dystrophy
- high throughput
- single cell
- crispr cas
- duchenne muscular dystrophy
- induced apoptosis
- clinical trial
- cell cycle arrest
- cell therapy
- genome editing
- stem cells
- signaling pathway
- transcription factor
- end stage renal disease
- ejection fraction
- endoplasmic reticulum stress
- sars cov
- cell death
- chronic kidney disease
- newly diagnosed
- machine learning
- skeletal muscle
- randomized controlled trial
- open label
- peritoneal dialysis
- pi k akt
- big data
- patient reported outcomes
- resistance training
- mass spectrometry
- deep learning
- high intensity