CRISPR/Cas9-mediated knock-out of AGXT1 in HepG2 cells as a new in vitro model of Primary Hyperoxaluria Type 1.

Abstract

AGXT1 encodes alanine:glyoxylate aminotransferase 1 (AGT1), a liver peroxisomal pyridoxal 5'-phosphate dependent-enzyme whose deficit causes Primary Hyperoxaluria Type 1 (PH1). PH1 is a rare disease characterized by overproduction of oxalate, first leading to kidney stones formation, and possibly evolving to life-threatening systemic oxalosis. A minority of PH1 patients is responsive to pyridoxine, while the option for non-responders is liver-kidney transplantation. Therefore, huge efforts are currently focused on the identification of new therapies, including the promising approaches based on RNA silencing recently approved. Many PH1-associated mutations are missense and lead to a variety of kinetic and/or folding defects on AGT1. In this context, the availability of a reliable in vitro disease model would be essential to better understand the phenotype of known or newly-identified pathogenic variants as well as to test novel drug candidates. Here, we took advantage of the CRISPR/Cas9 technology to specifically knock-out AGXT1 in HepG2 cells, a hepatoma-derived cell model exhibiting a conserved glyoxylate metabolism. AGXT1-KO HepG2 displayed null AGT1 expression and significantly reduced transaminase activity leading to an enhanced secretion of oxalate upon glycolate challenge. Known pathogenic AGT1 variants expressed in AGXT1-KO HepG2 cells showed alteration in both protein levels and specific transaminase activity, as well as a partial mitochondrial mistargeting when associated with a common polymorphism. Notably, pyridoxine treatment was able to partially rescue activity and localization of clinically-responsive variants. Overall, our data validate AGXT1-KO HepG2 cells as a novel cellular model to investigate PH1 pathophysiology, and as a platform for drug discovery and development.

• Authors: Leonardo Gatticchi, Silvia Grottelli, Giulia Ambrosini, Gioena Pampalone, Ottavia Gualtieri, Ilaria Dando, Ilaria Bellezza, Barbara Cellini
• Journal: Biochimie (Biochimie) Vol. 202 Pg. 110-122 (Nov 2022) ISSN: 1638-6183 [Electronic] France
• PMID: 35964771 (Publication Type: Journal Article)
• Copyright: Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Chemical References

• Pyridoxine
• Transaminases
• Oxalates
• Pyridoxal Phosphate

Topics

• Humans
• Hep G2 Cells
• CRISPR-Cas Systems
• Pyridoxine (pharmacology)
• Transaminases (genetics)
• Oxalates
• Pyridoxal Phosphate