Targeted gene therapy in human-induced pluripotent stem cells from a patient with primary hyperoxaluria type 1 using CRISPR/Cas9 technology.

Abstract

Primary hyperoxaluria type 1 (PH1) is an inherited metabolic disorder caused by a deficiency of the peroxisomal enzyme alanine-glyoxylate aminotransferase (AGT), which leads to overproduction of oxalate by the liver and results in urolithiasis, nephrocalcinosis and renal failure. The only curative treatment for PH1 is combined liver and kidney transplantation, which is limited by the lack of suitable organs, significant complications, and the life-long requirement for immunosuppressive agents to maintain organ tolerance. Hepatocyte-like cells (HLCs) generated from CRISPR/Cas9 genome-edited human-induced pluripotent stem cells would offer an attractive unlimited source of autologous gene-corrected liver cells as an alternative to orthotopic liver transplantation (OLT). Here we report the CRISPR/Cas9 nuclease-mediated gene targeting of a single-copy AGXT therapeutic minigene into the safe harbour AAVS1 locus in PH1-induced pluripotent stem cells (PH1-iPSCs) without off-target inserts. We obtained a robust expression of a codon-optimized AGT in HLCs derived from AAVS1 locus-edited PH1-iPSCs. Our study provides the proof of concept that CRISPR/Cas9-mediated integration of an AGXT minigene into the AAVS1 safe harbour locus in patient-specific iPSCs is an efficient strategy to generate functionally corrected hepatocytes, which in the future may serve as a source for an autologous cell-based gene therapy for the treatment of PH1.

• Authors: Julie Estève, Jean-Marc Blouin, Magalie Lalanne, Lamia Azzi-Martin, Pierre Dubus, Audrey Bidet, Jérôme Harambat, Brigitte Llanas, Isabelle Moranvillier, Aurélie Bedel, François Moreau-Gaudry, Emmanuel Richard
• Journal: Biochemical and biophysical research communications (Biochem Biophys Res Commun) Vol. 517 Issue 4 Pg. 677-683 (10 01 2019) ISSN: 1090-2104 [Electronic] United States
• PMID: 31402115 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2019 Elsevier Inc. All rights reserved.

Chemical References

• CRISPR-Associated Protein 9

Topics

• Animals
• Base Sequence
• CRISPR-Associated Protein 9 (metabolism)
• CRISPR-Cas Systems (genetics)
• Genetic Loci
• Genetic Therapy
• Genetic Vectors (metabolism)
• Hepatocytes (cytology)
• Humans
• Hyperoxaluria, Primary (genetics, therapy)
• Induced Pluripotent Stem Cells (pathology)
• Mice