1:Biomedical Innovation Unit, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) and Instituto de Investigación Sanitaria Fundación Jiménez. (IIS-FJD, UAM), 28040 Madrid, Spain; 2:Molecular Oncology Unit-CIEMAT and Centro de Investigación Biomédica en Red de Cancer (CIBERONC), 28040 Madrid, Spain; 3:Pathology Department, Hospital Universitario de Canarias, Universidad La Laguna. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 38320 Tenerife, Spain

     Nowadays the only curative treatment for many inherited metabolic liver disorders is liver transplantation. Due to the shortage of donor livers, new therapeutic approaches are needed. Liver cell replacement therapy appears as a promising alternative to organ transplant. Primary Hyperoxaluria Type 1 (PH1) is a rare genetic disorder characterized by the oxalate overproduction in the liver, resulting in renal damage. It is caused by mutations in the AGXT gene, codifying for alanine:glyoxylate aminotransferase (AGT), a hepatic enzyme involved in glyoxylate metabolism. We combined site-specific gene correction and hepatic direct cell reprogramming to generate autologous phenotypically healthy induced hepatocytes (iHeps) from PH1 patient-derived fibroblasts, avoiding the potentially tumorigenic step of iPSC.

     First, we obtained specific AGXT gene corrected cells using two different CRISPR/Cas9 based strategies: 1) Accurate point mutation correction (c.853T-C) by homology-directed repair with a ssODN harboring the wild-type sequence; 2) Knock-in of an enhanced version of AGXT cDNA near the start codon of the endogenous gene. Then, we generated iHeps by lentiviral overexpression of hepatic transcription factors, directly from AGXT-corrected fibroblasts. iHeps showed a hepatic gene expression profile and hepatocyte functionality. Most important, AGXT-corrected iHeps exhibited in vitro reversion of oxalate accumulation compared to non-edited PH1-derived iHeps.

     This innovative therapy set up a potential alternative cellular source to replace endogenous deficient hepatocytes with autologous functional corrected cells for genetic liver disorders. Moreover, patient-derived iHeps constitute a useful personalized in vitro disease modeling.