Gene editing and hepatic direct cell reprogramming for the treatment of Primary Hyperoxaluria type 1

Resumen

Primary Hyperoxaluria type 1 (PH1) is a rare autosomal recessive inherited metabolic liver disorder caused by mutations in the AGXT gene, coding for the hepatic peroxisomal enzyme alanine-glyoxylate aminotransferase (AGT). This enzyme catalyzes the conversion of glyoxylate to glycine in the liver. Due to the deficiency in the AGT enzyme, the excess of generated glyoxylate is converted to oxalate by the lactate dehydrogenase enzyme. Oxalate is an end-product metabolite that cannot be metabolized by mammals and is excreted by urine. The excessive oxalate forms non-soluble calcium oxalate crystals that accumulate mainly in the kidney resulting in progressive renal damage, but also in other tissues leading to systemic oxalosis. Nowadays the only definitive curative treatment is the combined liver-kidney transplantation. However, in addition to the shortage of donor livers, these patients must be under immunosuppressive treatment for their entire life. For these reasons, the development of alternative therapeutic approaches is needed. In this Doctoral Thesis, we explore the possibility of correcting fibroblasts derived from skin biopsies of PH1 patients by gene editing, followed by direct cell reprogramming of these AGXT-corrected cells to generate autologous healthy induced-Hepatocytes (iHeps). We conducted specific correction of AGXT gene at the endogenous locus by homology-directed repair (HDR) assisted by CRISPR/Cas9 nuclease, following two different approaches. On one hand, we performed precise correction of p.I244T point mutation in exon 7, the second most frequent mutation in PH1 patients and the most abundant in the Canary Islands, where a founder effect has been described. We demonstrated the feasibility of precise correction of this mutation in PH1 patient-derived fibroblasts by electroporation of a specific ribonucleoprotein (RNP) and a single-stranded oligodeoxynucleotide (ssODN) with the corrective sequence. On the other hand, we developed a knock-in strategy, applicable to almost all PH1 patients, with different mutations along the entire AGXT gene. This strategy consists in the targeted integration of an enhanced version of AGXT cDNA (hAGXT-RHEAM) at the ATG start codon of the endogenous locus. We obtained AGXT targeted knock-in clones by electroporation of a specific RNP and different designed plasmid donors in PH1 patient-derived fibroblasts. AGXT-corrected fibroblast clones were then directly reprogrammed into induced-Hepatocytes by lentiviral transduction of four hepatic transcription factors: FOXA2, HNF1α, HNF4α and TBX3. Fibroblasts from healthy donor and from uncorrected PH1 patient-derived fibroblasts were reprogrammed in parallel. Generated iHeps expressed specific hepatic genes involved in different liver functions and accumulated glycogen. We demonstrated in vitro reversion of oxalate accumulation in AGXT gene-corrected iHeps, in line with the induction of AGXT expression, when compared with their corresponding uncorrected PH1-derived iHeps. Our results indicate that homozygous knock-in correction with the enhanced hAGXT-RHEAM is the best option for the reversion of the PH1 pathological oxalate accumulation in gene edited iHeps. This work demonstrates the feasibility of generating induced-Hepatocytes with in vitro glyoxylate metabolic capacity by specific correction of AGXT gene and hepatic direct cell reprogramming of PH1 patient-derived fibroblasts. This cellular product will have to be evaluated in vivo to demonstrate their viability as an alternative cell source for liver cell replacement therapy for the treatment of PH1. In addition, these generated iHeps can be a valuable in vitro disease model, for the study of the disease as well as for other therapeutic approaches screening