Modelling and genetic correction of liver genetic diseases

The urea cycle is a set of biochemical reactions that converts highly toxic ammonia into urea for excretion. Deficiencies in any of the genes of the cycle can be life-threatening, with liver transplantation currently being the only definitive treatment. However, the scarcity of donor organs dictates the investigation of alternative treatments, which requires appropriate disease models, in vitro and in vivo, that faithfully recapitulate the disease pathology. Recent advancements in the field of genome engineering make interventions in the genetic code less challenging, thereby assisting in the generation of such tools, as well as raising the potential for genetic correction of these conditions. The research conducted in this thesis centres around two broad aims: the investigation of disease models and genetic correction of inherited liver disorders. Induced pluripotent stem cells (iPSC) hold great potential both for disease modelling and as a source of cells for cell therapy. However, their generation through cell reprogramming is sometimes challenging and inefficient. Therefore, in PAPER I we sought to optimize the reprogramming procedure by introducing modifications to the currently existing protocols, and managed to increase the reprogramming efficiency. IPSC could theoretically differentiate into any cell type, including hepatocytes. In order to assess the level of differentiation of the hepatocyte-like cells (HLC) generated from stem cell sources, comparisons with authentic primary liver tissues are necessary. To this end, in PAPER II we created gene expression profiles of fetal and mature (post-natal) liver tissues from a significant number of individuals. The dataset can serve as an accurate and simple assessment tool to evaluate and compare HLC, generated in different laboratories, to authentic human liver tissues. If HLC resemble the functions observed in mature primary hepatocytes, they could be used as in vitro disease models. In addition, programmable nucleases can be applied to either correct or introduce disease-causing of interest in the genome. In PAPER III, we generated iPSC from a patient with a pathogenic variant in the ornithine transcarbamylase (OTC) gene, the most common UCD, corrected the genetic defect and differentiated the cells into HLC. The correction was molecularly, as well as phenotypically confirmed by the restoration of urea cycle function. The thesis also focuses on the investigation of in vivo disease models of UCD. Specifically, in PAPERS IV and V we created liver-humanized mice with hepatocytes from patients with UCD, OTC deficiency (OTCD) or carbamoyl phosphate synthetase 1 deficiency (CPS1D). Highly repopulated animals faithfully recapitulated the clinical manifestations of the disease observed in patients, including hyperammonemia which is considered a hallmark of these UCD. Furthermore, in PAPER V, we investigated the efficacy and safety of ex vivo gene editing of primary OTCD hepatocytes. Ureagenesis was restored in vitro in edited cells, as well as in vivo as mice liver-repopulated with genetically engineered cells partially or completely reversed all markers of the disease investigated. Finally, extensive gene expression and deep sequencing analysis revealed no unspecific mutagenesis effected by the programmable nucleases, pointing out the safety of the application. In conclusion, the research work conducted in this thesis demonstrates the prospects that iPSC and humanized mice possess for the generation of models of liver genetic diseases, in vitro and in vivo. Furthermore, the emergence of genome editing technologies further enhances the aforementioned potentials, as well as raises possibilities for the treatment of liver genetic defects through genome manipulation

Correction of a urea cycle defect after ex vivo gene editing of human hepatocytes

Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient's own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective. Keywords: CRISPR; FRGN; ex vivo; genome editing; hepatocyte transplantation; liver-humanized mouse; primary hepatocytes; urea cycle disorder

A liver‐humanized mouse model of carbamoyl phosphate synthetase 1‐deficiency

A liver‐humanized mouse model for CPS1‐deficiency was generated by the high‐level repopulation of the mouse liver with CPS1‐deficient human hepatocytes. When compared with mice that are highly repopulated with CPS1‐proficient human hepatocytes, mice that are repopulated with CPS1‐deficient human hepatocytes exhibited characteristic symptoms of human CPS1 deficiency including an 80% reduction in CPS1 metabolic activity, delayed clearance of an ammonium chloride infusion, elevated glutamine and glutamate levels, and impaired metabolism of [15N]ammonium chloride into urea, with no other obvious phenotypic differences. Because most metabolic liver diseases result from mutations that alter critical pathways in hepatocytes, a model that incorporates actual disease‐affected, mutant human hepatocytes is useful for the investigation of the molecular, biochemical, and phenotypic differences induced by that mutation. The model is also expected to be useful for investigations of modified RNA, gene, and cellular and small molecule therapies for CPS1‐deficiency. Liver‐humanized models for this and other monogenic liver diseases afford the ability to assess the therapy on actual disease‐affected human hepatocytes, in vivo, for long periods of time and will provide data that are highly relevant for investigations of the safety and efficacy of gene‐editing technologies directed to human hepatocytes and the translation of gene‐editing technology to the clinic

Gene Editing Correction of a Urea Cycle Defect in Organoid Stem Cell Derived Hepatocyte-like Cells

Urea cycle disorders are enzymopathies resulting from inherited deficiencies in any genes of the cycle. In severe cases, currently available therapies are marginally effective, with liver transplantation being the only definitive treatment. Donor liver availability can limit even this therapy. Identification of novel therapeutics for genetic-based liver diseases requires models that provide measurable hepatic functions and phenotypes. Advances in stem cell and genome editing technologies could provide models for the investigation of cell-based genetic diseases, as well as the platforms for drug discovery. This report demonstrates a practical, and widely applicable, approach that includes the successful reprogramming of somatic cells from a patient with a urea cycle defect, their genetic correction and differentiation into hepatic organoids, and the subsequent demonstration of genetic and phenotypic change in the edited cells consistent with the correction of the defect. While individually rare, there is a large number of other genetic-based liver diseases. The approach described here could be applied to a broad range and a large number of patients with these hepatic diseases where it could serve as an in vitro model, as well as identify successful strategies for corrective cell-based therapy

Gene Editing Correction of a Urea Cycle Defect in Organoid Stem Cell Derived Hepatocyte-like Cells

Urea cycle disorders are enzymopathies resulting from inherited deficiencies in any genes of the cycle. In severe cases, currently available therapies are marginally effective, with liver transplantation being the only definitive treatment. Donor liver availability can limit even this therapy. Identification of novel therapeutics for genetic-based liver diseases requires models that provide measurable hepatic functions and phenotypes. Advances in stem cell and genome editing technologies could provide models for the investigation of cell-based genetic diseases, as well as the platforms for drug discovery. This report demonstrates a practical, and widely applicable, approach that includes the successful reprogramming of somatic cells from a patient with a urea cycle defect, their genetic correction and differentiation into hepatic organoids, and the subsequent demonstration of genetic and phenotypic change in the edited cells consistent with the correction of the defect. While individually rare, there is a large number of other genetic-based liver diseases. The approach described here could be applied to a broad range and a large number of patients with these hepatic diseases where it could serve as an in vitro model, as well as identify successful strategies for corrective cell-based therapy

Applying hydrodynamic pressure to efficiently generate induced pluripotent stem cells via reprogramming of centenarian skin fibroblasts.

Induced pluripotent stem cell (iPSC)-technology is an important platform in medicine and disease modeling. Physiological degeneration and disease onset are common occurrences in the aging population. iPSCs could offer regenerative medical options for age-related degeneration and disease in the elderly. However, reprogramming somatic cells from the elderly is inefficient when successful at all. Perhaps due to their low rates of replication in culture, traditional transduction and reprogramming approaches with centenarian fibroblasts met with little success. A simple and reproducible reprogramming process is reported here which enhances interactions of the cells with the viral vectors that leads to improved iPSC generation. The improved methods efficiently generates fully reprogrammed iPSC lines from 105-107 years old subjects in feeder-free conditions using an episomal, Sendai-Virus (SeV) reprogramming vector expressing four reprogramming factors. In conclusion, dermal fibroblasts from human subjects older than 100 years can be efficiently and reproducibly reprogrammed to fully pluripotent cells with minor modifications to the standard reprogramming procedures. Efficient generation of iPSCs from the elderly may provide a source of cells for the regeneration of tissues and organs with autologous cells as well as cellular models for the study of aging, longevity and age-related diseases

Applying hydrodynamic pressure to efficiently generate induced pluripotent stem cells via reprogramming of centenarian skin fibroblasts

Induced pluripotent stem cell (iPSC)-technology is an important platform in medicine and disease modeling. Physiological degeneration and disease onset are common occurrences in the aging population. iPSCs could offer regenerative medical options for age-related degeneration and disease in the elderly. However, reprogramming somatic cells from the elderly is inefficient when successful at all. Perhaps due to their low rates of replication in culture, traditional transduction and reprogramming approaches with centenarian fibroblasts met with little success. A simple and reproducible reprogramming process is reported here which enhances interactions of the cells with the viral vectors that leads to improved iPSC generation. The improved methods efficiently generates fully reprogrammed iPSC lines from 105–107 years old subjects in feeder-free conditions using an episomal, Sendai-Virus (SeV) reprogramming vector expressing four reprogramming factors. In conclusion, dermal fibroblasts from human subjects older than 100 years can be efficiently and reproducibly reprogrammed to fully pluripotent cells with minor modifications to the standard reprogramming procedures. Efficient generation of iPSCs from the elderly may provide a source of cells for the regeneration of tissues and organs with autologous cells as well as cellular models for the study of aging, longevity and age-related diseases