347. CRISPR/Cas9-Based Gene Correction of Arginase-Deficient Human Induced Pluripotent Stem Cells to Recover Enzyme Function

Urea cycle disorders (UCDs) are incurable genetic diseases that affect the body's ability to produce urea, leading to hyperammonemia due to a deficiency in any one of six enzymes in the cycle. For arginase deficiency, a mutation in the ARG1 gene, the final step of the cycle, results in hyperargininemia, developmental delays and disabilities, seizures, psychomotor function loss, and in serious cases, death. There is currently no completely effective treatment available. Advances in human induced pluripotent stem cell (hiPSC) research and genome-editing technologies have enabled the genetic modification of stem cells for potential cellular replacement therapies. In this study, we applied such technology to develop a stem cell-based approach for treating arginase deficiency applicable to all arginase deficient patients regardless of their mutation. Methods: Fibroblasts from three patients with arginase deficiency were obtained, defined for their mutation, and reprogrammed into hiPSCs. Selectable, full-length codon optimized human arginase cDNA (coARG) expression cassettes were then developed for site-specific integration into either the HPRT or albumin (ALB) locus. After confirming specificity by Sanger sequencing, genetically corrected hiPSCs were differentiated to hepatocyte-like cells. Results: Fibroblasts were reprogrammed into hiPSCs by applying a STEMCCA lentivirus-based method and were characterized for pluripotency by immunophenotyping for common stemness markers via ICC, alkaline phosphatase staining, and in vivo teratoma formation. Using a site-specific CRISPR/Cas9 nickase-mediated gene transferring system, we inserted coARG by two approaches. First, coARG was inserted into the HPRT locus under the control of constitutive hEF1α promoter (LEAPR); targeting HPRT allowed for positive clonal selection of successful on-target integration by 6-thioguanine treatment. Second, we inserted coARG into the ALB locus for expression under the endogenous ALB promoter (ALB-coARG) as ALB is highly expressed in human liver. After LEAPR and ALB-coARG modification and sequence confirmation, hiPSCs were differentiated to hepatocyte-like cells and characterized by immunophenotyping via ICC and RNA expression via RT-PCR for common hepatic markers; cells demonstrated more fetal-like characteristics. Moreover, LEAPR- and ALB-coARG-modified hepatocyte-like cells demonstrated 41% and 1% functional arginase activity recovery compared to human fetal liver, respectively. Discussion: In this study, we demonstrated the ability to genetically correct mutated ARG1 gene expression in hiPSCs derived from patients with hyperargininemia and restored arginase function in hiPSCs and hepatocyte derivatives by CRISPR/Cas9-based gene addition. As the LEAPR construct demonstrated marked coARG expression, recovery of arginase activity of ALB-ARG-modified hepatocyte-like cells was low; we expect significant arginase recovery after in vivo maturation of transplanted cells as ALB expression increases with hepatocyte maturation. Also, to demonstrate potential in vivo recovery of arginase deficiency pathogenesis, ongoing studies aim to transplant both cohorts of gene-corrected hepatocyte-like cells into an established arginase deficient immunosuppressed mouse model. Successful restoration of enzyme function in patient-specific hiPSCs and relevant cellular derivatives will highlight hiPSCs as a valuable tool in cell replacement therapies and advance applications of genetically modified hiPSCs to treat UCDs and other single enzyme liver deficiencies.