Arginase deficiency is a rare
Arginase deficiency is a rare urea cycle disorder with hyperargininemia and profound neurological impairment as hallmark features. Arginase-1-deficient mouse models have been created, and they exhibit a profound lethal phenotype approximately 2 weeks after birth in the global knockout (KO) mice or after induction of tamoxifen-induced gene KO in adult mice (about 2 weeks after induced KO).21, 22, 23 Recently, we generated induced pluripotent stem cells (iPSCs) from our inducible arginase-1-deficient mouse model carrying a deletion of Arg1 exons 7 and 8 (Arg1), which results in defective function of arginase-1. We showed that the deletion was repaired using CRISPR/Cas9, in combination with an excisable piggyBac transposon system to target corrective sequences to the endogenous Arg1 locus. In the current study, we report on TALEN-mediated correction of the Arg1 Rosiglitazone maleate in iPSCs and their successful differentiation to hepatocyte-like cells (HLCs) coupled with piggyBac transposon selection technology for seamless genetic manipulation, followed by secondary expansion to propagate the repaired iPSC-derived HLCs (named iHLCs hereafter). The genetically repaired iHLCs were transplanted to restore arginase-1 expression in Arg1 mice, and survival of the arginase-1-deficient mice was extended by up to a week in some mice. Although an elevation in arginase-1 expression was observed in the iHLC-transplanted mice, urea cycle function was still considerably lower than in wild-type control mice, thus necessitating further refinement in gene-edited iHLC transplantation cell therapy.
Discussion In this study, we demonstrate TALEN-mediated gene editing to repair the dysfunctional Arg1 allele in iPSCs, in concert with transplantation-based studies. Our data reveal that TALEN-mediated site-specific genome modification in mouse iPSCs was similar in efficiency with the CRISPR/Cas9 system. Our application of TALEN-mediated gene repair highlights the feasibility and potential for gene-editing strategies using engineered cell therapies, albeit with modest improvement in survival after iHLC transplantation in the arginase-1 KO mouse model. qPCR and western blot analyses verified the presence of repaired cells expressing arginase-1. Nevertheless, transplantation of repaired iHLCs only resulted in about 5% repopulation of livers, and the lifespan of the transplanted mice could only be modestly extended by up to a week in some mice. The transplanted cells failed to fully recapitulate the normal liver distribution of arginase-1 in the correct metabolic zones, hence leading to marginal urea cycle function and elevation of blood arginine. The major challenges highlighted by this study are the low rates of engraftment and the lack of hepatocyte repopulation in the correct liver zones. There are two main metabolic zonations in the liver. Metabolic activities such as glycolysis, lipogenesis, and xenobiotic disposal preferentially localized in perivenous (PV) areas encode glutamine synthetase, GLT1 (a glutamate transporter), and RHBG (an ammonia transporter). In contrast, key enzymes of the urea cycle such as ARG1 and carbamoyl-phosphate synthase (CPS1) are preferentially expressed in the periportal (PP) regions. Intrasplenic transplantation of hepatocytes, which we employed in this study, was reported to facilitate cell integration in PP locations. We also performed partial hepatectomy to create a growth advantage for transplanted cells. However, we could not achieve optimum engraftment and functional regeneration for long-term therapeutic effects. We are not certain as to why transplanted edited cells were distributed in a scattered pattern and not in clusters as seen in our previous work and by others, since similar protocols were used for transplantation studies. Further work is required to understand these phenomena. Taking our current study and previous observations together, we reasoned that proper integration of transplanted cells in the correct metabolic zone of liver parenchyma is critical for enhancing the metabolic urea cycle capacity of the liver. The success of the hepatocyte infusion protocol is likely to depend on engraftment of sufficient numbers of hepatocytes in the PP loci for optimum arginase-1 enzyme activity. There are several questions to be addressed: (1) What is the optimal differentiation stage of iHLCs to achieve the highest level of engraftment and urea cycle function? (2) As the remaining hepatocytes after partial hepatectomy still have extensive proliferative potential, will they constrain competitive advantage for the transplanted cells? (3) Is there senescence or any immunogenic response to the newly corrected gene product of transplanted iHLCs? One of the major concerns of iPSC development is the potential tumorigenicity of iPSCs and their progeny. However, recent findings using in vitro differentiated iPSC-derived cells have sparked optimism over their therapeutic potential. There was no evidence of immune rejection of iPSCs that have matured to an adult fate, including endothelial cells, hepatocytes, and neuronal cells upon transplantation into syngeneic mice.33, 34 The discrepancies between those studies may be attributable to different iPSC lines used in their experiments. In this regard, it should be noted that in vitro differentiated gene-edited cells may have distinctive immunogenicity due to genetic manipulation and their long culture time. It remains to be determined whether iHLCs derived from gene-edited iPSCs used in our transplantation studies were immunogenic and if transient immunosuppression is required following transplant. (4) Can zonal regeneration of gene-edited hepatocytes be manipulated to improve therapeutic efficacy? Several signaling pathways have been identified to direct zonal organization including Wnt/β-catenin35, 36 and its antagonistic pathway, Ras/MAPK/Erk. Further investigation is needed to refine our current understanding in shaping desired metabolic zonation.