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Investigating Nucleotide Deficiency-Based Genomic Instability and CRISPR/Cas9-mediated Genomic Addition for the Therapeutic Applications of Human Induced Pluripotent Stem Cells

  • Author(s): Lee, Patrick Chris
  • Advisor(s): Lipshutz, Gerald S
  • et al.
Abstract

Since the first reported generation of induced pluripotent stem cells (hiPSCs) from human somatic cells, the stem cell field has emerged as a promising avenue for both modeling and treating diseases. The difficulty in obtaining primary cell cultures has hindered the progress of disease research. Since patient-specific hiPSCs can be derived from accessible sources, such as dermal fibroblasts, and can differentiate into specialized cell types, it is possible to generate a substantial quantity of these patient-specific cells to overcome this obstacle. Furthermore, recent advances in genetic modification of hiPSCs make the goal of autologous cell transplantation even more attainable.

hiPSCs acquire genetic alterations, such as aneuploidy, through the reprogramming process and extensive passaging, jeopardizing their translation to clinical applications. We report that hiPSCs have an imbalance of deoxynucleotide triphosophate (dNTP) pools, which are required for DNA synthesis, potentially inducing replicative stress in hiPSCs. Additionally, hiPSCs exhibit higher levels of double-stranded breaks, indicating greater incidence of DNA damage. We demonstrate that genomic damage in hiPSCs can be alleviated when the cells are cultured with exogenous nucleosides, utilizing the cell’s nucleotide salvage pathway (NSP) to augment endogenous dNTP pools.

Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability.

Advances in human induced pluripotent stem cell research have allowed for the genetic modification of stem cells for potential cellular replacement therapies. We demonstrate a CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism.

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