Restoring Ureagenesis in Hepatocytes by CRISPR/Cas9-mediated Genomic Addition to Arginase-deficient Induced Pluripotent Stem Cells

Lee, Patrick; Truong, Brian; Vega-Crespo, Agustin; Gilmore, William B.; Hermann, Kip; Angarita, Stephanie; Tang, Jonathan K.; Chang, Katherine M; Wininger, Austin E.; Lam, Amanda; Schoenberg, Benjamen E.; Cederbaum, Stephen D.; Pyle, April D.; Byrne, James A.; Lipshutz, Gerald S.

doi:10.1038/mtna.2016.98

Cited by 29 publications

(14 citation statements)

References 54 publications

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“…The majority show excellent molecular correction, but fail to prove fully functional phenotypic correction resembling mature primary hepatocytes. Specifically, some researchers have used fetal, instead of adult, hepatocytes for comparison, while others fail to show the rescue of phenotype [ 40 , 41 ]. In this study, the ratio of labelled to total urea produced by unedited and edited iPSC-HLC was quantified by mass spectrometry.…”

Section: Discussionmentioning

confidence: 99%

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

Zabulica

Jakobsson

Ravaioli

et al. 2021

IJMS

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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.

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Section: Discussionmentioning

confidence: 99%

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

Zabulica

Jakobsson

Ravaioli

et al. 2021

IJMS

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“…After we submitted this manuscript, Lee et al . 41 reported a CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus to genetically modify and restore arginase activity using hyperargininemic patient-derived cells. In line with our current study, their differentiated hepatocyte-like cells co-express Afp and Alb, indicating an immature phenotype as opposed to adult hepatocytes, which is also typically found in many differentiation cultures of mouse and human cells to date.…”

Section: Discussionmentioning

confidence: 99%

Proof-of-Concept Gene Editing for the Murine Model of Inducible Arginase-1 Deficiency

Sin

Price

Ballantyne

et al. 2017

Sci Rep

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Arginase-1 deficiency in humans is a rare genetic disorder of metabolism resulting from a loss of arginase-1, leading to impaired ureagenesis, hyperargininemia and neurological deficits. Previously, we generated a tamoxifen-inducible arginase-1 deficient mouse model harboring a deletion of Arg1 exons 7 and 8 that leads to similar biochemical defects, along with a wasting phenotype and death within two weeks. Here, we report a strategy utilizing the Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system in conjunction with piggyBac technology to target and reincorporate exons 7 and 8 at the specific Arg1 locus in attempts to restore the function of arginase-1 in induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHLCs) and macrophages in vitro. While successful gene targeted repair was achieved, minimal urea cycle function was observed in the targeted iHLCs compared to adult hepatocytes likely due to inadequate maturation of the cells. On the other hand, iPSC-derived macrophages expressed substantial amounts of “repaired” arginase. Our studies provide proof-of-concept for gene-editing at the Arg1 locus and highlight the challenges that lie ahead to restore sufficient liver-based urea cycle function in patients with urea cycle disorders.

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“…Recently, conditions were refined for RGN-mediated AAVS1 -TI, and it was found that excluding Cas9 expression from G1 phase of the cell cycle significantly enhanced HDR-mediated integration at the locus [ 69 ]. The HPRT locus served as safe harbor for therapy in iPSCs of a patient with ARG1 -linked urea cycle disorder [ 70 ]. The strategy targeted exon 1 of HPRT for integration of a construct holding both a puromycin resistance expression cassette and an ARG1 cDNA driven by the EF1α promoter.…”

Section: Rare Repair: Employing the Molecular Toolkitmentioning

confidence: 99%

Rare Opportunities: CRISPR/Cas-Based Therapy Development for Rare Genetic Diseases

2019

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Rare diseases pose a global challenge, in that their collective impact on health systems is considerable, whereas their individually rare occurrence impedes research and development of efficient therapies. In consequence, patients and their families are often unable to find an expert for their affliction, let alone a cure. The tide is turning as pharmaceutical companies embrace gene therapy development and as serviceable tools for the repair of primary mutations separate the ability to create cures from underlying disease expertise. Whereas gene therapy by gene addition took decades to reach the clinic by incremental diseasespecific refinements of vectors and methods, gene therapy by genome editing in its basic form merely requires certainty about the causative mutation. Suddenly we move from concept to trial in 3 years instead of 30: therapy development in the fast lane, with all the positive and negative implications of the phrase. Since their first application to eukaryotic cells in 2013, the proliferation and refinement in particular of tools based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein (Cas) prokaryotic RNA-guided nucleases has prompted a landslide of therapy-development studies for rare diseases. An estimated thousands of orphan diseases are up for adoption, and legislative, entrepreneurial, and research initiatives may finally conspire to find many of them a good home. Here we summarize the most significant recent achievements and remaining hurdles in the application of CRISPR/Cas technology to rare diseases and take a glimpse at the exciting road ahead. Marina Kleanthous and Carsten W. Lederer contributed equally to this review.

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Restoring Ureagenesis in Hepatocytes by CRISPR/Cas9-mediated Genomic Addition to Arginase-deficient Induced Pluripotent Stem Cells

Cited by 29 publications

References 54 publications

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

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

Proof-of-Concept Gene Editing for the Murine Model of Inducible Arginase-1 Deficiency

Rare Opportunities: CRISPR/Cas-Based Therapy Development for Rare Genetic Diseases

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