Correction of a urea cycle defect after ex vivo gene editing of human hepatocytes.
Adult
Aged
Ammonia
/ metabolism
Animals
Cells, Cultured
Child
Disease Models, Animal
Female
Gene Editing
/ methods
Gene Expression Regulation
Hepatocytes
/ chemistry
Humans
Introns
Male
Mice
Mutation
Ornithine Carbamoyltransferase
/ genetics
Ornithine Carbamoyltransferase Deficiency Disease
/ genetics
Orotic Acid
/ urine
RNA Splicing
CRISPR
FRGN
ex vivo
genome editing
hepatocyte transplantation
liver-humanized mouse
primary hepatocytes
urea cycle disorder
Journal
Molecular therapy : the journal of the American Society of Gene Therapy
ISSN: 1525-0024
Titre abrégé: Mol Ther
Pays: United States
ID NLM: 100890581
Informations de publication
Date de publication:
05 05 2021
05 05 2021
Historique:
received:
12
07
2020
revised:
17
11
2020
accepted:
12
01
2021
pubmed:
24
1
2021
medline:
15
12
2021
entrez:
23
1
2021
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 33484963
pii: S1525-0016(21)00024-1
doi: 10.1016/j.ymthe.2021.01.024
pmc: PMC8116578
pii:
doi:
Substances chimiques
Orotic Acid
61H4T033E5
Ammonia
7664-41-7
Ornithine Carbamoyltransferase
EC 2.1.3.3
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1903-1917Commentaires et corrections
Type : CommentIn
Informations de copyright
Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of interests S.C.S. has stock in Yecuris (no Yecuris animals were used in these studies).
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