title: Generation of healthy mice from gene-corrected disease-specific induced pluripotent stem cells.
abstract: Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH⁻/⁻ mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH⁻/⁻-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH⁻/⁻ iPS cell lines, we aggregated FAH⁻/⁻-iPS cells with tetraploid embryos and obtained entirely FAH⁻/⁻-iPS cell-derived mice that were viable and exhibited the phenotype of the founding FAH⁻/⁻ mice. Then, we transduced FAH cDNA into the FAH⁻/⁻-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell-derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models.
authors.affiliation: Max-Planck-Institute for Molecular Biomedicine, Münster, Germany.
authors.forename: Guangming
authors.initials: G
authors.lastname: Wu
authors.forename: Na
authors.initials: N
authors.lastname: Liu
authors.forename: Ina
authors.initials: I
authors.lastname: Rittelmeyer
authors.forename: Amar Deep
authors.initials: AD
authors.lastname: Sharma
authors.forename: Malte
authors.initials: M
authors.lastname: Sgodda
authors.forename: Holm
authors.initials: H
authors.lastname: Zaehres
authors.forename: Martina
authors.initials: M
authors.lastname: Bleidissel
authors.forename: Boris
authors.initials: B
authors.lastname: Greber
authors.forename: Luca
authors.initials: L
authors.lastname: Gentile
authors.forename: Dong Wook
authors.initials: DW
authors.lastname: Han
authors.forename: Cornelia
authors.initials: C
authors.lastname: Rudolph
authors.forename: Doris
authors.initials: D
authors.lastname: Steinemann
authors.forename: Axel
authors.initials: A
authors.lastname: Schambach
authors.forename: Michael
authors.initials: M
authors.lastname: Ott
authors.forename: Hans R
authors.initials: HR
authors.lastname: Schöler
authors.forename: Tobias
authors.initials: T
authors.lastname: Cantz
mesh_terms: D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D002875:Chromosomes / Q000737:chemistry; D003512:Cyclohexanones / Q000494:pharmacology; D004195:Disease Models, Animal; D005260:Female; D005333:Fetus; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects*; D005816:Genetic Complementation Test / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000494:pharmacology*; D006801:Humans; D006867:Hydrolases / Q000172:deficiency / Q000235:genetics; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology; D016086:Lentivirus; D008099:Liver / Q000378:metabolism / Q000473:pathology; D051379:Mice; D018345:Mice, Knockout; D009579:Nitrobenzoates / Q000494:pharmacology; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D013155:Spleen Focus-Forming Viruses / Q000737:chemistry / Q000235:genetics; D057891:Tetraploidy; D020176:Tyrosinemias / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy
chemical_list: D003512:Cyclohexanones; D009579:Nitrobenzoates; D006867:Hydrolases; C020156:fumarylacetoacetase; C077073:nitisinone