Generation of healthy mice from gene-corrected disease-specific induced pluripotent stem cells

• Published in: PLoS biology Vol. 9; no. 7; p. e1001099
• Main Authors: Wu, Guangming, Liu, Na, Rittelmeyer, Ina, Sharma, Amar Deep, Sgodda, Malte, Zaehres, Holm, Bleidissel, Martina, Greber, Boris, Gentile, Luca, Han, Dong Wook, Rudolph, Cornelia, Steinemann, Doris, Schambach, Axel, Ott, Michael, Schöler, Hans R, Cantz, Tobias
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01.07.2011; Public Library of Science (PLoS)
• Subjects: Animal models; Animals; Biology; Cell culture; Cell Survival; Cells, Cultured; Chromosomes - chemistry; Cyclohexanones - pharmacology; Disease; Disease Models, Animal; Embryos; Epigenetics; Female; Fetus; Fibroblasts - cytology; Fibroblasts - drug effects; Genetic Complementation Test - methods; Genetic engineering; Genetic Therapy - methods; Genetic Vectors - pharmacology; Genomes; Genotype & phenotype; Humans; Hydrolases - deficiency; Hydrolases - genetics; Induced Pluripotent Stem Cells - cytology; Induced Pluripotent Stem Cells - physiology; Lentivirus; Liver - metabolism; Liver - pathology; Mice; Mice, Knockout; Nitrobenzoates - pharmacology; Pregnancy; Promoter Regions, Genetic; Spleen Focus-Forming Viruses - chemistry; Spleen Focus-Forming Viruses - genetics; Stem cells; Studies; Tetraploidy; Transplants & implants; Tyrosinemias - genetics; Tyrosinemias - metabolism; Tyrosinemias - pathology; Tyrosinemias - therapy; Genetic Therapy; Spleen Focus-Forming Viruses; Nitrobenzoates; Humans; Liver; Genetic Complementation Test; Tetraploidy; Fibroblasts; Female; Fetus; Chromosomes; Cyclohexanones; Disease Models, Animal; Promoter Regions, Genetic; Cell Survival; Cells, Cultured; Lentivirus; Tyrosinemias; Mice, Knockout; Pregnancy; Animals; Hydrolases; Mice; Genetic Vectors; Induced Pluripotent Stem Cells
• ISSN: 1545-7885; 1544-9173; 1545-7885
• DOI: 10.1371/journal.pbio.1001099

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.