Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells

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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. Pluripotent stem cells have unlimited self-renewing capability and the potential to differentiate into virtually all cell types of the body. Pluripotent stem cells are therefore of great interest for future cell-based therapies and are already in use today for studying diseases “in a dish” and screening for new drugs. After the seminal discovery that induced pluripotent stem cells (iPS cells) can be generated by the delivery of four transcription factors into non-pluripotent cells, a tremendous amount of enthusiasm arose about the idea that patient-derived pluripotent stem cells could be obtained and genetically corrected in order to develop customized therapies for regenerative medicine. Here, we present a mouse model of acute metabolic liver failure that fulfills such criteria. First, we demonstrated by stringent assays that disease-specific iPS cells exhibited full cellular and developmental potential and the iPS cell–derived mice reproduced the phenotypes of the founding FAH−/− mice faithfully. Then, we genetically repaired the disease-specific iPS cells by lentiviral delivery of an intact gene copy, and we investigated the impact of this additional genetic manipulation on these cells. With our analyses, we ruled out major, and even minor, chromosomal aberrations in the gene-corrected iPS cells. Most importantly, we demonstrated that the gene-corrected cells maintained their full potential and we generated viable mice that were completely derived from these repaired cells via tetraploid complementation approach, and these mice were healthy, without any signs of the metabolic liver disease.

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