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

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. © 2011 wu et al.