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

PLoS Biol. 2011 Jul;9(7):e1001099. doi: 10.1371/journal.pbio.1001099. Epub 2011 Jul 12.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Survival
  • Cells, Cultured
  • Chromosomes / chemistry
  • Cyclohexanones / pharmacology
  • Disease Models, Animal
  • Female
  • Fetus
  • Fibroblasts / cytology
  • Fibroblasts / drug effects*
  • Genetic Complementation Test / methods*
  • Genetic Therapy / methods*
  • Genetic Vectors / pharmacology*
  • 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
  • Tetraploidy
  • Tyrosinemias* / genetics
  • Tyrosinemias* / metabolism
  • Tyrosinemias* / pathology
  • Tyrosinemias* / therapy

Substances

  • Cyclohexanones
  • Nitrobenzoates
  • Hydrolases
  • fumarylacetoacetase
  • nitisinone