Hepatic lentiviral gene transfer is associated with clonal selection, but not with tumor formation in serially transplanted rodents.

UNLABELLED: Lentiviral (LV) vectors are promising tools for long-term genetic correction of hereditary diseases. In hematopoietic stem cell gene therapies adverse events in patients due to vector integration-associated genotoxicity have been observed. Only a few studies have explored the potential risks of LV gene therapy targeting the liver. To analyze hepatic genotoxicity in vivo, we transferred the fumarylacetoacetate hydrolase (FAH) gene by LV vectors into FAH((-/-)) mice (n = 97) and performed serial hepatocyte transplantations (four generations). The integration profile (4,349 mapped insertions) of the LV vectors was assessed by ligation-mediated polymerase chain reaction and deep sequencing. We tested whether the polyclonality of vector insertions was maintained in serially transplanted mice, linked the integration sites to global hepatocyte gene expression, and investigated the effects of LV liver gene therapy on the survival of the animals. The lifespan of in vivo gene-corrected mice was increased compared to 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) control animals and unchanged in serially transplanted animals. The integration profile (4,349 mapped insertions) remained polyclonal through all mouse generations with only mild clonal expansion. Genes close to the integration sites of expanding clones may be associated with enhanced hepatocyte proliferation capacity. CONCLUSION: We did not find evidence for vector-induced tumors. LV hepatic gene therapy showed a favorable risk profile for stable and long-term therapeutic gene expression. Polyclonality of hepatocyte regeneration was maintained even in an environment of enforced proliferation.

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.

Regulation of Liver Enriched Transcription Factors in Rat Hepatocytes Cultures on Collagen and EHS Sarcoma Matrices.

Liver-enriched transcription factors (LETF) play a crucial role in the control of liver-specific gene expression and for hepatocytes to retain their molecular and cellular functions complex interactions with extra cellular matrix (ECM) are required However, during cell isolation ECM interactions are disrupted and for hepatocytes to regain metabolic competency cells are cultured on ECM substrata. The regulation of LETFs in hepatocytes cultured on different ECM has not been studied in detail. We therefore compared two common sources of ECM and evaluated cellular morphology and hepatocyte differentiation by investigating DNA binding activity of LETFs at gene specific promoters and marker genes of hepatic metabolism. Furthermore, we studied testosterone metabolism and albumin synthesis to assess the metabolic competence of cell cultures. Despite significant difference in morphological appearance and except for HNF1ß (p<0.001) most LETFs and several of their target genes did not differ in transcript expression after Bonferroni adjustment when cultured on collagen or Matrigel. Nonetheless, Western blotting revealed HNF1ß, HNF3α, HNF3γ, HNF4α, HNF6 and the smaller subunits of C/EBPα and C/EBPß to be more abundant on Matrigel cultured cells. Likewise, DNA binding activity of HNF3α, HNF3ß, HNF4α, HNF6 and gene expression of hepatic lineage markers were increased on Matrigel cultured hepatocytes. To further investigate hepatic gene regulation, the effects of Aroclor 1254 treatment, e.g. a potent inducer of xenobiotic defense were studied in vivo and in vitro. The gene expression of C/EBP-α increased in rat liver and hepatocytes cultured on collagen and this treatment induced DNA binding activity of HNF4α, C/EBPα and C/EBPß and gene expression of CYP1A1 and CYP1A2 in vivo and in vitro. Taken collectively, two sources of ECM greatly affected hepatocyte morphology, activity of liver enriched transcription factors, hepatic gene expression and metabolic competency that should be considered when used in cell biology studies and drug toxicity testing.