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

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.

Dynamic clonal analysis of murine hematopoietic stem and progenitor cells marked by 5 fluorescent proteins using confocal and multiphoton microscopy

“…The strong internal viral promoter has been demonstrated to provide high-level constitutive expression in both hematopoietic and nonhematopoietic tissues. 13,[49][50][51] In contrast to prior studies, where serial sections were used in an attempt to colocalize hematopoietic and tissue-specific markers, such as CD45 and keratin, the use of confocal microscopy and 3D reconstruction gives much less ambiguous results. Thus far, we have not observed FPs introduced via Lin Ϫ BM cell transduction expressed in skeletal muscle fibers, cardiomyocytes, hepatocytes, epidermis, or other nonhematopoietic tissues.…”

A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny

“…However, the implementation of such strategies has been slowed down by the silencing of transgenic promoter/enhancer elements in iPSC/ESCs and their differentiated progeny [3][4][5][6][7][8]. This problem has become particularly obvious for transgenes expressed from c-retroviral or lentiviral vector constructs, which may be silenced by a number of epigenetic mechanisms such as DNA methylation or histone modification [9,10].…”

“…This problem has become particularly obvious for transgenes expressed from c-retroviral or lentiviral vector constructs, which may be silenced by a number of epigenetic mechanisms such as DNA methylation or histone modification [9,10]. Importantly, silencing of lentiviral transgenes has been observed not only in association with viral (spleen focus-forming virus, SFFV [6,11]) but also with housekeeping (truncated elongation factor 1a, EFS [12]) and even tissue-specific (transthyretin [7]) promoter elements. Ubiquitous chromatin opening elements (UCOEs) have been identified as regulators of heterochromatin structure [13,14] and specifically the A2UCOE derived from the heterogeneous nuclear ribonucleoproteins A2 B1/chromobox protein homolog 3 (HNRPA2B1-CBX3) housekeeping gene locus [13,15] has been demonstrated to effectively negate CpG methylationmediated transgene silencing of c-retro-and lentiviral gene therapy vectors in chinese hamster ovary (CHO) cells or the hypermethylating teratocarcinoma cell line P19 [16,17].…”

Stem Cell Therapy for Liver Diseases

“…Genetic liver disease can be cured by skin stem cells: Scientists believe to mend or repair the defective genes in stem cells, which create various types of tissues in the body, could acclaim a new era of therapies for a wide range of genetic disorders [34]. By producing a certain protein, the researchers targeted a gene that protects against inflammation in the liver.…”