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    • We next sought insight regarding the mechanism that

    2018-11-12

    We next sought insight regarding the mechanism that links regulation of pluripotency with that of enhanced maintenance of genetic integrity in pluripotent cells. The discovery that fully pluripotent PR619 can be derived by transduction of differentiated cells starting with four (Takahashi and Yamanaka, 2006), or fewer (Huangfu et al., 2008; Kim et al., 2009), key regulatory genes suggests that small numbers of factors control entire networks of downstream genes and gene products necessary to establish the fate of a cell. We tested the hypothesis that small numbers of pluripotency factors interact with large numbers of genetic integrity genes in pluripotent cells. We found evidence for extensive interactions between the pluripotency and genetic integrity gene networks. While further research will be needed to fully interrogate the functionality of each of the molecular interactions predicted by our analysis, the predicted interactions we report can account for the potential regulation of 22–50% of DNA repair gene and 21–35% of cell death gene differential expression in pluripotent cells. Undoubtedly, the extent of interactions between these two gene networks would be even greater if additional pluripotency factors and/or higher order interactions were considered. Indeed, the involvement of intermediary regulators linking the pluripotency and genetic integrity gene networks affords potential opportunities for additional levels of coordination of expression of downstream genetic integrity pathways.
    Conclusions In summary, our data indicate the Disposable Soma Theory (Kirkwood, 1977) applies to pluripotent cells as well as to germ cells. Further, we provide insight into the mechanism by which the pluripotency gene network interacts with the genetic integrity gene network to establish and maintain enhanced genetic integrity in pluripotent cells. This, in turn, suggests that key factors might be monitored and/or manipulated to maintain optimal genetic integrity in stem cells or their differentiated derivatives intended for use in therapeutic applications. The following are the supplementary data related to this article.
    Acknowledgments
    Introduction Duchenne muscular dystrophy (DMD) is characterized by a progressive muscle degeneration caused by mutations in one of the largest gene known, that of dystrophin (Koenig et al., 1987). There is no curative treatment at present and death usually occurs within 10 to 15years of symptom onset, typically from breathing complications and cardiomyopathy (Emery, 1993). Current promising clinical and pre-clinical approaches include gene editing, exon skipping, gene therapy and stem cell therapies (Fairclough et al., 2013). Of these, exon skipping is currently most advanced, where the delivery or expression of small RNAs affecting splicing is used to remove a mutated dystrophin mRNA exon. However, it may not offer a cure for all patients, depending on the type of the dystrophin gene mutation (Brolin and Shiraishi, 2011). Other gene therapy strategies aim at the delivery of a functional dystrophin coding sequence into the dystrophic muscle of DMD patients. Viral vectors are commonly used in gene therapy for their efficient gene delivery and integration in the myofiber genomes, but their use may be limited to the expression of truncated dystrophin variants, due to cargo-size limitations of the viral capsid (Phelps et al., 1995; Duan et al., 1998). In addition, safety concerns were linked to malignant cell transformation due to insertional mutagenesis linked to certain viral vectors (Hacein-Bey-Abina et al., 2008; Lewinski and Bushman, 2005), whereas the immunogenicity of the viral particles is also a concern (Raper et al., 2003; Yei et al., 1994). Thus, alternatives to viral vector gene transfer are also currently assessed. For instance, an alternative gene transfer method is the in vivo electroporation of naked plasmids containing the dystrophin gene, which has little transgene size constraint and low immunogenicity. Plasmids or synthetic chromosomes can persist in the muscle fiber without genome integration and transgene expression can be relatively stable in the non-dividing myofibers (Puttini et al., 2009; Tedesco et al., 2011). However, the long-term expression of the transgene may be reduced by gradual silencing effects and/or by the loss of the episomal DNA.