Abstract
A cell therapy strategy utilizing genetically-corrected induced pluripotent stem cells (iPSC) may be an attractive approach for genetic disorders such as muscular dystrophies. Methods for genetic engineering of iPSC that emphasize precision and minimize random integration would be beneficial. We demonstrate here an approach in the mdx mouse model of Duchenne muscular dystrophy that focuses on the use of site-specific recombinases to achieve genetic engineering. We employed non-viral, plasmid-mediated methods to reprogram mdx fibroblasts, using phiC31 integrase to insert a single copy of the reprogramming genes at a safe location in the genome. We next used Bxb1 integrase to add the therapeutic full-length dystrophin cDNA to the iPSC in a site-specific manner. Unwanted DNA sequences, including the reprogramming genes, were then precisely deleted with Cre resolvase. Pluripotency of the iPSC was analyzed before and after gene addition, and ability of the genetically corrected iPSC to differentiate into myogenic precursors was evaluated by morphology, immunohistochemistry, qRT-PCR, FACS analysis, and intramuscular engraftment. These data demonstrate a non-viral, reprogramming-plus-gene addition genetic engineering strategy utilizing site-specific recombinases that can be applied easily to mouse cells. This work introduces a significant level of precision in the genetic engineering of iPSC that can be built upon in future studies.
Highlights
One of the most exciting applications of our growing knowledge of stem cells is the potential to use them in cell therapy strategies for degenerative disorders
Reprogramming plasmids and strategy To achieve reprogramming, the classic Yamanaka reprogramming genes were integrated into mdx adult fibroblasts by conucleofection of two plasmids. pVI [22] encoded phiC31 integrase [23] to mediate genomic integration at native sequences. pCOBLW (Fig. 1a) contained the cDNA sequences for the murine Oct3/4, Sox2, Klf4, and cMyc genes driven by the CAG promoter and connected by 2A peptide sequences to facilitate polycistronic mRNA expression
Located loxP sites were included on pCOBLW and on the therapeutic donor plasmid, so that after integration of the therapeutic plasmid, Cre resolvase [29,30] could be used to excise the reprogramming cassette and other unnecessary sequences located between the two loxP sites (Fig. 1c)
Summary
One of the most exciting applications of our growing knowledge of stem cells is the potential to use them in cell therapy strategies for degenerative disorders. In considering which type of stem cells to employ in such therapies, pluripotent stem cells, including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) [1,2] are appealing, because they have an unlimited lifespan This feature would allow the cellular expansion needed to carry out genetic engineering methods to repair causative mutations, as well as permitting generation of the large numbers of cells needed to repair an extensive tissue target. While a variety of gene therapy and pharmacological approaches are being developed [9], the degenerative nature of muscular dystrophies makes a cell therapy approach attractive, because it has the potential to replace the muscle fibers that are lost during progression of these disorders [5]
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