1, step 2). Because the selection cassette was flanked by piggyBac TTAA recognition sequences,

most of the foreign DNA was simply removed by additional transfection of a plasmid expressing piggyBac transposase (Fig. 1, step 3). piggyBac-mediated loop out of the selection cassette left behind only a single foreign TTAA sequence Vincristine mw in the iPSC genome (Fig. 1, step 4). Importantly, Yusa et al. had positioned this TTAA sequence in the donor plasmid so that the resulting conversion from CTG to TTA in the iPSC genome maintained the wild-type A1AT amino acid code. After cells that had retained the drug selection cassette were eliminated, biallelic correction was detected in 11% of the iPSC colonies see more (Fig. 1, step 5). To establish that this strategy of PiZZ correction facilitated normal cell function, Yusa et al. differentiated the repaired iPSC lines into hepatocytes using a previously reported protocol.11 Indeed, the cells efficiently acquired characteristic functions of primary

hepatocytes and, importantly, secreted normal A1AT while lacking signs of accumulation of the mutant protein. In addition, after transplantation into immunodeficient mice with hepatocyte injury due to overexpression of urokinase plasminogen activator, hepatocytes derived from gene-corrected iPSCs formed clusters and secreted albumin into the mice’s serum. Finally, Yusa

et al. investigated the genomic integrity and thus the safety profile of corrected iPSC lines. Most of the amplifications, deletions, and mutations they detected had occurred in the process of reprogramming to pluripotency or subsequent cell culture, which is in accordance with previous reports.12-14 However, a few mutations manifested during the process of MCE gene correction. The nature of these mutations suggested that they were not the result of off-target cleavage, a known complication of ZFN-mediated gene correction,8 nor of piggyBac-mediated excision of the selection cassette. Furthermore, although these mutations occurred in protein-coding genes, they did not appear to affect the function of hepatocytes derived from the corrected iPSC lines. iPSC-derived hepatocytes also did not form tumors after transplantation, but larger numbers of recipient mice and longer observation periods are needed to conclude that these mutations do not impair safety. As a further step toward clinical application, Yusa et al. successfully used their method to correct the PiZZ genotype of iPSCs generated with Sendai viruses. In contrast to retroviruses, Sendai viruses do not integrate into the genome and are therefore considered a safer method of iPSC generation.