Individuals with the PiZZ genotype often show accumulation of the misfolded protein in hepatocytes.5 Over time, lack of A1AT in the blood leads to emphysema, whereas accumulation of misfolded A1AT in hepatocytes leads to liver fibrosis and cancer. To reduce progression of emphysema, patients can receive recombinant A1AT

protein. Strategies to reduce the accumulation of misfolded A1AT protein in hepatocytes, such as the autophagy-promoting Kinase Inhibitor Library cell line drug carbamazepine,6 are in development, but no definitive treatment is currently available. Therefore, A1AT deficiency is a promising target for hepatocyte replacement therapy with cells derived from gene-corrected autologous iPSCs. To develop a gene-correction strategy that would

be safe enough for clinical application, Yusa et al. relied on homologous recombination. Because spontaneous homologous recombination Selleckchem CH5424802 is inefficient in iPSCs,7 they used ZFNs to stimulate the process. ZFNs create double-stranded DNA breaks in a sequence-specific fashion.8 They are designed around two components, the zinc finger DNA binding motif and the FokI endonuclease. Recent insights into zinc finger DNA recognition have enabled targeting the activity of FokI to specific nucleotide sequences. Each zinc finger array recognizes approximately three base pairs but can be linked to additional arrays to recognize nine basepairs or more, thereby increasing sequence specificity. Because FokI is only active when dimerized, pairing ZFNs that recognize distinct, but adjacent sequences is typically used to further minimize off-target cleavage. ZFNs have been used to generate double-stranded DNA breaks to stimulate nonhomologous end-joining, or to induce homologous recombination with a donor sequence in a specific genomic locus. To allow specific expansion of iPSCs that underwent homologous recombination, Yusa et al. delivered a homologous

donor sequence in tandem with a drug selection cassette. Because their goal was to generate gene-corrected iPSCs with no or little additional genomic modification, they designed the selection cassette so that it could eventually be excised. For this purpose, they used piggyBac transposase. In contrast to genome MYO10 editing systems based on Cre recombinase or sleeping beauty transposase,9piggyBac affords site-specific excision without leaving behind a large footprint.10 Furthermore, piggyBac-mediated transposition is not associated with a high frequency of reintegration events.9 Yusa et al. started out with iPSC lines carrying the PiZZ genotype that were generated from patient fibroblasts by transduction with retroviruses expressing the four Yamanaka factors.11 They transfected the cells with plasmids expressing two ZFNs that targeted sequences immediately left and right of the Z mutation, respectively, and another plasmid encoding wild-type A1AT as donor sequence for homologous recombination (Fig. 1, step 1).