Over the last few years gene editing by homologous

Over the last few years, gene editing by homologous recombination (HR) has been widely used in human alizarin to avoid undesirable transgene insertion. HR efficacy is very limited in human cells, estimated at one HR event per 106 cells; however, the potential application of HR in human cells has been enhanced considerably by sequence-specific DNA nucleases (Carroll, 2011; Porteus and Carroll, 2005). Three different gene-editing strategies can be applied: gene correction, where a mutation is exchanged directly by the wild-type sequence; knockin, where a partial cDNA is inserted in the target locus to express a chimeric mRNA formed by endogenous first exons and partial cDNA under the endogenous promoter control; and safe harbor, in which the transgene is inserted by HR in a safe place in the genome, such as AAVS1 or CCR5 loci (Garate et al., 2013). Concurrent with the application of gene editing in human cells, the generation of human induced pluripotent stem cells (iPSCs) was described (Takahashi et al., 2007; Yu et al., 2007). iPSCs possess properties of self-renewal and pluripotency that are similar to those of embryonic stem cells (ESCs), but potential alloreactivity and ethical issues associated with human ESCs are avoided. The wide reproducibility of the iPSC technology, independent of cell type and reprogramming methods, has established their great potential for future cell therapies. Additionally, patient- or disease-specific iPSCs are becoming established as in vitro systems to model diseases and to explore new therapeutic approaches. Reprogramming of easily accessible cell sources such as skin fibroblasts (Park et al., 2008), keratinocytes (Aasen et al., 2008), or even peripheral blood mononuclear cells (PB-MNCs) (Loh et al., 2009; Ye et al., 2009) has been described, and many efforts are being made to improve the safety and efficacy of the reprogramming method. Recently, iPSC generation by a Sendai viral vector platform (SeV) (Fusaki et al., 2009; Nishimura et al., 2011), even from blood cells (Nishishita et al., 2011; Seki et al., 2010), has been described as a non-integrative and highly efficient platform. The correction of patient-specific iPSCs by homologous recombination has been explored in different pathologies (Garate et al., 2013; Karakikes et al., 2015; Rio et al., 2014; Sebastiano et al., 2011; Song et al., 2015), demonstrating its feasibility and setting up gene editing for other stem cells. Herein, we have assessed the combination of cell reprogramming and gene editing for PKD correction as a first example of the possible application of these advanced technologies to metabolic diseases affecting the erythroid lineage. PKD patient-specific iPSCs were efficiently generated from PB-MNCs by an SeV non-integrative system. The PKLR gene was edited by PKLR transcription activator-like effector nucleases (TALENs) to introduce a partial codon-optimized cDNA in the second intron by HR. Surprisingly, we found allelic specificity in the HR induced by the presence of a single nucleotide exchange (SNP), demonstrating the potential to select the allele to be corrected. Significantly, a high number of erythroid cells derived from PKDiPSCs was generated and displayed the energetic imbalance characteristic of PKD patients, which was corrected after gene editing.
Results
Discussion In this work, we have shown the potential to combine cell reprograming and gene editing as a therapeutic approach for PKD patients. We generated iPSCs from PB-MNCs taken from PKD patients using a non-integrating viral system. These PKDiPSC lines were effectively gene edited via a knockin strategy at the PKLR locus, facilitated by specific PKLR TALENs. More importantly, we have demonstrated the rescue of the disease phenotype in erythroid cells derived from edited PKDiPSCs by the partial restoration of the step of the glycolysis affected in PKD and the improvement of the total ATP level in the erythroid cells derived from PKDiPSCs. The restoration of the energetic balance in erythroid cells derived from PKD patients opens up the possibility of using gene editing to treat PKD patients.