enough Concurrent with the application of gene editing in hu

Concurrent with the application of gene editing in human cells, the generation of human induced pluripotent stem enough (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. To reprogram patient cells, we adopted the most feasible and safest protocol using a patient cell source that is easy to obtain, PB-MNCs, and an integration-free reprogramming strategy based on SeV vectors. PB-MNCs were chosen, as blood collection is common in patient follow-up and is minimally invasive. Additionally, it is possible to recover enough PB-MNCs from a routine blood collection to perform several reprogramming experiments. Finally, previous works showed that PB-MNCs could be reprogrammed, although at a very low efficiency (Staerk et al., 2010). On the other hand, the SeV reprogramming platform has been described as a very effective, non-integrative system for iPSC reprogramming with a wide tropism for the target cells (Ban et al., 2011; Fusaki et al., 2009). Reprogrammed SeVs are cleared after cell reprogramming due to the difference of replication between newly generated iPSCs and viral mRNA (Ban et al., 2011; Fusaki et al., 2009). However, reprogrammed T or B cells might be favored when whole PB-MNCs are chosen, as these are the most abundant nucleated cell type in these samples. Reprogramming T or B cells has the risk of generating iPSCs with either TCR or immunoglobulin rearrangements, decreasing the immunological repertoire of the hematopoietic cells derived from these rearranged iPSCs. In order to avoid this possibility, we have biased the protocol against reprogramming of either T or B lymphocytes by culturing PB-MNCs with essential cytokines to favor the maintenance and proliferation of hematopoietic progenitors and myeloid cells, as previously shown for retroviral reprogramming vectors (Staerk et al., 2010). This approach was supported here by the demonstration that SeV vectors preferentially transduced hematopoietic progenitors and myeloid cells under these specific conditions and consequently none of the iPSC lines analyzed had immunoglobulin or TCR rearrangements. We further demonstrated that the generation of iPSCs from PB-MNCs using SeV is feasible and simple and generates integration-free iPSC lines with all the characteristic features of true iPSCs that could be further used for research or clinical purposes.