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  • To reprogram patient cells we adopted the most feasible and

    2018-10-20

    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 alizarin 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. The next goal for gene therapy is the directed insertion of the therapeutic sequences in the cell genome (Garate et al., 2013; Genovese et al., 2014; Karakikes et al., 2015; Song et al., 2015). A number of different gene-editing strategies have been described, including gene modification of the specific mutation, integration of the therapeutic sequences in a safe harbor site, or knockin into the same gene locus. We directed a knockin strategy to insert the partial cDNA of a codon-optimized version of RPK in the second intron of the PKLR gene. If used clinically, this strategy would allow the treatment of up to 95% of the patients, those with mutations from the third exon to the end of the (cDNA)RPK (Beutler and Gelbart, 2000; Fermo et al., 2005; Zanella et al., 2005). Additionally, this approach retained the endogenous regulation of RPK after gene editing, a necessary factor as RPK is tightly regulated throughout the erythroid differentiation. This fine control would be lost if a safe-harbor strategy was chosen. The PKLR TALEN generated was very specific and very efficient. We did not find any mutation in any of the theoretical off-target sites defined by the off-site search algorithm and analyzed by PCR and gene sequenced. Moreover, we determined that 2.85 out to 100,000 electroporated PKDiPSCs, without considering the toxicity associated to nucleofection, were gene edited when the PKLR TALEN was used, reaching values similar to those previously published by others (Porteus and Carroll, 2005). Interestingly, 40% of the edited PKDiPSC clones presented indels in the untargeted allele or were biallelically targeted, which indicated that the developed TALEN are very efficient, cutting on the on-target sequence with a high frequency.