|dc.description.abstract||Fanconi anemia (FA) and PRKDC severe combined immunodeficiency (PRKDC-SCID) are inherited genetic diseases affecting different pathways responsible for DNA-damage signaling and repair. Fanconi anemia and PRKDC-SCID patients do not only exhibit an exacerbated sensitivity to DNA damaging agents, but also contain hematopoietic stem cells (HSCs) with defective proliferation and differentiation properties, resulting in bone marrow failure, in the case of FA patients, and severe immune defects, in PRKDC-SCID patients.
Herein, we investigated the efficacy and the safety of a self-inactivating (SIN) lentiviral vector harboring the FANCA gene (FANCA-LV) in a FA-A mouse model. The conditions for FANCA-LV transduction were optimized to minimize the ex vivo manipulation of Fanca-/- bone marrow (BM) HSCs, while maintaining a high transduction efficiency. Corrected cells were transplanted into irradiated Fanca-/- recipients, which were followed in the long-term. Thereafter, BM cells from primary recipients were serially transplanted to study the behavior of corrected HSCs capable of extensive repopulation. In these animals, analyses of vector copy number per cell, percentage of chimerism and sensitivity of transduced cells to inter-strand cross-linking drugs were conducted. Our results showed the efficient transduction and phenotype correction of true HSCs with the ability to generate a long-term engraftment of Fanca-/- recipients over serial BM transplantation. The use of lineal amplification mediated (LAM) PCR of the insertion sites coupled with next generation sequencing (NGS) allowed us to characterize the integrome of the hematopoietic system in peripheral blood and BM samples corresponding to different recipients at multiple post-transplantation periods. The overall insertional profile and clonal kinetics of gene corrected Fanca-/- HSCs showed a healthy polyclonal hematopoiesis in the transplanted recipients.
The generation of induced pluripotent stem cells (iPSCs) is postulated as an unlimited source of cells for regenerative therapies of numerous diseases. However, several syndromes still remain to be investigated with respect to the feasibility of this approach. By the use of viral and non-viral reprogramming vectors, we have attempted for the first time the reprogramming of Prkdcscid cells, deficient in the protein DNA-PKcs required for NHEJ.
In order to generate iPSCs from Prkdcscid fibroblasts, cells were either transduced with a CRE/LoxP polycistronic reprogramming LV (STEMCCA) or co-transfected with a reprogramming Sleeping Beauty (SB) transposon (T2/OSKM) and the SB100X hyperactive transposase. In control fibroblasts, we demonstrated the efficiency of the STEMCCA reprogramming LV to generate iPSCs. On the other hand, we observed that the reprogramming of Prkdcscid fibroblasts was markedly affected. However, we achieved the generation of Prkdcscid iPSCs when a more efficient reprogramming SB transposon/transposase system was used. The characterization of cells
undergoing cell reprogramming with the LV showed that Prkdcscid cells developed a senescent phenotype, with overexpression of p16/INK4a blocking the reprogramming process. The reprogramming SB transposon did not trigger such a senescent response, and thus, permitted the isolation of Prkdcscid iPSCs. Full description of Prkdcscid iPSCs demonstrated that these clones conserved the parental Prkdcscid mutation and the characteristic hypersensitivity to ionizing radiation as a consequence of their defect in NHEJ DNA repair.
Taken together, the results showed in this thesis evidence the efficiency and safety of the FANCA-LV, supporting its use for the gene therapy of FA-A patients. Besides, we also demonstrate the implications of the NHEJ DNA repair pathway in cell reprogramming. The use of highly efficient reprogramming SB transposons facilitated, however, the generation of Prkdcscid iPSCs, which will constitute a valuable tool to understand the role of NHEJ in cell differentiation, and to test new anticancer drugs in a cell model hypersensitive to DNA damage.||en_US