Thesis title: Gene Therapy for Ataxia-Telangiectasia
The general aim of the PhD project was to develop a gene therapy protocol for Ataxia-Telangiectasia; the specific aims were to generate a lentivirus vector containing wild-type Atm and to study the effects of enriched hematopoietic progenitor cells transplantation collected from wild-type mice, with focus in B and T cell progenitors, in Atm knock-out mouse model. The final goal being to transplant enriched HPCs from Atm knock-out mice after correction with lentivirus to tackle immunodeficiency, to recover the immunological phenotype and to prevent cancer onset/progression in Atm-/- mice.
Functional lentiviral vector (pLenti-EF1s-smURPF-FlagHisAtm-WPRE3) was generated, produced, and its transduction showed that the virus was able to be inserted and expressed into HEK293T cell lines and integrated into ATM-deficient lymphoblastoid cell lines. Atm-/- transplanted mice had an increase in the percentage of blood T cells, mainly CD4 single positive helper T cells, when compared to Atm-/- mice, 7 weeks after transplantation. Genomic analyses verified that transplanted Atm positive cells could be found in lymphoid organs but not in other tissue. Induced class switching, showed that transplanted mice were able to do B cell maturation. Thymocytes underwent maturation as it was observed the increase of CD4 positive cells and TCR highly expressing cells. Protein analysis of isolated T cells from transplanted mice, demonstrated that they expressed Atm and responded activating an Atm-dependent phosphorylation cascade after DNA damage in in vitro experiments. Transplanted Atm-/- mice had an extended lifespan compared to Atm-/- mice, due to thymoma prevention/delay, and showed signs of aging like white fur compared to wild-type mice. Moreover, thymocytes of transplanted mice revealed decreased trans-rearrangement at 7 weeks when compared with Atm-/- mice and even more at 6 months after transplantation. Dose-response experiments suggests that 0.5x106 cells are not enough to promote phenotype recover; however, 1x106 cells could be sufficient. In conclusion, by using pre-clinical models, this work demonstrates the feasibility of gene therapy for Ataxia-Telangiectasia.