Targeted gene therapy in a mouse model of fanconi anemia

• Autores: María José del Pino
• Directores de la Tesis: Juan Antonio Bueren Roncero (dir. tes.), Susana Navarro Ordoñéz (codir. tes.), Alberto Martínez Serrano (tut. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2016
• Idioma: español
• Tribunal Calificador de la Tesis: José María Almendral del Río (presid.), Paula Río Galdo (secret.), María Esther Grueso Hierro (voc.), Raúl Torres Ruiz (voc.), Massimo Bogliolo (voc.)
• Programa de doctorado: Programa de Doctorado en Biociencias Moleculares por la Universidad Autónoma de Madrid
• Calificación: Sobresaliente Cum Laude
• Materias:
  • Ciencias de la vida
    • Biología celular
      • Cultivo celular
    • Biología molecular
    • Radiobiología
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Fanconi anemia (FA) is an inherited disease associated with bone marrow failure and cancer predisposition. This disease is caused by mutations in any of the 21 FANC genes that belong to FA/BRCA DNA repair pathway. In this study, we have conducted a gene editing strategy aiming at the insertion of a reporter EGFP and a therapeutic hFANCA donor into the Mbs85 safe harbor locus, ortholog of the human AAVS1 locus. To achieve our goal, TALEN were nucleofected together with the different donor templates in Fanca-/- (FA-A) MEFs and also in mouse HSPCs from WT and FA-A mice. In these experiments, we demonstrated the cleavage activity of designed TALEN in the Mbs85 locus of different mouse cell types. Additionally, targeted integration (TI), as well as hFANCA expression and also phenotypic correction was demonstrated in gene-edited FA-A MEFs, as deduced from the reversion of their characteristic mitomycin C (MMC) hypersensitivity. Gene editing experiments with WT and FA-A HSPCs also allowed us to demonstrate TI in these cell types. As observed in FA MEFs evidence of phenotypic correction was observed in FA hematopoietic progenitor cells, although a significant toxicity was associated to the DNA nucleofection of these cells. Our results demonstrate for the first time the feasibility of performing a therapeutic targeted integration strategy in a safe harbor locus in cells from a mouse model of FA