Publication: Terapia génica dirigida en una nuevo "sitio seguro" en células progenitoras hematopoyéticas humanas para su aplicación en anemia de Fanconi
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2016-07-01
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Universidad Complutense de Madrid
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Abstract
La anemia de Fanconi es una enfermedad hereditaria de baja prevalencia, descrita por primera vez por el pediatra Guido Fanconi en 1927. Esta enfermedad se produce como consecuencia de mutaciones en cualquiera de los 19 genes de Fanconi descritos hasta la actualidad, y que participan en la ruta de Fanconi/BRCA. Esta ruta se encarga de la reparación de enlaces intercatenarios del ADN y de coordinar los distintos mecanismos de reparación de las dobles roturas en el ADN. La anemia de Fanconi está caracterizada por generar inestabilidad genómica, lo que da lugar a anomalías esqueléticas y predisposición al cáncer, si bien la principal causa de muerte de pacientes pediátricos es el fallo de médula ósea. Uno de los tratamientos alternativos al trasplante alogénico de progenitores hematopoyéticos de pacientes con anemia de Fanconi se basa en la reinfusión de células madre hematopoyéticas autólogas, tras su corrección con vectores lentivirales. Para limitar al máximo los riesgos de este tipo de terapias se están desarrollando nuevas tecnologías de edición génica basadas en la inserción dirigida de los genes terapéuticos. Esta nueva aproximación se fundamenta en la generación de dobles roturas en regiones específicas del genoma, cuya reparación por recombinación homóloga facilitaría la entrada de los genes terapéuticos aportados por ADNs donadores externos con homología por dicha región. En este trabajo se ha desarrollado una aproximación de edición génica en un nuevo “sitio seguro” del genoma denominado SH6. Para ello se ha trabajado con la línea celular HEK-293H, así como también con progenitores hematopoyéticos humanos purificados en base a la expresión del marcador CD34. Para su desarrollo se han utilizado nucleasas de edición, tales como meganucleasas y TALEN, en combinación con matrices donadoras portadoras del gen marcador EGFP (GM) o del gen terapéutico FANCA (TM). En todos los casos los genes marcadores y terapéuticos estaban regulados por el promotor EF1α, y flanqueados por dos brazos de homología para el sitio SH6. Estos plásmidos han servido como molde para realizar la terapia génica de edición en el sitio seguro SH6...
Fanconi anemia is an inherited disease of low prevalence that was first described by the pediatrician Guido Fanconi in 1927. This disease occurs as a result of mutations in any of the 19 Fanconi genes so far described, and involved in the Fanconi/BRCA pathway. This pathway is responsible for the repair of the DNA interstrand cross-links and coordinates different repair mechanisms of DNA double strand breaks. Fanconi anemia is characterized by genome instability, and results in skeletal abnormalities and predisposition to cancer, although the main cause of death in pediatric patients is the bone marrow failure. One of the alternative treatments to allogeneic hematopoietic stem cell transplantation in Fanconi anemia patients is based on the reinfusion of autologous hematopoietic stem cells, after correction with lentiviral vectors. In order to minimize the risks of such therapies, new gene editing technologies based on the targeted integration of the therapeutic genes are under development. These new approaches are based on the generation of double strand breaks in specific regions of the genome that facilitates the homologous recombination of therapeutic genes provided by external DNA homology donors. In this work we have developed an approach for gene editing in a new "safe harbor" of the genome called SH6. To achieve this goal, we have worked with the HEK-293H cell line, as well as with purified human hematopoietic progenitors that expressed the CD34 marker. For this development, specific nucleases such as meganucleases and TALEN were used in combination with donor matrixes that carried the EGFP marker gene (GM) or the therapeutic FANCA gene (TM). In all cases the marker and therapeutic genes were regulated by the EF1α promoter, and flanked by two homology arms for SH6 site. These plasmids have served as templates for targeted gene therapy on the safe harbor SH6...
Fanconi anemia is an inherited disease of low prevalence that was first described by the pediatrician Guido Fanconi in 1927. This disease occurs as a result of mutations in any of the 19 Fanconi genes so far described, and involved in the Fanconi/BRCA pathway. This pathway is responsible for the repair of the DNA interstrand cross-links and coordinates different repair mechanisms of DNA double strand breaks. Fanconi anemia is characterized by genome instability, and results in skeletal abnormalities and predisposition to cancer, although the main cause of death in pediatric patients is the bone marrow failure. One of the alternative treatments to allogeneic hematopoietic stem cell transplantation in Fanconi anemia patients is based on the reinfusion of autologous hematopoietic stem cells, after correction with lentiviral vectors. In order to minimize the risks of such therapies, new gene editing technologies based on the targeted integration of the therapeutic genes are under development. These new approaches are based on the generation of double strand breaks in specific regions of the genome that facilitates the homologous recombination of therapeutic genes provided by external DNA homology donors. In this work we have developed an approach for gene editing in a new "safe harbor" of the genome called SH6. To achieve this goal, we have worked with the HEK-293H cell line, as well as with purified human hematopoietic progenitors that expressed the CD34 marker. For this development, specific nucleases such as meganucleases and TALEN were used in combination with donor matrixes that carried the EGFP marker gene (GM) or the therapeutic FANCA gene (TM). In all cases the marker and therapeutic genes were regulated by the EF1α promoter, and flanked by two homology arms for SH6 site. These plasmids have served as templates for targeted gene therapy on the safe harbor SH6...
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Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, Departamento de Bioquímica y Biología Molecular, leída el 02/02/2016