Gene therapy and cell reprogramming in mouse models of monogenic hematopoietic stem cell diseases

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 20-11-2013

Detalles Bibliográficos
Autor: Molina Estévez, Francisco Javier
Tipo de recurso: tesis doctoral
Fecha de publicación:2013
País:España
Institución:Universidad Autónoma de Madrid
Repositorio:Biblos-e Archivo. Repositorio Institucional de la UAM
Idioma:inglés
OAI Identifier:oai:repositorio.uam.es:10486/660256
Acceso en línea:http://hdl.handle.net/10486/660256
Access Level:acceso abierto
Palabra clave:Terapia génica - Tesis doctorales
Enfermedades raras - Tesis doctorales
Biología y Biomedicina / Biología
id ES_7a7c53f7dc5c753fa644cd5633e1ac13
oai_identifier_str oai:repositorio.uam.es:10486/660256
network_acronym_str ES
network_name_str España
repository_id_str
dc.title.none.fl_str_mv Gene therapy and cell reprogramming in mouse models of monogenic hematopoietic stem cell diseases
title Gene therapy and cell reprogramming in mouse models of monogenic hematopoietic stem cell diseases
spellingShingle Gene therapy and cell reprogramming in mouse models of monogenic hematopoietic stem cell diseases
Molina Estévez, Francisco Javier
Terapia génica - Tesis doctorales
Enfermedades raras - Tesis doctorales
Biología y Biomedicina / Biología
title_short Gene therapy and cell reprogramming in mouse models of monogenic hematopoietic stem cell diseases
title_full Gene therapy and cell reprogramming in mouse models of monogenic hematopoietic stem cell diseases
title_fullStr Gene therapy and cell reprogramming in mouse models of monogenic hematopoietic stem cell diseases
title_full_unstemmed Gene therapy and cell reprogramming in mouse models of monogenic hematopoietic stem cell diseases
title_sort Gene therapy and cell reprogramming in mouse models of monogenic hematopoietic stem cell diseases
dc.creator.none.fl_str_mv Molina Estévez, Francisco Javier
author Molina Estévez, Francisco Javier
author_facet Molina Estévez, Francisco Javier
author_role author
dc.contributor.none.fl_str_mv Güenechea Amurrio, Guillermo
Bueren Roncero, Juan Antonio
Departamento de Biología Molecular
Facultad de Ciencias
dc.subject.none.fl_str_mv Terapia génica - Tesis doctorales
Enfermedades raras - Tesis doctorales
Biología y Biomedicina / Biología
topic Terapia génica - Tesis doctorales
Enfermedades raras - Tesis doctorales
Biología y Biomedicina / Biología
description Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 20-11-2013
publishDate 2013
dc.date.none.fl_str_mv 2013
2013-11-20
dc.type.none.fl_str_mv doctoral thesis
http://purl.org/coar/resource_type/c_db06
NA
http://purl.org/coar/version/c_be7fb7dd8ff6fe43
dc.type.openaire.fl_str_mv info:eu-repo/semantics/doctoralThesis
format doctoralThesis
dc.identifier.none.fl_str_mv http://hdl.handle.net/10486/660256
url http://hdl.handle.net/10486/660256
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:Biblos-e Archivo. Repositorio Institucional de la UAM
instname:Universidad Autónoma de Madrid
instname_str Universidad Autónoma de Madrid
reponame_str Biblos-e Archivo. Repositorio Institucional de la UAM
collection Biblos-e Archivo. Repositorio Institucional de la UAM
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869411441672454144
spelling Gene therapy and cell reprogramming in mouse models of monogenic hematopoietic stem cell diseasesMolina Estévez, Francisco JavierTerapia génica - Tesis doctoralesEnfermedades raras - Tesis doctoralesBiología y Biomedicina / BiologíaTesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 20-11-2013Fanconi 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 2 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.Güenechea Amurrio, GuillermoBueren Roncero, Juan AntonioDepartamento de Biología MolecularFacultad de Ciencias20132013-11-20doctoral thesishttp://purl.org/coar/resource_type/c_db06NAhttp://purl.org/coar/version/c_be7fb7dd8ff6fe43info:eu-repo/semantics/doctoralThesisapplication/pdfhttp://hdl.handle.net/10486/660256reponame:Biblos-e Archivo. Repositorio Institucional de la UAMinstname:Universidad Autónoma de MadridInglésengopen accesshttp://purl.org/coar/access_right/c_abf2info:eu-repo/semantics/openAccessoai:repositorio.uam.es:10486/6602562026-06-23T12:46:27Z
score 15,300719

Similares en LA Referencia