Gene editing mediated by non-homologous end-joining: a versatile approach for the gene therapy of hematopoietic stem cells from fanconi anemia patients
Autor (es)
Román Rodríguez, Francisco JoséEntidad
UAM. Departamento de Biología Molecular; Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT); Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER); Instituto de Investigación Sanitaria de la Fundación Jiménez DíazFecha de edición
2018-11-27Financiado por
El trabajo de investigación descrito en esta memoria ha sido realizado en la División de Terapias Innovadoras en el Sistema Hematopoyético del Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) / Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) / Unidad Mixta de Terapias Avanzadas CIEMAT/Instituto de Investigación Sanitaria de la Fundación Jiménez Díaz (IIS-FJD). Para su ejecución, esta tesis doctoral ha contado con la colaboración de los siguientes Programas de Investigación: Ministerio de Economía, Comercio y Competitividad y Fondo Europeo de Desarrollo Regional (FEDER) (SAF2015-68073-R y SAF2015-64152-R). Séptimo Programa Marco de la Comisión Europea (HEALTH-F5-2012-305421; EUROFANCOLEN). Ministerio de Sanidad, Servicios Sociales e Igualdad (EC11/060 y EC11/550) Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III (RD12/0019/0023). Programa de transferencia de tecnología en el campo de la terapia génica de la Fundación Botín. Francisco José Román Rodríguez ha disfrutado de una beca de Formación de Personal Investigador (FPI) del Ministerio de Economía y Competitividad (BES-2013-063397) vinculada al proyecto SAF2012-39834: “Terapia celular y génica dirigida en Anemia de Fanconi: un paso adelante”, y de un contrato del CIBERER.Materias
Anemia - Terapia génica - Tesis doctorales; Ingeniería genética - Tesis doctorales; Biología y Biomedicina / BiologíaNota
Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27-11-2018Esta tesis tiene embargado el acceso al texto completo hasta el 27-05-2020

Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Resumen
Preclinical as well as clinical studies suggest that gene therapy based on the infusion of
autologous hematopoietic progenitor and stem cells (HSPCs) previously corrected with
lentiviral vectors may constitute a future alternative for treating Fanconi anemia (FA) patients.
In this context, gene editing appears to be a new step in the development of safe and precise
gene therapy approaches. Since non-homologous end-joining (NHEJ) is the preferred
mechanism to repair DNA double-strand breaks in quiescent cells, such as HSPCs, and given
that this pathway has been reported to be enhanced in FA cells, we have tested the feasibility of
using NHEJ to generate compensatory mutations that may restore the function of FANCA
protein, thus mimicking the spontaneous reversions reported in some FA mosaic patients.
Our hypothesis was initially tested in two FA-A patient-derived lymphoblastic cell lines
carrying different mutations in FANCA using the CRISPR/Cas9 system. Analyses of the targeted
sites by next generation sequencing (NGS) revealed the presence of cells harbouring potentially
therapeutic repair events whose frequency increased over time. Functional analyses confirmed
the re-expression of a new functional FANCA protein capable of correcting the FA cell
phenotype. Importantly, transplantation of healthy donor HSPCs after NHEJ-editing in
immunodeficient (NSG) mice showed the feasibility of efficiently targeting long-term
repopulating HSCs. Moreover, when FA-A patient HSPCs were targeted by our CRISPR/Cas9
nuclease, therapeutic NHEJ-repair events were identified by NGS, showing up to 50-fold in
vitro expansion after only 9 days in culture. Furthermore, the transplantation of limited
numbers of FA-edited hCD34+ cells into an NSG mouse showed a remarkable in vivo expansion
of corrected cells. Additionally, corrected cells showed the reversion of the hypersensitivity to
mitomycin C, defined as a hallmark of FA cells. All together, these results demonstrate for the
first time the NHEJ-mediated phenotypic correction of FA HSPCs. The NGS analyses of the topfive
in silico predicted off-target loci in edited FA HSPCs showed no unspecific activity,
confirming the safety of this new approach.
Moving forward to in vivo applications of NHEJ-mediated repair approaches, serotype 6
adeno-associated viral vectors (AAVs) were tested in vitro and also in vivo, demonstrating the
possibility of transducing hematopoietic progenitor cells in both settings. Moreover, the
delivery of the CRISPR/Cas9 system via an all-in-one AAV confirmed the feasibly of these
vectors to edit human HSPCs, opening the possibility of future in vivo NHEJ-mediated gene
editing approaches in FA
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