Novel CRISPR/Cas9 approaches targeting chromosomal aberrations for efficient and selective elimination of cancer cells
EntityUAM. Departamento de Bioquímica; Centro Nacional de Investigaciones Oncológicas (CNIO)
Funded byesta tesis doctoral ha contado con la financiación de los siguien¬tes Programas de Investigación: Proyectos de Investigación en Salud (AES 2019). Modalidad Proyectos de Desarrollo Tecnológico en salud. (DTS19/00111) Programa Caixa Impulse (CI18-00017)
SubjectsBiociencias moleculares; Biología y Biomedicina / Biología; Medicina
NoteTesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de Lectura: 21-01-2022
Esta tesis tiene embargado el acceso al texto completo hasta el 21-07-2023
Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Cancer is one of the leading causes of human death worldwide and chromosome abnormalities play a causal role in tumorigenesis, mainly by contributing to the generation of novel fusion oncogenes (FOs) or oncogene amplifications (OAs). Despite improvements in treatments that have increased the survival of many patients in recent years, there are still many types of cancer that need the development of new and more effective therapies, particularly for the cases that generate resistance that produce relapses and metastases. This highlights the need for developing novel targeted therapies with improved efficacy and fewer side effects. This work is especially focused on the development of innovative approaches to specifically target FOs or OAs associated with tumour development. CRISPR/Cas9 system has been utilized since it allows the modification of genomic sequences in a precise and efficient way, opening the possibility of developing new therapies directed against specific genetic targets. In this thesis project, we have developed two approaches based on CRISPR/Cas9 genome editing. One is focused on targeting FOs by inducing a deletion involving two introns – one in each gene involved in the rearrangement that gives rise to the FO–, which results in the elimination of the FO in cancer cells without altering exonic sequences or the expression of wild-type genes in healthy cells and functioning irrespective of the FO isoform or the patientspecific breakpoint. We have validated this strategy in Ewing sarcoma and chronic myeloid leukaemia cells, characterized by the EWSR1-FLI1 and BCR-ABL1 FOs respectively, and also in animal models. The second approach consists of the elimination of cancer cells harbouring OAs by targeting an intron of the amplified gene, thus generating multiple double-strand breaks and activation of apoptosis pathways. The OA approach was validated in neuroblastoma cells with MYCN amplification and in xenograft model. We confirmed that both approaches, based on a highly efficient non-homologous end joining strategy, are efficient in the reduction of cell proliferation, clonogenicity capacity, tumour growth and animal mortality. To improve efficacy, a combination of the gene editing approach with standard-of-care chemotherapies was analysed and the results revealed an increased effectiveness. Genomic analysis of the most probable off-targets as well as detectable cytogenetic alterations demonstrated that the approaches seem highly specific. In conclusion, we describe the development of two novel approaches for the elimination of cancer cells by targeting cancer FOs and OAs based on CRISPR/Cas9 gene editing that could represent a promising strategy with therapeutic potential
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Google Scholar:Martínez-Lage García, Marta
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