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Master Thesis

Alternative à la thérapie génique additive par utilisation du système CRISPR-Cas9 pour la correction génique des maladies héréditaires du globule rouge

Abstract : Introduction: congenital erythropoietic porphyria (CEP) is a severe autosomal recessive disease. The most frequent mutation is c.217T>C on exon 4 of the UROS gene (p. C73R). To date, allogeneic cell transplantation of hematopoietic stem cells is the only effective treatment for CEP. Proof of disease cells correction by integrative lentiviral vectors in CEP mice was performed by additive gene therapy "ex vivo" but insertional mutagenesis risk persists. Our objective is to evaluate the feasibility of a directed, effective and definitive homologous recombination gene correction using the non-integrative and transient CRISPR-Cas9 technology in CEP. Method: First, we optimized the transfection by nucleofection in HEK cells and used tools to evaluate edition of the UROS gene: capillary electrophoresis (quantitative) and sequencing (qualitative). Results: We demonstrated the importance of single strand donor oligonucleotide concentration and created a CEP cell line that we rescued by homology directed repair. Then we demonstrated its effectiveness in primary cells by editing normal hematopoietic stem cells and correcting CEP deficient fibroblasts. Conclusion: finally, it remains to be confirmed that the system is transient and to extend our results in a more common hereditary hematologic disease such as sickle cell anemia.
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Master Thesis
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https://dumas.ccsd.cnrs.fr/dumas-01567122
Contributor : Bu Carreire Université de Bordeaux <>
Submitted on : Friday, July 21, 2017 - 5:25:33 PM
Last modification on : Thursday, October 26, 2017 - 1:13:02 AM

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Julian Boutin. Alternative à la thérapie génique additive par utilisation du système CRISPR-Cas9 pour la correction génique des maladies héréditaires du globule rouge. Médecine humaine et pathologie. 2017. ⟨dumas-01567122⟩

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