Développement d'un traitement thérapeutique pour la dystrophie musculaire de Duchenne à l'aide des protéines TALENs ou Cas9

Authors: Agudelo, Daniel
Advisor: Tremblay, Jacques-P.
Abstract: Duchenne muscular dystrophy (DMD) is an hereditary disease linked to chromosome X. It is mainly caused by the deletion of one or more exons of the DMD gene, which causes a change in the reading frame, obtaining a truncated and inactive protein. Genome editting by TALEN or CRISPR/cas9 systems has become in the recent years a powerfull tool for developing treatments for this type of disease. However, the use of plasmids encoding these systems leads to a prolonged expression, which may increase the off-target risk. Thus, it is important to note that today, viruses vectors remain the most effective delivery system for these plasmids, which always entails a risk of integration into the genome, increasing the probability of side effects for a treatment. In this work, we illustrate the development of a genome edditing treatment for DMD, but using purified protein TALENs or Cas9. These proteins are transduced in order to generate double strand breaks in the genomic DNA. Thus, the correction of this mutation by non-homologous end joining can correct the reading frame of the gene, producing a functional Dystrophin protein, as for Becker patients. Although TALEN proteins show a good activity in vitro, the cut-effectiveness has not been observed in the cells. It would indicate a defect in the protein transduction. However, in the case of CRISPR/cas9 system, we have obtained the expected cleavage products during the transduction with cationic lipids in both cell lines. These results are similar with those obteined when the plasmids coding for both systems were transfected. This indicates that the CRISPR/cas9 system can be used effectively in protein form while targeting a gene specifically. Protein therapy using the CRISPR/cas9 system can be a promising method in order to develop an alternative treatment for genetic diseases. Finally, in order to confirm that this system can be used in vivo, we will soon test it in the hDMD mouse model, containing the complete human DMD gene.
Document Type: Mémoire de maîtrise
Issue Date: 2017
Open Access Date: 24 April 2018
Permalink: http://hdl.handle.net/20.500.11794/27455
Grantor: Université Laval
Collection:Thèses et mémoires

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