Correction du gène de la dystrophine avec la méthode CRISPR induced deletion (CinDel)

Authors: Iyombe, Jean-Paul
Advisor: Tremblay, Jacques-P.
Abstract: Duchenne Muscular Dystrophy (DMD) is an X-linked genetically recessive genetic disorder. It affects 1 boy out of 3500 male births. The boy with the disorder presents walking disorders at the age of 3-4 years and loses it around the age of 11. Death occurs around 18-30 years of age from cardiopulmonary complications. To date, there is no effective cure for this serious disease. We have developed a gene therapy approach called CRISPR-induced deletion (CinDel) to correct the mutated DMD gene. It uses two gRNAs that target the exons preceding and following the deletion responsible for the frame shift. The recognition of the target sites by the two gRNAs allows the recruitment of the Cas9 nuclease, which generates double-strand breaks. The exonic and intronic sequences located between the two cuts are then deleted and the remains of the exons are fused by Non-Homologous End Joining (NHEJ) to produce a hybrid exon and restore the reading frame and to allow the synthesis of the truncated dystrophin with correct SLR structure and heptads. The CinDel approach was used in this project to correct the mutated DMD gene in the myoblasts of a patient with a 51-53 deletion. Exons 50 and 54 were targeted by SpCas9 and two gRNAs and to produce double strand breaks, delete the sequences between the two cleavage sites and produce a hybrid exon 50-54 by NHEJ. This restored the normal reading frame and allowed the expression of truncated dystrophin in the patient's myotubes. The approach also made it possible to correct in vivo the mutated DMD gene in the animal model, the transgenic mouse with a human DMD gene having a deletion of exon 52 (del52hDMD) using an AAV9 viral vector containing the SpCas9 gene and two ARNgs. To verify the location with respect to the sarcolemma of truncated dystrophin with or without a correct SLR structure and heptads, we electroporated the Tibialis anterior muscles of mdx/mdx mice with the plasmids encoding the normal or the truncated dystrophin gene fused with the eGFP gene. The results of this experiment show that truncated and normal dystrophins were well localized under sarcolemma. In order to effectively repress the SpCas9 gene and avoid its prolonged expression that may be the basis of random and unexpected (off-target effects) cuts in the genome, we have developed a method of repression called molecular Hara-Kiri. It uses the CinDel method and consists of targeting two regions of the SpCas9 gene with two gRNAs. Recruiting nuclease allows it to cut its own gene (Hara-Kiri). The sequence between the two cleavage sites is deleted. The residues of the SpCas9 gene are then joined by NHEJ generating a TAA stop codon at the junction point. This approach effectively repressed the SpCas9 gene in vitro and in vivo.
Document Type: Thèse de doctorat
Issue Date: 2019
Open Access Date: 29 May 2019
Grantor: Université Laval
Collection:Thèses et mémoires

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