139. Development of a Therapy for Duchenne Muscular Dystrophy Using Either TALEN of Cas9 Proteins

Abstract

Duchenne muscular dystrophy (DMD) is a hereditary disease due to a mutation of the DMD gene located in the X chromosome. It is mainly caused by the deletion of one or more exons of the DMD gene, which changes the reading frame and results in a premature stop codon and a truncated and inactive dystrophin protein. Nowadays, genome editing by programmable endonucleases like TALENs or the CRISPR/Cas9 systems is a powerful method for developing a treatment for this type of disease. However, the use of DNA encoding these systems leads to a prolonged expression, which may increase the off-target activity of these nucleases. Despite the risk of integration into the genome hence increased probability of side effects, viral vectors remain the most effective delivery system for the nucleases and the sgRNA. The objective of this work was to develop a novel approach using purified TALEN proteins or Cas9 complex (Cas9 protein with crRNA and tracrRNA) for genome editing as potential treatment for DMD. TALEN proteins or Cas9 complex were transduced to generate double strand breaks (DSBs) in the dystrophin gene. Repair of this DSB by non-homologous end joining (NHEJ) could restore the normal reading frame of the DMD gene producing a functional dystrophin protein as already described for Becker patients (Koenig et al. 1989). To develop this approach, DNA coding for a pair of TALEN proteins targeting exon 54 of the DMD gene were engineered and cloned in the pet16b expression vector. The resulting TALEN proteins contained a His-Tag for purification purposes. The proteins were produced in bacteria and the TALEN-His-Tag proteins were purified using a Nickel column. The purity was analyzed by SDS-PAGE electrophoresis. The Cas9 protein, the crRNA and the tracrRNA were obtained commercially. To verify the cleavage activity of the programmable nuclease-complexes, the target genomic region including exon 54 was PCR amplified and the resulting amplicon was incubated with either the TALEN proteins or the Cas9 complex. Cleavage products were analysed by gel electrophoresis. Both types of proteins were able to cut 100% of the amplicons in vitro. The TALEN proteins or the Cas9 complex were tested in Hela IR8 and in human myoblast cell lines. The TALEN proteins were transduced with Cys-(Npys)-(d-Arg)9, Bioporter or the iTop delivery system (propanebetaine). The Cas9 complex was transduced with RNAiMax. The presence of DSBs was evaluated using the Surveyor assay. The Cas9 complex generated the expected cleavage products. These results are similar to those obtained when the plasmids coding for both systems were transfected. This indicates that the Cas9 complex could be used effectively to target a specific gene. In contrary, the TALEN proteins did not induce INDELs detectable by the surveyor assay. This could be due to an insufficient protein transduction. In conclusion, protein therapy using the CRISPR/Cas9 complex could be a promising approach to develop alternative treatments for genetic diseases. We are currently validating this approach in vivo using the hDMD mouse model containing the complete human DMD gene. (‘t Hoen et al. 2008).