Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disorder with a prevalence ranging from 0.5 to 18.1 per 100,000 individuals. Typically, DM1 patients suffer from progressive myopathy and myotonia, cardiac conduction defects and cognitive impairments. DM1 is caused by the presence of expanded trinucleotide CTG repeats (CTGexp) in the 3’UTR of DMPK gene on ch19q13.3. Expression of the DMPK gene with mutated 3’UTR gives rise to an expanded CUG repeat RNA transcript (CUGexp), which is pathogenic to cells and is the primary factor in DM1 pathogenesis. For the treatment of DM1 patients there are numerous therapeutic strategies that have been proposed, but very few have been effective. Therefore, we intend to develop a novel therapeutic approach to correct the disease mutation by targeting the pathogenic mutated locus at the DNA level. In our study, we derived induced pluripotent stem cells from DM1 patients (DM1-iPSCs) and differentiated them into myogenic cells (DM1-iPSC-Myo). This represents an invaluable in vitro DM1 myogenic model mimicking the aberrant DM1 alternate splicing and biomarkers. Thereafter, we developed a dual guide RNA (gRNA) based CRISPR/Cas9 correction approach to excise the expanded CTG repeats and validated our correction approach in our DM1 myogenic model. The CRISPR/Cas9 correction approach yielded a relatively robust correction efficiency of up to 52% in DM1-iPSC-Myo obviating the need for any selection step, consistent with the disappearance of ribonuclear foci, reversal in alternate splicing of SERCA and redistribution of MBNL1 proteins. In anticipation of future clinical translation, we subsequently used a non-viral, non-DNA based “hit and go” approach to correct the DM1-iPSCs using CRISPR/Cas9 ribonucleoprotein (RNP) complexes, obtaining up to 90% efficiency of correction. The repeat excision was further confirmed by TP-PCR, Southern blot analysis and ribonuclear foci analysis of individual iPSC clones. Additionally, we evaluated the alternate splicing of the corrected DM1-iPSCs post cardiomyogenic differentiation, which showed a reversal in the splicing pattern in the corrected cells as compared to their non-corrected counterpart. Altogether, the proof of concept study demonstrates that CRISPR/Cas9 can mediate relatively efficient correction of the pathogenic DM1 mutation, which represents an essential step towards development of novel and clinically relevant therapeutic strategies for DM1 disease. Additionally, the in vitro DM1 myogenic and cardiomyogenic disease models generated in the current study provide invaluable tools for DM1 research for developing novel therapies and to further study the pathophysiological mechanisms of DM1.
Datum Prijs21 mei 2019
Toekennende instantie
  • Vrije Universiteit Brussel
BegeleiderMarinee Chuah (Promotor), Thierry VandenDriessche (Promotor), Manuel Goncalves (Jury), Nicole Déglon (Jury), Maurilio Sampaolesi (Jury), Luc Bouwens (Jury), Ivan Bautmans (Jury) & Tamara Vanhaecke (Jury)

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