Syracuse

DMPK promoter silencing by transcriptome editing as a new therapeutic strategy in myotonic dystrophy type 1

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Porquet, Florent | Weidong, L | Jehasse, K | Blacher, S | Massotte, L | Gazon, H | Di Valentin, E | Furling, Denis | Gillet, N | Klein, Arnaud, F | Willems, Luc | Seutin, Vincent

Edité par CCSD -

International audience. "a. IntroductionType 1 myotonic dystrophy (DM1) is one of the most prevalent muscular dystrophies. DM1 is also a life threatening disease and causes severe multisystem symptoms. Specifically, this disease originates from an increase of CTG triplet localized in the DMPK gene. Its transcription leads to a pathologic mRNA, inducing a general dysregulation of gene expression. Unfortunately, there are currently only symptomatic treatments. In addition, various therapeutic strategies have already been tested in order to neutralize the pathologic DMPK mRNA or its consequences. Nevertheless, they all have at least one serious drawback that limits their clinical applicability. Therefore, our team aims at elaborating a new curative approach which consists in the DMPK promoter silencing by the CRISPRi transcriptome editing.b. MethodsThe ability of our CRISPRi transcriptome editing to inhibit DMPK promoter was tested in immortalized myoblasts from a DM1 patient (myoDM1) or cells from a healthy donor (myoWT). 13 sgRNAs against the DMPK promoter (sgDMPK) and 3 scrambled sgRNAs (sgNT) were individually cloned into different CRISPRi plasmids used to produce respective lentiviral vectors. Next, the myoblasts were transduced and selected with blasticidin to produce stable cell lines. From these cell lines, the myoblasts were differentiated during 3-5 days. Next, their cellular and molecular characteristics were assessed by FISH, RT-qPCR and RT-PCR.c. ResultsThe density of nuclear DMPK RNA foci determined by FISH was used to screen the sgDMPK. Three out of the 13 sgDMPK candidates sharply reduced by up to 80% the density of foci in the nuclei. Next, the DMPK promoter silencing was evaluated by quantifying the amounts of DMPK RNA using RT-qPCR. The DMPK expression was inhibited by up to 80% in both myoDM1 and myoWT myocytes. Finally, RNA splicing of genes involved in muscle physiology was studied using RT-PCR. Indeed, these genes have been shown to undergo aberrant splicing in DM1 patients. The CRISPRi treatment leads to a strong correction of the splicing for these studied genes.d. ConclusionsOur CRISPRi transcriptome editing system is able to efficiently inhibit the formation of foci by preventing the production of DMPK mRNA in a relevant cell model. As a consequence, it is also able to correct the splicing defects of important genes."

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