顔面肩甲上腕型筋ジストロフィーの遺伝学的多様性を包括するゲノム編集治療法の開発

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This project is to develop genome editing therapy that encompassing all FSHD by targeting abnormal DUX4 expression. Last fiscal year, our PolyA knockout strategy cannot fully suppress DUX4. This year we change the strategy to investigate the DUX4 regulators by enzyme-binding-mediated biotin labelling system to find the potential therapy molecular targets. We have successfully made stable labelling protocol for targeting D4Z4 labelling system. We examined the normalization way for proteomics data of biotin labelled proteins. By endogenous protein normalization, we successfully validated positive controls of protein list which were reported to be located at D4Z4 repeat locus. Next for picking up the candidates, the further normalization of each condition will be investigated.On the other hand, aberrant expression of DUX4 gene is also due to DNA hypomethylation of the D4Z4 repeat in the 4q35 region in both types of FSHD. So, we considered changing to dCas9-mediated epigenetic editing strategy for DUX4 silencing. The suppression effect on DUX4 gene was successfully validated in the patient-derived iPSC model. For future clinical application, we also established dCas9-mediated RNA epigenetic editing system delivered by lipid particles. The DUX4 suppression effect was also confirmed in our vitro differentiated myocyte. FSHD mouse model was introduced to our lab and the phenotype was successfully validated. Now we are trying to in vivo proof of concept of this editing strategy to proceed clinical application.