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

该项目旨在通过靶向异常DUX4表达，开发包含所有FSHD的基因组编辑疗法。上个财政年度，我们的PolyA敲除策略不能完全抑制DUX4。今年我们改变策略，通过酶结合介导的生物素标记系统来研究DUX4调节因子，以寻找潜在的治疗分子靶点。我们成功地制定了针对D4Z4标记系统的稳定标记方案。我们研究了生物素标记蛋白的蛋白质组学数据的归一化方法。通过内源蛋白归一化，我们成功验证了位于D4Z4重复位点的阳性对照蛋白列表。接下来选取候选项时，将研究每个条件的进一步归一化。另一方面，DUX4基因的异常表达也是由于两种类型FSHD中4q35区域D4Z4重复序列的DNA低甲基化。因此，我们考虑改变dcas9介导的表观遗传编辑策略来沉默DUX4。在患者来源的iPSC模型中成功验证了对DUX4基因的抑制作用。为了将来的临床应用，我们还建立了dcas9介导的脂质颗粒递送的RNA表观遗传编辑系统。在体外分化的肌细胞中也证实了DUX4的抑制作用。我们将FSHD小鼠模型引入实验室，并成功验证了表型。现在我们正试图对这种编辑策略的概念进行体内验证，以进行临床应用。

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.