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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

开发涵盖面肩肱型肌营养不良症遗传多样性的基因组编辑疗法

基本信息

批准号：
21J11349
负责人：
何君潔
金额：
$ 0.96万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for JSPS Fellows
财政年份：
2021
资助国家：
日本
起止时间：
2021-04-28 至 2023-03-31
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-21J11349/
关键词：
therapy genome editing FSHD ゲノム編集治療標的病態解析

项目摘要

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.

该项目旨在通过靶向异常DUX4表达来开发涵盖所有FSHD的基因组编辑疗法。上个财政年度，我们的PolyA淘汰策略无法完全抑制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。我们已经成功地建立了稳定的标记方案，针对D4Z4标记系统。我们研究了生物素标记蛋白质组学数据的标准化方法。通过内源性蛋白质标准化，我们成功地验证了蛋白质列表的阳性对照，这些蛋白质列表被报道位于D4Z4重复位点。另一方面，DUX4基因的异常表达也是由于两种FSHD中4q35区域D4Z4重复序列的DNA低甲基化所致。因此，我们考虑改变为dCas9介导的表观遗传编辑策略用于DUX4沉默。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，我们还建立了由脂质颗粒递送的dCas9介导的RNA表观遗传编辑系统。DUX4抑制作用也在我们的体外分化的肌细胞中得到证实。本实验室引进FSHD小鼠模型，并成功验证了其表型。目前我们正在尝试对这种编辑策略进行体内概念验证，以进行临床应用。