Robinson Meghan, Fraser Ian, McKee Emily, Scheck Kali, Chang Lillian, Willerth Stephanie M
Division of Medical Sciences, University of Victoria, Victoria, BC, Canada.
Biomedical Engineering Program, University of Victoria, Victoria, BC, Canada.
Front Bioeng Biotechnol. 2018 Nov 21;6:173. doi: 10.3389/fbioe.2018.00173. eCollection 2018.
Cellular transdifferentiation changes mature cells from one phenotype into another by altering their gene expression patterns. Manipulating expression of transcription factors, proteins that bind to DNA promoter regions, regulates the levels of key developmental genes. Viral delivery of transcription factors can efficiently reprogram somatic cells, but this method possesses undesirable side effects, including mutations leading to oncogenesis. Using protein transduction domains (PTDs) fused to transcription factors to deliver exogenous transcription factors serves as an alternative strategy that avoids the issues associated with DNA integration into the host genome. However, lysosomal degradation and inefficient nuclear localization pose significant barriers when performing PTD-mediated reprogramming. Here, we investigate a novel PTD by placing a secretion signal sequence next to a cleavage inhibition sequence at the end of the target transcription factor-achaete scute homolog 1 (ASCL1), a powerful regulator of neurogenesis, resulting in superior stability and nuclear localization. A fusion protein consisting of the amino acid sequence of ASCL1 transcription factor with this novel PTD added can transdifferentiate cerebral cortex astrocytes into neurons. Additionally, we show that the synergistic action of certain small molecules improves the efficiency of the transdifferentiation process. This study serves as the first step toward developing a clinically relevant transdifferentiation strategy for converting astrocytes into neurons.
细胞转分化通过改变成熟细胞的基因表达模式，使其从一种表型转变为另一种表型。操纵转录因子（即与DNA启动子区域结合的蛋白质）的表达可调节关键发育基因的水平。通过病毒递送转录因子能够有效地对体细胞进行重编程，但这种方法存在不良副作用，包括导致肿瘤发生的突变。利用与转录因子融合的蛋白质转导结构域（PTD）来递送外源性转录因子是一种替代策略，可避免与DNA整合到宿主基因组相关的问题。然而，在进行PTD介导的重编程时，溶酶体降解和低效的核定位构成了重大障碍。在此，我们通过在靶转录因子——achaete scute同源物1（ASCL1，一种强大的神经发生调节因子）末端的切割抑制序列旁边放置一个分泌信号序列，研究了一种新型PTD，从而实现了更高的稳定性和核定位。添加了这种新型PTD的由ASCL1转录因子氨基酸序列组成的融合蛋白能够将大脑皮质星形胶质细胞转分化为神经元。此外，我们还表明某些小分子的协同作用可提高转分化过程的效率。这项研究是朝着开发一种将星形胶质细胞转化为神经元的临床相关转分化策略迈出的第一步。
