Targeted and directed degradation of oncogenic MYC mRNA with non-enzymatic peptide nucleic acid degraders (degPNAs)

在超过50%的人类癌症中，MYC癌基因被解除了调控。然而，由于MYC蛋白对小分子抑制剂的“不可药物”性质，到目前为止还没有临床治疗方法。一种有希望的替代方案是通过使用反义寡核苷酸(ASO)在mRNA水平上干扰其翻译。ASO是一种与靶基因反义的短核酸，可诱导酶降解或立体阻断翻译或剪接。以前针对MYC的ASO已经显示出初步的有希望的临床结果，但最终失败了。为了克服过去基于ASO的策略的缺点，该项目提出了利用化学降解机制开发和应用基于多肽核酸的MYC mRNA降解器(DECPNAs)。去核糖核酸通过连接在肽骨架上的降解物碱基柄在氢化时降解目标mRNA，从而诱导碱基介导的磷酸二酯键的断裂。这项建议描述了基于结构的设计和合成的MYC信使核糖核酸靶向去核糖核酸，以及它们对高效RNA降解的优化。此外，描述了与细胞穿透肽(CPPs)的结合，随后进行了荧光团标记和显微镜观察以评估细胞摄取。最后，通过RNA和蛋白质定量以及整体转录，研究了MYC在几个癌细胞系中的体外靶向。综上所述，所提出的去核糖核酸结合了电荷中性ASO的优点和酶招募ASOS灵活的设计和催化作用模式，为靶向RNA降解提供了一种新的方法，并可能为通用型化学基因沉默平台铺平道路。

The MYC oncogene is deregulated in more than 50% of human cancers. However, no clinical treatment is available to date due to the "undruggable" nature of the MYC protein towards small molecule inhibitors. A promising alternative represents the interference with its translation on the mRNA level through the use of antisense oligonucleotides (ASOs). ASOs are short nucleic acids with antisense to the targeted mRNA that induce enzymatic degradation or sterically block translation or splicing. Previous ASOs targeting MYC have shown initial promising clinical results but eventually failed. To overcome the shortcomings of past ASO-based strategies, this project proposes the development and application of peptide nucleic acids-based degraders (degPNAs) of the MYC mRNA utilising a chemical degradation mechanism. The degPNAs degrade the target mRNA upon hydridisation through attached degrader base handles on the peptide backbone, which induce base-mediated cleavage of the phosphodiester bond. This proposal describes the structure-based design and synthesis of MYC mRNA-targeting degPNAs and their optimisation towards efficiency RNA degradation. Further, the conjugation to cell-penetrating peptides (CPPs) is described followed by fluorophore labeling and microscopy to evaluate cell uptake. Finally, in vitro MYC targeting in several cancer cell lines is investigated through RNA and protein quantification as well as global transcriptomics. In conclusion, the proposed degPNAs combine the advantages of charge-neutral ASOs with the flexible design and catalytic mode of action of enzyme-recruiting ASOs, which offers a novel approach for targeted RNA degradation and could pave the way towards a general chemical gene silencer platform.