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

MYC癌基因在超过50%的人类癌症中失调。然而，由于MYC蛋白对小分子抑制剂的“不可用药”性质，迄今为止没有临床治疗可用。一个有前途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是与靶mRNA反义的短核酸，其诱导酶降解或空间阻断翻译或剪接。先前靶向MYC的ASO显示出初步有希望的临床结果，但最终失败。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的MYC mRNA的基于肽核酸的降解剂（degPNAs）的开发和应用。degPNA通过肽骨架上连接的降解剂碱基柄（其诱导磷酸二酯键的碱基介导的裂解）在双链化后降解靶mRNA。该提案描述了基于结构的MYC mRNA靶向degPNA的设计和合成及其对有效RNA降解的优化。此外，共轭细胞穿透肽（CPP）的描述，然后通过荧光标记和显微镜检查，以评估细胞的摄取。最后，通过RNA和蛋白质定量以及全局转录组学研究了几种癌细胞系中的体外MYC靶向。总之，所提出的degPNAs联合收割机结合了电荷中性ASO的优点和酶募集ASO的灵活设计和催化作用模式，这为靶向RNA降解提供了一种新的方法，并可能为通用化学基因沉默剂平台铺平道路。

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.