Development of new approaches for anti-cancer therapy: “smart”, responsive therapeutics against highly oncogenic microRNAs

Abstract

Triple-negative breast cancer (TNBC) remains a clinical challenge owing to its aggressive nature and lack of receptor targets for conventional therapies. MicroRNA-155 (miR-155) has been identified as a key oncogenic driver in TNBC, making it a promising therapeutic target. This thesis presents a new potential therapeutic strategy for disease-modifying therapy of TNBC through the design, synthesis, and biological evaluation of a novel class of synthetic catalytic agents, peptidyl oligonucleotide conjugates (POCs), engineered to selectively bind to and cleave miR-155 in TNBC cell model. The work began with the synthesis and purification of arginine-rich and histidine-rich catalytic peptides library, which were then conjugated to oligonucleotides using aminohexyl linkers. Conjugates were designed in mono- and bis-formats with variations in the peptide attachment sites (at the terminal 5′-, or 3’-position, ′ and/or at the internal abasic sites) to enable structural and functional diversity. Characterisation by ¹H NMR and MALDI-TOF MS confirmed the high level of purity and integrity of the conjugates, thus establishing a reliable synthetic platform. Fluorescence-based cleavage assays in a cell-free system demonstrated effective hybridisation and catalytic degradation of miR-155 for most of the studied conjugates, but with the bis-conjugate “BC6-4His” showing superior performance. Comparative studies revealed that conjugate with a dual 4-histidine-containing peptide achieved a higher cleavage efficiency than those using 4-arginine peptide. FRET-based cleavage assays and kinetic modelling validated the reaction mechanism and turnover capacity, as well as the synergy upon recruitment of RNase H, under physiologically relevant conditions. In-vitro evaluation in TNBC cell model confirmed the biological efficacy of BC6-4His. The conjugate significantly reduced miR-155 levels, restored the expression of the tumour suppressor SOCS1, and inhibited STAT3 phosphorylation, thereby disrupting the oncogenic miR-155/SOCS1/STAT3 axis. ELISA assays further revealed the suppression of pro-inflammatory cytokines (IL-6, IFN-γ, TNF-α, and IL-1β) and elevated intracellular ROS, indicating both anti-tumour and stress activation responses. Collectively, this work provides a proof-of-concept for catalytic miRNA inhibition using structurally tunable POCs. These conjugates exhibit potent, selective, and catalytic degradation of oncogenic miRNAs both in vitro and in cellular systems. These findings pave the way for further development of POCs as RNA-targeted therapeutics, with future work focusing on in vivo delivery, structural optimisation and expansion to other clinically relevant miRNA targets.