A Divalent and Chemically Modified Microrna-34A with Enhanced Circulation Kinetics and Improved Endosomal Escape for the Treatment of Triple-Negative Breast Cancer

Treatment of triple-negative breast cancer (TNBC) remains highly challenging due to the absence of HER2/hormonal receptors and its resistance to conventional therapies. Although surgery, chemotherapy, and radiotherapy are available treatment options, these broad-spectrum therapies are limited by their non-specific targeting and severe side effects, underscoring the need for more precise therapeutic options. Targeted micro RNA (miRNA) therapeutics have gained significant attention in this regard. In most cancers including TNBC, tumor suppressive miRNAs are often lost, leading to unchecked oncogene expression. One such miRNA, miR-34a which is transcriptionally activated by p53, regulates several oncogenic pathways by down-regulating genes like MET, AXL, and C-MYC. Restoring miR-34a in TNBC is therefore being explored as a potential therapeutic strategy. However, clinical use of miRNAs has been limited by their susceptibility to nucleases, immunogenicity, and lack of targeted delivery methods. To address these limitations, our lab developed a chemically modified, folate-conjugated miRNA-34a (FM-FolamiR-34a) that is highly stable and can be specifically delivered to TNBC cells overexpressing folate receptor alpha (FRα). FM-FolamiR-34a effectively downregulated its targets and demonstrated promising antitumor activity. However, due to its small size (∼14 kDa), FM-FolamiR-34a was subject to rapid renal clearance, limiting its therapeutic availability. Additionally, endosomal entrapment restricted its cytoplasmic abundance, further reducing its mRNA-targeting efficiency. To overcome these challenges, we developed a novel, chemically modified, folate-conjugated divalent miR-34a with enhanced circulation kinetics and improved endosomal escape. This divalent molecule includes two miR-34a duplex copies linked by cleavable deoxythymidine linkers (dT-linkers), which are susceptible to enzymatic cleavage by DNAse II in late endosomes. The dT-linker facilitates miR-34a release from the receptor promoting endosomal escape and increasing cytosolic availability. The dual nature of our construct also raises its molecular weight above the kidney clearance threshold (∼30 kDa), preventing rapid excretion and providing additional time for engagement with FRα-expressing cancer cells. Furthermore, delivering two miR-34a strands per receptor doubles the intracellular miR-34a copy number, enhancing cellular availability. We anticipate that our divalent miR-34a will demonstrate superior antitumor efficacy in TNBC compared to the monovalent version (FM-FolamiR-34a). This innovative approach addresses key critical barriers in miRNA-based therapeutics - specifically endosomal entrapment and rapid renal clearance - presenting an effective strategy to target FRα overexpressing TNBC. Sharjeel Anjum, Shreyas Iyer, Digambar Waiker, Andrea Kasinski. A divalent and chemically modified microRNA-34a with enhanced circulation kinetics and improved endosomal escape for the treatment of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6691.