Next Generation Oncogenic MicroRNAs Inhibitors for Cancer Therapeutics

Objective This study was focused on designing, synthesizing, and characterizing next-generation of Peptide Nucleic Acids (PNAs) targeting oncogenic miRNAs for therapeutic use in different cancers. We tested short PNA probes (anti-seed PNAs) to target seed regions of miR 155 and other oncomiR clusters. We hypothesized that the short PNA antimiR possesses numerous attractive features; ease of synthesis and quality control analysis, flexibility to conjugate large fluorophores as theranostic agents, and conducive physical-biochemical attributes; solubility, permeability, and non-self-aggregating-features. Methods We synthesized PNAs using solid-phase synthesis and further used reverse-phase HPLC for PNA purification followed by quality control analysis by mass spectrometry. To confirm the binding of short anti-seed PNAs to target miRNAs, we performed gel shift assays and thermal melting analysis. Tumor targeting characteristic was achieved by conjugating the short PNAs to pH low insertion peptide (pHLIP). We also confirmed the cellular uptake of synthesized PNAs by flow cytometry and confocal microscopy. The efficacy of these anti-seed PNA constructs was evaluated in cancer cells by gene expression, western blot analysis, and cell viability assays and further in vivo efficacy was evaluated in a mouse model. Results Anti-seed PNAs showed superior efficacy as compared to the efficacy of regular PNAs and scrambled controls. Our results show superior binding, efficacy, and minimal off-target effects with the short anti-seed PNAs. Conclusions This work outlines a promising approach to selectively deliver antimiR PNA probes to the tumor cells and inhibit the oncogenic microRNAs. Overall, these studies establish the use of short antimiR PNAs as a novel therapeutic strategy for miRNA silencing. This study was focused on designing, synthesizing, and characterizing next-generation of Peptide Nucleic Acids (PNAs) targeting oncogenic miRNAs for therapeutic use in different cancers. We tested short PNA probes (anti-seed PNAs) to target seed regions of miR 155 and other oncomiR clusters. We hypothesized that the short PNA antimiR possesses numerous attractive features; ease of synthesis and quality control analysis, flexibility to conjugate large fluorophores as theranostic agents, and conducive physical-biochemical attributes; solubility, permeability, and non-self-aggregating-features. We synthesized PNAs using solid-phase synthesis and further used reverse-phase HPLC for PNA purification followed by quality control analysis by mass spectrometry. To confirm the binding of short anti-seed PNAs to target miRNAs, we performed gel shift assays and thermal melting analysis. Tumor targeting characteristic was achieved by conjugating the short PNAs to pH low insertion peptide (pHLIP). We also confirmed the cellular uptake of synthesized PNAs by flow cytometry and confocal microscopy. The efficacy of these anti-seed PNA constructs was evaluated in cancer cells by gene expression, western blot analysis, and cell viability assays and further in vivo efficacy was evaluated in a mouse model. Anti-seed PNAs showed superior efficacy as compared to the efficacy of regular PNAs and scrambled controls. Our results show superior binding, efficacy, and minimal off-target effects with the short anti-seed PNAs. This work outlines a promising approach to selectively deliver antimiR PNA probes to the tumor cells and inhibit the oncogenic microRNAs. Overall, these studies establish the use of short antimiR PNAs as a novel therapeutic strategy for miRNA silencing.