Abstract WP315: Pharmacological Validation of Advanced Gamma Peptide Nucleic Acid (PNA) Based miR Inhibitors for Ischemic Stroke Treatment

Abstract

Backgrounds: Our prior work demonstrated the potential of targeting miR-141-3p to mitigate ischemic stroke damage. In this study, we aimed to synthesize and validate more advanced and efficacious gamma peptide nucleic acid (PNA) based anti-miR 141-3p inhibitors, comparing them with regular PNA and phosphothiorate (PS) based counterparts. These inhibitors were subsequently encapsulated in poly(lactide-co-glycolide) (PLGA)-based nanoparticles (NPs) for treatment in a mouse model of ischemic stroke. Methods: In-house synthesis yielded PS, PNA, and gamma-PNA anti-miR 141-3p, encapsulated in PLGA NPs via double emulsion solvent evaporation. After physiochemical characterization, in vitro safety and efficacy were evaluated in HEK293 cells via MTT, LDH cytotoxicity assays, and qPCR for gene expression. In vivo efficacy studies employed the most potent gamma-PNA based anti-miR 141-3p, administered intraperitoneally 4 hours after 60-minute of transient middle cerebral artery occlusion (MCAO). Mice were sacrificed at 3 (acute) or 30 (chronic) days post-stroke. Acute cohort brain tissues were analysed for miRNA and target mRNA levels, along with infarct volume. Chronic cohort mice were subjected to weekly behavioral task to major sensorimotor deficit or recovery. Results: twenty-four-hour exposed HEK-293 cells were no sign of mortality and toxicity at concentration range of 0.015-1.5ug/mL of various anti-miR 141-3p. Gamma-PNA based anti-miR exhibited notably higher efficacy in inhibiting miR-141-3p (IC50=0.05ug/mL) compared to PS (IC50>1.5ug/mL) or regular PNA (IC50>1.5ug/mL). A single dose of gamma-PNA-based anti-miR significantly reduced (P<0.05 vs. Scramble control) infarct injury and brain tissue miR-141-3p levels (>2-fold vs. scramble control). Gamma-PNA treatment also led to swift improvement in sensorimotor deficits in the rotarod task. Conclusion: NPs of gamma-PNA-based anti-miR 141-3p were both safe and potent in vitro and in vivo. They effectively mitigated infarct injury, improved neurobehavioral deficits, demonstrating promising translational potential for ischemic stroke treatment. In future we aim to explore more mechanistic studies to explore it mode of action.