Prophylactic oligonucleotide-mediated enhancement of host acetylcholinesterase protects from organophosphate poisoning

Abstract

Acute organophosphate (OP) poisoning is lethal due to irreversible acetylcholinesterase (AChE) inhibition in neuromuscular junctions (NMJ). Recently, we discovered that AChE levels are down-regulated by microRNA-132 (miR-132) and that blocking this down-regulation can significantly elevate endogenous AChE levels [1]. Here, we report effective prophylactic protection from paraoxon poisoning of BalbC mice injected intravenously (IV) 24 hours pre-exposure with 3.3 mg/kg AM132, a 22-mer antisense chemically protected from nuclease degradation by 2′-O-methyl modified bases on a phosphorothioate backbone and conjugated to cholesterol at its 3' end for enhanced penetration. When exposed to 0.9LD50 paraoxon, all naive but none of the Chol-AM132-protected mice exhibited severe poisoning symptoms (Mann-Whitney U-test, P=0.028, N=4/group). 1.3LD50 exposure caused poisoning symptoms in all mice, but 40% of the Chol-AM132-treated mice survived and recovered, while naive ones all showed severe poisoning symptoms and died within 3 minutes post-exposure (ANOVA P=6.62E-12, N=4-7/group). No mice survived exposure to 10LD50 Paraoxon, but Chol-AM132 treatment conspicuously extended survival time compared to controls (ANOVA P=6.8E-9, N=5/group). To identify the underlying mechanism, we measured AChE activity in NMJs of diaphragm muscle from mice that had been injected with either Chol-AM132 or PBS for 3 successive days. Chol-AM132 treated mice had 10.9±4.2 compared to 6.3±2.7 stained NMJs/ field in control mice (t test, P=0.0069, N=5 mice/group and 18 fields/specimen), possibly reflecting increased expression of AChE in pre-existing NMJs, development of new ones or both. Additionally, Chol-AM132 treatment elevated AChE activity in bone marrow macrophages (29.0±15.0 vs. 14.1±2.6 compared to 15.0±6.4 vs. 9.8±1.2 nmol substrate hydrolyzed/mg protein/min for 3 and 1 day treatment, respectively; Student's t-test: <0.0065), further predicting potential protection from post-exposure inflammatory insults. Importantly, Chol-AM132 can be synthesized in unlimited amounts using standard equipment, exhibits long-term stability at room temperature and field conditions, and unlike pyridostigmine, does not inhibit pre-existing AChE. Our findings open new venues for prophylactic protection against nerve gas poisoning.