| Organophosphate nerve agents exert their acute toxicity by the inhibition of acetylcholinesterase at cholinergic synapses and neuromuscular junctions. Current therapies can reduce mortality, but fail to prevent the debilitating symptoms associated with organophosphate toxicity. As an alternative, prophylactic protection and postexposure treatment with human cholinesterases has been proposed. This "cholinesterase therapy" has been shown to prevent all symptoms associated with organophosphate poisoning, however, the cholinesterases are necessary in stoichiometric rather than catalytic quantities. This requirement has exceeded all known production platforms. As a solution, I have proposed using plant biotechnology to produce these valuable therapeutics. Plants enjoy near limitless scalability and freedom from the safety concerns that plague mammalian-based production systems. In this work, I report molecular cloning, production, and purification of plant-derived acetylcholinesterase and butyrylcholinesterase, a related serum cholinesterase. I show that conjugation of these cholinesterases to long chain polyethylene glycol is sufficient to provide the required biological half-life for therapeutic administration. I demonstrate that these recombinant cholinesterases are capable of preventing all acute mortality and morbidity associated with organophosphate toxicity. In addition to these acute symptoms, organophosphates also induce severe long-term consequences, including neuromuscular junction dysmorphology, immunosupression, and significant upregulation of a soluble acetylcholinesterase variant called acetylcholinesterase-R, which itself is a potent inflammatory mediator and modulator of stress responses. With transient prophylactic administration of plant-produced acetylcholinesterase-R, I am capable of conferring protection from all of these deleterious consequences. Interestingly, I see soluble acetylcholinesterase-R and its C-terminal peptide derivative acetylcholinesterase readthrough peptide as an effective means to increase levels of pro-inflammatory cytokines such as tumor necrosis factor-alpha and interleukin 6 that have been reduced by organophosphate poisoning. Furthermore, I propose a novel molecular mechanism for the immunosupression induced by the organophosphate toxins that causes induction of the cholinergic anti-inflammatory response. Thus, I have demonstrated a multivalent approach for the prevention of both the acute and chronic symptoms associated with organophosphate toxicity. |
