Research Project
7271629
[unreadable] DESCRIPTION (provided by applicant): P.I. Rosenberg, Y.J. ABSTRACT Organophosphorus (OPs) compound are potent neurotoxic chemicals that are widely used in medicine, industry and agriculture; most notably as insecticides. The neurotoxicity may take the form of cholinergic crisis and death as a consequence of acute exposure or psychiatric symptoms and delayed neuropathy following chronic exposure. Inhibition of the targeted acetylcholinesterase (AChE) by OP insecticides may be prevented by high levels of serum butyrylcholinesterase (BChE) which is a potent bioscavenger and detoxicant of OP. Currently, treatment of insecticide poisoning consists of supportive care and specific therapy e.g. atropine and oximes, which often fail to prevent morbidity or death. The long term goal of this project is to develop an efficacious recombinant (r) BChE bioscavenger as pre- and post exposure treatments of insecticide toxicity. This will involve two stages: (1) Production of macaque (Ma) rBChE using the plant expression system which is versatile and inexpensive due to its ability to yield a large biomass in GMP conditions (Phase I) and (2) as a result of their unsialyted and atypical glycans, these recombinant plant proteins will exhibit poor bioavailability and must undergo post-translational modification (Phase II) to create stable native-like molecules. Thus, in Phase I, plants will be engineered to eliminate the expression of [unreadable] 1,2-xylose and core a l,3-fucose and remove potential immunogenic epitopes. This will be achieved by (i) directing rMaBChE protein synthesis to the endoplasmic reticulum (ER) in tobacco cells by means of a 4 aa KDEL tag which will result in only "high mannose" type glycans or (ii) using mutant Arabidopsis plants in which genes encoding for [unreadable]1,2-xylosyltransferase and a1,3-fucosyltransferase have been knocked out: each method thereby avoiding the downstream plant specific glycans. In Phase II studies, chemical modification of engineered BChE will be performed, either by further in vitro glycoslyation or by PEG-ylation to yield a protein with the best bioavailability. Pharmacokinetics, immunogenicity, and activity of the modified rMaBChE will be assessed using mice and homologous macaques. The use of MaBChE will establish the plausibility of using plants for the scaled-up production of complex glycoproteins and to provide expedited preclinical safety data in an homologous system for the subsequent production of a HuBChE treatment for insecticide-exposed humans. Many glycoproteins exhibit very important physiological functions but cannot be used as therapeutic treatments because they are quickly removed from the circulation. The aim of this project is to develop an inexpensive plant-derived pre- or post- exposure butyrylchol- inesterase treatment, to prevent organophosphate insecticide toxicity; which is fast becoming a significant public health issue. Thus, by modifying the sugar side chains on the genetically engineered form of enzyme, it will be retained in the blood longer and have an enhanced capacity to neutralize the neurotoxins toxins before they reach the nervous system. [unreadable] [unreadable] [unreadable]
