Generation of Recombinant Human Ache Op-Scavengers With Extended Circulatory Longevity

Abstract

Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of infomation. Send co.mments regarding this burden estimate or any other aspect of this colledion of information, including suggestions for reducing this burden to Washington Headquarters Sen/ices, Directorate Ibr Infbmialion Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302, and to the Office of Management and Budget, Papenvork Reduction Project (0704-01881, Washington, DC 20503 Agency Use Only (Leave blank). 11. Supplementary Notes (i.e., report contains color photos, report contains appendix in non-print form, etc.)Original contains color plates: All DTIC reproductions will be in black and white. 12a. Distribution/Availability Statement)Approved for public release; distribution unlimited Abstract {Maximum 200 Words) 12b. Distribution Code (Leave Blank)The use of recombinant human acetylcholinesterase as an effective bioscavenger of organophosphorus compounds requires that it reside in the circulation for sufficiently long periods of time. We have demonstrated in the past that extension of the circulatory lifetime of AChE can be achieved either by optimizing post-translation-related enzyme processing or by chemical conjugation of polyethylene glycol (PEG) moieties to the enzyme. To delineate the interrelationship between these two modes of AChE modulation with regard to pharmacokinetic behavior, an array of AChE molecules differing in their post-translation-related processing was subjected to PEG-conjugation and monitored for their pharmacokinetic performance. In all cases tested to date, PEG-conjugation was found to give rise to enzyme species which reside for very long periods of time in the circulation of mice in a nearly equal manner. We have now found a novel mode of AChE circulatory elimination based on the recognition of specific amino acid-related epitopes. This unique clearance mechanism can also be efficiently overcome by enzyme PEGylation. These findings indicate that the circulatory residence is dictated primarily by the PEG appendage, and that cost-effective microorganisms-based production systems which do not support animal-cell-related enzyme processing, may be utilized for large-scale AChE production. In line with this notion, we have constructed a synthetic human AChE gene designed for optimal expression in microorganism-based production systems, which will now be tested for its ability to sustain human AChE production.14. Subject Terms (keywords previously assigned to proposal abstract or terms which apply to this award) I. GENERAL INTRODUCTIONThe primary role of acetylcholinesterase (acetylcholine acetylhydrolase 3.1.1.7, AChE) is the termination of impulse transmission in cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylchoUne (ACh). The enzyme has been th...