A Plant-Derived Recombinant Bioscavenger to Prevent Insecticide Neurotoxicity

描述（由申请人提供）：P.I.摘要有机磷化合物是一种具有神经毒性的化合物，广泛应用于医药、工业和农业，尤其是作为杀虫剂。神经毒性可能表现为胆碱能危象和急性接触导致的死亡或精神症状，以及慢性接触后的迟发性神经病。高水平的血清丁酰胆碱酯酶（BChE）是OP的有效生物清除剂和解毒剂，可以防止OP杀虫剂对目标乙酰胆碱酯酶（AChE）的抑制。目前，杀虫剂中毒的治疗包括支持性护理和特定治疗，如阿托品和肟，这往往不能防止发病或死亡。该项目的长期目标是开发一种有效的重组（r）BChE生物清除剂，作为杀虫剂毒性的暴露前和暴露后处理。这将包括两个阶段：（1）使用植物表达系统生产猕猴（Ma）rBChE，由于其在GMP条件下产生大生物量的能力，该系统是通用的和廉价的（阶段I），和（2）由于其非唾液酸化和非典型聚糖，这些重组植物蛋白将表现出差的生物利用度，并且必须经历翻译后修饰（阶段II）以产生稳定的天然样分子。因此，在阶段I中，植物将被工程化以消除1，2-木糖和核心α 1，3-岩藻糖的表达并去除潜在的免疫原性表位。这将通过以下方式实现：（i）通过将仅产生“高甘露糖”型聚糖的4 aa KDEL标签将rMaBChE蛋白合成引导至烟草细胞中的内质网（ER），或（ii）使用其中编码α 1，2-木糖基转移酶和α 1，3-岩藻糖基转移酶的基因已被敲除的突变拟南芥植物：每种方法由此避免下游植物特异性聚糖。在II期研究中，将通过进一步体外糖基化或PEG化对工程化BChE进行化学修饰，以产生具有最佳生物利用度的蛋白质。将使用小鼠和同源猕猴评估修饰的rMaBChE的药代动力学、免疫原性和活性。MaBChE的使用将确立使用植物扩大生产复杂糖蛋白的可行性，并在同源系统中提供加速的临床前安全性数据，用于随后生产暴露于二甲双胍的人类的HuBChE治疗。许多糖蛋白具有非常重要的生理功能，但不能用作治疗性治疗，因为它们很快从循环中除去。该项目的目的是开发一种廉价的植物源性暴露前或暴露后丁酰胆碱酯酶治疗，以防止有机磷杀虫剂毒性;这正在迅速成为一个重要的公共卫生问题。因此，通过修改酶的基因工程形式上的糖侧链，它将在血液中保留更长时间，并具有增强的能力，在神经毒素到达神经系统之前中和神经毒素。

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 ¿ 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 ¿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.