A Plant-Derived Recombinant Bioscavenger to Prevent Insecticide Neurotoxicity

描述(申请人提供)：P.I.Rosenberg，Y.J.摘要有机磷化合物(OPS)是一种强有力的神经毒性化学物质，广泛用于医药、工业和农业；最主要的是用作杀虫剂。神经毒性可能采取胆碱能危象和死亡的形式，作为急性暴露或精神症状的结果，以及慢性暴露后的迟发性神经病变。OP杀虫剂对靶向乙酰胆碱酯酶(AChE)的抑制可通过高水平的血清丁酰胆碱酯酶(BChE)来阻止，BChE是OP的有效生物清除剂和解毒剂。目前，杀虫剂中毒的治疗包括支持性护理和特殊治疗，如阿托品和肟类药物，但往往无法预防发病率或死亡。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，用于杀虫剂毒性暴露前后的治疗。这将包括两个阶段：(1)利用植物表达系统生产猕猴(Ma)rBChE，该系统因其在GMP条件下能够产生大量生物量而具有通用性和廉价(阶段I)和(2)由于它们的非唾液酸化和非典型的多糖，这些重组植物蛋白将表现出较差的生物利用度，必须经过翻译后修饰(阶段II)以产生稳定的天然类分子。因此，在第一阶段，植物将被改造成消除1，2-木糖的表达，并以L，3-岩藻糖为核心，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4个氨基酸KDEL标签将rMaBChE蛋白质合成引导到烟草细胞的内质网(ER)，这将导致只产生“高甘露糖”型多糖，或(Ii)使用突变的拟南芥植物，其中编码1，2-木糖转移酶和1，3-岩藻糖基转移酶的基因已经被敲除：每种方法都避免了下游植物特有的多糖。在第二阶段的研究中，将对工程BChE进行化学修饰，要么通过进一步的体外糖基化，要么通过聚乙二醇化，以产生具有最佳生物利用度的蛋白质。修饰的rMaBChE的药代动力学、免疫原性和活性将在小鼠和同源猕猴身上进行评估。MaBChE的使用将确立利用植物扩大生产复杂糖蛋白的可能性，并在相应的系统中提供快速的临床前安全数据，用于随后为暴露于杀虫剂的人类生产HuBChE治疗。许多糖蛋白具有非常重要的生理功能，但不能用于治疗，因为它们很快就被从循环中移除。该项目的目的是开发一种廉价的植物来源的暴露前或暴露后丁酰胆醇酯酶处理方法，以防止有机磷杀虫剂中毒；这正迅速成为一个重大的公共卫生问题。因此，通过修改基因工程形式的酶上的糖侧链，它将在血液中保留更长时间，并在神经毒素到达神经系统之前具有更强的中和能力。

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.