A Brain-Targeted Organophosphorus Hydrolase Fusion Protein Mediated by Angiopep-2 Neutralizes Central Organophosphates and Alleviates Neurotoxicity

Yanwei Xie,1,2 Yanan Zhai,1,2 Jiaxuan Shang,1 Ming Ma,1 Dan Wang,1 Shengye Xu,1 Yuhua Ran,1,2 Zhiping Li,1,2 Xiang Gao,1,2 Jing Gao1,2 1State Key Laboratory of National Security Specially Needed Medicines, Beijing, 100039, People’s Republic of China; 2Academy of Military Medical Sciences, Beijing, 100850, People’s Republic of ChinaCorrespondence: Xiang Gao, Email gaoxiang609@126.com Jing Gao, Email gjsmmu@126.comPurpose: Organophosphates (OPs) exert neurotoxicity by inhibiting acetylcholinesterase (AChE) activity, leading to acetylcholine accumulation, excessive nervous system stimulation, and even fatal outcomes. Current clinical countermeasures focus on AChE reactivation and symptom antagonism but lack the ability to efficiently clear OPs in vivo, especially in the brain, failing to mitigate central neurotoxicity and resulting in persistent neuronal damage and permanent disability in survivors. Organophosphorus hydrolase (OPH) has robust OP clearance capacity but is hindered by poor blood-brain barrier (BBB) penetration. Thus, the purpose of this study is to address the BBB penetration challenge of OPH and develop an effective strategy for efficient clearance of brain-invading OPs to improve the prognosis of patients with OP-induced central neurotoxicity.Patients and Methods: A fusion protein ANG-OPHDS5 was constructed by conjugating OPH mutant OPHDS5 with Angiopep-2 (ANG), a brain-targeting ligand that mediates transcytosis across the BBB via specific binding to low-density lipoprotein receptor-related protein 1 (LRP1). In vivo imaging and pharmacodynamic studies were performed to evaluate the brain-targeting ability, OP clearance efficiency, and neurotoxicity alleviation effect of ANG-OPHDS5.Results: In vivo imaging and pharmacodynamic studies confirmed that the constructed fusion protein ANG-OPHDS5 efficiently achieved targeted delivery to the brain, effectively cleared OPs residing in the central nervous system (CNS), and significantly alleviated neurotoxicity induced by OPs.Conclusion: Our strategy of centrally targeted delivery of OPH-based bioscavengers (ANG-OPHDS5) overcomes the limitations of current OP poisoning treatments, which lack efficient brain OP clearance capacity. This strategy holds great potential for improving the prognosis of patients with OP-induced central neurotoxicity.Plain Language Summary: 1. Organophosphate poses a risk of brain damage, which can be effectively countered via cerebral delivery of organophosphorus hydrolase. 2. A brain-targeting recombinant fusion protein termed ANG-OPHDS5 was successfully constructed for efficient brain delivery. 3. ANG-OPHDS5 demonstrated potent antagonistic ability against lethal ethyl paraoxon toxicity and significantly alleviated accompanying central nervous system injury. The infographic consists of four sections. The first section, ’Fusion protein expression’ shows a gene construct with ligand, linker and protein components. It illustrates the process in E. coli BL21(DE3) leading to the production of ANG-OPHDS5 fusion protein. The second section, ’Poisoning treatment,’ depicts organophosphate exposure in mice, followed by intravenous administration of OPHDS5, leading to recovery. The third section, ’Central rescue,’ illustrates a synaptic gap with acetylcholine (ACh) and acetylcholinesterase (AChE) activity, highlighting the stopping of harmful effects. The fourth section, ’Blood-brain barrier delivery,’ shows a blood vessel with the delivery of substances across the blood-brain barrier into brain tissue, focusing on the synaptic gap area.Infographic on fusion protein expression, poisoning treatment, central rescue and blood-brain barrier delivery.