Stroke neuroprotection with a recombinant fusion protein

描述（由申请人提供）：美国每年约有80万人患有急性中风。目前还没有神经保护剂可以给急性中风患者，中风是第三大死亡原因。在美国，中风患者的康复费用每年超过400亿美元。尽管制药行业付出了巨大的努力，但迄今为止还没有开发出有效的神经保护药物，原因是大多数药物不能穿过血脑屏障（BBB）。神经营养因子是潜在的大分子神经保护剂，但它们也不能穿过血脑屏障。本研究计划旨在将神经营养因子药物发现与血脑屏障药物靶向技术相结合，从而将重组神经营养因子重新配制为基因工程融合蛋白，使神经营养因子在人体延迟静脉给药后能够通过血脑屏障在体内运输。这种药物在静脉给药后会对中风患者产生神经保护作用，因为这种神经保护剂是专门设计用来穿过血脑屏障的。这项工作将制备一种基因工程融合蛋白，其中神经营养因子变体与靶配体融合，在体内经历受体介导的跨血脑屏障运输。这种血脑屏障运输载体先前已经过基因工程改造，使其能够在没有免疫反应的情况下用于人类。融合蛋白将是一种双功能分子，不仅可以穿过血脑屏障，还可以与神经元上特定的神经营养因子受体结合，从而引起神经元的神经保护。在第一阶段，一个表达载体被设计用于细胞的永久转染。免疫细胞化学和Western blotting显示永久转染的细胞表达融合蛋白。第二阶段的工作将产生一个分泌高水平融合蛋白的细胞系，这种蛋白的生产将扩大规模，用于生产。II期工作的完成将使FDA能够准备IND，以测试这种新型神经保护剂在人类急性中风中的作用。

DESCRIPTION (provided by applicant): Approximately 800,000 persons in the U.S. suffer an acute stroke each year. There presently is no neuroprotective agent that can be given to patients with acute stroke, and stroke is the third leading cause of death. The costs for rehabilitation of patients that survive a stroke are greater than $40 billion per year in the U.S. The reason that an effective neuroprotective drug has not been developed so far, despite intensive efforts in the pharmaceutical industry, is that most of the drugs do not cross the blood-brain barrier (BBB). Neurotrophins are potential large molecule neuroprotectives but these, too, do not cross the BBB. The present research plan aims at a merger of neurotrophin drug discovery and BBB drug targeting technology, so that a recombinant neurotrophin can be re-formulated as a genetically engineered fusion protein to enable transport of the neurotrophin through the BBB in vivo following delayed intravenous administration in humans. Such a drug will cause neuroprotection in stroke patients following intravenous administration, because the neuroprotective agent will have been specifically designed to cross the BBB. This work will prepare a genetically engineered fusion protein wherein the neurotrophin variant is fused to a targeting ligand that undergoes receptor-mediated transport across the BBB in vivo. This BBB transport vector has been previously genetically engineered to enable use in humans without immunological reaction. The fusion protein will be a bi-functional molecule that not only crosses the BBB, but also binds to the specific neurotrophin receptor on neurons to cause neuronal neuroprotection. In phase I, an expression vector was engineered for permanent transfection of cells. Immunocytochemistry and Western blotting demonstrated expression of the fusion protein by the permanently transfected cells. The phase II work will produce a cell line secreting high levels of the fusion protein, and the production of this protein will be scaled up for manufacturing. The completion of the phase II work will enable the preparation of an IND to the FDA for testing of this novel neuroprotective agent in acute stroke in humans.