Targeting the Blood-Brain Barrier in Neuroinflammation

描述：[申请人摘要]这项建议探索了转铁蛋白受体介导的药物传递的一种新用途。一种基于寡核苷酸(ODN)的药物将针对脑微血管内皮细胞，目的是抑制这些细胞的炎症反应。这种方法可能对影响血脑屏障和中枢神经系统的疾病具有广泛的适用性。在这里，我们关注实验性自身免疫性脑脊髓炎(EAE)，这是一种多发性硬化症的动物模型，以证明这种血管靶向策略的可行性。该病发病机制中的关键事件影响血脑屏障的功能和完整性。形成血脑屏障的内皮细胞表达影响淋巴细胞迁移的黏附分子，以及促进炎症的酶，如诱导型一氧化氮合酶和环氧合酶-2。这些蛋白的表达受转录因子NF-kappaB的控制。因此，阻止核因子-kappaB的激活可能是一种很有前途的治疗策略。抑制转录因子的一种很好的方法是细胞递送诱骗ODN，即包含该因子的共识结合序列的短双链ODN。不幸的是，诱骗寡核苷酸面临着与相关反义方法类似的问题，即通过细胞膜的渗透性差。转铁蛋白受体系统提供的受体介导的内吞作用可以克服这一障碍。在这里，针对小鼠转移受体的8D3抗体将作为载体传递核因子-kappaB诱骗ODN。聚阴离子ODN将被阳离子聚合物聚乙烯亚胺结合在一个络合物中，然后通过亲和素-生物素桥偶联到8D3抗体上。聚乙烯亚胺是一种很有吸引力的DNA载体，因为它具有介导“内体逃逸”的能力，从而使ODN能够到达它们的胞浆或核作用部位。该项目的具体目标包括：(I)在小鼠脑内皮细胞系的体外模型中测定ODN递送系统的细胞摄取和药理活性；(Ii)测定该递送系统在正常小鼠体内的药代动力学；(Iii)评估载体介导的ODN递送系统在小鼠EAE模型中的体内药理作用。

DESCRIPTION: [Applicant's Abstract] This proposal explores a novel use of transferrin receptor mediated drug delivery. An oligonucleotide (ODNbased drug will be targeted to brain microvascular endothelial cells, with the goal to inhibit inflammatory responses in these cells. The approach could have broad applicability for diseases affecting the blood-brain barrier and the central nervous system. Here, we focus on experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, to demonstrate the feasibility of this vascular targeting strategy. Crucial events in the pathogenesis of that disease affect function and integrity of the BBB. The endothelial cells forming the BBB express adhesion molecules affecting lymphocyte transmigration, and enzymes such as inducible NO-synthase and cyclooxygenase-2 contributing to inflammation. Expression of these proteins is under control of the transcription factor NF-kappaB. Therefore, preventing NF-kappaB activation could be a promising therapeutic strategy. An elegant way to inhibit a transcription factor is the cellular delivery of decoy ODNs, short double stranded ODNs containing the consensus binding sequence of that factor. Unfortunately, decoy ODNs face similar problems as related antisense approaches, namely poor permeability through cell membranes. The receptor-mediated endocytosis provided by the transferrin receptor system can overcome that obstacle. Here, the 8D3 antibody specific for the mouse transferring receptor will serve as the vector for delivery of a NF-kappaB decoy ODN. The polyanionic ODN will be bound in a complex by the cationic polymer polyethylenimine, and then coupled to the 8D3 antibody via an avidin-biotin bridge. Polyethylenimine is an attractive carrier for DNA due to its capacity to mediate "endosomal escape", thus enabling the ODNs to reach their cytosolic or nuclear sites of action. The Specific Aims of the project comprise (i) the determination of the cellular uptake and pharmacological activity of the ODN delivery system in an in vitro model with a murine brain endothelial cell line; (ii) determining the in vivo pharmacokinetics of the delivery system in normal mice; and (iii) evaluating in vivo pharmacological effects of vector-mediated ODN delivery in the mouse EAE-model.