Bioengineering of the blood-brain barrier permeability

描述（由申请人提供）：各种药物通过血脑屏障（BBB）的通透性显著依赖于位于脑毛细血管内皮细胞（BCEC）膜上的特异性外流转运蛋白的表达和功能活性。转运蛋白的选择性瞬时下调将导致对中枢神经系统的脑肿瘤或病毒感染应用更有效和毒性更小的治疗药物。以前，反义抑制剂已被证明可以暂时阻止主要多药耐药因子膜p糖蛋白的合成，并促进耐药细胞表型的逆转。最近发现了一种更有效的RNA干扰机制，可以选择性地关闭各种基因的表达。短发夹RNA （siRNA）可以利用非病毒基因治疗的方法通过质粒DNA前体导入靶细胞。然而，将pDNA靶向递送到血脑屏障细胞需要良好的系统性载体和与血脑屏障结合的选择性载体。作为这样一种载体，用脑特异性归巢肽修饰的聚合物交联纳米凝胶颗粒被选择用于shrna编码质粒DNA转染血脑屏障，最终目的是抑制血脑屏障中的特异性膜蛋白，即药物外排转运体。从噬菌体展示文库中的大量肽中选择了附着在纳米凝胶表面并靶向递送到血脑屏障的特异性BSHPs。纳米凝胶是一种无毒、高效的转染剂，可用于多种细胞系，是一种具有全身给药潜力的载体。载体RNA干扰产生系统（RIP系统）可用于被特异性药物外排转运体阻碍的治疗药物血脑屏障通透性的生物工程。该提案的中心假设是，在中枢神经系统相关疾病的化疗期间，通过使用靶向rnai产生系统全身转染脑内皮，暂时抑制血脑屏障中选定的药物外流转运蛋白，可以显著增强药物向脑的转运。我们的具体目标1是开发靶向bcec的纳米凝胶载体，用于将质粒DNA系统递送到血脑屏障。特异性目的2是增强bcec靶向纳米凝胶载体在体内和体外的转染效能。具体目的3是在体内抑制血脑屏障中选定的药物外排转运蛋白，并暂时增加药物向大脑的转运。在本研究中，几种具有代表性的核苷类似物药物的脑运输将在动物模型中被基于纳米凝胶的rnai产生系统在血脑屏障中药物外排转运蛋白的短暂下调后进行评估。

DESCRIPTION (provided by applicant): Permeability of various drugs across the blood-brain barrier (BBB) is significantly dependent on the expression and functional activity of specific efflux transporters located in the membrane of brain capillary endothelial cells (BCEC). Selective transient downregulation of the transporters will lead to the application of more effective and less toxic doses of therapeutic drugs against brain tumors or viral infections in CNS. Previously, antisense inhibitors have been shown to temporarily arrest the synthesis of major multidrug resistance agent, membrane P-glycoprotein, and promote reversal of the resistant cell phenotype. The more effective RNA interference mechanism has been recently discovered for selective switching off expression of various genes. Short hairpin RNA (siRNA) could be introduced into target cells through a plasmid DNA precursor using methods of non-viral gene therapy. However, targeted delivery of the pDNA to the cells of the BBB requires a good systemic carrier and selective vectors that bind to the BCEC. As such a carrier, polymer crosslinked Nanogel particles modified with the brain-specific homing peptides (BSHP) have been chosen for tranfection of the BBB by shRNA-encoding plasmid DNA with an ultimate goal suppressing the specific membrane proteins, drug efflux transporters, in the BBB. Specific BSHPs to be attached to the surface of the Nanogel and target delivery to the BBB have been selected in vivo from a vast amount of peptides in the phage display library. Nanogel is non-toxic and highly effective as a transfection agent in many cell lines and evidently, one of the carriers with great potential for systemic administration. The vectorized RNA Interference-Producing system (RIP system) could be used for bioengineering of the BBB permeability for therapeutic agents whose brain accessibility was hampered by specific drug efflux transporters. The central hypothesis of the proposal is that transient suppression of selected drug efflux transporters in the BBB via systemic transfection of brain endothelium using targeted RNAi-producing systems can result in significant enhancement of drug transport to the brain during chemotherapy of the CNS-related diseases. Our Specific aim 1 is to develop the BCEC-targeted Nanogel carriers for systemic delivery of plasmid DNA to the BBB. Specific aim 2 is the enhance transfection efficacy of the BCEC-targeted Nanogel carriers in vitro and in vivo. Specific aim 3 is to suppress selected drug efflux transporters in the BBB in vivo and temporary increase drug transport into the brain. In this Aim brain transport of several representive nucleoside analogue drugs will be assessed in animal model following the transient downregulation of drug efflux transporters in the BBB by the Nanogel-based RNAi-producing systems.