Bioengineering of the blood-brain barrier permeability

描述(由申请人提供)：各种药物对血脑屏障(BBB)的通透性在很大程度上取决于位于脑毛细血管内皮细胞(BCEC)膜上的特定外流转运体的表达和功能活性。选择性的瞬时下调转运蛋白将导致应用更有效和毒性更低的治疗药物来治疗中枢神经系统的脑瘤或病毒感染。以前，反义抑制剂被证明可以暂时阻止主要的多药耐药药物--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，用于选择性地关闭各种基因的表达。短发夹状RNA(SiRNA)可以通过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靶向输送到BBB的细胞需要一个良好的系统载体和与BCEC结合的选择性载体。作为这样一种载体，经脑特异性归巢肽(BSHP)修饰的聚合物交联纳米凝胶颗粒被选为通过shRNA编码的质粒DNA来转导血脑屏障，最终目的是抑制血脑屏障中的特异性膜蛋白-药物外排转运体。从噬菌体展示文库中的大量多肽中，已经在体内选择了结合到纳米凝胶表面的特定BSHP和靶向BBB的递送。纳米凝胶在许多细胞系中作为转染剂是无毒和高效的，显然是具有巨大全身给药潜力的载体之一。矢量化RNA干扰产生系统(RIP系统)可用于治疗药物的血脑屏障通透性的生物工程，这些药物的脑可及性因特定的药物外流转运体而受阻。该方案的中心假设是，通过使用靶向RNAi产生系统的系统转染脑内皮细胞，瞬时抑制血脑屏障中选定的药物外流转运体，可以在中枢神经系统相关疾病的化疗过程中显著增强药物向大脑的转运。我们的具体目标1是开发以BCEC为靶点的纳米凝胶载体，用于系统地将质粒DNA输送到血脑屏障。具体目的2是提高BCEC靶向纳米凝胶载体的体内外转染率。具体目的3是抑制体内血脑屏障中选定的药物外流转运体，并暂时增加药物进入大脑的转运量。在这一目标中，几种具有代表性的核苷类似物药物的脑转运将在动物模型中进行评估，因为基于纳米凝胶的RNAi产生系统瞬时下调了血脑屏障中的药物外流转运体。

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.