AIRWAY KININ RECEPTORS AS TARGETS FOR ADENOVIRAL VECTORS

囊性纤维化（CF）肺部疾病的成功基因治疗需要有效的体内基因转移到气道上皮。基因转移治疗CF肺部表现的靶组织是高分化（WD）纤毛气道上皮。在CF基因治疗中最有前途的载体是腺病毒载体（AdV）。然而，AdV是体内呼吸上皮的低效基因转移载体，人们正在努力提高AdV介导的基因转移效率。我们推测，与PD气道细胞相比，基因转移差异是由于WD的表型差异造成的，对AdV与气道上皮细胞早期相互作用的分析表明，基因转移效率的降低是由于AdV穿过WD细胞的顶膜的有限进入，反映了顶表面多达三个独立的限速步骤：(1)减少了特异性AdV附着；(2)缺失alphavbeta3/5整合素；(3)风险内部化率降低。为了提高基因向气道上皮的转移效率，该建议将AdV重新定位于“新型”受体类型，这些受体类型存在于气道上皮的体内顶端表面，经激动剂激活后内化到网格蛋白包被的囊泡中。本提案将重点关注b2 -激肽受体（B2k）作为候选靶受体，因为除了存在于气道中外，该受体还利用非amer肽，缓激肽（BK）作为激动剂。我们建议在验证b2 -激肽受体作为有效靶标后，设计AdV在病毒衣壳上表达BK，以测试这种靶向策略。本建议的具体目的是：1。验证B2k作为气道上皮管腔表面的有效靶点，能够通过双特异性抗体连接体介导adv介导的有效基因转移；, 2。产生包含BK的转基因AdV，从而将AdV的向性重定向到B2k。

Successful gene therapy for cystic fibrosis (CF) lung disease requires efficient in vivo gene transfer to the airway epithelium. The target tissue for gene transfer approaches for the treatment of the pulmonary manifestations of CF is the well- differentiated (WD) ciliated airway epithelium. Among the most promising vectors for CF gene therapy are the adenoviral vectors (AdV). However, AdV are inefficient gene transfer vectors for the respiratory epithelium in vivo and efforts are being directed at increasing the efficiency of AdV-mediated gene transfer. We have speculated that the gene transfer discrepancy is due to phenotypic differences for WD compared to PD airway cells and analyses of the early interactions of AdV with airway epithelial cells shows that decreased efficiency of gene transfer is due to a limited entry of AdV across the apical membrane of WD cells reflecting as many as three independent rate-limiting steps at the apical surface: (1) reduced specific AdV-attachment; (2) the absence of alphavbeta3/5 integrins; and (3) a reduced rate of AdV-internalisation. In an effort to increase gene transfer efficiency to airway epithelial this proposal will re-target AdV to 'novel' receptor-types, present on the apical surface of airway epithelium in vivo, which on activation by agonist internalise into clathrin-coated vesicles. This proposal will focus on the B2-kinin receptor (B2k) as a candidate target receptor since in addition to being present in the airway, this receptor utilises the nonamer peptide, bradykinin (BK), as an agonist. It is proposed that after validation of the B2-kinin receptor as a utile target, AdV will be engineered to express BK on the virus capsid coat in order to test this targeting strategy. The specific aims of this proposal are: 1. To validate B2k as an effective target on the lumenal surface of airway epithelium that is capable of mediating efficient AdV-mediated gene transfer with bi-specific antibody linkers; and, 2. To generate genetically modified AdV encompassing BK, so redirecting AdV tropism to B2k.