Development of topical gene delivery system for clinical application

基因治疗是治疗先天性和后天性疾病的有效手段。眼睛作为基因递送的靶器官具有许多优势。11非病毒载体的使用由于其不存在病毒载体系统固有的一些风险而引起了人们对体内基因传递的极大兴趣。在各种脂质体的转染实验中，与DOTMA/Chol脂质体复合的pDNA在玻璃体内注射后显示出最高的转染活性。电荷比为+2.0的复合物在眼睛中产生最大的基因表达。因此，优化阳离子脂质体的脂质组成和pDNA/阳离子脂质体复合物的电荷比可以提高基因转染效率。我们研究了血液成分对聚乙烯亚胺（PEI）基因转染人内皮细胞系的影响。当存在胎牛血清、牛血清白蛋白、人血、人血浆、胆固醇和脂肪酸时，pDNA/PEI复合物的转染活性显著降低，但卵磷脂和胆汁盐对pDNA/PEI复合物的转染活性无影响。聚阴离子三元配合物可降低血液成分对基因表达的影响。另一方面，葡萄糖的存在使pDNA/PEI复合物的转染活性增加2-3倍。我们还发现pDNA/PEI复合物和pDNA/脂质体复合物的基因表达受肝炎等疾病的影响，在此基础上建立了模型药物布那唑嗪的眼药动学/药效学（PK/PD）模型。PK/PD模型可预测布那唑嗪滴眼和眼内注射后眼组织中的药物浓度及其消肿作用。这一PK/PD模型对基因传递系统的设计具有一定的指导意义。

Gene therapy is a useful treatment for curing inborn and acquired diseases. The eye has a number of advantages as a target organ for gene delivery. The use of non-viral vectors attracts great interests for in vivo gene delivery because they lack some of the risks inherent in viral vector systemsWe have investigated the transfection activity of plasmid DNA (pDNA)/cationic liposome complexes to the eye in rabbits. Among the transfection experiments with various lipids, pDNA complexed with DOTMA/Chol liposomes showed the highest transfection activity after intravitreal injection. The complexes at a charge raio of +2.0 produced maximal gene expression in the eye. In conlusion, optimizing the lipid composition of cationic liposomes and the charge ratio of pDNA/cationic liposome complexes should enhance the transfection efficiency to the eye.We have examined the effect of blood components on gene delivery with polyethyleneimine (PEI) in a human endothelial cell line. The transfection activity with pDNA/PEI complex was markedly decreased in the presence of fetal bovine serum, bovine serum albumin, human blood, human plasma, cholesterol and fatty acids, although it was not affected by lecithin and bile salts. Ternary complexes using polyanion could decrease the influence of blood components on gene expression. On the other hand, the transfection activity with pDNA/PEI complex was increased 2-3 times by the presence of glucose. We also found that the gene expression of pDNA/PEI complex and pDNA/liposomes complex was influenced by the disease such as hepatitis.We have developed the ocular pharmacokinetic/pharmacodynamic (PK/PD) model of model drug, bunazosin, on the basis of the concentration-time profiles and hypotensive profiles of bunazosin. The PK/PD models can predict the concentrations of bunazosin in the ocular tissues and its hypotensive effect after instillation and ocular injection. This PK/PD model must be useful for designing the gene delivery system.