VECTOR MEDIATED ODN DELIVERY FOR ANTIVIRAL CHEMOTHERAPY

针对病毒基因的反义寡核苷酸 参与疾病发病机制是一种很有前途的方法 发展合理的抗病毒化疗。使用寡聚脱氧- 甲基膦(d-OMPS)我们证明了对疱疹的抑制作用 单纯疱疹病毒(HSV)IE基因抑制病毒体外和体外生长 感染动物(小鼠耳模型)。新开发的OMPS具有 交替的2‘-O-甲基核糖核苷甲基膦和 磷酸二酯键(ALT-MR-OMP)对其具有较高的亲和力 靶向mRNAs，并显著提高抗病毒活性。我们最近 构建了一种无核酸载体，由腺病毒和 流感多肽参与细胞穿透和 内溶，并表明它增加了OMP的抗病毒活性。 在这里，我们建议检查KALM介导的交付是否改变了OMP 细胞内定位，并增加其细胞内 生物分布和抗病毒活性。由KALM矢量传递的OMP 它缺乏融合性流感蛋白成分以及游离 OMP和与BSA络合的OMP将作为对照。FITC/[32P]-标记 OMPS将用于检查摄取、细胞内定位和 细胞内生物利用度的持续时间。分析和量化 荧光图像将由共聚焦显微镜完成。要检查OMP，请执行以下操作 FITC标记的OMP与蛋白质的摄取/转运、共定位 包被的内吞囊泡、小凹和β-COP将由 罗丹明标记抗体的双重免疫荧光染色 囊泡蛋白。靶向HSV基因表达的抑制将是 用各自的抗体进行免疫印迹测定，结果 与抗病毒活性相比。在体内的摄取和生物分布 作为KALM络合物递送的OMP及其抗病毒活性将是 在小鼠耳朵模型中确定。将研究KALM交付的OMPS 因为它们有能力干扰HSV基因表达和病毒生长 在培养的原代神经细胞中，病毒基因的表达 在首次感染时和再次感染时，受到不同的监管 启动病毒复制。

Antisense oligonucleotides (ODNs) that target viral genes specifically invOlved in disease pathogenesis are a promising approach to the development of rational antiviral chemotherapy. Using oligodeoxy- methylphosphonates (d-OMPs) we demonstrated that inhibition of Herpes simplex virus (HSV) IE genes reduces virus growth in vitro and in infected animals (mouse ear model). Newly developed OMPs with alternating 2'-O-methylribonucleoside methylphosphonate and phosphodiester bonds (alt-mr-OMPs) have a higher affinity for their target mRNAs and significantly improved antiviral activity. We recently developed a nucleic acid free vector (KALM) consisting of adenovirus and influenza peptides which are involved in cell penetration and endosomolysis, and showed that it increases OMP antiviral activity. Here we propose to examine whether KALM-mediated delivery alters OMP intracellular localization and increases its intracellular biodistribution and antiviral activity. OMP delivered by a KALM vector which lacks the fusogenic influenza protein component as well as free OMP and OMP complexed to BSA will serve as controls. FITC/[32P]-labeled OMPs will be used to examine uptake, intracellular localization and duration of intracellular bioavailability. Analysis and quantitation of fluorescent images will be done by confocal microscopy. To examine OMP uptake/trafficking, colocalization of the FITC labeled OMP with proteins of coated endocytic vesicles, caveolae and beta-COP will be examined by double immunofluorescent staining with rhodamine-labeled antibodies to vesicle proteins. Inhibition of targeted HSV gene expression will be determined by immunoblotting with respective antibodies and the results compared to antiviral activity. The in vivo uptake and biodistribution of OMPs delivered as KALM complexes and their antiviral activity will be determined in the mouse ear model. KALM-delivered OMPs will be studied for their ability to interfere with HSV gene expression and virus growth in cultured primary neuronal cells in which virus gene expression is differently regulated, at the time of first infection and at re- initiation of virus replication.