CYTOPLASMIC DELIVERY OF OLIGONUCLEOTIDES

将反义寡核苷酸输送到靶细胞胞浆，a 他们活动的必要条件，一直是最多的 发展中的挑战性任务。文献证据表明 细胞通过内吞作用摄取寡核苷酸。因为管腔内 内涵体的pH可低至5.0，与pKa 6形成弱酸性 质子化成为内体中的中性物种，并在 细胞质，pH值7.2。目前的研究将检验这样一种假设 弱酸可以作为胞质递送的载体 寡核苷酸。 在本研究中，pKa在5到7范围内的弱酸将 作为载体的一部分。它不仅会被添加到5‘-或3’- 末端，但也插入在寡核苷酸的中间。为了 使共轭的化学与固相相容 合成方面，我们计划合成一系列合成子。类似地，一个 含胺的合成子将准备将荧光团连接到 齐聚物的5‘-末端。 载体/寡核苷酸结合物将通过三种方法进行表征 系统。首先，寡核苷酸结合物在单个 具有跨膜pH梯度的脂质体双层将被 监视器。第二，荧光偶联物的细胞配置将 在多种细胞系中进行研究，如RAW 264.7、CHO、CACO2和MDCK 通过共聚焦荧光显微镜观察。第三，反义 抗c-myc原癌基因偶联弱酸载体(S) 用于功能分析。在整个研究过程中，甲基膦 将使用不同大小的脱氧寡核苷酸，最大可达30聚体。这个 给定条件下弱酸性载体(S)的数量和/或位置 寡核苷酸也会发生变化，以获得有效的细胞质 结合的寡核苷酸的递送。总括而言，建议的 这项研究旨在检验一种假设，即pH梯度存在 穿过早期的内体膜可在细胞质中被利用 寡核苷酸的运送。如果实现，这项技术还应该 在其他类型的细胞质递送中找到了普遍的应用 大分子药物。

Delivery of antisense oligonucleotides to the target cell cytoplasm, a necessary condition for their activity, has been one of the most challenging tasks in the development. Literature evidence suggests that cells take up oligonucleotides via endocytosis. Since the intraluminal pH of endosomes can be as low as 5.0, a weak acid with pKa 6 will be protonated to become a neutral species in endosomes and ionized in the cytoplasm at pH 7.2. The present study will test a hypothesis that such a weak acid can serve as a carrier for cytoplasmic delivery of oligonucleotides. In the present study, weak acid with a pKa in the range of 5 to 7 will serve as the carrier moiety. It will be added not only to the 5'- or 3'- end but also inserted in the middle of an oligonucleotide. In order to render the chemistry of the conjugation compatible with the solid-phase synthesis, we plan to synthesize a series of synthons. Similarly an amine-containing synthon will be prepared to link a fluorophore to the 5'-end of an oligomer. Carrier/oligonucleotide conjugates will be characterized in three assay systems. First, uptake of oligonucleotide conjugates across a single liposomal lipid bilayer with a transmembrane pH gradient will be monitors. Second, cellular disposition of fluorescent conjugates will be studied in various cell lines such as RAW 264.7, CHO, Caco2, and MDCK by means of the confocal fluorescence microscopy. Third, antisense against c-myc protooncogene conjugated with the weak-acid carrier(s) will be used in a functional assay. Throughout the study, methylphosphonate deoxyoligonucleotides of different sizes up to 30mer will be used. The number and/or sites of the weak-acid carrier(s) for a given oligonucleotide will also vary to obtain an effective cytoplasmic delivery of the conjugated oligonucleotides. In summary, the proposed study is designed to test a hypothesis that the pH gradient existing across the early endosomal membrane can be exploited in cytoplasmic delivery of oligonucleotides. If realized, the technology should also find a general application in cytoplasmic delivery of other types of macromolecular drugs.