CYTOPLASMIC DELIVERY OF OLIGONUCLEOTIDES

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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.