Cationic Oligospermine-Oligonucleotide Conjugates Provide Carrier-free Splice Switching in Monolayer Cells and Spheroids

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We report the evaluation of 18-mer 2′-O-methyl-modified ribose oligonucleotides with a full-length phosphorothioate backbone chemically conjugated at the 5′ end to the oligospermine units (Sn-: n = 5, 15, 20, 25, and 30 [number of spermine units]) as splice switching oligonucleotides (SSOs). These conjugates contain, in their structure, covalently linked oligocation moieties, making them capable of penetrating cells without transfection vector. In cell culture, we observed efficient cytoplasmic and nuclear delivery of fluorescein-labeled S20-SSO by fluorescent microscopy. The SSO conjugates containing more than 15 spermine units induced significant carrier-free exon skipping at nanomolar concentration in the absence and in the presence of serum. With an increasing number of spermine units, the conjugates became slightly toxic but more active. Advantages of these molecules were particularly demonstrated in three-dimensional (3D) cell culture (multicellular tumor spheroids [MCTSs]) that mimics living tissues. Whereas vector-complexed SSOs displayed a drastically reduced splice switching in MCTS compared with the assay in monolayer culture, an efficient exon skipping without significant toxicity was observed with oligospermine-grafted SSOs (S15- and S20-SSOs) transfected without vector. It was shown, by flow cytometry and confocal microscopy, that the fluorescein-labeled S20-SSO was freely diffusing and penetrating the innermost cells of MCTS, whereas the vector-complexed SSO penetrated only the cells of the spheroid’s outer layer.
We already reported the use of DMT-spermine phosphoramidite as a versatile reagent compatible with solid-phase oligonucleotide synthesis for the attachment of the desired number of spermine moieties to oligonucleotides, which allowed us to synthesize a variety of oligospermine-oligonucleotide conjugates (zip nucleic acids [ZNAs]). When ZNAs are hybridized to their complementary strands, the cationic oligospermine tail acts as a zipper to neutralize the polyanionic internucleotidic phosphates, thus enhancing binding affinity and binding kinetics. These biophysical properties can be finely tuned according to the number of attached spermine units, making ZNA a versatile PCR probe. ZNA is commercially available (number of spermine units <10) and used in numerous nucleic-acid-based diagnostic applications. Cationic oligospermines covalently attached to oligonucleotides can also act similarly to the polyamine-type delivery vectors. We described small interfering RNA (siRNA)-oligospermine conjugates containing 30 spermine units that induced an efficient carrier-free luciferase gene silencing. Locked nucleic acid (LNA)-oligospermine conjugates with nine spermine units were also reported as active cell-permeable oligonucleotides for antisense and antigene inhibition of gene expression. More recently, the oligospermine with 15 spermine units was attached to cyclic RGD (cRGD)-siRNA conjugate, thus enhancing the tumor cell-specific delivery. (Mol Ther Nucleic Acids. 2018 Dec 7; 13: 483–492.)

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