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

Abstract

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 (S_n-: 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.

Keywords: MCTS; SSO; carrier-free oligonucleotide delivery; cationic oligonucleotide-oligospermine conjugate; exon skipping; multicellular tumor spheroids; splice switching oligonucleotides.

Graphical abstract

Figure 1

Solid-Phase Synthesis of Oligospermine-Oligonucleotide Conjugates (S)_n-[X]

Figure 2

Carrier-free Cellular Uptake of (S)₂₀-[ON705]-(F) in Monolayer HeLa pLuc/705 Cells Fluorescently labeled (S)₂₀-[ON705]-(F) were incubated at the concentrations indicated on the top of images during (A, right panels) 45 min, (B, right panels) 2 hr, and (C, right panels) 4 hr in serum-free medium. Naked [ON705]-(F) was used as a control in the same conditions (A–C, left panels). Images were taken by fluorescent microscopy using 488-nm laser. (D) Zoom of the right panel in (C).

Figure 3

Time-Course Profile of Carrier-free Splice Switching in Monolayer HeLa pLuc/705 Cells Using (S)_n-[ON705] (S)_n-[ON705] (n = 15, 20, and 30) were added to the cells initially in serum-free DMEM, and FBS was added to 10% after 4 hr. Luciferase reporter gene expression levels were periodically measured as RLU/mg of cell proteins and normalized against the levels of untreated cells. All data are presented as mean ± SEM of n = 3 separate experiments.

Figure 4

Carrier-free Splice Switching in Monolayer HeLa pLuc/705 Cells by (S)_n-[ON705] (n = 15, 20, and 25) Luciferase activity was determined after 48 hr of incubation. (A) (S)_n-[ON705] were incubated for 4 hr in serum-free conditions; then FBS (10%) was added. (B) (S)_n-[ON705] were added in serum-containing DMEM (10%). The rhombi indicate the total protein measurement. All data are presented as mean ± SEM of n = 3 separate experiments. JM, JetMessenger.

Figure 5

Morphology of HeLa pLuc/705 MCTS Two-day-old spheroids were incubated with (S)₁₅-[ON705] 0.7 μM in serum-containing DMEM (10%) for 46 hr. Images were periodically taken by microscopy. (Left) Untreated spheroids. (Right) Spheroids treated with (S)₁₅-[ON705].

Figure 6

Carrier-Free Splice Switching in 3D Culture of HeLa pLuc/705 Cells by (S)₁₅- and (S)₂₀-[ON705] (S)₁₅- and (S)₂₀-[ON705] were added to 2-day-old HeLa pLuc/705 spheroids in serum-containing DMEM (10%). Luciferase activity was determined after 48 hr of incubation. The rhombi indicate the total protein measurement. All data are presented as mean ± SEM of n = 3 separate experiments. JM, JetMessenger.

Figure 7

Delivery of Fluorescently Labeled SSOs in 3D Culture of HeLa pLuc/705 Cells Analyzed by Flow Cytometry Untreated spheroids (red, control). [ON705]-(F) (0.2 μM, yellow) complexed with JM (JetMessenger) and (S)₂₀-[ON705]-(F) (0.7 μM, blue) were added to 2-day-old HeLa pLuc/705 spheroids in serum-containing DMEM (10%). After 48 hr of incubation, cellular uptake was analyzed using a 488-nm laser.

Figure 8

Delivery of Fluorescently Labeled SSOs in 3D Culture of HeLa pLuc/705 Cells Analyzed by Confocal Microscopy To 2-day-old HeLa pLuc/705 spheroids in serum-containing DMEM (10%) was added [ON705]-(F) complexed with JM (JetMessenger) or (S)₂₀-[ON705]-(F). Images were taken after 48 hr. (A) Fluorescein (488 nm). (B) Nuclei (405 nm). (C) Merge.