Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents

Abstract

For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called 'gymnosis') that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.

Figure 1.

Gymnotic delivery leads to long-term Bcl-2 silencing in 518 A2 melanoma cells. (A) Time course of Bcl-2 protein silencing in 518A2 melanoma cells after gymnotic delivery of SPC2996. Maximum protein silencing was observed after ∼6–7 days for SPC2996. C, untreated control. (B) RT–PCR evaluation of Bcl-2 mRNA levels in 518A2 melanoma cells demonstrating silencing after 6 days in the gymnotic delivery experiment in Figure 1A. C, untreated control; C*, single primer only. (C) Western blots in 518A2 melanoma cells demonstrating silencing of Bcl-2 protein by gymnotic delivery (6 days) of the sequence-specific anti-Bcl-2 phosphorothioate LNA gap-mer SPC2996. Three control oligomers with scrambled sequence, of which one (SPC3053) has identical chemistry to SPC2996, were inactive. (D) Iterative re-addition of SPC2996 (2.5 µM) produces long-term silencing. 518A2 melanoma cells were treated with SPC2996 at a plating density to ensure that the cells were confluent only by Day 10, and then re-plated at that density in the presence of 2.5 µM oligomer. This process was repeated after 10 additional days. Seventeen repetitions were performed. Bcl-2 protein silencing was continuously maintained after 180 consecutive days. However, if the oligomer was removed after 180 days, baseline levels of Bcl-2 protein were restored after 3 days.

Figure 2.

Gymnotic delivery and silencing is observed in many cell lines. (A) Silencing of Bcl-2 protein expression in the 591.8, 1036 and 1000.36 melanoma cell lines by 2.5 and 5 µM SPC2996. Cells were treated for 10 days by gymnotic delivery before western blotting was performed. C, untreated control. (B) Separate experiments were performed to examine silencing of Bcl-2 protein expression in 591.8, 1036 and 1000.36 melanoma cell lines by 5 µM SPC2996 and the scrambled control oligomer SPC3046. Cells were treated for 6 days by gymnotic delivery before western blotting was performed. (C) Top: gymnotic delivery (10 days) and differential silencing of Bcl-2 protein expression in HT1080 fibrosarcoma cells by SPC2996. To the right, separate experiments were performed to examine gymnotic silencing of Bcl-2 (6 days) by SPC2996 and the scrambled control oligomer SPC3046; bottom: gymnotic delivery and silencing (10 days) of Bcl-2 protein expression in LNCaP cells, which can be accomplished with minimum toxicity, as compared with lipofection. (D) In vitro effects of the gymnotic delivery of SPC2996 on the expression of the Bcl-2 mRNA in Namalwa Burkitt’s lymphoma cells. qPCR analysis of Bcl-2 mRNA expression was performed after exposure to SPC2996 or SPC3088, the control oligomer (10 µM, final concentrations) for the indicated times. qPCR values represent the mean ± SD (n = 3).

Figure 3.

Off-target effects after gymnotic delivery. Off-target effects of the gymnotic silencing of Bcl-2 by 2.5 µM SPC2996 in 518A2 melanoma cells (6 days). As seen in these western blots, no changes in the expression of Bcl-xL, tubulin, Mcl-1, PKC-α or survivin proteins were observed. Furthermore, unlike after lipofection of SC2996, Bcl-2 silencing was not associated with either PARP-1 or pro-caspase-3 cleavage.

Figure 4.

Co-localization of LNA oligonucleotides with gw182, a GW/P body marker. Confocal microscopic imaging of Huh-7 cells after gymnotic delivery of either a 12-mer (SPC5024) or a 16-mer (SPC4007) anti-ApoB 5′-FAM-3′, 5′-end-capped LNA phosphorothioate gap-mer. After 1 day (A) or 3 days (B) of delivery, cells were fixed and stained with an anti-gw182 primary antibody and visualized with a rhodamine-conjugated secondary antibody. After image merging, the oligomers were observed co-localizing with the GW/P body marker. The 12-mer accumulated more rapidly than the 16-mer after 1 day, but after 3 days of gymnotic delivery, the accumulation in the GW/P bodies are approximately equivalent.

Figure 5.

In vitro gymnotic delivery and silencing correlates with in vivo gene silencing. (A) Increased potency of an anti-ApoB 3′,5′-end-capped LNA–phosphorothioate 12-mer gap-mer (SPC3716) versus a 16-mer (SPC3302) after 6 days of in vitro gymnotic delivery to Huh-7 cells. ApoB mRNA silencing data are presented as the average ± SD (n = 3; *P < 0.0001 by Student’s t-test assuming unequal variances). (B) In vivo silencing of intra-hepatic ApoB mRNA expression in female NMRI mice after intravenous delivery (5 mg/kg i.v., Days 0, 1 and 2), demonstrating the increase in potency for the 12-mer versus the 16-mer. Data are presented as the average ± SD (n = 3; **P < 0.02 by Student’s t-test assuming unequal variances). (C) In vitro lipofection (Lipofectamine 2000) of the 12-mer and 16-mer anti-ApoB oligomers into Huh-7 human hepatoma cells, demonstrating the increased potency of the latter compared with its 12-mer counterpart. In contrast to the in vitro gymnotic delivery data, these results are inversely correlated with the in vivo data. Data are presented as the average ± SD (n = 3).