Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality

Abstract

Therapeutic application of the recently discovered small interfering RNA (siRNA) gene silencing phenomenon will be dependent on improvements in molecule bio-stability, specificity and delivery. To address these issues, we have systematically modified siRNA with the synthetic RNA-like high affinity nucleotide analogue, Locked Nucleic Acid (LNA). Here, we show that incorporation of LNA substantially enhances serum half-life of siRNA's, which is a key requirement for therapeutic use. Moreover, we provide evidence that LNA is compatible with the intracellular siRNA machinery and can be used to reduce undesired, sequence-related off-target effects. LNA-modified siRNAs targeting the emerging disease SARS, show improved efficiency over unmodified siRNA on certain RNA motifs. The results from this study emphasize LNA's promise in converting siRNA from a functional genomics technology to a therapeutic platform.

Figure 1

Serum stability of siRNA and siLNA. The different siLNA/siRNA were incubated in 10% foetal bovine serum at 37°C and withdrawn at indicated time points. (a) The oligos were separated by PAGE and visualized with SYBR gold. ‘ds’ depicts double-stranded siRNA marker and ‘ss’ single-stranded. (b) Quantification of the bands. The mean and SD values are from three independent experiments. (c) Same layout as with foetal bovine serum but with 100% human serum or (d) 100% mouse serum.

Figure 2

Effect of different LNA loads on the activity of siRNAs against exogenous (a) firefly luciferase gene, (c) Renilla luciferase or (d) endogenous NPY gene. (a) Firefly luciferase activity. HEK293 cells were co-transfected with firefly luciferase and plasmids, and siLNA (13 nM) and luciferase activity was assessed 24 h later. The firefly luciferase activity was normalized to the Renilla luciferase activity and the uninhibited activity (plasmids alone) was set to 100%. (b) Dose–response curves on selected siLNA targeting firefly luciferase. The total siLNA/siRNA concentration was kept constant at 13 nM; the ratio of effective and irrelevant siLNA/siRNA was varied. The graph shows the log concentration of the effective siLNA from one of two representative experiments, with mean and SD derived from duplicate samples. (c) Renilla luciferase activity was assessed as for firefly luciferase. The Renilla activity was normalized to the firefly luciferase activity. (d) NPY mRNA levels. Rat PC12 cells, endogenously expressing NPY, were transfected with siLNA (100 nM). NPY mRNA was measured 24 h later by quantitative PCR. The NPY mRNA levels were normalized to cyclophilin A. The uninhibited normalized NPY mRNA level was set to 100%. The mean and SD values in the case of luciferase are from two independent experiments performed in triplicate, and from two independent experiments performed in duplicate in the case of NPY.

Figure 3

Effect on the activity of single RNA to LNA substitutions in the antisense strand. Mean and SD values are derived from two or more experiments.

Figure 4

Sense and antisense strand activity of siRNA and 5′ sense end modified siLNA. (a) Schematic representation of the SARS 3 target cloned in the sense (pS3Xs) or the antisense (pS3Xas) direction behind firefly luciferase. Also shown is the parental luciferase plasmid without the SARS 3 target (pGL3). (b) Activity of siRNA and siLNA against sense (pS3Xs), antisense (pS3Xas) or control target (pGL3-Control). Mean and SD values are from two experiments performed in duplicate.

Figure 5

LNA improvement of medium-efficient siRNAs. (a) Renilla luciferase activity. Effect of siRNA and siLNA depending on the target sequence (siRNA4 and 5 have different target sequences, accordingly also siLNA40 and 41). (b) Effect of siRNA and siLNA on SARS-induced cytotoxicity depending on the target sequence (SARS 1–3). Vero cells were transfected with 85 nM siRNA or siLNA and then infected with 6000 TCID50 SARS-CoV. SARS-induced cytotoxicity was assessed 50 h later. The untransfected but infected sample was set to 100% cytotoxicity. Mean and SD values in the Renilla case are from two experiments performed in triplicate, and in the SARS case from three experiments performed in quadruplicate.