Stability study of unmodified siRNA and relevance to clinical use

Abstract

RNA interference offers enormous potential to develop therapeutic agents for a variety of diseases. To assess the stability of siRNAs under conditions relevant to clinical use with particular emphasis on topical delivery considerations, a study of three different unmodified siRNAs was performed. The results indicate that neither repeated freeze/thaw cycles, extended incubations (over 1 year at 21 degrees C), nor shorter incubations at high temperatures (up to 95 degrees C) have any effect on siRNA integrity as measured by nondenaturing polyacrylamide gel electrophoresis and functional activity assays. Degradation was also not observed following exposure to hair or skin at 37 degrees C. However, incubation in fetal bovine or human sera at 37 degrees C led to degradation and loss of activity. Therefore, siRNA in the bloodstream is likely inactivated, thereby limiting systemic exposure. Interestingly, partial degradation (observed by gel electrophoresis) did not always correlate with loss of activity, suggesting that partially degraded siRNAs retain full functional activity. To demonstrate the functional activity of unmodified siRNA, EGFP-specific inhibitors were injected into footpads and shown to inhibit preexisting EGFP expression in a transgenic reporter mouse model. Taken together, these data indicate that unmodified siRNAs are viable therapeutic candidates.

FIG. 1.

Tissue culture-based assay for assessing functional siRNA activity. (A) Schematic representation of K6a, hepatitis C virus (HCV), and EGFP bicistronic reporter constructs [all linked to firefly luciferase (fLuc)] and the target sequences and locations for each siRNA. The K6a and EGFP coding regions are separated from the fLuc coding region by the foot and mouth disease virus (FMVD) 2A element [shaded gray, (Wang et al., ; Hickerson et al., 2008)]. (B) 293FT cells were cotransfected with each reporter plasmid and the indicated concentration of cognate siRNA. fLuc activity was measured following addition of luciferin substrate using the IVIS imaging system. The data were normalized and then corrected against cells transfected with a nonspecific control siRNA.

FIG. 2.

SiRNA stability under conditions relevant to clinical use, including incorporation into cream formulations. Ten freeze/thaw cycles (A) were performed by alternating cycles of freezing at −20°C for 12 hours, followed by thawing at 21°C for 30 minutes. SiRNAs were stored at 21°C for 28 days (B) or alternatively at 37°C (C) or 95°C (D) for the indicated times. An aliquot of each sample (50 pmoles siRNA per lane) was analyzed by native 10% PAGE. Gels were stained with ethidium bromide and visualized by phosphorimager. Functional activity was determined following cotransfection of 1 nM siRNA (final concentration) with 16 ng fLuc fusion construct in 293FT cells as described in Figure 1.

FIG. 3.

Stability of siRNA exposed to skin, hair, saliva, and sera. SiRNAs (10 μM final concentration) were subjected to potential RNase degradation by incubation in 95% fetal bovine serum (A), 95% human serum (B), PBS exposed to human skin (C), or hair (D), or 95% saliva (E) for the indicated times, or RNase A (F) for 10 minutes at the indicated concentrations. Treated siRNAs were analyzed as in Figure 2.

FIG. 4.

L2G85 EGFP transgenic mouse model. L2G85 mice (which express EGFP under the control of the modified chicken beta actin promoter; Cao et al., 2005) were assayed for GFP expression using the CRi Maestro in vivo imaging system. (A) Image following illumination with full-spectrum light. (B) EGFP-specific emission following excitation with blue light is pseudo-colored green (following un-mixing from background spectra, see Materials and Methods). Left mouse: nontransgenic control mouse. Right mouse: L2G85 mouse (expresses EGFP). Note that the fur blocks detection of fluorescence. Shaved L2G85 mice show EGFP expression in shaved regions (data not shown). (C) EGFP expression (antibody staining, see Materials and Methods) of a skin section prepared from the footpad of an L2G85 mouse (bar = 50 μm). The section was counterstained with DAPI to allow visualization of nuclei (purple).

FIG. 5.

SiRNA-mediated inhibition of preexisting gene expression in an EGFP transgenic mouse model. Treatment of L2G85 mice with unmodified EGFP-specific siRNA leads to decreased fluorescence. EGFP-specific or nonspecific control (NSC4) siRNAs were intradermally injected (100 μg/injection) into footpads of L2G85 mice (three injections per mouse footpad spaced over 1 week, n = 4). At the indicated times, the mice were imaged for EGFP expression using the Maestro (CRi) imaging system (EGFP expression is pseudo-colored white in these images; fluorescence is not observed in areas of fur) as described in Figure 4. This experiment has been independently repeated three times.