Application of Locked Nucleic Acid Oligonucleotides for siRNA Preclinical Bioanalytics

Abstract

Despite the exquisite potential of siRNA as a therapeutic, the mechanism(s) responsible for the robust indirect exposure-response relationships have not been fully elucidated. To understand the siRNA properties linked to potent activity, requires the disposition of siRNA to be characterized. A technical challenge in the characterization is the detection and quantitation of siRNA from biological samples. Described herein, a Locked Nucleic Acid (LNA) Hybridization-Ligation ECL ELISA was designed for ultra-sensitive quantification of both sense and antisense strands of siRNA independent of structural modifica-tions. This assay was applied to measure siRNA in serum and tissue homogenate in preclinical species. We observed rapid clearance of siRNA from the systemic circulation which contrasted the prolonged accumulation within the tissue. The assay was also able to distinguish and quantify free siRNA from RNA-induced silencing complex (RISC) and Argonaute 2 (Ago2) associated with therapeutic siRNA. We utilized an orthogonal method, LC-MS, to investigate 3' exonuclease activity toward the antisense strand metabolism. Taken together, we have demonstrated that the LNA Hybridization-Ligation ECL ELISA is arobust analytical method with direct application to measuring the exposure of siRNA therapeutics seamlessly across biological matrices.

Figure 1

Illustration of the hybridization-ligation ECL ELISA including sequences used for the biotinylated and digoxygenin conjugated probes.

Figure 2

LNA Hybridization-Ligation ECL ELISA assay sensitivity and specificity. (A) The assay demonstrates pM sensitivity in both rat serum and liver homogenate (n = 2). The lower limit of quantification with this assay in both serum and liver is 1.0 pM. (B) The assay is selective for both 3′ n-1 and n-2 metabolites with a response of 133% and 95% reactivity when comparing the EC50 values back to the intact siRNA molecule (n = 2). The 3′ n-3 metabolite displayed a right-shifted EC50 with a reactivity of 25% compared to full-length siRNA molecule (n = 2). Graphs fitted in SoftMax Pro (Molecular Devices, LLC. San Jose, CA) with a 4-parameter fit with fixed weighting.

Figure 3

Extracted ion chromatogram of the full length antisense strand and metabolites in NHP liver. With LC-MS analysis, we observed the full siRNA antisense strand and down to six nucleotide base subtractions from the 3′ end.

Figure 4

Measurement of siRNA molecule in vivo. (Serum exposures of siRNA molecule in NHPs (n = 6) after a single subcutaneous dose of with 0.1, 1 or 10 mg/kg. Solid lines represent sense strand and dashed lines represent antisense strand. Liver results 28 days post-dose (B). Ago2 pulldown in liver homogenate (n = 3) to measure siRNA amount bound (C). Cropped blot of pulldown displayed within inset (Full blot image provided in Supplementary Information).