(T1430-02-08) A Sensitive, Specific and High-throughput Assay to Quantify an Oligonucleotide Therapeutics in Monkey Liver using Stem‑Loop RT-qPCR Platform

Purpose: Oligonucleotide therapeutics (OT) represent a novel class of drugs composed of short strands of synthetic DNA or RNA designed to treat a variety of diseases by silencing specific genes. RBM-Oligo-202503 is a small interfering RNA (siRNA) therapeutic with the potential to silence transthyretin (TTR) mRNA in hepatocytes, thereby reducing the production of both mutant and wild-type TTR proteins and alleviating the symptoms of hereditary transthyretin-mediated amyloidosis (hATTR). Traditionally, liquid chromatography coupled with mass spectrometry (LC-MS) and ligand-binding assays (LBA) platforms have been extensively used for the bioanalysis of OT. However, stem-loop RT-qPCR is particularly suited for projects requiring very low sample concentrations and quantities due to its high specificity and sensitivity. This is achieved through uniquely designed stem-loop primers that selectively bind to the target small RNA, enabling precise reverse transcription and amplification while minimizing non-specific interactions. In this study, we aim to develop a fast, specific, and sensitive stem-loop RT-qPCR assay to quantify RBM-Oligo-202503 in monkey liver tissue.

Methods: Liver tissue samples (100 mg) were homogenized in 1 mL of PBS containing 0.1% BSA and 0.01% Tween-80. The samples were heated at 95°C for 5 minutes, briefly vortexed, and then placed on ice for 5 minutes. Following this, the samples were centrifuged at 21,000 × g for 5 minutes at 4°C. The siRNA-containing supernatants were quantified using stem-loop reverse transcription followed by TaqMan PCR. Stem-loop reverse transcription primers and TaqMan qPCR probes/primers targeting the antisense strand of RBM-Oligo-202503 were designed using the Custom TaqMan® Small RNA Assays online tool (Thermo Fisher). Reverse transcription was performed using the TaqMan™ MicroRNA Reverse Transcription Kit (Thermo Fisher, 4366596), followed by qPCR on 384-well plates using TaqPath™ ProAmp™ Master Mix (Thermo Fisher, A30865), following the manufacturer’s protocols. Data analysis was performed using QuantStudio™ 7 Pro Design & Analysis Software.

Results: During the method development stage, several challenges were encountered, including sample/reagent contamination and assay buffer optimization. To address contamination issues, reverse transcription (RT) reactions and qPCR reaction preparations were conducted in physically segregated areas to prevent cross-contamination. Assay buffer selection was guided by a systematic evaluation of performance metrics, ensuring optimal sensitivity and specificity. The method was successfully developed and fully qualified through six accuracy and precision runs. The calibration curves demonstrated a wide dynamic range from 1.00 × 10⁶ to 1.00 femtomolar (fmol) with a sensitivity of 1.00 fmol. Calibration standards exhibited accuracy ranging from -6.3% to 10.0% and precision from 0.5% to 22.8%. Five levels of quality control (QC) samples—upper limit of quantification (ULOQ, 1 × 10⁶ fmol), high QC (HQC, 7.5 × 10⁵ fmol), middle QC (MQC, 1 × 10³ fmol), low QC (LQC, 3 fmol), and lower limit of quantification (LLOQ, 1 fmol)—were evaluated in triplicate across six runs. The standard curves exhibited excellent linearity, with an average 𝑅² value of 0.997 and PCR efficiencies of 101.328%. The QC samples demonstrated accuracy and precision ranging from 4.4% to 45.3% for LLOQ and 0.8% to 35.8% for all other levels. Additionally, the matrix effect, dilution linearity, prozone effect, selectivity, and stability were thoroughly evaluated and met validation criteria.

Conclusion: A sensitive, high-throughput and specific stem–loop RT-qPCR assay was first published for oligo, RBM-Oligo-202503, in monkey liver using stem–loop RT-qPCR platform.