In vivo quantification of formulated and chemically modified small interfering RNA by heating-in-Triton quantitative reverse transcription polymerase chain reaction (HIT qRT-PCR)

Here, we describe a novel method, heating-in-Triton quantitative reverse transcription PCR (HIT qRT-PCR) that improves upon the stem-loop RT-PCR technique for the detection of formulated and chemically modified siRNAs from plasma and tissue. The broad dynamic range of this assay spans five orders of magnitude and can detect as little as 70 pg duplex in 1 g of liver or in 1 ml of plasma. We have used this assay to quantify intravenously administrated siRNA in rodents and have reliably correlated target reduction with tissue drug concentrations. We were able to detect siRNA in rat liver for at least 10 days post injection and determined that for a modified factor VII (FVII) siRNA, on average, approximately 500 siRNA molecules per cell are required to achieve a 50% target reduction.HIT qRT-PCR is a novel approach that simplifies the in vivo quantification of siRNA and provides a highly sensitive and reproducible tool to measure the silencing efficiency of chemically modified and formulated siRNAs.Several small interfering RNA (siRNA)-based therapeutics are currently in various phases of preclinical and clinical development [1-3]. There is an unmet need to develop sensitive methods to detect and quantify siRNAs in cells and tissues. Therapeutic siRNAs for systemic delivery are typically chemically modified and formulated. Chemical modifications of the sugar-phosphate backbone stabilise siRNA duplexes by enhancing their nuclease resistance and increasing their specificity by reducing off-target effects [4]. Of all delivery systems described, the formulation of siRNAs within cationic lipid nanoparticles (LNPs) is the most validated method for delivery to liver and possibly to other organs [5]. While chemical modifications contribute to increased siRNA stabilisation and specificity, they can significantly increase duplex melting temperature. Moreover, although formulations enhance siRNA tissue delivery and cellular uptake, they can inhibit siRNA release and detection. Curr