RNA-Induced Silencing Complex-Bound Small Interfering RNA Is a Determinant of RNA Interference-Mediated Gene Silencing in Mice

Abstract

Deeper knowledge of pharmacokinetic and pharmacodynamic (PK/PD) concepts for RNA therapeutics is important to streamline the drug development process and for rigorous selection of best performing drug candidates. Here we characterized the PK/PD relationship for small interfering RNAs (siRNAs) targeting luciferase by examining siRNA concentration in plasma and liver, the temporal RNA-induced silencing complex binding profiles, mRNA reduction, and protein inhibition measured by noninvasive bioluminescent imaging. A dose-dependent and timerelated decrease in bioluminescence was detected over 25 days after a single treatment of a lipid nanoparticle-formulated siRNA targeting luciferase messenger RNA. A direct relationship was observed between the degree of in vivo mRNA and protein reduction and the Argonaute2 (Ago2)-bound siRNA fraction but not with the total amount of siRNA found in the liver, suggesting that the Ago2-siRNA complex is the key determinant of target inhibition. These observations were confirmed for an additional siRNA that targets endogenously expressed Sjö gren syndrome antigen B (Ssb) mRNA, indicating that our observations are not limited to a transgenic mouse system. Our data provide detailed information of the temporal regulation of siRNA liver delivery, Ago2 loading, mRNA reduction, and protein inhibition that are essential for the rapid and cost-effective clinical development of siRNAs therapeutics.

Non-linear pharmacokinetics of octaarginine-modified lipid nanoparticles: Barriers from in vitro to in vivo

“…A strong in vitro and in vivo correlation was seen in the relationship between the number of siRNA molecules per cell and mRNA knockdown (Figure 3a, b). Although these results were different from the fact that the Argonaute 2 (Ago2)-bound siRNA fraction, but not the total amount of siRNA found in the liver were correlated with target mRNA inhibition [11], we speculate that the capacity of siRNA to bind Ago2 was not saturated at our doses. The other possible explanation may be that most of the siRNAs in the cytosol are present in condensed form.…”

“…We initially focused on the PD, which can be analyzed based on the relationship between the number of cytosolic siRNA molecules and its gene silencing activity, because previous reports suggested that PD contributed to the non-linear relationship in both pDNA and siRNA delivery systems [8][9][10][11]. In this study, the number of siRNA molecules in the cells was used to estimate PD of siRNA.…”

“…In a previous study, we reported on a remarkable non-linearity between the number of pDNA molecules delivered to the nucleus and their gene expression activity in the case of LipofectAMINE and the PLUS reagent [8,9] and R8-MEND [10]. In addition, Wei J et al demonstrated that a saturated PD effect existed in the case of an siRNA delivery system [11]. These results suggest that PD rather than PK is a determining factor that governs transfection efficiency, however, the rate limiting step for creating a huge gap between in vitro and in vivo still remains unclear.…”

Technologies for Investigating the Physiological Barriers to Efficient Lipid Nanoparticle–siRNA Delivery

“…This is the reason that total siRNA in tissues does not always generally agree with efficacy Seitzer et al 2011). Wei et al (2011 demonstrated that target mRNA silencing in LNP-siRNA-treated livers correlated with the amount of siRNA bound to Ago2 but not with the siRNA measured in total liver lysates.…”

Biodistribution and Metabolism Studies of Lipid Nanoparticle–Formulated Internally [3H]-Labeled siRNA in Mice

“…Therefore, as highlighted in a recent PK/PD relationship study by Wei et al (2011), high concentrations of siRNA at the desired target organ do not necessarily correlate with a high knock-down level of the desired mRNA. Factors that are also likely to be needed for successful RNAi are as follows: number of Ago2-siRNA complexes, turnover of the Ago2 complex, stability of the siRNA, target mRNA half-life, mRNA expression levels, cell division and turnover, or a combination thereof.…”