Profiling RNA metabolism to understand tissue and subcellular clearance of therapeutic RNA

Recent clinical success for RNA therapeutics supports the promise of these modalities to unlock additional drug targets with pharmacodynamic responses that are considerably more durable than other drug classes. Metabolic clearance in tissue is a key determinant of dosing requirements and therapeutic durability. To build our understanding of the mechanisms of RNA metabolic clearance, we quantified the catalytic efficiency of 3'- and 5'-exonucleases, and endonucleases in S9 fractions from various rat tissues using novel fluorescent RNA probes. We validated the specificity of these probes using recombinant nucleases and rat liver S9 fractions, demonstrating their ability to accurately report enzyme activity without cross-reactivity between nuclease classes. Key experimental parameters, such as solution pH and enzyme-substrate ratios, were optimized to maximize dynamic range. Profiling nuclease activity across various rat tissues revealed tissue-specific variations, with kidney, muscle, and plasma showing the highest catalytic efficiency for 3' exonuclease, 5' exonuclease, and endonuclease, respectively. Using a model siRNA targeting hypoxanthine phosphoribosyltransferase, comparative degradation studies in rat liver homogenate, liver S9 fractions, and liver tritosomes revealed divergent metabolic profiles; S9 fractions and homogenate processed both double-stranded siRNA and single-stranded antisense RNA to a similar extent, whereas in tritosomes single-stranded RNA was observed to be degraded more rapidly than a double-stranded form. These differences are consistent with distinct nuclease activities in each compartment, reflecting both enzyme identity and relative abundance as revealed by the probe analyses. Collectively, these findings offer critical mechanistic insights into RNA metabolism and establish a robust platform to improve the development and predictability of siRNA therapeutics. SIGNIFICANT STATEMENT: Metabolic clearance of RNA therapeutics is a critical determinant of their pharmacological durability and potency. This work quantitatively describes the activity of 3 major classes of ribonucleases within various tissues and subcellular compartments, revealing specific patterns of RNA metabolism and enable discovery of novel RNA therapeutics with improved pharmacokinetic properties.