Understanding small interfering RNA (siRNA) fraction unbound (f u) in relevant physiologic compartments is critical for establishing pharmacokinetic-pharmacodynamic relationships for this emerging modality. In our attempts to isolate the equilibrium free fraction of N-acetylgalactosamine-conjugated siRNA using classic smallmolecule in vitro techniques, we found that the hydrodynamic radius was critical in determining the size exclusion limit requirements for f u isolation, largely validating the siRNA "rigid rod" hypothesis. With this knowledge, we developed an orthogonally validated 50 kDa molecular-mass cutoff ultrafiltration assay to quantify f u in biologic matrices including human, nonhuman primate, rat, and mouse plasma, and human liver homogenate. To enhance understanding of the siRNA-plasma interaction landscape, we examined the effects of various common oligonucleotide therapeutic modifications to the ribose and helix backbone on siRNA f u in plasma (f u,plasma) and found that chemical modifications can alter plasma protein binding by at least 20%. Finally, to gain insight into which specific plasma proteins bind to siRNA, we developed a qualitative screen to identify binding "hits" across a panel of select purified human plasma proteins. All authors are employees and stock holders of Amgen, Inc.

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.

After two decades teetering at the intersection of laboratory tool and therapeutic reality, with two siRNA drugs now clinically approved, this modality has finally come into fruition. Consistent with other emerging modalities, initial proof-of-concept efforts concentrated on coupling pharmacologic efficacy with desirable safety profiles. Consequently, thorough investigations of siRNA absorption, distribution, metabolism, and excretion (ADME) properties are lacking. Advancing ADME knowledge will aid establishment of in vitro–in vivo correlations and pharmacokinetic–pharmacodynamic relationships to optimize candidate selection through discovery and translation. Here, we outline the emerging siRNA design principles and discuss the consequences for siRNA disposition and biotransformation. We propose a conceptual framework for siRNA ADME evaluation, contextualizing the site of biotransformation product formation with PK–PD modulation, and end with a discussion around safety and regulatory considerations and future directions for this modality.

The potential repertoire of short interfering RNA (siRNA) therapeutics is expanding as targeting strategies evolve. One approach to enable organ-specific delivery has been to directly conjugate siRNA to a monoclonal antibody (siRNA-mAb), analogous to antibody-drug conjugates. Detection of intact siRNA-mAb conjugates presents a bioanalytical challenge given that certain synthetic nucleotide chemical modifications and lowtemperature requirements render common oligonucleotide detection assays, such as reverse transcriptionpolymerase chain reaction, incompatible with the immunoassay component. To circumvent these issues, we developed a triplex-forming oligonucleotide ELISA using locked nucleic acid (LNA) containing oligonucleotide probes. We demonstrate that the incorporation of these LNAs allow for an enrichment and immobilization of siRNA directly conjugated to an antibody at nondenaturing temperatures. Without further requirement for extraction or amplification, we can sensitively and specifically detect intact siRNA-mAb conjugates in complex matrices such as serum and tissue homogenate.

Oligonucleotide therapeutics use short interfering RNA (siRNA) or antisense oligonucleotide (ASO) molecules to exploit endogenous systems—neutralizing target RNA to prevent subsequent protein translation. While the potential clinical application is vast, delivery efficiency and extrahepatic targeting is challenging. Bioanalytical assays are important in building understanding of these complex relationships. The literature currently lacks description of robust and sensitive methods to measure siRNA and ASOs in complex biological matrices. Described herein is a non-enzymatic hybridization-based immunoassay that enables quantification of individual siRNA strands (antisense or sense) in serum, urine, bile, and liver and kidney homogenates. Assay utility is also demonstrated in ASOs. The assay improves upon previous works by abolishing enzymatic steps and further incorporating Locked Nucleic Acid (LNA) nucleotide modifications to increase analyte hybridization affinity and improve sensitivity, specificity, and robustness. We report an assay with an ultrasensitive dynamic range of 0.3 to 16,700 pM for siRNA in serum. The assay was submitted to full qualification for accuracy and precision in both serum and tissue matrices and assay performance was assessed with single and mixed analytes. The reliable LNA-hybridization-based approach removes the need for matrix sample extraction, enrichment or amplification steps which may be impeded by more advanced chemical modifications.