Plasma and Liver Protein Binding ofN-Acetylgalactosamine–Conjugated Small Interfering RNA

Understanding small interfering RNA (siRNA) fraction unbound (fu) 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 small-molecule in vitro techniques, we found that the hydrodynamic radius was critical in determining the size exclusion limit requirements for fu 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 fu 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 fu in plasma (fu,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.