10074412Investigation of Mergo? mechanism of action by quantitative serum protein profiling.ClosedCollaborative R&DInnovate UKSixfold Bioscience, a London-based biotech SME, are developing Mergo?, RNA-based delivery systems designed to carry oligonucleotides therapeutics to extrahepatic tissues. The Mergo? technology allows the development of a platform that can rapidly be engineered to explore and screen a large and diverse chemical space. Indeed, Mergos can be easily chemically modified to change their physicochemical properties. Sixfold has shown that Mergos with altered chemical composition can distribute to extrahepatic tissues _in vivo_ (mice models). More interestingly, certain Mergo? candidates display cell tropism, opening the way to cell-specific drug delivery. Yet, the mechanism by which Mergo? distribute to certain cells specifically has not been addressed. The current leading hypothesis for altered distribution is based on protein fingerprinting. We postulate that modifying the chemical identities of Mergo? results in altered biological identity (protein binders), leading to differences in distribution, cellular uptake and therapeutic effect. Indeed, upon injection to biological fluids (_e.g_ serum or cerebral spinal fluid), proteins are shown to bind Mergo?, forming a protein corona. Moreover, Mergo? with different _in vivo_ distribution profiles have been qualitatively shown to display different protein binding (PB) profiles. For example, whole affinity to serum changed, and the higher-order structures formed (protein-Mergo? complexes) have different shapes and sizes. While pulldown experiments analyzed by SDS-PAGE have revealed that protein binders differ between Mergo? candidates, their identities have not been yet elucidated. Resolving PB fingerprints would help Sixfold, and more globally the RNAi drug delivery field, to link chemical identity and biological outcomes. More specifically, the extracellular protein binders can be used to understand _in vivo_ distribution, while intracellular PB might inform on toxicity and therapeutic activity. Additionally, understanding whether protein binders are conserved from one species to another would help understanding whether results observed in small animals (mouse) can be translated to bigger species (NHP, human), accelerating preclinical phase and platform development. Finally, the development of a robust, potentially high-throughput method to identify protein binders could help to rationally design Mergo? candidates, and be integrated within the Sixfold AI/ML chemical design loop. Currently, the choice of the modifications added to Mergo? candidates is based on ADMET properties of small molecules. We believe that PB can be used to better predict _in vivo_ behaviour. In summary, protein binders identification could be used to understand mechanism of action (MOA), toxicity, cross-species differences and inform better the design of next-generation Mergo? candidates.