Nanoparticles That Deliver RNA to Bone Marrow Identified by in Vivo Directed Evolution

Bone marrow endothelial cells (BMECs) regulate their microenvironment, which includes hematopoietic stem cells. This makes BMECs an important target cell type for siRNA or gene editing (e.g., CRISPR) therapies. However, siRNA and sgRNA have not been delivered to BMECs using systemically administered nanoparticles. Given that in vitro nanoparticle screens have not identified nanoparticles with BMEC tropism, we developed a system to quantify how >100 different nanoparticles deliver siRNA in a single mouse. This is the first barcoding system capable of quantifying functional cytosolic siRNA delivery (where the siRNA drug is active), distinguishing it from in vivo screens that quantify biodistribution (where the siRNA drug went). Combining this approach with bioinformatics, we performed in vivo directed evolution, and identified BM1, a lipid nanoparticle (LNP) that delivers siRNA and sgRNA to BMECs. Interestingly, chemical analysis revealed BMEC tropism was not related to LNP size; tropism changed with the structure of poly­(ethylene glycol), as well as the presence of cholesterol. These results suggest that significant changes to vascular targeting can be imparted to a LNP by making simple changes to its chemical composition, rather than using active targeting ligands. BM1 is the first nanoparticle to efficiently deliver siRNA and sgRNA to BMECs in vivo, demonstrating that this functional in vivo screen can identify nanoparticles with novel tropism in vivo. More generally, in vivo screening may help reveal the complex relationship between nanoparticle structure and tropism, thereby helping scientists understand how simple chemical changes control nanoparticle targeting.