Correlating the Structure and Gene Silencing Activity of Oligonucleotide-Loaded Lipid Nanoparticles Using Small-Angle X-ray Scattering

With three FDA-approvedproducts, lipid nanoparticles (LNPs) areunder intensive development for delivering wide-ranging nucleic acidtherapeutics. A significant challenge for LNP development is insufficientunderstanding of structure-activity relationship (SAR). Smallchanges in chemical composition and process parameters can affectLNP structure, significantly impacting performance in vitro and in vivo. The choice of polyethylene glycollipid (PEG-lipid), one of the essential lipids for LNP, has been provento govern particle size. Here we find that PEG-lipids can furthermodify the core organization of antisense oligonucleotide (ASO)-loadedLNPs to govern its gene silencing activity. Furthermore, we also havefound that the extent of compartmentalization, measured by the ratioof disordered vs ordered inverted hexagonal phases within an ASO-lipidcore, is predictive of in vitro gene silencing. Inthis work, we propose that a lower ratio of disordered/ordered corephases correlates with stronger gene knockdown efficacy. To establishthese findings, we developed a seamless high-throughput screeningapproach that integrated an automated LNP formulation system withstructural analysis by small-angle X-ray scattering (SAXS) and in vitro TMEM106b mRNA knockdown assessment. We appliedthis approach to screen 54 ASO-LNP formulations while varying thetype and concentration of PEG-lipids. Representative formulationswith diverse SAXS profiles were further visualized using cryogenicelectron microscopy (cryo-EM) to help structural elucidation. Theproposed SAR was built by combining this structural analysis with in vitro data. Our integrated methods, analysis, and resultingfindings on PEG-lipid can be applied to rapidly optimize other LNPformulations in a complex design space.