With three FDA-approved
products, lipid nanoparticles (LNPs) are
under intensive development for delivering wide-ranging nucleic acid
therapeutics. A significant challenge for LNP development is insufficient
understanding of structure–activity relationship (SAR). Small
changes in chemical composition and process parameters can affect
LNP structure, significantly impacting performancein vitroandin vivo. The choice of polyethylene glycol
lipid (PEG-lipid), one of the essential lipids for LNP, has been proven
to govern particle size. Here we find that PEG-lipids can further
modify the core organization of antisense oligonucleotide (ASO)-loaded
LNPs to govern its gene silencing activity. Furthermore, we also have
found that the extent of compartmentalization, measured by the ratio
of disordered vs ordered inverted hexagonal phases within an ASO-lipid
core, is predictive ofin vitrogene silencing. In
this work, we propose that a lower ratio of disordered/ordered core
phases correlates with stronger gene knockdown efficacy. To establish
these findings, we developed a seamless high-throughput screening
approach that integrated an automated LNP formulation system with
structural analysis by small-angle X-ray scattering (SAXS) andin vitro TMEM106bmRNA knockdown assessment. We applied
this approach to screen 54 ASO-LNP formulations while varying the
type and concentration of PEG-lipids. Representative formulations
with diverse SAXS profiles were further visualized using cryogenic
electron microscopy (cryo-EM) to help structural elucidation. The
proposed SAR was built by combining this structural analysis within vitrodata. Our integrated methods, analysis, and resulting
findings on PEG-lipid can be applied to rapidly optimize other LNP
formulations in a complex design space.