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

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 performance in vitro and in 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 of in vitro gene 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) and in vitro TMEM106b mRNA 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 with in vitro data. Our integrated methods, analysis, and resulting findings on PEG-lipid can be applied to rapidly optimize other LNP formulations in a complex design space.

Individual ASO-LNP samples and the screening sample plates were prepared by a robotic liquid handler-assisted, high-throughput solvent-injection method that we developed previously. Briefly, a liquid handler (TECAN EVO, NC, USA) was used to dispense the ASO dissolved in a citrate buffer (25 mM, pH 4) into a 96-well sample plate, as well as to mix individual lipid stocks at certain molar ratios to generate different lipid mixtures. For the PEG-lipid screening, LNPs were designed with the lipid composition with molar percent of MC3 : DSPC : cholesterol : PEG-lipids at 40 : 10 : (100 - X) : X, where X = 1, 3, or 5, a total lipid concentration of 1 mM, and a N/P ratio of 2. The prepared lipid mixtures were rapidly mixed with the ASO aqueous phase at a volume ratio of 1 : 3 (50 μL : 150 μL) in the sample plate using the robot, allowing for self-assembly of ASO-loaded LNPs, which were then characterized for particle structure by SAXS in the preparation buffer.In separate experiments, certain LNPs were processed through ultracentrifugation using an Amicon filter with the MWCO of 10 kD (Millipore, MA, USA) for purification and buffer exchange to PBS. Next we tested how modifying formulation parameters, including the PEG-lipid concentration and N/P ratio, affected these SAXS parameters. We varied the PEG-lipid concentration from 2 to 5% and N/P ratios of ASO-LNPs from 2 to 5. We next examined the potential of LNP morphology changes after purification. During the purification process, the pre-purified LNPs are buffer exchanged into a physiologically relevant phosphate-buffered saline (PBS) buffer with pH 7.4. SAXS data were collected in the high throughput mode (HT-SAXS) using the Advanced Light Source SIBYLS beamline 12.3.1 at the Lawrence Berkeley National Laboratory (CA, USA). X-ray wavelength was set at λ = 1.216 Å, and the sample-to-detector distance was 2070 mm, resulting in a scattering vector, q, ranging from 0.01 Å-1 to 0.45 Å-1. The scattering vector is defined as q = 4πsinθ/λ, where 2θ is the scattering angle. Experiments were performed at 20 °C as described elsewhere 29. Briefly, the sample was exposed for 10 s with the detector framing at 0.3 s to maximize the signal while merging the SAXS signal using the SAXS FrameSlice application (https://bl1231.als.lbl.gov/ran). No radiation damage was observed during the 10 s exposure, and all collected frames were merged. We used ASO-LNPs composed of 40 mol% MC3, 2 mol% DMG-PEG-2k, 10 mol% DSPC, and 48 mol% cholesterol at an N/P ratio of 2 for SAXS features assignments. The LNPs in pre-purification conditions (1:3 volume ratio of ethanol : 25 mM citrate buffer, pH 4) exhibited sharp SAXS features, indicating highly-ordered LNP morphology. The 4 mM total lipid concentration showed two overlapping but distinguishable peaks at q = 0.126 Å-1 and 0.139 Å-1. Using the relationship d = 2π/q, where d is the distance between lipid/ASO/water repeated structures, the peaks show organized structures spaced at d = 50 Å and 45 Å, respectively. The maxima at d = 50 Å is associated with the hexagonal phase (HII). Confirming this assignment are the two apparent ancillary peaks at q = 0.218 Å-1 and 0.251 Å-1 that would be expected for hexagonal packing at a 50 Å distance, although the main peak splitting between the 50 and 45 Å are less significant at lower (2 and 1 mM) total lipid concentrations.

PEGx_y.dat = a final merged SAXS profile, where x is a #ID of specific PEG-lipid and y is the molar ratio of PEG-lipid. MC3, DSPC, cholesterol, and PEG-lipid analogs under a molar ratio of 40:10:(50-y):y. 1 mM total lipids, N/P = 2. #1 DMPE (C14:0)-PEG0.55k #2 DMPE(C14:0)-PEG1k #3 DMPE(C14:0)-PEG2k #4 DPPE(C16:0)-PEG1k #5 DPPE(C16:0)-PEG2k #6 DSPE(C18:0)-PEG0.55k #7 DSPE(C18:0)-PEG1k #8 DSPE(C18:0)-PEG2k #9 DSPE(C18:0)-2arm-PEG2k #10 DOPE(C18:1)-PEG0.55k #11 DOPE(C18:1)-PEG1k #12 DOPE(C18:1)-PEG2k #13 DMG(C14:0)-PEG2k #14 DSG(C18:0)-PEG2k #15 Ceramide(C8)-PEG0.75k #16 Ceramide(C8)-PEG2k #17 Ceramide(C16)-PEG0.75k #18 Ceramide(C16)-PEG2k HTS1_pre_purification.dat = SAXS curve of ASO-LNP composed of 40 mol% MC3, 2 mol% DMG-PEG-2k, 10 mol% DSPC, and 48 mol% cholesterol at an N/P ratio of 2 in a citrate buffer (25 mM, pH 4). HTS2_pre_purification.dat = SAXS curve of ASO-LNP composed of 40 mol% MC3, 5 mol% DMG-PEG-2k, 10 mol% DSPC, and 45 mol% cholesterol at an N/P ratio of 2 in a citrate buffer (25 mM, pH 4). HTS3_pre_purification.dat = SAXS curve of ASO-LNP composed of 40 mol% MC3, 2 mol% DMG-PEG-2k, 10 mol% DSPC, and 48 mol% cholesterol at an N/P ratio of 5 in a citrate buffer (25 mM, pH 4). HTS1_post_purification.dat = SAXS curve of ASO-LNP composed of 40 mol% MC3, 2 mol% DMG-PEG-2k, 10 mol% DSPC, and 48 mol% cholesterol at an N/P ratio of 2 in PBS buffer. HTS2_post_purification.dat = SAXS curve of ASO-LNP composed of 40 mol% MC3, 5 mol% DMG-PEG-2k, 10 mol% DSPC, and 45 mol% cholesterol at an N/P ratio of 2 in a PBS buffer. HTS3_post_purification.dat = SAXS curve of ASO-LNP composed of 40 mol% MC3, 2 mol% DMG-PEG-2k, 10 mol% DSPC, and 48 mol% cholesterol at an N/P ratio of 5 in a PBS buffer. HTS1_1mM_LNP.dat, HTS1_2mM_LNP.dat, and HTS1_4mM_LNP.dat are final merged SAXS curves of HTS1 ASO-LNP at 1, 2 and 4 mM total lipid concentration.

2023-02-07

2023-10-20

HT-SAXS

1.216 Å

2.07 m

Sample:

• Macromolecule 1: ASO-LNP
• Sample Full Name: LNP with antisense oligonucleotide
• Sample Type: Lipid Nanoparticle
• Source Organism:
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• Expression System:
• Expression NCBI Taxonomy ID:
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