Lipid nanoparticle screening in nonhuman primates with minimal loss of life

mRNA synthesis

The template for in vitro transcription (IVT) of glycosylphosphatidylinositol-aVHH was purchased from Integrated DNA Technologies. The sequences (Supplementary Table 1) were codon-optimized for mouse (primary species) and human (secondary species) using the GenSmart Codon Optimization tool (GenScript). The linear IVT templates were amplified by PCR using forward and T120 extended reverse primers, then purified with the DNA Clean & Concentrator Kit (Zymo Research). The MEGAscript T7 Transcription Kit (Invitrogen) was used for IVT for 2 h at 37 °C. During the reaction, mRNA was capped with Cap 1 (BOC Sciences) and all UTPs were modified with N¹-methylpseudouridine-5′-triphosphate (N¹-MeΨTP, BOC Sciences). After 2 h, DNase I was treated to remove the template DNA for 15 min at 37 °C. The resulting mRNA was purified by lithium chloride precipitation. Purified RNA products were analyzed by gel electrophoresis to ensure purity.

Nanoparticle formulation

LNPs were formulated with aVHH mRNA^23,24,25. The lipid components were diluted in 100% ethanol. Seventy-two different compositions are listed in Supplementary Fig. 2. DNA barcodes (Integrated DNA Technologies; sequences listed in Supplementary Table 1) were mixed in the nucleic acid phase at a 1:10 ratio of DNA barcodes to mRNA in 25 mM sodium acetate buffer. The phases were then microfluidically mixed^32,33 using Ignite (Precision Nanosystems) at a flow rate of 3:1 (nucleic acid:lipid phases) at a total flow rate of 12 ml min⁻¹. LNPs were diluted 1:42 in sterile 10 mM Tris buffer and dialyzed by centrifugation in 100-kDa ultracentrifuge tubes (Amicon) at 4 °C. Then the LNPs were sterile-filtered with a 0.22-µm filter before administration or downstream analysis.

LNP characterization

Hydrodynamic diameters and PDIs of LNPs were measured using high-throughput dynamic light scattering (DynaPro Plate Reader II, Wyatt). DYNAMICS software (version 8.3.1.1145, Wyatt) was used for data collection. Endotoxin levels were measured with the Pierce Chromogenic Endotoxin Quant Kit (Thermo Scientific) before administration (Supplementary Fig. 4). The standard range of 0.01–0.1 endotoxin units (EU) ml⁻¹ was used following the manufacturer’s protocol. Encapsulated mRNA and encapsulation efficiency were evaluated using Quant-iT RiboGreen RNA assay kit (Thermo Fisher Scientific). In brief, an equal volume of 6 ng μl⁻¹ LNP and 1× Tris–EDTA (TE; Thermo Fisher) or 2% Triton X-100 (Sigma-Aldrich) in 1× TE were mixed. After incubation at 37 °C for 10 min, an equal volume of 1:100 of RiboGreen reagent (Thermo Fisher) was added to each well. The fluorescence was quantified using a plate reader (PerkinElmer Victor X4 Microplate Reader) at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. PerkinElmer 2030 Workstation software 4.0 (version 4.00.0.15) was used for data collection. The zeta potential was measured using a Malvern Zetasizer Nano Z. Zetasizer software (version 8.02) was used for data collection. The measurement was done with the refractive index of 1.4, dispersant viscosity of 0.882 cP and refractive index of 1.33.

Freeze–thawing of LNPs

Sucrose (National Formulary, European Pharmacopoeia, Japanese Pharmacopoeia, Chinese Pharmacopoeia, high purity, low endotoxin; Fisher Scientific) was dissolved in a sterile 10 mM Tris buffer at 60% (w/v). LNPs in 10 mM Tris buffer were mixed with 60% (w/v) sucrose solution at 5:1 (v/v LNP:sucrose) for a final concentration of sucrose at 10% (w/v). LNPs were then flash-frozen in liquid nitrogen. Hydrodynamic diameter, PDI and encapsulation efficiency were measured before and after freeze–thawing. Frozen LNPs were stored at ≤−120 °C in the vapor phase of liquid nitrogen. For thawing, LNPs were incubated at room temperature for 20 min.

LNP selection for pooling

After formulation, LNPs with PDI under 0.4 were selected (71 out of 72 LNPs). Then, LNPs with encapsulation efficiency above 85% were selected (50 out of 71 LNPs). As a third step, we measured the PDI and hydrodynamic diameter of these 50 LNPs after they were mixed with 10% sucrose, frozen and thawed. Forty-five formulations remained monodisperse with a hydrodynamic diameter between 50 nm and 150 nm and a PDI below 0.3. The frozen aliquots of selected 45 LNPs were thawed and pooled to assess the physical characteristics. Hydrodynamic diameter, PDI, zeta potential, encapsulation efficiency, encapsulated mRNA, endotoxin level and pK_a were measured for the LNP pool. The LNP pool was also imaged by cryo-TEM. Before each administration, 1-ml aliquots of those 45 frozen LNPs were thawed at room temperature and pooled. Each time, the hydrodynamic diameter, PDI, zeta potential, encapsulation efficiency, encapsulated mRNA and endotoxin level were measured.

Cryo-TEM

LNP samples were placed on 300-mesh copper grids, with a holey carbon substrate (1.2-µm holes spaced by 1.3 µm; C-flat, Electron Microscopy Sciences). Grids were glow-discharged (negative charge) for 15 s using a GloQube Plus glow discharge system (Quorum Tech). Samples (3 µl) were blotted with filter paper for 1 s at room temperature and 100% humidity and plunge-frozen into liquid ethane using a Vitrobot Mark IV (Thermo Scientific). Cryo-EM grids were stored in liquid nitrogen until cryo-TEM data acquisition. Cryo-EM grids were loaded under liquid nitrogen temperatures, using a Gatan 914 cryo-TEM sample holder, into a JEOL JEM-2200FS TEM (JEOL) operating at 200 keV. Micrographs were acquired under low-dose conditions using the Serial EM software (v4.1.6)⁵⁷ with a DE20 direct electron detector device (Direct Electron), at nominal magnifications of ×40,000 or ×80,000, yielding pixel sizes of 1.4 Å and 0.8 Å per pixel, respectively.

pK
_a assay

A stock solution of 10 mM HEPES (Sigma-Aldrich), 10 mM 2-(N-morpholino)ethanesulfonic acid (Sigma-Aldrich), 10 mM sodium acetate (Sigma-Aldrich) and 140 mM sodium chloride (Sigma-Aldrich) was prepared and pH adjusted with hydrogen chloride and sodium hydroxide (pH 3–10). Using four replicates for each pH, 140 μl pH-adjusted buffer was added to a 96-well plate, followed by adding 5 μl of 2-(p-toluidino)-naphthalene-6-sulfonic acid (60 μg ml⁻¹). LNPs (5 µl) were added to each well. After 5 min of incubation at 300 rpm, fluorescence (excitation 325 nm, emission 435 nm) was measured using a plate reader.

Mouse experiments

All mouse experiments were performed in accordance with the approval of the Emory University School of Medicine’s Institutional Animal Care and Use Committee. All animals were housed in the Emory University animal facilities. C57BL/6J mice were purchased from The Jackson Laboratory. All mice were housed in the Emory University animal facilities in conventional cages with a 12:12 h light–dark cycle and ad libitum access to food and water. The vivarium was kept at 23 °C with 50% humidity. Mice (N = 3) were injected intravenously with LNP pool (0.5 mg kg⁻¹) in the lateral tail vein.

Mouse cell isolation and staining

Mice were euthanized by CO₂ asphyxiation, followed by cervical dislocation and perfusion with 5 ml of 1× phosphate-buffered saline (PBS) through the right atrium. Liver tissues were finely minced and then placed in a digestive enzyme solution including collagenase type I (Sigma-Aldrich), collagenase XI (Sigma-Aldrich) and hyaluronidase (Sigma-Aldrich). The mixture was then incubated at 37 °C, 550 rpm for 45 min (refs. ^58,59). For bone marrow, both ends of the femur were cut and 5 ml of RPMI1640 (Sigma-Aldrich) per femur was flushed using a 25-gauge needle. Spleens were finely minced and placed in RPMI1640. Cell suspensions were filtered through a 70-µm mesh. Blood was collected from terminal cardiac puncture, then red blood cells were lysed by ACK lysing buffer (Quality Biological) following the manufacturer’s protocol. Cells were stained to identify specific cell populations and sorted using the Cytek Aurora cell sorter. SpectroFlo software (version 1.3.1, Cytek) was used for data collection. The anti-mouse antibody clones and dilutions used were the following: anti-mouse erythroid cells (TER119, BioLegend, 1:500), anti-CD31 (390, BD, 1:500), anti-CD45.2 (104, BioLegend, 1:500), anti-CD19 (6D5, BioLegend, 1:500), anti-CD3 (17A2, BioLegend, 1:500), anti-CD11c (N418, BioLegend, 1:500) and anti-CD11b (M1/70, BioLegend, 1:500). LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Invitrogen) was used for live cell gating (dilution 1:5000). Flow cytometry staining panels and gating strategies are shown in Supplementary Figs. 6 and 7. Mice injected with 10 mM Tris buffer were used as gating controls.

Rhesus monkey studies

All animal procedures conformed to the requirements of the Animal Welfare Act, and protocols were approved before implementation by Emory University School of Medicine’s Institutional Animal Care and Use Committee. The LNPs were thawed, screened (for example, sterility and endotoxin) and then aliquoted for intravenous injection under aseptic conditions in rhesus monkeys (N = 6; 1–17 years of age, 2.3–6 kg; 3 males and 3 females) comparing the two clinical statuses (healthy versus gastrointestinal inflammation); one uninjected control animal (11 years, 7.2 kg, female) and one uninjected EoL animal (17 years, 9 kg, male) were included. Animals were sedated with telazol (5–8 mg kg⁻¹ intramuscularly), a blood sample was collected at time point 0 and a slow intravenous injection of LNPs was performed using the aseptic technique. Animals were pretreated with diphenhydramine, and no adverse reactions were observed. Complete blood counts and clinical chemistry panels were evaluated before and after administration and found to be within normal limits. Animals were sedated 24 h after administration and weighed, their final blood samples were collected (serum and peripheral blood mononuclear cells) and then the animals were euthanized (overdose of pentobarbital). Tissues were collected and weighed including the liver, spleen and bone marrow from all long bones.

Rhesus cell isolation and staining

Liver tissues were cut into 0.5-cm cubes, placed into a digestive enzyme solution containing 12 ml of R5 medium (RPMI1640 supplemented with 5% fetal bovine serum) supplemented with 150 µg ml⁻¹ of collagenase type I (Sigma-Aldrich), and processed using a GentleMACS Octo Dissociator with heaters (Miltenyi Biotec). The samples were processed using the following protocol: incubation at 37 °C, two sets of clockwise–counterclockwise agitations at 300 rpm for 20 s, two sets of clockwise–counterclockwise agitations at 50 rpm for 40 min and two sets of clockwise–counterclockwise agitations at 300 rpm for 20 s. Spleen tissues were cut into 0.5-cm cubes, placed into 5 ml of R5 medium and processed using a GentleMACS Octo Dissociator with heaters (Miltenyi Biotec) using the m_spleen_1 program. Liver and spleen tissues were taken off the GentleMACS, and bone marrow samples were resuspended in 10 ml of R5 medium. All samples were passed through a 70-µm mesh prewet with 1 ml of R5 medium. After centrifugation at 500g for 10 min, all samples were incubated with ACK lysis buffer for 5 min. Single-cell suspensions were then washed twice with PBS. Forty million cells were incubated with Fc receptor binding inhibitor polyclonal antibody (Invitrogen) for 20 min at 4 °C. Cells were stained to identify specific cell populations and sorted using the Cytek Aurora cell sorter. SpectroFlo software (version 1.3.1, Cytek) was used for data collection. The antibody clones and dilutions used were the following: anti-human CD31 (WM59, BD, 1:50), anti-human ASGPR (REA608, Miltenyi, 1:50), anti-NHP CD45 (D058-1283, BD, 1:50), anti-NHP CD3 (10D12, Miltenyi, 1:50), anti-NHP CD20 (2H7, BioLegend, 1:50), anti-NHP HLA-DR (G46-6/L243, BD, 1:50), anti-NHP CD16 (3G8, BD, 1:50) and anti-NHP CD14 (M5E2, BD, 1:50). The LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Invitrogen) was used for live cell gating (dilution 1:5,000). Anti-camelid VHH antibody (clone 96A3F5, GenScript) was used for detecting aVHH expression. Flow cytometry staining panels and gating strategies are shown in Supplementary Figs. 8 and 9. All remaining cells were aliquoted into 10 million cells and cryopreserved in CryoStor CS10 (StemCell Technologies). In all cases, cryopreserved samples from the respective uninjected control animals were used as gating controls.

Rhesus cytokine analysis

Rhesus monkey sera were sent to IDEXX BioAnalytics and tested on the Milliplex MAP Non-Human Primate Cytokine Magnetic Bead Panel (Millipore, cat. no. PCYTMG-40K-PX23) according to the manufacturer’s protocol. Data were collected by xPONENT 4.3 (Luminex), and data analysis was completed using BELYSA 1.1.0 software. The data collected by the instrument software are expressed as median fluorescence intensity. Analyte standards, quality controls and sample median fluorescence intensity values were adjusted for background. Calibrator data were fit to either a five-parameter logistic or four-parameter logistic model depending on best fit to produce accurate standard curves for each analyte. Quality control and sample data were interpolated from the standard curves and then adjusted according to dilution factor to provide calculated final concentrations of each analyte present in the sample. Qualified assay ranges are presented in Supplementary Table 1.

Barcode counts

For FACS-sorted aVHH⁺ cells from mice and NHPs, DNA barcodes were isolated by QuickExtract DNA Extraction Solution (Lucigen) following the manufacturer’s protocol. The samples were then amplified using KAPA HiFi HotStart Ready Mix (Roche), following the manufacturer’s protocol. Next-generation sequencing runs were performed using multiplexed runs on Illumina MiniSeq^23,60. In brief, the results were processed using a custom Python-based tool to extract raw barcode counts for each cell type. These raw counts were then normalized using R before further analysis. Counts for each particle were normalized to the barcoded LNP mixture injected into mice.

Single-cell multiomics preparation

The BD Rhapsody Single Cell Analysis System was used for single-cell multiomics. Single-cell suspensions were stained with oligo-tagged anti-camelid VHH antibodies (5′-CCTTGGCACCCGAGAATTCCAAAGTATGCCCTACGABAAAAAAAAAAAAAAAAAAAAAAAAAAAA*A*A-3′ chemically conjugated to MonoRab rabbit anti-camelid VHH antibody, mAb, GenScript; the asterisks denote phosphorothioate bonds). The final concentration was 0.5 mg ml⁻¹ by protein weight. The dilution for staining was 1:2,000. After washing the labeled cells twice with PBS, the cell viability and numbers were recorded for each sample. The cells were then pooled at the same ratio, and a BD Rhapsody cartridge was loaded with 60,000 cells. cDNA libraries were prepared using the BD Rhapsody Whole Transcriptome Analysis Amplification Kit following the BD Rhapsody System mRNA Whole Transcriptome Analysis and Sample Tag Library Preparation protocol (BD Biosciences). The final libraries were quantified using a Qubit fluorometer and sent to Novogene for sequencing. After the assessment of library quality using Bioanalyzer (Agilent), sequencing was performed by Illumina NovaSeq X Plus PE150 at Novogene.

Processing of single-cell multiomics sequencing data

The data were processed using STARsolo (v2.7.9a)⁶¹ in R (v4.3.1). All samples were mapped to rheMac10, and only exonic regions were counted. All output files were loaded into Seurat (v5.0.1). Global-scaling normalization was used for the aVHH expression assay in a Seurat object. The process normalizes the feature expression measurements for each cell by the total expression, multiplies this by a scale factor 10,000 and log-transforms the result. DoubletFinder (v3) was used to identify doublets⁶². This was followed by principal component analysis dimensional reduction and UMAP clustering. aVHH oligo-tag counts were combined with RNA counts in Seurat and treated similarly to other multimodal datasets such as cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq)⁶³. Visualization of gene and aVHH expression levels was performed using R/Seurat commands (FeaturePlot and DotPlot). For the integration of multiple Seurat objects, Harmony algorithm (v.1.2.1) was used. For UMAP representation in Fig. 3a,d,g, cell clusters were manually annotated in Seurat with marker genes listed in Supplementary Figs. 16–18.

Statistics and reproducibility

A minimum of three biological replicates were analyzed for quantification, except for untreated rhesus monkeys (N = 1). For the representative cryo-TEM image (Fig. 1i), four wide-field images and at least ten high-magnification images (×40,000) were collected from one batch of LNP pool with a representative size distribution. All data are presented as the mean ± standard deviation or standard error of mean. Statistical analysis between groups was performed using GraphPad Prism (versions 9.5 and 10). For data with multiple groups, the statistically significant differences were assessed using two-way analysis of variance with Tukey’s multiple-comparison test. For comparisons between two groups, unpaired two-tailed Student’s t-tests were used. The sample sizes (biological replicates), specific statistical tests and main effects of our statistical analyses for each experiment are detailed in each figure legend. A P value of less than 0.05 was considered significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.