Ethics statement and description of the animals

Female BALB/c ByJ mice (6 weeks old) were purchased from Charles River Laboratories, (Les Oncins, France). Animals were housed in the R&D animal facility of Sanofi (Campus Mérieux, 1541 Av Marcel Mérieux, 69280 Marcy l’Etoile, France), accredited by AAALAC International (Association for Assessment and Accreditation of Laboratory Animal Care) since 2011, and in accordance with European Directive 2010/63/EU. These studies were included in the project APAFIS#18358-2019010817217725v2 reviewed by the French Animal Ethics Committee number 11 and approved by the Research, Innovation and Education Ministry. Mice were anesthetized using isoflurane (ISOFLURIN 100%, Alcyon), then euthanized by a cervical dislocation. Endpoints used were general ones like 20% weight loss or decreased locomotion, but they were not reached as the protocol did not induce any noticeable adverse event.

Cynomolgus macaques (Macaca fascicularis), aged 35 to 42 months (13 males) and originating from Mauritian AAALAC-certified breeding centers, were used in this study. All animals were housed in the IDMIT facilities (CEA, Fontenay-aux-roses, Animal facility authorization #D92-032-02, Préfecture des Hauts de Seine, France) in compliance with European Directive 2010/63/EU, French regulations, and the Standards for the Humane Care and Use of Laboratory Animals of the Office for Laboratory Animal Welfare (OLAW, assurance number #A 5826-01, US). The protocols were approved by the institutional ethics committee “Comité d’Ethique en Expérimentation Animale du Commissariat à l’Energie Atomique et aux Énergies Alternatives” (CEtEA #44) under statement number A20-001. The study was authorized by the “Research, Innovation and Education Ministry” under registration number APAFIS#24620-2020031115133688 v1. For all handling procedures, animals were anesthetized using ketamine hydrochloride (Imalgen® 1000 5 mg/kg) associated with medetomidine hydrochloride (Domitor® 0.05 mg/kg) by i.m. route. Animals were not euthanized.

mRNA labeling and LNP formulation

1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), PEG2000-PE, and cholesterol were obtained from Avanti Polar Lipids (Alabaster, AL, USA). MC3 was obtained from SAI Life Sciences (Hyderabad, India).

The synthetic dioleoyl lipid bearing an imidazolium head group, DOG-IM4, was synthesized as described in ref. 12 and obtained from SAI Life Sciences (Hyderabad, India).

High purity, custom-synthesized, non-replicative, non-modified mRNA encoding the hemagglutinin (HA) of the influenza strain A/Netherlands 2009 (H1N1) was obtained from TriLink (San Diego, CA). The Ulysis™ Alexa Fluor™ 647 Nucleic Acid Labeling Kit was obtained from Invitrogen. The fluorescent labeling of mRNA, based on the universal linkage system, was adapted according to the commercial protocol. Small scale purification of the labeled mRNA was carried out using a spin-column (Princeton separations Inc, New Jersey, USA) and larger scale purification of the nucleic acid by successive precipitation in ethanol.

LNPs were prepared as described in ref. 12. Briefly, an ethanol phase containing the lipids at molar ratios of 50:10:38.5:1.5 (ionizable lipid/phospholipid/cholesterol/PEGLipid), at a total concentration of 20 mg/mL, and an aqueous phase containing mRNA dissolved in 50 mM citrate buffer (pH 4.0), at 0.265 mg/mL for the preparation of DOG-IM4 or 0.305 mg/mL for the preparation of the MC3 LNPs, were prepared. The two phases were then rapidly mixed in a microfluidic mixer (NanoAssemblR, Precision Nanosystems, Vancouver, BC) at a ratio of 1:3, respectively, with a final combined flow rate of 4 mL/min. The formulations were then dialyzed (Spectrum Labs, Rancho Dominguez, CA) in 50 mM citrate buffer (pH 4.0) for at least 4 h and then in phosphate-buffered saline (pH 7.4) for 24 h. The nanoparticles were stored at 4 °C under an inert atmosphere after sterile filtration.

The size and polydispersity index of the LNPs were measured by dynamic light scattering (Zetasizer Nano ZSP, Malvern Instruments). The mRNA encapsulation efficiency was determined by mRNA accessibility to Ribogreen using the QuantiT Ribogreen RNAassay (Life Technologies, Burlington, ON).

Administration of LNPs to mice

To evaluate mRNA delivery, mice were anesthetized using 3% isoflurane (1818290, ISOFLURIN 100%, Alcyon) and injected via the i.m. route with various LNPs (50 µl) containing 5 µg AF647 labeled mRNA-HA with 3/10 cc insulin syringes (15638708, Fisher, France), in accordance with the Sanofi internal guideline for the care and use of laboratory animals. Following LNP administration, the mice were placed on an imaging platform under continuous anesthesia (isoflurane 2%) and imaged at various time points after injection.

Assessment of the local reaction in mice

The injected muscles of mice were collected at D1 and D7 following LNP/mRNA injection. The tissues were fixed in formalin for 24 h at RT. After fixation, the dehydrated tissues were embedded in paraffin and sections (4 μm) were stained with hematoxylin eosin and examined by a pathologist. A graduated scale from 0 to 4 based on severity for myofiber regeneration and cell infiltration within the muscle was established.

Fluorescence imaging studies in mice

Fluorescence imaging was performed by IVIS Spectrum CT (In Vivo Imaging System, Perkin Elmer).

Epi-fluorescence imaging (2D) and 3D fluorescence imaging tomography were used in this study. For the 3D study, a trans-illumination protocol, associated with an X-ray scan of the whole body of the mouse, was used, allowing precise 3D location of the fluorescent signal. Six excitation sites were selected to trans-illuminate the mouse and thus excite the AF647-mRNA vaccine (excitation: 625 nm, emission: 650 nm).

All data were analyzed using Living Image software. The 2D fluorescence signal was quantified over a region of interest (ROI) applied to the injected area and is expressed as the total radiant efficiency (photons/s)/(μW/cm2). For the 3D fluorescence study, the fluorescent signal was quantified at three different ROIs consisting of the injected muscle (quadriceps), iliac LN, and popliteal LN. Quantification of the source fluorescence voxels is expressed in pmol and normalized by the subtraction of the signal background quantified in the PBS control group.

In vivo imaging in macaques

LNPs were injected i.m. into the biceps of the animals at an interval of four weeks (D0 and D28). Then the macaques received 500 µl of LNPs containing 50 µg AF647-mRNA and 10 µg anti-HLA-DR/DP/DQ labeled with AF488. A group of three macaques, used as controls, received a solution of 500 µl PBS containing 50 µg of the labeled mRNA and 10 µg of the labeled antibody.

Monoclonal anti-human HLA-DR, DP, DQ, purchased from BDbiosciences (Cat. No 550853, cloneTu39), was covalently conjugated to the fluorophore using the Alexa Fluor™ 488 Microscale Protein Labeling Kit (A30006, Invitrogen), as described in the provided protocol. The concentrations of the protein and dye were measured using a NanoDrop 2000 spectrophotometer (Thermo ScientificTM). Imaging sessions were performed 24 h after each injection of LNP/mRNA + anti-HLA-DR/DP/DQ. A near-infrared (NIR) imaging system FluobeamTM 700 (λex: 680 nm/λem: >700 nm; Fluoptics, Grenoble, France), already described in the study of ref. 19, was used to screen mRNA fluorescence at the injection site and the axillary dLNs. All images were acquired using a camera exposure time of 100 ms. Then, in vivo FCFM (Cellvizio® Dual Band, Mauna Kea Technologies, France) was performed. Briefly, an incision of the skin was made under sterile conditions in the area of interest. The UltraMiniO probe was applied directly to the muscle or the axillary dLN and moved continuously to record the distribution of the fluorescence signal from the AF647-mRNA and AF488-HLA-DR18.

Quantification of fluorescent mRNA and HLA-DR and co-localization

Films were selected and exported using IC-Viewer software. Fifty images per imaging session were randomly chosen from the recorded movies. An automatic algorithm was applied to quantify the mRNA and HLA-DR fluorescence, as well as their co-localization, using ImageJ 1.6 software (National Institute of Mental Health, Bethesda, USA). Briefly, the threshold was set to reduce the background signal and the size of the selected objects ranged from 10 to 2000 pixels.

The number of fluorescent spots of mRNA (AF647-positive spot) and HLA-DR-positive cells (AF488-positive spot) was quantified. The number of spots is expressed as fluorescent pixels per mm2 in muscle and fluorescent cells per mm2 in the dLNs. For estimations of the percentage of colocalization, 50 randomized images were obtained from a pool of images showing colocalization of the mRNA and HLA-DR signals.

Hematological parameters and inflammation

Blood cell counts and hemoglobin levels were determined from EDTA blood using a DHX800 analyzer (Beckman Coulter). Cytokines were quantified in sera using NHP Milliplex (Millipore) and a Bioplex 200 analyzer (Bio-Rad) according to the manufacturer’s instructions.

Immunohistofluorescence of muscle biopsies and LNs

For two animals per group, muscle biopsies and axillary LNs were harvested 24 h after the second injection of LNP/mRNA. Fresh tissues were immediately fixed with 0.05 M phosphate buffer containing 0.1 M L-lysine (pH 7.4), 2 mg/ml NaIO4, and 4% paraformaldehyde for 6 h at 4 °C and then incubated with 30% sucrose in PBS overnight at 4 °C. The LNs were embedded in an optimal cutting temperature compound. The tissues were snap-frozen in liquid nitrogen-cooled isopentane and stored at −80 °C. The frozen tissues were cut into 10 µm-thick sections for the staining of immune cells.

Following rehydration in PBS, tissues were permeabilized for 30 min in PBS/Triton 0.3% and then blocked with PBS/BSA 10% for 30 min. For muscle staining, sections were incubated overnight at 4 °C with a mix of PBS/0.2% BSA containing anti-desmin (Clinisciences Cat. No Mob060-01; Dilution 1:500) and anti-CD66 abce (Miltenyi Cat. No 130-095-212; Dilution 1/100) antibodies. Tissue sections were then incubated with secondary antibodies Goat anti-mouse IgG1, Alexa Fluor™ 555 (Life Technologies Cat. No A-21127; Dilution 1:500) and Goat anti-mouse IgG2b, Alexa Fluor™ 594 (Life Technologies Cat. No A-21145; Dilution 1:500) for 1 h at room temperature before being immunostained for 3 h at room temperature with anti-CD68 (Biolegend Cat. No 14-0688-82; Dilution 1:200) and anti-CD163 (Biolegend Cat. No 333602; Dilution 1:71) conjugated antibodies.

For LN staining, the same protocol was used with anti-CD20 (Dako Cat. No M0755; Dilution 1:64) and anti-CD3 (BD Biosciences Cat. No 557705; Dilution 1:100) as the primary antibodies and anti-CD68 (Biolegend Cat. No 14-0688-82 Dilution 1:180), anti-CD163 (Biolegend Cat. No 333602; Dilution 1:66), anti-HLA DR, DP, DQ (BD Biosciences Cat. No 550853 Dilution 1:126) as the conjugated antibodies.

All tissue sections were stained with DAPI and mounted with Vectashield medium (H-1000, Eurobio Scientific).

We used a Leica SP8 tiling confocal microscope equipped with a 40X oil immersion objective for image acquisition and IMARIS software (Bitplane Scientific Software) for analysis.

Data analysis

Data were collected using Excel files (Microsoft Excel 2016). Differences between unmatched groups were compared using the Mann-Whitney U test and Kruskal-Wallis test. The two-tailed Wilcoxon signed-rank test was used to compare paired conditions (clinical parameters over time in each animal group) (GraphPad Prism 8.0). ANOVA multiple comparison tests were used to compare the mean values of experimental groups together or to those of control groups (Fluorescence imaging mice data). Data are presented as mean values ± SD.

Reporting summary

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