Nonclinical safety assessment of an mRNA Covid‐19 vaccine candidate following repeated administrations and biodistribution

Messenger RNA (mRNA) vaccines have demonstrated efficacy against Severe Acute Respiratory Syndrome Coronavirus‐2 (SARS‐CoV‐2) in humans. mRNA technology holds tremendous potential for rapid control and prevention of emergencies due to its flexibility with respect to production, application, and design for an efficacious and safe use in humans. We assessed the toxicity and biodistribution of MRT5500, an mRNA vaccine encoding for the full‐length of the SARS‐CoV‐2 spike protein and delivered by lipid nanoparticles (LNPs) containing a novel ionizable lipid, Lipid‐1 in preclinical animal models. In the repeated dose toxicity study, rabbits received three intramuscular (IM) injections of MRT5500 at 3‐week interval followed by a 4‐week observation period. In an exploratory biodistribution study in mice receiving a single IM injection of an mRNA encoding luciferase encapsulated in an LNP containing Lipid‐1, the expression of the luciferase protein was monitored in vivo and ex vivo at several time points. In the regulatory biodistribution study in rabbits receiving a single IM injection of MRT5500, the quantification of the mRNA and the ionizable Lipid‐1 were monitored in the same organs and time points as in the exploratory biodistribution study. MRT5500 was safe and well‐tolerated with a transient acute phase response/inflammation and an expected vaccine‐related immunological response, typical of those observed following a vaccine administration. The biodistribution data demonstrated that the mRNA and Lipid‐1 components of the vaccine formulations were mainly detected at the injection site and in the draining lymph nodes. These results support the use of MRT5500 and its deployment into clinical trials.

observation period.In an exploratory biodistribution study in mice receiving a single IM injection of an mRNA encoding luciferase encapsulated in an LNP containing Lipid-1, the expression of the luciferase protein was monitored in vivo and ex vivo at several time points.In the regulatory biodistribution study in rabbits receiving a single IM injection of MRT5500, the quantification of the mRNA and the ionizable Lipid-1 were monitored in the same organs and time points as in the exploratory biodistribution study.MRT5500 was safe and well-tolerated with a transient acute phase response/inflammation and an expected vaccine-related immunological response, typical of those observed following a vaccine administration.The biodistribution data demonstrated that the mRNA and Lipid-1 components of the vaccine formulations were mainly detected at the injection site and in the draining lymph nodes.These results support the use of MRT5500 and its deployment into clinical trials.

| INTRODUCTION
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is responsible for the current worldwide pandemic of Coronavirus disease 2019 (Covid-19).Since its first appearance in China (Wuhan, Hubei Province) in December 2019, SARS-CoV-2 has infected more than 600 million people worldwide and caused the deaths of more than 6 million people (WHO, 2022).Several strategies, including development of vaccines and therapeutics, have been pursued to contain the devastating impact of the Covid-19 pandemic (Sempowsk et al., 2020).Among these, messenger RNA (mRNA) vaccine technology has proven to be a very effective strategy in the event of a pandemic-thanks to its flexibility with respect to production and application and efficiency in immunogen design and manufacturing (Kis, Kontoravdi, Dey, Shattock, & Shah, 2020;Sahin & Türeci, 2018).
Several Covid-19 mRNA vaccines have been developed shortly after the identification of the SARS-CoV-2 sequence (Zhou et al., 2020), and two of them were the first to receive market authorization (Baden et al., 2021;Polack et al., 2020).Both these vaccines consisted in mRNA molecules encapsulated in lipid nanoparticles (LNPs) and induced antibody responses against the trimeric SARS-CoV-2 spike (S) protein responsible for facilitating the binding and fusion of the viral particle to the surface of the cell membrane of the host-cell (Jackson et al., 2022;Yi et al., 2020).The development of SARS-CoV-2 mRNA vaccines continues because the emergence of new SARS-CoV-2 variants of concerns evading the protection afforded by current vaccines remains a possibility (Sabino et al., 2021;Tegally et al., 2021;Zhang et al., 2021).MRT5500 is a SARS-CoV-2 mRNA vaccine.Its efficacy has been assessed in three preclinical animal models.MRT5500 vaccine elicited potent neutralizing antibodies in mice and nonhuman primates (NHP) and T helper type 1 (T H 1)-biased cellular immune responses.In addition, the immunization was effective in preventing Covid-19 in a hamster model (Kalnin et al., 2021).Moreover, a third dose of MRT5500 administered as a booster to the currently approved two doses regimens (Jackson et al., 2020;Polack et al., 2020) has shown to effectively promote broadly neutralizing antibodies against all variants of concerns in NHPs (Kalnin et al., 2022).
We recently characterized the genotoxic potential of the novel ionizable Lipid-1 and its in vivo safety as part of the LNP vehicle for mRNA delivery (Broudic et al., 2022).To further support the development of the MRT5500 vaccine, we assessed its safety profile and biodistribution in mice and rabbits.In a pivotal repeated dose toxicity study in rabbit (Study 1), the systemic toxicity and local tolerance of MRT5500 vaccine following three intramuscular (IM) injections were investigated.The reversibility and/or delayed occurrence of any effects during a 4-week observation period were also evaluated.
The evaluation of the organ/tissue distribution of the mRNA and/or the ionizable lipid (Lipid-1) was performed in two studies.An exploratory biodistribution study was conducted in mice receiving a single IM injection of an mRNA encoding the firefly enzyme luciferase (mRNA-FFLuc) encapsulated in the same LNP as MRT5500 and containing Lipid-1 (Study 2).The luciferase protein expression and distribution were followed using bioluminescence readouts.A subsequent regulatory biodistribution study was conducted in rabbits receiving a single IM injection of MRT5500 (Study 3).In this study, mRNA biodistribution was followed by a reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) method, and the presence of the ionizable lipid, Lipid-1, was followed using a liquid chromatographic separation followed by a mass spectrometer detection (LC-MS/MS) in several collected organs.Males and females New Zealand White [Crl:KBL] (NZW) rabbits were obtained from Charles River Laboratories (Châtillon sur Chalaronne, France).Rabbits were individually housed in environmentally controlled rooms with a mean temperature ranging between 15 C and 23 C, relative humidity above 35%, and 10 or more air changes per hour.All rabbits received a complete rabbit pellet (Kliba) diet and had free access to softened and filtered drinking water.The rabbits were allowed to acclimatize to the study housing conditions for 12 or 13 days before the study start.The animals were aged between 11 and 14 weeks and weighed between 2.2 and 3.4 kg at the initiation of dosing.Before arrival at the test facility, rabbits were prescreened for Immunoglobulin G (IgG) binding to SARS-CoV-2 S protein.Seronegative rabbits or very low responders (i.e., with optical density [OD] < 0.400 and OD < 0.761, respectively) were selected for the study.

| General animal conditions
Female BALB/cByJ mice were obtained from Charles River Laboratories (Les Oncins, France).The mice were group-housed by three or five in individual standard cages.Water was supplied ad libitum, and they were fed with a commercial low fluorescence diet (SAFE).
Mice were allowed to acclimatize to the study housing conditions for 1 week before the study start.The animals were 8 weeks old and weighed between 15.4 and 20.8 g at the initiation of dosing.

| Control and test items
For studies performed in the rabbits, the test item was MRT5500, which is a SARS-CoV-2 mRNA vaccine, composed of an mRNA construct incorporating a coding sequence containing both the prefusion mutation (2P) (Wrapp et al., 2020) and the furin cleavage site mutation (GSAS) (Rabaan et al., 2020;Xing, Li, Gao, & Dong, 2020) to make the double mutant (2P/GSAS) of the full-length SARS-CoV-2 S protein encapsulated in a LNP as the delivery system.The LNP contains a novel ionizable lipid, Lipid-1, and three well-known components such as 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene (DMG-PEG-2 K) (Kalnin et al., 2021).MRT5500 was supplied by Sanofi as white to translucent suspension and at a concentration of 1 mg/mL of mRNA and stored at À80 C. Dose formulations were prepared before each injection.The low and mid concentrations of MRT5500 were obtained using 1.5X phosphate buffered saline (PBS), while the high concentration was obtained using 2.2X PBS.Preparations were made under aseptic conditions in a laminar flow cabinet and under RNAsefree conditions.A saline solution (0.9% NaCl) was used as a negative control.
For the study in the mice, the test item was an unmodified mRNA-FFLuc encapsulated in an LNP, containing Lipid-1, cholesterol, DMG-PEG-2 K, and DOPE.Dosing formulations were prepared using the PBS 1X to obtain 2 μg mRNA/30 μL.Luciferin K + salt (Interchim ® ) was prepared at the initial concentration of 150 mg/mL and stored at À20 C. A negative control was also added in this study to quantify the nonspecific bioluminescent signal.This includes an mRNA encoding a hemagglutinin H3 antigen from the Singapore influenza strain encapsulated in an LNP composed of another ionizable lipid, different from Lipid-1, and the same three other components (cholesterol, DMG-PEG-2 K, and DOPE) at the dose level of 2 μg mRNA/30 μL.Dose formulations were used within their established stability period.

| Study design
This pivotal repeated dose toxicity study was performed in accordance with the World Health Organization (WHO) guidelines on nonclinical evaluation of vaccines (WHO, 2005(WHO, , 2014) ) and in compliance with Good Laboratory Practices (GLP).
On Day 1, 80 NZW rabbits were randomized into four groups: a negative control (0.9% NaCl, Group 1), MRT5500 15 μg mRNA/dose (Group 2), MRT5500 45 μg mRNA/dose (Group 3), and MRT5500 135 μg mRNA/dose (Group 4).Each group was composed of 10 male and 10 female rabbits.They received three IM injections (0.5 mL each) at 3 weeks apart (Days 1, 22, and 43) (Table 1).A different injection site was used for each injection.The fur at the injection site was clipped 1 or 2 days before each injection and then as necessary during the study to allow appropriate evaluation of local effects.
Following the last injection on Day 43, half of the rabbits in each group were kept for a 2-day observation period and sacrificed on Day 45, while the remaining half were kept for a 4-week observation period and sacrificed on Day 71.

| Clinical examinations
All rabbits were checked for general health, mortality, and moribundity at least twice daily.After arrival, each animal was observed within its cage for the recording of clinical signs at least once daily.A detailed clinical examination was performed at least once a week, from 2 weeks before the beginning of the treatment period until the end of the study.Rabbits were observed for a 2-h period after each injection.
Individual body weights were recorded daily during the week following each injection and twice weekly during any other week from 2 weeks before study start.A fasted weight was recorded on the day of necropsy.Food consumption was quantitatively measured daily from 2 weeks before the beginning of the treatment period.Body temperature was measured using an implanted microchip before the first vaccination (pretest) and on each dosing days (before injection and 3, 6, and 24 h after the injection).Ophthalmic evaluations were conducted once at pretest, on Day 3, and before each sacrifice, using indirect ophthalmoscopy following instillation of a mydriatic agent (Tropicamide ® ).  and on Days 3, 20 (prior to the second injection), and 45.Blood samples were collected from the ear artery without anesthesia.Hematology blood samples (0.5 mL) were collected in EDTA-K 2 tubes and analyzed using ADVIA 120 or ADVIA 2120 blood hematology analyzer.Coagulation blood samples (0.9 mL) were collected into trisodium citrate tubes and analyzed using Star-Max Analyzer or with STAGO Coag Expert.Blood samples (0.7-1 mL) for evaluating clinical chemistry were collected, processed to serum via centrifugation at 1800g for 10 min at +4 C, and analyzed using AU680 clinical chemistry analyzer.Blood samples (0.2-0.5 mL) for evaluating CRP were centrifuged within 1 h of blood collection at 1800g for 10 min at +4 C.

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Blood samples were processed to serum within 1 h, and serum was collected on ice after centrifugation.CRP was measured by enzymelinked immunosorbent assay (ELISA) (CRP-10 kit from Life Diagnostics).Additional blood samples (1 mL + 2.5 mL) for immunogenicity investigations were collected from all animals at pretest, on Day 45 and on Day 71 (recovery animals only), centrifuged at 1800 g for 10 min at +4 C, and the collected serum was stored at À80 C.

| Terminal procedures
Animals were euthanized on Day 45 or Day 71 by intravenous injection of sodium pentobarbital followed by exsanguination.All rabbits were fasted overnight before necropsy and had a terminal body weight recorded.They were subjected to a complete necropsy examination, which included evaluation of carcass and musculoskeletal system, injection sites, all external surfaces and orifices, cranial cavity and external surfaces of the brain, and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.An extended list of organs (Table S2) was collected, weighed, and evaluated.Organto-body weight and organ-to -brain weight ratios were calculated.
Representative samples of the tissues identified in Table S2 were collected from all animals and preserved in 10% neutral buffered formalin, except for eyes and optic nerves, testes, and epididymides, which were preserved in modified Davidson's fixative.Tissues were dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

| Study design
The aim of this non-GLP study was to determine the biodistribution of an unmodified mRNA-FFLuc, encoding for a luciferase protein, and encapsulated in a LNP as the delivery system.On dosing day (Day 0), a total of 32 mice were immunized intramuscularly in the quadriceps of the right hind leg with 30 μL of either Lipid-1/mRNA-FFLuc or the Singapore H3 negative control (2 μg mRNA/dose) (Hassett et al., 2019).Before injection, mice were anesthetized with isoflurane at 2% in the XGI-8 anesthesia system (at a flow rate of oxygen of 2 L per minutes to the induction chamber).In the treatment group, there were five mice for the in vivo part of the study, and a total of 24 mice (three mice per timepoint) for the ex vivo part.Three mice were used in the control group.At each acquisition timepoint, mice received an intraperitoneal administration of luciferin (150 mg/kg) (Jelicks et al., 2013).Ten minutes after luciferin injection, mice were anesthetized and placed immediately inside an In Vivo Imaging System (IVIS) SpectrumCT imaging system and then imaged with the 2D modality option of the IVIS SpectrumCT optical device to determine the in vivo and the ex vivo bioluminescence level (Andreu et al., 2011;Dothager et al., 2009).When imaging was completed, the mouse was removed from the IVIS SpectrumCT and returned to its cage, where it was monitored until full recovery from the anesthesia.Images were analyzed with the Living Image software.The bioluminescent signal was considered as specific if it was twofold superior to control group (including standard deviations); otherwise, the signal was noted as below detection.At the end of the study, the mice were euthanized by cervical dislocation under anesthesia (isoflurane) and according to internal standard operating procedures (SOP).

| Clinical examination
Mice received a cage-side health check observation at least once daily.The injection site was observed clinically in all mice on Days 1, 2, and 3 postinjections.Their body weight was recorded on Days À1, 1, 2, 3, 7, 14, 21, and 28.

| Study design
The objective of this non-GLP study was to evaluate the biodistribution, local tolerance, and systemic toxicity of MRT5500 in rabbits after a single IM administration.A total of 98 NZW rabbits were randomized into three groups to receive 1 IM injection (0.5 mL) of the diluent (PBS; Group 1) or MRT5500 15 μg mRNA/dose (Group 2) or 135 μg mRNA/dose (Group 3).Groups 2 and 3 were composed of 21 male and 21 female rabbits, while Group 1 (control) was composed of 7 male and 7 female rabbits.Three males and three females per treatment group (one male and one female for the control group) were necropsied at 1, 6, 24, and 72 h, D7, D14, or D28 postinjection.The kinetic assessment of migration to different organs/tissues was evaluated at the same early, mid, and late time points as in Study 2. Rabbits were injected in the dorsal lumbar muscle as described in Study 1.

| Clinical examinations
Each rabbit was observed within its cage at least twice daily, beginning upon arrival through termination.On injection day, rabbits were observed approximately 2 h after injection.Local reactions were scored according to an adapted Draize scale (Table S1).Detailed clinical observations were performed weekly from at least 1 week before the study start and throughout the study.Individual body weight and food consumption were recorded at least twice weekly from the acclimation period and throughout the study.

| Blood sampling: Hematology, coagulation, clinical chemistry, and CRP
Blood samples for hematology (0.5 mL), coagulation (0.9 mL), clinical chemistry (0.7 mL), and CRP (0.5 mL) analysis were collected and analyzed as described in Study 1, with the difference that the blood samples for clinical chemistry were processed for plasma instead of serum.Blood samples were collected at pretest and at 24 and 72 h and 7, 14, and 28 days after injection.

| Evaluation of blood/tissue samples for biodistribution by RT-qPCR
Blood samples (minimum 1.6 mL) were obtained from the vena cava on EDTA-K2 tubes at necropsy for all euthanized animals.Samples The assay was characterized by the lower limit of quantification (LLOQ) that was determined to be 50 copies/well, and the upper limit of quantitation (ULOQ) was determined to be 1 Â 10 8 copies/well.
The limit of detection (LOD) was 12.5 copies/well.

| Bioanalytical samples analysis
This analysis was performed at Charles River Laboratories (Saint- The assay for the determination of Lipid-1 was developed in rabbit for each of the above-mentioned tissue type and qualified in a multiple tissue pooled matrix, over the concentration range of 20 to 10,000 ng/mL, with an assay volume of 50 μL.

| Terminal procedures
Animals were euthanized and subjected to a complete necropsy examination as described in Study 1. Animals were not fasted overnight before necropsy.Representative samples of the following tissues were collected at 48 and 72 h, preserved in 10% neutral buffered formalin, mounted on glass slides, and stained with hematoxylin and eosin for histopathological evaluation: brain, draining lymph node (inguinal), heart, injection site (dorsal lumbar muscle), kidneys, lacrimal gland, liver, lungs with bronchi, ovary, spleen, testis (preserved in modified Davidson's fixative), and thymus.

| Statistical analysis
For Studies 1 and 3, all statistical tests were conducted at the 5% significance level.All pairwise comparisons were conducted using twosided tests and were reported at the 1% and 5% levels.showed the electrons present into the imaging chamber (background signal) during acquisitions as there was no enzymatic reaction.

| Clinical observations
There were neither unscheduled deaths nor vaccine-related systemic clinical signs in any group.
The main observation was edema noted at the injection sites in all the study groups (Table 2).Very slight to well-defined edema (Grade 1 or Grade 2) was noted after each injection at a comparable incidence (1/10 to 3/10 animals per sex) in the control group and in 15 or 45 μg mRNA/dose groups.The incidence and severity were slightly higher in animals given 135 μg mRNA/dose (up to 4/10 animals per sex and up to moderate/severe edema [Grade 3 or Grade 4]).
Generally, edema got resolved within a few days and before the following injection (maximum of 19 days in the control group).Other local reactions were considered not to be related to the vaccine but rather secondary to the administration procedure.
All treated rabbits from both sexes lost weight or had a lower body weight gain in a dose-related manner on the day following each injection, when compared with the control group (Table 3).Rabbits recovered in the following days, but the overall mean body weight gain remained slightly lower from 45 μg mRNA/dose than in the control group throughout the observation period.
In correlation with body weight changes, a dose-related reduced food consumption was noted from 45 μg mRNA/dose mainly on the day following each injection (Table 3).This effect was transient, and food consumption was similar between the treatment and control groups thereafter.
A dose-related increase in mean body temperature was noted at 6 h after each injection in the 15 or 45 μg mRNA/dose groups (respective maximum mean increase: +0.9 C and +1.8 C vs. control) and from 3 h after each injection in the 135 μg mRNA/dose group, reaching a peak at +6 h postinjection (maximum mean increase: +1.9 C vs. control) (Table 4).Body temperature returned to basal values 24 or 48 h after each injection in all the treatment groups.The increase in body temperature was consistent with the injection of a vaccine and its associated inflammatory reaction.
There were no vaccine-related ophthalmic findings in any groups 2 days after the first and third injections (Days 3 and 45).

| Hematology, coagulation, clinical chemistry parameters, and CRP
Vaccine-related effects were observed in some hematology, coagulation, and clinical chemistry parameters 2 days after the first and the third injections (Table 5).A dose-related increase in mean neutrophil counts was observed from 45 μg mRNA/dose, accompanied by an increase in mean monocyte counts, and to a lesser extent by a slight decrease in eosinophil counts, when compared with the control group and pretest values.These effects led to an increase in total white blood cell counts from 45 μg mRNA/dose.A slight decrease in red blood cell count, hematocrit, and hemoglobin concentration was noted mainly at 135 μg mRNA/dose (with a maximum decrease of 10%).This decrease was associated with a reduced absolute reticulocyte count at all dose levels in males and at 135 μg mRNA/dose in females (with a maximum decrease of 38%).Two days after the first injection, a decrease in mean platelet count was observed from 45 μg mRNA/dose in males and at 135 μg mRNA/dose in females.This effect was more pronounced 2 days after the third injection with a dose-related mean decrease noted in all the treatment groups.All other changes in hematology parameters were considered incidental since they were due to isolated variations already noted during pretest and/or were within historical control data range.
Minor changes in coagulation parameters were observed in all treatment groups when compared with the control group and pretest values.An increase in mean fibrinogen concentration was noted at all dose levels without a clear dose relationship.This change was similar in magnitude after the first and third injections and was consistent with an inflammatory reaction secondary to the vaccine injection.A shorter mean prothrombin time (PT) was noted from 45 μg mRNA/ dose in both sexes.These variations in PT could be secondary effects to the inflammatory reactions seen in the treatment groups.
T A B L E 2 Repeated dose toxicity study in rabbits (Study 1): Summary of local reactions at the injection site.during pretest and/or were within historical control data range and were therefore considered incidental.
All the above-described changes were no longer observed before the second injection (Day 20) (i.e., 3 weeks after the first injection) and 4 weeks after the third injection.

| Organ weights
At terminal euthanasia (Day 45), mean absolute and relative vaccinerelated increases in spleen weight were noted in males from 45 μg mRNA/dose (+33% and +35% at 45 μg mRNA/dose and +35% and +38% at 135 μg mRNA/dose, respectively) and in females with 135 μg mRNA/dose (+39% and +46%, respectively).At recovery euthanasia (Day 71), mean absolute and relative vaccine-related increases in spleen weights were still observed in males previously administered from 45 μg mRNA/dose (+32% and +35% at 45 μg mRNA/dose, and +38% and +41% at 135 μg mRNA/dose, respectively).These higher mean absolute and relative spleen weights, compared with controls, correlated with the increased germinal center cellularity of the lymphoid follicles observed at microscopic examination in males receiving 45 μg or 135 μg mRNA/dose.All other organ weight differences were considered incidental or related to difference of sexual maturity and unrelated to administration of the vaccine.

| Pathology
No vaccine-related gross findings were noted, both at terminal and at recovery euthanasia.
At terminal euthanasia (Day 45), vaccine-related subcutaneous and/or muscular inflammation, hemorrhage, muscle fiber necrosis/ regeneration, fibrosis, and subcutaneous basophilic material accumulation were noted at the injection sites in all the treatment groups.
Minimal-to-mild inflammation was noted in the subcutis and/or in the injected muscle across all the vaccine doses, with a trend towards higher incidence and severity from 45 μg mRNA/dose.It was chronic or subacute at sites 1 and 2 (injected on Day 1 or Day 22, respectively), where the change was characterized by mononuclear cell infiltrates composed of macrophages and lymphocytes, accompanied by fewer heterophils.
At the injection site 3 (injected on Day 43, i.e., 2 days before necropsy), the inflammation was mainly acute or subacute and contained more heterophils, associated with fibrin and cellular debris.Minimal-to-moderate necrosis of muscle fibers was observed at the three injection sites from control and dosed groups, with a dose-related higher incidence and severity at injection site 3. Minimal subcutaneous fibrosis was noted only at injection sites 1 and 2 (with a trend towards higher incidence in the 135 μg mRNA/dose group), as well as minimal-to-mild subcutaneous and/or muscular hemorrhage at all injection sites (with a marginally higher incidence and/or severity at site 3 in the treatment groups, when compared to controls).The type of inflammation and the differences in incidence of necrosis, hemorrhage, and fibrosis in the muscle and subcutis at each injection site were considered to reflect the expected evolution of the inflammatory process over time.In addition, minimal dark basophilic extracellular material was noted in the subcutis of all the treatment groups, mainly at injection site 3.Although the exact pathogenesis remains unclear, this basophilic material was considered not to be directly related to the vaccine but rather to accompany the local acute or subacute subcutaneous inflammation.In addition, the iliac, subiliac, and, to a lesser extent, sacral lymph nodes draining the injected muscles, as well as the spleen showed a dose-related increase in the cellularity of the germinal centers of the lymphoid follicles with 135 μg mRNA/dose, which was considered to be secondary to the stimulation of the immune system by the vaccine.
At recovery euthanasia (Day 71), vaccine-related findings were still noted at all the three injection sites in all the treatment groups and were mainly composed of subcutaneous and/or muscular chronic inflammation and fibrosis.Minimal chronic inflammation, sometimes accompanied by minimal fibrosis, was noted in the subcutis and/or in the muscle of animals across all doses and was characterized by mononuclear cell infiltrates occasionally associated with fewer heterophils.These changes showed no clear dose-response relationship, but their incidences were higher at injection site 3 than at injection sites 1 and 2. Some marginal findings were also noted on isolated occasions: minimal muscle fiber necrosis (injection site 2 in one female in the 15 μg mRNA/dose group), muscle fiber regeneration (injection site 3 in one female in the 45 μg mRNA/dose group), and minimal basophilic granule accumulation in the subcutis (injection site 3 in one male in the 45 μg mRNA/dose group).The nature of the microscopic changes in the muscle and subcutis at each injection site and the overall lower incidences and severities recorded at the end of the observation period, compared with the main study phase, were considered to reflect the resolution of the inflammatory process over time, indicating partial reversibility.The iliac, subiliac, and sacral lymph nodes draining the injected muscles, as well as the spleen, still showed a vaccine-related increase in the cellularity of the germinal centers of the lymphoid follicles in all the treatment groups but with a lower incidence and severity than at the end of the observation phase.
Other microscopic findings observed either at terminal or at recovery euthanasia were considered incidental.The nature, incidence, and severity of these findings were similar in the control and the treatment groups and were therefore considered as unrelated to the vaccine.

| Immunogenicity
At pretest, 9 out of 80 rabbits from the control group (all time points) or the vaccine group showed low titers ranging from 1.6 to 2.2 log 10 EU, most likely due to the presence of antibodies cross-reacting with, or nonspecifically binding to, the SARS-CoV-2 S protein (Figure S1).
Following the 3 IM injections, high specific IgG titers were detected in all samples from those treated with MRT5500, with a mean titer ranging between 4.0 and 5.0 log 10 EU on Day 45 and close to or above 5.0 log 10 EU on Day 71 as compared with the pretest values.Titers increased from Days 45 to 71 in a dose-dependent manner.

| In vivo results
In vivo luciferase expression was detected at the injection site in the liver, and in the spleen across different timepoints, indicating drainage to these organs.At the injection site, the Lipid-1/mRNA-FFLuc group showed a very high, specific, and localized bioluminescent signal at Day 0 (6 h) (Figure 1A).This signal decreased slowly until Day 14 and was comparable with background bioluminescence beyond that time point (Figure 1B).In the liver, the bioluminescent signal peaked 6 h postinjection.Then, there was a fast elimination of the luciferase protein until Day 3, with a complete elimination of the bioluminescent signal from the liver at Day 7. Beyond Day 7, the in vivo bioluminescent signal was comparable with the background signal (Figure 1C).In the spleen, less luciferase protein expression was observed at 6 h after immunization compared with expression in the liver and at the injection site.The protein expression in the spleen decreased until Day 2 and was comparable between the vaccine and the control group beyond Day 2 (Figure 1D).

| Ex vivo results
In the Lipid-1/mRNA-FFLuc group, a bioluminescence signal was detected in several organs between 6 h and 28 days postinjection (Table S3).In accordance with the in vivo results, a specific signal was predominantly detected between 6 h (peak of expression) and Day 1 at the injected site, in the draining lymph nodes, spleen, and liver and, to a lesser extent, in the lacrimal glands, brain, thymus, lungs, and adrenal glands (Figure 2).Thereafter, the bioluminescence signal decreased.At Day 28, under study conditions, the luciferase protein was no longer detected in vivo and ex vivo.

| Clinical observations
There were no unscheduled deaths during the study or vaccinerelated systemic clinical signs.A few local reactions (erythema and/or hematoma) were observed in animals of both groups and were assessed as procedure-related.There were no vaccine-related body weight changes during the observation period in either sex.A decrease in mean food consumption (up to À37.2% vs. control value) was reported in the 135 μg mRNA/dose group during the first 3 to 4 days postinjection with a return towards control values thereafter.

| Hematology, coagulation, clinical chemistry parameters, and CRP
Vaccine-related effects were observed in some hematology, coagulation, and CRP parameters (Table 6).An increase in fibrinogen concentrations was detected on Day 2 in males and females at both dose levels, returning to control and/or pretest values by Day 8.A dose-related increase in mean CRP levels was also noted in both sexes on Day 2, when compared with the control group and pretest values.CRP levels progressively returned to pretest/control range values generally by Day 4 for those administered 15 μg mRNA/dose and by Day 8 for those receiving 135 μg mRNA/dose.Overall, these changes in hematology, coagulation, and CRP values were consistent with an acute-phase response, secondary to vaccine administration; they were considered as nonadverse as they were transient, of small magnitude, and within the historical control data range.In addition, there were slight increases in alanine aminotransferase and aspartate aminotransferase activities (outside the range of historical data) in two males and one female receiving 135 μg mRNA/dose on Day 2 and whose relationship with the vaccine treatment was regarded as equivocal.These slightly higher liver enzyme values observed in isolated cases were no longer observed on Day 4.

| RT-qPCR
mRNA from the test item was not detected nor quantified in any sample from control animals.No PCR inhibition was detected in any sample.
Among rabbits receiving 15 μg mRNA/dose, MRT1500 was quantified in at least one sample per tissue/organ type from 1 hour after injection (Figure 3).A main peak was detected at 1 h for iliac lymph nodes (draining lymph nodes) and liver, at 6 h for injection site and spleen, and at 24 h for adrenal glands, bone marrow, brain, heart, kidneys, lungs, ovaries, testes, thymus, lachrymal glands, and plasma samples.In the liver, the amount of MRT1500 detected remained similar for the first 24 h, but by Day 14, it had decreased to levels below the LLOQ.The maximal mean increase in MRT1500 was observed in plasma (3.04 Â 10 8 copies/mL of plasma for females and 2.51 Â 10 8 copies/mL of plasma for males), injection site (6.40 Â 10 7 copies/μg of RNA for females and 9.46 Â 10 7 copies/μg of RNA for males), and iliac lymph nodes (draining lymph nodes) (3.61 Â 10 6 copies/μg of RNA for females and 2.43 Â 10 7 copies/μg of RNA for males).MRT1500 could also be quantified in all other organs (adrenals glands, spleen, heart, lungs, kidneys, bone marrow, brain, gonads, thymus, and lachrymal glands) with the number of copies ranging from 6.00 Â 10 2 to 7.23 Â 10 5 copies/μg of RNA at the peak, corresponding to 1.58 Â 10 5 to 1.31 Â 10 2 times less than the maximum injection site level.The number of positive tissues (i.e., above 50 copies/well), as well as MRT1500 levels drastically decreased until Day 14 in all tissue/organ samples, except for some plasma, iliac lymph node, and injection sites samples, which remained positive.MRT1500 was no longer quantified at Day 28.
Similar observations were made among those receiving 135 μg mRNA/dose (Group 3), where MRT1500 was quantified in all tissues from 1 h after injection (Figure S3).A main peak was detected at 6 h for iliac lymph nodes, injection site, liver, spleen, and thymus samples and at 24 h for adrenal glands, bone marrow, brain, heart, kidneys, lacrimal glands, lungs, ovaries, testes, and plasma samples.The maximal mean increase was observed in plasma (9.63 Â 10 8 copies/mL of plasma for females and 6.37 Â 10 8 copies/mL of plasma for males), injection site (1.49Â 10 8 copies/μg of RNA for females and 2.15 Â 10 9 copies/μg of RNA for males), and iliac lymph node (1.21 Â 10 8 copies/μg of RNA for females and 2.51 Â 10 8 copies/μg of RNA for males).MRT1500 was also quantified in all other organs (adrenals glands, spleen, kidneys, liver, bone marrow, heart, lungs, ovaries, testes, thymus, brain, and lacrimal glands), and the mean number of copies ranged between 6.00 Â 10 2 and 4.69 Â 10 6 copies/μg of RNA at the peak, corresponding to 3.10 Â 10 6 to 4.58 Â 10 2 times less than the maximum injection site level.The number of positive tissues (i.e., above 50 copies/well) as well as MRT1500 levels decreased until Day 28.At that point, a reduced number of copies-up to 97% lower than the numbers observed at Day 14-could be observed only in the plasma, iliac lymph node, and injection site samples.
There was a tendency for dose relationship in MRT1500 levels in the spleen, bone marrow, and liver, but no real dose relationship was observed in other tissues/organs due to intervariability in the tissues, mainly at the injection site.No evident difference was seen in MRT1500 levels between males and females in both the groups.

| Determination of Lipid-1 concentrations
Lipid-1 was not detected or not quantified (BLQ, 20 ng/mL) in all the samples from control animals.However, it was quantified in the plasma 6 h after the injection of 15 μg mRNA/dose or from 1 to 24 h following the injection of the 135 μg mRNA/dose, with a main peak observed at 6 h.
After injection with 15 μg mRNA/dose, muscle (injection site) and sacral lymph nodes (draining lymph nodes) were exposed to Lipid-1, with a peak between 1 and 168 h after the injection, with concentrations up to 2480 and 592 ng/mL, respectively.In the other organs/tissues, Lipid-1 was below or close to the limit of quantification (LOQ).Lipid-1 was not quantified or close to the LOQ on Day 28 in the muscle and sacral lymph nodes.
After injection with the 135 μg mRNA/dose, Lipid-1 was quantified in the same tissues/organs, with a concentration up to 5220 ng/ mL in the muscle, 3750 ng/mL in the sacral lymph nodes, 193 ng/mL in the liver, 218 ng/mL in the adrenal glands, and 352 ng/mL in the spleen.Moreover, Lipid-1 was quantified in the liver, adrenal glands, and spleen with a maximal mean concentration observed between 6 and 72 h.At later timepoints, Lipid-1 was still quantifiable in these organs/tissues, but with a decrease in concentration from Day 7 in the muscle and adrenal glands and from Day 14 in the spleen, liver, and sacral lymph nodes.At Day 28, concentrations of Lipid-1 in the liver were close to the LOQ, while in other organs (brain, bone marrow, heart, kidneys, lacrimal glands, lungs, ovaries, and testis), they were below or close to the LOQ.

| Pathology
Vaccine-related macroscopic observations were noted at the injection sites and in the inguinal lymph nodes (draining lymph nodes).
Red discoloration at the injection site was observed in rabbits receiving either the 15 or 135 μg mRNA/dose during the necropsies conducted between 1 h and 15 days postadministration.This correlates with microscopic hemorrhage in the treated animals euthanized at 24 or 72 h.In the inguinal lymph nodes, enlargement was noted in rabbits receiving 135 μg mRNA/dose during the necropsies conducted at 6 h and Day 8 postadministration.At 24 and 72 h, it correlated microscopically with increased cellularity of those lymph nodes.
Vaccine-related microscopic observations were noted in the liver, spleen, inguinal lymph nodes, thymus, bone marrow, and at the injection sites.In the liver, minimal-to-mild hepatocyte single cell necrosis was observed 72 h postinjection in those receiving 135 μg mRNA/ F I G U R E 3 Regulatory biodistribution study in rabbits (Study 3): biodistribution evaluation by RT-qPCR.Mean quantity of the positive samples in copies/μg of RNA or copies/mL of plasma (i.e., above 50 copies/well) in Group 2 (animals administered with 15 μg mRNA/dose) and (B) Group 3 (animals administered with 135 μg mRNA/dose).
dose.This was sometimes associated with a minimal increase in hepatocellular mitotic figures.These findings were no longer observed after 7 days.In the spleen, minimally decreased lymphoid cellularity was noted at 24 h postinjection, regardless of dose level.This change was characterized by multifocal areas of decreased lymphoid cellularity in the white pulp (periarteriolar lymphoid sheaths and sometimes extending in the marginal zone) and associated with apoptotic cell debris.In the inguinal lymph nodes, cellularity of the paracortex was minimally to mildly increased in animals administered 135 μg mRNA/ dose at 72 h.In the thymus, an increase in tingible body macrophages was noted at 24 h after either the 15 or 135 μg mRNA/dose and up to 72 h in the 135 μg mRNA/dose.This increase was associated with a decrease in the lymphoid cellularity of the thymus in rabbits administered 135 μg mRNA/dose.In the bone marrow, a minimal increased cellularity of the myeloid cell line was noted after 15 and 135 μg mRNA/dose at 72 h.
At the injection sites, a variable combination of minimal-to-mild hemorrhage, acute to subacute inflammation, and necrosis was noted in the muscle and/or the subcutaneous tissue after 15 and 135 μg mRNA/dose at 24-and 72-h postdose.These findings were of higher magnitude in treated animals when compared with controls but with no clear dose relationship between 15 and 135 μg mRNA/dose.None of the remaining microscopic findings were considered as vaccine-related since they were consistent with spontaneously occurring findings described in the literature (Bradley, 2012).Overall, the observed findings were similar in frequency and appearance across treatment groups.

| DISCUSSION
Since the outbreak of the novel coronavirus SARS-CoV-2 and associated Covid-19 disease, a remarkable scientific effort led to the rapid development and approval of highly effective LNP-formulated mRNA vaccines (Anderson et al., 2020;Baden et al., 2021;Polack et al., 2020;Sahin et al., 2014).However, different types and numerous doses of vaccines are still needed to meet the global demand and to cope with new emerging variants (Sabnis et al., 2018;Tegally et al., 2021;Zhang et al., 2021).
In the present work, we described the nonclinical safety assessment of the SARS-CoV-2 mRNA vaccine MRT5500.The MRT5500 mRNA vaccine was evaluated preclinically for its potential systemic toxicity and local tolerance following three IM injections in NZW rabbits.Regression and/or delayed occurrence of any signs during a 4-week observation period was also evaluated.
The design of the study was aligned with the dose regimens intended to be evaluated in a Phase I/II clinical trial, which consisted of either one IM injection or two IM injections at 3 weeks apart and, as per the N + 1 rule, included one additional injection compared with the planned clinical schedule to maximize animal exposure to the vaccine (ClinicalTrials.gov-identifier:NCT04798027).The rabbit model was chosen as it is an accepted species for preclinical toxicity testing by regulatory agencies, and a full-human dose can be injected allowing a proper evaluation of the potential local reactions (Namdari et al., 2021).Moreover, our choice was supported by a previous dose range finding toxicity study in NZW rabbits, in which an mRNA encoding the influenza hemagglutinin H3 antigen was encapsulated in the same LNP vehicle (Broudic et al., 2022), based on the assumption that the safety profile of LNP/mRNA-based vaccines is mainly driven by the LNP component rather than by the mRNA (Hassett et al., 2019;Lutz et al., 2017;Marlowe et al., 2017;Sabnis et al., 2018;Sedic et al., 2018).In this study, the No Observed Adverse Effect Level (NOAEL) was determined at 250 μg mRNA/ dose, and rabbits developed an immune response to the vaccine formulation, demonstrating that the LNP could efficiently deliver the encapsulated mRNA into cells, leading to the production of specific antibodies (Broudic et al., 2022).An increase in CRP, neutrophil counts, and fibrinogen levels is known to be related to local inflammation (Destexhe et al., 2013).
Moreover, an increase in CRP level is also known to be a good indicator of vaccine-induced cytotoxic T-cell activation or T H 1 immune response involvement (Green, 2015).The establishment of an immune response was further confirmed by the activated microscopic appearance of the draining lymph nodes and spleen only in the treated animals, with higher incidence or severity after 135 μg mRNA/ dose.This effect correlated with the higher spleen weight noted in males receiving 45 or 135 μg mRNA/dose and in females receiving 135 μg mRNA/dose.Four weeks after the last injection, the microscopic changes at the injection sites, spleen, and draining lymph nodes were still observed but at a lower incidence and/or severity.This reflects the resolution of the inflammatory process and the evolution of the immune reaction and indicates ongoing reversibility.The overall changes observed in our study were in line with those seen after the administration of conventional and/or adjuvanted vaccines.This includes raised neutrophil counts, the presence of mixed inflammatory cell infiltration at the injection site, lymphoid hyperplasia, and increased weights of spleen and draining lymph nodes (Baldrick, 2016).Moreover, specific IgG against SARS-CoV-2 S protein was induced in all rabbits receiving MRT5500 vaccine, and the IgG response increased with the dose and between Days 45 and 71.
Messenger RNA therapeutics contain many components and are processed at different levels (Pardi et al., 2018;Sahin et al., 2014).
In addition, the chemical structure and physical properties of the LNPs influence both their biodistribution in tissues and the efficiency of delivery (Kauffman et al., 2015;Reichmuth et al., 2016).Given the novelty and the limited information available on LNPs biodistribution, we investigated both the potential toxic effect and the biodistribution of the MRT5500 vaccine's mRNA component in target tissues and organs by RT-qPCR and the concentration of the novel ionizable Lipid-1 by LC-MS/MS.We used live animal optical imaging to observe the biodistribution of the luciferase protein in mice.The kinetic assessment and migration of Lipid-1/mRNA-FFLuc in different organs and tissues at several time points were observed in vivo and ex vivo.In vivo, the luciferase expression was detected at different timepoints at the injection site, liver, and spleen indicating drainage to these organs.As expected with an mRNA product, the luciferase expression was transient and decreased over time.Luciferase signals at the injection sites, most likely reflecting distribution to the lymph nodes draining the injection sites, peaked 6 h postinjection with a bioluminescence approximately 12,000 times higher than that observed in the control group.The signal decreased slowly during the first 72 h, and after 7 days, its intensity was only 11 times that of the signal obtained in the control group.In accordance with the in vivo results, the ex vivo bioimaging showed that LNP-formulated RNAs were predominately detected at the injection site (quadriceps muscle from 6 h until Day 21 postinjection), in liver and spleen (at 6 h and 24 h postinjection), and in the draining lymph nodes (at 6 and 24 h and Day 21 postinjection).To a lesser extent, the luciferase protein was also expressed in several other organs such as the lacrimal and adrenal glands (up to Day 21 postinjection), thymus, lungs, brain, kidneys, and heart but was not detected in bone marrow and ovaries.These results confirmed several literature reports (Hassett et al., 2019;Kauffman et al., 2015;Reichmuth et al., 2016) indicating that LNP-formulated RNAs can spread rather nonspecifically to several organs such as spleen, liver, kidney, lungs, and brain.
Finally, a single IM administration of MRT5500 vaccine at 15 or 135 μg mRNA/dose to rabbits was well-tolerated, inducing a spectrum of nonadverse changes consistent with the expected local and systemic immune response to injection of immunogenic material (Baldrick, 2016).Similar to the repeated dose toxicity study (study 1), findings from study 3 mainly included inflammatory changes at the Regarding the mRNA analysis by RT-qPCR, MRT1500 was quantified in all tissues/organs from 1 or 6 h postinjection and peaked between 6 and 24 h after the injection, depending on tissues/organs, with both mRNA doses.The maximal amount of mRNA was observed in plasma, in the injection site muscle samples, and in the draining lymph nodes.The number of positive samples (above 50 copies/well) as well as MRT1500 levels decreased until Day 28, with undetectable levels for all tissues/organs except for plasma, draining lymph nodes, and the injection site, which remained positive only in animals having been injected with 135 μg mRNA/dose.
A tendency for a dose relationship was observed in bone marrow, liver, and spleen only, and no evident difference was seen between males and females.Lipid-1 analysis by LC-MS/MS revealed that the main exposed tissues were the muscle at injection site and the draining lymph nodes, with decreased concentrations from 7 days after the injection.Lipid-1 was also detected in the liver, adrenal gland, and spleen with decreased concentrations from 14 days after the injection.On Day 28, Lipid-1 levels were undetectable or close to the lower limit of quantitation.There were no MRT5500 dose levels and Lipid-1 tissues/organs concentration relationship and no differences among sexes.
Results from the two biodistribution studies with mRNA encapsulated in the same LNP (i.e., containing Lipid-1), conducted either with a luciferase surrogate in mice or with a SARS CoV-2 mRNA vaccine in rabbits, showed that the components of the mRNA-LNP vaccine formulation were mainly detected at the injection site and in the draining lymph nodes.
Testing was consistent with current guideline recommendations (WHO, 2021).Our strategy was in line with other SARS-CoV-2 mRNA vaccines already on the market (Baldrick, 2022) and consisted in the selection of Lipid-1 through in silico and in vitro analyses to evaluate its genotoxicity potential, followed by a non-GLP preliminary dose range finding toxicity study to confirm the safety of Lipid-1 in vivo determining the NOAEL (Broudic et al., 2022) and moved forward in the present study with a repeated dose toxicity study characterizing the safety profile of the mRNA vaccine containing Lipid-1 and Lipid -2 biodistribution studies determining the distribution and the persistence of the mRNA and Lipid-1.However, as the available guidelines only give broad recommendations for general toxicity testing, vaccine testing packages use different study designs including the species used, dosing, frequency, and number of doses and recovery period making difficult the comparison between studies and the translation into the human situation.Nevertheless, this platform approach allows the rapid development of vaccines with standardized components which are consistent across target vaccines such that the only change is in antigen or nucleic acid sequence for antigen expression (Bennet et al., 2020).
In conclusion, in this study, we characterized the safety and biodistribution profiles of MRT5500 SARS-CoV-2 mRNA vaccine in rabbits and mice.MRT5500 vaccine was safe and well-tolerated and elicited an IgG immune response in rabbits, using worst-case experimental conditions as compared to human use.The biodistribution data from both studies demonstrated that the components of the MRT5500 vaccine formulation are predominantly observed at the injection site and in the draining lymph nodes following a single IM injection.Some potential systemic dissemination was also detected, supporting the idea that exploratory studies such as Study 2 could be predictive of regulatory ones in different animal models (Study 3).Moreover, the route of administration, dose levels, and dose volume (0.5 mL) intended to be used in the Phase I/II clinical trial (ClinicalTrials.gov-identifier:NCT04798027) were used, supporting the administration of up to two IM injections, 3 weeks apart, of the MRT5500 vaccine in healthy adults.
Three studies were conducted either at Charles River Laboratories (Les Oncins and Evreux, France) or at Sanofi (Marcy l'Etoile, France) and sponsored by Sanofi (Marcy L'Etoile, France).Studies were carried out in compliance with animal health regulations (Council Directive No. 2010/63/EU of 22 September 2010 and French decree No. 2013-118 of 01 February 2013 on the protection of animals used for scientific purposes) in AAALAC-accredited laboratories.
Blood sampling: Hematology, coagulation, clinical chemistry, and CRP Hematology, coagulation, clinical chemistry parameters, and C-reactive protein (CRP) were evaluated on all rabbits at pretest T A B L E 1 Repeated dose toxicity study in rabbits (Study 1): Experimental design.
were gently mixed and centrifuged at 1800 g for 10 min at +4 C within 30 min of sampling.The resultant plasma was separated, transferred into duplicate cryotubes, snap frozen in liquid nitrogen, and stored at À80 C pending RNA extraction.A clean set of disposable instruments was used for each animal and for each organ.Tissues were harvested in the following order: brain, lungs, heart, kidneys, gonads, liver, spleen, draining lymph nodes (iliac), adrenal glands, thymus, bone marrow, lacrimal gland, and injection site.Tissues were rinsed with clean PBS without calcium and magnesium.Care was taken to remove adipose tissues around the sampled organs.Three samples per organ were collected when the size of the organ allowed it, except for the injection site where five samples were collected.Tissue samples were placed into cryotubes, snap frozen in liquid nitrogen, and stored at À80 C pending RNA extraction.The PureLink RNA mini Kit (Ambion) was used to extract RNA from tissues which were weighted before the extraction, and then 50 μL of RNase-free water was used to elute the RNA.For plasma samples, RNA was extracted using the QIAamp viral kit (Qiagen) and then eluted in 70 μL RNase/DNase free water.After determination of RNA concentration and verification of RNA integrity, the samples were subjected to RT-qPCR.
On the day following the injection (Day 2), mean platelet counts were decreased in both sexes at both dose levels.Platelet counts returned to normal by Day 4, with values close to control and pretest values.An increase in mean platelet counts was observed at both dose levels on Day 8, possibly related to overcompensation.Mean neutrophil counts were increased on Day 2 in both sexes and at both dose levels, with counts returning to control and/or pretest values by Day 4 in rabbits receiving 15 μg mRNA/dose and by Day 15 in those administered 135 μg mRNA/dose.On Day 2, these increases in mean neutrophil counts were associated with decreased mean lymphocyte (within the historical control data range), monocyte and eosinophil (outside the range of historical data), and basophil counts (within the historical control data range) in males and females receiving 135 μg mRNA/dose.Other apparent differences in hematology between control and treatment groups were considered to be incidental and not vaccine-related, as they were isolated, already observed during F I G U R E 1 Exploratory biodistribution study in mice (Study 2): LNP formulated with mRNA encoding for firefly luciferase.(A) Representative IVIS images at 6 h postadministration of Lipid-1/mRNA-FFLuc.Bioluminescent signal at the injection site.The color gradient is indicated on the right.Luciferase expression in the injection site (B), liver (C), and spleen (D) over time.Results are expressed as total flux photons per seconds (p/s).F I G U R E 2 Exploratory biodistribution study in mice (Study 2): Ex vivo bioluminescence quantification.(A) Organs sampled.(B) Luciferase expression 6 h postadministration of Lipid-1/mRNA-FFLuc in ex vivo organs.(C) Luciferase expression 6 h postadministration of Lipid-1/mRNA-FFLuc (green) versus negative control (black).Results are expressed as photons per seconds per square centimeter per steradian (ph/s/cm 2 /sr).T A B L E 6 Regulatory biodistribution study in rabbits (Study 3): Main hematology, coagulation, and CRP changes (fold change or % from control group).
After repeated IM administrations, MRT5500 vaccine at 15, 45, and 135 μg mRNA/dose was locally well-tolerated with only transient edema observed at the injection site.The incidence and severity were slightly higher in animals receiving 135 μg mRNA/dose compared with the other treatment and control groups.Local reactions were resolved within few days and before the following injection.An increase in body temperature was noted 3 (135 μg mRNA/dose) or 6 h (15 and 45 μg mRNA/dose) after each injection, which returned to basal values during the following 24 to 48 h.Body weight changes and reduced food consumption were also noted on the day after each injection (45 and 135 μg mRNA/dose).These effects were transient and did not affect the health status of the animals.Inflammatory reactions, including increases in CRP, fibrinogen, globulin, and shortened PT, were observed in both sexes 2 days after the first and third injections (Days 3 and 45).Slight changes were noted among hematology parameters mainly after the 45 or 135 μg mRNA/dose, including slightly increased neutrophil and monocyte counts, a slight decrease in eosinophil counts, slightly reduced red blood cell parameters, and reduced reticulocyte and platelet counts.The above-mentioned changes noted on Day 3 were no longer observed before the second injection (Day 20) (i.e., 3 weeks after the first injection) and 4 weeks after the third injection.The changes were generally slight and transient, and therefore considered as nonadverse.
injection site, correlating with transient increases in neutrophil counts, fibrinogen, and CRP concentrations and associated with increased cellularity of the draining lymph nodes and bone marrow.Additional findings included transient decreases in platelet counts at all dose levels and in lymphocyte, monocyte, eosinophil, and basophil counts at 135 μg mRNA/dose, along with microscopic changes of low magnitude in the liver (reversible, minimal, or slight single cell necrosis and increased mitotic figures with 135 μg mRNA/dose at 72 h postadministration), spleen, and thymus (decreased cellularity with 15 and/or 135 μg mRNA/dose).
Repeated dose toxicity study in rabbits (study 1): body weight gain (g) and food consumption (g).Repeated dose toxicity study in rabbits (Study 1): Main hematology, coagulation, clinical chemistry, and CRP changes (fold change or % from control group).
A dose-related increase in mean CRP concentrations was noted in both sexes 2 days after the first and second injections.This change was consistent with an acute phase response following the administration of a vaccine.A slight increase in mean globulin concentration was noted at all dose levels 2 days after the first and third injections without a clear relationship.This change is an expected pharmacodynamic response to the vaccine.In addition, a slight decrease in mean albumin concentration was noted 2 days after the first injection in rabbits receiving 135 μg mRNA/dose.All other changes in clinical chemistry parameters were due to isolated variations already noted T A B L E 3 Note: -: No noteworthy finding.*p ≤ 0.05, and **p ≤ 0.01.T A B L E 4 Repeated dose toxicity study in rabbits (Study 1): Mean body temperature.T A B L E 5