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Pharmaceutical Pathobiology–Original Article
Safety Evaluation of Lipid Nanoparticle–
Formulated Modified mRNA in the Sprague-
Dawley Rat and Cynomolgus Monkey
Maja Sedic1, Joseph J. Senn1, Andy Lynn1, Michael Laska1, Mike Smith1,
Stefan J. Platz2, Joseph Bolen3, Stephen Hoge1, Alex Bulychev1,
Eric Jacquinet1, Victoria Bartlett4, and Peter F. Smith5
Abstract
The pharmacology, pharmacokinetics, and safety of modified mRNA formulated in lipid nanoparticles (LNPs) were evaluated afterrepeat intravenous infusion to rats and monkeys. In both species, modified mRNA encoding the protein for human erythropoietin(hEPO) had predictable and consistent pharmacologic and toxicologic effects. Pharmacokinetic analysis conducted following thefirst dose showed that measured hEPO levels were maximal at 6 hours after the end of intravenous infusion and in excess of 100-fold the anticipated efficacious exposure (17.6 ng/ml) at the highest dose tested.
24hEPO was pharmacologically active in both the
rat and the monkey, as indicated by a significant increase in red blood cell mass parameters. The primary safety-related findingswere caused by the exaggerated pharmacology of hEPO and included increased hematopoiesis in the liver, spleen, and bonemarrow (rats) and minimal hemorrhage in the heart (monkeys). Additional primary safety-related findings in the rat includedmildly increased white blood cell counts, changes in the coagulation parameters at all doses, as well as liver injury and release ofinterferon g–inducible protein 10 in high-dose groups only. In the monkey, as seen with the parenteral administration of cationic
LNPs, splenic necrosis and lymphocyte depletion were observed, accompanied with mild and reversible complement activation.These findings defined a well-tolerated dose level above the anticipated efficacious dose. Overall, these combined studies indicatethat LNP-formulated modified mRNA can be administered by intravenous infusion in 2 toxicologically relevant test species and
generate supratherapeutic levels of protein (hEPO) in vivo.
Keywords
modified mRNA, lipid nanoparticle, toxicology, pharmacokinetics, drug discovery
The promise of mRNA as a novel modality to deliver thera-
peutic proteins in humans is vast, as evidenced by the growth
and success of recombinant human therapeutic proteins over
the last 3 decades, such as recombinant human insulin for thetreatment of diabetes mellitus.
17Protein expression directed by
exogenous mRNA offers many advantages over other nucleicacid–based concepts, as well as recombinant proteins. Potentialadvantages of mRNA over DNA-based technology include (1)no integration into the host genome thereby circumventing therisk of deleterious chromosomal changes, and (2) faster and
more efficient expression with proper modifications, since
mRNA therapeutics only require access to the cytoplasm. Incomparison with recombinant proteins, mRNA would havelower manufacturing costs and could enable access to intracel-lular as well as cell membrane–bound therapeutic targets. Thebiggest challenges of mRNA technology are its potential forimmunogenicity and its relatively poor in vivo stability. Thesechallenges have been addressed through progress in chemistry
and sequence engineering (eg, optimization of the 5
0cap, 50-,and 30-untranslated regions and coding sequences) and through
the use of specific nucleotide modifications.16,21,29
Nucleotide-modified mRNA is nearly identical to naturally
occurring mammalian mRNA, with the exception that certain
nucleotides, normally present in mammalian mRNA, arepartially or fully replaced with nucleosides, such as pyrimidinenucleosides—specifically, pseudouridine, 2-thiouridine,
1Moderna Therapeutics, Cambridge, MA
2AstraZeneca, Melbourn, Royston, UK
3PureTech Health, Boston, MA
4Akcea Therapeutics, Cambridge, MA
5Alnylam Pharmaceuticals Inc, Cambridge, MA
Supplementary material for this article is available online.
Corresponding Author:
Joseph J. Senn, Moderna Therapeutics, 200 Technology Square, Cambridge,
MA 02139, USA.Email: [email protected] Pathology
2018, Vol. 55(2) 341-354
ªThe Author(s) 2017
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DOI: 10.1177/0300985817738095
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5-methyl cytosine, or N1-methyl-pseudouridine.2,14,16,21,29
These naturally occurring pyrimidine nucleotides are present
in mammalian tRNA, rRNA, and small nuclear RNAs.20Incor-
poration of these nucleotides in place of the normal pyrimidine
base has been shown to minimize the indiscriminate recogni-tion of exogenous mRNA by pathogen-associated molecularpattern receptors, such as toll-like receptors, retinoic acid–inducible gene 1, melanoma differentiation-associated protein5, nucleotide-binding oligomerization domain-containing pro-tein 2, and protein kinase R.
7
Given the lability of a naked mRNA molecule, the devel-
opment of mRNA therapeutics has been further hampered bythe lack of appropriate formulations for delivery and poten-tially as a targeting mechanism to a diseased organ or tissue.
12
However, the application of lipid-based nanoparticle deliverysystems, initially developed for the in vivo delivery of siRNA,has enabled systemic administration of modified mRNA.
22
Adequate delivery of mRNA with lipid nanoparticles (LNPs)
has been demonstrated for mRNA-based vaccines, where
intramuscular injection of low doses of mRNA formulatedin either LNPs or nanoemulsion induced immune protectionfrom influenza and respiratory syncytial virus in mice, as wellas cytomegalovirus and respiratory syncytial virus in mon-keys.
9Furthermore, a single administration of modified
mRNA-LNP complexes in mice by various routes resultedin high, sustained protein production.
19Finally, Thess
et al25reported that repeated administration of unmodified
mRNA in combination with the nonliposomal polymericdelivery system ( Trans IT) induced high systemic protein lev-
els and strong physiologic respon ses in mice. These authors
also noted similar observations following single-dose admin-istration of erythropoietin (EPO)–mRNA in LNPs to pigs andmonkeys.
LNPs have been reported to be clinically effective for the
delivery of siRNA.
6The LNP vehicle is currently in late-phase
clinical trials of a synthetic siRNA in patients suffering fromtransthyretin amyloidosis and has been well tolerated in thispopulation.
6Therefore, considerable work has been done to
understand the safety profile of systemic administration ofsiRNA-LNPs.
3Here, we set out to describe, for the first time,
the pharmacology and toxicologic effects of repeated adminis-tration of hEPO-mRNA in LNPs in male Sprague-Dawley rats
and female cynomolgus monkeys.
Materials and Methods
Animals and Husbandry
The study plan and any amendments or procedures involving
the care and use of animals in these studies were reviewed and
approved by the Institutional Animal Care and Use Committee
of Charles River Laboratories Preclinical Services (Montrealand Sherbrooke, Canada). During the study, the care and use ofanimals were conducted according to the guidelines of the USNational Research Council and the Canadian Council on Ani-mal Care.Male Sprague-Dawley rats (Charles River Laboratories)
were 11 to 12 weeks old and weighed between 390 and 497
g at dose initiation. Animals were group housed in polycarbo-
nate bins and separated during designated procedures. Thetemperature of the animal room was kept between 19
/C14C and
25/C14C, with humidity between 30 %and 70%. The light cycle
was 12 hours light and 12 hours dark, except during designatedprocedures. Animals were fed PMI Nutrition International Cer-tified Rodent Chow No. 5CR4 (14 %protein) ad libitum
throughout the in-life studies, except during designated proce-
dures. Municipal tap water treated by reverse osmosis and
ultraviolet irradiation was freely available to each animal viaan automatic watering system. Environmental enrichment wasprovided to animals per standard operating procedures ofCharles River Laboratories (Montreal, Canada), except duringstudy procedures and activities.
Female cynomolgus monkeys (Charles River Laboratories)
were 1.5 to 6 years old and weighed 2.5 to 5.1 kg at the initia-
tion of dosing. Animals were housed in stainless-steel cages
and separated during designated procedures. The temperatureof the animal room was kept between 20
/C14C and 26/C14C, with
humidity between 30 %and 70%. The light cycle was 12 hours
light and 12 hours dark except during designated procedures.Animals were fed PMI Nutrition International Certified Pri-mate Chow No. 5048 (25 %protein). Municipal tap water
treated by reverse osmosis and ultraviolet irradiation was freely
available to each animal via an automatic watering system.
Psychological and environmental enrichment was provided toanimals per standard operating procedures of Charles RiverLaboratories (Montreal, Canada) except during study proce-dures and activities.
Control, Test, and Reference Items
An 850-nucleotide messenger RNA was prepared by in vitrotranscription from a linearized DNA template with T7 RNAPolymerase. The DNA template encoded the T7 promoter, a 5
0
untranslated region, the 579-nuc leotide open reading frame
encoding human EPO (hEPO) mature protein with signalsequence, a 3
0untranslated region, and a polyadenylated tail.
The in vitro transcription was performed with the canonicalnucleotides adenosine triphosphate and guanosine triphosphate
and the modified nucleotides 1-methylpseudouridine tripho-
sphate and 5-methylcytidine triphosphate. The mRNA containsa5
0Cap 1 structure, which consisted of 7-methylguanosine
linked to the 50nucleoside of the mRNA chain through a 50–
50triphosphate bridge and 20-O-methyl group present on the
first nucleotide of the mRNA.23The messenger RNA was pur-
ified and buffer exchanged into low ionic strength buffer forformulation.
18The final mRNA had a calculated molecular
weight of 277 786 Da.
The mRNA-loaded LNPs were generated via stepwise etha-
nol dilution, with an approach adapted from previously demon-strated methods.
13,30The LNP formulation was prepared by
dissolving the lipids (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3),342 Veterinary Pathology 55(2)
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1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), choles-
terol, and 1,2-dimyristoyl-rac-glycerol, methoxypolyethyleneglycol (PEG2000-DMG) in ethanol. The 4 lipids were preparedas a combined stock, with a total concentration of 12.5 mM(molar ratio of 50:10:38.5:1.5, MC3:DSPC:cholesterol:PEG2000-DMG). In brief, the solution containing lipids wasmixed with an acidic aqueous buffer containing mRNA (0.18
mg/ml, pH 4.0) in a T-mixer device. The resulting LNP
dispersion was diluted and subsequently purified and concen-trated by tangential flow filtration. The formulation wasfiltered through a clarification filter (0.8/0.2 mm nominal).
Prior to storage, the formulation was additionally filteredthrough 2 in-line sterile filters (0.2 mm) and aseptically filled
into sterilized vials, stoppered, and capped. Empty LNPs weregenerated with a similar approach, whereby mRNA was
excluded from the process.
The final LNP lipid concentration was determined with an
ultraperformance liquid chromatography system with online
charged aerosol detection. The total concentrations of lipidsin the final mRNA-LNPs and empty LNPs were 22.0 and13.1 mg/ml, respectively. The final mRNA content in hEPOLNPs was quantified by ultraviolet analysis, resulting in anmRNA concentration of 1.2 mg/ml. Measured lipid and mRNA
concentration values enabled dilution with phosphate-buffered
saline (PBS) to target levels for dosing (Tables 1, 2). Particlehydrodynamic diameters were determined by dynamic lightscattering. Resulting diameters for mRNA and empty LNPswere 81 nm (0.08 polydispersity index) and 61 nm (0.10 poly-dispersity index), respectively. Total mRNA encapsulation wasquantified with the Ribogreen assay (ThermoFisher Scientific).The final value for hEPO-mRNA in LNP encapsulation was
97%. Additional information for the control, test, and reference
items is provided in Supplemental Table 1.
Male Rat Study Design
Only male rats were used for this study, as there was no
expected sex-specific differences in metabolism, distribution,or toxicity. The negative control, test, or reference items wereadministered over the course of 2 weeks in a 10-minute intra-
venous (IV) infusion via a caudal vein at a dose level, dose
volume, and frequency listed in Table 1. Dose levels for eachstudy were based on previous pharmacology data demonstrat-ing production of efficacious levels of hEPO in the rat andcynomolgus monkey at doses /C200.03 mg/kg of mRNA. Based
on pharmacokinetic (PK) data indicating predictable increasesin protein expression with dose, the mid- and high doses forthese studies were selected to achieve significant multiples of
the efficacious dose level. Since PK behavior and physiologic
consequences are well defined for EPO therapy, we employed asimilar approach in our study of hEPO-mRNA in LNPs.
15,16,25
Each infused dose was administered with a temporary indwel-ling catheter inserted in a caudal vein connected to an injectionset and infusion pump. The animals were temporarilyrestrained for the dose administration and not sedated. The dosevolume for each animal was based on the most recent body
weight measurement. The first day of dosing was designated as
day 1. Six males per group were used for toxicity assessment,12 males per group for immunology assessment, and 6 malesper group for PK / pharmacodynamic (PD) assessment. Thefollowing end points were evaluated: clinical signs (includingobservations of the infusion sites), body weights, food con-sumption, PK/PD, clinical pathology (hematology, coagula-tion, and clinical chemistry), macro- and microscopic
examination of tissues, and immunotoxicology markers: hista-
mine, interleukin 6 (IL-6), interferon g–induced protein 10 (IP-
10), tumor necrosis factor a(TNF- a), interferon a(IFN-a), and
complement (C3).
Blood samples were collected from nonfasted animals and
analyzed for hematology on day 9 and from fasted animals forhematology, coagulation, and clinical chemistry on day 16 (atnecropsy). For PD (hEPO) or PK (hEPO-mRNA), blood sam-
ples were collected and processed to plasma prestudy and at 2,
6, 24, and 48 hours after the end of injection/infusion on days 1and 15. After processing, the plasma samples were stored in afreezer set to maintain /C080
/C14C until analyzed. For cytokines (ie,
IL-6, IP-10, TNF- a), histamine, and complement (C3) analysis,
blood samples were collected prestudy and at 5 minutes and 2,Table 1. Experimental Design for Safety Study: Rat.a
Group
No.TestMaterialDose
Level,
mg/kg
bIntravenous
AdministrationDose
Concentration
mg/ml
1 PBS 0 10 min infusion, 2/C2/wk 0
2 mRNA EPO 0.03 10 min infusion, 2/C2 /wk 0.006
3 mRNA EPO 0.1 10 min infusion, 2/C2 /wk 0.02
4 mRNA EPO 0.3 10 min infusion, 2/C2 /wk 0.06
5 mRNA EPO 0.3 10 min infusion, 1/C2 /wk 0.06
6 Empty LNP 0.3 10 min infusion, 2/C2 /wk 0.06
Abbreviations: EPO, erythropoietin; LNP, lipid nanoparticle; PBS, phosphate-
buffered saline.
aNo. of males per group, n24. Dose volume per group, 5 ml/kg. Dose rate
per group, 30 ml/kg/h.
bDose levels in terms of mRNA content. For group No. 6, the dose level is
listed in terms of the same amount of lipid:mRNA ratio (by weight).Table 2. Experimental Design for Safety Study: Monkey.a
Group
No.TestMaterialDose
Level
(mg/kg)
bIntravenous
AdministrationDose
Concentration
(mg/mL)
1 PBS 0 60 min infusion (2/C2 /wk) 0
2 mRNA EPO 0.03 60 min infusion (2/C2 /wk) 0.006
3 mRNA EPO 0.1 60 min infusion (2/C2 /wk) 0.02
4 mRNA EPO 0.3 60 min infusion (2/C2 /wk) 0.06
5 mRNA EPO 0.3 60 min infusion (1/C2 /wk) 0.06
6 Empty LNP 0.3 60 min infusion (2/C2 /wk) 0.06
Abbreviations: EPO, erythropoietin; LNP, lipid nanoparticle; PBS, phosphate-
buffered saline.
aNo. of females per group, n3. Dose volume per group, 5 ml/kg. Dose rate
per group, 5 ml/kg/h.
bDose levels in terms of mRNA content. For group No. 6, the dose level is
listed in terms of the same amount of lipid to mRNA ratio (by weight).Sedic et al 343
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6, and 24 hours after the end of injection/infusion on days 1 and
15 in K 3EDTA tubes and processed to plasma or serum (no
anticoagulant) for IFN- aanalysis.
Female Monkey Study Design
Female monkeys were used for this study, as there was noexpected sex-specific differences in metabolism, distribu-tion, or toxicity. The negative control, test, and referenceitems were administered over the course of 2 weeks in a 60-minute IV infusion via an appropriate peripheral vein (eg,saphenous or brachial) at the dose level, dose volume, and
frequency listed in Table 2. The dose volume for each ani-
mal was based on the most recent body weight measure-ment. The animals were temporarily restrained (on a slingor a chair) for the dose administration and not sedated. Eachinfused dose was administered with a temporary indwellingcatheter inserted in a peripheral vein connected to an injec-tion set and infusion pump. The first day of dosing wasdesignated as day 1. The end points in this study included
clinical signs (including observation of the infusion sites),
body weights, food consumption, PK/PD, clinical pathology(hematology, coagulation, and serum chemistry), macro-and microscopic examination of tissues, and selected cyto-kines (interleukin 1 b[IL-1b], IL-6, TNF- a, and IP-10) and
complement (C3a and C5b-9).
Blood samples were collected from overnight-fasted ani-
mals for hematology, coagulation, and clinical chemistry
parameters at predose (baseline) and on day 16. Addition-
ally, blood was analyzed on day 8 for hematology para-meters only. For PK/PD assessments, blood samples werecollected and processed to plasma at the following timepoints: predose; 2, 6, 24, and 48 hours after the first dose;and 6 hours after subsequent dosing occasions. After pro-cessing, the plasma samples were stored in a freezer set to/C080
/C14C until analyzed. Blood samples were collected in
K3EDTA tubes and processed to plasma for analysis of
cytokines (ie, IL-1 b, IL-6, TNF- a) and complement (ie,
C3a and C5b-9) or to serum for analysis of IFN- aand IP-
10 at the following time points for all groups: predose; at 2,6, and 24 hours after the end of infusion on day 1; and at 2,6, and 24 hours after the end of infusion on day 15. Addi-tionally, for complement analysis only, blood samples werecollected 2, 6, and 24 hours after the end of infusion on day
4 (groups 1–4 and 6).
Clinical Pathology
Hematology parameters were measured with Bayer Advia 120
Automated Hematology Analyzer (Siemens Healthcare). Stan-dard coagulation parameters were measured on a START 4Compact Stago Analyzer (Diagnostica Stago). Standard clini-cal chemistry parameters were measured with Modular Analy-tics (Roche/Hitachi).Histamine, Cytokine and Complement Levels
Histamine levels in the rat plasma were determined with theHistamine EIA Kit (IM-2015; Immunotech). Serum levels ofIFNawere determined with the Rat IFN aELISA Kit (KT-
60242; Kamiya Biomedical Company) and the Human IFN a
Multi-subtype ELISA Kit (41105-1 or 41105-2; PBL Biome-dical Laboratories). IL-6, IP-10, and TNF ain rat plasma were
determined with the Rat Cytokine/Chemokine Magnetic PanelKit (RECYMAG-65K; Millipore). IL1 b, IL-6, and TNF ain
monkey plasma were determined with the Non-Human PrimateCytokine/Chemokine Magnetic Panel Kit (PRCYTOMAG-40K;Millipore). IP-10 in the monkey serum was determined withthe Monkey IP-10 Singleplex Magnetic Kit (LHB0001;Invitrogen). C3 levels in the rat plasma were determined withthe Rat C3 ELISA Kit (GWB-A8B8AF; Genway). C3a levelsin the monkey plasma were determined with the Human C3aEIA Kit (A031; Quidel). C5b-9 levels in the monkey plasma
were determined with the Human C5b-9 ELISA Kit (558315;
BD Bioscience).
Histopathology
Representative samples of the following tissues from all ani-mals were preserved in 10 %neutral buffered formalin: bone
marrow (sternum), heart, infusion site (last dose), kidney, liver,
lung, spleen, and thymus. Tissues were embedded in paraffin,
sectioned, mounted on glass slides, and stained with hematox-ylin and eosin. The histopathologic evaluation was internallypeer reviewed.
hEPO bDNA
The bioanalysis of plasma samples for quantification of hEPO-
mRNA levels was conducted at AxoLabs according to the
bDNA method for mRNA detection developed by QuantiGene(Affymetrix).
26Briefly, plasma samples were directly diluted
in lysis buffer. On each bDNA plate, including a customizedassay-specific set of probes, a dilution curve was pipetted withspiked standards into untreated plasma. Signal amplificationwas carried out with oligonucleotides bound to the enzymealkaline phosphatase. The calculated amount in picograms was
normalized to the amount of plasma in the lysate and to the
amount of lysate applied to the plate. Since measurements inthe PBS-treated control group were within the backgroundlevel range, cross-reactivity of hEPO-mRNA to rat or monkeyEPO mRNA was considered negligible.
hEPO ELISA
hEPO levels were measured with a human EPO SandwichELISA Kit (01630; Stemcell Technologies). For this assay, thelower and upper limits of quantitation were 12 and 800 pg/ml,respectively. Since predose measurements were within thebackground level range, cross-reactivity of hEPO to rat ormonkey EPO was considered negligible.344 Veterinary Pathology 55(2)
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Data Analysis and Reporting
The toxicokinetic parameters of human modified hEPO-
mRNA and its expressed protein in plasma were calculatedwith a noncompartmental approach in WinNonlin Phoenix64, version 6.3 (Pharsight). Dose-normalized maximum serumconcentration (C
max/dose) and area under the curve (AUC/
dose) were determined by dividing the respective parametersby dose and calculated by either WinNonlin or Excel. Themean, standard deviation, and percentage coefficient of varia-
tion of the toxicokinetic parameters were calculated in Win-
Nonlin. All reported values were rounded to either 3 significantfigures or 1 decimal place (time to reach maximum serumconcentration [T
max], half-life [t 1/2]).
Results
Administration of hEPO-mRNA in LNP Results in
Detection of Significant Serum hEPO Levels andCorresponding PD Effects in the Rat and Monkey
Toxicokinetic analysis in the rat revealed that hEPO-mRNA
had a moderate half-life (2.9–5.7 hours) and low clearance(49.0–97.2 ml/h/kg; Fig. 1, Table 3). The C
max/dose values
were consistent among the 4 dose groups, ranging from 2270to 3320 ng/ml/mg/kg (Table 3). Measured hEPO levels were
maximal approximately 6 hours after the 10-minute infusion
(Fig. 2, Table 4). The AUC values (for hEPO-mRNA andhEPO) increased in more than a dose-proportional manner,between 0.03 and 0.3 mg/kg (Table 4). Plasma samples col-lected at 6 hours after each dose indicated that hEPO levelswere constant at C
maxat all dose levels until day 15, when
measured hEPO levels were significantly decreased in the mid-and high-dosed groups (Fig. 3). Consistent with literature data,
1
peak reticulocytosis (PD marker described later) was observedby day 9, and levels remained elevated during the 15-dayperiod. Overall, these results indicate that plasma concentra-tions of hEPO were mostly consistent throughout the study andexhibited greater-than-dose-proportional increases in AUCafter IV administration.
Significant increases were noted in red blood cell and asso-
ciated parameters (hemoglobin, hematocrit) in all male rat
groups dosed with hEPO-mRNA in LNPs as compared with
the PBS group and the group dosed with empty LNPs. Inter-estingly, the changes in red blood cell parameters (except meancorpuscular volume) were similar across all hEPO-mRNA-dosed groups and did not seem to be dose related (Fig. 4, Suppl.Fig. 1). In addition, dose-dependent increases in platelet countsand reticulocytes were noted, particularly at the highest dosesadministered twice weekly (Suppl. Fig. 1). Overall, these
results indicate that repeated administration of hEPO-mRNA
in LNPs achieves physiologically relevant and persistent hEPOlevels that result in significant changes in precursor cells andmature red blood cell count at doses as low as 0.03 mg/kg.
Like in male rats, toxicokinetic findings in female monkeys
indicated that the total exposure (AUC) to hEPO-mRNA and
Figures 1–3. Plasma concentration of hEPO mRNA (ng/ml; Fig. 1)
and hEPO (ng/ml; Figs. 2, 3) in rats. Graphs represent mean values ( n¼
6); error bars indicate SD. Following a 10 minute infusion, peak plasma
concentrations of hEPO mRNA appear to occur at approximately 2
hours, while peak plasma concentrations of hEPO are approximately
at 6 hours. Note that hEPO levels appear constant at all dose levels
until day 15. hEPO, human erythropoietin; Q7D, 1 dose per week.Sedic et al 345
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hEPO increased in a dose-related manner after IV administra-
tion of 0.1 to 0.3 mg/kg/d of hEPO-mRNA in LNPs (Figs. 5, 6;Tables 5, 6). There was a notable difference in hEPO-mRNAexposure after the first dose between the groups administered0.3 mg/kg twice and once per week, which could be due to thesmall sample size and large intragroup variability (Table 5). Inaddition, the results showed that hEPO-mRNA had a relatively
long half-life (5.9–9.3 hours) and low clearance (9.03–27.0 ml/
h/kg; Table 5). After a 60-minute infusion of hEPO-mRNA inLNPs, maximum plasma concentration of hEPO was estimatedto occur between 6 and 24 hours (Table 6). The delay in esti-mated T
maxobserved in group 5 could be a consequence ofTable 3. Toxicokinetic Values for hEPO mRNA in the Rat.a
Dose, mg/kg t 1/2, h Cmax, ng/ml Cmax/Dose, ng/ml AUC, h /C2ng/ml AUC/Dose, h /C2ng/ml Cl, ml/h/kg
0.03 5.7 92.6 3090 309 10,300 97.2
0.1 4.3 227 2270 1460 14,600 68.5
0.3 4 902 3010 5450 18,200 55.1
0.3b2.9 995 3320 6120 20,400 49
Abbreviations: AUC, area under the curve; Cl, clearance; Cmax, maximum serum concentration; hEPO, human erythropoietin; t 1/2, half-life; Tmax, time to reach
maximum serum concentration.
aTmax per dose, 2 hours.
bOne dose per week.
Table 4. Toxicokinetic Values for hEPO in the Rat.a
Dose, mg/kg Cmax, ng/ml AUC, h /C2ng/ml t 1/2,h
0.03 77.1 1590 6.4
0.1 154 4480 8.7
0.3 3540 44 400 6.1
0.3b2340 34 100 6.6
Abbreviations: AUC, area under the curve; Cmax, maximum serum
concentration; hEPO, human erythropoietin; t 1/2, half-life; Tmax, time to reach
maximum serum concentration.
aTmax per dose, 6 hours.
bOne dose per week.
Figure 4. Red blood cell (RBC) mass parameters in rats: RBCs ( /C2106cells/ml), hemoglobin (HGB; g/dl), hematocrit (HCT; %), and erythrocyte
distribution width (RDW; %). Graphs represent mean values ( n¼6); error bars indicate SD. Ordinary one way analysis of variance (multiple
comparisons) was used to calculate the Pvalues. * P< .05. LNP, lipid nanoparticle; PBS, phosphate buffered saline; Q7D, 1 dose per week.346 Veterinary Pathology 55(2)
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increased hEPO-mRNA exposure in this particular group or, as
mentioned before, could be an artifact of high variability within
that group. Also, as in male rats, serum hEPO concentrations at
Cmaxwere well maintained at all dose levels throughout the
study until day 15, when measured hEPO levels significantlydecreased in the mid- and high-dose groups (Fig. 7).
As expected, hEPO expression in monkeys led to significant
changes in red blood cell mass. By day 8 of the study, absolutereticulocyte counts increased in all dose groups (Fig. 8). By theend of the study, a significant spike was noted in all other red
blood cell parameters, such as absolute red blood cells, hema-
tocrit, hemoglobin, and erythrocyte distribution width, whereasreticulocytes returned to baseline levels. Like in rats, theincreases in red blood cell parameters were similar across allgroups and did not seem to be dose related. These changes werenot observed in the group dosed with empty LNPs.
Tolerability of hEPO-mRNA in LNPs in Male Rats
There were no notable clinical observations or body weights/food consumption changes in any group (data not shown).Slight changes were observed in coagulation parameters. Spe-cifically, activated partial thromboplastin time was prolongedin all animals dosed with hEPO-mRNA in LNPs, and pro-thrombin time was prolonged in all animals dosed withhEPO-mRNA twice per week. In addition, fibrinogen levels
were elevated for animals receiving 0.3 mg/kg of hEPO-
mRNA once and twice per week (Suppl. Fig. 2).
Assessment of hematologic parameters on days 9 and 16 of
the study indicated that white blood cells increased at doses/C210.1 mg/kg of hEPO-mRNA in LNPs given twice weekly.
Consistent with this, neutrophil, monocyte, and atypical lym-phocyte counts were elevated across all groups dosed withhEPO-mRNA in LNPs. Interestingly, administration of
hEPO-mRNA in LNPs once weekly or empty LNPs did not
elicit the same increase in white blood cells (Fig. 9).
In addition, histamine release and serum levels of cytokines
were evaluated. IP-10 was elevated 6 to 24 hours postdose ondays 1 and 15 only in the groups dosed with 0.3 mg/kg ofhEPO-mRNA in LNPs once and twice weekly (Fig. 10). Nochanges in IP-10 or histamine were seen in the group givenempty LNPs (Fig. 10 and data not shown). No changes in other
cytokines (IL-6, TNF- a, IFN-a) or complement (C3) were
observed in any study group (data not shown).
Administration of hEPO-mRNA in LNPs at doses /C210.03
mg/kg resulted in several macro- and microscopic findings in
the spleen, bone marrow, liver, lungs, and stomach. Primaryfindings were considered to be related to increased hEPOexpression and included an increase in extramedullary hema-topoiesis in the spleen, liver, and bone marrow (Figs. 11–16).
1
In addition, macroscopic enlargement of the spleen was noted.These findings correspond to the hematologic changes (reticu-locyte counts, red blood cells, and red cell mass parameters).Minimal hemorrhage in the lung and glandular stomach wasnoted at all doses of hEPO-mRNA in LNPs, which correspondsto macroscopic observations of dark foci in these tissues.
Figures 5–7. Plasma concentrations of hEPO mRNA (ng/ml; Fig. 5)
and hEPO (ng/ml; Figs. 6, 7) in monkeys. Graphs represent mean values
(n¼3); error bars indicate SD. Following a 60 minute infusion, peak
plasma concentrations of hEPO mRNA appear to be at 2 hours, whilepeak plasma concentrations of hEPO are approximately at 6 hours.
Note that hEPO levels appear relatively constant at all dose levels until
day 15. hEPO, human erythropoietin; Q7D, 1 dose per week.Sedic et al 347
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Additional findings observed in groups dosed with hEPO-
mRNA in LNPs or empty LNPs included increased mononuc-lear cell infiltration and a minimal to moderate extent of
single-cell necrosis in the liver at doses /C210.1 mg/kg (Figs. 17,
18). At 0.3 mg/kg per dose of hEPO-mRNA, minimal to mild
hypertrophy/hyperplasia of the sinusoidal endothelial cells wasobserved in the liver. These liver findings were accompaniedwith mild elevations in alanine aminotransferase (ALT) andaspartate aminotransferase (AST; Fig. 19). Overall, these datasuggest that doses /C210.1 mg/kg result in minor liver injury that
seems to be primarily driven by the vehicle (LNPs).
Tolerability of hEPO-mRNA in LNPs in Female Monkey
There were no clinical signs or effects on body weights andfood consumption related to hEPO-mRNA in LNPs (data notshown). Mild decreases in phosphorus and albumin levels werenoted only in animals given 0.3 mg/kg of hEPO-mRNA inLNPs once or twice weekly (Suppl. Fig. 3) and were likelyrelated to the mild proinflammatory changes observed.
There were no changes in leukocytes when compared with
PBS in all groups throughout the duration of the study (data not
shown). No significant changes in cytokine release (IL-1 b,
IL-6, TNF- a, IP-10) were observed (data not shown). However,
complement activation (C3a, C5b-9) was detected in the mid-
and high-dose groups given hEPO-mRNA in LNPs once andtwice a week. The magnitude of complement change appears toincrease with repeat dosing; significant changes were observedat 2 to 6 hours postdosing on day 1, 6 hours postdosing on day4, and at 2 to 6 hours postdosing on day 15. By day 15 (2–6
hour postdosing), a mild but not statistically significant trend in
complement activation was observed in all groups dosed withhEPO-mRNA in LNPs as well as empty LNPs (Fig. 20).
Macro- and microscopic pathology findings were minimal
and mainly present in the heart and spleen. Minimal hemor-rhage was noted in the heart at doses /C210.1 mg/kg and was
consistent with suprapharmacologic effects of hEPO.
1Findings
in the spleen were primarily related to administration of the
drug product (hEPO-mRNA in LNPs) once weekly at 0.3 mg/
kg and included minimal lymphoid depletion in the periarter-iolar lymphoid sheaths in the white pulp of the spleen in 3animals, as well as mild multifocal white pulp necrosis anddecreased cellularity of the red pulp in only 1 animal (data notshown).
Discussion
The ability to deliver therapeutic levels of proteins with
modified mRNA opens the door to potential life-saving
therapies in various indications. The modified mRNA plat-form has the potential to enable the development of singleas well as combination therapeutic agents. However, likesiRNA, mRNA is a labile biological molecule and thereforerequires the use of protective delivery systems to effectivelyharness its potential. Indeed, systemic administration ofunformulated and unmodified RNA molecules leads to
degradation by RNAses, rapid renal clearance, and potential
stimulation of an immune response, resulting in very shorthalf-lives (<5 minutes postdosing).
5,11,16,27Therefore, LNPs
were utilized in our studies to improve the PK profile andcellular uptake of mRNA.
10
In our studies, plasma AUC of hEPO-mRNA increased in a
dose-proportional manner in monkeys and in a greater-than-dose-proportional manner in rats. In addition, hEPO-mRNA
had moderate to long half-life and relatively low clearance in
both species. Finally, hEPO expression resulted in suprather-apeutic levels ( *100-fold the projected clinically efficacious
dose) in rats and monkeys.
24The protein produced had
expected PK and PD properties (t 1/2) as evidenced by exagger-
ated pharmacologic effects associated with supratherapeuticTable 5. Toxicokinetic Values for hEPO mRNA in the Monkey.a
Cmax, ng/mL AUC, h /C2ng/ml t1/2, h Cl, mL/h/kg
Dose, mg/kg Mean SD Mean Cmax/Dose, ng/mL Mean SD Mean AUC/Dose, h /C2ng/ml Mean SD Mean SD
0.03 715 379 23 800 4090 1870 136 000 5.89 0.554 9.03 5.57
0.1 882 904 8820 4440 3140 44 400 9.31 8.12 27 13
0.3 3270 2620 10 900 19 900 15 400 66 300 6.99 1.22 21.3 12.8
0.3b9240 1860 30 800 49 900 13 600 166 000 7.36 6.51 6.33 1.87
Abbreviations: AUC, area under the curve; Cl, clearance; Cmax, maximum serum concentration; hEPO, human erythropoietin; t 1/2, half-life; Tmax, time to reach
maximum serum concentration.
aMean Tmax per dose, 2 hours.
bOne dose per week.
Table 6. Toxicokinetic Values for hEPO in the Monkey.
Cmax, ng/ml AUC, h /C2ng/ml
Dose, mg/kg Mean Tmax, h Mean SD Mean SD
0.03 6 30.6 7.42 600 39.8
0.1 6 210 187 4240 2530
0.3 6 283 111 6660 915
0.3a24 253 49.4 8170 1550
Abbreviations: AUC, area under the curve; Cmax, maximum serum
concentration; hEPO, human erythropoietin; Tmax, time to reach maximumserum concentration.
aOne dose per week.348 Veterinary Pathology 55(2)
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doses of hEPO observed in both species (increased red blood
cell mass parameters; mild hemorrhages in the heart, stomach,
and lungs; extramedullary hematopoiesis in the liver andspleen; and increased hematopoiesis in bone marrow). These
data suggest that hEPO-mRNA (and putatively modified
mRNA in general) administered in LNPs has acceptable
Figure 8. Red blood cell (RBC) mass parameters in monkeys: RBCs ( /C2106cells/ml), hemoglobin (HGB; g/dl), hematocrit (HCT; %), erythrocyte
distribution width (RDW; %), and reticulocytes (RETIC; /C2109cells/ml). Graphs represent mean values ( n¼3); error bars indicate SD. Ordinary
one way analysis of variance (multiple comparisons) was used to calculate the Pvalues. * P< .05. LNP, lipid nanoparticle; PBS, phosphate buffered
saline; Q7D, 1 dose per week.Sedic et al 349
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Figure 9. Leukocyte parameters in rats: white blood cells (WBC; /C2103cells/mL), neutrophils (NEUT; /C2103cells/mL), monocytes (MONO;
/C2103/mL), and large unstained cells (LUC; /C2103/mL). Graphs represent mean values ( n¼6); error bars indicate SD. Ordinary one way analysis of
variance (multiple comparisons) was used to calculate the Pvalues. * P< .05. LNP, lipid nanoparticle; PBS, phosphate buffered saline; Q7D, 1 dose
per week.
Figure 10. Plasma interferon g–inducible protein 10 (IP 10) levels in rats. Graph represents mean values ( n¼6); error bars indicate SD.
Ordinary one way analysis of variance (multiple comparisons) was used to calculate the Pvalues. * P< .05. LNP, lipid nanoparticle; PBS,
phosphate buffered saline; Q7D, 1 dose per week.350 Veterinary Pathology 55(2)
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properties for a drug product at least when administered over a
2-week period.
A marked decrease of the plasma concentration of hEPO
was observed by day 15 in the mid- and high-dose groups inboth species. This reduction in hEPO levels may have beenmediated by antibodies against the expressed protein sinceanimals were exposed to a nonhomologous protein.
4,8To fur-
ther investigate for the presence of antiprotein antibodies, wehave subsequently developed appropriate bioanalytical meth-ods, and the data collected from subsequent in vivo studiesexploring the levels of species-specific anti-hEPO antibodiessupport this hypothesis (data not shown).
Figures 11–16. Histologic findings in control rats and in rats dosed with 0.3 mg/kg of hEPO mRNA twice a week. Hematoxylin and eosin.
Figure 11. Liver, control rat. Figure 12. Liver, rat dosed with hEPO mRNA. There is extramedullary hematopoiesis. Figure 13. Spleen,
control rat. Figure 14. Spleen, rat dosed with hEPO mRNA. There is increased extramedullary hematopoiesis. Figure 15. Bone marrow
(sternal), control rat. Figure 16. Bone marrow (sternal), rat dosed with hEPO mRNA. There is increased hematopoiesis. hEPO, human
erythropoietin.Sedic et al 351
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From a safety perspective, modified mRNA delivered in
LNPs is a complex molecule that requires the assessment of
safety of the delivery system (LNPs), mRNA, and the trans-lated protein product. Overall, administration of 0.3 mg/kg ofLNPs alone via IV infusion was well tolerated. In male rats,increases in neutrophils and monocytes were observed, and
microscopic changes in liver were noted that correlated with
mild and monitorable increases in liver enzymes (ALT andAST). In female monkeys, only minimal and transient comple-ment activation was induced by LNPs alone. Similar changeshave been reported for LNP vehicle.
3
IV infusion of hEPO-mRNA in LNPs was well tolerated in
both species. A mild and reversible proinflammatory profilewas noted that consisted of elevations in IP-10 (rat) and detec-
tion of complement cleavage products (monkey). These find-
ings correlated with slight changes in both species in terms ofhematology parameters (white blood cell counts, lymphocytes,neutrophils, and monocytes) and coagulation parameters (acti-vated partial thromboplastin time, prothrombin time, and fibri-nogen), although more consistently observed in the rat. In therat, liver microscopic changes with elevations of serum ALTand/or AST were similar to those observed in the group dosedwith empty LNPs. In addition, changes in the spleen (depletionin the periarteriolar lymphoid sheaths and splenic necrosis)were observed in monkeys. These findings suggest that theadministration of mRNA in LNPs at doses as high as 0.3 mg/
kg per dose induces a mild to moderate and reversible proin-
flammatory response. Furthermore, it is evident that thisimmune activation can be mitigated by either lowering the doseor decreasing the frequency of dosing.
These findings (serum complement activation, cytokine ele-
vation, and potential liver and spleen effects) are in agreementwith those previously reported for LNPs loaded with siRNA innonclinical and clinical studies.
3,6Comparable toxicologic pro-
files of modified siRNA and mRNA in LNPs suggest that thetoxicologic effects and possibly the distribution properties (pri-marily liver and monocyte phagocytic system) of the drugproduct are predominantly vehicle driven. Note that an emptyLNP may have slightly different surface properties than the oneloaded with mRNA; thus, small differences in magnitude or
Figures 17 and 18. Histologic findings in control rat and in rat dosed with 0.3 mg/kg of empty lipid nanoparticles twice a week. Figure 17.
Liver, control rat. Figure 18. Liver, rat dosed with empty empty lipid nanoparticles. There is single cell necrosis of hepatocytes.
Figure 19. Serum levels of liver enzymes in rats. Graphs represent mean values ( n¼6); error bars indicate SD. Ordinary one way analysis of
variance (multiple comparisons) was used to calculate the Pvalues. *P < .05. ALT, alanine aminotransferase; AST, aspartate aminotransferase;
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effects may occur.28Therefore, a preferred control instead of
the empty LNPs would be LNPs loaded with nonsense mRNA
with equivalent base modifications.
In conclusion, the PK, PD, and toxicologic properties of
modified mRNA loaded in LNPs are generally consistentbetween species. The primary toxicologic findings ofhEPO-mRNA in LNPs are related to the supratherapeuticexposure to hEPO and inflammatory findings that aremainly LNP driven. Given the similarities in LNP-relatedtoxicities between the rat and the monkey, it is likely thatsimilar effects will translate to the clinic and be monitorablewith the parameters identified in nonclinical species.
Although the studies were not conducted to support
advancement of a clinical candidate, they suggest that themRNA-in-LNP approach might be feasible to safely delivertherapeutic levels of an exogenous protein. This wouldenable novel therapies for a variety of indications. Futurework will be geared toward evaluating different routes ofadministration, the effects of chronic dosing, and the risk tojuvenile animals, as juveniles may be particularly important
in the setting of rare disease.Acknowledgements
We thank everyone involved in execution, analysis, and discussion of
these studies at Charles River Laboratories, Moderna, AxoLabs, and
AstraZeneca.
Declaration of Conflicting Interests
The author(s) declared the following potential conflicts of interest with
respect to the research, authorship, and/or publication of this article: Allauthors except Stefan J. Platz are employed by Moderna or were employed
by Moderna at the time when these studies were conducted. Stefan J. Platz
is employed by AstraZeneca, Moderna’s collaborator on the project.
Funding
The author(s) received the following financial support for the
research, authorship, and/or publication of this article: Moderna and
AstraZeneca provided financing for this project.
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