Original Article
`
`Safety Evaluation of Lipid Nanoparticle–
`Formulated Modified mRNA in the Sprague-
`Dawley Rat and Cynomolgus Monkey
`
`Veterinary Pathology
`1-14
`ª The Author(s) 2017
`Reprints and permission:
`sagepub.com/journalsPermissions.nav
`DOI: 10.1177/0300985817738095
`journals.sagepub.com/home/vet
`
`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 after
`repeat 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 the
`first 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.24 hEPO 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 findings
`were caused by the exaggerated pharmacology of hEPO and included increased hematopoiesis in the liver, spleen, and bone
`marrow (rats) and minimal hemorrhage in the heart (monkeys). Additional primary safety-related findings in the rat included
`mildly increased white blood cell counts, changes in the coagulation parameters at all doses, as well as liver injury and release of
`interferon 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 indicate
`that 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 the
`treatment of diabetes mellitus.17 Protein expression directed by
`exogenous mRNA offers many advantages over other nucleic
`acid–based concepts, as well as recombinant proteins. Potential
`advantages of mRNA over DNA-based technology include (1)
`no integration into the host genome thereby circumventing the
`risk of deleterious chromosomal changes, and (2) faster and
`more efficient expression with proper modifications, since
`mRNA therapeutics only require access to the cytoplasm. In
`comparison with recombinant proteins, mRNA would have
`lower manufacturing costs and could enable access to intracel-
`lular as well as cell membrane–bound therapeutic targets. The
`biggest challenges of mRNA technology are its potential for
`immunogenicity and its relatively poor in vivo stability. These
`challenges have been addressed through progress in chemistry
`and sequence engineering (eg, optimization of the 50 cap, 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, are
`partially or fully replaced with nucleosides, such as pyrimidine
`nucleosides—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: Joe.Senn@modernatx.com
`
`ARBUTUS - EXHIBIT 2026
`Moderna Therapeutics, Inc. v. Arbutus Biopharma Corporation
`IPR2019-00554
`
`

`

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`
`Veterinary Pathology XX(X)
`
`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.20 Incor-
`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 molecular
`pattern receptors, such as toll-like receptors, retinoic acid–
`inducible gene 1, melanoma differentiation-associated protein
`5, 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 by
`the 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 delivery
`systems, 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 formulated
`in either LNPs or nanoemulsion induced immune protection
`from influenza and respiratory syncytial virus in mice, as well
`as cytomegalovirus and respiratory syncytial virus in mon-
`keys.9 Furthermore, a single administration of modified
`mRNA-LNP complexes in mice by various routes resulted
`in high, sustained protein production.19 Finally, Thess
`et al25 reported that repeated administration of unmodified
`mRNA in combination with the nonliposomal polymeric
`delivery system (TransIT) induced high systemic protein lev-
`els and strong physiologic responses in mice. These authors
`also noted similar observations following single-dose admin-
`istration of erythropoietin (EPO)–mRNA in LNPs to pigs and
`monkeys.
`LNPs have been reported to be clinically effective for the
`delivery of siRNA.6 The LNP vehicle is currently in late-phase
`clinical trials of a synthetic siRNA in patients suffering from
`transthyretin amyloidosis and has been well tolerated in this
`population.6 Therefore, considerable work has been done to
`understand the safety profile of systemic administration of
`siRNA-LNPs.3 Here, 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 (Montreal
`and Sherbrooke, Canada). During the study, the care and use of
`animals were conducted according to the guidelines of the US
`National 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. The
`temperature of the animal room was kept between 19C and
`25C, 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 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 via
`an automatic watering system. Environmental enrichment was
`provided to animals per standard operating procedures of
`Charles River Laboratories (Montreal, Canada), except during
`study 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 temperature
`of the animal room was kept between 20C and 26C, 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 to
`animals per standard operating procedures of Charles River
`Laboratories (Montreal, Canada) except during study proce-
`dures and activities.
`
`Control, Test, and Reference Items
`
`An 850-nucleotide messenger RNA was prepared by in vitro
`transcription from a linearized DNA template with T7 RNA
`Polymerase. The DNA template encoded the T7 promoter, a 50
`untranslated region, the 579-nucleotide open reading frame
`encoding human EPO (hEPO) mature protein with signal
`sequence, a 30untranslated region, and a polyadenylated tail.
`The in vitro transcription was performed with the canonical
`nucleotides adenosine triphosphate and guanosine triphosphate
`and the modified nucleotides 1-methylpseudouridine tripho-
`sphate and 5-methylcytidine triphosphate. The mRNA contains
`a 50 Cap 1 structure, which consisted of 7-methylguanosine
`linked to the 50 nucleoside of the mRNA chain through a 50–
`50 triphosphate bridge and 20-O-methyl group present on the
`first nucleotide of the mRNA.23 The messenger RNA was pur-
`ified and buffer exchanged into low ionic strength buffer for
`formulation.18 The 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.13,30 The LNP formulation was prepared by
`dissolving the lipids (6Z,9Z,28Z,31Z)-heptatriaconta-
`6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3),
`
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`Sedic et al
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`3
`
`Table 1. Experimental Design for Safety Study: Rat.a
`
`Table 2. Experimental Design for Safety Study: Monkey.a
`
`Dose
`Level,
`mg/kgb
`
`Test
`Material
`
`Intravenous
`Administration
`10-min infusion, 2/wk
`PBS
`0
`mRNA EPO 0.03 10-min infusion, 2/wk
`10-min infusion, 2/wk
`mRNA EPO 0.1
`10-min infusion, 2/wk
`mRNA EPO 0.3
`10-min infusion, 1/wk
`mRNA EPO 0.3
`10-min infusion, 2/wk
`Empty LNP
`0.3
`
`Dose
`Concentration
`mg/ml
`
`Group
`No.
`
`Test
`Material
`
`Dose
`Level
`(mg/kg)b
`
`0
`0.006
`0.02
`0.06
`0.06
`0.06
`
`1
`2
`3
`4
`5
`6
`
`0
`PBS
`mRNA EPO 0.03
`mRNA EPO 0.1
`mRNA EPO 0.3
`mRNA EPO 0.3
`Empty LNP
`0.3
`
`Intravenous
`Administration
`60-min infusion (2/wk)
`60-min infusion (2/wk)
`60-min infusion (2/wk)
`60-min infusion (2/wk)
`60-min infusion (1/wk)
`60-min infusion (2/wk)
`
`Dose
`Concentration
`(mg/mL)
`
`0
`0.006
`0.02
`0.06
`0.06
`0.06
`
`Group
`No.
`
`1
`2
`3
`4
`5
`6
`
`Abbreviations: EPO, erythropoietin; LNP, lipid nanoparticle; PBS, phosphate-
`buffered saline.
`aNo. of males per group, n ¼ 24. 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).
`
`Abbreviations: EPO, erythropoietin; LNP, lipid nanoparticle; PBS, phosphate-
`buffered saline.
`aNo. of females per group, n ¼ 3. 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).
`
`1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), choles-
`terol, and 1,2-dimyristoyl-rac-glycerol, methoxypolyethylene
`glycol (PEG2000-DMG) in ethanol. The 4 lipids were prepared
`as 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 was
`mixed 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 was
`filtered through a clarification filter (0.8/0.2 mm nominal).
`Prior to storage, the formulation was additionally filtered
`through 2 in-line sterile filters (0.2 mm) and aseptically filled
`into sterilized vials, stoppered, and capped. Empty LNPs were
`generated 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 lipids
`in the final mRNA-LNPs and empty LNPs were 22.0 and
`13.1 mg/ml, respectively. The final mRNA content in hEPO
`LNPs was quantified by ultraviolet analysis, resulting in an
`mRNA 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). Particle
`hydrodynamic diameters were determined by dynamic light
`scattering. Resulting diameters for mRNA and empty LNPs
`were 81 nm (0.08 polydispersity index) and 61 nm (0.10 poly-
`dispersity index), respectively. Total mRNA encapsulation was
`quantified 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 were
`
`administered 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 each
`study were based on previous pharmacology data demonstrat-
`ing production of efficacious levels of hEPO in the rat and
`cynomolgus monkey at doses 0.03 mg/kg of mRNA. Based
`on pharmacokinetic (PK) data indicating predictable increases
`in protein expression with dose, the mid- and high doses for
`these 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 a
`similar 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 injection
`set and infusion pump. The animals were temporarily
`restrained for the dose administration and not sedated. The dose
`volume 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 males
`per group for PK / pharmacodynamic (PD) assessment. The
`following end points were evaluated: clinical signs (including
`observations 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 for
`hematology, coagulation, and clinical chemistry on day 16 (at
`necropsy). 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 1
`and 15. After processing, the plasma samples were stored in a
`freezer set to maintain 80C 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,
`
`

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`
`Veterinary Pathology XX(X)
`
`6, and 24 hours after the end of injection/infusion on days 1 and
`15 in K3EDTA tubes and processed to plasma or serum (no
`anticoagulant) for IFN-a analysis.
`
`Female Monkey Study Design
`
`Female monkeys were used for this study, as there was no
`expected sex-specific differences in metabolism, distribu-
`tion, or toxicity. The negative control, test, and reference
`items 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 sling
`or a chair) for the dose administration and not sedated. Each
`infused dose was administered with a temporary indwelling
`catheter inserted in a peripheral vein connected to an injec-
`tion set and infusion pump. The first day of dosing was
`designated 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 were
`collected and processed to plasma at the following time
`points: 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
`80C until analyzed. Blood samples were collected in
`K3EDTA tubes and processed to plasma for analysis of
`cytokines (ie, IL-1b, IL-6, TNF-a) and complement (ie,
`C3a and C5b-9) or to serum for analysis of IFN-a and 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 were
`collected 2, 6, and 24 hours after the end of infusion on day
`4 (groups 1–4 and 6).
`
`Histamine, Cytokine and Complement Levels
`
`Histamine levels in the rat plasma were determined with the
`Histamine EIA Kit (IM-2015; Immunotech). Serum levels of
`IFNa were determined with the Rat IFNa ELISA Kit (KT-
`60242; Kamiya Biomedical Company) and the Human IFNa
`Multi-subtype ELISA Kit (41105-1 or 41105-2; PBL Biome-
`dical Laboratories). IL-6, IP-10, and TNFa in rat plasma were
`determined with the Rat Cytokine/Chemokine Magnetic Panel
`Kit (RECYMAG-65K; Millipore). IL1b, IL-6, and TNFa in
`monkey plasma were determined with the Non-Human Primate
`Cytokine/Chemokine Magnetic Panel Kit (PRCYTOMAG-40K;
`Millipore). IP-10 in the monkey serum was determined with
`the Monkey IP-10 Singleplex Magnetic Kit (LHB0001;
`Invitrogen). C3 levels in the rat plasma were determined with
`the Rat C3 ELISA Kit (GWB-A8B8AF; Genway). C3a levels
`in the monkey plasma were determined with the Human C3a
`EIA 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 internally
`peer 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).26 Briefly, plasma samples were directly diluted
`in lysis buffer. On each bDNA plate, including a customized
`assay-specific set of probes, a dilution curve was pipetted with
`spiked standards into untreated plasma. Signal amplification
`was carried out with oligonucleotides bound to the enzyme
`alkaline 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 in
`the PBS-treated control group were within the background
`level range, cross-reactivity of hEPO-mRNA to rat or monkey
`EPO mRNA was considered negligible.
`
`Clinical Pathology
`
`hEPO ELISA
`
`Hematology parameters were measured with Bayer Advia 120
`Automated Hematology Analyzer (Siemens Healthcare). Stan-
`dard coagulation parameters were measured on a START 4
`Compact Stago Analyzer (Diagnostica Stago). Standard clini-
`cal chemistry parameters were measured with Modular Analy-
`tics (Roche/Hitachi).
`
`hEPO levels were measured with a human EPO Sandwich
`ELISA Kit (01630; Stemcell Technologies). For this assay, the
`lower and upper limits of quantitation were 12 and 800 pg/ml,
`respectively. Since predose measurements were within the
`background level range, cross-reactivity of hEPO to rat or
`monkey EPO was considered negligible.
`
`

`

`Sedic et al
`
`5
`
`Data Analysis and Reporting
`
`The toxicokinetic parameters of human modified hEPO-
`mRNA and its expressed protein in plasma were calculated
`with a noncompartmental approach in WinNonlin Phoenix
`64, version 6.3 (Pharsight). Dose-normalized maximum serum
`concentration (Cmax/dose) and area under the curve (AUC/
`dose) were determined by dividing the respective parameters
`by dose and calculated by either WinNonlin or Excel. The
`mean, 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 significant
`figures or 1 decimal place (time to reach maximum serum
`concentration [Tmax], half-life [t1/2]).
`
`Results
`
`Administration of hEPO-mRNA in LNP Results in
`Detection of Significant Serum hEPO Levels and
`Corresponding 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 Cmax/dose values
`were consistent among the 4 dose groups, ranging from 2270
`to 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 and
`hEPO) 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 levels
`were constant at Cmax at 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 observed
`by day 9, and levels remained elevated during the 15-day
`period. Overall, these results indicate that plasma concentra-
`tions of hEPO were mostly consistent throughout the study and
`exhibited greater-than-dose-proportional increases in AUC
`after 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 mean
`corpuscular 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 counts
`and reticulocytes were noted, particularly at the highest doses
`administered twice weekly (Suppl. Fig. 1). Overall, these
`results indicate that repeated administration of hEPO-mRNA
`in LNPs achieves physiologically relevant and persistent hEPO
`levels that result in significant changes in precursor cells and
`mature 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 (n ¼
`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.
`
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`Veterinary Pathology XX(X)
`
`Table 3. Toxicokinetic Values for hEPO-mRNA in the Rat.a
`
`Dose, mg/kg
`
`t1/2, h
`
`Cmax, ng/ml
`
`Cmax/Dose, ng/ml
`
`AUC, h  ng/ml
`
`AUC/Dose, h  ng/ml
`
`Cl, ml/h/kg
`
`0.03
`0.1
`0.3
`0.3b
`
`5.7
`4.3
`4
`2.9
`
`92.6
`227
`902
`995
`
`3090
`2270
`3010
`3320
`
`309
`1460
`5450
`6120
`
`10,300
`14,600
`18,200
`20,400
`
`97.2
`68.5
`55.1
`49
`
`Abbreviations: AUC, area under the curve; Cl, clearance; Cmax, maximum serum concentration; hEPO, human erythropoietin; t1/2, 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
`AUC, h  ng/ml
`
`Cmax, ng/ml
`
`Dose, mg/kg
`
`0.03
`0.1
`0.3
`0.3b
`
`77.1
`154
`3540
`2340
`
`1590
`4480
`44 400
`34 100
`
`t1/2, h
`
`6.4
`8.7
`6.1
`6.6
`
`Abbreviations: AUC, area under the curve; Cmax, maximum serum
`concentration; hEPO, human erythropoietin; t1/2, half-life; Tmax, time to reach
`maximum serum concentration.
`aTmax per dose, 6 hours.
`bOne dose per week.
`
`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-mRNA
`exposure after the first dose between the groups administered
`0.3 mg/kg twice and once per week, which could be due to the
`small sample size and large intragroup variability (Table 5). In
`addition, 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 in
`LNPs, maximum plasma concentration of hEPO was estimated
`to occur between 6 and 24 hours (Table 6). The delay in esti-
`mated Tmax observed in group 5 could be a consequence of
`
`Figure 4. Red blood cell (RBC) mass parameters in rats: RBCs (106 cells/ml), hemoglobin (HGB; g/dl), hematocrit (HCT; %), and erythrocyte
`distribution width (RDW; %). Graphs represent mean values (n ¼ 6); error bars indicate SD. Ordinary one-way analysis of variance (multiple
`comparisons) was used to calculate the P values. *P < .05. LNP, lipid nanoparticle; PBS, phosphate-buffered saline; Q7D, 1 dose per week.
`
`

`

`Sedic et al
`
`7
`
`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
`Cmax were well maintained at all dose levels throughout the
`study until day 15, when measured hEPO levels significantly
`decreased 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, absolute
`reticulocyte counts increased in all dose groups (Fig. 8). By the
`end 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, whereas
`reticulocytes returned to baseline levels. Like in rats, the
`increases in red blood cell parameters were similar across all
`groups and did not seem to be dose related. These changes were
`not 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 prolonged
`in all animals dosed with hEPO-mRNA in LNPs, and pro-
`thrombin time was prolonged in all animals dosed with
`hEPO-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
`0.1 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 with
`hEPO-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 on
`days 1 and 15 only in the groups dosed with 0.3 mg/kg of
`hEPO-mRNA in LNPs once and twice weekly (Fig. 10). No
`changes in IP-10 or histamine were seen in the group given
`empty 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 0.03
`mg/kg resulted in several macro- and microscopic findings in
`the spleen, bone marrow, liver, lungs, and stomach. Primary
`findings were considered to be related to increased hEPO
`expression 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 was
`noted at all doses of hEPO-mRNA in LNPs, which corresponds
`to 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
`(n ¼ 3); error bars indicate SD. Following a 60-minute infusion, peak
`plasma concentrations of hEPO-mRNA appear to be at 2 hours, while
`peak 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.
`
`

`

`8
`
`Veterinary Pathology XX(X)
`
`Table 5. Toxicokinetic Values for hEPO-mRNA in the Monkey.a
`AUC, h  ng/ml
`
`Cmax, ng/mL
`
`Dose, mg/kg Mean
`
`SD
`
`Mean Cmax/Dose, ng/mL Mean
`
`SD
`
`t1/2, h
`Mean AUC/Dose, h  ng/ml Mean
`
`SD
`
`Cl, mL/h/kg
`
`Mean
`
`SD
`
`0.03
`0.1
`0.3
`0.3b
`
`715
`882
`3270
`9240
`
`379
`904
`2620
`1860
`
`23 800
`8820
`10 900
`30 800
`
`4090
`4440
`19 900
`49 900
`
`1870
`3140
`15 400
`13 600
`
`136 000
`44 400
`66 300
`166 000
`
`5.89
`9.31
`6.99
`7.36
`
`0.554
`8.12
`1.22
`6.51
`
`9.03
`27
`21.3
`6.33
`
`5.57
`13
`12.8
`1.87
`
`Abbreviations: AUC, area under the curve; Cl, clearance; Cmax, maximum serum concentration; hEPO, human erythropoietin; t1/2, 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.
`AUC, h  ng/ml
`
`Cmax, ng/ml
`
`Dose, mg/kg Mean Tmax, h Mean
`
`SD
`
`Mean
`
`SD
`
`0.03
`0.1
`0.3
`0.3a
`
`6
`6
`6
`24
`
`30.6
`210
`283
`253
`
`7.42
`187
`111
`49.4
`
`600
`4240
`6660
`8170
`
`39.8
`2530
`915
`1550
`
`Abbreviations: AUC, area under the curve; Cmax, maximum serum
`concentration; hEPO, human erythropoietin; Tmax, time to reach maximum
`serum concentration.
`aOne dose per week.
`
`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 0.1 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 was
`observed in the liver. These liver findings were accompanied
`with mild elevations in alanine aminotransferase (ALT) and
`aspartate aminotransferase (AST; Fig. 19). Overall, these data
`suggest that doses 0.1 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