Poster #281

Figure 5. Repeated dosing of MRT5201 shows sustained effects of targeted CO-hOTC mRNA therapy in OTCspf-ash miceOTCspf-ash or WT mice received one to five IV doses of buffer or 0.10 mg/kg MRT5201 and were challenged with NH4Cl on Day 2, 16, 30, 44 or 58 (n=8 mice/group). Urine samples were collected 24 h following each dose on Days 2, 16, 30, 44 and 58, and prior to NH4Cl challenge. Blood and liver samples were harvested 40 min after NH4Cl challenge. (A) RT-qPCR results of hOTC mRNA detected in the liver. Data represent copies of CO-hOTC mRNA per μg of total RNA. (B) ELISA results of hOTC protein detected in the liver. Data shown as ng-hOTC per mg of total protein. (C) OTC activity in the liver. Data shown as Log10 μmol citrulline/h/mg total protein. (D) Blood ammonia levels depicted as μmol/L of NH3. (E) Urinary orotic acid levels depicted as μmol orotic acid (OA)/mmol creatinine. (A-E) ***p < 0.001, **p < 0.01, *p < 0.05 compared to untreated animals.

SIMD 2019 (Society for Inherited Metabolic Disorders), April 6-9, Bellevue, WA

TREATMENT OF ORNITHINE TRANSCARBAMYLASE DEFICIENCY WITH A TARGETED mRNA THERAPEUTIC (MRT)Kim Askew, Shrirang Karve, Anusha Dias, Lianne Smith, Jonathan Abysalh, Zarna Patel, Kate Lane, Mike Heartlein, Frank DeRosaTranslate Bio, Lexington, Massachusetts

Introduction• Ornithine transcarbamylase deficiency (OTCD) is a rare, X-linked

disorder characterized by complete or partial lack of the enzyme ornithine transcarbamylase (OTC), a component of the urea cycle.

• MRT5201 is a codon-optimized human OTC (hOTC) mRNA encapsulated in lipid nanoparticles (LNPs) and formulated for intravenous (IV) administration.

• IV administration of MRT5201 in mice leads to deposition of hOTC mRNA in the liver where the endogenous cellular machinery translates the mRNA to produce functional OTC enzyme restoring deficits in the urea cycle.

• MRT5201 is designed for biweekly IV administration.

Objective• To demonstrate the ability of LNPs to deliver hOTC mRNA to the liver restoring

functional OTC enzyme activity.

Methods and Materials• A widely accepted mouse model of OTCD, B6EiC3Sn a/A-OTC sparse fur –

abnormal skin and hair (OTCspf-ash), was used to test the efficacy of MRT5201.

• An ammonium chloride (NH4Cl) challenge in the OTCspf-ash mice leads to elevated blood ammonia (NH3) levels.

• Mice were treated by IV administration of MRT5201 and then subjected to NH4Cl challenge.

• Blood NH3 levels were analyzed using an IDEXX Catalyst Dx analyzer.

• hOTC mRNA and protein were measured by RT-qPCR and ELISA, respectively.

• OTC enzyme activity was determined by measuring the conversion of carbamoyl phosphate and ornithine to citrulline.

• Urinary orotic acid, a biomarker for OTCD, was measured using Rapid-Fire mass spectrometry.

Dose-Dependent Expression of hOTC and Protection from Hyperammonemia Following NH4Cl Challenge

Durability of Efficacy Following Repeated Dosing with MRT5201

Results

Figure 3. MRT5201 drives functional hOTC expression in the liver of OTCspf-ash miceOTCspf-ash mice received a single IV injection of MRT5201 at a dose of 0.5, 0.16, 0.05, or 0.016 mg/kg (n=6 mice/group). Controls included WT and untreated OTCspf-ash mice (n=6 mice per group) that received a sham injection with buffer. NH4Cl challenge was performed 24 h post-treatment and urine samples were collected prior to NH4Cl challenge. Blood samples were harvested 40 min after NH4Cl challenge and livers were harvested 24 h after NH4Cl challenge. (A) RT-qPCR results of hOTC mRNA detected in the liver. Data represent copies of CO-hOTC mRNA per µg of total RNA. (B) ELISA results of hOTC protein detected in the liver. Data represent ng-hOTC per mg of total protein. (C) OTC enzymatic activity in the liver. Data represent µmol citrulline/h/mg total protein. (D) Blood ammonia levels depicted in µmol/L. (E) Urinary orotic acid levels depicted as µmol orotic acid (OA)/mmol creatinine. (A-E) ***p < 0.001, **p < 0.01 compared to untreated animals.

Summary and Conclusions• A codon-optimized hOTC mRNA formulated in LNPs was efficiently delivered to the liver of OTCspf-ash mice following IV administration.

• Functional OTC enzyme activity protected OTCspf-ash mice from hyperammonemia following an NH4Cl challenge in a dose-dependent manner.

• A single IV dose of 0.15 mg/kg was sufficient to protect against hyperammonemia for up to 4 weeks.

• Efficacy was maintained following repeat dosing of MRT5201.

fig. 2fig.1

Figure 1. Levels of hOTC Protein Following a Single IV Dose of MRT5201Expression of hOTC protein following a single dose of MRT5201. CD-1 mice (n=40) received a single IV tail-vein injection of MRT5201 at a dose of 0.50 mg/kg on Day 1 and were then harvested at the indicated time points to determine hOTC protein levels by ELISA. Control mice received no treatment and were euthanized on day 2 of the study (NT, n=4). Protein levels are graphed as ng hOTC/mg total protein.

Figure 2. Blood Ammonia Levels Post Ammonium Chloride ChallengeWild-type C57BL/6 mice (n=8) and OTCspf-ash mice (n=24) were given a single IP bolus injection of 5mmol/kg NH4Cl, and blood samples were taken at the indicated times. Data represent blood ammonia levels in OTCspf-ash (red squares) or WT mice (blue circles). NH3 levels are graphed as μmol/L. ***p < 0.001, **p < 0.01 compared to WT.

In vivo hOTC Protein ProductionNH4Cl Challenge in OTCspf-ash Mice Leads to Elevated Blood NH3 Levels

Figure 4. MRT5201 yields durable levels of functional hOTC expression in the liver of OTCspf-ash miceOTCspf-ash mice were dosed with MRT5201 at 0.15 mg/kg IV, and then challenged with NH4Cl at 1, 2, 3, or 4 weeks post dosing (n=6 mice per group). Controls included WT and untreated OTCspf-ash mice (n=6 mice per group) that received a sham injection with buffer. Urine samples were collected prior to NH4Cl challenge. Blood and liver samples were harvested 40 min post NH4Cl challenge. (A) RT-qPCR results of hOTC mRNA detected in the liver. Data represent copies of CO-hOTC mRNA per µg of total RNA. (B) ELISA results of hOTC protein detected in the liver. The level of protein in the control groups (WT and untreated) was below the lower limit of quantitation of the assay. Data are shown as ng-hOTC per mg of total protein. (C) OTC enzymatic activity in the liver. Data represent µmol citrulline/h/mg total protein. (D) Blood ammonia levels are shown as µmol/L. (E) Urinary orotic acid levels depicted as µmol orotic acid (OA)/mmol creatinine. (A-E) ***p < 0.001, *p < 0.05 compared to untreated animals.

Duration of Action of a Single Dose of MRT5201

MRT5201 is Designed to Treat Patients with OTC Deficiency

2

1

OTC Deficiency is a Metabolic Liver Enzyme Disorder that Results from Mutations in the OTC Gene

WT

Untreate

d

0.016 m

g/kg

0.05 mg/kg

0.16 mg/kg

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101

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0

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ng O

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05

1015202550

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OTC Activity

µmol

citr

ullin

e / h

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g *****A B C

Figure 3

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0.016 mg/kg

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Blood NH3

NH3 (µm

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*** ***

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0.016 mg/kg

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Urinary Orotic Acid

nmol

OA/m

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reati

nine *** *** *** ***

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Figure 3

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5060708090

OTC Activity

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Figure 4

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NH3

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Urinary Orotic Acid

µmol

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mm

ol C

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inin

e *** *********D E

Figure 4

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1×107

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ies

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TC/µ

g to

tal R

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Figure 5

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Log 1

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Figure 5

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Log 10

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mRN

A (c

opie

s of

OTC

/μg

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l RNA

)