Skip to main content
NCBI home page
Molecular Therapy logoLink to Molecular Therapy
. 2018 Jan 4;26(3):801–813. doi: 10.1016/j.ymthe.2017.12.024

Targeted mRNA Therapy for Ornithine Transcarbamylase Deficiency

Mary G Prieve 1,∗, Pierrot Harvie 1, Sean D Monahan 1, Debashish Roy 1, Allen G Li 1, Teri L Blevins 1, Amber E Paschal 1, Matt Waldheim 1, Eric C Bell 1, Anna Galperin 1, Jean-Rene Ella-Menye 1, Michael E Houston 1
PMCID: PMC5910669  PMID: 29433939

Abstract

We describe a novel, two-nanoparticle mRNA delivery system and show that it is highly effective as a means of intracellular enzyme replacement therapy (i-ERT) using a murine model of ornithine transcarbamylase deficiency (OTCD). Our Hybrid mRNA Technology delivery system (HMT) comprises an inert lipid nanoparticle that protects the mRNA from nucleases in the blood as it distributes to the liver and a polymer micelle that targets hepatocytes and triggers endosomal release of mRNA. This results in high-level synthesis of the desired protein specifically in the liver. HMT delivery of human OTC mRNA normalizes plasma ammonia and urinary orotic acid levels, and leads to a prolonged survival benefit in the murine OTCD model. HMT represents a unique, non-viral mRNA delivery method that allows multi-dose, systemic administration for treatment of single-gene inherited metabolic diseases.

Keywords: hybrid mRNA technology delivery system, lipid nanoparticle, LNP, ornithine transcarbamylase deficiency, OTCD, inherited metabolic disorder, intracellular enzyme replacement therapy, i-ERT, mRNA delivery, Otcspf -ash mice

In this issue of Molecular Therapy, Prieve et al. describe a novel, targeted mRNA delivery system that corrects a rare liver disease in a murine model of ornithine transcarbamylase deficiency having a single enzyme defect. This non-viral delivery method allows multi-dose, systemic administration for treatment of inherited metabolic diseases.

Introduction

Limited treatment options are available for many inherited metabolic disorders (IMDs), which are typically caused by single gene defects. Ornithine transcarbamylase deficiency (OTCD), which results from the loss of a key enzyme in the urea cycle that is predominantly expressed in the liver,1 is an example of such an IMD. Because the function of the urea cycle is to metabolize ammonia, a by-product of protein metabolism, OTCD causes ammonia levels to rise in the bloodstream (hyperammonemia), which can result in neurological impairment, coma, and even death. Currently available treatments such as a protein-restricted diet and ammonia scavengers do not address the underlying cause of the disease. Nucleic acid-based therapies could be an appropriate treatment option for IMDs like OTCD because they replace the missing or defective enzyme inside the affected cell, which we refer to as intracellular enzyme replacement therapy (i-ERT), and thus correct the disease.

We, and others,2, 3 have been developing mRNA delivery systems to treat IMDs. Delivery of mRNA has multiple potential advantages over other nucleic acid-based therapies, such as plasmid DNA and viral vectors. To date, delivery of plasmid DNA has resulted in low levels of activity potentially because of the barriers to entering the nucleus.4 In contrast, mRNA only needs to reach the cytoplasm to allow almost immediate initiation of protein translation. With mRNA, there is no risk of insertional mutagenesis, a risk for both plasmid DNA and viral vectors.5, 6, 7 Likewise, mRNA delivery can avoid the side effects that are frequently associated with viral delivery,8, 9 such as immune-mediated hepatitis. Delivery of mRNA allows control of pharmacokinetics and dosing10 because it does not result in irreversible changes to the cell or genome.11

The therapeutic use of mRNA also has potential advantages over the delivery of therapeutic proteins. Conventional protein-based enzyme replacement therapy (ERT), as used in lysosomal storage diseases such as Pompe disease, has a number of limitations including anaphylactoid reactions, a high rate of antibody formation, and the consequent requirement for slow infusion over 4 hr.12 In contrast, the intracellular delivery of biosynthetic mRNA takes advantage of the endogenous translational machinery and post-translational mechanisms to produce the intended therapeutic protein, eliminating many of the typical complications of recombinant protein production.

In spite of its potential advantages, use of mRNA as a therapeutic modality has faced challenges compared with other nucleic acid technologies. It is an inherently unstable molecule due to ubiquitous RNases.13 Because mRNA is a charged macromolecule, it cannot freely cross the cell membrane;14 hence the levels of naked mRNA that can be taken up are low.15 To address the challenges of mRNA delivery, we have developed our Hybrid mRNA Technology delivery system (HMT): a novel two-nanoparticle mRNA delivery system comprising a N-acetylgalactosamine (GalNAc)-targeted polymer micelle for hepatocyte-specific delivery and endosomal escape, and an inert lipid nanoparticle (LNP) that protects the mRNA from nucleases during delivery and entry into the liver. Here we show HMT delivery results in liver-specific expression of firefly luciferase mRNA with a rapid onset of expression that is dependent on both nanoparticles. We then show that intrahepatic delivery of an mRNA encoding human OTC has a prolonged therapeutic benefit and a good safety profile in a hyperammonemic murine model of OTCD.

Results

Hybrid mRNA Technology Delivery System

HMT comprises two nanoparticles: a di-block polymer micelle that allows liver-specific targeting and facilitates endosomal release of the mRNA into the cytoplasm, and an inert LNP that protects the mRNA from nucleases (Figure 1A). The di-block polymer is derived from a family of polymers used as carriers of small interfering RNA (siRNA)16, 17, 18 and possesses pH-sensitive chemical functionalities that mediate increased membrane permeation in the acidic pH environment of the endosome. The polymer has three functional domains. First, GalNAc serves as a targeting ligand for uptake specifically in the liver. GalNAc is commonly used to target the liver as it binds to the asialoglycoprotein receptor (ASGPR) that is abundantly expressed in hepatocytes.19, 20 The second domain comprises a hydrophilic polymer block that maintains polymer solubility. The third domain is a polymer block containing butyl methacrylate (BMA; a hydrophobic monomer), 2-propylacrylic acid (PAA; a carboxylic acid monomer), and 2-(dimethylamino)ethyl methacrylate (DMAEMA; a tertiary amine monomer), which allows mRNA release from the endosome in a pH-dependent manner. Because the polymer is not active at physiological pH, it does not require any masking21, 22 to prevent premature membrane destabilization (hemolysis data not shown, but similar to that described by Convertine et al.17). The combination of the endosomolytic polymer block with its increased hydrophobic content preceded by the first hydrophilic polymer block spontaneously induces micelle formation (∼16 nm in size; Table S1; Figure 1A).

Figure 1.

Figure 1

Open in a new tab

The Hybrid mRNA Technology Delivery System

(A) Key components of HMT include a polymer micelle and LNP. (B) Graphic illustrates the in vivo delivery protocol and release of mRNA into the cytoplasm of hepatocytes for protein production.

The LNP component is similar in composition and function to those used in siRNA delivery systems.23 It is composed of (2,3-dioleoyloxy-propyl)-trimethylammonium chloride (DOTAP; a cationic lipid), cholesteryl hemisuccinate (CHEMS; an anionic lipid that is pH sensitive), cholesterol (a helper lipid that stabilizes the lipid bilayer), and a polyethylene glycol (PEG) lipid. The mRNA/LNP is passively targeted to the liver through particle size and surface charge because of the discontinuous endothelium in liver.24, 25 For effective uptake in liver, the particle size of LNPs should be <100 nm. Because the stability of the mRNA is conferred by the LNP, its particle size, surface charge, and the encapsulation and integrity of the mRNA are routinely assessed (see typical results in Table S1). For in vivo administration, the mRNA/LNP and polymer micelles are prepared as separate nanoparticle solutions and mixed before intravenous (i.v.) administration (Figure 1B).

We have several lines of evidence that the polymer and mRNA/LNP distribute independently to the liver, but that effective mRNA expression is dependent on both components. In situ hybridization detection of firefly luciferase (luc) mRNA showed similar localization in liver tissue with luc mRNA/LNP alone compared with luc mRNA/LNP + GalNAc-targeted polymer (luc mRNA/HMT) (Figure S1A), suggesting that the mRNA/LNP is distributed independently of the polymer micelle. Similar luciferase activity in vivo was observed whether luc mRNA/LNP and polymer were co-administered or sequentially administered 30 min apart (Figure S2), again supporting independent distribution. However, addition of the polymer results in a dramatic increase in mRNA expression in the liver. Expression of mRNA with luc mRNA/HMT showed a 2,000-fold greater luciferase activity in the liver than luc mRNA/LNP alone (Figure S1B). These data suggest that the polymer and mRNA/LNP likely localize independently to the same endosomes in hepatocytes where the polymer mediates endosomal escape of mRNA into the cytoplasm and possibly disassembly of the LNP once inside the endosome.

Firefly Luciferase Expression in Mice

Liver-Specific Expression

To confirm liver-specific expression of our HMT formulation and the importance of the GalNAc-targeting component, we gave mice an i.v. bolus of a luc mRNA/LNP formulation plus a GalNAc-targeted polymer (i.e., luc mRNA/HMT) or one of two control polymers (a mannose-targeted polymer21 or a non-targeted polymer) to demonstrate targeting specificity. Luminescence measurements were performed 6 hr after dosing. Luminescence imaging in vivo (Figures 2A and 2C), as well as tissue-specific imaging ex vivo (Figure 2B), showed that expression was 5 logs above background only with luc mRNA/HMT and limited solely to the liver with no expression in spleen, pancreas, heart, kidney, uterus/ovary, and lung. The luciferase expression with luc mRNA/HMT showed an ∼2,000-fold increase in expression over both of the control formulations (Figure 2C), while there was virtually no difference between the expression of the controls. To confirm this, we conducted subsequent studies and showed that over 95% of the GalNAc-targeted polymer dose was detected in liver 2 hr after administration (Figure S3A). Immunofluorescence demonstrated that mRNA expression in the liver following luc mRNA/HMT administration occurred specifically in cells having the typical morphology of hepatocytes (Figure 2D), as would be expected based on the hepatocyte-specific expression of the GalNAc receptor, ASGPR.19, 20

Figure 2.

Figure 2

Open in a new tab

Liver-Specific Expression of luc mRNA/HMT

(A–C) Luminescence in vivo (A and C) or ex vivo (B) detected 6 hr after injection of buffer or luc mRNA/LNP plus GalNAc-targeted polymer (i.e., luc mRNA/HMT), mannose-targeted polymer, or non-targeted polymer (all 0.5 mg/kg mRNA + 25 mg/kg polymer). (C) Bars are mean of n = 5, with error bars as SD; ***p < 0.001. Student’s t test with two tails; data are representative of two independent studies. (D) Immunofluorescent detection of luciferase in liver tissue collected 6 hr after a single injection of luc mRNA/HMT (1 mg/kg mRNA + 75 mg/kg polymer). Inset shows liver section from a buffer-treated animal. A rabbit anti-luc antibody was used to detect luciferase protein (green) in conjunction with Alexa Fluor 488-conjugated donkey anti-rabbit IgG. Cells were counterstained with DAPI (blue). Data are representative of n = 3 mice and are from four different regions of tissue section.

Dose Optimization

Optimal dosing levels were determined by evaluating expression following the administration of increasing mRNA dose levels (0.5, 1.0, and 5 mg/kg) of luc mRNA/LNP with 30 mg/kg polymer (Figure 3A) and increasing polymer dose levels (10, 20, 25, and 30 mg/kg) with 0.5 mg/kg luc mRNA/LNP (Figure 3B). A dose-responsive increase in luciferase activity was observed with increasing mRNA dose levels (Figure 3A). Similarly, a dose-responsive increase in luciferase activity was observed with increasing polymer dose levels (Figure 3B), with activity plateauing between 25 and 30 mg/kg polymer. Additional experiments showed that luciferase expression could be detected 30 min after injection (Figure 3C), and that it could still be detected 96 hr after dosing (Figure 3D). High levels of expression were also maintained during a multiple-dosing regimen. Mice received weekly i.v. injections of luc mRNA/HMT for a total of 12 weeks. This multiple-dosing regimen maintained a constant level of luciferase expression following each dose (Figure 3E), suggesting an absence of any neutralizing antibody formation. Mice receiving only a single injection included at most time points showed comparable levels of luciferase expression (data not shown).

Figure 3.

Figure 3

Open in a new tab

Dose Optimization of luc mRNA/HMT

(A) Luminescence in vivo 6 hr following the administration of increasing doses of luc mRNA/LNP (0.5, 1.0, or 5.0 mg/kg mRNA) with 30 mg/kg polymer (**p < 0.01; ***p < 0.001; mean of n = 5, error bars show SD). Data are representative of three independent studies. (B) Luminescence in vivo following the administration of increasing doses of polymer (10, 20, 25, and 30 mg/kg) with 0.5 mg/kg luc mRNA/LNP (**p < 0.01; ***p < 0.001; mean of n = 5, error bars show SD). Data are representative of three independent studies. n.s., not significant. (C and D) Time course of luminescence in vivo following a single injection of luc mRNA/HMT (1 mg/kg mRNA + 45 mg/kg polymer) (***p < 0.001 relative to buffer group; mean of n = 5, with error bars as SD). Data are representative of two independent experiments. (E) Luminescence measured 6 hr after each weekly dose of luc mRNA/HMT (0.5 mg/kg mRNA + 30 mg/kg polymer) for 12 weeks (mean of n = 5, error bars as SD). There were no significant differences between groups at each weekly repeat dose relative to the first dose. All analyses: Student’s t test with two tails. Data are representative of two independent studies.

Evaluation of Possible Side Effects

In addition to the expression analyses, we also measured serum liver alanine transaminase (ALT) levels and CXCL10 cytokine levels to ensure that HMT treatment was well-tolerated and that it did not elicit an innate immune response. These data showed that there was no increase in ALT levels (Figure S4) 24 hr after a single dose of luc mRNA/HMT at the dosing levels used in most of our studies (0.5–1.0 mg/kg luc mRNA); higher doses (5 mg/kg luc mRNA) showed a slight, but not significant, elevation in ALT levels. Likewise, there were no significant mRNA dose-related changes in serum CXCL10 levels measured 3 hr after a single dose of luc mRNA/HMT (Table S2). Although CXCL10 levels from luc mRNA/HMT-treated mice are above buffer-treated mice, the levels are substantially lower (∼50-fold lower) than the level induced by an immunostimulatory oligoribonucleotide.26

Expression, Efficacy, and Safety in OTC-Deficient Mice

We investigated the therapeutic effectiveness of HMT for OTCD, a life-threatening, inherited liver disease. OTC is one of six enzymes in the urea cycle that helps break down and remove nitrogen from the body (Figure 4). OTCD is an X-linked disorder that causes elevated ammonia levels in the blood (hyperammonemia) and high levels of urinary orotic acid.

Figure 4.

Figure 4

Open in a new tab

Graphic of the Urea Cycle during OTC Deficiency

Metabolites that accumulate are shown in red. ARG, arginase; ASL, argininosuccinate lyase; ASS1, argininosuccinate synthase; CPSI, carbamoyl phosphate synthetase I; NAGS, N-acetylglutamate synthase.

Otcspf-ash Murine Model of OTCD

To model OTCD in mice, we used the Otcspf-ash mouse model27, 28 in which mice have an R129H mutation that causes inefficient splicing resulting in 5%–10% of residual OTC enzyme activity. To create a model that is clinically relevant, hyperammonemia is induced by using an AAV vector that expresses an Otc short hairpin RNA (shRNA) to knock down the residual mouse Otc mRNA and protein. Hyperammonemia develops within days, and the mice die without treatment.29 This model closely parallels the human clinical condition.30 The details of model testing and optimization are described in Supplemental Materials and Methods and Figure S5. Biomarkers in this OTCD model include elevations in plasma ammonia and urinary orotic acid; body weights and long-term survival are also greatly reduced.

Single-Dose Expression of hOTC mRNA/HMT

Prior to running efficacy studies, we measured human OTC protein expression and enzyme activity levels following a single injection of human OTC (hOTC) mRNA/HMT into Otcspf-ash mice that had not received the AAV-Otc shRNA and compared them with activity levels in hOTC mRNA/HMT-treated and untreated CD1 mice. hOTC protein levels were detected by western blotting 2, 4, 7, and 10 days after dosing Otcspf-ash mice (Figure 5A). OTC enzyme activity in Otcspf-ash mice reached peak levels that were 14% of normal CD1 mouse levels (4-fold higher than buffer-treated mice) (Figure 5B). OTC enzyme activity remained elevated through 10 days after dosing. In contrast, OTC enzyme activity in normal CD1 mice reached 200% of normal activity following a single dose (Figure 5C). Reduced OTC activity in Otcspf-ash mice was not unexpected because control experiments comparing luc mRNA/HMT expression also showed 8-fold lower luciferase activity in affected Otcspf-ash mice compared with normal C57BL/6 mice and heterozygous littermates (see Figure S6).

Figure 5.

Figure 5

Open in a new tab

OTC Enzyme and Activity Levels after a Single Injection of hOTC mRNA/HMT

Non-AAV-treated Otcspf-ash or normal CD1 mice were given a single bolus dose of buffer or hOTC mRNA/HMT (3 mg/kg mRNA + 25 mg/kg polymer [n = 3/time point]). (A) Western blot using an OTC antibody that preferentially detects hOTC over mouse Otc (see second and third lanes from left). Samples 1–3 show endogenous OTC protein levels are below the level of detection in Otcspf-ash mice injected with buffer alone. Samples 4–15 show that elevated levels of OTC protein can be readily detected in hOTC mRNA/HMT-treated mice through day 10 after dosing. (B and C) OTC enzyme activity (residual mouse and human) is elevated through 10 days after dosing. OTC activity following treatment of Otcspf-ash mice (B) and OTC activity following treatment of normal CD1 mice (C) are shown (Student’s t test with two tails; *p < 0.05; **p < 0.01 relative to buffer group; data are mean of n = 3, with error bars as SD). (A–C) Data are representative of two independent studies. (D) Immunofluorescent analysis of liver from Otcspf-ash mice dosed with hOTC mRNA/HMT (panels 1 and 2), Otcspf-ash mice dosed with buffer (panel 3), or untreated wild-type littermate mice (panel 4). A human-specific anti-OTC mouse monoclonal antibody was used to detect hOTC protein (green) in conjunction with Alexa Fluor 546-conjugated donkey anti-mouse IgG. Cells were counterstained with DAPI (blue). n = 3 per treatment. Images are representative of at least four different regions in the tissue section. C, central vein region; P, portal triad region. (E) Quantitation of hOTC-positive cells from four separate regions of each tissue section.

Evaluating expression using immunofluorescence showed that there were numerous hepatocytes positive for hOTC protein (Figure 5D, panels 1 and 2) in Otcspf-ash mice dosed with hOTC mRNA/HMT. Under 40× magnification with image deconvolution, the positive signal appears to be punctate and cytosolic, correlating with mitochondrial localization of the OTC protein (Figure 5D, panel 1). There was no difference in the distribution of hOTC protein expression between the central vein and portal regions (Figure 5D, panel 2); both had ∼80% hepatocytes positive for hOTC protein (Figure 5E). In contrast, there are essentially no positive cells for hOTC found in the liver of either Otcspf-ash mice dosed with buffer or in wild-type littermates (Figure 5D, panels 3 and 4). Subsequent experiments, conducted to confirm the liver specificity of hOTC mRNA/HMT, using qPCR showed that there was a 40-fold higher level of mRNA uptake by the liver relative to kidney and spleen (Figure S3B).

Repeat-Dose Efficacy of hOTC mRNA/HMT

We next evaluated the efficacy in a repeat-dosing study in the OTCD model. Otcspf-ash male mice were administered the AAV-Otc shRNA on day 0. The hOTC mRNA/HMT formulation, the control mRNA/HMT formulation, or buffer was administered once or twice a week starting on day 4. Analyses included daily body weights, plasma ammonia levels, urinary orotic acid levels, and hOTC protein expression in liver. Dosing continued for 35 days after which half of the animals in the twice-a-week hOTC mRNA/HMT group (6 of 12) were maintained to determine survival rates post-treatment, and the other 6 of 12 were sacrificed on day 37 to assess remaining hOTC protein levels and endogenous mouse Otc mRNA in liver to confirm the continued knockdown activity of the Otc shRNA. There were no surviving animals in the buffer and control groups at day 35, and the remaining animals in the once-per-week hOTC mRNA/HMT treatment group were sacrificed. Analysis of plasma ammonia and urinary orotic acid levels showed that they were normalized with both hOTC mRNA/HMT treatment regimens (Figures 6A and 6B) in comparison with buffer or control mRNA/HMT-treated mice. Orotic acid levels in the urine were significantly reduced relative to control animals within 24 hr of the first dose of hOTC mRNA/HMT and remained similar to normal mice throughout the study in the twice-per-week hOTC mRNA/HMT group (Figure 6B). Orotic acid levels in the once-per-week hOTC mRNA/HMT treatment group began to show elevated levels by day 20. The once-per-week hOTC mRNA/HMT treatment group also began to lose weight at day 23, while the twice-a-week group had a significant increase in body weight compared with the control groups, demonstrating both efficacy and good tolerability with this dosing regimen (Figure 6C).

Figure 6.

Figure 6

Open in a new tab

Repeat-Dose Efficacy Study in the Otcspf-ash OTCD Model

Otcspf-ash mice were given 100 μL of AAV 2/8/Otc shRNA, 1 × 1011 genome copies (GCs) per mouse on day 0. The mice were then given a formulation consisting of buffer (twice a week), hOTC mRNA/HMT (once a week or twice a week), or control mRNA/HMT (once a week) (both at 3 mg/kg mRNA and 25 mg/kg polymer) via i.v. bolus into the tail vein starting on day 4 (n = 12 per group). (A) Plasma ammonia (Student’s t test with two tails; *p < 0.05; **p < 0.01; comparison with normal mice; bars show averages with SD error bars) measured on days 14, 21, 28, and 35. Note the large error bar with control mRNA/HMT on day 21 was due to one out of eight surviving animals with a very high plasma ammonia level. (B) Urinary orotic acid levels (two-way ANOVA; ***p < 0.001; comparison with control mRNA; data are shown as geomean, and error bars are SEM) measured on days 5, 6, 7, 10, 13, 20, 27, and 34. (C) Body weights were measured daily (two-way ANOVA; ***p < 0.001; comparison with control mRNA; data are averages with SD error bars). (D) Kaplan-Meyer survival curve (log rank test; ***p < 0.001; comparison with control mRNA). (E) Western blot showing hOTC expression in liver from representative mice with buffer, control mRNA/HMT, and hOTC mRNA/HMT twice-a-week treatment. hOTC mRNA/HMT mice were sacrificed on day 37, 48 hr after the last dose. The rest were sacrificed when mice met their endpoint of ≥20% body weight loss. Normal human and mouse livers were included as controls. Weak (non-significant) expression of OTC was detected by the anti-OTC antibody in the treatment with control mRNA/HMT. This may be because of a low level of non-AUG translation initiation.31 (F) Western blot quantitation. Data are shown relative to OTC in normal human liver (Student’s t test with two tails; *p < 0.05; ***p < 0.001; comparison with buffer; data are averages with SD error bars). Data are representative of two independent experiments.

All animals in the control mRNA formulation and buffer treatment groups succumbed to hyperammonemia or met protocol criteria for sacrifice before the end of the dosing period (Figure 6D). The control mRNA group showed a slight delay in body weight loss, orotic acid elevation, and lower plasma ammonia levels on day 14 compared with the buffer group (Figures 6A–6C). This may be because of a low-level expression of hOTC from control mRNA/HMT treatment as a result of non-AUG translation initiation31 (Figure 6E). Animals treated once per week with hOTC mRNA/HMT began to die on day 21, about the same time weight loss and the increases in orotic acid were measured. We did not measure increases in plasma ammonia at that time because blood samples from mice that died were unavailable for analysis. All mice in the twice-per-week hOTC mRNA/HMT treatment group survived the 35-day dosing period. In addition, all six of the hOTC mRNA/HMT mice that were maintained after the termination of dosing survived for at least 3 additional weeks (Figure 6D). The six mice sacrificed 48 hr after the final dose had approximately 35% of normal OTC protein compared with a normal human liver tissue control and had approximately 12% of normal protein 3–4 weeks later (Figures 6E and 6F). From these data, a 12-day half-life for OTC protein can be calculated. Measurements of endogenous mouse Otc mRNA levels showed that they remained low, indicating that the increased survival was not due to loss of the AAV-Otc shRNA construct (Figure S7).

Repeat-Dose Safety of hOTC mRNA/HMT

We measured the safety profile of hOTC mRNA/HMT in a repeat-dosing study in Otcspf-ash male mice that had not received the AAV-Otc shRNA. The hOTC mRNA/HMT formulation, the control mRNA/HMT formulation, or buffer was administered twice a week and continued for 4 weeks for a total of nine repeat doses. Normal ALT and aspartate aminotransferase (AST) levels were observed 24 hr after the first dose and 24 hr after the final dose (Figure 7A). Cytokines levels were monitored 3 and 24 hr after the final dose. No increase was observed with interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, tumor necrosis factor alpha (TNF-α), or monocyte chemoattractant protein-1 (MCP-1) levels. Minimal increases (<5-fold) were observed with IL-12 and CXCL10 levels (Figures 7B and 7C). Histopathology of internal organs (liver, kidney, heart, lung, and spleen) was examined 48 hr after the final dose (Figure S8). No significant pathological findings were observed in any of the tissues from the treated animals.

Figure 7.

Figure 7

Open in a new tab

Repeat-Dose Safety in Otcspf-ash Mice

Normal serum liver transaminases and low cytokine levels were observed following repeat administration of buffer, control mRNA/HMT, or OTC mRNA/HMT in Otcspf-ash mice (both at 3 mg/kg mRNA and 25 mg/kg polymer) via i.v. bolus into the tail vein. (A) ALT and AST levels were measured 24 hr after the first and final ninth repeat dose (administered 2×/week) of buffer or mRNA/HMT. Student’s t test with two tails showed no significance between buffer (n = 8) versus control mRNA/HMT (n = 7) or versus OTC mRNA/HMT group (n = 7). Cytokine levels were measured (B) at 3 or (C) 24 hr after nine repeat doses of buffer or mRNA/HMT. Student’s t test with two tails showed significance between buffer versus control mRNA/HMT or versus OTC mRNA/HMT group only with IL-12, but no other cytokines; **p < 0.01. Bars represent mean with SD error bars.

Discussion

We demonstrated the application of HMT in a model of OTCD, a urea cycle disorder that has devastating patient consequences. Patients are at a constant risk for ammonia toxicity, which can cause cumulative and permanent neurological impairment, and even death. Current therapies include protein-restricted diets and ammonia scavenger drugs. Liver transplantation has also been performed, but it has its own set of problems including the requirement for lifelong immunosuppression.32 i-ERT using the HMT delivery system may be a preferable means to reinstate the normal physiology and correct this devastating disease. Indeed, we demonstrated that hOTC mRNA/HMT administration not only reduces ammonia levels but also leads to significant survival benefit in the murine OTCD disease model. The hOTC protein accumulated to ∼35% of normal human levels and had a long duration of activity (estimated protein half-life of 12 days), with the mice living an additional 3–6 weeks after the dosing period in an aggressive hyperammonemic OTCD model. The rapid onset of expression, as well as the tolerability of repeat dosing, suggests clinical utility.

We saw better survival and normalization of plasma ammonia and orotic acid levels during the dosing period using twice-per-week dosing than once-per-week dosing. We have shown that affected Otcspf-ash mice have reduced expression of exogenous mRNA (both luciferase and OTC activity; Figure S6; Figures 5B and 5C) compared with normal mice. We believe this is the reason two times per week dosing frequency is needed for extended therapeutic efficacy in Otcspf-ash mice. As a potential therapeutic, a less frequent dosing interval would be preferable, and we believe reduced dosing is likely possible based on the 100% increase in OTC activity in normal mice following a single dose of hOTC mRNA/HMT (Figure 5C). In the case of OTCD, we have the advantage that the OTC protein has a long half-life, thus making biweekly to monthly dosing a possibility. We are continuing to evaluate optimal dosing regimens.

This is the first demonstration of a systemically administered mRNA therapy with repeat dosing for correction of OTCD in a therapeutically relevant model. This is also the first demonstration of efficacy in an OTCD model using a non-viral delivery mechanism for OTC mRNA. Several studies have shown that AAV can also be used to deliver OTC mRNA in the murine OTCD model.33, 34 Higher OTC protein and enzyme activity levels are typically observed in AAV studies because AAV directs long-term transgene expression in comparison with transient expression using non-viral mRNA delivery. However, several concerns have been raised with regard to the use of AAV in OTCD patients. First is its limited durability in pediatric OTCD patients. Reports from two groups suggest that gene therapy using non-integrating viruses such as AAV would be lost in neonatal livers because of proliferation of the developing hepatocytes.35, 36 Second, AAV vectors are susceptible to pre-existing neutralizing antibodies37 and can trigger cytotoxic T cell responses to the viral vector (e.g., capsid protein)38 that limit its effectiveness, and can elevate liver transaminases and potentially cause hepatic failure.6, 7, 39, 40 Finally, AAV is known to cause immune-mediated hepatitis, which is treated with glucocorticoid drugs.40 But glucocorticoids cause catabolism, which can trigger a hyperammonemic crisis and are therefore contraindicated in OTCD.1

The HMT delivery system also demonstrates advantages over other mRNA delivery modalities. Multiple delivery methods have been tried with mRNAs, including lipoplexes/polyplexes,41, 42 polymers,43 and ionizable LNPs,2, 44, 45 with varying levels of success. Most of these alternative delivery methods rely on ionizable LNPs, which results in expression predominately in the liver, but also in spleen and pancreas.44, 46 In contrast, with HMT the inert LNP in combination with the GalNAc-targeted polymer that is highly specific for hepatocytes directs protein expression specifically in the liver with no detectable expression in any other tissue examined (Figure 2). While luciferase activity using HMT is comparable with activity with ionizable LNPs,45, 47 the safety profile by cytokine induction is quite different between these mRNA delivery modalities. Cytokine levels following repeat administration of hOTC mRNA/HMT (3 mg/kg mRNA) are low with no increase in IL-6, TNF-α, GM-CSF, IFN-γ, or MCP-1 levels and minimal increase in IL-12 and CXCL10 levels (<5-fold) (Figures 7B and 7C). In comparison, a single dose of 2 mg/kg mRNA delivered with an ionizable LNP showed ∼150-fold increase in IL-6 and ∼20-fold increase in TNF-α.48

The choice of the polymer used in the HMT delivery system also gives it advantages over other systems that use a polymer-based delivery approach and require masking.21, 22, 49 The HMT polymer is not active at physiological pH and does not require any masking because the components in its third domain (endosome-release block) neutralize each other (the tertiary amine of DMAEMA is neutralized by the carboxylates in polymeric PAA). Thus, mRNA release occurs only in the acidic environment of the endosome.

It is our belief that HMT represents an attractive option for sustained enzyme replacement in OTC deficiency, as well as other IMDs, which continue to have a critical need for effective therapeutics. Although most inherited metabolic diseases are rare, the total incidence of IMDs is estimated to be 1 in 5,000 births,50, 51 with many having devastating consequences to patients and families. The estimated frequency of OTC deficiency alone is 1/50,000–80,000 births, and because urea cycle disorders often go unrecognized and/or infants born with the disorders die without a definitive diagnosis, it may be under-diagnosed.52 By delivering the mRNA encoding the missing enzyme into the cell, i-ERT reinstates the normal intracellular physiology of the cell and offers the potential to correct these diseases.

Materials and Methods

Polymer Synthesis

A detailed description of polymer synthesis can be found in the Supplemental Materials and Methods. In brief, synthesis of the di-block polymer was conducted using standard RAFT (reversible addition-fragmentation chain-transfer) polymerization techniques.53 For the block 1 polymerization reaction, 70%–100% poly(ethylene glycol) methyl ether methacrylate (PEGMA) monomer was polymerized together with 0%–30% of a hydrophobic monomer. The resulting polymer was purified by precipitation and dialysis. For block 2 polymerization, DMAEMA, BMA, and PAA were co-polymerized. The di-block polymer was purified by precipitation and dialysis. Polymers were analyzed by proton nuclear magnetic resonance (1H-NMR) (Figure S9A). Monomer incorporation for each block was determined by analytical high-performance liquid chromatography (HPLC). Polymer molecular weight and polydispersity were determined by gel permeation chromatography (GPC) (Figure S9B).

Synthetic mRNAs

Firefly luciferase (luc), human ornithine transcarbamylase (OTC), and hOTC control mRNAs were synthesized at TriLink Biotechnology (San Diego, CA, USA). All mRNAs have a 5′-cap (Cap 1) and a 3′ poly A tail (size ranging from 120 to 220 A’s) and are modified with pseudouridine. The incorporation of modified nucleosides suppresses recognition by TLRs.54, 55 5′ and 3′ UTRs flank the coding sequence in each mRNA. The OTC mRNA coding sequence comprises a mouse mitochondrial leader sequence encoding amino acids 1–32 and the first two amino acids of the mature enzyme (amino acids 33–34) followed by the hOTC sequence (amino acids 35–354), which has been codon optimized for expression in mice (GenScript). The mouse mitochondrial leader sequence was used to facilitate the import of the OTC protein to mouse mitochondria.56 The control mRNA is the same OTC mRNA sequence but with mutations in all in-frame and out-of-frame AUGs. HPLC purification of mRNAs to remove truncated transcripts and/or double-stranded RNA contaminants arising during mRNA synthesis was conducted similarly to the method described by Karikó et al.57

HMT Formulation and Characterization

To prepare the mRNA/LNP formulations for administration in vivo, we diluted the mRNA in formulation buffer (300 mM sucrose, 20 mM phosphate [pH 7.4]) at 0.45 mg/mL. For the LNP, DOTAP (Corden Pharma International, Woburn, MA, USA), CHEMS (Avanti Polar Lipids, Alabaster, AL, USA), cholesterol (Corden Pharma International), and a PEG 2000-lipid (Avanti Polar Lipids) were each solubilized in ethanol at 25–50 mg/mL. The four lipids were mixed together to prepare a lipid stock in ethanol. The mRNA/LNP was assembled by quickly mixing the lipid stock in ethanol with the mRNA stock in buffer at a 1:3 ratio (lipid:mRNA; 33% ethanol final) and a nitrogen-to-phosphate ratio of 7. After a 1-hr incubation at room temperature, the mRNA/LNP was dialyzed overnight against 100 vol of formulation buffer at 4°C. Prior to each administration, the particle size (at pH 7.4), the surface charge (at pH 7.4), mRNA encapsulation (measured by Quant-iT RiboGreen RNA Assay for Dye Accessibility; Thermo Fisher), and mRNA integrity (using Agilent 2100 Bioanalyzer) were evaluated. Details of these methods can be found in the Supplemental Materials and Methods. Both HMT formulation components can be stored frozen. When thawed, mixed, and injected, the same activity was observed as freshly prepared formulations (data not shown).

The polymers used for co-injection were solubilized at 20 mg/mL in formulation buffer with agitation at 400 rpm for 1 hr at room temperature and then stored overnight at 4°C. The di-block polymer was diluted to 2–15 mg/mL in buffer prior to injection. Prior to dosing, the mRNA/LNP and polymer solutions were mixed and injected by bolus i.v. administration. We have also detected good in-use stability and similar activity of the formulation mixture when the mixture was incubated for 24 hr compared with 5 min before injection (data not shown).

IACUC Protocol

PhaseRx Institutional Animal Care and Use Committee (IACUC) approval was obtained for all procedures and protocols involving animals. Quotient Bioresearch’s (Rushden, UK) IACUC approved all animal procedures for the 14C-labeled polymer distribution study.

Requirement for Polymer and LNP Components

See Supplemental Materials and Methods.

Luminescence Studies

Tissue Localization

Female CD1 mice (n = 3–5; 7–10 weeks; Charles River Laboratories, Wilmington, MA, USA) were used in all tissue localization studies. Mice were given a single i.v. bolus of buffer or a formulation consisting of luc mRNA/HMT (0.5–1.0 mg/kg mRNA plus 25–75 mg/kg polymer; 10 mL/kg dosing volume by weight). Luminescence and immunofluorescence studies were conducted 6 hr after injection. Luciferase activity in the liver was measured by luminescent imaging in vivo. Immediately after imaging, mice were euthanized and tissues were removed for imaging ex vivo. Luminescence measurements were made using an IVIS Lumina II Imaging System (PerkinElmer, Hopkinton, MA, USA) in connection with Living Image Software (version 4.3; PerkinElmer). Details of the luminescence protocols can be found in the Supplemental Materials and Methods. For immunofluorescence, a piece of liver was embedded in O.C.T. compound for cryosectioning. A rabbit anti-luciferase antibody (catalog [Cat] no. PA5-28197; Sigma) was diluted 1:1,000 with 12% BSA in PBS and used to detect luciferase protein in conjunction with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Cat no. A21206, 1:1,000 dilution in 12% BSA in PBS; Life Technologies). See Supplemental Materials and Methods for details.

Dose Response

Female CD1 mice (n = 5; 7–10 weeks) were given a single i.v. bolus injection of buffer or a formulation consisting of luc mRNA/LNP (0.5, 1.0, or 5.0 mg/kg mRNA) and 30 mg/kg polymer (10 mL/kg dosing volume by weight). For polymer dose response, mice were given a single injection of 0.5 mg/kg luc mRNA/LNP with 10, 20, 25, or 30 mg/kg polymer. Luminescence in vivo was measured in the liver 6 hr after injection.

Expression Duration

To evaluate the duration of expression, we gave female CD1 mice (n = 5; 7–10 weeks) a single i.v. bolus injection of a formulation consisting of luc mRNA/HMT (1 mg/kg mRNA plus 45 mg/kg polymer; 10 mL/kg dosing volume by weight). Luminescence in vivo was measured in the liver 0.5, 1, 3, 6, 24, 48, 72, and 96 hr after injection.

Repeat Dosing

Female CD1 mice (n = 5; 7–10 weeks) were given a weekly i.v. bolus of a formulation consisting of luc mRNA/HMT (0.5 mg/kg mRNA and 30 mg/kg polymer; 10 mL/kg dosing volume by weight). Luminescence in vivo was performed 6 hr after each dose. Naive mice were included at most time points to compare repeat-dosed mice with naive mice that received only one dose on that day.

Serum Cytokine Assays

See Supplemental Materials and Methods.

OTCD Model

Otcspf-ash mice were obtained from Jackson Laboratories (Stock 001811; Bar Harbor, ME, USA). Breeding took place at PhaseRx to generate hemizygous males containing the Otcspf-ash mutation. In some cases, wild-type or heterozygous littermates were used as controls. A hyperammonemia model in Otcspf-ash mice using AAV/Otc shRNA to knock down residual Otc expression was conducted essentially as described previously.29 The details of model optimization and testing are described in the Supplemental Materials and Methods and Figure S5.

Single-Dose Study

Naive Otcspf-ash male mice (7–10 weeks; n = 3) or CD1 female mice (6–8 weeks; n = 3) were given a single i.v. bolus of buffer or a formulation consisting of hOTC mRNA/HMT (3 mg/kg mRNA plus 25 mg/kg polymer; 10 mL/kg dosing volume by weight) on day 0. We selected a dose of 3 mg/kg mRNA based on initial studies where 1 mg/kg dosing did not show therapeutic effects. 25 mg/kg was selected as the polymer dose because maximal expression was achieved with luc mRNA/HMT at this dose level (Figure 3B). OTC protein expression (by western blot) and OTC enzyme activity were measured in the liver at 2, 4, 7, and 10 days after dosing. Detailed protocols for western blotting and OTC enzyme activity measurements can be found in the Supplemental Materials and Methods. Immunofluorescence was conducted at endpoint as described in the Supplemental Materials and Methods.

Repeat-Dose OTCD Efficacy Study

For the repeat-dose efficacy study, Otcspf-ash male mice (8–12 weeks) were given 100 μL of AAV2/8/Otc shRNA at 1 × 1011 genome copies (GCs) per mouse prepared by RegenXBio (Rockville, MD, USA) on day 0. The AAV2/8/Otc shRNA construct was a gift from I. Alexander. The mice were then given a formulation consisting of buffer (n = 12; twice per week), hOTC mRNA/HMT (n = 12; once per week or twice per week), or hOTC control mRNA/HMT (n = 12; once per week) (all at 3 mg/kg mRNA and 25 mg/kg polymer [10 mL/kg dosing volume by weight]) via i.v. bolus into the tail vein starting on day 4. Analyses included daily body weights, urinary orotic acid levels (days 5, 6, 7, 10, 13, 20, 27, and 34; see Supplemental Materials and Methods for procedural details), and plasma ammonia levels (days 14, 21, 28, and 35; see Supplemental Materials and Methods for procedural details). Mice were sacrificed if they had ≥20% body weight loss or signs of morbidity/ataxia. Animals removed at sacrifice were considered as deaths in survival analyses. One mouse in the buffer group and one mouse in the hOTC mRNA/HMT twice per week group were excluded from analysis because they succumbed to an anesthetic event during blood collection. Because mice treated with hOTC mRNA/HMT twice per week had extended survival, formulation dosing was stopped after day 35. Half of the hOTC mRNA/HMT mice were sacrificed on day 37 (48 hr after the last dose) to examine hOTC protein expression and endogenous mouse Otc mRNA in liver to confirm the knockdown activity of the Otc shRNA (see Supplemental Materials and Methods). The remaining half of the hOTC mRNA/HMT mice were continued to be monitored daily for body weight change and any signs of ataxia.

Repeat-Dose Safety Study in Otcspf-ash Mice

Naive Otcspf-ash male mice (8–12 weeks; n = 7–8) were given a formulation consisting of buffer (n = 8), hOTC control mRNA/HMT (n = 7), or hOTC mRNA/HMT (n = 7) administered two times per week (every 3–4 days) for nine repeat doses (all at 3 mg/kg mRNA and 25 mg/kg polymer [10 mL/kg dosing volume by weight]) via i.v. bolus into the tail vein. Mice were analyzed for ALT/AST serum chemistry parameters at 24 hr after the first dose and the final dose. A panel of cytokines was measured at 3 and 24 hr following the final dose. Histopathology was examined at 48 hr after the final dose in liver, kidney, heart, lung, and spleen tissues. Methods for serum chemistry, cytokine analysis, and histopathology are described in the Supplemental Materials and Methods.

Statistical Analyses

To detect statistical differences between various mRNA-treated groups and control groups, we performed Student’s t tests based on a two-tailed test using Excel. For statistical comparison of luminescence data, the log of the luminescence values was determined and the t test was run on the log values. For statistical comparison of groups responding over a time course, we performed two-way ANOVA using GraphPad Prism software, version 7.02 (La Jolla, CA, USA). Kaplan-Meier analysis was performed to plot the probability of survival in treated and control Otcspf-ash mice. The log rank test was used to determine the statistical significance of survival differences among treated and control groups. p < 0.05 was considered statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001). Excel and GraphPad Prism software were used to prepare graphs. Given the small sample size, tests for normality of distribution and variance were not performed.

General Methods for Animal Models

Formal sample size determination was not performed. For mouse studies examining luciferase activity, typically five animals per group were used. For mRNA and polymer distribution studies in mice and rats, typically three animals per group were used. For therapeutic effects in the Otcspf-ash mouse model, 12 animals per group were used. This is based on literature for a similar type of study.29

For studies using CD1 mice or Sprague-Dawley rats, animals were randomized by indiscriminate draw as they were removed from shipping crates and assigned to groups. For Otcspf-ash mouse studies, animals were age-matched and randomized by body weight. Hemizygous affected male littermates were typically co-housed and assigned to the same group.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Author Contributions

M.G.P. designed and supervised the studies and wrote the manuscript; P.H. prepared HMT formulations for studies; S.D.M. designed and supervised polymer synthesis; D.R. designed and performed polymer synthesis; A.G.L., T.L.B., and E.C.B. performed in vivo experiments; A.G.L., A.E.P., and M.W. performed in vitro experiments; M.W. and A.G. purified mRNA for studies; J.R.E.-M. prepared reagents for polymer synthesis; M.E.H. designed and supervised studies.

Conflicts of Interest

All authors are employees of PhaseRx, Inc. and own shares of PhaseRx.

Acknowledgments

We thank D. Durnam for assistance in preparing the manuscript, M. Rogers for help in preparing figures, and R. Overell for technical input and critical review of manuscript. We thank G. Brandt, R. Palmiter, P. Johnson, J. Schmidt, P. Stayton, and N. Sherbina for critically reading the manuscript. We thank I. Alexander for providing the AAV2/8/Otc shRNA construct. This study was funded by PhaseRx, Inc.

Footnotes

Supplemental Information includes Supplemental Materials and Methods, nine figures, and two tables and can be found with this article online at https://doi.org/10.1016/j.ymthe.2017.12.024.

Supplemental Information

Document S1. Supplemental Materials and Methods, Figures S1–S9, and Tables S1 and S2
mmc1.pdf (3.9MB, pdf)
Document S2. Article plus Supplemental Information
mmc2.pdf (12.3MB, pdf)

References

  • 1.Lichter-Konecki U., Caldovic L., Morizono H., Simpson K. Ornithine transcarbamylase deficiency. In: Adam M., Ardinger H., Pagon R., Wallace S., Bean L., Stephens K., editors. GeneReviews(R) University of Washington; Seattle: 1993. [Google Scholar]
  • 2.Ramaswamy S., Tonnu N., Tachikawa K., Limphong P., Vega J.B., Karmali P.P., Chivukula P., Verma I.M. Systemic delivery of factor IX messenger RNA for protein replacement therapy. Proc. Natl. Acad. Sci. USA. 2017;114:E1941–E1950. doi: 10.1073/pnas.1619653114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.DeRosa F., Guild B., Karve S., Smith L., Love K., Dorkin J.R., Kauffman K.J., Zhang J., Yahalom B., Anderson D.G., Heartlein M.W. Therapeutic efficacy in a hemophilia B model using a biosynthetic mRNA liver depot system. Gene Ther. 2016;23:699–707. doi: 10.1038/gt.2016.46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Hardee C.L., Arévalo-Soliz L.M., Hornstein B.D., Zechiedrich L. Advances in non-viral DNA vectors for gene therapy. Genes (Basel) 2017;8:65. doi: 10.3390/genes8020065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Hacein-Bey-Abina S., von Kalle C., Schmidt M., Le Deist F., Wulffraat N., McIntyre E., Radford I., Villeval J.L., Fraser C.C., Cavazzana-Calvo M., Fischer A. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N. Engl. J. Med. 2003;348:255–256. doi: 10.1056/NEJM200301163480314. [DOI] [PubMed] [Google Scholar]
  • 6.Mingozzi F., High K.A. Immune responses to AAV vectors: overcoming barriers to successful gene therapy. Blood. 2013;122:23–36. doi: 10.1182/blood-2013-01-306647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Calcedo R., Morizono H., Wang L., McCarter R., He J., Jones D., Batshaw M.L., Wilson J.M. Adeno-associated virus antibody profiles in newborns, children, and adolescents. Clin. Vaccine Immunol. 2011;18:1586–1588. doi: 10.1128/CVI.05107-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Raper S.E., Chirmule N., Lee F.S., Wivel N.A., Bagg A., Gao G.P., Wilson J.M., Batshaw M.L. Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol. Genet. Metab. 2003;80:148–158. doi: 10.1016/j.ymgme.2003.08.016. [DOI] [PubMed] [Google Scholar]
  • 9.Vannucci L., Lai M., Chiuppesi F., Ceccherini-Nelli L., Pistello M. Viral vectors: a look back and ahead on gene transfer technology. New Microbiol. 2013;36:1–22. [PubMed] [Google Scholar]
  • 10.Vallazza B., Petri S., Poleganov M.A., Eberle F., Kuhn A.N., Sahin U. Recombinant messenger RNA technology and its application in cancer immunotherapy, transcript replacement therapies, pluripotent stem cell induction, and beyond. Wiley Interdiscip. Rev. RNA. 2015;6:471–499. doi: 10.1002/wrna.1288. [DOI] [PubMed] [Google Scholar]
  • 11.Weissman D. mRNA transcript therapy. Expert Rev. Vaccines. 2015;14:265–281. doi: 10.1586/14760584.2015.973859. [DOI] [PubMed] [Google Scholar]
  • 12.El-Gharbawy A.H., Mackey J., DeArmey S., Westby G., Grinnell S.G., Malovrh P., Conway R., Kishnani P.S. An individually, modified approach to desensitize infants and young children with Pompe disease, and significant reactions to alglucosidase alfa infusions. Mol. Genet. Metab. 2011;104:118–122. doi: 10.1016/j.ymgme.2011.07.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Houseley J., Tollervey D. The many pathways of RNA degradation. Cell. 2009;136:763–776. doi: 10.1016/j.cell.2009.01.019. [DOI] [PubMed] [Google Scholar]
  • 14.Whitehead K.A., Langer R., Anderson D.G. Knocking down barriers: advances in siRNA delivery. Nat. Rev. Drug Discov. 2009;8:129–138. doi: 10.1038/nrd2742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Su X., Fricke J., Kavanagh D.G., Irvine D.J. In vitro and in vivo mRNA delivery using lipid-enveloped pH-responsive polymer nanoparticles. Mol. Pharm. 2011;8:774–787. doi: 10.1021/mp100390w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Convertine A.J., Benoit D.S., Duvall C.L., Hoffman A.S., Stayton P.S. Development of a novel endosomolytic diblock copolymer for siRNA delivery. J. Control. Release. 2009;133:221–229. doi: 10.1016/j.jconrel.2008.10.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Convertine A.J., Diab C., Prieve M., Paschal A., Hoffman A.S., Johnson P.H., Stayton P.S. pH-responsive polymeric micelle carriers for siRNA drugs. Biomacromolecules. 2010;11:2904–2911. doi: 10.1021/bm100652w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Lundy B.B., Convertine A., Miteva M., Stayton P.S. Neutral polymeric micelles for RNA delivery. Bioconjug. Chem. 2013;24:398–407. doi: 10.1021/bc300486k. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Ashwell G., Morell A.G. The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv. Enzymol. Relat. Areas Mol. Biol. 1974;41:99–128. doi: 10.1002/9780470122860.ch3. [DOI] [PubMed] [Google Scholar]
  • 20.Schwartz A.L., Marshak-Rothstein A., Rup D., Lodish H.F. Identification and quantification of the rat hepatocyte asialoglycoprotein receptor. Proc. Natl. Acad. Sci. USA. 1981;78:3348–3352. doi: 10.1073/pnas.78.6.3348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Rozema D.B., Lewis D.L., Wakefield D.H., Wong S.C., Klein J.J., Roesch P.L., Bertin S.L., Reppen T.W., Chu Q., Blokhin A.V. Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes. Proc. Natl. Acad. Sci. USA. 2007;104:12982–12987. doi: 10.1073/pnas.0703778104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Rozema D.B., Blokhin A.V., Wakefield D.H., Benson J.D., Carlson J.C., Klein J.J., Almeida L.J., Nicholas A.L., Hamilton H.L., Chu Q. Protease-triggered siRNA delivery vehicles. J. Control. Release. 2015;209:57–66. doi: 10.1016/j.jconrel.2015.04.012. [DOI] [PubMed] [Google Scholar]
  • 23.Adami R.C., Seth S., Harvie P., Johns R., Fam R., Fosnaugh K., Zhu T., Farber K., McCutcheon M., Goodman T.T. An amino acid-based amphoteric liposomal delivery system for systemic administration of siRNA. Mol. Ther. 2011;19:1141–1151. doi: 10.1038/mt.2011.56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Mishra N., Yadav N.P., Rai V.K., Sinha P., Yadav K.S., Jain S., Arora S. Efficient hepatic delivery of drugs: novel strategies and their significance. BioMed Res. Int. 2013;2013:382184. doi: 10.1155/2013/382184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Immordino M.L., Dosio F., Cattel L. Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential. Int. J. Nanomedicine. 2006;1:297–315. [PMC free article] [PubMed] [Google Scholar]
  • 26.Jurk M., Chikh G., Schulte B., Kritzler A., Richardt-Pargmann D., Lampron C., Luu R., Krieg A.M., Vicari A.P., Vollmer J. Immunostimulatory potential of silencing RNAs can be mediated by a non-uridine-rich toll-like receptor 7 motif. Nucleic Acid Ther. 2011;21:201–214. doi: 10.1089/nat.2011.0298. [DOI] [PubMed] [Google Scholar]
  • 27.Rosenberg L.E., Kalousek F., Orsulak M.D. Biogenesis of ornithine transcarbamylase in spfash mutant mice: two cytoplasmic precursors, one mitochondrial enzyme. Science. 1983;222:426–428. doi: 10.1126/science.6623083. [DOI] [PubMed] [Google Scholar]
  • 28.Hodges P.E., Rosenberg L.E. The spfash mouse: a missense mutation in the ornithine transcarbamylase gene also causes aberrant mRNA splicing. Proc. Natl. Acad. Sci. USA. 1989;86:4142–4146. doi: 10.1073/pnas.86.11.4142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Cunningham S.C., Kok C.Y., Dane A.P., Carpenter K., Kizana E., Kuchel P.W., Alexander I.E. Induction and prevention of severe hyperammonemia in the spfash mouse model of ornithine transcarbamylase deficiency using shRNA and rAAV-mediated gene delivery. Mol. Ther. 2011;19:854–859. doi: 10.1038/mt.2011.32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.García-Pérez M.A., Sanjurjo P., Rubio V. Demonstration of the spf-ash mutation in Spanish patients with ornithine transcarbamylase deficiency of moderate severity. Hum. Genet. 1995;95:183–186. doi: 10.1007/BF00209398. [DOI] [PubMed] [Google Scholar]
  • 31.Asano K. Why is start codon selection so precise in eukaryotes? Translation (Austin) 2014;2:e28387. doi: 10.4161/trla.28387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Youssef D., Niazi A., Alkhouri N. Long term complications in pediatric liver transplant recipients: what every pediatrician should know. Curr. Pediatr. Rev. 2016;12:209–221. doi: 10.2174/1573396312666160627124242. [DOI] [PubMed] [Google Scholar]
  • 33.Wang L., Morizono H., Lin J., Bell P., Jones D., McMenamin D., Yu H., Batshaw M.L., Wilson J.M. Preclinical evaluation of a clinical candidate AAV8 vector for ornithine transcarbamylase (OTC) deficiency reveals functional enzyme from each persisting vector genome. Mol. Genet. Metab. 2012;105:203–211. doi: 10.1016/j.ymgme.2011.10.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Cunningham S.C., Spinoulas A., Carpenter K.H., Wilcken B., Kuchel P.W., Alexander I.E. AAV2/8-mediated correction of OTC deficiency is robust in adult but not neonatal Spf(ash) mice. Mol. Ther. 2009;17:1340–1346. doi: 10.1038/mt.2009.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Yang Y., Wang L., Bell P., McMenamin D., He Z., White J., Yu H., Xu C., Morizono H., Musunuru K. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat. Biotechnol. 2016;34:334–338. doi: 10.1038/nbt.3469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Cunningham S.C., Kok C.Y., Spinoulas A., Carpenter K.H., Alexander I.E. AAV-encoded OTC activity persisting to adulthood following delivery to newborn spf(ash) mice is insufficient to prevent shRNA-induced hyperammonaemia. Gene Ther. 2013;20:1184–1187. doi: 10.1038/gt.2013.51. [DOI] [PubMed] [Google Scholar]
  • 37.Vandamme C., Adjali O., Mingozzi F. Unraveling the complex story of immune responses to AAV vectors trial after trial. Hum. Gene Ther. 2017;28:1061–1074. doi: 10.1089/hum.2017.150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Gernoux G., Wilson J.M., Mueller C. Regulatory and exhausted T cell responses to AAV capsid. Hum. Gene Ther. 2017;28:338–349. doi: 10.1089/hum.2017.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Manno C.S., Pierce G.F., Arruda V.R., Glader B., Ragni M., Rasko J.J., Ozelo M.C., Hoots K., Blatt P., Konkle B. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat. Med. 2006;12:342–347. doi: 10.1038/nm1358. [DOI] [PubMed] [Google Scholar]
  • 40.Nathwani A.C., Tuddenham E.G., Rangarajan S., Rosales C., McIntosh J., Linch D.C., Chowdary P., Riddell A., Pie A.J., Harrington C. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N. Engl. J. Med. 2011;365:2357–2365. doi: 10.1056/NEJMoa1108046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Uchida S., Itaka K., Uchida H., Hayakawa K., Ogata T., Ishii T., Fukushima S., Osada K., Kataoka K. In vivo messenger RNA introduction into the central nervous system using polyplex nanomicelle. PLoS ONE. 2013;8:e56220. doi: 10.1371/journal.pone.0056220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Baba M., Itaka K., Kondo K., Yamasoba T., Kataoka K. Treatment of neurological disorders by introducing mRNA in vivo using polyplex nanomicelles. J. Control. Release. 2015;201:41–48. doi: 10.1016/j.jconrel.2015.01.017. [DOI] [PubMed] [Google Scholar]
  • 43.Cheng C., Convertine A.J., Stayton P.S., Bryers J.D. Multifunctional triblock copolymers for intracellular messenger RNA delivery. Biomaterials. 2012;33:6868–6876. doi: 10.1016/j.biomaterials.2012.06.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Kauffman K.J., Dorkin J.R., Yang J.H., Heartlein M.W., DeRosa F., Mir F.F., Fenton O.S., Anderson D.G. Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs. Nano Lett. 2015;15:7300–7306. doi: 10.1021/acs.nanolett.5b02497. [DOI] [PubMed] [Google Scholar]
  • 45.Pardi N., Tuyishime S., Muramatsu H., Kariko K., Mui B.L., Tam Y.K., Madden T.D., Hope M.J., Weissman D. Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes. J. Control. Release. 2015;217:345–351. doi: 10.1016/j.jconrel.2015.08.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Fenton O.S., Kauffman K.J., McClellan R.L., Appel E.A., Dorkin J.R., Tibbitt M.W., Heartlein M.W., DeRosa F., Langer R., Anderson D.G. Bioinspired alkenyl amino alcohol ionizable lipid materials for highly potent in vivo mRNA delivery. Adv. Mater. 2016;28:2939–2943. doi: 10.1002/adma.201505822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Pardi N., Secreto A.J., Shan X., Debonera F., Glover J., Yi Y., Muramatsu H., Ni H., Mui B.L., Tam Y.K. Administration of nucleoside-modified mRNA encoding broadly neutralizing antibody protects humanized mice from HIV-1 challenge. Nat. Commun. 2017;8:14630. doi: 10.1038/ncomms14630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Thran M., Mukherjee J., Pönisch M., Fiedler K., Thess A., Mui B.L., Hope M.J., Tam Y.K., Horscroft N., Heidenreich R. mRNA mediates passive vaccination against infectious agents, toxins, and tumors. EMBO Mol. Med. 2017:e201707678. doi: 10.15252/emmm.201707678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Wong S.C., Klein J.J., Hamilton H.L., Chu Q., Frey C.L., Trubetskoy V.S., Hegge J., Wakefield D., Rozema D.B., Lewis D.L. Co-injection of a targeted, reversibly masked endosomolytic polymer dramatically improves the efficacy of cholesterol-conjugated small interfering RNAs in vivo. Nucleic Acid Ther. 2012;22:380–390. doi: 10.1089/nat.2012.0389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Martins A.M. Inborn errors of metabolism: a clinical overview. Sao Paulo Med. J. 1999;117:251–265. doi: 10.1590/s1516-31801999000600006. [DOI] [PubMed] [Google Scholar]
  • 51.Seymour C.A., Thomason M.J., Chalmers R.A., Addison G.M., Bain M.D., Cockburn F., Littlejohns P., Lord J., Wilcox A.H. Newborn screening for inborn errors of metabolism: a systematic review. Health Technol. Assess. 1997;1:i–iv. 1–95. [PubMed] [Google Scholar]
  • 52.Mew, N. (2017). Ornithine transcarbamylase deficiency. https://rarediseases.org/rare-diseases/ornithine-transcarbamylase-deficiency/.
  • 53.Chiefari J., Chong Y.K., Ercole F., Krstina J., Jeffery J., Le T.P.T., Mayadunne R.T.A., Meijs G.G., Moad C.L., Moad G. Living free-radical polymerization by reversible addition−fragmentation chain transfer: the RAFT process. Macromolecules. 1998;31:5559–5562. [Google Scholar]
  • 54.Karikó K., Buckstein M., Ni H., Weissman D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity. 2005;23:165–175. doi: 10.1016/j.immuni.2005.06.008. [DOI] [PubMed] [Google Scholar]
  • 55.Sahin U., Karikó K., Türeci Ö. mRNA-based therapeutics—developing a new class of drugs. Nat. Rev. Drug Discov. 2014;13:759–780. doi: 10.1038/nrd4278. [DOI] [PubMed] [Google Scholar]
  • 56.Ye X., Zimmer K.P., Brown R., Pabin C., Batshaw M.L., Wilson J.M., Robinson M.B. Differences in the human and mouse amino-terminal leader peptides of ornithine transcarbamylase affect mitochondrial import and efficacy of adenoviral vectors. Hum. Gene Ther. 2001;12:1035–1046. doi: 10.1089/104303401750214267. [DOI] [PubMed] [Google Scholar]
  • 57.Karikó K., Muramatsu H., Ludwig J., Weissman D. Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Res. 2011;39:e142. doi: 10.1093/nar/gkr695. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Supplemental Materials and Methods, Figures S1–S9, and Tables S1 and S2
mmc1.pdf (3.9MB, pdf)
Document S2. Article plus Supplemental Information
mmc2.pdf (12.3MB, pdf)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from Molecular Therapy are provided here courtesy of The American Society of Gene & Cell Therapy

RESOURCES