Targeted mRNA Therapy for Ornithine Transcarbamylase Deficiency Mary G. Prieve, Pierrot Harvie, Sean D. Monahan, Debashish Roy, Allen G. Li, Teri L. Blevins, Amber E. Paschal, Matt Waldheim, Eric C. Bell, Anna Galperin, Jean-Rene Ella-Menye, Michael E. Houston  Molecular Therapy  Volume 26, Issue 3, Pages 801-813 (March 2018) DOI: 10.1016/j.ymthe.2017.12.024 Copyright © 2018 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 1 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. Molecular Therapy 2018 26, 801-813DOI: (10.1016/j.ymthe.2017.12.024) Copyright © 2018 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 2 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. Molecular Therapy 2018 26, 801-813DOI: (10.1016/j.ymthe.2017.12.024) Copyright © 2018 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 3 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. Molecular Therapy 2018 26, 801-813DOI: (10.1016/j.ymthe.2017.12.024) Copyright © 2018 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 4 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. Molecular Therapy 2018 26, 801-813DOI: (10.1016/j.ymthe.2017.12.024) Copyright © 2018 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 5 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. Molecular Therapy 2018 26, 801-813DOI: (10.1016/j.ymthe.2017.12.024) Copyright © 2018 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 6 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. Molecular Therapy 2018 26, 801-813DOI: (10.1016/j.ymthe.2017.12.024) Copyright © 2018 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 7 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. Molecular Therapy 2018 26, 801-813DOI: (10.1016/j.ymthe.2017.12.024) Copyright © 2018 The American Society of Gene and Cell Therapy Terms and Conditions