Volume 22, Issue 10, Pages 1792-1802 (October 2014) Myocyte-mediated Arginase Expression Controls Hyperargininemia but not Hyperammonemia in Arginase-deficient Mice  Chuhong Hu, Jennifer Kasten, Hana Park, Ragini Bhargava, Denise S Tai, Wayne W Grody, Quynh G Nguyen, Stephen D Hauschka, Stephen D Cederbaum, Gerald S Lipshutz  Molecular Therapy  Volume 22, Issue 10, Pages 1792-1802 (October 2014) DOI: 10.1038/mt.2014.99 Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Codon-optimized arginase cDNA improves arginase expression levels. HEK293 cells were transfected with plasmids to assess the function of a codon-optimized version of murine arginase compared to the wild-type cDNA; studies were performed in duplicate. Plasmid DNA was transfected into 293 cells and arginase activity was examined 2 days later. Molecular Therapy 2014 22, 1792-1802DOI: (10.1038/mt.2014.99) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Whole-mouse luciferase expression to assess promoter activity and tissue specificity. Mice were injected intravenously with 3 × 1012 genome copies/kg of AAV serotype rh10 expressing luciferase on neonatal day 2. At 3 weeks of age, whole-animal bioluminescent imaging and tissue luminometry was performed to assess expression in different tissues in wild-type animals. (a) In vivo bioluminescent imaging demonstrates photon diffusion patterns among representative images. Left panel: CBA promoter exhibits expression in the heart (red arrow) and the liver (white arrow). Middle panel: Liver-specific thyroxine-binding globulin promoter exhibits expression in the liver (white arrow). Right panel: The striated muscle-specific CK8 regulatory cassette exhibits expression in the heart (red arrow) and skeletal muscle (orange arrow). For all groups, images were acquired with the mice in the ventral position. Images were set with the same references such that side-by-side comparison can be made. After removal of individual tissues, levels of luciferase protein expressed as relative light units (RLU) per µg protein was compared in skeletal muscle (b), the heart (c), and the liver (d). (Data is presented as mean + SD with n of 3–5 per tissue.) Molecular Therapy 2014 22, 1792-1802DOI: (10.1038/mt.2014.99) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Rescue of ARG1−/−mice. Survival comparisons in days between untreated ARG1−/− mice (n = 38), littermate controls (n = 34) (heterozygotes), and ARG1−/− mice injected with rAAVrh10-CBA-ARG1-WPRE (n = 45), rAAVrh10-TBG-coARG1 (n = 42), rAAVrh10-CK8-coARG1 (n = 8), rAAVrh10-TBG-luciferase (n = 4), and rAAVrh10-CK8-luciferase (n = 5). (*= end of study). Molecular Therapy 2014 22, 1792-1802DOI: (10.1038/mt.2014.99) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Arginase activity in different tissues depends on the promoter. Total protein was isolated from liver, heart, kidney, and skeletal muscle of either control littermate heterozygotes (a) or ARG1−/− mice that were injected intravenously on neonatal day two with AAV carrying either the (b) chicken β-actin promoter/CMV enhancer or (c) the liver-specific thyroxine-binding globulin promoter linked to the codon-optimized ARG1 cDNA. Arginase activity was measured with a colorimetric assay determining the quantity of urea converted from arginine by each tissue lysate. Results are expressed as mean ± SD. (Heterozygote, CBA, TBG, respectively: 7 days: n = 23, 9, 12; 1 month: n = 8, 9, 6; 2 months: n = 10, 7, 4; 4 months: n = 4, 4, 4; 8 months: n = 4, 4, 5.) Molecular Therapy 2014 22, 1792-1802DOI: (10.1038/mt.2014.99) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Immunohistochemical analysis of cardiac and skeletal muscle expression of arginase following rAAV-ARG1 administration to ARG1−/−neonatal mice. Neonatal ARG1−/− mice were intravenously injected with AAV-TBG-coARG1, AAV-CK8-coARG1 or AAV-CBA-ARG1. Immunostaining for murine arginase is shown for cardiac muscle: (a) AAV-TBG-coARG1; (b) AAV-CK8-coARG1; (c) AAV-CBA-ARG1) and skeletal muscle: (d) AAV-TBG-coARG1; (e) AAV-CK8-coARG1; (f) AAV-CBA-ARG1). TBG and CBA mice were 4 months old when killed and CK8 mice were 2 weeks old when killed due to their hyperammonemic symptoms. (g) AAV genome copy numbers in liver, skeletal and cardiac muscle were analyzed at selected time points, and are plotted as the mean ± SD (n = 3–5 per group). AAV, adeno-associated viral vector. Molecular Therapy 2014 22, 1792-1802DOI: (10.1038/mt.2014.99) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Improvement of plasma urea cycle metabolite levels following neonatal delivery of rAAV-TBG-coARG1. Plasma metabolite levels were measured at ~1 and 3 weeks, and then monthly thereafter in the AAV-TBG-coARG1-injected ARG1−/− animals. Metabolites examined were: (a) ammonia, (b) arginine, and (c) glutamine. In (a) ammonia levels in untreated ARG1−/− mice were measured in clinically ill animals as these mice all died from hyperammonemia by day 18. Heterozygous controls were included for comparisons. All samples levels are plotted as mean ± SD, n = 3–4 per group. AAV, adeno-associated viral vector. Molecular Therapy 2014 22, 1792-1802DOI: (10.1038/mt.2014.99) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Immunohistochemical detection of arginase expression in hepatocytes at progressive times following neonatal delivery of rAAV-TBG-coARG1 to ARG1−/−mice. Representative images show arginase in the hepatocytes of: (a) untreated wild type age day 7 and (b) untreated ARG1−/− day 7 mice and in AAV-TBG-treated ARG1−/− mice: (c) AAV-TBG-treated ARG1−/− day 7 of age, (d) AAV-TBG-treated ARG1−/− 1 month of age, (e) AAV-TBG-treated ARG1−/− 2 months of age, (f) AAV-TBG-treated ARG1−/− 4 months of age, and (g) AAV-TBG-treated ARG1−/− 8 months of age following rAAV-TBG-coARG1 delivery. AAV, adeno-associated viral vector. Molecular Therapy 2014 22, 1792-1802DOI: (10.1038/mt.2014.99) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 Plasma ammonia and arginine and arginase activity in selected organs after AAV-CK8-coARG1 delivery to neonatal mice. Animals were examined 2 weeks after intravenous administration of AAV-CK8-coARG1. Plasma arginine (a) and ammonia (c) in these mice was compared to untreated ARG1−/− and heterozygote littermate control mice. Selected organs and tissues (b) were examined for arginase expression (heterozygotes: liver, n = 5; heart, n = 9; skeletal muscle of abdomen, n = 9, lower extremity n = 5, upper extremity n = 5; and AAV-CK8-coARG1-injected ARG1−/−: liver, n = 6; heart, n = 6; skeletal muscle of abdomen, n = 9, lower extremity, n = 6, upper extremity, n = 6). Hepatic arginine was examined also (d) and compared with untreated ARG1−/−, heterozygote, and ARG1−/− mice treated with AAV-TBG-coARG1 (heterozygotes n = 5, ARG1−/− untreated n = 4, ARG1−/− AAV-TBG-treated n = 5, AAV-CK8-treated n = 6). All samples are expressed as mean ± SD. AAV, adeno-associated viral vector. Molecular Therapy 2014 22, 1792-1802DOI: (10.1038/mt.2014.99) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions