Volume 17, Issue 7, Pages 1155-1163 (July 2009) Short-term Correction of Arginase Deficiency in a Neonatal Murine Model With a Helper- dependent Adenoviral Vector  Chia-Ling Gau, Robin A Rosenblatt, Vincenzo Cerullo, Fides D Lay, Adrienne C Dow, Justin Livesay, Nicola Brunetti-Pierri, Brendan Lee, Stephen D Cederbaum, Wayne W Grody, Gerald S Lipshutz  Molecular Therapy  Volume 17, Issue 7, Pages 1155-1163 (July 2009) DOI: 10.1038/mt.2009.65 Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 1 Schematic of HDV-mAI. The figure (not drawn to scale) shows the relevant portions of the helper-dependent adenoviral vectors containing (a) the mAI cDNA or (b) LacZ cDNA downstream of the liver specific PEPCK promoter and the ApoAI intron. ApoAI, ApoAI intron; HDV, helper-dependent adenoviral vector; hGH pA, human growth hormone polyA; ITR, inverted terminal repeat; LacZ, β-galactosidase; mAI, murine arginase I; WPRE, woodchuck postregulatory enhancer; ψ, packaging signal. Molecular Therapy 2009 17, 1155-1163DOI: (10.1038/mt.2009.65) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 2 Survival of AI knockout mice. Arg+/− mice were mated to produce litters containing arg+/+, arg+/−, and arg−/− mAI mice. All pups were injected intravenously via the superficial temporal vein between 2 and 3 days of age with either pharmaceutical grade saline or 5 × 109 viral particles/g (5 × 1012 vp/kg) of HDV-mAI. AI knockout mice injected with HDV-mAI survived up to 27 days compared to saline-injected knockout mice, which survive up to 14 days postnatally. HDV, helper-dependent adenoviral vector. Molecular Therapy 2009 17, 1155-1163DOI: (10.1038/mt.2009.65) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 3 Distribution of HDV after neonatal intravenous injection is widespread. (a) Biodistribution of helper-dependent adenoviral vector (HDV). Arg+/− mice were injected on the second day of life with 5 × 109 viral particles/g HDV-LacZ. Two days later, mice (n = 4) were euthanized and tissues were removed. Biodistribution of the hepatotropic HDV vector was determined by PCR. (b) Histochemical hepatic staining for helper-dependent adenoviral vector. X-gal staining of the liver was performed on the eighteenth day of life from mice that received a neonatal injection of HDV-LacZ. X-gal staining is detected in hepatocytes with no preference for any specific lobular location. Molecular Therapy 2009 17, 1155-1163DOI: (10.1038/mt.2009.65) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 4 Growth of mouse and liver from neonate to adult. Both saline-injected and HDV-injected mice were euthanized at predetermined time points (n = 3–8 per time point). (a) Animals and (b) livers, after removal, were weighed. The mouse liver generally represents 4.5–5.5% of the weight of a lean C57BI/6 mouse (g = grams). HDV, helper-dependent adenoviral vector. Molecular Therapy 2009 17, 1155-1163DOI: (10.1038/mt.2009.65) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 5 Change in viral copies and message over time. (a) Viral copy number per cell was determined by quantitative real-time PCR, whereas (b) mRNA copies were similarly determined after reverse transcription. Molecular Therapy 2009 17, 1155-1163DOI: (10.1038/mt.2009.65) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 6 Arginase expression by HDV-mAI decreases with time. (a) Tissue expression of viral RNA over time. Total RNA was isolated from tissues of HDV-mAI injected heterozygous mice at various ages (d = day). cDNA was synthesized and amplified using primers specific to the viral cDNA and analyzed by ethidium bromide-stained gel electrophoresis. The expected fragment sizes for HDV-mAI and β-actin cDNA are 750 and 350 bp, respectively; β-actin gDNA fragment size is 1 Kb. HDV-mAI cDNA was expressed only in the liver (1) and not in the brain (2). (b) Protein expression of HDV-mAI. Total protein was isolated from mice injected with HDV-mAI at postnatal days 14 or 25 (d = day). Western blot analysis demonstrated that arg−/− mice injected with HDV-mAI expressed subendogenous levels of arginase (arginase molecular size is 35 kd). Protein expression was greatly reduced when assayed on day 25. (c) Liver arginase activity of HDV-mAI-injected mice. Total protein was isolated from the livers of mice injected with either saline or HDV-mAI. Arginase activity was measured with a colorimetric assay determining the quantity of urea converted from arginine by each tissue lysate. Saline-injected arg−/− mice had virtually no arginase activity, whereas the livers of HDV-mAI-injected arg−/− mice at postnatal day 14 have ∼20% of the arginase activity of heterozygous mice. By day 26, when the HDV-mAI-injected arg−/− mice have a short remaining lifespan, the hepatic arginase activity is <10% of endogenous activity. Error bars represent mean ± SD. HDV, helper-dependent adenoviral vector; KO, knockout; mAI, murine AI. Molecular Therapy 2009 17, 1155-1163DOI: (10.1038/mt.2009.65) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 7 Antibodies against adenoviral capsid proteins and against arginase. (a) Antiadenoviral-specific ELISA was performed on day 25 of mice injected with either saline or HDV-LacZ on the second day of life. There were no differences between the virus-injected or saline-injected groups. (b) Antiarginase-specific ELISA was performed on day 25 of arg−/− mice injected as neonates on the second day of life. There was no development of antiarginase antibodies (n ≥ 3 per group). Error bars represent mean ± SD. HDV, helper-dependent adenoviral vector; mAI, murine AI. Molecular Therapy 2009 17, 1155-1163DOI: (10.1038/mt.2009.65) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 8 Near correction of metabolic abnormalities occurs at day 14 of life in HDV-injected mice, but is lost by day 26. (a) Plasma ammonia of HDV-mAI injected mice. Plasma of mice injected with saline or HDV-mAI was collected at postnatal day 14 or 27 by cardiac puncture or retro-orbital bleeding (n = 9) for saline injected arg−/− mice at day 14, n = 6 for HDV-mAI injected arg−/− mice at day 14, n = 4 for HDV-mAI injected arg−/− mice at day 27, and n = 3 for arg+/− mice at each time point). At postnatal day 14, saline-injected arg−/− mice are on the verge of death and exhibit hyperammonemia, whereas arg−/− mice injected with HDV-mAI have levels of ammonia similar to those in uninjected heterozygous mice. By day 26, arg−/− mice that had received HDV-mAI, but which did not yet exhibit a tremor phenotype, had increased ammonia levels over heterozygous controls. Error bars represent mean ± SD. (b,c) Hepatic arginine and ornithine levels in HDV-mAI injected mice. Liver tissue was collected from HDV-mAI-injected mice at postnatal day 14 and 26 (n = 6 mice of each genotype at each time point). Arg−/− mice injected with HDV-mAI exhibited lower levels of arginine (b) and normalization of ornithine (c) at postnatal day 14 compared to saline-injected mice. At day 26, arginine levels have increased, indicating the accumulation of arginine with the loss of the viral arginase activity. Ornithine levels at day 26 are greatly increased, which is the opposite of uninjected AI-deficient mice that exhibit ornithine deficiency. (d) Relative OAT expression in HDV-mAI- and saline-injected mice. Liver tissue was collected from arg+/− mice at day 14 and day 26, along with arg−/− saline-injected at day 14 of life. OAT expression by RT-qPCR was compared with HDV-mAI-injected mice at day 14 and day 26. Day 14 HDV-mAI-injected mice demonstrate lower levels of OAT expression likely due to near normalization of hepatic arginase activity. Although OAT expression at day 26 is less than two times that of day 26 arg+/− mice, the mechanism of hepatic hyperornithine levels remains unexplained. HDV, helper-dependent adenoviral vector; Het, heterozygous; KO, knockout; mAI, murine AI; OAT, ornithine aminotransferase. Error bars represent mean ± SD. Molecular Therapy 2009 17, 1155-1163DOI: (10.1038/mt.2009.65) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions