Volume 18, Issue 6, Pages 1076-1084 (June 2010) Complete Normalization of Hepatic G6PC Deficiency in Murine Glycogen Storage Disease Type Ia Using Gene Therapy  Wai Han Yiu, Young Mok Lee, Wen-Tao Peng, Chi-Jiunn Pan, Paul A Mead, Brian C Mansfield, Janice Y Chou  Molecular Therapy  Volume 18, Issue 6, Pages 1076-1084 (June 2010) DOI: 10.1038/mt.2010.64 Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Histochemical analysis of G6Pase-α activity in the liver of AAV-GPE and AAV-CBA infused G6pc−/− mice. Freshly sectioned liver specimens were analyzed for G6Pase-α activity using the method of lead trapping of phosphate generated by G6P hydrolysis.29 The age of the mice when infused is indicated at the far left (2 days, 2 weeks, or 4 weeks). The age of the mice at the time of sectioning is indicated by the labeling closest to the panels: 2W, 2 weeks; 4W, 4 weeks; 6W, 6 weeks; 24W, 24 weeks of age. Treatments are indicated by: (−/−) untreated G6pc−/−; (+/+) untreated G6pc+/+; (−/− GPE) G6pc−/− mice infused with 1.2 × 1011 vg/mouse of AAV-GPE; (−/− CBA) G6pc−/− mice infused with 4.8 × 1011 vg/mouse of AAV-CBA. In each pair of images, the left panel is ×50 magnification and the right panel ×200. AAV, adeno-associated virus; CBA, chicken β-actin; GPE, G6PC promoter/enhancer; G6P, glucose-6-phosphate. Molecular Therapy 2010 18, 1076-1084DOI: (10.1038/mt.2010.64) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Phenotype analysis of AAV-GPE-infused G6pc−/− mice. (a) Postnatal development. Data for female G6pc−/− mice infused with 1.2 × 1011 vg/mouse of AAV-GPE and their female G6pc+/+/G6pc+/− littermates are shown. The age at infusion is shown above the graph. (open circles), G6pc+/+/G6pc+/− mice; (closed circles), AAV-GPE-infused G6pc−/− mice. (b) Blood glucose, cholesterol, triglyceride, uric acid, and lactic acid levels. Because of the similarities of the respective metabolites in each group, data shown are pooled data of age 6–24 weeks. (+/+ & +/−), G6pc+/+/G6pc+/−, (−/−), G6pc−/−, or (−/− GPE), G6pc−/− mice infused with AAV-GPE at age 2 days (n = 36), 2 weeks (n = 24), or 4 weeks (n = 9). Data are presented as mean ± SEM. *P < 0.05 and **P < 0.005. AAV, adeno-associated virus; GPE, G6PC promoter/enhancer. Molecular Therapy 2010 18, 1076-1084DOI: (10.1038/mt.2010.64) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Histological and liver glycogen and lipid analyses in wild-type and AAV-GPE-infused G6pc−/− mice at 24 weeks of age. Representative results are shown. (+/+), G6pc+/+/G6pc+/− mice (n = 5) and (−/− GPE) G6pc−/− (−/−) mice infused with 1.2 × 1011 vg/mouse of AAV-GPE mice at age 2 days (n = 3), 2 weeks (n = 3), or 4 weeks (n = 3). (a) H&E stained liver sections at original magnifications of ×200 and hepatic glycogen contents. Each plate represents an individual mouse, so two mice are shown for each age of infusion. For quantitation, the data for glycogen are presented as mean ± SEM. **P < 0.005. (b) Oil red O staining at original magnifications of ×400 and quantitative histochemical measurement of lipid deposition. For quantitation, the lipid imaged with Oil red O staining was converted into pixel density units using Adobe Photoshop and data are presented as mean ± SEM. AAV, adeno-associated virus; GPE, G6PC promoter/enhancer. Molecular Therapy 2010 18, 1076-1084DOI: (10.1038/mt.2010.64) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Glucose homeostasis in AAV-GPE-infused G6pc−/− mice. G6pc−/− mice were infused with 1.2 × 1011 vg/mouse of AAV-GPE at age 2 weeks or 4 weeks and fasting blood glucose or glucose tolerance analyses were conducted at 10 weeks post-infusion at age 12 weeks (n = 4) and 14 weeks (n = 4), respectively. Age matched G6pc+/+/G6pc+/− mice (n = 4) were used as controls. (a) Fasting blood glucose analysis. (b) Glucose tolerance test. G6pc+/+/G6pc+/− or AAV-GPE-infused G6pc−/− mice were fasted for 6 hours, injected subcutaneously with 0.25 ml of 10% dextrose, then sampled for blood every 30 minutes via the tail vein. Data are presented as mean ± SEM. (open circles), G6pc+/+/G6pc+/− mice; (closed circles), AAV-GPE-infused G6pc−/− mice. AAV, adeno-associated virus; GPE, G6PC promoter/enhancer. Molecular Therapy 2010 18, 1076-1084DOI: (10.1038/mt.2010.64) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Analysis of anti-G6Pase-α antibodies and hepatic infiltration of CD8+ lymphocytes. Microsomal proteins from Ad-human G6Pase-α or Ad-mouse G6Pase-α infected COS-1 cells were electrophoresed through a single 12% polyacrylamide-SDS gel and transferred onto a PVDF membrane. Membrane strips, representing individual lanes on the gel were individually incubated with the appropriate antiserum. The antigen–antibody complex was visualized as described under Materials and Methods. (a) Antibodies against human G6Pase-α. Lanes 1 and 2: anti-human (h) G6Pase-α antiserum (1:3000 dilution); lanes 3 and 16: serum samples (1:200 dilution) from G6pc−/− mice (−/−), lanes 4, 6, 8, 10, 12, and 14: serum samples (1:200 dilution) from 24-week-old G6pc+/+/G6pc+/− (+/+) mice, or serum samples (1:200 dilution) from 24-week-old G6pc−/− mice infused with AAV-GPE (−/− GPE) at age 2 days (lanes, 5 and 7), 2 weeks (lanes 9 and 11), or age 4 weeks (lanes 13 and 15). (b) Antibodies against mouse G6Pase-α. Lanes 1 and 3: serum samples (1:200 dilution) from 24-week-old G6pc+/+/G6pc+/− mice, lanes 2 and 4: serum samples (1:200 dilution) from 24-week-old G6pc−/− mice infused with AAV-CBA (−/− CBA) at age 2 weeks, lane 5, serum sample (1:200 dilution) from G6pc−/− mice, lanes 6 and 7; anti-hG6Pase-α antiserum (1:3000 dilution) that recognizes murine G6Pase-α.25 Each lane denotes an individual mouse. The arrow denotes G6Pase-α. (c) Quantification of hepatic CD8+ lymphocyte infiltration. CD8+ lymphocytes were scored at age 2 weeks in G6pc−/− mice infused with AAV-GPE (−/− GPE) or AAV-CBA (−/− CBA) at age 2 days or at age 4 weeks in G6pc−/− mice infused with AAV-GPE or AAV-CBA at age 2 weeks; 2- and 4-week-old G6pc+/+/G6pc+/− (+/+) and G6pc−/− (−/−) mice were used as controls. Results are the mean ± SEM. Each point represents the average of three to four animals. **P < 0.005. AAV, adeno-associated virus; CBA, chicken β-actin; GPE, G6PC promoter/enhancer; PVDF, polyvinylidene fluoride; SDS, sodium dodecyl sulfate. Molecular Therapy 2010 18, 1076-1084DOI: (10.1038/mt.2010.64) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions