Volume 9, Issue 6, Pages 866-875 (June 2004) Correction of metabolic, craniofacial, and neurologic abnormalities in MPS I mice treated at birth with adeno-associated virus vector transducing the human α-l-iduronidase gene  Seth D Hartung, Joel L Frandsen, Dao Pan, Brenda L Koniar, Patrick Graupman, Roland Gunther, Walter C Low, Chester B Whitley, R.Scott McIvor  Molecular Therapy  Volume 9, Issue 6, Pages 866-875 (June 2004) DOI: 10.1016/j.ymthe.2004.03.011 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Plasma IDUA activity and urine GAG in AAV-IDUA-treated mice. (A) AAV-IDUA plasmid pTRCA1, constructed as described in Materials and Methods. TR, AAV inverted terminal repeat; CMV/BA, CMV early enhancer/chicken β-actin promoter; G, chicken β-globin intron; IDUA, human α-l-iduronidase cDNA; pA, bovine growth hormone polyadenylation signal. (B) Plasma IDUA activity in control-treated normal and affected MPS I littermates. One-day-old mice were injected via the superficial temporal vein with 1010 particles of control AAV-gfp vector. Blood plasma was obtained as described in Materials and Methods and tested for IDUA activity at the indicated times after vector injection. (○) Animals 62, (▵) 63, Idua+/+; (□) 37, (♦) 38, Idua+/−; (⊞) 36, 39, 65, 70, Idua−/−. (C) Plasma IDUA activity in vTRCA1-treated Idua−/− animals. One-day-old mice were injected via the superficial temporal vein with 1010 particles of vTRCA1. Blood plasma was obtained as described in Materials and Methods and tested for IDUA activity at the indicated times after vector injection. (□) Animals 26, ( ) 27, (▪) 28, (▵) 30, (⊞) 31, (♦) 33, (⊕) 35, (▿) 41, (□) 44, (♦) 45. ( ) Mean IDUA activity. (D) Urine GAG. GAG, glycosaminoglycan; cr, creatinine. Beginning 1 month after vector injection, urine was collected and GAG was quantitated as described in Materials and Methods. For Idua−/− and Idua+/−, graph depicts the mean (+SD) of n = 4 animals. For vTRCA1-treated Idua−/− animals, graph depicts the mean (+SD) of n = 9 animals through month 3 and depicts the mean of n = 7 animals for months 4 and 5. *P < 0.05 between Idua−/− and vTRCA1-treated Idua−/− animals. **P < 0.005 between Idua−/− and vTRCA1-treated Idua−/− animals. Idua+/−, untreated heterozygous controls; Idua−/−, AAV-gfp-treated homozygous null controls. Molecular Therapy 2004 9, 866-875DOI: (10.1016/j.ymthe.2004.03.011) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Effects of AAV-IDUA treatment on Idua−/− weights. Animals were weighed every 4 weeks beginning 1 month after vector injection. Bars depict the mean percentage difference from same sex Idua+/− control (±SEM) for n = 6 animals per group. At each time point, the heterozygous control group is defined as 100%. Molecular Therapy 2004 9, 866-875DOI: (10.1016/j.ymthe.2004.03.011) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Histopathological analysis of tissues for storage material. Group designations are as described for previous figures. One square millimeter, 1-μm-thick sections were stained with toluidine blue, observed at 100× magnification, and scored for pathology as described in Materials and Methods. For liver, heart, and brain, sections from animals 35 and 27 are depicted as examples of uncorrected and corrected pathology, respectively, in the vTRCA1-treated group. In the brain, sections from animals 26 and 45 are depicted as examples of uncorrected and corrected pathology, respectively, in the vTRCA1-treated group. (A–D) Histopathology of liver. H, hepatocyte; Ku, Kupffer cell. Arrows indicate extensive cytoplasmic vacuolization of hepatocytes and Kupffer cells. (E–H) Histopathology of heart. (I–L) Histopathology of lung. (M–P) Histopathology of cerebral cortex. Arrow indicates perivascular cells distended by vacuoles. (Q–T) Histopathology of cerebellum. Arrow indicates large vacuoles in condensed Purkinje cells. (U–X) Histopathology of hippocampus. Arrow indicates perivascular cells distended by vacuoles. Molecular Therapy 2004 9, 866-875DOI: (10.1016/j.ymthe.2004.03.011) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Effects of AAV-IDUA treatment on craniofacial parameters. At 20 weeks after vector injection, mice were anesthetized, computed tomography was performed, and radiological distances were calculated as described in Materials and Methods. Graphs depict the means of n = 12 (±SEM) for Idua+/− and Idua−/− control groups and n = 4 for the vTRCA1-treated Idua−/− group. (A and C) P < 0.05 between Idua−/− control and vTRCA1-treated Idua−/− animals. Molecular Therapy 2004 9, 866-875DOI: (10.1016/j.ymthe.2004.03.011) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Open field test of habituation. At 16 weeks post-vector injection, animals were tested in three trials of 5 min per trial in the open field as described under Materials and Methods. Percentage difference describes the % change between the third and the first trial. Bars depict the means (±SEM) of n = 12 for all control groups and n = 7 for the vTRCA1-treated Idua−/− group. *P < 0.05 between Idua−/− control and vTRCA1-treated Idua−/− animals. Molecular Therapy 2004 9, 866-875DOI: (10.1016/j.ymthe.2004.03.011) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions