Volume 9, Issue 2, Pages 231-240 (February 2004) AAV2 Vector Harboring a Liver-Restricted Promoter Facilitates Sustained Expression of Therapeutic Levels of α-Galactosidase A and the Induction of Immune Tolerance in Fabry Mice  Robin J Ziegler, Scott M Lonning, Donna Armentano, Chester Li, David W Souza, Maribeth Cherry, Christine Ford, Christine M Barbon, Robert J Desnick, Guangping Gao, James M Wilson, Richard Peluso, Simon Godwin, Barrie J Carter, Richard J Gregory, Samuel C Wadsworth, Seng H Cheng  Molecular Therapy  Volume 9, Issue 2, Pages 231-240 (February 2004) DOI: 10.1016/j.ymthe.2003.11.015 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Efficacy of administration of AAV2/CMVHI-αgal into Fabry mice. Seven-month-old immune-suppressed Fabry mice were administered 5 × 1011 particles of AAV2/CMVHI-αgal via the tail vein. Animals were killed at 1, 2, and 3 months posttreatment and their organs analyzed for the levels of (A) α-galactosidase A and (B) GL-3. An ELISA specific for human α-galactosidase A was used to measure the enzyme levels in the different tissue homogenates. The shaded area within the graph represents the range of α-galactosidase A levels observed in normal (C57BL/6) mouse tissues. To measure the GL-3 levels, an ELISA that was based on the affinity of Escherichia coli verotoxin to bind the glycosphingolipid was used. Data are expressed as means ± SEM (n = 4 animals/time point). Molecular Therapy 2004 9, 231-240DOI: (10.1016/j.ymthe.2003.11.015) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Expression of α-galactosidase A from a CMV and a liver-restricted promoter. Six-week-old male BALB/c mice were injected intravenously with vehicle or with 5 × 1011 particles of either AAV2/CMVHI-αgal or AAV2/DC190-αgal. Animals were killed at 1, 2, and 3 months postinjection and various organs collected for analysis. Blood was also collected by eye bleed at the same time intervals. An ELISA specific for human α-galactosidase A was used to detect protein levels in the different tissue homogenates and serum samples. The shaded areas within the graphs represent the range of α-galactosidase A levels observed in normal (C57BL/6) mouse tissues. Data are expressed as means ± SEM (n = 5 animals/time point). Molecular Therapy 2004 9, 231-240DOI: (10.1016/j.ymthe.2003.11.015) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Longevity of expression of α-galactosidase A from the liver-restricted promoter. Six-week-old male BALB/c mice were injected intravenously with 3 × 1011 particles of AAV2/DC190-αgal. Blood was collected periodically by eye bleed starting at day 30 until day 340 posttreatment. An ELISA specific for human α-galactosidase A was used to detect protein levels in the serum samples. Open circles represent values in individual mice and the closed circles, the mean (n = 5 animals/time point). Molecular Therapy 2004 9, 231-240DOI: (10.1016/j.ymthe.2003.11.015) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Quantitation of viral DNA and α-galactosidase A mRNA in various tissues following intravenous administration of the recombinant AAV vectors. Six-week-old male BALB/c mice were injected intravenously with 3 × 1011 particles of either AAV2/CMVHI-αgal or AAV2/DC190-αgal. Animals were killed 30 days later; their organs were harvested and then assayed for the presence of (A) AAV genomes and (B) human α-galactosidase A mRNA. The shaded areas within the graphs represent values that are below the range of reliable quantitation. Data are expressed as means ± SEM (n = 5 animals). Molecular Therapy 2004 9, 231-240DOI: (10.1016/j.ymthe.2003.11.015) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Titers of antibodies to human α-galactosidase A and AAV2 in BALB/c mice. Groups of 4 male BALB/c mice were injected intravenously with vehicle or 3 × 1011 particles of either AAV2/CMVHI-αgal or AAV2/DC190-αgal. Mice were bled prior to treatment and subsequently on months 1, 2, and 3 posttreatment. Antibodies to (A) human α-galactosidase A and (B) AAV2 were quantitated using an ELISA as described under Materials and Methods. Data are expressed as means ± SEM (n = 5 animals/time point). Molecular Therapy 2004 9, 231-240DOI: (10.1016/j.ymthe.2003.11.015) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Serum levels of α-galactosidase A and antibodies to α-galactosidase A following administration of AAV2/DC190-αgal and subsequent challenge with purified recombinant enzyme. Groups of 4 male BALB/c mice were administered increasing amounts of AAV2/DC190-αgal via the tail vein. (A) Mice were bled 88 days later and the serum levels of α-galactosidase A quantitated by ELISA. (B) At 6 months posttreatment, the mice were challenged intraperitoneally with 50 μg of purified recombinant human α-galactosidase A that had been emulsified in Complete Freund's adjuvant. The animals were bled 38 days following the challenge and the serum levels of anti-α-galactosidase A antibodies determined. The open circles represent individual animals and the closed circles the means of all the values. No PreRx refers to the group of mice that had not been treated with AAV2/DC190-αgal. IV, intravenous; IP, intraperitoneal. Molecular Therapy 2004 9, 231-240DOI: (10.1016/j.ymthe.2003.11.015) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 7 Efficacy of administration of AAV2/DC190-αgal into Fabry mice. Four-month-old male Fabry mice were injected intravenously with 5 × 1011 particles of AAV2/DC190-αgal via the tail vein. Animals were killed at 1, 2, and 3 months posttreatment and their organs analyzed for the levels of (A) α-galactosidase A and (B) GL-3. An ELISA specific for human α-galactosidase A was used to measure the enzyme levels in the different tissue homogenates. The shaded area within the graph represents the range of α-galactosidase A levels observed in normal (C57BL/6) mouse tissues. To measure the GL-3 levels, an ELISA that was based on the affinity of E. coli verotoxin to bind the glycosphingolipid was used. Data are expressed as means ± SEM (n = 4 animals/time point). Molecular Therapy 2004 9, 231-240DOI: (10.1016/j.ymthe.2003.11.015) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions