Volume 13, Issue 6, Pages 1085-1092 (June 2006) Adeno-associated Virus Vector Serotypes Mediate Sustained Correction of Bilirubin UDP Glucuronosyltransferase Deficiency in Rats  Jurgen Seppen, Conny Bakker, Berry de Jong, Cindy Kunne, Karin van den Oever, Kristin Vandenberghe, Rudi de Waart, Jaap Twisk, Piter Bosma  Molecular Therapy  Volume 13, Issue 6, Pages 1085-1092 (June 2006) DOI: 10.1016/j.ymthe.2006.01.014 Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Correction of serum bilirubin by injection of AAV vectors. Male Gunn rats were injected with CMV-UGT1A1 vectors and serum bilirubin was measured. Dashed lines in each graph represent the same group of control or sham-operated rats, N = 14. The solid lines show the serum bilirubin concentrations of animals injected with the indicated AAV serotypes. Each AAV-treated group consisted of five animals up to 1 year, later time points are from fewer than five animals. All serotypes lowered serum bilirubin significantly over this period. Data shown are mean and standard deviation per time point. Molecular Therapy 2006 13, 1085-1092DOI: (10.1016/j.ymthe.2006.01.014) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Biodistribution of AAV-mediated UGT1A1 gene transfer. High-molecular-weight DNA was isolated between 14 and 20 months after administration of AAV vectors for serotypes 1, 2, and 8 and at 12 months for serotype 6. DNA samples were analyzed for the presence of human UGT1A1 sequence by PCR as described. As loading control endogenous GAPDH was amplified. M, marker; Li, liver; Lu, lung; Ki, kidney; Sp, spleen; St, stomach; Du, duodenum; Il, ileum; Je, jejunum; Co, colon; Pa, pancreas; He, heart; Mu, skeletal muscle; Te, testis; Th, thymus; Br, brain. Molecular Therapy 2006 13, 1085-1092DOI: (10.1016/j.ymthe.2006.01.014) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 3 HPLC analysis of bilirubin glucuronides in bile. Bile was collected and analyzed for the presence of bilirubin glucuronides. HPLC traces of representative bile samples from AAV 1, 2, 6, and 8 are shown. For comparison, a trace from an untreated control Gunn rat is also shown. BDG, bilirubin diglucuronide; BMG, bilirubin monoglucuronide; UCB, unconjugated bilirubin. Bilirubin glucuronides are present only in bile from rats injected with CMV AAV vectors. Bile from control rats contains only unconjugated bilirubin. Molecular Therapy 2006 13, 1085-1092DOI: (10.1016/j.ymthe.2006.01.014) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Histological demonstration of AAV gene transfer in liver. Gunn rats were injected with GFP AAV vectors and GFP was detected by immunohistochemistry. The different images show that GFP-positive hepatocytes (darkly stained cells) are present in animals injected with all AAV serotypes. Sections from AAV-injected rat livers are shown at original magnification of 10×. To show accurately the specificity of the staining, a section of an untreated control rat liver is shown at original magnification of 5×. Molecular Therapy 2006 13, 1085-1092DOI: (10.1016/j.ymthe.2006.01.014) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Use of a liver-specific promoter abrogates immune response to UGT1A1. Male Gunn rats were injected with CMV-UGT1A1 and ALB-UGT1A1 vectors. Serum bilirubin and antibody titers were measured as described. Both AAV1 ALB-UGT1A1 (N = 2) and CMV-UGT1A1 (N = 5) reduce serum bilirubin levels in Gunn rats as shown on the left. The dashed line represents control animals (N = 14). On the right, titrations of sera from ALB-UGT1A1- (N = 2) and CMV-UGT1A1- (N = 2) injected animals are shown. A strong immunoreactivity is seen in sera from CMV-UGT1A1-injected animals only. AAV1 ALB UGT1A1 administration did not lead to the formation of UGT antibodies. Titrations of sera 10 and 17 weeks after administration of AAV vector are shown. Molecular Therapy 2006 13, 1085-1092DOI: (10.1016/j.ymthe.2006.01.014) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Liver histology 2 years after AAV vector administration. Gunn rats were sacrificed 2 years after the start of the experiment and tissues were processed for immunohistochemistry. (A) A macroscopic image of the left median lobes from two sham-operated control rats and AAV1-, 2-, and 8-injected animals. On the surface of the AAV-injected rat livers large white spots can be seen. These spots are absent from the age-matched control livers. (B and C) Hematoxylin/phloxin staining of sham-operated control and AAV1-injected rat livers, respectively. A large lesion can be seen in the liver from the AAV1-injected rat. (D) A spontaneous liver tumor in an untreated control rat. (E and F) Oil red O staining of livers from control and AAV1-injected rats, respectively. The strong red staining in the liver from the AAV1-injected rat identifies the lesions as fatty deposits. Molecular Therapy 2006 13, 1085-1092DOI: (10.1016/j.ymthe.2006.01.014) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions