Volume 12, Issue 6, Pages 1043-1051 (December 2005) Local Delivery of a Viral Vector Mitigates Neutralization by Antiviral Antibodies and Results in Efficient Transduction of Rabbit Liver  Bradley L. Hodges, Kristin M. Taylor, Qiuming Chu, Samantha E. Scull, Rebecca G. Serriello, Scott C. Anderson, Fei Wang, Ronald K. Scheule  Molecular Therapy  Volume 12, Issue 6, Pages 1043-1051 (December 2005) DOI: 10.1016/j.ymthe.2005.06.475 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Fluoroscopic spot image of balloon catheter administration of adenovirus through a hepatic vein in the rabbit model. The head of the animal is toward the top of this image. The vena cava and four major hepatic veins are outlined by dotted lines. The catheter can be seen descending through the superior vena cava into the right lateral lobe, where the occlusion balloon, inflated with contrast agent, blocks outflow from the vein. The injected solution, which contains contrast, can be seen highlighting the venous branching of this lobe (dotted red circle). This image was captured just prior to virus injection (see Materials and Methods) and illustrates the patency of the balloon-mediated occlusion of the selected hepatic vein. Molecular Therapy 2005 12, 1043-1051DOI: (10.1016/j.ymthe.2005.06.475) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Optimizing injection volume for local delivery. The distribution of β-galactosidase expression 3 days after administration of 1.5 × 1012 vp/kg of Ad2β-gal in a (A, B, C) 3, (D, E, F) 8, or (G, H, I) 20 ml injection volume using a balloon catheter and rabbits naive to Ad2 is shown. Expression as evaluated by immunohistochemistry (see Materials and Methods) is shown at 10× original magnification for both the injected (lobe 1) and an uninjected (lobe 4) lobe (A, B, D, E, G, H). For a given volume, all uninjected lobes had similar immunohistochemical localizations. Photomicrographs are typical of 15 sections in each of two injected animals. Expression of bacterial β-galactosidase as determined by a luminescence assay (see Materials and Methods) in three cores per lobe is shown schematically (C, F, I); the injected lobe is shaded. The numbers represent expression in relative light units of β-galactosidase/pg protein. For simplicity, lobes are numbered. Note that the injected lobe (lobe 1) is the circumscribed lobe shown in Fig. 1. Molecular Therapy 2005 12, 1043-1051DOI: (10.1016/j.ymthe.2005.06.475) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 3 The distribution of β-galactosidase expression 3 days after systemic administration of 1.5 × 1012 vp/kg of Ad2β-gal in a volume of 8 ml to (A, C, E) rabbits naive to Ad2 and to (B, D, F) rabbits passively immunized with human serum containing anti-Ad2 antibodies. Immunohistochemical localization of β-galactosidase expression in lobe 1 is shown at 10× and 40× original magnification. All four lobes had similar immunohistochemical localizations. Photomicrographs are representative of two or three injected animals. Schematic of the distribution of β-galactosidase expression determined by ELISA is shown for naive (E) and passively immunized (F) rabbits (because this is a systemic administration, there is no distinction between lobes). The numbers represent the average expression from three tissue cores per lobe in units of pg β-galactosidase/μg protein. Black arrows indicate double and single nuclei of hepatocytes (C). White arrows indicate nuclei of nonhepatocytes (D). Molecular Therapy 2005 12, 1043-1051DOI: (10.1016/j.ymthe.2005.06.475) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 4 The distribution of β-galactosidase expression 3 days after administration of 1.5 × 1012 vp/kg of Ad2β-gal using a balloon catheter in a volume of 8 ml to (A–E) naive rabbits and to (F–K) rabbits passively immunized against Ad2. Expression as evaluated by immunohistochemistry (see Materials and Methods) is shown at 10× and 40× original magnification for both the injected (lobe 1; A, C, F, H) and an uninjected (lobe 4; B, D, G, I) lobe. All uninjected lobes had similar immunohistochemical localizations. Photomicrographs are representative of three injected rabbits. Expression in each lobe as determined by ELISA (see Materials and Methods) is shown schematically; the injected lobe is shaded. The numbers represent the average expression from three tissue cores per lobe in units of pg β-galactosidase/μg protein. Flushing the injected lobe with 20 ml of saline immediately prior to virus injection resulted in the distribution of expression shown in (K). Molecular Therapy 2005 12, 1043-1051DOI: (10.1016/j.ymthe.2005.06.475) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 5 MetaMorph quantitation (see Materials and Methods) of the number of β-galactosidase-positive hepatocytes (filled bars) and nonhepatocytes (open bars) per 1-mm2 field of each liver lobe following (A, B) systemic or (C–E) local delivery of an identical amount of virus (1.5 × 1012 vp/kg of Ad2β-gal) in an identical volume (8 ml) into naive rabbits (A, C) or rabbits passively immunized with human serum containing anti-Ad2 antibodies (B, D, E). Numbers in parentheses represent the fraction of β-gal-positive hepatocytes among all β-gal-positive cells within each given lobe. Lobes 1 and 4 correspond to those shown schematically in Figs. 2–4 (A, C, D, N = 45 fields; B, E, N = 30 fields). Molecular Therapy 2005 12, 1043-1051DOI: (10.1016/j.ymthe.2005.06.475) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions