Volume 17, Issue 8, Pages 1427-1433 (August 2009) Evaluation of Vascular Delivery Methodologies to Enhance rAAV6-mediated Gene Transfer to Canine Striated Musculature  Paul Gregorevic, Brian R Schultz, James M Allen, Jeffrey B Halldorson, Michael J Blankinship, Norman A Meznarich, Christian S Kuhr, Caitlin Doremus, Eric Finn, Denny Liggitt, Jeffrey S Chamberlain  Molecular Therapy  Volume 17, Issue 8, Pages 1427-1433 (August 2009) DOI: 10.1038/mt.2009.116 Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Inflammation and tissue clearance in the absence of immune suppression. Two dogs were administered 2 × 1013 vg rAAV6-CMV-hPLAP via the femoral artery, with the same infusion protocol. At week 6 after infusion, the animals were euthanized and tissues examined for transgene expression and inflammation. (a,b) Mosaic images of serial gastrocnemius sections for each animal. (a) Sections stained for the presence of hPLAP. (b) Sections stained by hematoxylin and eosin. (c) Higher magnification of the section in b, located at the arrowhead in b. The arrowheads in a demark the same region of each respective section. (d) Serial gastrocnemius sections with hematoxylin and eosin staining and immunofluorescent detection of CD4+, CD8a+, and CD11b+ cells in the immune suppressed (+) and nonimmune suppressed (−) animals. Bar = (a,b) 2 mm, (c,d) 100 µm. Molecular Therapy 2009 17, 1427-1433DOI: (10.1038/mt.2009.116) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Effect of compression wrap and dwell time on rAAV6-CMV-hPLAP transduction. Four dogs were each administered 2 × 1013 vg rAAV6-CMV-hPLAP into the femoral artery at the femoral triangle. In animals with a vector dwell time, blood flow into and out of the leg was occluded before infusion and restored after the noted time. Depending on the animal, an external compression wrap was applied to the leg, and either removed before infusion, or maintained during vector dwell time. Tissues were harvested and stained for the presence of hPLAP. Bar = 200 µm. TC, tibialis cranialis; FHL, flexor hallucis longus. Molecular Therapy 2009 17, 1427-1433DOI: (10.1038/mt.2009.116) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Effect of removing compression wrap before vector infusion of hindlimb. Animals underwent application of an external compression wrap followed by occlusion of the major femoral vessels. With the wrap either in place or removed, and the blood flow still occluded, rAAV6-CMV-hPLAP was injected into the femoral artery at a dose of 2 × 1013 vg. Tibialis cranialis and extensor digitorum longus cross sections were placed on slides together and stained for the presence of hPLAP. Bar = 5 mm. Molecular Therapy 2009 17, 1427-1433DOI: (10.1038/mt.2009.116) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 rAAV6-CMV-hPLAP expression at varying levels of hindlimb muscles. Dog 9997 was injected with 2 × 1013 vg rAAV6-CMV-hPLAP via the femoral artery, with a preinfusion compression wrap and a 15-minute vector dwell time. Three weeks after infusion, the animal was euthanized and tissues harvested. Each muscle was sectioned at 25% (proximal), 50% (middle), and 75% (distal) of its length, and stained for the presence of hPLAP. Bar = 200 µm. TC, tibialis cranialis; FHL, flexor hallucis longus. Molecular Therapy 2009 17, 1427-1433DOI: (10.1038/mt.2009.116) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 rAAV6 transduction of heart and diaphragm via jugular vein infusion. A dog was infused with 2 × 1013 vg rAAV-RSV-hPLAP via the jugular vein. An un-injected dog served as a negative control. Three weeks after vector administration, both animals were euthanized, tissues were harvested and stained for the presence of hPLAP. (a) Three sections each of heart and diaphragm from the injected animal are shown, alongside a counterpart section from the un-injected animal (Neg). (b) Sections stained by hematoxylin and eosin and for the presence of hPLAP. Bar = (a) 200 µm, (b) 100 µm. H&E, hematoxylin and eosin. Molecular Therapy 2009 17, 1427-1433DOI: (10.1038/mt.2009.116) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions