Volume 11, Issue 3, Pages 444-451 (March 2005) Increased fluid secretion after adenoviral-mediated transfer of the human aquaporin-1 cDNA to irradiated miniature pig parotid glands  Z. Shan, J. Li, C. Zheng, X. Liu, Z. Fan, C. Zhang, C.M. Goldsmith, R.B. Wellner, B.J. Baum, S. Wang  Molecular Therapy  Volume 11, Issue 3, Pages 444-451 (March 2005) DOI: 10.1016/j.ymthe.2004.11.007 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 1 The effects of adenoviral vector delivery on salivary secretion from irradiated minipig parotid glands. (A) Time line of the procedures conducted for this study. Three separate cohorts of minipigs were tested in this manner. (B) Effects of irradiation on minipig parotid salivary flow. A single parotid gland of each (n = 18) minipig was exposed to one dose of 20 Gy. The results shown were obtained 16 weeks after the irradiation exposure. Data shown are the means ± SEM of the parotid salivary output (μl/10 min) for the targeted and the contralateral glands. (C) Effects of hAQP1 gene transfer on minipig parotid salivary output. The irradiated parotid glands of minipigs were administered one of two adenoviral vectors at either 108 (n = 2/vector) or 109 (n = 7 for AdhAQP1 or 5 for AdCMVluc) pfu/gland. Three days following vector administration parotid saliva was collected. Data shown are the means ± SEM of the increase in parotid salivary output (μl/10 min) after vector delivery. (D) Pattern of parotid salivary flow following irradiation and adenoviral vector administration. Parotid salivary flow rates (μl/10 min) were obtained in minipigs as indicated in A. The flow rate prior to irradiation (average of two separate determinations) was set at 100% and the salivary flow rates obtained at times thereafter are represented as a percentage of this initial value. The arrow indicates the time point when adenoviral vectors (109 pfu/gland) were administered. Data shown are the means ± SEM for minipigs administered either AdCMVluc or AdhAQP1 (n = 6/vector). Molecular Therapy 2005 11, 444-451DOI: (10.1016/j.ymthe.2004.11.007) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Immunochemical detection of AQP1 expression in minipig parotid glands. Parotid glands were obtained on day 3 from an animal administered 109 pfu/gland AdCMVluc (A; 40× original magnification) or 109 pfu/gland AdhAQP1 (B; 40× original magnification). Tissue samples were processed for immunocytochemical detection of hAQP1 as described under Materials and Methods. Arrow in B points to positive immunoreactivity indicating the presence of the hAQP1 transgene product in ductal (d), but not acinar (a), cells. In addition, in both A and B, the small arrowheads point to immunoreactivity in endothelial cells of capillaries and venules (comparable with results shown in Fig. 5 of Ref. [6]). Note that the cellular organization in both tissues appears to be abnormal and they manifest atrophy or atalectasia. (C) 10 μg of crude membrane protein was subjected to SDS–gel electrophoresis and Western blotting as described under Materials and Methods. Lane 1, positive control membranes from rat kidney; lane 2, membranes from AdCMVluc-infected minipig parotid gland; lane 3, membranes from AdhAQP1-infected minipig parotid gland. The migration position of the nonglycosylated AQP1 monomer (28 kDa [6,8]) is shown to the right. Molecular Therapy 2005 11, 444-451DOI: (10.1016/j.ymthe.2004.11.007) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Effects of irradiation and adenoviral vector administration to minipig parotid glands on circulating white blood cell and salivary K+ levels. (A) Effects on circulating white blood cells. Animals were irradiated at time 0 and administered either 109 pfu/gland AdCMVluc or 109 pfu/gland AdhAQP1 at week 17. Circulating levels of white blood cells were determined at various times as indicated in Fig. 1A. Data shown are the means ± SEM for minipigs administered AdCMVluc or AdhAQP1 (n = 6/time point for both vectors). (B) Effects on parotid salivary K+ levels. Animals were irradiated at time 0 and administered either 109 pfu/gland AdCMVluc or 109 pfu/gland AdhAQP1 at week 17. Parotid salivary K+ levels were determined at various times as indicated in Fig. 1A. Data shown are the means ± SEM for minipigs administered AdCMVluc (n = 4–6/time point) or AdhAQP1 (n = 4–7/time point). Molecular Therapy 2005 11, 444-451DOI: (10.1016/j.ymthe.2004.11.007) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions