Volume 18, Issue 6, Pages 1165-1172 (June 2010) Expression of Human α1-Antitrypsin in Mice and Dogs Following AAV6 Vector-mediated Gene Transfer to the Lungs  Christine L Halbert, David K Madtes, Andrew E Vaughan, Zejing Wang, Rainer Storb, Stephen J Tapscott, A Dusty Miller  Molecular Therapy  Volume 18, Issue 6, Pages 1165-1172 (June 2010) DOI: 10.1038/mt.2010.51 Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Human AAT expression in normal and immunodeficient mice. (a) hAAT in blood plasma from C57BL/6 mice that received 5 × 1010 (closed symbols) or 1 × 1011 vg (open symbols) of the ACAGhAAT(AAV6) vector. (b) Plasma hAAT in C57BL/6 Rag2 immunodeficient mice that received 1011 (open symbols) or 2 × 1011 vg (closed diamond) of the ACAGhAAT(AAV6) vector, or 1011 vg of an AAV6 vector encoding alkaline phosphatase [ARAP4(AAV6)] (closed circles) as a negative control. AAT, α1-antitrypsin; AAV, adeno-associated virus; hAAT, human AAT. Molecular Therapy 2010 18, 1165-1172DOI: (10.1038/mt.2010.51) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Immunostain for hAAT expression in mouse lungs. Immunodeficient C57BL/6 Rag2 mice were given 1011 vg of ACAGhAAT(AAV6) or ARAP4(AAV6) as a negative control and lungs were harvested at 3 months and stained with an antibody specific for hAAT in formalin-fixed mouse lung. Expression (purple stain) was seen in airway epithelium (top row), alveolar cells (bottom row), and in some vascular cells (data not shown). AAV6, adeno-associated virus type 6; hAAT, human α1-antitrypsin. Molecular Therapy 2010 18, 1165-1172DOI: (10.1038/mt.2010.51) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 AP expression in lungs of dogs and mice following administration of ACAGAP(AAV6). ACAGAP(AAV6) or saline was administered to lungs of animals and lungs were stained for AP expression (purple/black stain) 2 months later. AAV6, adeno-associated virus type 6; AP, alkaline phosphatase. Molecular Therapy 2010 18, 1165-1172DOI: (10.1038/mt.2010.51) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Immune response to AAV vector and AP protein. (a) Neutralization of an AAV6 vector by 1:100 dilutions of blood plasma and (b) measurement of antibodies against AP by ELISA of plasma harvested at the indicated times from a dog given ACAGAP(AAV6). AAV6, adeno-associated virus type 6; AP, alkaline phosphatase. Molecular Therapy 2010 18, 1165-1172DOI: (10.1038/mt.2010.51) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Expression of hAAT in normal and immunosuppressed dogs. (a) Serum samples from normal and immunosuppressed dogs taken at various times after vector administration were analyzed by ELISA for hAAT levels. (b) Expression of hAAT in ELF of the immunosuppressed dog. Three BAL samples from each lung at each time point were analyzed by ELISA to determine hAAT concentration, and ELF levels were calculated from urea assays to determine fold dilution of ELF in BAL fluid. The means ± SD are shown. (c) BAL fluids from immunosuppressed dogs given ACAGhAAT(AAV6) or ACAGAP(AAV6) were obtained at day 44 and day 60, respectively, and western analysis was done to detect expression of hAAT. Ten µl portions of three BAL fluid samples obtained sequentially from the right (R) and left (L) lungs were analyzed. Ten nanogram of an hAAT standard is shown in the left lane. The arrow marks the position of hAAT. Canine AAT is shown in the lane containing the sample from the ACAGAP(AAV6)-treated dog. The rabbit antibody is specific for human AAT in ELISA. In denaturing conditions such as that used for the western analysis, the antibody also recognizes canine AAT. AAT, α1-antitrypsin; AAV6, adeno-associated virus type 6; BAL, bronchial alveolar lavage; ELF, epithelial lining fluid; ELISA, enzyme-linked immunosorbent assay; hAAT, human AAT. Molecular Therapy 2010 18, 1165-1172DOI: (10.1038/mt.2010.51) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Expression of hAAT in two immunosuppressed dogs. (a) Expression of hAAT in serum. (b,c) Expression of hAAT in epithelial lining fluid (ELF). hAAT, human α1-antitrypsin. Molecular Therapy 2010 18, 1165-1172DOI: (10.1038/mt.2010.51) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions