Volume 16, Issue 6, Pages 1073-1080 (June 2008) Analysis of AAV Serotypes 1–9 Mediated Gene Expression and Tropism in Mice After Systemic Injection  Carmela Zincarelli, Stephen Soltys, Giuseppe Rengo, Joseph E Rabinowitz  Molecular Therapy  Volume 16, Issue 6, Pages 1073-1080 (June 2008) DOI: 10.1038/mt.2008.76 Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 1 Study design. (a) Diagram of packaging vector containing AAV2-ITRs and the cytomegalovirus (CMV)-driven luciferase transgene. This transgene was packaged within adeno-associated virus (AAV) serotypes 1–9. (b) After tail vein injection, the mice were imaged according to the above schedule. Most of the mice were killed at 100 days post injection; however, some mice were imaged up to 9 months after the injection. ITR, inverted terminal repeat. Molecular Therapy 2008 16, 1073-1080DOI: (10.1038/mt.2008.76) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 2 Kinetics of adeno-associated virus (AAV) serotype-mediated luciferase expression. Total flux (TF) (photons/sec/cm2/radian) released by luciferase activity at different time-points. For each serotype the mean flux was determined by averaging the flux emitted from each animal at each time-point. Luciferase expression from the tails of the animals was excluded while measuring TF. The values used are from the ventral images and are shown as mean values ± SEM. Molecular Therapy 2008 16, 1073-1080DOI: (10.1038/mt.2008.76) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 3 In vivo imaging of luciferase after tail vein (TV) injection of adeno-associated virus (AAV) serotypes 1–9. (a) Low-expression group. For AAV serotypes 2–5 the expression range was 5,000–25,000 photons/second/cm2. The images are shown at 29, 56, and 100 days post-injection (dpi). In order to discount AAV3 expression in the tail, a mask was placed over the tail in the last image. (b) Medium-expression group including AAV 1, 6, and 8. In this group, the range of expression was 15,000–200,000 photons/second/cm2. (c) High-expression group: for AAV7 and 9 the expression range was 10,000–1,000,000 photons/second/cm2. In both medium- and high-expression groups the images are shown at 14, 29, 56, and 100 dpi in a ventral view, and at 100 dpi in a dorsal view (bottom row). (d) Luciferase detection at 9 months after TV injection of AAV1–9. Molecular Therapy 2008 16, 1073-1080DOI: (10.1038/mt.2008.76) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 4 Luciferase protein expression profile of adeno-associated virus (AAV) serotypes 1–9. The levels of luciferase activity [in relative light units (RLU) per mg protein] were determined in selected tissue at 100 days after intravenous injection of 1 × 1011 particles of AAV1–9 into adult mice. The data are presented as mean values ± SEM. Molecular Therapy 2008 16, 1073-1080DOI: (10.1038/mt.2008.76) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 5 Vector genome copy numbers in selected tissues. Luciferase genome copy numbers/μg of genomic DNA. Persistence of viral genomes in selected tissues 100 days after tail vein injection of 1 × 1011 particles of adeno-associated virus (AAV) serotypes 1–9. Genomic DNA was isolated from the indicated tissues and 100 ng of each was used in triplicate to determine vector genome copies. Levels of significance were determined using one-way analysis of variance. The data are shown as mean values ± SEM. *P < 0.05 versus AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8. #P < 0.05 versus all. **P < 0.05 versus all. Molecular Therapy 2008 16, 1073-1080DOI: (10.1038/mt.2008.76) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions