Volume 12, Issue 5, Pages 867-875 (November 2005) Efficient Hepatic Delivery and Expression from a Recombinant Adeno-associated Virus 8 Pseudotyped α1-Antitrypsin Vector  Thomas J. Conlon, Travis Cossette, Kirsten Erger, Young-Kook Choi, Tracy Clarke, Marda Scott-Jorgensen, Sihong Song, Martha Campbell-Thompson, James Crawford, Terence R. Flotte  Molecular Therapy  Volume 12, Issue 5, Pages 867-875 (November 2005) DOI: 10.1016/j.ymthe.2005.05.016 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Transduction of hepatocytes by AAV serotype 8 produces high levels of α1-antitrypsin and more genome copies. (A) Packaging plasmids encoding the capsid sequences for AAV serotypes 1, 2, 5, and 8 were cotransfected with pCBAT into 293 cells to generate complete virions. The array of viruses generated was titered and injected into the portal vein of C57Bl/6 mice to target the liver with equal doses of 9.6 × 1010 vector particles per animal. At time points of 0, 1, 3, 5, 8, 12, 16, 20, and 24 weeks postinjection, serum samples were analyzed for transgene (AAT) expression, and means at each time point are graphed. A similar pattern of heightened expression by 1 week can be detected for each serotype and stable transgene levels are detected throughout the study. (B) Twenty-four weeks after being injected through the portal vein with 1 × 1011 particles, animals underwent a partial hepatectomy. Liver biopsies were collected and genomic DNA was extracted. Purified DNA was assayed by quantitative RT-PCR for AAV copies using the pCBAT plasmid as the standard and using the 166,000 mouse genomes per microgram of genomic DNA standard. The mean ± standard deviation of copies per cell for each animal is plotted for comparison. Molecular Therapy 2005 12, 867-875DOI: (10.1016/j.ymthe.2005.05.016) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Immunostaining for hAAT in the murine liver. All sections were incubated with a rabbit anti-human AAT antibody as the primary antibody. (A) Liver section of a PBS-injected mouse to serve as a negative control reference showing no positive staining for hAAT. (B) AAV2/1-CBAT, (C) AAV2-CBAT, (D) AAV5-CBAT, and (E) AAV8-CBAT liver sections from mice administered with 1 × 1013 vector particles via the portal vein. Staining of livers from each serotype reveals an overall panlobular blush, midzonal, periportal, and perivenous detection of hAAT. Central veins (CV) and portal veins (PV) are noted. (F) 40× magnification of outlined portion in (E) demonstrating staining around the central vein. Tissues were harvested at 24 weeks postinjection during a partial hepatectomy procedure. Original magnifications: A–E, 20×; F, 40×. Molecular Therapy 2005 12, 867-875DOI: (10.1016/j.ymthe.2005.05.016) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Decreased levels of hAAT and AAV genome copies after partial hepatectomy for serotypes 1, 2, and 8. (A) At 24 weeks after portal vein injection, the animals in Fig. 2 underwent a partial hepatectomy that could remove up to 85% of the total liver weight. Starting 1 week after surgery serum sampling resumed until week 8, when the animals were sacrificed. AAT transgene means for each serotype cohort determined by ELISA are plotted as a percentage of the AAT levels obtained at week 24. A minor decrease at week 1 reflects the 2-week half-life of AAT in circulation. The increase in levels for the serotype 5 group may reflect a propensity toward integration compared to other serotypes. (B) This graph represents the AAV copy number as detected by real-time PCR before and after surgery removing three lobes of the liver. Each bar representing the mean and standard deviation of a serotype cohort is a percentage of the total copy number obtained the same way before surgery 8 weeks prior. Again, a trend toward copy regeneration within the proliferating liver is seen as well as the expected drop in copies for AAV8. Molecular Therapy 2005 12, 867-875DOI: (10.1016/j.ymthe.2005.05.016) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Southern blot for genomic persistence of AAV serotypes in the liver. Total cellular genomic DNA was extracted from frozen liver tissue. Forty micrograms of genomic DNA was digested with either HindIII (one-cutter within the vector) or ApaI (two-cutter within the vector) overnight and separated on agarose gel. (A) Schematic of the restriction sites and expected band sizes and annealing location of the hAAT probe. (B) After blotting to nitrocellulose, a 32P-labeled probe to the hAAT sequence was hybridized. Endogenous bands not vector related, head-to-tail (H-T) concatemers, and the double-digested internal fragments are noted. Molecular Therapy 2005 12, 867-875DOI: (10.1016/j.ymthe.2005.05.016) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Dose response in AAV8-injected mice. High titer AAV8/CBAT virus was produced and injected via the portal vein into C57Bl/6 mice to determine a dose–response relationship. Three doses of AAV8, 1.65 × 109 (low, N = 3), 1.65 × 1010 (medium, N = 5), and 1.65 × 1011 (high, N = 4), were injected and serum AAT levels determined at weeks 0, 1, 3, 5, 8, 12, 16, 20, and 24. Means ± standard deviations are plotted on a log scale. An approximately 50-fold increase for each 10-fold higher dose of virus is observed. Molecular Therapy 2005 12, 867-875DOI: (10.1016/j.ymthe.2005.05.016) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Demonstration of AAV serotype 8 genome persistence as episomes. The previously used AAV8 dose–response animals were subjected to a partial hepatectomy. hAAT levels present in the serum were quantitated by ELISA. The resulting data were plotted on a weekly timeline for visualization after control means were subtracted. The previously used “low dose” AAV8 group is not shown because of low levels difficult to distinguish from controls after partial hepatectomy. Molecular Therapy 2005 12, 867-875DOI: (10.1016/j.ymthe.2005.05.016) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 7 Total AAV copy number decreases after partial hepatectomy in mice administered three doses of AAV2/8. This graph represents the AAV copy number as detected by real-time PCR before and after surgery removing three lobes of the liver. Each bar represents the mean and standard deviation of a serotype cohort as a percentage of the total copy number obtained the same way before surgery 8 weeks prior. A comparable reduction in total copy is seen with each dose group. Molecular Therapy 2005 12, 867-875DOI: (10.1016/j.ymthe.2005.05.016) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions