Volume 13, Issue 1, Pages 77-87 (January 2006) Biology of AAV Serotype Vectors in Liver-Directed Gene Transfer to Nonhuman Primates  Guangping Gao, You Lu, Roberto Calcedo, Rebecca L. Grant, Peter Bell, Lili Wang, Joanita Figueredo, Martin Lock, James M. Wilson  Molecular Therapy  Volume 13, Issue 1, Pages 77-87 (January 2006) DOI: 10.1016/j.ymthe.2005.08.017 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Kinetics of rhCG expression in different AAV serotype vector-mediated gene transfers to the NHP liver. The study animals were bled at different time points post-vector infusion for measurement of serum rhCG levels. Animal ID numbers and vectors received are indicated in each graph. Molecular Therapy 2006 13, 77-87DOI: (10.1016/j.ymthe.2005.08.017) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Comparative analysis of the transgene expression and liver function test (LFT) data in the first 2 months of the AAV-mediated rhCG gene transfer to the NHP liver. AST (the left y axes) and rhCG (the right y axes) levels at different time points after vector infusion from each of eight study subjects are presented. Molecular Therapy 2006 13, 77-87DOI: (10.1016/j.ymthe.2005.08.017) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 3 RhEpo expression profiles and hematocrit levels in the cynomolgus macaques that received different AAV serotype vectors. The study animals were bled at different time points post-vector infusion for measurement of serum rhEpo and hematocrit levels. Animal ID numbers and vectors received are indicated in each graph. Molecular Therapy 2006 13, 77-87DOI: (10.1016/j.ymthe.2005.08.017) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Molecular status of AAV serotype vector genomes in cynomolgus macaque livers. Liver tissues were harvested at the end of the study by necropsy for total DNA extraction. Ten micrograms each of DNA from different animal livers, which were either (A) untreated or (B) digested with XhoI, a single cutter, or (C) HpaI, a double cutter in the vector genome, was subjected to Southern blot DNA hybridization analysis (RDC, relaxed double-strand circles; MSC, monomeric supercoiled double-strand double circles; LDC, linearized monomeric double-strand circles; HMW, high molecular weight; H-T, head-to-tail; T-T, tail-to-tail). The vector genome plasmid, pAAVTBGrhEpo, was digested with HpaI and used as a copy number control. A 1.2-kb HpaI fragment from vector genome plasmid was used as the probe for hybridization. The data of vector genome copy numbers per cell as quantified by TaqMan real-time PCR and serum rhEpo levels at the peak expression and the end of the study are also presented. Molecular Therapy 2006 13, 77-87DOI: (10.1016/j.ymthe.2005.08.017) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Comparative analysis of the transgene expression and LFT data in the first 2 months of the AAV-mediated rhEpo gene transfer to the NHP liver. AST (the left y axes) and rhEpo (the right y axes) levels at different time points after vector infusion from each of 10 study subjects are presented. Molecular Therapy 2006 13, 77-87DOI: (10.1016/j.ymthe.2005.08.017) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Biodistribution of vector genomes following AAV TBGrhEpo serotype vector infusion into cynomolgus livers. Ten distant organs and targeted liver tissue were harvested from the nine study animals at the end of the study by necropsy. The quantity of vector genomes within each tissue was analyzed by TaqMan PCR using a probe/primer set to amplify the vector genome. The abundance of vector genomes is illustrated as copies per diploid cell (F, female; M, male). BM, bone marrow; B, brain; C, colon; H, heart; K, kidney; LI, liver; Lu, lung; LN, lymph node; GD, gonadal tissue; SB, small bowel; S, spleen. Molecular Therapy 2006 13, 77-87DOI: (10.1016/j.ymthe.2005.08.017) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions