1. Volume 15, Issue 5, Pages 912-920 (May 2007)
Rapamycin-regulated Control of Antiangiogenic Tumor Therapy Following rAAV- mediated Gene Transfer 
Minh Nguyen, Guang Huan-Tu, Melissa Gonzalez-Edick, Victor M Rivera, Tim Clackson, Karin U Jooss, Thomas C Harding 
Molecular Therapy 
Volume 15, Issue 5, Pages (May 2007)
DOI: /mt.sj
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Schematic representation of the angiostatin-regulated AAV constructs. In the bi-directional constructs, AATBi-Angio and AATBi-Ci-Angio, the mouse TTR promoter drives the expression of the activation domain FRB-p65, whereas the minimal SV40 promoter drives the expression of the DNA-binding domain, ZFHD1-2×FKBP. The TTR promoter has been replaced by the thyroid hormone-binding globulin promoter with two copies of α1-microglobulin/bikunin enhancer (LSP) in the AALBi-Ci-Angio construct. In the IRES constructs, AATI-Angio and AATI-Ci-Angio, the TTR promoter drives the expression of a transcriptional unit encoding for FRB-p65, and ZFHD1-2×FKBP is translated from encephalomyocarditis virus IRES. The human angiostatin gene was cloned downstream of the inducible promoter—eight ZFHD1 recognition sites with a minimal IL-2 promoter (Z8IL-2p)—with or without a chimeric intron and upstream of the minimal simian virus 40 polyadenylation sequences. ITRs represent the AAV-2 inverted terminal repeats.
Molecular Therapy  , DOI: ( /mt.sj )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
In vitro analysis of regulated angiostatin AAV constructs. (a) HuH7 cells were transduced with AAV-2 pseudotyped vectors in triplicate, and the medium was replaced the following day with medium with or without 30 nm rapamycin. Angiostatin concentration was measured by ELISA 48 h after media change. Fold induction was calculated by dividing the angiostatin level with rapamycin by the baseline level without rapamycin. For baseline levels below the limit of detection, 0.1 ng/ml was used to calculate the fold induction. (b) HuH7 cells infected with vectors, as described in a, were lyzed 8 days after transduction for Western blot analysis, and stained with anti-p65 or anti-FKBP12 antibodies. Anti-p65 antibodies detect FRB-p65 (activation domain) and endogenous p65 as well, thus providing a loading control. Anti-FKBP12 antibodies detect ZFHD1-2×FKBP (DNA-binding domain) and endogenous FKBP12 as well, thus providing a loading control. Lane 1, AATI-Angio; lane 2, AATI-Ci-Angio; lane 3, AATBi-Angio; lane 4, AATBi-Ci-Angio; lane 5, AALBi-Ci-Angio; and lane 6, non-transduced HuH7 cells.
Molecular Therapy  , DOI: ( /mt.sj )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
In vivo analysis of regulated angiostatin AAV vectors. (a) Diagram of experimental parameters illustrates rapamycin dosing and blood collection schedules in days. Top arrows represent time points for blood collection, while bottom arrows denote time of rapamycin injections, and bottom dotted line represents time of vector administration. (b) Rapamycin-regulated angiostatin expression in NCR-Nu nude mice following AAV vector administration. Mice received 4×1011 vg/mouse of rAATBi-Angio or rAATBi-Ci-Angio by intravenous injection on day 1. On days 26 and 27, days 47 and 48, and days 69 and 70, transgene expression was induced by a 3 mg/kg rapamycin injection. Serum levels of angiostatin were quantified by ELISA 2 days following induction. Values are mean±SEM for five mice per group. P-values were calculated for AATBi-Ci-Angio compared to AATBi-Angio for each given time point.
Molecular Therapy  , DOI: ( /mt.sj )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Induction of soluble VEGF receptor expression delays tumor growth in 4C8 glioblastoma model. (a) Schematic representation of regulated soluble VEGF receptor constructs used in this study. The angiostatin gene in the bi-directional vectors has been replaced by sVEGFR1/R2 to produce the single-intron AATBi-sVEGFR1/R2 and the double-intron AATBi-Ci-sVEGFR1/R2 vectors. (b) Diagram of experimental parameters illustrates rapamycin dosing and blood collection schedules in days. Top arrows represent time points for blood collection, while bottom arrows denote time of rapamycin injections. Top dotted line represents time of tumor challenge, while bottom dotted line represents time of vector administration. (c) NCR-Nu nude mice received 5×1011 vg/mouse of rAATBi-sVEGFR1/R2 or rAATBi-Ci-VEGFR1/R2 vector. 3 mg/kg of rapamycin was administered on days 21, 22, and 28, and serum was assayed for sVEGFR1/R2 expression by ELISA. Values are mean±SEM for six mice per group. P-values were calculated for AATBi-Ci-sVEGFR1/R2 compared to AATBi-sVEGFR1/R2. (d) 4C8 tumor cells were implanted subcutaneously 44 days after vector administration, and 3 mg/kg of rapamycin was injected twice weekly, starting 4 days after tumor implant. Tumor volumes are mean±SEM for eight or nine mice per group. P-values were calculated for AATBi-Ci-sVEGFR1/R2-injected animals with rapamycin compared to vector control with rapamycin.
Molecular Therapy  , DOI: ( /mt.sj )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Induction of soluble VEGF receptor expression delays tumor growth in U-251 glioblastoma model. (a) Diagram of experimental parameters illustrates rapamycin dosing and blood collection schedules in days. Top arrows represent time points for blood collection, whereas bottom arrows denote time of rapamycin injections. Top dotted line represents time of tumor challenge, while bottom dotted line represents time of vector administration. (b) NCR-Nu nude mice received 1×1012 AAV-8 pseudotyped vectors on day 1. Rapamycin administrations were initiated at day 22 post-vector administration and given weekly at 3 mg/kg. Values represent mean serum sVEGFR1/R2 levels±SEM for nine to 10 mice per group. (c) U-251 tumor cells were implanted on day 1 post-vector administration, and rapamycin injections were initiated on day 22 and given once a week at 3 mg/kg. Tumor volumes are mean±SEM for nine or 10 mice per group. P-values were calculated for AATBi-Ci-sVEGFR1/R2-injected animals with rapamycin compared to control animals with rapamycin alone.
Molecular Therapy  , DOI: ( /mt.sj )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions