1. Volume 13, Issue 5, Pages 956-966 (May 2006)
AAV Serotype 8-Mediated Gene Delivery of a Soluble VEGF Receptor to the CNS for the Treatment of Glioblastoma 
Thomas C. Harding, Alshad S. Lalani, Byron N. Roberts, Satya Yendluri, Bo Luan, Kathryn E. Koprivnikar, Melissa Gonzalez-Edick, Guang Huan-Tu, Randy Musterer, Melinda J. VanRoey, Tomoko Ozawa, Richard A. LeCouter, Dennis Deen, Peter J. Dickinson, Karin Jooss 
Molecular Therapy 
Volume 13, Issue 5, Pages (May 2006)
DOI: /j.ymthe
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Bioavailability and VEGF binding affinity of soluble VEGF receptors following rAAV-mediated gene transfer. (A) Schematic diagram of rAAV vectors used within this study. Soluble VEGF receptors were cloned downstream of the constitutive CAG promoter and upstream of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and the bovine growth hormone polyadenylation sequence 3′ (bGHpA): sVEGFR-1(d1-7), soluble VEGF receptor 1 decoy encoding all of the seven extracellular IgG-like domains of the Flt-1 receptor fused to the human IgG1 Fc heavy chain region; sVEGFR-1(d1-3), soluble VEGF receptor 1 decoy encoding N-terminal domains 1–3 of the Flt-1 receptor fused to IgG1 Fc; and sVEGFR1/R2, soluble chimeric receptor decoy composed of IgG-like domain 2 of the Flt-1 receptor fused to the IgG-like domain 3 of the KDR receptor followed by the human IgG1 Fc heavy chain region. ITRs represent the AAV-2 inverted terminal repeats. (B) Bioavailability of soluble VEGF receptors in vivo following rAAV mediated gene transfer. Female NCRnu.nu mice (n = 8/group) were injected with a single dose of 1 × 1011 vg of rAAV vectors encoding sVEGFR-1(d1-7), sVEGFR-1(d1-3), or sVEGFR1/R2 by tail-vein injection. Mice were bled by retro-orbital puncture on selected intervals up to 6 weeks postadministration and assayed for mean circulating sVEGF receptor levels (±SEM) using a capture sandwich ELISA. (C) sVEGFR1/R2-mediated inhibition of VEGF-stimulated human microvascular endothelial cell (HMVEC) proliferation in vitro. HMVECs were stimulated with 25 ng/ml VEGF in the presence of increasing concentrations of sVEGFR-1(d1-7) or sVEGFR1/R2 derived from rAAV-transduced 293 cell conditioned media. After 48 h of incubation, cell proliferation was measured by tetrazolium salt conversion. The percentage of inhibition was determined by comparison to proliferation in the presence and absence of VEGF alone. IC50 represents the concentration of soluble receptor required for half-maximal inhibition.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Expression of sVEGFR1/R2 following rAAV-mediated gene transfer decreases tumor growth and extends median survival time in a subcutaneous C6 glioblastoma model. (A) Dose-dependent sVEGFR1/R2 expression following rAAV-mediated gene transfer. Female NCRnu.nu mice (n = 10/gp) were iv injected with a single dose of rAAV-sVEGFR1/R2 vector at 1 × 1011, 3.33 × 1010, or 1 × 1010 vg/animal. Following injection, mice were bled by retro-orbital puncture on a weekly schedule and assayed for mean circulating sVEGFR1/R2 levels (±SEM) by using a capture sandwich ELISA. (B) Efficacy of rAAV-mediated sVEGFR1/R2 expression on subcutaneous C6 tumor growth. Two weeks following rAAV administration, mice (n = 10/group) were challenged with a subcutaneous injection of 2 × 105 C6 cells into the right flank. Following tumor implantation, tumor volume was monitored using a caliper measurement at biweekly intervals. The mean tumor volume (±SEM) is presented, with an asterisk indicating statistical significance as defined by linear regression (P = <0.001) comparing rAAV-sVEGFR1/R2 animals injected at the 1 × 1011 and 3.33 × 1010 doses to rAAV-Control -injected animals. (C) Kaplan–Meier survival curves showing an increase in the MST in the rAAV-sVEGFR1/R2-treated mice at the 3.33 × 1010 and 1 × 1011 doses (MST 35 days) compared to the low dose (1 × 1010 vg/animal) rAAV-sVEGFR1/R2-injected, rAAV-Control -injected, and PBS-injected animals (MST 26 days). Mice (n = 10/group) were sacrificed when the C6 glioblastoma tumor size reached a volume of >2000 mm3 or displayed significant necrosis. A log-rank test performed on the Kaplan–Meier curves showed that the 3.33 × 1010 and 1 × 1011 dose groups of rAAV-sVEGFR1/R2 treatment are significant (P = <0.0001) compared to the rAAV-Control vector-injected group for increasing MST.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
rAAV-sVEGFR1/R2 inhibits murine 4C8 and human U-251 MG and U-87 MG glioblastoma tumor growth in a dose-dependent manner. Female NCRnu.nu mice (n = 12/group) were challenged with a subcutaneous injection of (A) 4C8, (B) U-251, or (C) U-87 MG tumor cells. One day following tumor implantation animals were iv injected with a single dose of rAAV-sVEGFR1/R2 vector at 1 × 1011, 3.33 × 1010, or 1 × 1010 vg/animal. Following tumor implantation, tumor volume was monitored using a caliper measurement at biweekly intervals. The mean tumor volume (±SEM) is presented, with an asterisk indicating statistical significance (P < 0.01) as determined by linear regression analysis.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
rAAV-mediated sVEGFR/R2 expression effectively reduces blood vessel growth in the U-251 MG model as assessed by immunohistochemistry. Subcutaneous U-251 MG tumors were excised, sectioned, and stained with Texas red-conjugated antibodies to detect CD-31/platelet-endothelial cell adhesion molecule-1 (PECAM-1) (left) and mounted with a DAPI counterstain (right) at 58 days post-tumor implantation. U-251 MG tumors processed from rAAV-Control and the low dose (1 × 1010 vg/animal) of rAAV-sVEGFR1/R2 vector displayed a high degree of vascularization, with PECAM-1-positive endothelial cells surrounding the vessel perimeter, in comparison to U-251 MG tumors from animals treated at the 1 × 1011 or 3.33 × 1010 vg/animal dose of rAAV-sVEGFR1/R2, which had a dose-dependent decrease in PECAM-1 staining.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
rAAV mediated sVEGFR1/R2 and GFP transgene expression in the CNS and a murine glioblastoma model. (A) Immunohistochemical detection of sVEGFR1/R2 expression in murine CNS following striatal injection of rAAV-sVEGFR1/R2 vector. B6D2F1 mice were stereotactically injected in the right striatum with 1 × 109 vector genomes of rAAV-sVEGFR1/R2 vector. Three weeks following injection animals were euthanized and brains immunohistochemically processed for sVEGFR1/R2 detection using a fluorescence-conjugated anti-human Fc antibody with a DAPI counterstain. (B) Photomicrograph of sVEGFR1/R2 expression within an orthotopic 4C8 tumor 2 weeks following rAAV-sVEGFR1/R2 stereotactic injection. (C and D) rAAV-mediated green fluorescent protein (GFP) expression in an orthotopic 4C8 murine glioblastoma tumor. B6D2F1 mice were orthotopically implanted in the right striatum with 4C8 murine glioblastoma cells. Seven days following implantation, animals were stereotactically injected with rAAV encoding GFP (rAAV-GFP). Two weeks following rAAV-GFP vector injection, brains examined for GFP expression. The orthotopic tumor mass (T) and surrounding striatum (S) are indicated in addition to the tumor/striatum border (arrow).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
rAAV-sVEGFR1/R2 decreases tumor growth and extends survival in orthotopic GBM models following local delivery. B6D2F1 mice (n = 6/group) were implanted with 2 × 105 4C8 murine glioblastoma cells in the right striatum. Seven days following tumor injection, animals were administered 1.0 × 1010 (A, B, and D) or 1.0 × 109 vector genomes (C and E) of rAAV-sVEGFR1/R2 or rAAV-Control directly into the tumor site by stereotactic injection using the same coordinates as were used for tumor cell implantation. A separate group received 1.0 × 1010 vector genomes of rAAV-sVEGFR1/R2 by tail vein injection 7 days following 4C8 injection. (A) MR images of representative mouse brains locally treated with rAAV-Control (left) or rAAV-sVEGFR1/R2 (right) taken at 36 days post-tumor implantation to assess tumor volume. Images represent 1.2-mm longitudinal sections through individual mouse brains. Arrows indicate 4C8 tumor (dark) within the murine brain (white). (B and C) Tumor volume as assessed using MR imaging and quantified by image analysis at 26, 34, 46, 57, and 69 days following 4C8 implantation. The mean tumor volume (±SEM) is presented, with an asterisk indicating statistical significance as defined by linear regression (P < 0.01) comparing rAAV-sVEGFR1/R2-injected animals with rAAV-Control-injected. (D and E) Kaplan–Meier survival curves showing an increase in the MST in the intracranially injected rAAV-sVEGFR1/R2-treated mice compared to the rAAV-Control- and systemically injected rAAV-sVEGFR1/R2 animals. Mice were euthanized and scored as a cancer death when they displayed significant adverse neurological symptoms as assessed by ACUC institutional guidelines. A log-rank test performed on the Kaplan–Meier curves showed that the locally injected rAAV-sVEGFR1/R2 treatment is significant (P < 0.001) compared to the rAAV-Control vector-injected group for increasing MST. (F) Kaplan–Meier survival curves showing the prolongation of survival following rAAV-VEGFR1/R2 treatment in an established orthotopic U-251 MG model. Athymic rats (n = 13/group) were implanted with U-251 MG human glioblastoma cells in the caudate-putamen on day 0. Fifteen days following tumor injection, animals were administered either rAAV-sVEGFR1/R2 or rAAV-Control directly into the tumor site by surgical implantation of an osmotic minipump draining into the same coordinates as were used for tumor cell implantation. Rats were euthanized and scored as a cancer death when they displayed significant adverse neurological symptoms as assessed by ACUC institutional guidelines. A log-rank test performed on the Kaplan–Meier curves showed that the locally injected rAAV-sVEGFR1/R2 treatment (MST 47 days) is significant (P = 0.019) compared to the rAAV-Control (MST 37 days) vector-injected group for increasing MST.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions