AAV Serotype 8-Mediated Gene Delivery of a Soluble VEGF Receptor to the CNS for the Treatment of Glioblastoma

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doi:10.1016/j.ymthe.2006.02.004

AAV Serotype 8-Mediated Gene Delivery of a Soluble VEGF Receptor to the CNS for the Treatment of Glioblastoma Thomas C. Harding,1,* Alshad S. Lalani,1 Byron N. Roberts,2 Satya Yendluri,1 Bo Luan,1 Kathryn E. Koprivnikar,1 Melissa Gonzalez-Edick,1 Guang Huan-Tu,1 Randy Musterer,1 Melinda J. VanRoey,1 Tomoko Ozawa,3 Richard A. LeCouter,2 Dennis Deen,3 Peter J. Dickinson,2 and Karin Jooss1 2

1 Cell Genesys, Inc., 500 Forbes Boulevard, South San Francisco, CA 94080, USA Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California at Davis, Davis, CA 95616, USA 3 Brain Tumor Research Center, University of California at San Francisco, San Francisco, CA 94143, USA

*To whom correspondence and reprint requests should be addressed. Fax: +1 650 266 2900. E-mail: [email protected].

The presence of the blood–brain barrier complicates drug delivery in the development of therapeutic agents for the treatment of glioblastoma multiforme (GBM). The use of local gene transfer in the brain has the potential to overcome this delivery barrier by allowing the expression of therapeutic agents directly at the tumor site. In this study, we describe the development of a recombinant adeno-associated (rAAV) serotype 8 vector that encodes an optimized soluble inhibitor, termed sVEGFR1/R2, of vascular endothelial growth factor (VEGF). VEGF is an angiogenic factor highly up-regulated in GBM tumor tissue and correlates with disease progression. In subcutaneous models of GBM, VEGF inhibition following rAAV-mediated gene transfer significantly reduces overall tumor volume and increases median survival time following a single administration of vector. Using orthotopic brain tumor models of GBM, we find that direct intracranial administration of the rAAV-sVEGFR1/R2 vector to the tumor site demonstrates anti-tumor efficacy at doses that are not efficacious following systemic delivery of the vector. We propose that rAAVmediated gene transfer of a potent soluble VEGF inhibitor in the CNS represents an effective antiangiogenic treatment strategy for GBM. Key Words: antiangiogenesis, adeno-associated virus, serotype 8, glioblastoma, brain cancer, vascular endothelial growth factor, gene therapy

INTRODUCTION Glioblastoma multiforme (GBM; grade IV astrocytoma) is the most aggressive form of primary brain cancer, with an incidence of 3.2 per 100,000 persons/year in the United States (www.CBTRUS.org). The median survival time (MST) following diagnosis is 9–12 months with the current standard of care involving surgery followed by radiation and/or chemotherapy. One of the hallmarks of GBM is the high degree of neovascularization manifested by vasoproliferation and endothelial cell hyperplasia that is often observed within the tumor site [1,2]. This intense neovascularization is correlated with an upregulation of proangiogenic genes, such as vascular endothelial growth factor (VEGF), which are secreted by tumor and stromal cells into the surrounding tissue [3–5]. In addition to VEGF, other angiogenic factors are frequently overexpressed in GBM, including plateletderived growth factor [6,7], epidermal growth factor [8],

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hepatocyte growth/scatter factor [9], and fibroblast growth factor [10,11]. One of the barriers to the development of successful therapies for the treatment of GBM is the delivery of therapeutic agents to the central nervous system (CNS). Drug delivery to most tumors is difficult, first, because of the tortuous and aberrant nature of the vasculature that characterizes most tumors [12] and second, because drug penetration into tumor tissue is diminished due to an elevated interstitial pressure within the tumor, owing to severe leakage from the vasculature [13]. These effects in part can be attributed to the action of VEGF on increasing vascular permeability [14]. In the case of GBM, drug delivery is particularly compounded by the presence of the blood–brain barrier (BBB) that limits the delivery of large or charged macromolecules to the tumor site in the CNS. The BBB is composed of multiple cell layers, including capillary endothelial cells, astroglia, pericytes,

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and microglia, in a basal lamina that serves to regulate the delivery of agents to the brain [15]. Due to the selective penetration of drugs into the brain across the BBB, many systemically administered antineoplastic drugs that are in use for tumors located outside the CNS have a limited application for GBM [16–18]. To overcome the limitation of drug delivery in GBM, direct infusion of therapeutic agents into the tumor site or more advanced local delivery techniques such as convectionenhanced delivery are being evaluated to help achieve effective drug concentrations over larger areas within the CNS [19]. Furthermore, the use of vector-mediated gene transfer to the brain following both systemic and direct administration to establish local production of therapeutic proteins is under investigation [20,21]. Recombinant adeno-associated viral (rAAV) vectors are currently being evaluated for a number of human gene therapy applications. rAAV vectors have been shown to transduce quiescent cells of the liver, muscle, brain, and eye, leading to long-term expression of therapeutic transgenes [22]. AAV vectors are currently under clinical evaluation for cystic fibrosis [23], hemophilia B [24], Canavan disease [25], and Parkinson disease [26]. Here, we have investigated the use of rAAV gene transfer to the brain by intracranial injection to establish local and longterm production of an optimized soluble VEGF receptor for the treatment of GBM. In multiple subcutaneous models of GBM, we show that the optimized soluble VEGF receptor expressed systemically via rAAV-mediated gene transfer to the liver potently decreased tumor growth rate and significantly increased the survival time of tumorbearing animals. Furthermore, we demonstrate that local delivery of the soluble VEGF receptor to the CNS of mice challenged orthotopically with a murine 4C8 tumor effectively reduces tumor growth and extends MST at doses that are not efficacious following systemic delivery of the vector. In addition, we were able to demonstrate efficacy of locally administered rAAV-sVEGFR1/R2 in an established human U-251 MG orthotopic athymic rat model. These findings suggest a promising new therapeutic approach for the treatment of GBM.

RESULTS sVEGFR1/R2 Displays Favorable VEGF Binding and in Vivo Bioavailability To evaluate the therapeutic effects of a soluble VEGF decoy receptor in preclinical GBM tumor models we constructed recombinant AAV serotype 8 vectors that encode VEGF receptor/IgG1 Fc fusion proteins derived from the highaffinity VEGF receptor Flt-1 (VEGFR1) under the transcriptional control of the constitutive CAG promoter (Fig. 1A). We evaluated the soluble receptor constructs initially for in vivo bioavailability following liver-directed rAAV-mediated gene transfer. The parental construct, rAAVsVEGFR1(d1-7), encodes a soluble decoy receptor encom-

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passing all seven of the extracellular immunoglobulin (Ig)like domains of the Flt-1 receptor, fused to the human IgG1 heavy chain (Fc). We also generated a truncated Flt-1 receptor construct, rAAV-sVEGFR1(d1-3), that contains only the first three N-terminal Ig-like domains of the receptor fused to Fc. In addition, based upon the results of Holash and colleagues [27], we constructed a chimeric soluble receptor termed sVEGFR1/R2 that links the Ig-like domain 2 of Flt-1 to domain 3 of KDR (VEGFR2) followed by the Fc fusion. We inoculated NCRnu.nu mice with a single tail-vein administration of 1  1011 vector genomes (vg) of rAAV vectors pseudotyped with the AAV serotype 8 capsid [28] encoding the various soluble VEGF receptors and monitored serum levels of the receptors by ELISA (Fig. 1B). We observed low serum levels of the soluble receptor sVEGFR1(d1-7), which averaged approximately 150 ng/ml by 6 weeks post-vector administration, in animals that had received the rAAV-sVEGFR1(d1-7) vector. Animals injected with rAAV-sVEGFR1(d1-3) demonstrated slightly higher serum levels of this soluble VEGF receptor, reaching about 600 ng/ml at the same time point. In contrast, animals injected with a vector expressing the chimeric soluble receptor vector, sVEGFR1/R2, had extremely high sVEGFR1/R2 serum levels 6 weeks p.i. of N100,000 ng/ml. These levels of transgene expression remained stable for N10 weeks (Fig. 1B). To verify the biological activity of the soluble VEGF receptors generated from the rAAV vectors, we evaluated the ability of the soluble VEGF decoy receptors to bind and block VEGF-A-stimulated human microvascular endothelial cell (HMVEC) proliferation (Fig. 1C). VEGF-stimulated HMVEC proliferation was equally blocked with an estimated IC50 of 5–10 ng/ml for all receptors derived from Flt-1, including the sVEGFR1/R2 KDR/Flt-1 chimera. The ability of the soluble receptors to bind and block VEGF-mediated effects was additionally verified in the chick chorioallantoic membrane assay (Supplemental Fig. 1). Taken together, these results demonstrate that the soluble chimeric VEGF receptor, sVEGFR1/R2, has an equivalent affinity for binding and blocking the action of VEGF as the parental soluble Flt-1 receptor when assessed by standard angiogenic assays in vitro and in vivo. However, the hybrid sVEGFR1/R2 protein displays an improved bioavailability in vivo following rAAV-mediated gene transfer. On the basis of an equivalent VEGF affinity and the improved pharmacokinetic profile, we selected sVEGFR1/R2 for further preclinical evaluation of its anti-tumor efficacy in murine tumor models. rAAV-sVEGFR1/R2 Anti-tumor Efficacy in a Murine Subcutaneous C6 Glioma Model We evaluated the anti-tumor efficacy of systemic rAAVmediated sVEGFR1/R2 expression in a variety of subcutaneous (sc) tumor models of glioblastoma in mice, initially using the rat C6 xenograft model, which is responsive to recombinant sVEGFR1/R2 protein therapy

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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 3V (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(d13), 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 (FSEM) 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 halfmaximal inhibition.

[27]. Given the delayed onset of transgene expression following rAAV-mediated gene transfer and the rapid growth of the C6 tumor, we administered rAAVsVEGFR1/R2 vectors first to establish transgene expression prior to tumor challenge. We injected NCRnu.nu mice via the tail vein with three different doses of rAAVsVEGFR1/R2: 1  1011, 3.33  1010, and 1  1010 vg/ animal. We monitored serum levels of sVEGFR1/R2 on a weekly basis (Fig. 2A). We observed a clear dose response between input vector genomes and serum sVEGFR1/R2 levels, with 1  1011 vg producing a steady-state level of approximately 300 Ag/ml (high dose), 3.33  1010 vg producing 20 Ag/ml (medium dose), and 1  1010 vg producing 3 Ag/ml (low dose) of serum sVEGFR1/R2 at 5 weeks post-rAAV vector administration. Two weeks following vector administration, we implanted the animals with C6 cells in the dorsal flank (2  105 cells/ site) and monitored tumor growth rate by caliper volume measurement. Mice treated with rAAV-Control vector displayed a tumor growth curve (Fig. 2B) and MST (Fig. 2C and Table 1) that were consistent with PBS-injected control animals, indicating no impact on tumor progression from the injection of the rAAV vector alone. In contrast, rAAV-sVEGFR1/R2-injected mice treated at the medium (3.33  1010 vg/animal) and high (1  1011 vg/ animal) doses showed a dose-dependent decrease in

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tumor growth rate (Fig. 2B) and a corresponding increase in the MST (Fig. 2C and Table 1). The reduction in tumor growth observed in the C6 model by rAAV-sVEGFR1/R2 treatment correlated with the induction of necrosis in the tumor mass (T.C.H. and M.G., unpublished data), as expected from successful angiogenic blockade, at a relatively smaller tumor volume than in rAAV-Controltreated mice, resulting in euthanization according to ACUC guidelines. Treatment using the low dose of rAAVsVEGFR1/R2 (1  1010 vg/animal) had no apparent therapeutic benefit. These data indicate that in the C6 model, serum levels of sVEGFR1/R2 at z20 Ag/ml after a single systemic administration of rAAV-sVEGFR1/R2 vector had a significant effect in delaying tumor growth and prolonging the MST of mice challenged with tumors. We also examined the efficacy of the parental rAAV-sVEGFR1(d1-7) vector in the sc C6 model and observed no significant impact on tumor growth or survival in this model (T.C.H. and A.S.L., unpublished data), consistent with the decreased bioavailability data described above. rAAV-sVEGFR1/R2 Anti-tumor Efficacy in Murine sc 4C8, U-251 MG, and U-87 MG Models To test the anti-tumor efficacy of rAAV-sVEGFR1/R2 in multiple sc models of glioblastoma, we evaluated the

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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 retroorbital puncture on a weekly schedule and assayed for mean circulating sVEGFR1/R2 levels (FSEM) 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 (FSEM) is presented, with an asterisk indicating statistical significance as defined by linear regression ( P = b0.001) 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/R2injected, 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 N2000 mm3 or displayed significant necrosis. A logrank 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 = b0.0001) compared to the rAAV-Control vector-injected group for increasing MST.

anti-tumor activity of the vector in sc murine 4C8 [29,30], human U-251 MG, and human U-87 MG murine models. In contrast to the C6 tumor study, in which animals were preinjected with the rAAV vector before tumor challenge, we conducted the 4C8, U-251 MG, and U-87 MG experiments as treatment models in which vector was not injected until after the tumors were implanted. We inoculated NCRnu.nu mice sc with 4C8 cells (2  106 cells/site), U-251 MG cells (5  106 cells/ site), or U-87 MG cells (5  106 cells/site) in the dorsal

flank. One day following tumor implantation, mice received rAAV vectors intravenously via the tail vein. We evaluated three different doses of rAAV-sVEGFR1/R2, 1  1011, 3.33  1010, and 1  1010 vg/animal, to determine an effective level of circulating sVEGFR1/R2 required for anti-tumor activity. Serum expression levels of sVEGFR1/R2 following rAAV injection in the three tumor models were comparable to the time course of expression in the C6 tumor dose response. The medium (3.3  1010 vg/animal) and high doses (1  1011 vg/

TABLE 1: rAAV-sVEGFR1/R2 increases median survival time in subcutaneous GBM models in a dose-dependent manner

Tumor model C6 4C8 U-251 MG U-87 MG

rAAV-Control 1  1011 vg/animal MSTa

rAAV-sVEGFR1/R2 1  1010 vg/animal MST

26 37 65 44

26 44 72 46

rAAV-sVEGFR1/R2 3.33  1010 vg/animal MST 35 (P b 0.0001b) 77.5 (P b 0.0001) N80 (P b 0.0001) 60 (P = 0.0262)

rAAV-sVEGFR1/R2 1  1011 vg/animal MST 35 (P N80 (P N80 (P 67 (P

b 0.0001) b 0.0001) b 0.0001) b 0.0001)

a

Median survival time expressed in days post-tumor implantation as determined from Kaplan–Meier survival curves. Mice were euthanized as a cancer death when tumors reached a volume of N2000 mm3 (n = 10/group for C6 model; n = 12/group for 4C8, U-251 MG, and U-87 MG models). Significance determined by a log-rank test performed on the Kaplan–Meier curves.

b

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animal) of rAAV vector had a significant impact on the tumor growth rate in all three tumor models (Fig. 3), which correlated with an increase in MST (Table 1) compared to the rAAV-Control- injected group. No significant anti-tumor efficacy was observed in animals treated with the low vector dose (1  1010 vg/animal). Thus, circulating sVEGFR1/R2 levels of z20 Ag/ml are required following rAAV-mediated gene transfer for demonstrating a significant impact on tumor growth rate and increase in MST in these tumor models. To evaluate the effect of rAAV-mediated sVEGFR1/R2 expression on tumor-associated angiogenesis, we pro-

FIG. 3. rAAV-sVEGFR1/R2 inhibits murine 4C8 and human U-251 MG and U87 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 (FSEM) is presented, with an asterisk indicating statistical significance (P b 0.01) as determined by linear regression analysis.

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cessed U-251 MG tumors at 58 days post-tumor implantation for immunohistochemical staining with an antibody to PECAM-1/CD31 to visualize the vasculature (Fig. 4). Tumors from the rAAV-Control group and low dose of rAAV-sVEGFR1/R2 vector displayed a high vascular density throughout the tissue (Figs. 4A, 4B, 4G, and 4H), averaging 237 (F22) and 246 (F46) vessels/mm2, respectively, as assessed by image analysis. In comparison, animals treated at the medium or high dose of rAAVsVEGFR1/R2 (Figs. 4C, 4D, 4E, and 4F) had a dosedependent decrease in PECAM-1/CD31-positive staining, averaging 42 (F21) and 10 (F9) vessels/mm2, respectively. Tumors harvested from animals treated at the high dose of rAAV-sVEGFR1/R2 vector also displayed large central areas of necrosis (M.G., unpublished data). rAAV-Mediated sVEGFR1/R2 Expression within the Murine CNS Following demonstration of the therapeutic efficacy of rAAV-VEGFR1/R2 in sc glioma models using systemic administration, we evaluated the anti-tumor efficacy of rAAV-sVEGFR1/R2 in orthotopic glioma models following local gene transfer. Initially, we examined soluble VEGF receptor expression histologically following stereotactic injection into the murine brain (Figs. 5A and 5B). We injected B6D2F1 mice in the left striatum with 1  109 vg/animal of rAAV-sVEGFR1/R2 vector. Three weeks following injection we euthanized the animals and immunohistochemically processed their brains for sVEGFR1/R2 detection. We detected expression of the sVEGR1/R2 protein in the cytoplasm of cells around the injection site within the striatum (Fig. 5A). We also examined expression in vivo after rAAV-mediated gene transfer of sVEGFR1/R2 to an orthotopically implanted tumor. We implanted B6D2F1 mice intracranially with 4C8 murine glioblastoma cells in the left striatum. Seven days following implantation, animals received 1  109 vg of rAAV-sVEGFR1/R2 vector into the same coordinates used for tumor implantation by stereotactic injection. Two weeks following rAAV vector injection, we euthanized the animals and excised and processed their brains for sVEGFR1/R2 immunohistochemistry. Positive cells expressing sVEGFR1/R2 were evident throughout the tumor site (Fig. 5B) and in normal host neurons and astrocytes that surrounded the 4C8 tumor within the brain (T.C.H., unpublished data). We also evaluated the transduction efficiency of rAAV vectors pseudotyped with the AAV serotype 8 capsid following intratumoral injection of vector expressing green fluorescent protein (GFP) under the control of the CAG promoter into the naive murine brain and an established 4C8 orthotopic tumor (Figs. 5C and 5D; Supplemental Figs. 2 and 3). Two weeks following administration of 1.0  1010 vg/animal of rAAV-GFP, 21.3% (F3.6%) of the cells present within the tumor mass were GFP+. Cells transduced by the AAV8 vector in the orthotopic 4C8 tumor mass represented

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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.

both Her2/Neu and GFAP costaining tumor cells and astrocytes, respectively (Supplemental Fig. 4). rAAV-sVEGFR1/R2 Efficacy Following Local Delivery in a 4C8 Orthotopic Mouse Model To evaluate the anti-tumor efficacy of rAAV-sVEGFR1/R2 in an orthotopic glioma model, we implanted B6D2F1 mice with 2  105 4C8 murine glioma cells into the left striatum. Seven days following 4C8 tumor implantation, we injected two doses (1  109 or 1  1010 vg/animal) of rAAV-sVEGFR1/R2 or rAAV-Control stereotactically at the same coordinates used for tumor implantation. We evaluated the comparative efficacy of systemic sVEGFR1/ R2 expression on orthotopic 4C8 tumor growth by tail-

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vein injection of rAAV-sVEGFR1/R2 vector at an identical dose (1.0  1010 vg) 7 days following tumor implantation. As shown in Figs. 6A, 6B, and 6C animals treated with stereotactic brain injection of rAAVsVEGFR1/R2 at either dose exhibited a significant inhibition of tumor growth, as monitored by MR imaging of tumor volume in comparison to rAAV control-treated animals. Tumor growth inhibition by local delivery of rAAV-sVEGFR1/R2 correlated with a distinct survival advantage as calculated from Kaplan– Meier curves (Figs. 6D and 6E). In contrast, systemic delivery of the rAAV-sVEGFR1/R2 vector at the 1  1010 vg/animal dose did not reduce tumor volume or increase MST in the 4C8 orthotopic glioma model.

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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 rAAVsVEGFR1/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 fluorescenceconjugated 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) rAAVmediated 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).

Local treatment of a 4C8 orthotopic glioma model with rAAV-VEGFR1/R2 significantly reduces tumor growth in the brain and prolongs MST; however, tumors were subsequently found to progress following sVEGFR1/ R2 blockade (Figs. 6B and 6C). We evaluated the expression of the target ligand for the soluble VEGFR1/ R2 receptor, VEGF-A, in orthotopic 4C8 tumors following escape from inhibition by quantitative PCR analysis of tumor mRNA. Following euthanization of the animals at their endpoint, we harvested 4C8 tumors and extracted mRNA and then processed it for quantitative PCR using primer/probe sets specific for mVEGF. mVEGF mRNA expression was up-regulated 2.12-fold (F0.2) in tumors treated with the rAAV-VEGFR1/R2 therapy in comparison to rAAV-Control-treated tumors, suggesting a potential mechanism for tumor progression following treatment with a VEGF inhibitor. In addition to VEGF, additional angiogenic factors such as members of the fibroblast growth factor and platelet-derived growth factor families and midkine and chromogranin-A were also identified by microarray and quantitative PCR to be up-regulated by rAAV-VEGFR1/R2 treatment (Supplemental Fig. 5; Supplemental Table 1).

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rAAV-Mediated sVEGFR1/R2 Expression Prolongs Survival Following Local Delivery in an Established Orthotopic U-251 MG Athymic Rat Model We investigate the efficacy of rAAV-mediated local gene transfer for the treatment of glioblastoma further in an established orthotopic U-251 MG GBM model. We implanted athymic rats stereotactically with 5  106 U251 MG human glioblastoma cells in the caudate putamen on day 0. Following implantation, we allowed U-251 tumors to develop to an established size of ~20 mg (day 15) before rAAV treatment. Animals (n = 13/group) received 2  1011 vg of rAAV-sVEGFR1/R2 or rAAVControl locally into the tumor site by an osmotic minipump. Implantation of the minipump allows the slow infusion of rAAV vector over a 24-h period (8 Al/h) to transduce more effectively the intracranial tumor mass. We euthanized the animals when they developed significant adverse neurological signs associated with the tumor burden. Animals treated with the rAAV-VEGFR1/ R2 vector displayed an increase in MST over the rAAVControl-treated group of 47 days vs 37 days, respectively (Fig. 6F). In addition, 13.3% of the animals treated with the rAAV-VEGFR1/R2 vector survived to the termination of the study (day 89) without neurological symptoms of significant U-251 MG progression.

DISCUSSION Frequent or prolonged administration of recombinant antiangiogenic agents is usually required to achieve optimal therapeutic efficacy in chronic diseases such as cancer [31]. In the case of GBM, repeated local administration of antiangiogenic agents directly into the brain is technically difficult. To overcome this limitation, we have explored the use of local delivery of a potent soluble antiangiogenic agent using recombinant AAV vectors. rAAV offers many advantages as a vector delivery system for antiangiogenic therapy for GBM. rAAV vectors are replication defective and offer stable, long-term transgene expression without any evidence of vector-related toxicity [22]. In addition, rAAV vectors are also being examined in phase I clinical trials for Canavan [25] and Parkinson disease [26] following direct CNS injection. In our studies we have used rAAV vectors pseudotyped with the capsid protein derived from the recently identified AAV serotype 8. Gene expression from the liver following administration of serotype 8-pseudotyped rAAV vectors is improved (1–2 logs) over the more established AAV-2 serotype vector [28]. Compared to rAAV vectors based upon AAV serotype 2, gene transfer using serotype 8 vectors is much improved in both the normal brain and an orthotopic 4C8 tumor (Supplemental Figs. 2 and 3), providing an effective gene transfer platform to deliver therapeutic genes for the treatment of glioblastoma. Furthermore, the in situ production of a soluble VEGF receptor that is secreted into the surrounding tissue from

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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 posttumor 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 (FSEM) is presented, with an asterisk indicating statistical significance as defined by linear regression (P b 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 b 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.

rAAV-transduced cells allows the efficient targeting of the dispersed GBM tumor mass. In comparison, previous clinical gene transfer strategies for GBM have been limited by the use of therapeutic transgenes that act

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intracellularly and/or on neighboring tumor cells via gap junctions and therefore cannot effectively target tumor cells that have invaded tissue sites separated from the site of transgene production [32]. Previous studies have also

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applied the use of a rAAV vector encoding a soluble VEGF receptor derived from the mouse Flt-1 or Flk-1 for tumor treatment [33,34]. However, in these studies, either tumor cells were transduced directly with the rAAV vector in vitro prior to implantation [33] or the rAAV vector was administered 10 weeks before tumor implantation [34] to achieve efficacy. In comparison, the use of an optimized soluble VEGF receptor coupled with the high efficiency of gene transfer of the rAAV serotype 8 capsid [28] used in our studies allows potent therapeutic efficacy in subcutaneous and orthotopic GBM tumor models when a single-dose of the vector is given posttumor implantation. Following demonstration of anti-tumor efficacy in subcutaneous glioma models we then evaluated sVEGFR1/R2 in established orthotopic models. In the murine 4C8 model we compared the relative efficacy of the sVEGFR1/R2 expressed continuously at the tumor site or systemically following a single intravenous administration of recombinant AAV vector to the liver. A single administration of 1  109 or 1  1010 vector genomes delivered locally into preestablished brain tumors provides a significant delay in tumor growth and increases the overall MST in tumor-bearing mice, whereas systemic delivery at the same dose does not offer an equivalent therapeutic benefit in the orthotopic 4C8 tumor model that was examined. However, it should be noted that in preliminary studies it has been possible to achieve anti-tumor responses in the orthotopic B6D2F1/4C8 model using significantly higher doses (z5  1010 vg/animal) of rAAV-sVEGFR1/R2 delivered systemically that provide serum VEGFR1/R2 expression levels N0.5 mg/ml (T.C.H., B.N.R., R.A.L., P.J.D., and K.J., unpublished results). The increased efficacy following local expression of sVEGFR1/R2 compared to systemic administration of the same dose could be due to higher local concentrations of the soluble VEGF inhibitor within the tumor site, which can effectively block microenvironment concentrations of VEGF that are known to be critical for stimulating angiogenesis and subsequent tumor growth [35]. These data support the rationale of using local gene delivery for sustained expression of soluble antiangiogenic agents for achieving an enhanced therapeutic effect in the treatment of GBM. In addition to increased efficacy, local delivery of a therapeutic agent to the CNS also has the potential to avoid systemic treatment-related toxicities that have been observed with other anti-VEGF agents. Side effects of the anti-VEGF antibody bevacizumab (Avastin; Genentech) have included serious tumorrelated bleeding episodes, hypertension, thrombosis, proteinuria (with occasional nephrotic syndrome), and epistaxis following systemic delivery of the agent [36]. Following treatment with rAAV-sVEGFR1/R2, orthotopic 4C8 tumors displayed a significant decrease in tumor growth in comparison to control animals; how-

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ever, tumors were observed to progress eventually, requiring euthanization of animals due to neurological symptoms. Expression from rAAV vectors, unlike other vector platforms, is stable and long term within the CNS [22] and therefore progression of the orthotopic 4C8 tumor occurred in the presence of continued VEGF inhibition by rAAV-mediated VEGFR1/R2 expression. We hypothesized, therefore, that changes in angiogenic gene expression in response to long-term VEGF inhibition may be responsible in part for tumor progression and analyzed a panel of candidate angiogenic factors by microarray and quantitative RT-PCR (Supplemental Fig. 5; Supplemental Table 1). In agreement with Gerber et al. [37], we find that VEGF mRNA expression was upregulated (approximately twofold) in tumors treated with a VEGF inhibitor therapy in comparison to control treated tumors, suggesting a potential feedback mechanism in response to anti-VEGF therapy. The up-regulation of tumor-associated VEGF may be a consequence of tumor hypoxia associated with rAAV-sVEGFR1/R2 inhibition [38]. In addition, the associated VEGF receptor, neuropilin-1, was also demonstrated to be up-regulated in rAAV-sVEGFR1/R2-treated tumors. However, while we find that VEGF and associated receptors are induced, additional angiogenic factors are up-regulated, such as members of the fibroblast growth factor family, plateletderived growth factors A and B, midkine, and vasostatin, or down-regulated, such as erb-2, angiopoeitin-2, and TIMP2, in response to sVEGFR1/R2 therapy. Thus, although our studies demonstrate that VEGF is an early critical ligand required for GBM growth, targeting angiogenic factors in addition to VEGF may provide increased efficacy for the antiangiogenic treatment of GBM and additional tumor types. Interestingly, PDGF-A was also recently described to be an important chemotactic factor produced by VEGF-null fibrosarcoma cells for the recruitment of tumor-associated fibroblasts [39], highlighting the role of additional angiogenic factors in tumor progression in the presence of VEGF inhibition. In summary, a single injection of rAAV-sVEGFR1/R2 administered directly into the tumor site effectively blocks tumor-associated angiogenesis, impedes tumor growth, and prolongs survival as demonstrated in several preclinical murine models of glioblastoma. These studies demonstrate for the first time the preclinical efficacy of using localized rAAV-mediated gene transfer for the sustained expression of a potent soluble VEGF inhibitor in the CNS as a novel antiangiogenic treatment strategy for GBM.

MATERIALS

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METHODS

Recombinant AAV vector construction. Full details of recombinant vector construction are provided in the Supplemental Materials and Methods. In brief, to create sVEGFR1/R2, individual Ig-like domains of the parental VEGF receptors (VEGFR1 IgG-like domain 2 and VEGFR2 IgG-like domain 3) were amplified using the Expand High Fidelity PCR Kit (Roche Applied

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Science, Mannheim, Germany) and then joined using a secondary PCR step. The IgG1 FC region was PCR amplified and fused to the 3V end of the VEGFR1 domain 2/VEGFR2 domain 3 fusion using a third round of PCR. This final PCR fusion was cloned into pBluescript SK(+) (Stratagene, La Jolla, CA, USA) and the VEGFR1 signal sequence was added as a synthetic oligonucleotide. A rAAV plasmid, termed pTR-CAG-sVEGFR1/R2-WPREBGHpA, encoding the sVEGFR1/R2 soluble receptor was constructed by cloning the hybrid soluble VEGF receptor into pTR-CAG-VEGFR-3-WPREBGHpA. Construction of rAAV vector plasmids encoding domains 1–3 and 1–7 of VEGFR1 fused to IgG1 Fc was done by PCR amplification of the specific domains from pBLAST-hFLT-1 (Invivogen). The resulting PCR fragments were ligated into pTR-CAG-sVEGFR1/R2-WPRE-BghpA to create pTR-CAG-sVEGFR-1(d1-3)-WPRE-BGHpA and pTR-sVEGFR-1(d1-7)WPRE-BGHpA, respectively. All constructs were fully sequenced using an ABI Prism 3100 (Applied Biosystems, Foster City, CA, USA). Pseudotyped rAAV serotype 8 vectors were produced in HEK 293 cells using calcium phosphate triple transfection of the rAAV vector expression plasmid of interest in combination with the AAV-8 serotype helper plasmid p5e18-VD2/8 [28] and pXX-6 [40]. Virions were isolated on two sequential CsCl gradients and titers determined by dot blot using radioactive probe specific for the rAAV transgene. Cells. Human embryonic kidney 293 (ATCC, Manassas, VA, USA), human U-87 MG (ATCC), rat C6 (ATCC), murine 4C8 (C.A. Dyer, Children’s Hospital of Philadelphia, PA, USA), and U-251 MG (Department of Neurological Surgery Tissue Bank, University of California at San Francisco, CA, USA) cells were maintain in DMEM supplemented with 10% FBS. Adult HMVECs were purchased from Cambrex (East Rutherford, NJ, USA) and maintained in EGM-MV culture medium according to the manufacturer’s recommendations. VEGF inhibition bioassay. HMVECs were seeded in 96-well flat-bottom plates at a density of 5  103 cells/well and cultured overnight at 378C in a humidified incubator. The next day, the medium was replaced with EBM2 basal medium containing 5% FBS and incubated for 6 h to deprive the cells of mitogenic growth factors. The cells were then stimulated with 20 ng/ml recombinant human VEGF (R&D Systems, Minneapolis, MN, USA) in the presence, or absence, of conditioned medium containing the indicated amounts of rAAV-produced soluble VEGF decoy receptors. After 72 h, cell proliferation was measured using a Cell Counting Kit (Dojindo Laboratories, Gaithersburg, MD, USA). Quantification of soluble VEGF receptors by ELISA. Soluble sVEGFR1 (d1–7) and (d1–3) was quantified using a commercially available sandwich ELISA kit (R&D Systems). Soluble VEGFR1/R2 was quantified using a sandwich ELISA technique using a goat anti-human IgG-Fc polyclonal antibody (Sigma Chemical Co., St. Louis, MO, USA) followed by a HRP-conjugated anti-human IgG-Fc antibody (Bethyl Laboratories, Montgomery, TX, USA). Purified sVEGFR1/R2 protein from plasmidtransfected HEK 293 cells was used for standard curves. Samples were detected using ABTS peroxidase detection substrate at 450 nm optical density.

conditions. For tumor implantation, mice were anesthetized and stereotactically implanted (David Kopf Instruments, Tujunga, CA, USA) with 4C8 cells (1  106 cells in 5 Al) in the left cerebral cortex. Seven days following 4C8 implantation, rAAV vector was administered by either tail vein (as described above) or intratumoral injection. For tumor size assessment, sequential MR images of 4C8 orthotopic tumors were acquired under general anesthesia using a Bruker Biospec DBX scanner (Bruker Medical, Billerica, MA, USA) interfaced to an Oxford 7.0 T/183 clear-bore magnet (Oxford Instruments, Oxford, UK) and tumor area for each slice was calculated using NIH Image 1.62 software (NIH). Mice were euthanized and scored as a cancer death when they displayed significant adverse neurological signs as assessed by UC Davis ACUC institutional guidelines. Orthotopic U-251 MG glioblastoma model. Six-week-old male athymic rats were purchased from Harlan (Indianapolis, IN, USA) and housed under SPF conditions. U-251 MG tumor cells were implanted as previously described [41]. Fifteen days post-U-251 MG implantation, a 200-Al Alzet osmotic minipump was inserted into a subcutaneous pocket in the midscapular region on the back and a catheter was connected between the pump and a brain infusion cannula. Osmotic minipumps were loaded to administer 2  1011 virus genomes over a 24-h period (8 Al/h). Following virus delivery animals were monitored for survival and scored as a cancer death when they displayed significant adverse neurological symptoms as assessed by UCSF ACUC institutional guidelines. Immunohistochemistry. Tissues harvested from animals were fixed in 4% paraformaldehyde, sectioned by cryostat, and incubated with primary antibodies goat polyclonal anti-PECAM-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) to detect endothelial cells or rabbit polyclonal anti-human IgG (DAKO, Carpinteria, CA, USA) to detect sVEGFR1/R2. The corresponding secondary antibodies, goat anti-rabbit Alexa 594 and rabbit anti-goat Alexa 594 (Molecular Probes, Eugene, OR, USA) were used and tissues analyzed by fluorescence microscopy using a Zeiss Axioplan microscope equipped with a SPOT RT Slider digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA). Quantification was done using Image Pro Plus (MediaCybernetics, Silver Springs, MD, USA) software. Quantitative PCR analysis. Pieces (50–100 mg) of frozen tumors were homogenized in TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and genomic DNA was removed from the samples using DNA-free (Ambion, Austin, TX, USA). Total RNA was converted to cDNA and analyzed by quantitative PCR using ABI’s TaqMan One-Step RT-PCR Master Mix Reagents Kit (Applied Biosystems) and Assay-on-Demand reagents for mVEGF-A and mGAPDH (Applied Biosystems). The murine GAPDH (Assay Mm99999915 _ g1) probe sequence was 5V-TGAACGGATTTGGCCGTATTGGGCG-3V and murine VEGF-A (assay Mm00437304_m1) probe sequence was 5V-CCACCATGCCAAGTGGTCCCAGGCT-3V. VEGF-A values were normalized with GAPDH values. Results were analyzed for relative expression using the DDC t method (ABI User Bulletin 2, P/N 4303859).

Subcutaneous tumor studies. Six- to eight-week-old female NCR-Nu nude mice were obtained from Taconic and housed under SPF conditions. For systemic gene transfer studies, rAAV vectors were administered at the indicated rAAV vector genome dose by a single tail-vein injection using a 200-Al dosing volume over a 30-s period. Mice were bled by alternate retro-orbital puncture on scheduled intervals to measure the serum levels of circulating soluble VEGF receptors by ELISA. For subcutaneous glioma tumor models C6 (2  105 cells/site), 4C8 (2  106 cells/site), U-251 MG (5  106 cells/site), or U-87 MG (5  106 cells/ site) tumor cells were diluted in 100 Al of sterile basal medium and injected sc into the right dorsal flank. Tumor volumes (as cubic millimeters) were calculated as volume = length  width2  0.5. Mice were euthanized as a bcancer deathQ when the sc tumor volume exceeded 2000 mm3 or when the tumor became excessively necrotic. Studies running longer than 80 days were actively terminated.

Statistical analysis and data presentation. Data are presented as mean tumor volumes (FSEM) over time. Statistical analysis for tumor volume growth curves were evaluated by linear regression analysis comparing the slopes of the mean tumor volume for each treatment group over time using GraphPad Prism Software, and differences with P b 0.05 were considered statistically significant as indicated by an asterisk. Multiparameter statistics for the Kaplan–Meier survival curves were performed by a log-tank test using GraphPad Prism software. Relative mRNA expression from individual animals is presented and differences were considered significant at P b 0.05 as determined by a Student t test.

Orthotopic 4C8 murine glioblastoma model. Six-week-old, male, B6D2F1 mice were obtained from The Jackson Laboratory and housed under SPF

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ymthe.2006.02.004.

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APPENDIX A. SUPPLEMENTARY DATA

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RECEIVED FOR PUBLICATION JUNE 22, 2005; REVISED FEBRUARY 6, 2006; ACCEPTED FEBRUARY 6, 2006.

REFERENCES 1. Brem, S., Cotran, R., and Folkman, J. (1972). Tumor angiogenesis: a quantitative method for histologic grading. J. Natl. Cancer Inst. 48: 347 – 356. 2. Burger, P. C., and Vogel, F. S. (1991). Brain: tumors. In Surgical Pathology of the Nervous System and Its Coverings (P. C. Burger, B. W. Scheithauer, F. S. Vogel, Eds.), pp. 193 – 405. Churchill Livingstone, New York. 3. Plate, K. H., Breier, G., Weich, H. A., and Risau, W. (1992). Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 359: 845 – 848. 4. Berkman, R. A., et al. (1993). Expression of the vascular permeability factor/vascular endothelial growth factor gene in central nervous system neoplasms. J. Clin. Invest. 9: 153 – 159. 5. Godard, S., et al. (2003). Classification of human astrocytic gliomas on the basis of gene expression: a correlated group of genes with angiogenic activity emerges as a strong predictor of subtypes. Cancer Res. 63: 6613 – 6625. 6. Nister, M., Heldin, C. H., Wasteson, A., and Westermark, B. (1984). A glioma-derived analog to platelet-derived growth factor: demonstration of receptor competing activity and immunological crossreactivity. Proc. Natl. Acad. Sci. USA 81: 926 – 930. 7. Nister, M., et al. (1988). Expression of messenger RNAs for platelet-derived growth factor and transforming growth factor-alpha and their receptors in human malignant glioma cell lines. Cancer Res. 48: 3910 – 3918. 8. Libermann, T. A., et al. (1985). Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin. Nature 313: 144 – 147. 9. Moriyama, T., Kataoka, H., Tsubouchi, H., and Koono, M. (1995). Concomitant expression of hepatocyte growth factor (HGF), HGF activator and c-met genes in human glioma cells in vitro. FEBS Lett. 372: 78 – 82. 10. Libermann, T. A., et al. (1987). An angiogenic growth factor is expressed in human glioma cells. EMBO J. 6: 1627 – 1632. 11. Takahashi, J. A., et al. (1990). Gene expression of fibroblast growth factors in human gliomas and meningiomas: demonstration of cellular source of basic fibroblast growth factor mRNA and peptide in tumor tissues. Proc. Natl. Acad. Sci. USA 87: 5710 – 5714. 12. Deane, B. R., and Lantos, P. L. (1981). The vasculature of experimental brain tumours. J. Neurol. Sci. 49: 55 – 77. 13. Boucher, Y., Salehi, H., Witwer, B., Harsh, G. R., and Jain, R. K. (1997). Interstitial fluid pressure in intracranial tumours in patients and in rodents. Br. J. Cancer 75: 829 – 836. 14. Senger, D. R., Connolly, D. T., Van de Water, L., Feder, J., and Dvorak, H. F. (1990). Purification and NH2-terminal amino acid sequence of guinea pig tumor-secreted vascular permeability factor. Cancer Res. 50: 1774 – 1778. 15. Pardridge, W. M. (2003). Molecular biology of the blood–brain barrier. Methods Mol. Med. 89: 385 – 399. 16. Rich, J. N., and Bigner, D. D. (2004). Development of novel targeted therapies in the treatment of malignant glioma. Nat. Rev. Drug Discovery 3: 430 – 446. 17. Bendell, J. C., et al. (2003). Central nervous system metastases in women who receive trastuzumab-based therapy for metastatic breast carcinoma. Cancer 97: 2972 – 2977. 18. Leis, J. F., et al. (2004). Central nervous system failure in patients with chronic myelogenous leukemia lymphoid blast crisis and Philadelphia chromosome positive acute lymphoblastic leukemia treated with imatinib (STI-571). Leuk. Lymphoma 45: 695 – 698. 19. Bobo, R. H., Laske, D. W., Akbasak, A., Morrison, P. F., Dedrick, R. L., and Oldfield, E. H. (1994). Convection-enhanced delivery of macromolecules in the brain. Proc. Natl. Acad. Sci. USA 91: 2076 – 2080. 20. Chen, S. H., Shine, H. D., Goodman, J. C., Grossman, R. G., and Woo, S. L. (1994). Gene therapy for brain tumors: regression of experimental gliomas by adenovirusmediated gene transfer in vivo. Proc. Natl. Acad. Sci. USA 91: 3054 – 3057.

966

doi:10.1016/j.ymthe.2006.02.004

21. Nilaver, G., et al. (1995). Delivery of herpesvirus and adenovirus to nude rat intracerebral tumors after osmotic blood–brain barrier disruption. Proc. Natl. Acad. Sci. USA 92: 9829 – 9833. 22. Grimm, D., and Kay, M. A. (2003). From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy. Curr. Gene Ther. 3: 281 – 304. 23. Moss, R. B., et al. (2004). Repeated adeno-associated virus serotype 2 aerosolmediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis: a multicenter, double-blind, placebo-controlled trial. Chest 125: 509 – 521. 24. Manno, C. S., et al. (2003). AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B. Blood 101: 2963 – 2972. 25. Janson, C., et al. (2002). Gene therapy of Canavan disease: AAV-2 vector for neurosurgical delivery of aspartoacylase gene (ASPA) to the human brain. Hum. Gene Ther. 13: 1391 – 1412. 26. Luo, J., et al. (2002). Subthalamic GAD gene therapy in a Parkinson’s disease rat model. Science 298: 425 – 429. 27. Holash, J., et al. (2002). VEGF-Trap: a VEGF blocker with potent antitumor effects. Proc. Natl. Acad. Sci. USA 99: 11393 – 11398. 28. Gao, G. P., Alvira, M. R., Wang, L., Calcedo, R., Johnston, J., and Wilson, J. M. (2002). Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc. Natl. Acad. Sci. USA 99: 11854 – 11859. 29. Dyer, C. A., and Philibotte, T. (1995). A clone of the MOCH-1 glial tumor in culture: multiple phenotypes expressed under different environmental conditions. J. Neuropathol. Exp. Neurol. 54: 852 – 863. 30. Weiner, N. E., et al. (1999). A syngeneic mouse glioma model for study of glioblastoma therapy. J. Neuropathol. Exp. Neurol. 58: 54 – 60. 31. Drixler, T. A., Rinkes, I. H., Ritchie, E. D., van Vroonhoven, T. J., Gebbink, M. F., and Voest, E. E. (2000). Continuous administration of angiostatin inhibits accelerated growth of colorectal liver metastases after partial hepatectomy. Cancer Res. 60: 1761 – 1765. 32. Lang, F. F., et al. (2003). Phase I trial of adenovirus-mediated p53 gene therapy for recurrent glioma: biological and clinical results. J. Clin. Oncol. 21: 2508 – 2518. 33. Hasumi, Y., et al. (2002). Soluble FLT-1 expression suppresses carcinomatous ascites in nude mice bearing ovarian cancer. Cancer Res. 62: 2019 – 2023. 34. Davidoff, A. M., Nathwani, A. C., Spurbeck, W. W., Ng, C. Y., Zhou, J., and Vanin, E. F. (2002). rAAV-mediated long-term liver-generated expression of an angiogenesis inhibitor can restrict renal tumor growth in mice. Cancer Res. 62: 3077 – 3083. 35. Ozawa, C. R., et al. (2004). Microenvironmental VEGF concentration, not total dose, determines a threshold between normal and aberrant angiogenesis. J. Clin. Invest. 113: 516 – 527. 36. Sandler, A. B., Johnson, D. H., and Herbst, R. S. (2004). Anti-vascular endothelial growth factor monoclonals in non-small cell lung cancer. Clin. Cancer Res. 10: 4258 – 4262. 37. Gerber, H. P., Kowalski, J., Sherman, D., Eberhard, D. A., and Ferrara, N. (2000). Complete inhibition of rhabdomyosarcoma xenograft growth and neovascularization requires blockade of both tumor and host vascular endothelial growth factor. Cancer Res. 60: 6253 – 6258. 38. Pugh, C. W., and Ratcliffe, P. J. (2003). Regulation of angiogenesis by hypoxia: role of the HIF system. Nat. Med. 9: 677 – 684. 39. Dong, J., et al. (2004). VEGF-null cells require PDGFR alpha signaling-mediated stromal fibroblast recruitment for tumorigenesis. EMBO J. 23: 2800 – 2810. 40. Xiao, X., Li, J., and Samulski, R. J. (1998). Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J. Virol. 72: 2224 – 2232. 41. Ozawa, T., Wang, J., Hu, L. J., Bollen, A. W., Lamborn, K. R., and Deen, D. F. (2002). Growth of human glioblastomas as xenografts in the brains of athymic rats. In Vivo 16: 55 – 60.

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