ABSTRACT
Regulated gene expression may be required for the clinical development of certain gene therapies. Several approaches have been developed that allow pharmacologic control of transgene expression, including the dimerizer-regulated transcriptional system in which rapamycin or its analogs function as transcriptional inducers. These compounds can also act as direct antitumor agents via inhibition of mammalian target of rapamycin (mTOR). We describe the development of an optimized recombinant adeno-associated virus (AAV) expression cassette that allows dimerizer-regulated gene expression from a single vector in vitro and in vivo. After demonstrating multiple cycles of rapamycin-dependent transgene induction following a single administration of an AAV vector in vivo, application of this regulated AAV gene expression system to the pharmacologic control of antiangiogenic therapy was evaluated in preclinical tumor models. Dimerizer-regulated vectors were constructed encoding a soluble inhibitor of the vascular endothelial growth factor (VEGF) pathway. In two subcutaneous models of glioblastoma, regulated expression of the VEGF inhibitor via recombinant AAV-mediated gene transfer, in combination with rapamycin, was shown to decrease tumor growth rate significantly. The dual properties of rapamycin--as a transcriptional inducer and mTOR inhibitor--are exploited in combination with an AAV-encoded antiangiogenic agent to provide a novel approach for the treatment of malignant diseases.
Subject(s)
Dependovirus/genetics , Genetic Therapy/methods , Neoplasms/therapy , Sirolimus/pharmacology , Angiostatins/genetics , Angiostatins/metabolism , Animals , Blotting, Western , Cell Line , Cell Line, Tumor , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Female , Gene Expression Regulation/drug effects , Gene Transfer Techniques , Genetic Vectors/genetics , Glioblastoma/genetics , Glioblastoma/metabolism , Glioblastoma/therapy , Humans , Immunosuppressive Agents/pharmacology , Mice , Mice, Nude , Neoplasms/blood supply , Neoplasms/genetics , Prealbumin/genetics , Promoter Regions, Genetic/genetics , Receptors, Vascular Endothelial Growth Factor/genetics , Receptors, Vascular Endothelial Growth Factor/metabolism , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: The purpose of the present study was to evaluate granulocyte macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapy in combination with vascular endothelial growth factor (VEGF) blockage in preclinical models. EXPERIMENTAL DESIGN: Survival and immune response were monitored in the B16 melanoma and the CT26 colon carcinoma models. VEGF blockade was achieved by using a recombinant adeno-associated virus vector expressing a soluble VEGF receptor consisting of selected domains of the VEGF receptors 1 and 2 (termed sVEGFR1/R2). Dendritic cell and tumor infiltrating lymphocyte activation status and numbers were evaluated by fluorescence-activated cell sorting analysis. Regulatory T cells were quantified by their CD4+CD25hi and CD4+FoxP3+ phenotype. RESULTS: The present study established that GM-CSF-secreting tumor cell immunotherapy with VEGF blockade significantly prolonged the survival of tumor-bearing mice. Enhanced anti-tumor protection correlated with an increased number of activated CD4+ and CD8+ tumor-infiltrating T cells and a pronounced decrease in the number of suppressive regulatory T cells residing in the tumor. Conversely, overexpression of VEGF from tumors resulted in elevated numbers of regulatory T cells in the tumor, suggesting a novel mechanism of VEGF-mediated immune suppression at the tumor site. CONCLUSION: GM-CSF-secreting cancer immunotherapy and VEGF blockade increases the i.t. ratio of effector to regulatory T cells to provide enhanced antitumor responses. This therapeutic combination may prove to be an effective strategy for the treatment of patients with cancer.
Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Immunotherapy/methods , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/therapy , T-Lymphocytes, Regulatory/drug effects , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Animals , Apoptosis , Carcinoma/therapy , Cell Count , Colonic Neoplasms/therapy , Combined Modality Therapy , Dendritic Cells/cytology , Fas Ligand Protein/physiology , Gene Expression Regulation, Neoplastic , Genetic Therapy/methods , Granulocyte-Macrophage Colony-Stimulating Factor/therapeutic use , Lymphocytes, Tumor-Infiltrating/cytology , Melanoma, Experimental/therapy , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Neoplasms, Experimental/mortality , Receptors, Vascular Endothelial Growth Factor/genetics , Receptors, Vascular Endothelial Growth Factor/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/therapeutic use , Survival Analysis , T-Lymphocytes, Regulatory/cytology , Treatment Outcome , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A/metabolism , fas Receptor/analysisABSTRACT
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 rAAV-mediated gene transfer of a potent soluble VEGF inhibitor in the CNS represents an effective antiangiogenic treatment strategy for GBM.
