The presence of metastases in regional lymph nodes is a strong indicator of poor patient survival in many types of cancer. It has recently been shown that the lymphangiogenic growth factor, vascular endothelial growth factor-C (VEGF-C), and its receptor, VEGF receptor-3 (VEGFR3), may play a pivotal role in the promotion of metastasis to regional lymph nodes. In this study, human prostate and melanoma tumor models that preferentially metastasize to the lymph nodes following s.c. tumor cell implantation were established from lymph node metastases via in vivo selection. Melanoma tumor cell sublines established from lymph node metastasis express higher amounts of VEGF-C than the parental tumor cells. The inhibition of tumor-derived VEGF-C with a soluble VEGFR3 decoy receptor, sVEGFR3-Fc, expressed via a recombinant adeno-associated viral vector, potently blocks tumorassociated lymphangiogenesis and tumor metastasis to the lymph nodes, when the treatment was initiated before the tumor implantation. In addition, sVEGFR3-Fc serum levels required for efficient blockade of lymph node metastases are strictly dependent on the VEGF-C levels generated by the primary tumor. Recombinant adeno-associated virusmediated gene transfer of sVEGFR3-Fc may represent a feasible therapeutic strategy for blockade of lymphogenous metastasis. (Cancer Res 2005; 65(15): 6901-9)
Purpose: The purpose of the present study was to evaluate granulocyte macrophage colonystimulating 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 solubleVEGF 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 Tcells 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 Tcells in the tumor, suggesting a novel mechanism ofVEGF-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.
AAV producer cell lines can be readily scaled to meet the needs of clinical trials. One 500 L bioreactor of these producer cells can produce the equivalent of 2500 high capacity roller bottles or 25 000 T-175 tissue culture flasks.
Treacher Collins syndrome is an autosomal dominant disorder of craniofacial development, which has been localized to chromosome 5q32-33.1. In the present study, the isolation of new polymorphic markers has allowed the identification of overlapping recombination events in two affected individuals. Extension of the transcription map of the critical region proximally has resulted in the isolation of a new gene (which has been named Treacle) of unknown function. The identification of different mutations in five unrelated families, all of which would result in premature termination of the predicted protein, indicates that the Treacher Collins syndrome gene has been positionally cloned.
Previous experiments have demonstrated the stable expression of factor IX (FIX) protein in mice and canine models of hemophilia B following portal vein gene transfer with a recombinant adeno-associated virus (rAAV) vector encoding FIX. Here, we present the results of studies that further optimized the rAAV vector transgene cassette used to express FIX and explored the use of the less-invasive intravenous (i.v.) route of vector administration for the treatment of hemophilia B. First, a liver-specific promoter was evaluated in conjunction with cis-acting regulatory elements in mice. Constructs that included both the b-globin intron and the woodchuck hepatitis virus post-transcriptional regulatory element resulted in the highest level of FIX expression in vivo. Using this optimized vector, we demonstrate that i.v. injection was feasible for hepatic gene transfer in mice, achieving 70-80% of portal vein expression levels of FIX. In further studies using the Chapel Hill strain of hemophilia B dogs, we demonstrate for the first time FIX expression and partial correction of the bleeding disorder following i.v. administration of an AAV vector.
A critical factor in clinical development of cancer immunotherapies is the identification of tumor-associated antigens that may be related to immunotherapy potency. In this study, protein microarrays containing >8,000 human proteins were screened with serum from prostate cancer patients (N = 13) before and after treatment with a granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting whole cell immunotherapy. Thirty-three proteins were identified that displayed significantly elevated (P
Osteogenesis imperfecta type II was diagnosed prenatally by analysis of DNA obtained from chorionic villus sampling (CVS) performed at 12 weeks of gestation in a woman who previously had had an affected child. The father had been shown to be mosaic for a mutation in the gene (COL1A2) which encodes the alpha 2(I) chain of type I collagen. An affected fetus was predicted by detection of the mutation in amplified chorionic villus genomic DNA. Ultrasound examination at 13 weeks 4 days demonstrated femoral deformity and virtual absence of calvarial mineralization. In pregnancies at risk for osteogenesis imperfecta type II, sonographic evidence of skeletal abnormalities may be evident by 13 weeks' gestation.
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.
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