Changchun Ren

Cancer gene therapy using mesenchymal stem cells expressing interferon-β in a mouse prostate cancer lung metastasis model

Cell-based therapy for cancer is a promising new field. Among cell types that can be used for this purpose, mesenchymal stem cells (MSCs) appear to hold great advantage for reasons including easier propagation in culture, possible genetic modification to express therapeutic proteins and preferential homing to sites of cancer growth upon in vivo transfer. The present study evaluated the potential of genetically modified MSC, constitutively expressing interferon (IFN)-β, in an immunocompetent mouse model of prostate cancer lung metastasis. A recombinant adeno-associated virus (rAAV) encoding mouse IFN-β was constructed and initially tested in vitro for high-level expression and bioactivity of the transgenic protein. MSCs were transduced by the rAAV-IFN-β or green fluorescent protein ex vivo and used as cellular vehicles to target lung metastasis of TRAMP-C2 prostate cancer cells in a therapy model. Cohorts of mice were killed on days 30 and 75 to determine the effect of therapy by measurement of tumor volume, histology, immunohistochemistry, enzyme-linked immunosorbent assay and flow cytometry. Results indicated a significant reduction in tumor volume in lungs following IFN-β-expressing MSC therapy. Immunohistochemistry of the lung demonstrated increased tumor cell apoptosis and decreased tumor cell proliferation and blood vessel counts. A significant increase in the natural kill cell activity was observed following IFN-β therapy correlating the antitumor effect. Systemic level of IFN-β was not significantly elevated from this targeted cell therapy. These data demonstrate the potential of MSC-based IFN-β therapy for prostate cancer lung metastasis.

Therapeutic Potential of Mesenchymal Stem Cells Producing Interferon‐α in a Mouse Melanoma Lung Metastasis Model

Adult stem cells represent a potential source for cell‐based therapy of cancer. The present study evaluated the potential of bone marrow‐derived mesenchymal stem cells (MSC), genetically modified to express interferon (IFN)‐α, for the treatment of lung metastasis in an immunocompetent mouse model of metastatic melanoma. A recombinant adeno‐associated virus (rAAV) 6 vector encoding IFN‐α was used to transduce mouse bone marrow‐derived MSC ex vivo. Expression and bioactivity of the transgenic protein from rAAV‐transduced MSC were confirmed prior to in vivo studies. A lung metastasis model of melanoma was developed by i.v. injection of B16F10 cells into 8‐week‐old C57BL/6 mice. Ten days later, MSC transduced with rAAV‐IFN‐α or green fluorescent protein were intravenously injected. One cohort of mice was sacrificed to determine the effects of the therapy at an earlier time point, and another cohort was observed for long‐term survival. Results indicated that systemic administration of MSC producing IFN‐α reduced the growth of B16F10 melanoma cells and significantly prolonged survival. Immunohistochemistry analysis of the tumors from MSC‐IFN‐α‐treated animals indicated an increase in apoptosis and a decrease in proliferation and blood vasculature. These data demonstrate the potential of adult MSC constitutively producing IFN‐α to reduce the growth of lung metastasis in melanoma.

Enhanced transduction of mouse bone marrow-derived dendritic cells by repetitive infection with self-complementary adeno-associated virus 6 combined with immunostimulatory ligands

The potential of adeno-associated virus (AAV)-based vectors in human gene therapy is being explored for several diseases. Although sustained transgene expression and low vector-associated cellular immunity are attractive features of recombinant (r) AAV, the wider application of rAAV vectors encapsidated in serotype 2 capsid is hampered by poor transduction efficiency in many target tissues. These include ex vivo-generated dendritic cells (DC), which have demonstrated promising immunotherapeutic activity. We report here that efficient transduction of mouse bone marrow-derived DC can be achieved with self-complementary (sc) rAAV encapsidated in serotype 6 capsid. Sequential exposure of DC precursor cultures to IL-4 and GM-CSF with sc rAAV6 encoding the human tumor antigen, carcinoembryonic antigen (CEA), for 7 days followed by activation with CpG oligodeoxynucleotides (ODN) and anti-mouse CD40 antibody resulted in highly efficient transduction of DC. DC surface markers as determined by flow cytometry analysis of sc rAAV6-transduced DC were comparable to nontransduced DC. Efficiency of vector transduction and transgene expression were confirmed by immunostaining and real-time PCR. Microarray analysis of RNA from CpG ODN and CD40 antibody stimulated sc AAV6-transduced DC revealed upregulation of transcription factors and cytokines involved in immune activation and downregulation of inhibitory factors, suggesting a possible role of transcriptional activation in the observed effect. The adoptive transfer into syngeneic mice of the ex vivo-transduced and activated DC resulted in the development of CEA-specific antibody and T-helper 1-associated immune responses. Immunized mice also developed antibody to AAV6 capsid protein, which did not crossreact with AAV2 capsid protein. These studies demonstrate the potential utility of sc rAAV serotype 6-based vectors in transduction of DC for genetic vaccination approaches.

Free fatty acids enhance breast cancer cell migration through plasminogen activator inhibitor-1 and SMAD4

Obesity is a risk factor for breast cancer and is associated with increased plasma concentrations of free fatty acids (FFAs). We and others have demonstrated that FFA induces plasminogen activator inhibitor-1 (PAI-1) expression in a variety of cells. Emerging evidence supports elevation of PAI-1 as a prognostic marker for breast cancer. Therefore, we hypothesized that FFAs might increase expression of PAI-1 in breast cancer cells and facilitate breast cancer progression. Secreted PAI-1 was higher in invasive and metastatic MDA-MB-231 cells compared with less invasive and non-metastatic Hs578T cells. Utilizing FFAs with different saturation and chain lengths, we demonstrated that linoleic acid induced expression of PAI-1 in MDA-MB-231 cells. Linoleic acid also induced in vitro migration of MDA-MB-231. By contrast, other FFAs tested had little or no effect on PAI-1 expression or migration. Linoleic acid-induced breast cancer cell migration was completely inhibited by virally expressed antisense PAI-1 RNA. Furthermore, increased expression of PAI-1 by FFAs was not detected in the SMAD4-deficient MDA-MB-468 breast carcinoma cells. Electrophoretic mobility-shift assay confirmed that linoleic acid-induced expression of PAI-1 was mediated, at least in part, by SMAD4 in MDA-MB-231 cells. That linoleic acid induces PAI-1 expression in breast cancer cells through SMAD4 provides a novel insight into understanding the relationships between two migration-associated molecules, FFAs, and PAI-1.

Intraperitoneal gene therapy by rAAV provides long-term survival against epithelial ovarian cancer independently of survivin pathway

Epithelial ovarian carcinoma is the leading cause of death from gynecological malignancies. Owing to the lack of an effective screening method, insidious onset, and non-specific symptoms, a majority of women present with advanced stage disease. Despite improvements from cytoreductive surgery and chemotherapy, recurrent disease remains a formidable challenge. In the present study, we demonstrate for the first time that stable intra-abdominal genetic transfer of endostatin and angiostatin (E+A) by recombinant adeno-associated virus (rAAV) provides sustained antitumor effects on the growth and dissemination of epithelial ovarian cancer in a mouse model. Further, when combined with paclitaxel (taxol), the effect of this therapy was dramatically increased and resulted in long-term tumor-free survival overcoming prior limitations of chemotherapy and gene therapy. The combined effects of angiosuppressive therapy and chemotherapy were found to be independently of survivin pathway. Evidence for the superior effects of the combination therapy was indicated by significantly lower ascites volume with less hemorrhage and tumor conglomerates, lower ascites vascular endothelial growth factor, higher tumor cell apoptosis and decreased blood vasculature, and long-term disease-free survival. Histopathology of visceral organs and liver enzyme assays indicated no toxicity or pathology.

Notch1 Augments Intracellular Trafficking of Adeno-Associated Virus Type 2

ABSTRACT We report here the significance of the Notch1 receptor in intracellular trafficking of recombinant adeno-associated virus type 2 (rAAV2). RNA profiling of human prostate cancer cell lines with various degrees of AAV transduction indicated a correlation of the amount of Notch1 with rAAV transgene expression. A definitive role of Notch1 in enhancing AAV transduction was confirmed by developing clonal derivatives of DU145 cells overexpressing either full-length or intracellular Notch1. To discern stages of AAV2 transduction influenced by Notch1, competitive binding with soluble heparin and Notch1 antibody, intracellular trafficking using Cy3-labeled rAAV2, and blocking assays for proteasome and dynamin pathways were performed. Results indicated that in the absence or low-level expression of Notch1, only binding of virus was found on the cell surface and internalization was impaired. However, increased Notch1 expression in these cells allowed efficient perinuclear accumulation of labeled capsids. Nuclear transport of the vector was evident by transgene expression and real-time PCR analyses. Dynamin levels were not found to be different among these cell lines, but blocking dynamin function abrogated AAV2 transduction in DU145 clones overexpressing full-length Notch1 but not in clones overexpressing intracellular Notch1. These studies provide evidence for the role of activated Notch1 in intracellular trafficking of AAV2, which may have implications in the optimal use of AAV2 in human gene therapy.

849. Transduction of Mouse Dendritic Cells Is Enhanced by Repetitive Infection with Self-Complementary Adeno-Associated Virus 6 Combined with Immunostimulatory Ligands

The potential of adeno-associated virus (AAV)-based vectors in human gene therapy is being explored for several diseases. Although sustained transgene expression and low vector-associated cellular immunity are attractive features of recombinant (r) AAV, the wider application rAAV vectors encapsidated in serotype 2 capsid, the most commonly applied rAVV, is hampered by poor transduction efficiency in many target tissues. These include ex vivo generated dendritic cells (DC), which have demonstrated promising immunotherapeutic activity. We report here that efficient transduction of mouse bone marrow-derived DC can be achieved with self-complementary (sc) rAAV encapsidated in serotype 6 capsid. Sequential exposure of DC precursor cultures to IL-4 and GM-CSF with sc rAAV6 encoding the human tumor antigen, carcinoembryonic antigen (CEA), for seven days followed by activation with CpG oligodeoxynucleotides (ODN) and anti-mouse CD40 antibody resulted in highly efficient transduction of DC. DC surface markers as determined by flow cytometry analysis of sc rAAV6-transduced DC were comparable to non-transduced DC. Efficiency of vector transduction and transgene expression were confirmed by immunostaining and real-time PCR. Microarray analysis of RNA from CpG-ODN and CD40 antibody stimulated sc AAV6-transduced DC revealed upregulation of transcription factors and cytokines involved in immune activation and down-regulation of inhibitory factors, suggesting a possible role of transcriptional activation in the observed effect. The adoptive transfer into syngeneic mice of the ex vivo transduced and activated DC resulted in the development of CEA-specific antibody. Immunized mice also developed antibody to AAV-6 capsid protein which did not cross-react with AAV-2 capsid protein. These studies demonstrate the potential utility of sc rAAV serotype 6-based vectors in transduction of DC for genetic vaccination approaches.

746. Genomic Stability of Self-Complementary Recombinant Adeno-Associated Virus 2 in Mouse Muscle

Recent studies have demonstrated that packaging of recombinant adeno-associated virus 2 (rAAV) genome as a self-complementary duplex strand (ds) results in early transgene expression, possibly overcoming the rate-limiting second-strand synthesis. In the present study, we evaluated the molecular organization and stability of the ds-AAV genome in 293 and Hela cells in vitro and in the quadriceps muscle of C57/BL/6J mice in vivo as compare to single-strand (ss) AAV-2 genome. The studies were carried out with rAAV containing GFP gene as either ds or as ss structure encapsidated in AAV-2 capsids. Cohorts of mice were injected with 1 × 1011 particles of rAAV-GFP (ds/ss) in quadriceps muscle and the injected tissues were harvested 1, 2 and 3 weeks later. The tissue from each animal was cut into two and processed directly for total DNA isolation or paraffin-embedded for immunohistochemistry and in situ hybridization. The total DNA was subjected to Southern blot analysis and PCR using primers specific for the amplification of linear and/or circular concatemers. Immunohistochemistry was performed with anti-GFP antibody and in situ hybridization using a DIG-labeled CMV probe. Results of Southern blot and PCR analysis indicate increased disintegration of ss-AAV vector genome over time in vivo compared to ds-AAV vector genome. Southern blot and PCR analysis also indicated efficient conversion of the vector genome to concatemers in ds vector. Approximately 30–40% of ds-AAV genome was detected as concatemers as early as three weeks. As expected, transgene expression was detected at early time point with ds vector and persisted stably compared to ss vector. These data demonstrate that ds AAV vectors provide better stability for transgene structure and expression and may have better utility in human gene therapy applications.