442 Baculovirus Mediated miR 122 and miR 151 Modulation Suppresses Mahlavu Cell Proliferation/Migration In Vitro and Tumor Growth In Vivo Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright[.]
CANCER-TARGETED GENE AND CELL THERAPY: CELLULAR AND VIRAL VECTOR-BASED APPROACHES, AND BIOLOGICAL ASPECTS OF TARGETED THERAPIES B cells in association with T cells, and the biological effects observed in vitro suggest that B lymphocytes may play a role in T cell-based antitumor immunity Cancer-Targeted Gene and Cell Therapy: Cellular and Viral Vector-Based Approaches, and Biological Aspects of Targeted Therapies 440 Sequence-Defined Nanocarriers for c-Met Receptor-Directed Gene Transfer In Vitro and In Vivo Petra Kos,1,2 Ulrich Lächelt,1,2 Annika Herrmann,1 Dongsheng He,1 Ernst Wagner.1,2 Pharmaceutical Biotechnology, Center for System-Based Drug Research, Ludwig-Maximilians-University, Munich, Germany; Nanosystems Initiative Munich, Munich, Germany The overexpression of a variety of surface receptors in cancer tissues offers the possibility to selectively address tumor cells by applying different targeting ligands which recognize and bind to such receptors and therewith help to overcome the side effects related to classical cancer therapy Diverse ligands, such as antibodies, carbohydrates, vitamins and peptides have been conjugated to nonviral polymeric carriers and exploited for targeted gene delivery In this respect, ligands binding to growth factor receptors, e.g epidermal growth factor receptor (EGFR) or vascular endothelial growth factor receptor (VEGFR) have shown vast potential Herein, a phage display-derived hepatocyte growth factor receptor (c-Met/ HGFR) binding peptide (cMBP2) was for the first time applied to a gene delivery system by being conjugated to monodisperse sequencedefined oligomers which facilitate crucial steps in pDNA delivery, such as polyplex formation, cellular uptake and endosomal escape Methods: The cMBP2 ligand was covalently attached to artificial oligo(ethanamino)amides synthesized via solid-phase peptide synthesis (SPPS) Apart from the targeting moiety, the resulting oligomers comprised a shielding PEG domain, protonable pH-tuned oligoamines and histidines necessary for nucleic acid compaction and endosomal escape, and cysteines for additional polyplex stabilization Different cMBP2-targeted oligomers were evaluated for their biophysical characteristics, cellular binding, cellular internalization, receptor activation, and gene transfer in vitro and in vivo Results and Discussion: cMBP2-equipped shielded oligomers directed towards tyrosine kinase receptor c-Met were pinpointed as a novel potent gene delivery approach All synthesized oligomers displayed satisfactory pDNA compaction ability that slightly decreased with increasing PEG amount and implementation of histidines in the oligomer backbone The cMBP2-targeted pDNA polyplexes revealed specific receptor-dependent targeting effect in different cancer cell lines in vitro The transfection efficiency was significantly further improved by incorporation of the “proton sponge” promoting histidines, whereas the addition of further PEG units beyond the optimal amount led to a reduced gene transfer The cytotoxicity of the polyplexes was found to be negligible Importantly, the absence of c-Met receptor activation and subsequent downstream signaling upon polyplex attachment to the cell surface was confirmed A clear targeting effect was demonstrated in vivo both after intratumoral local and intravenous systemic injection in Huh7 hepatocellular carcinoma tumor-bearing mice Conclusion: The designed precise pH-revised c-Met receptortargeted oligomers present new promising and well biocompatible tumor-specific gene delivery systems The novel formulations highlight the necessity of PEGylation on the one hand and a sufficient polycationic part on the other hand for tumor-targeted delivery, and set the stage for further evolvement of defined, efficient and safe nucleic acid carriers Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy 441 Suppression of Hypoxia/HIF-1α Promoted Breast Cancer Malignant Progression by p16/ INK4a Jun Zhang,1 Liyuan Li,1 Yi Lu.1 Pathology and Laboratory Medicine, University of Tennessee Health Science Center, Memphis, TN While gene therapy effects on tumor cells have been widely studied, few studies have been explored on effects of tumor suppressor genes on tumor-surrounding cells and effects of tumor-surrounding cells on tumor progression Accumulated evidences demonstrate that tumor-surrounding stromal cells (such as fibroblasts) are integrated components of the tumor environment that play critical roles in the tumor progression In this study, we investigated whether HIF-1α from tumor-surrounding fibroblasts plays a critical role in breast cancer (BCa) malignant progression, and whether p16 gene transfer neutralizes HIF-1α promoted migration and invasion of cancer cells We co-cultured BCa 4T1/GFP cells with MEF fibroblasts that either expressed or lacked the HIF-1α gene in a 3D culture system established in our lab The mixed cells (BCa:MEF) grew in a suspension manner and formed aggregates/spheroids We found that HIF-1α plays a role under hypoxia in the 3D BCa cells’ protrusion formation, an established marker for invasive, aggressively malignant tumor cells that start to invade the surrounding matrix Moreover, we performed transwell overnight invasion assay of BCa LM2 cells with and without cocultured MEF under both normoxia (21% O2) and hypoxia (1% O2) We found that MEF significantly promoted BCa invade through matrigel, and HIF-1α WT MEF promoted BCa invasion more than HIF-KO MEF did under hypoxia A similar pattern of HIF-1α-dependent, hypoxia-stimulated cell migration was observed in fibroblasts when we analyzed HIF-1α WT and KO MEF cells, demonstrating that hypoxia stimulated a HIF-1α-dependent cell migration in both BCa and MEF cells By using a Tet-on induced system, we found p16-expressing BCa cells have significantly reduced invasion and migration abilities under both normoxia and hypoxia compared to their p16-nonexpressing counterparts p16 also significantly inhibit hypoxia-induced MEF migration and MEFassisted BCa invasion Together, these results indicate that p16 has a novel property (besides its well-known inhibition of cell proliferation) to neutralize hypoxia/HIF-1α-promoted malignant progression of BCa from both tumor-surrounding fibroblasts and cancer cells These new findings should further support p16 gene therapy as a conjugate therapeutics to combat BCa metastasis 442 Baculovirus-Mediated miR-122 and miR-151 Modulation Suppresses Mahlavu Cell Proliferation/Migration In Vitro and Tumor Growth In Vivo Chiu-Ling Chen,1 Pei-Hsiang Yuan,1 Guan-Yu Chen,1 Kuei-Chang Li,1 Yu-Chen Hu.1 Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan Baculovirus is non-pathogenic to humans and has emerged as a promising gene delivery vector in cancer therapy Although the design of microRNA precursors and sponges have been reported, the antitumor effects by simultaneously modulating microRNAs (miRs) during hepatocellular carcinogenesis have yet to be explored Here, we employed a recombinant Sleeping Beauty (SB)-based baculovirus vector to deliver miR-122 precursors and miR-151 sponges into HCC (heptocellular carcinoma) cells and aimed to evaluate the feasibility of baculovirus-mediated miRNA modulation for HCC therapy To this end, we constructed baculovirus vectors: Bac-s151 expressed miR-151-specific sponges; Bac-m122 harbored a cluster comprising copies of pre-miR-122 while Bac-Dual accommodated both cassettes By co-transducing Mahlavu cells with another SB S169 CANCER-TARGETED GENE AND CELL THERAPY: CELLULAR AND VIRAL VECTOR-BASED APPROACHES, AND BIOLOGICAL ASPECTS OF TARGETED THERAPIES transposase-expressing Bac-SB, the SB/Dual group simultaneously enhanced miR-122 expression and suppressed miR-151 in transduced Mahlavu cells, yet SB/m122 and SB/s151 group only persistently regulated single miRNA In addition, the modulation of miR-122 and miR-151 led to enhanced apoptosis, inhibition of cell motility, cell cycle and proliferation Western blot analysis also showed that the baculovirus-mediated miRNA modulation suppressed the protein levels (ADAM10, ADAM17, Bcl-w, cyclin G1) downstream of miR122, as well as regulated the protein levels (Rho GTPases family, RhoGDIA) associated with the miR-151 pathway Intratumoral injection of the baculovirus into the mice bearing Mahlavu tumor effectively repressed the tumor growth and enhanced the survival rate The simultaneous miRNA modulation also suppressed the cell cycle, metastasis and triggered apoptosis in vivo Altogether, the baculovirus-mediated, miRNA-based genetic modulation may serve as a means of therapy to block the proliferation and migration of Mahlavu cells 443 Mesenchymal Stem Cells as a Delivery System for Anti-Fibrotic Genes to Areas of Irradiation-Induced Pulmonary Fibrosis Byung Rhieu,1 Ashwin Shinde,1 Ronny Kalash,1 Julie Goff,1 Darcy Franicola,1 Tracy Dixon,1 Xichen Zhang,1 Michael Epperly,1 Peter Wipf,2 Song Li,3 Joel S Greenberger.1 Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA; 2Chemistry, University of Pittsburgh Cancer Institute, Pittsburgh, PA; 3Pharmaceutical Science, University of Pittsburgh Cancer Institute, Pittsburgh, PA Irradiation of the lung during radiation therapy of lung cancer results in damage of normal lung tissue leading to development of pulmonary fibrosis Fibrosis is characterized by an accumulation of fibroblasts and deposition of collagen, which results in lungs with limited expansion capacity We developed a model for using mesenchymal stem cells (MSC) to deliver antioxidant genes to the site of developing fibrosis to remove the reactive oxygen species that stimulate the formation of fibrosis Using C57BL/6NHsd mouse model, pulmonary fibrosis is induced beginning at day 150 after 20 Gy pulmonary irradiation We irradiated C57BL/6NHSD mice to 20 Gy to the pulmonary cavity shielding the remainder of the body and intraperitoneally injected MSC isolated from a luciferase positive transgenic mouse on day 2, 60 or 127 following irradiation A subgroup of the mice were placed on water containing the antioxidant MMS350 which is a water soluble DMSO analogue Live mice were imaged at serial time points following injection of D-luciferin using an IVIS(r) 200 Optical Imaging System The bioluminescent signal for each mouse was quantitated As a control similar experiments were performed with fibrosis resistant C3H/ HeNHsd mice Irradiated C57BL/6NHsd or C3H/HeNHSD injected with Luc+ MSC at day or day 60 after irradiation demonstrated no migration of Luc+ cells to the lungs However, irradiated C57BL/6NHsd mice that were injected on day 127 with Luc+ MSC cells demonstrated significant migration of Luc+ MSC to the lungs In contrast, fibrosis resistant C3H/HeNHsd mice showed no detectable migration of Luc+ MSCS to the lungs C57BL/6NHsd mice receiving daily MMS350 beginning on day 80 after irradiation, then injected with Luc+ MSC on day 127, showed significantly decreased migration of LUC+ MSC to the lungs Mice treated with MMS350 demonstrated significantly decreased less pulmonary fibrosis at day 200 after irradiation Thus, delivery of antioxidants prevents the development of pulmonary fibrosis In a proposed model, MSC cells would be isolated from mice and transfected with an antioxidant enzyme such as manganese superoxide dismutase At the time of development S170 of pulmonary fibrosis injection of MnSOD-transgene-transfectedMSC migrate to the lungs will be tested for capacity to reduce the development of fibrosis Supported by NIAID /NIH grant U191A168021-08 444 A Small Molecule That Inhibits p53 Degradation Influences Cytotoxicity of Ad-p53 in INK4A/ARF-Defective Mesothelioma with WildType p53 Gene Masatoshi Tagawa,1 Kiyoko Kawamura,1 Shinya Okamoto,2 Yuanyuan Jiang,1 Zhihan Li,1 Shuji Kubo,3 Masato Shingyoji,4 Yuji Tada,2 Yuichi Takiguchi,5 Koichiro Tatsumi,2 Hideaki Shimada,6 Kenzo Hiroshima.7 Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, Chiba, Japan; 2Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan; 3Department of Genetics, Hyogo College of Medicine, Nishinomiya, Japan; 4Department of Thoracic Diseases, Chiba Cancer Center, Chiba, Japan; 5Department of Medical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan; Department of Surgery, School of Medicine, Toho University, Tokyo, Japan; 7Department of Pathology, Tokyo Women’s Medical University Yachiyo Medical Center, Yachiyo, Japan A majority of mesothelioma has a homologous deletion in the INK4A/ARF locus that contains the p14 and the p16 genes, but the specimens with the deletion show that the p53 gene is wildtype The characteristic genetic alterations lead to decrease of p53 functions because loss of p14 protein cannot inhibit p53-degradating Mdm-2 functions, and further promote cell cycle progression by phosphorylated pRb that is caused by failure of p16-induced suppression of CDK4/6 functions Forced expression of p53 is a rational strategy for mesothelioma by restoring the p53 levels and dephosphorylating pRb through p53-induced p21 functions In fact, transduction of p14/p16-defective mesothelioma with adenoviruses expressing the wild-type p53 gene (Ad-p53) induced the p53 phosphorylation at Ser 15 and Ser 46, both of which were markers of p53 activation, and dephosphorylated pRb at Ser 795 The transduced cells were then subjected to apoptosis with caspases activations Moreover, Ad-p53 also augmented susceptibility of mesothelioma to the current first-line chemotherapeutic agents, cisplatin and pemetrexed We then investigated a possible strategy to augment the Ad-p53 effects with a chemical agent in terms of p53 regulations Nutlin-3a inhibits interactions between Mdm-2 and p53 and subsequently augmented p53 levels in p53-wild-type mesothelioma Heat shock protein 90 (Hsp90) inhibitors, 17AAG and 17-DMAG, suppress Mdm-4 functions that facilitate p53 degradation, and accordingly increased p53 expression in the mesothelioma These agents augmented p53 phosphorylation and induced cell death A combinatory use of nutlin-3a and Ad-p53 produced synergistic cytotoxicity, however, the combination of Ad-p53 and the Hsp90 inhibitors was rather antagonistic to Ad-p53mediated cytotoxicity The inhibitory action of Hsp90 inhibitors was not due to suppression of Ad-mediated infection or Ad-mediated transcription since Hsp90 inhibitors did not decrease fluorescence of cells infected with Ad bearing green fluorescent protein gene The Hsp90-mediated inhibition was due to suppressed chaperon activity of Hsp90 that targets p53 stability These data collectively indicates that up-regulation of p53 in mesothelioma is a therapeutic strategy but a combination of Ad-p53 with an agent needs to consider a fine tuning of p53 metabolic pathways Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy ... expression and suppressed miR- 151 in transduced Mahlavu cells, yet SB/m122 and SB/s151 group only persistently regulated single miRNA In addition, the modulation of miR- 122 and miR- 151 led to... Bcl-w, cyclin G1) downstream of miR1 22, as well as regulated the protein levels (Rho GTPases family, RhoGDIA) associated with the miR- 151 pathway Intratumoral injection of the baculovirus into the... bearing Mahlavu tumor effectively repressed the tumor growth and enhanced the survival rate The simultaneous miRNA modulation also suppressed the cell cycle, metastasis and triggered apoptosis in