236 Microwave Ablation Assisted Liver Gene Transfection in Rats Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy S92 CanCer – TargeTed gene a[.]
Cancer – Targeted Gene and Cell Therapy I fibroblasts has respectively profound effects on the invasive ability of breast cancer cells By applying both HIF-1α wild-type (WT) and knockout (KO) fibroblasts and breast cancer cells and using an in vitro invasion assay through commercially precoated invasion inserts, we found that fibroblasts, regardless their HIF-1α status, stimulated cell invasion of breast cancer cells that have intact HIF-1α In contrast, only HIF-1α-expressing fibroblasts, not HIF-1α-nonexpressing ones, stimulated HIF-1α KO breast cancer cell invasion These results suggest the complicated and respective HIF-1α contribution from breast cancer per se and surrounding fibroblasts to the tumor invasion and malignancy Moreover, by using a Tet-on inducible system, we found that ectopic p16 gene transfer is capable of inhibiting hypoxia-induced cell migration and invasion of breast cancer cells, and suppressing cocultured fibroblast-stimulated invasiveness of breast cancer cells These results suggest that p16, in addition to its well-known anti-tumor proliferation function, has novel anti-cancer properties capable of suppressing hypoxia/HIF-1α-mediated cancer cell migration and invasion Furthermore, our current ongoing study implies an interaction between p16 and HIF-1α that may elucidate a novel p16-mediated tumor suppression pathway via HIF-1α-regulated signaling These studies together may provide new information of the convergence of several important cell-growth regulation pathways on tumor invasion that would yield potential novel targets for effective cell- or gene-therapy strategy for treatment of breast cancer 234 Hydrodynamic Cell Delivery for Assessment of Tumor Growth and Drug Sensitivity in Different Organs and Microenvironment Mohammad H Alsaggar,1 Qian Yao,1 Dexi Liu.1 Pharmaceutical & Biomedical Sciences, University of Georgia, Athens, GA Tumor metastasis often confers poor prognosis for cancer patients because successful treatment of metastatic tumors remains as an unmet need in clinic Currently used subcutaneous and orthotopic models have failed to truly recapitulate metastatic tumor biology, particularly tumor-stroma interactions within tumor microenvironment in different organs This certainly accounts for insufficient therapeutic outcomes of current antimetastatic therapies that were designed assuming metastatic tumors behave the same in all organs Therefore, relevant tumor models are urgently needed for reliable anticancer drug screening and development, and assessment of tumor-environment interactions in different metastatic sites Toward this end, we have developed a multi-organ tumor growth model using hydrodynamic cell delivery method to establish simultaneous growth in liver, lung and kidney in mice by hydrodynamic tail vein injection of cell suspension We hypothesized that tumors growing in different organs are biologically heterogeneous in growth characters, as well as in tumor response to a given anti-cancer therapy Using luciferase-expressing murine melanoma cells that can be quantified by luciferase assay, our results showed that the liver is the primary organ to be seeded with tumor cells upon hydrodynamic injection, with 35% of tumor cells in liver, 21% in the lungs, and 8% in kidneys Growth rate assessment revealed that liver microenvironment was the most supportive to melanoma tumor growth compared to lungs and kidneys; where tumors grew minimally at early phase, and aggressively thereafter Tumors in different organs were also heterogeneous in tumor response to chemotherapy and immune gene therapy using dacarbazine and interferon beta gene, respectively While lung tumors responded to chemotherapy better than tumors in liver, lung tumors showed minimal response to interferon beta, with around 25% tumor suppression in lung, compared to more than 80% in liver and kidneys On the other hand, tumors in liver showed superior response to interferon beta compared to dacarbazine (81% vs 45% suppression) Differential behavior of metastatic tumors was S92 also confirmed using metastatic colon cancer model established in different organs These results point out the increasing need for better understanding of differences in tumor microenvironment in different organs, as it greatly determines therapeutic outcomes of antimetastatic therapies Moreover, the biological heterogeneity of metastatic tumors necessitates precluding of “one size fits all” therapies, and rethinking of organ-specific therapies and combined treatments to act globally on metastatic tumors in different organs 235 Anticancer Effectiveness of Mesenchymal Stem Cells Transfected With Thymidine Phosphorylase for Colorectal Cancer Cells Nasrin Salehi,1 Ching-An Peng.1 Michigan Technological University, Houghton, MI Gene-directed enzyme prodrug therapy (GDEPT) consists of targeted delivery to tumor cells with a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites One of the major bottlenecks of GDEPT is to have the therapeutic gene specifically target on cancer cells prior to the administration of prodrug Among various gene delivery methods, mesenchymal stem cells (MSCs) have emerged as potential cellular vehicles for gene delivery They exhibit remarkable tumor-tropic and low immunogenic capacities In this study, MSCs was engineered to express thymidine phosphorylase (TP) which has the capability of converting prodrug 5’-deoxy-5-fluorouridine into toxic 5-fluorouracil TP expression in the MSCs post-transfection of TP cDNA was confirmed by immunoblotting analysis and its activity was determined by spectrophotometric assay Our results showed the anticancer effectiveness of human HT29 colorectal cells co-cultured with TPexpressing MSCs was enhanced with the addition of various dosages of 5’-deoxy-5-fluorouridine 236 Microwave Ablation Assisted Liver Gene Transfection in Rats Xianghui He,1 Ruoyu Jiang,1 Lingkai Meng,1 Xiaoyu Liang,1 Jie Zhang,1 Zhixiang Zhang,1 Liwei Zhu.1 Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, China BACKGROUNDS: Thermal ablation has been widely used to manage liver tumor This study was designed to determine histological change of rat liver after microwave ablation, investigate the effect of microwave ablation on gene transfection METHODS: In this study, microwave ablation was applied to rat liver, tissue demarcation was evaluated Green fluorescent protein (GFP) and Renilla luciferase-expressing plasmids were administrated to the liver by percutaneous or intravenous injection pre-ablation The expression of transgene in the reactive inflammatory zone was evaluated GFP expression was determined with the inverted fluorescence microscope in frozen section, and Renilla luciferase expression in target tissue was determined using Renilla luciferase plasmid kit The effect of hydrodynamic portal vein injection was also evaluated RESULTS: GFP expression was observed in the tissue of reactive inflammatory zone after microwave ablation and plasmid injection Green fluorescence in targeted area was visible on till the seventh day post ablation under inverted fluorescence microscope On the first day postablation and Renilla luciferase plasmid injection, luminescence value of the experimental group was higher than the control group, but lower than the portal hypertension injection group CONCLUSIONS: Direct injection of plasmid vector to inflammatory reaction zone after microwave ablation can achieve effective gene transfection, but the transfection efficiency is lower Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy Hematologic and Immunologic Diseases I than hydrodynamic portal injection These results provide some experimental basis for exploring microwave ablation assistant target gene transfer for cancer gene therapy Hematologic and Immunologic Diseases I 237 Genetic Therapy for Perforin Deficiency Associated Hemophagocytic Lymphohistiocytosis Requires High Level Expression of the Perforin Gene for Adequate Correction Swati Tiwari,1 Adrianne Hontz,3 Catherine Terrell,4 Paritha Arumugam,5 Marlene Carmo,2 Bobby H Gaspar,2 Kimberly Risma,3 Michael Jordan,4 Punam Malik.5 Mol Genetics, UC, Cincinnati, OH; 2Mol Immunology Unit, UCL, London, United Kingdom; 3Allergy, CCHMC, Cincinnati, OH; 4Immunobiology, CCHMC, Cincinnati, OH; 5Exp Hematology, CCHMC, Cincinnati, OH Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal immune regulatory disorder, whereby heightened immune activation following viral infections causes hypercytokinemia, pancytopenia and end-organ damage The majority of familial HLH is caused by Perforin-1 (prf1) gene mutations Perforin is expressed in CD8+ T cells and natural killer (NK) cells and is critical for lymphocyte cytotoxicity Currently, immunosuppressive therapy followed by allogeneic hematopoietic stem cell (HSC) transplant is the only curative option for HLH, but is restricted by availability of matched donors and a high (20-50%) transplant related mortality We posited that perforin gene transfer into autologous HSC may be curative, have lower mortality, and not be limited by donor availability We recently reported significant, but partial, correction of HLH symptoms in prf1-/- mice using lentivirus vectors (LV) expressing perforin from a ubiquitous phosphoglycerate kinase (PGK) promoter or a tissue specific perforin gene promoter (PRF) We hypothesized that negative selection of high perforin expressing HSC and the perforin gene dosage delivered by the LV contributes to the partial correction We redesigned and developed a series of LV and compared prf1 expression from the PGK/PRF promoters to that from the MND promoter/ enhancer; additionally we restricted perforin expression from PGK and MND in HSC with repeats of miR126 (4T) target sequence, a miRNA specifically expressed in HSCs but not in lymphocytes, thus, circumventing the negative selection of high perforin expressing HSC Improved perforin expression and cytotoxicity was observed with the ‘HSC-restricted’ MND4T LV in the KHYG1 human NK cell line, and in prf1-/- P14 mice, transgenic for a T-cell receptor that recognizes a lymphocytic choriomeningitis virus (LCMV) glycoprotein These LV were compared in prf1-/- mice that were challenged with LCMV following transplant of gene-modified HSC High gene marked NK chimerism was achieved: 87.6±1.7%, 83.1±2.4% and 58.1±4.6% with the MND4T, PGK4T and PRF0T LV Following LCMV challenge, γ-IFN levels in MND4T, PRF0T and PGK4T mice were 1.7±0.3, 1.5±0.5 and 5.9±2.1 ng/ml, respectively, compared to 1.7±0.5 ng/ml in prf1-/- mice receiving WT HSC Similarly, the degree of anemia in WT and MND4T mice was comparable 15 days after LCMV challenge: hemoglobins declined by 4.3±0.3, 3.7±0.3,, 5.6±0.3 and 5.8±0.7 g/dL in WT, MND4T, PGK4T and PRF0T mice, respectively Most importantly, 66.7% of the mice in the MND4T group survived the LCMV challenge, while only 10% and no mice survived in PGK4T and PRF0T groups, suggesting that perforin expression from PGK4T and PRF0T were insufficient to prevent fatal HLH in the majority of animals Overall, we show that perforin deficient HLH requires a robust perforin expression in cytotoxic T and NK cells for adequate correction of the disease phenotype Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy 238 BTK-Promoter LV Vectors Utilizing Conserved Intron Element Mediate Functional Rescue in Murine XLA Swati Singh,1 Brenda Seymour,1 Karen Sommer,1 Iram Khan,1 Socheath Khim,1 Malika Hale,1 Courtnee Clough,1 David J Rawlings.1,2 Center for Immunity and Immunotherapies and Program for Cell and Gene Therapies, Seattle Children’s Research Institute, Seattle, WA; 2Department of Pediatrics and Immunology, University of Washington, Seattle, WA X-linked agammaglobulinema (XLA) is a rare immunodeficiency disorder caused by a mutation in the gene coding Bruton’s tryosine kinase (BTK) BTK is required for signal transduction downstream of the B cell antigen receptor (BCR) and is essential for normal B cell development, activation, and survival Patients lacking BTK have very few mature B cells, markedly reduced antibody production, and suffer from recurrent and life-threatening infections XLA is an ideal candidate for hematopoietic gene therapy, as B cells that express BTK are positively selected during development We previously developed a SIN-lentiviral (LV) construct that used the human BTK promoter (BTKp) to drive BTK expression We also incorporated a truncated, 0.7 kb, fragment derived from the HNRPA2B1/CBX3 ubiquitously acting chromatin opening element (UCOE) upstream of the BTKp to facilitate stable BTK expression over time Bone marrow transplantation in a murine XLA model (BTK-/-Tec-/- mice) using LV transduced hematopoietic stem cells (HSCs) reconstituted myeloid and B cell numbers, BCR-dependent B cell proliferation and T-dependent antibody responses The goal of the current study was to further optimize BTK expression in B and myeloid cells while reducing the viral copy number (VCN) required for efficient functional reconstitution Using the ENCODE database, we identified several highly conserved upstream and intronic regions exhibiting histone modifications and transcription factor binding profiles in B cells consistent with enhancer regions These conserved elements were introduced individually or in tandem into the LV construct between the UCOE and BTKp and tested for their ability to rescue BTK expression and B cell development in the murine XLA model Addition of two key intronic regions led to consistent BTK expression and rescued the development and function of B cells, despite a >5fold reduction in VCN per splenic B cell Our findings illustrate the usefulness of this strategy for improving the efficacy of endogenous promoter-based genetic therapies This new LV vector, UCOE I4-5.BTKpro-hBTK, is currently being evaluated using mobilized CD34+ stem cells isolated from XLA patients and in additional pretoxicology studies designed to support its application in a future LV clinical trial for XLA 239 Preclinical Studies for the First Hematopoietic Stem Cell (HSC) Gene Editing Trial: Phase Study of Beta-Thalassemia With Autologous Transplantation of Zinc Finger Nuclease-Treated HSC To Upregulate Fetal Hemoglobin Dale Ando,1 Mark C Walters,2 Kai-Hsin Chang,3 Andreas Reik,1 Fyodor D Urnov,1 Gary Lee,1 Kaye S Spratt,1 Edward J Rebar,1 Philip D Gregory,1 Winson Tang,1 Thalia Papayannopoulou,3 George Stamatoyannopoulos,3 Geoffrey Nichol.1 Sangamo BioSciences, Inc, Richmond, CA; 2UCSF Benioff Children’s Hospital Oakland, Oakland, CA; 3University of Washington, Seattle, WA Beta-thalassemia (β-thal) and sickle cell disease (SCD) are monogenic diseases caused by mutations in the adult β-globin gene Allogeneic hematopoietic cell transplantation (HCT) is the only S93 ... Malik.5 Mol Genetics, UC, Cincinnati, OH; 2Mol Immunology Unit, UCL, London, United Kingdom; 3Allergy, CCHMC, Cincinnati, OH; 4Immunobiology, CCHMC, Cincinnati, OH; 5Exp Hematology, CCHMC, Cincinnati,... Immunology, University of Washington, Seattle, WA X-linked agammaglobulinema (XLA) is a rare immunodeficiency disorder caused by a mutation in the gene coding Bruton’s tryosine kinase (BTK) BTK is required... activation following viral infections causes hypercytokinemia, pancytopenia and end-organ damage The majority of familial HLH is caused by Perforin-1 (prf1) gene mutations Perforin is expressed in CD8+