623 Inhibition of NF kB in Fusogenic Membrane Glycoprotein Causing HL 60 Cell Death Implications for Acute Myeloid Leukemia Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American[.]
CANCER - TARGETED GENE THERAPY II adenoviral E2-late promoter plays an important role in the adenoviral life cycle (Mantwill et al., Cancer Research, 2006) With this knowledge we have developed different oncolytic adenoviral vectors in which the transactivation domain CR3 of E1A was ablated to target YB-1 positive tumor cells The application of these vectors causes a strong antitumor effect in glioma cell lines which could be achieved at lower pfu/cell, when combined with chemotherapy Additionally, a glioma xenograft mouse model with U87 tumors was established Animals treated with the YB-1 dependent oncolytic adenovirus showed significantly smaller tumors than untreated controls and the effect was even enhanced by the combination with temozolomide, leading to complete regression of two from six examined tumor bearing mice which was verified by bioluminescence imaging and histological studies In addition, histological evaluation concerning lymphocytic infiltration and induction of apoptosis were used to characterise the mode of action of the different treatments Our results have shown the feasibility of YB-1 dependent virotherapy and form the basis for a phase I/II clinical study 621 Clinical Application of Telomerase-Specific Oncolytic Adenovirs to Ex Vivo Biological Imaging of Human Circulating Tumor Cells Futoshi Uno,1 Yuuri Hashimoto,2 Toru Kojima,1 Shunsuke Kagawa,1 Hiroshi Tazawa,1 Yasuo Urata,2 Noriaki Tanaka,3 Toshiyoshi Fujiwara.1 Center for Gene and Cell Therapy, Okayama University Hospital, Okayama, Japan; 2Oncolys BioPharma, Inc, Tokyo, Japan; Department of Surgery, Okayama University Graduate School, Okayama, Japan The presence of circulating tumor cells (CTC) in the peripheral blood is associated with short survival and, therefore, the detection of CTC is clinically useful as prognostic factors of disease outcome and/or surrogate markers of treatment response Recent technical advances including immunocytometric analysis and quantitative real-time PCR assay have made possible to detect a few CTC in the blood; however, there is no sensitive assay for detecting viable CTC Here we report a new approach to visually detect live CTC among millions of peripheral blood leukocytes using telomerase-specific replication-selective adenovirus expressing green fluorescent protein (GFP) We constructed a GFP-expressing attenuated adenovirus, in which the telomerase promoter regulates viral replication (OBP-401, TelomeScan) We used OBP-401 to establish a simple ex vivo method for detecting viable human circulating tumor cells in the peripheral blood The detection method involves a three-step procedure including the lysis of red blood cells, the subsequent addition of OBP-401 to the cell pellets, and the automated scan under the fluorescent microscope OBP-401 infection increases the signal-to-background ratio as a tumor-specific probe, because the fluorescent signal can be amplified only in tumor cells by viral replication We evaluated 23 samples obtained from histologically-confirmed gastric cancer patients Although the CTC level varied widely, ranging from to 47 in 5-ml samples, 14 patients had more than CTC In the two cases that underwent surgery, the CTC level dropped after weeks of complete resection These results suggest that enumeration of CTC might be useful for monitoring the efficacy of treatment This GFP-expressing virus-based simple method has the potential to allow physicians to assess the response to treatment as well as disease outcome as a relevant clinical parameter, especially in patients without elevated levels of tumor markers Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy 622 Potential Uses of MicroRNA in Lung Cancer Diagnosis, Prognosis, and Therapy Ruian Xu,1,2 Qizhao Wang,1,2 Yong Diao,1,2 William Xu,3 Habib Nagy,4 Weidong Xiao.5 Engineering Research Center of Molecular Medicine, Ministry of Education, Quanzhou, Fujian, China; 2Institute of Molecular Medicine, Huaqiao University, Quanzhou, Fujian, China; Faculty of Science, University of New South Wales, Sydney, NSW, Australia; 4Children’s Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA Lung cancer is the leading cause of death from cancer in the world Although the molecular network of lung carcinogenesis has been partly known at the levels of genes and proteins, and personalized therapy based on the genetic changes has made considerable progress in the last decade, the high mortality rate is not markedly changed microRNAs (miRNAs), a class of short endogenous RNAs, acting as post-transcriptional regulators of gene expression, are similar with siRNAs in both the biosynthesis and the function steps While, miRNAs mostly silence gene expression by binding imperfectly matched sequences in the 3’ UTR of target mRNA, which is different with siRNAs by targeting ORF of mRNA with a perfectly complementary manner miRNAs have multiple functions in lung development, and abnormal expression of miRNAs could lead to lung tumorigenesis The different expression profiles of miRNAs in lung cancer, and the stability of miRNAs in serum, all together make them as new potentially clinical biomarkers for diagnosis and prognosis Moreover, miRNAs may serve as either novel potential targets acting directly as oncogenes (e.g miR-17-92 cluster) or directly therapeutic molecules working as tumor suppressor genes (e.g let-7 family) RNAi technology based on miRNAs has many advantages over siRNAs, such as in vivo stablility, highly RNA promoter-compatiblity and no overt toxicity Eventually, it might overcome the present disadvantages, and is used for lung cancer therapy 623 Inhibition of NF-kB in Fusogenic Membrane Glycoprotein Causing HL-60 Cell Death: Implications for Acute Myeloid Leukemia Li Tan,1 Wenlin Huang,1 Jiangxue Wu,1 Hongyun Jia,1 Yufang Zuo.1 State Key Laboratory, Cancer Center, Sun Yat-sen University, Guangzhou, China Acute myeloid leukemia (AML) is a serious hematologic cancer with the accumulation of abnormally differentiated myeloid cells that are not mature Although, the development of better chemotherapy regimens has improved remission induction and overall survival, relapse is common and long-term survival rates remain low Resistance to standard chemotherapeutic drugs is an important cause of the relapsed, refractory leukemia to which most patients succumb, so relapse and resistance remain a significant problem A unique gene therapy approach for human cancers is transduction with viral FMGs Viral fusogenic membrane glycoproteins (FMGs) are new therapeutic genes for the control of tumor growth, the cellular mechanisms mediating cell death is non-apoptotic FMG transfection is a much more effective treatment for killing human tumor lines in vitro than commonly used suicide genes, such as melanoma tumors, glioma cell lines and hepatocellular carcinoma cell lines Here, we showed FMG expression in HL-60 cells leaded to the formation of multinucleated syncytia and cell death, the main death mode of cells is necrosis Overexpression of HSP70 in HL-60 cells mediated by Gibbon Ape leukemia virus hyperfusogenic envelope protein (GALVFMG) inhibited the nuclear translocation of p65, the transcriptive activity of NF-kB and prevented the degradation of I-kB NF-kB may negatively regulate HSP70 expression, which made a positive feed back loop for expression of HSP70 Overexpression of HSP70 S237 HEMATOLOGIC-HEMOPHILIA in tumor cells may increase their intrinsic immunogenicity So this form of cell death should be effective in vivo, gene therapy basing on FMG deserve further study for the treatment of AML Hematologic-Hemophilia 625 Nonviral Gene Transfer of a Plasmid Encoding a B Domain Variant FVIII/N6 cDNA Reduced Inhibitory Anti-FVIII Antibody Titer in Hemophilia A Mice Dominika Jirovska,1 Peiqing Ye,1 Carol H Miao.1,2 Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA; 2Pediatrics, University of Washington School of Medicine, Seattle, WA 624 Creation and Characterization of a New Large Animal Model for Hemophilia A Gene Therapy Christopher D Porada,1 Chad Sanada,1 Josh A Wood,1 Jay N Lozier,3 Charles Long,4 Mark Westhusin,4 Duane Kraemer,4 Graỗa Almeida-Porada.1 Anim Biotech., Univ of Nevada, Reno, NV; 2NIH Clinical Center, Bethesda, MD; 3Texas A&M Univ., College Station, TX Hemophilia A (HA) is the most common severe hereditary coagulation disorder, affecting in 5000 males Animal models in dog and mouse have been developed and used to study FVIII function and to evaluate gene therapy-based treatments Unfortunately, for unknown reasons, results obtained using these models hasn’t always resulted in successful therapies when applied to humans Due to its striking physiological and anatomical similarities to humans, sheep are considered an ideal model to study a vast array of pathologies We employed a variety of reproductive technologies to re-establish an extinct line of sheep with a bleeding disorder that closely mimics severe human HA These animals exhibited prolonged bleeding from the umbilical cord that promptly stopped upon administration of purified human FVIII (hFVIII) concentrate Blood collected prior to FVIII administration showed that these animals had almost nonexistent levels of FVIIIc and extremely prolonged PTT, with normal levels of platelets, fibrinogen, FVII, FIX, and vWF All animals that survived birth have developed clinical symptomatology closely mimicking that of severe human HA, with each of these animals exhibiting multiple episodes of severe spontaneous bleeding including hemarthroses, muscle hematomas, and hematuria, all of which have responded to hFVIII Thus far, inhibitors to hFVIII have been detected in treated animals, further validating the clinical relevance of this model RT-PCR of mRNA from the spleen of normal sheep, followed by overlapping sequence analysis allowed us to walk along the mRNA and obtain the sequence of the complete 6765 nucleotide coding sequence for ovine FVIII, which is translated into a 2254 amino acid protein BLAST alignment to hFVIII protein revealed a high degree of identity in all regions except the B domain (which is dispensable for clotting activity in humans) Specifically, the A1 domain showed 81% identity, A2: 88%, A3: 87%, C1: 90%, and C2: 86%, while the B domain exhibited only 47% identity Analysis of mRNA isolated from the spleen of a deceased HA sheep identified an 11bp region in exon 14 that differed between the wild type and the hemophiliac Importantly, this difference introduced a premature stop codon at base position 3112-4 in exon 14, as is seen in some human patients with HA This mutation also included a single nucleotide insertion, introducing a frame shift which created additional stop codons within the next 183bp A PCR-based RFLP analysis allows us to unequivocally distinguish sheep that are wild-type, heterozygous, or homozygous for the HA mutation, greatly facilitating studies using these sheep Given the close physiologic similarity between sheep and humans, the high degree of identity in their FVIII protein, and the decades of experience using the sheep to study both normal physiology and a wide array of diseases, we hope that this large animal model will contribute to a better understanding of HA and the development of novel gene therapy-based approaches that can directly translate to human patients with HA S238 Due to the large size of FVIII, a B-domain deleted FVIII (BDDFVIII) cDNA is usually used for developing gene therapy protocols for treating hemophilia A Inefficient transcription of wild type (WT) FVIII cDNA can be overcome by deletion of the heavily glycosylated B-domain encoding portion of the gene BDD-FVIII is as clinically efficacious and not more immunogenic than full-length recombinant FVIII More recently, it was demonstrated that a partial deletion of the B-domain leaving an N-terminal 226 amino acid stretch containing putative asparagine-linked glycosylation sites intact (FVIII/N6) was able to increase in vitro and in vivo secretion of FVIII by 10-15 fold We have inserted this B domain variant FVIII/N6 cDNA into our liver-specific gene expression vector The resulting construct, FVIII/N6 plasmid was delivered into the hemophilia A mouse liver by the hydrodynamic method In control mice treated with FVIII plasmid containing the BDD-FVIII cDNA, FVIII expression levels dropped to undetectable levels at weeks post injection and hightiter anti-FVIII antibodies were generated in all the mice However, in mice treated with FVIII/N6 plasmid, one out of five mice never developed inhibitory antibodies and still had some FVIII gene expression (∼10%) at weeks post gene transfer Except for one mouse which developed high-titer inhibitory antibodies, all other FVIII/N6 plasmid-treated mice developed anti-FVIII antibodies with significantly reduced inhibitor titer The CD4+ T cells isolated from mice injected with FVIII/N6 constructs proliferated less in response to FVIII stimulation than those from mice injected with BDD-FVIII These results indicated that FVIII/N6 protein is less immunogenic than BDD-FVIII Interestingly, both BDD-FVIII and FVIII/N6 constructs produced similar levels of FVIII gene expression following nonviral gene transfer These findings suggest that in combination with a FVIII/N6 construct, induction of long term tolerance against FVIII may be achievable with a reduced dosage and duration of the immunomodulation protocol 626 Induction of Immuno Tolerance to Canine FVIII (cFVIII) Using Liver Directed AAV Mediated Expression of cFVIII in Hemophilia A Dogs with Inhibitor Antibodies Jonathan D Finn,1 Denise E Sabatino,2 Margareth C Ozelo,3 ShangZhen Zhou,1 David Lillicrap,3 Timothy C Nichols,4 Valder R Arruda.1,5 Children’s Hospital of Philadelphia, Philadelphia, PA; 2University of Pennsylvania School of Medicine, Philadelphia, PA; 3Queen’s University, Kingston, ON, Canada; 4University of North Carolina, Chapel Hill, NC; 5Hematology, University of Pennsylvania, Philadelphia, PA The formation of antibodies (or inhibitors) to FVIII is a major complication in the treatment of humans with hemophilia A (HA), affecting up to 30% of individuals with severe or moderate disease Inhibitors develop mainly in young boys, and render the treatment with infused protein ineffective Although liver-directed gene therapy has been used to deliver therapeutic transgenes and can induce tolerance to the expressed protein, to date there have been no large animal studies using liver gene therapy to eradicate inhibitors to FVIII We hypothesize that sustained expression of cFVIII will eradicate inhibitors to canine FVIII in dogs that have a history of inhibitors, thus demonstrating a potential alternative to the current immune tolerance Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy ...HEMATOLOGIC-HEMOPHILIA in tumor cells may increase their intrinsic immunogenicity So this form of cell death should be effective in vivo, gene therapy basing on FMG deserve further study for the treatment of AML... N-terminal 226 amino acid stretch containing putative asparagine-linked glycosylation sites intact (FVIII/N6) was able to increase in vitro and in vivo secretion of FVIII by 10-15 fold We have inserted... obtain the sequence of the complete 6765 nucleotide coding sequence for ovine FVIII, which is translated into a 2254 amino acid protein BLAST alignment to hFVIII protein revealed a high degree of