856 Clinical Gene Transfer in the Community Ten Years of Local IBC Review Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S330 CANCER TARGET[.]
CANCER-TARGETED GENE & CELL THERAPY III 854 Adenovirus-Delivered Short HairpinStructured RNA for Microphage Migration Inhibitory Factor Silencing Inhibits Human Prostate Cancer Cell Proliferation Ling Zhang,1 Anqi Wang,1 Chengyao Li,1 Hongwei Li,1 Leah N Braseth,2 Arseniy E Shabashvili,2 Colin Sumners.2 School of Biotechnology, Southern Medical University, Guangzhou, Guangdong, China; 2Dept of Physiology and Functional Genomics, Univ of Florida, Gainesville, FL Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine involved in a wide range of pathophysiologic events in association with inammation, innate and acquired immune responses, and cell growth More recently it has begun to be recognized as a pro-tumorigenic factor and has been dened as a novel oncogene Increased expression of MIF has been demonstrated in human melanomas, breast carcinomas, adenocarcinomas of the lung, bladder cancer, hepatocellular carcinomas, gastric cancer and neuroblastoma The expression of MIF is also increased in prostate cancer (PCa), one of the most common cancers in men Existing therapies and surgical interventions have not been able to effectively manage this form of cancer and, therefore, efforts are ongoing to explore novel targets and strategies for the management of PCa We previously demonstrated that silencing the MIF using small interfering RNAs induces apoptotic cell death in prostate cancer cells To develop this MIF siRNA technique into a therapy for prostate cancers, we generated an adenovirus to stably express a short hairpin-structured RNA (shRNA) against the MIF gene in vivo We transduced the androgen-independent prostate cancer cell line Du145 with the MIF shRNA recombinant adenovirus, which resulted in a signicant inhibition of MIF expression from 24h to 72 h post-transduction MIF depletion resulted in a decrease in cell proliferation in Du-145 cells Downregulation of MIF expression signicantly increased the expression of p53 at both the mRNA level and the activated p53 level These studies may help in understanding the molecular mechanism associated with MIF function in prostate cancer cells In summary, our data demonstrate that blocking MIF expression via shRNA is a potential gene therapeutic strategy for androgen-independent prostate cancer Key Words: prostate cancer, macrophage migration inhibitory factor, short hairpin-structured RNA, human prostate cancer cell proliferation effect was achieved using a DT-A plasmid under the control of a strong non-specic CMV promoter To reach specicity for prostate cancer cells, a prostate specic androgen enhancer/promoter (PSE/ PSA) will be investigated rst in vitro using C4-2 as positive and PC-3 as a PSA negative prostate cancer cell line According to the results obtained, in vivo studies will be performed in SCID mice as before After introduction of plasmid by in vivo EP in experimental tumors, we will assess apoptosis immunohistochemically by TUNEL assay in a quantitative manner using stereology and IMARIS evaluation There is evidence from preliminary data that apoptotic cells are observed more frequently in DT-A treated tumors compared to controls treated with Mock plasmid For conrmation, quantication of apoptosis in tumor sections is performed at present 856 Clinical Gene Transfer in the Community: Ten Years of Local IBC Review Jan P Vleck.1 IBC Services, Western IRB, Olympia, WA INTRODUCTION Since 2000, we have provided administrative support for local IBCs mainly serving community-based hospitals or clinics in the US (including 46 states) and other countries These IBCs have reviewed 126 clinical gene transfer trials, about 8% of the worldwide total recorded in the database of the Journal of Gene Medicine This community IBC experience can be compared semi-quantitatively with the worldwide experience OBSERVATIONS Indications: The community IBCs have seen a lower proportion of trials directed at cancer and monogenic disease, and a comparatively higher proportion of trials directed at “healthy volunteers” (prophylactic vaccines) and cardiovascular disease 855 Monitoring of Electroporation-Mediated Diphtheria Toxin A Expression in Prostate Cancer Cells Grown in SCID Mice Christine Goepfert,1 Felix J Frey,1 Brigitte M Frey.1 Departments of Nephrology and Hypertension and Clinical Research, University of Berne, Berne, Switzerland Prostate cancer is the second leading cause of death among men in most Western countries and often common therapies not lead to the designated effects Since very low concentrations of diphtheria toxin A (DT-A) lead to protein synthesis abrogation and apoptosis of cells, DT-A might serve as an efcient killer in cancer gene therapy Here, we investigated in a quantitative manner the apoptotic effect of DT-A plasmid expression in a prostate cancer tumor model Initially, we injected Mio of androgen sensitive prostate cancer cells (C4-2) together with Matrigel sc into each ank of SCID mice Tumor size was monitored every 4th day by caliper measurements Tumors with an approximate size of 150mm³ were injected either with DT-A plasmid or Mock plasmid in various concentrations (15-75 µg plasmid/tumor) and application frequencies (1-3 times/9 days) in order to ascertain an optimal setting Comparing different transfection methods, electroporation (EP) and lipid formulation (DMRIE-C), we observed higher transfection efciency and in parallel a reduced tumor growth applying EP than DMRIE-C This S330 Phase: The community IBCs see more later phase trials (14.3%, versus 4.2% worldwide with phase III component) However, rstin-human studies are common in community settings (about a quarter of the total) [data not shown] Vector: The top three vector categories seen by community IBCs are adenovirus, plasmids, and vaccinia Worldwide, the top three are adenovirus, retrovirus, and plasmids About half of the vector categories recorded worldwide have been presented to these community IBCs Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY III DISCUSSION Data integrity: Non-clinical and registry protocols (where no rDNA was given to humans in the protocol) are not included in the data The list of protocols reviewed by the community IBCs is complete, but not all of these protocols are listed in the Journal of Gene Medicine database Spot checking of trials against the ClinicalTrials.gov database suggests that many of the listings at ClinicalTrials.gov are out-of-date, or not entirely in agreement with the protocols For example, clinical phases were sometimes more expansive (phase I/II) on the public database than in the protocol itself (phase I) Sample bias: Protocols seen by community IBCs have passed through an economic lter because most of them are commercially sponsored Therefore, they represent a biased subset –protocols that have attracted private funding The 2009 local data are limited, but may suggest fewer phase I trials opened in that year of economic difculty First-in-Human studies: Determining which trials represent rstin-humans use of a new construct is difcult and time consuming However, their presence in the community experience indicates that non-university IBCs must be prepared to independently evaluate novel protocols CONCLUSION Community-based, non-academic IBCs are active in review of gene transfer clinical trials The IBCs represented in this sample have seen a representative distribution of the worldwide activity, in terms of indications, clinical trial phase, and vectors used Hematologic and Immunologic Gene & Cell Therapy III 857 High Incidence of Vector Integration near Cancer-Related Genes within Primitive Hematopoietic Stem Cells (HSC) after Fetal Gene Transfer with Ȗ-Retroviral Vectors Joe Tellez,1 Josh Wood,1 Graỗa Almeida-Porada,1 Chris D Porada.1 Animal Biotech., Univ of Nevada, Reno, NV We have previously reported the efcient transduction of primitive HSC following the direct intraperitoneal injection of γ-retroviral vectors into early gestation sheep fetuses Further studies revealed that earlier gestational ages facilitated higher levels of hematopoietic gene transfer Given the inherent risk of insertional mutagenesis with these vectors, we performed studies to: 1) determine the mechanism of the age-related alteration in hematopoietic transduction; and 2) assess the relative safety of GT as a function of fetal age by comparing the proximity of vector integration sites to genes implicated in cancer We collected marrow CD34+ cells from year-old sheep that received in utero GT at varying gestational ages LM-PCR was then performed to map the vector integration sites and assess whether the enhanced levels of gene transfer at earlier gestational ages were due to transduction of larger numbers of HSC clones Sequencing of LM-PCR products revealed a similar number of integration sites in each animal, suggesting that similar numbers of Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy HSC clones were transduced regardless of recipient age at the time of GT Thus, the higher levels of transduced hematopoietic cells present following GT at earlier gestational ages were likely due to expansion of transduced clones during fetal growth Furthermore, several common integration sites were observed within the CD34+ cells from these animals, a nding of pronounced signicance given the presumed semi-random nature of γ-retroviral vector integration Further analysis of the integration sites within these CD34+ cells present at years post-GT revealed a remarkable prevalence of integrants in the neighborhood of genes which have previously been implicated in cancer Specically, of the 84 integration sites mapped from animals, the nearest neighboring gene was cancer-related in 51 of these sites The frequency of integrations near cancer-related genes did not differ as a function of fetal age at the time of vector administration, suggesting this potential genotoxicity did not vary with the ontogenic “age” of the HSC Given that these recipients were analyzed at years post-GT, all of the integration sites we observed were the result of transduction of highly primitive HSC Thus, our studies show, for the rst time, that γ-retroviral vectors exhibit a marked preference for integrating near cancer-related genes within primitive HSC following vector delivery in vivo in a large animal model Moreover, these ndings suggest that the most primitive HSC may be at higher risk of insertional mutagenesis than has previously been predicted by in vitro studies in which the vast majority of transduction occurs in more mature progeny It is important to note, however, that despite the high percentage of integrants near cancerrelated genes, hematologic malignancy was never observed during the 3-year course of this study The present ndings thus highlight the need for further carefully designed experiments in large animals to determine the precise risk of insertional mutagenesis with HSCbased GT protocols, especially if the GT is designed to target the most primitive, long-term reconstituting HSC 858 Successful Treatment of Canine Leukocyte Adhesion Deciency Using a MSCV-Promoter Based Foamy Viral Vector and Low-Dose Busulfan Conditioning Thomas R Bauer, Jr.,1 Erik M Olson,2 Laura M Tuschong,1 James M Allen,2 Tanya H Burkholder,3 David W Russell,2 Dennis D Hickstein.1 Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD; Department of Hematology, University of Washington, Seattle, WA; 3Division of Veterinary Services, Ofce of Research Services, National Institutes of Health, Bethesda, MD We investigated the efcacy of low-dose busulfan conditioning in facilitating the engraftment of foamy viral (FV) vector transduced CD34+ cells from animals with canine leukocyte adhesion deciency (CLAD) Dogs with CLAD, like children with LAD, suffer from recurrent bacterial infections and early death due to the lack of the CD18 leukocyte integrin on the neutrophil surface; CD18 decient neutrophils fail to adhere and migrate to sites of infection We previously published successful results treating four CLAD pups using FV vector-mediated gene therapy with a non-myeloablative conditioning regimen of 200 cGy total body irradiation All four dogs lived greater than years post-infusion of genetically-corrected cells In the current study, four CLAD pups received a reduced intensity conditioning regimen of mg/kg Busulfex given IV two days prior to infusion of genetically-corrected cells We selected this regimen to demonstrate efcacy of Busulfex IV in preparation for a clinical human gene therapy trial for leukocyte adhesion deciency type (LAD-1) Autologous CLAD CD34+ bone marrow cells were transduced in an overnight exposure to FV vector FV-MSCV-cCD18 on Retronectin in the presence of 50 ng/mL human G-CSF, human Flt3L, and canine SCF, and infused the following day Transduction S331 ... non-academic IBCs are active in review of gene transfer clinical trials The IBCs represented in this sample have seen a representative distribution of the worldwide activity, in terms of indications, clinical. .. protocols reviewed by the community IBCs is complete, but not all of these protocols are listed in the Journal of Gene Medicine database Spot checking of trials against the ClinicalTrials.gov database... GENE & CELL THERAPY III DISCUSSION Data integrity: Non -clinical and registry protocols (where no rDNA was given to humans in the protocol) are not included in the data The list of protocols reviewed