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219 Angiotensin II Type 2 Receptor Gene Mediated Apoptosis of Human Lung Adenocarcinoma A549 Cells Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell T[.]
CANCER-TARGETED GENE & CELL THERAPY I 217 Prostate Cancer Gene Therapy Using Adenovirus Expressing a Novel ApoptosisInducing Gene and Its Conjugation with Chemotherapy Xiongwen Zhang,1 Ben Beheshti,2 Yi Lu.1 Pathology and Urology, University of Tennessee Health Science Center, Memphis, TN; 2Ontario Cancer Institute, Princess Margaret Hospital, Toronto, ON, Canada A novel gene, rat pHyde, and its human homologue, hpHyde, have been cloned from prostate cancer cells Database search and FISH analysis consistently indicated that hpHyde gene localizes at human chromosome 2q14 Protein sequence analysis suggests that hpHyde may be a plasma membrane protein with calcium channel function hpHyde is differentially expressed in various normal human tissues and organs, suggesting that hpHyde may play roles in development and differentiation A recombinant adenovirus containing pHyde cDNA gene (AdRSVpHyde) showed signicant growth inhibition on human prostate cancer cells, both in vitro and in vivo, through a caspase-3 dependent apoptosis induction The anti-prostate cancer effects of pHyde in conjunction with chemotherapy agent were analyzed by in vitro and in vivo assays using AdRSVpHyde in combination with DNA damaging chemotherapeutic agent, cisplatin, and docetaxel, respectively Growth suppression and induction of apoptosis were additively greater in DU145 human prostate cancer cells treated with AdRSVpHyde and cisplatin than either agent alone both in vitro and in vivo Moreover, AdRSVpHyde and docetaxel also have a similar additively inhibitory effect on DU145 cell growth Taken together, these results support the potential use of pHyde for prostate cancer gene therapy coupled with chemotherapy to improve therapeutic index Our results also suggest that pHyde may play important physiological and pathological roles in normal prostate development and prostate carcinogenesis 218 Development and Preclinical Testing of Large Batches of SNS01-A – An eIF5A-Based Gene Therapy Nanoparticle Designed for the Treatment of Multiple Myeloma Catherine A Taylor,1 Zhongda Liu,1 Terence Tang,1 Bin Ye,1 Bruce Galton,2 Kathleen A Donovan,3 John A Lust,3 John E Thompson,1,2 Richard Dondero.2 Biology, University of Waterloo, Waterloo, ON, Canada; 2Senesco Technologies Inc., New Brunswick, NJ; 3Mayo Clinic, Rochester, MN INTRODUCTION: The eukaryotic translation initiation factor 5A (eIF5A) is the only known protein to be regulated by post-translational addition of a hypusine residue Both hypusinated eIF5A and the enzyme that mediates eIF5A hypusination have been identied as markers of neoplastic growth and metastasis However, recent studies have indicated that, in its unhypusinated form, eIF5A is pro-apoptotic and thus functionally distinct from hypusine-modied eIF5A In vitro cell studies and in vivo xenograft studies have demonstrated that simultaneous suppression of eIF5A expression and over-expression of a non-hypusinable mutant of eIF5A (eIF5AK50R) potently induces apoptosis in multiple cancer cell types SNS01-A is a polyethylenimine (PEI) nanoparticle that combines: 1) a DNA plasmid that expresses the pro-apoptotic eIF5AK50R mutant; and 2) an siRNA that targets the native hypusinated eIF5A that promotes growth of cancer cells In order to be viable in the clinic, one must be able to produce batches large enough for a clinical study In this study we describe the formulation and testing of clinical-size batches of SNS01-A METHODS: Dynamic light scattering was used to monitor changes in size distribution In vitro biological activity was assessed using RT-qPCR to measure eIF5A and eIF5AK50R transgene expression Anti-tumoral activity of SNS01-A was evaluated using Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy a human myeloma tumor model treated with twice-weekly intravenous injections RESULTS: Formation of nanoparticles in dilute volumes of % glucose was found to give rise to signicantly smaller particles with in vivo biological activity equivalent to more concentrated formulations For instance, PEI nanoparticles that were formed using a standard protocol [SNS01; 30 µg nucleic acid; N/P = 6; 0.1 mL] had an average zeta diameter of 120.6 nm +/- 18.0 nm and inhibited myeloma tumor growth by 94.9 % (p = 0.009), while those made using a 4x dilution [SNS01-A; 7.5 µg nucleic acid; N/P = 6; 0.1 mL] had an average zeta diameter of 77.6 nm +/- 11.2 nm and inhibited myeloma tumor growth by 96.9 % (p = 0.021) The more dilute SNS01-A formulation was found to be more amenable to largescale production than SNS01 as determined by the size distribution (215 nm vs 557 nm) Reversal of the combining steps (ie addition of nucleic acid to PEI) during the preparation of 50 mL batches of SNS01-A was found to reduce the zeta diameter from 215.0 nm to 71.58 nm The addition of HEPES to SNS01-A was found to improve the nanoparticle characteristics by neutralizing the pH, reducing the polydispersity and increasing the zeta potential CONCLUSION: Large-scale production of the multiple myeloma gene therapy product SNS01-A was made possible by dilution of the nanoparticles to create smaller, biologically-active particles and has the added advantages of using less material and having a more favorable safety prole These results indicate that smaller PEI nanoparticles may have greater in vivo biological activity 219 Angiotensin II Type Receptor Gene Mediated Apoptosis of Human Lung Adenocarcinoma A549 Cells Yanling Zhang,1 Ling Zhang,1 Yongxin Gao,2 Baihong Chen,1 Hongwei Li.1 School of Biotechnology, Southern Medical University, Guangzhou, China; 2Dept of Physiology and Functional Genomics, Univ of Florida, Gainesville, American Samoa Lung cancer is one of the main causes of cancer death worldwide The disease is more common in countries with high tobacco consumption, including China The high mortality often is due to the presence of advanced-stage metastasis at the initial diagnosis This grim prognosis indicates a continued need for novel therapeutic approaches to reduce lung cancer mortality Angiotensin II (AII) is a multifunctional bioactive peptide, and host renin-angiotensin system (RAS) is closely associated with tumor growth Recent reports have described that AII is a proangiogenic growth factor, and that Angiotensin II type (AT1) receptor antagonists reduce tumor growth and tumor-associated angiogenesis Further, numerous studies have demonstrated antigrowth and antiproliferative effects of Ang II via AT2R, in opposition to actions of this peptide via the AT1R The present studies report apoptosis of lung cancer cells induced by AT2R overexpression A recombinant adenoviral vector expressing AT2R (Ad-G-AT2R-EGFP) was transduced into lung cancer A549 cells Following AT2R transduction, apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining (TUNEL staining) and cell apoptosis DAPI detection kit The results indicate transduction of A549 cells with Ad-G-AT2R-EGFP for days resulted in a large number of cells that exhibited apoptotic-like morphological characteristics when compared with Ad-CMV-EGFP treated A549 cells For example, the AT2R-expressing A549 cells exhibited irregular shaped nuclei and a clear boundary between nuclei and cytoplasm when compared with the controls The apoptotic action following AT2R transduction was conrmed by the nding that incubation of A549 cells with Ad-G-AT2R-EGFP produced a signicant increase in TUNEL labeling, compared with the Ad-CMVEGFP treated cells; and nuclear alterations were evidenced by the DAPI uorescence microscopy The results indicate that increased expression of AT2R alone induced apoptosis in the lung cancer line S83 HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY I A549 The observations presented here suggest that the ability of increased AT2R expression to induce apoptosis in lung cancer cells may have potential therapeutic implications for this disease, and suggest that AT2R is a promising novel target gene for lung cancer gene therapy Keyword:angiotensin II type receptor, apoptosis, human lung adenocarcinoma Hematologic and Immunologic Gene & Cell Therapy I 220 Modeling X-Linked Chronic Granulomatous Disease Using Neutrophils Differentiated from Patient-Derived Induced Pluripotent Stem Cells Colin L Sweeney,1 Jizhong Zou,2 Uimook Choi,1 Bin-Kuan Chou,2 Linzhao Cheng,2 Harry L Malech.1 Laboratory of Host Defenses, NIAID, NIH, Bethesda, MD; 2Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD X-linked chronic granulomatous disease (X-CGD) is characterized by defective neutrophil function due to mutations in the CYBB gene involved in production of microbicidal reactive oxygen species (ROS) for phagocytosis X-CGD hematopoietic stem cells (HSCs) are a target for gene therapy and transplant; however, obtaining sufcient numbers of gene-corrected cells for treatment is hampered by limited cell availability and expansion potential ex vivo Induced pluripotent stem cells (iPSCs) generated from adult somatic cells exhibit a pluripotent state resembling human embryonic stem cells (hESCs), and could provide an alternate source of patient-derived cells with the capacities for unlimited proliferation and differentiation into all cell lineages, including hematopoietic cells To establish X-CGD iPSCs, we rst obtained bone marrow mesenchymal stem cells (MSCs) from an X-CGD patient with an L153R mutation in CYBB The MSCs were transduced with retroviruses encoding human Oct3/4, Sox2, c-Myc, and Klf4 transcription factors, and the resulting X-CGD iPS cell lines were screened for pluripotency markers and the capacity to differentiate into cells of all three germ layers A major issue for clinical use of pluripotent stem cells is the development of methods for directing differentiation to produce the relevant mature cells Protocols for the efcient, directed production of functional neutrophils from pluripotent hESCs have recently been established by Yokoyama et al (Blood 2009) and Saeki et al (Stem Cells 2009) In our hands, differentiation of H9 hESCs by Yokoyama’s protocol produced functional neutrophils which were 96% positive for CD45 hematopoietic marker, 30% positive for CD13 myeloid marker, and 82% positive for production of ROS by DHR assay Since hESC neutrophil differentiation protocols had not previously been tested on iPSCs, we next applied this protocol both to normal iPSCs and X-CGD iPSCs After differentiation, both normal and X-CGD cells expressed CD45 (97% or 99% positive, respectively), and subsets also expressed CD16 neutrophil marker (43% or 50% positive, respectively), or exhibited neutrophil morphology Neutrophils from both normal and X-CGD iPSCs could uptake zymosan (75% or 74% positive, respectively), a measure of bacterial ingestion for phagocytosis However, while neutrophils from normal iPSCs exhibited functional production of microbicidal ROS (39% positive by DHR), neutrophils from X-CGD iPSCs did not produce ROS (0.6% positive by DHR), reproducing the functional defect observed in peripheral blood neutrophils of X-CGD patients In conclusion, we have demonstrated for the rst time the directed differentiation of normal human iPSCs to produce functional neutrophils, and that neutrophils from X-CGD patient-derived iPSCs model the functional defect characteristic of this disease, a necessary step towards testing a gene therapy approach for X-CGD using patient iPSCs The next step will involve CYBB gene correction in X-CGD iPSCs to restore neutrophil function S84 221 Use of the Natural Human Artemis Promoter To Regulate Human Artemis Expression Prevents Toxicity and Corrects Artemis Decient SCID after Ex Vivo Lentiviral Transduction in a Murine Model of the Disease Megan M Multhaup,1 Kelly Podetz-Pedersen,1 Sweta Gurram,1 Andrea Karlen,1 Debra Swanson,1 Nikunj V Somia,1 Bruce R Blazar,2 Morton J Cowan,3 R Scott McIvor,1 McIvor Lab Dept of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN; 2Dept of Pediatrics, Heme/ Oncology/Bone and Marrow Transplant Division and Cancer Center, University of Minnesota, Minneapolis, MN; 3Dept of Pediatrics, University of California, San Francisco, CA Artemis protein is an endonuclease characterized as a key factor involved in both non-homologous end joining (NHEJ) and V(D) J recombination Mutations in the gene encoding Artemis result in a radiation-sensitive form of severe combined immunodeciency found at a high incidence in Athabascan-speaking Native Americans (SCID-A) and characterized by the absence of mature B and T lymphocytes Early treatment is critical since the disease results in severe infection ultimately leading to fatality at a young age The current therapy for SCID-A is allogeneic hematopoeitic cell transplantation (HCT); however, HCT often results in incomplete reconstitution of B lymphocytes and may lead to complications such as graft versus host disease Transplantation with genetically corrected autologous cells is an alternative approach that may provide improved treatment of SCID-A A murine model of Artemis deficiency backcrossed onto C57BL/6 exhibiting no leakiness (mArt-/-) was recently reported (Xiao, Z et al Biol Blood Marrow Transplant 15(1) 1-11, 2009) We previously described cytotoxicity associated with Artemis over-expression (Mol Ther 16(S1) S226, 2008), demonstrating the necessity of establishing conditions that provide Artemis expression at a level that is non-toxic yet sufcient to complement Artemis deciency To this end, we subsequently recovered and characterized the endogenous human Artemis promoter (APro) as a one-kilobase region located directly upstream of the human Artemis translational start site APro conferred a moderate level of reporter gene expression in vitro and in vivo, including secondary mouse transplant recipients, thus demonstrating reliable expression after lentiviral gene transfer into hematopoeitic stem cells (HSCs) (Mol Ther 17 (S1) S252-S253) In this study, we employed innate regulation of the human Artemis cDNA using its own endogenous promoter sequence for correction of a murine model of SCID-A A lentiviral vector containing these sequences (pOK-APro-hArt) was assembled, packaged by VSV-G pseudotyping, and then used to transduce mArt-/- donor marrow with subsequent transplantation into mildly pre-conditioned (500 cGy) mArt-/- recipient mice Recipient animals were evaluated on a monthly basis post-transplant, and beginning at weeks were found to have normal levels of CD3+CD4+ and CD3+CD8+ T lymphocytes and B220+NK1.1- B lymphocytes, thus providing evidence for immune reconstitution resulting from APro-hArt transduction of HSCs These results demonstrate that the endogenously regulated Artemis lentiviral vector effectively complemented murine SCID-A, contributing to the development and advancement of gene transfer as a clinically relevant and feasible approach for treatment of SCID-A in humans Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy ... AT2R is a promising novel target gene for lung cancer gene therapy Keyword :angiotensin II type receptor, apoptosis, human lung adenocarcinoma Hematologic and Immunologic Gene & Cell Therapy I 22 0...HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY I A549 The observations presented here suggest that the ability of increased AT2R expression to induce apoptosis in lung cancer cells may have potential... reliable expression after lentiviral gene transfer into hematopoeitic stem cells (HSCs) (Mol Ther 17 (S1) S2 52- S253) In this study, we employed innate regulation of the human Artemis cDNA using its own