340 a multi use GMP facility for cell processing in an academic center designed for the implementation of new regulatory requirements

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CANCER TARGETED GENE THERAPY I would provide new insights into the mechanism of tumor angiogenesis in general and may have the potential for the development of novel anti-angiogenic gene therapy approaches for prostate cancer in particular 338 Genetic Regulation of iNOS Expression as a Potential Therapy for Cancer Rana S Al-Assah, Rachel L Cowen, Edwin C Chinje, Kaye J Williams, Brian A Telfer, Ian J Stratford Experimental Oncology Group -Pharmacy Department, Manchester University, Manchester, United Kingdom Tumor growth is a complex phenomenon regulated by a variety of molecules including Nitric Oxide (NO) NO is produced during the conversion of L-arginine to citrulline, which is catalyzed, by the Nitric Oxide Synthase (NOS) family of enzymes in particular inducible NOS (iNOS) The role of NO in tumor growth remains controversial: low levels promote tumor growth and angiogenesis while high levels can be cytotoxic through the induction of apoptosis Moreover, iNOS has high sequence homology to P450 Reductase and we have shown that like P450 Reductase, iNOS can metabolise prodrugs like tirapazamine (TPZ) Thus, because of the great potential for iNOS as a therapeutic in a gene therapy approach, we have harnessed iNOS expression using the ecdysone inducible system This will allow us to tightly control the levels of iNOS expression and exclude its unwanted pro-tumor effects The inducible system was first tested by generating stable HT1080 clones (human fibrosarcoma) expressing inducible lac Z Low basal levels and high fold induction in response to Ponasterone A (Pon A) addition (max 17 fold induction with 20μM Pon A) were achieved in vitro Also, this clone was grown in nude mice which were later injected with Pon A (1mg/mouse) After staining with Xgal, xenografts of the clone turned blue throughout Following this evaluation, stable clones of HT-1080 encoding for inducible iNOS expression were generated This clone has been fully evaluated (max iNOS activity 21pmole/min/mg with 20μM Pon A versus 1.83 pmole/min/mg without Pon A) and in vivo(6.57 pmole/min/mg with mg Pon A /mouse compared to undetectable levels in uninduced tumors) Using this xenograft model we are studying the effects of inducible iNOS expression on tumor vascularity and the impact of this on tumor growth and bioreductive drug distribution Moreover, adenoviruses encoding for iNOS expression (lac Z) in the ecdysone cassette have been generated allowing us to extend our work to a variety of cell-lines We aim to use these viruses to induce high levels of iNOS expression in vitro and in vivo allowing us to pulse high levels of NO and induce apoptosis In addition these tumor cells will be sensitised to killing by TPZ and radiation 339 Delivery of Small DNA Fragments to Human Airway Epithelial Cells Via Arginine-Rich Polypeptides James Lausier,1 Kevin Foley,1 Emanuela Bruscia,1 Wolfgang Dostmann,1 Dieter C Gruenert.1 Departments of Medicine and Pharmacology, University of Vermont, Burlington, VT One obstacle to transfecting DNA into eukaryotic cells is the delivery of DNA to the nucleus This feature of DNA transfection has important implications for in vitro gene expression and in vivo gene therapy Numerous DNA delivery systems, both viral and non-viral, have been developed to overcome this obstacle with varying degrees of success A number of non-viral systems have been based on cationic polypeptides This study evaluates the potential of arginine rich-polypeptides (DT-5 and DT-6) to facilitate transfer and release of DNA in the nucleus or human airway epithelial cells These polypeptides are based of sequences derived from the HIV Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy Tat protein and have been shown to facilitate both trans-cytoplasmic and trans-nuclear transport of nucleic acids To test the potential of this DNA delivery system to facilitate transport of small DNA fragments (SDF) into the nucleus of these cultured epithelial cells, SDF were mixed at different charge ratios of peptide to DNA Gel retardation analysis showed that the complexes were stable After delivery into the cells, confocal analysis of differentially labeled DNA (red fluorescence) and peptide (green fluorescence) demonstrated that the SDF were transferred to the nucleus within 4-8 hrs and then release within the nucleus as free DNA Further studies are underway to evaluate whether the SDF can mediate modifications in genomic DNA both in vitro and in vivo 340 A Multi-Use GMP Facility for Cell Processing in an Academic Center - Designed for the Implementation of New Regulatory Requirements Gerhard Bauer,1 Jon E Walker,1 Jan A Nolta,1 Steven Devine,1 John F DiPersio.1 Oncology, Washington University School of Medicine, St Louis, MO, United States Introduction: Over the recent years clinical applications of cellular therapy, particularly gene therapy, have come under much scrutiny Many academic centers, until recently, have only used dedicated lab spaces or even only dedicated biosafety cabinets to perform clinical grade tissue manipulations This kind of practice is prone to contamination, particularly when an open system (flask system) for tissue culturing is applied New and stricter regulatory requirements have recently been established for researchers who want to translate laboratory research into Phase I and II clinical trials These new regulations already mandated or to be mandated in the near future by the regulatory agencies will require many academic centers to rethink their approach towards clinical trials of gene - or cellular therapy, and even tissue processing for transplantation Objective: To be in compliance with current Good Laboratory Practice (GLP), Good Tissue Practice (GTP), and Good Manufacturing Practice (GMP), Washington University School of Medicine in St Louis undertook the difficult task of designing and constructing a new GMP laboratory for cellular therapy Six Class 10,000 manufacturing labs are the core of this 2,615 square foot facility Class 100,000 intermediate exit and entry rooms separate the manufacturing rooms from other areas, and serve as the only access for the individual Class 10,000 labs There is no personnel or air exchange directly from one manufacturing lab to another This makes the facility completely versatile in terms of production of separate cellular products without cross-contamination from personnel or air Personnel flow is unidirectional only, from a gowning room into an intermediate entry room, from intermediate entry room into one manufacturing lab at one time only To exit, personnel enters an intermediate exit room and proceeds into the de-gowning room The highest air pressure is maintained in the manufacturing rooms, while the gowning areas have negative air pressure towards the hallway This prevents contaminants from the outside air to enter the manufacturing rooms, and protects the outside environment from contamination with material that will be manufactured in the GMP facility Since one of the major requirements of a GMP facility is to operate it under GMP conditions, a strict Quality Control and Quality Assurance program has been put in place Environmental cleaning and monitoring is addressed in a range of Standard Operating Procedures (SOPs) written specifically for this GMP lab Summary: A GMP facility must provide an environment for the safe processing and manipulation of tissues for use in humans under the new and stricter regulations Its versatility in an academic center S133 CANCER TARGETED GENE THERAPY I should allow for new protocols to be developed and readily applied in the ever changing and more demanding field of cellular and gene therapy 341 Large Scale Manufacturing of Plasmid DNA for Pharmaceutical Applications: Current Plasmid Quality Standards, Contaminations, Process Difficulties and Solutions Markus Mueller,1 John Vu,2 Wayne Tvrdik,2 Astrid Breul,1 Joachim Schorr.1 QIAGEN GmbH, Hilden, Germany; 2QIAGEN,Inc., Valencia, CA, United States A prerequisite for the use of plasmid DNA in clinical trials for gene therapy and genetic vaccination is a manufacturing process that is suitable to generate the pharmaceutical product in a “stateof-the-art” quality Also, such a manufacturing process needs to meet the requirements concerning batch scale, robustness, reproducability and regulatory compliance As the efficacy of a (plasmid) drug determines the commercial success or failure of the complete project, the feasability of the manufacturing process has a similar impact This presentation will illustrate the plasmid quality standards as they are currently accepted for use in clinical trials by regulatory authorities around the world Critical contaminations and their causes will be discussed and the challenge of the removal thereof These considerations will be done viewing on pilot to large scale manufacturing processes Knowing the hurdles, preventive actions can be implemented in the manufacturing to improve the quality of the plasmid product used in clinical trials and therewith enhancing the probability to succesfully bring a product from the research and clinical phases into the commercial market 342 Toxicology of Oncolytic Measles after Intraperitoneal Administration in MeaslesSusceptible Mice Rae Myers,1 Marie Frenzke,1 Suzanne Greiner,1 Mary Harvey,1 Diane Soeffker,1 Evanthia Galanis,2 Kah-Whye Peng,3 Stephen Russell.1,3 Toxicology Core, Mayo Foundation, Rochester, MN; 2Medical Oncology, Mayo Foundation, Rochester, MN; 3Molecular Medicine Program, Mayo Foundation, Rochester, MN MV-CEA is an attenuated oncolytic measles virus engineered to express the soluble extracellular domain of human carcinoembryonic antigen Viral gene expression can be followed noninvasively in MV-CEA treated tumor bearing animals by monitoring the plasma concentrations of CEA Based on the promising antitumor efficacy of MV-CEA in a human ovarian xenograft model, we have developed a phase I/II clinical protocol in which we propose to test the agent in patients with advanced, treatment refractory ovarian cancer Increasing doses of MV-CEA, harvested by lysis of virus-infected Vero cells, will be administered by intraperitoneal infusion to successive cohorts of three patients until dose-limiting toxicity is encountered In support of the proposed clinical study we have evaluated the toxicity of the agent in CD46 receptor-transgenic, interferon receptor knockout mice that were previously shown to be susceptible to measles virus infection Groups of to week old mice were challenged intraperitoneally with a single dose of MVCEA (10e5, 10e6, or 10e7pfu) or with six doses of 10e7 pfu administered over a two-week period Control mice were either uninjected or injected with Vero cell lysate containing no virus Animals were individually microchipped and monitored regularly out to 90 days for changes in general wellbeing, coat condition, activity level, body weight, plasma chemistry profile, complete S134 blood count and blood coagulation Plasma CEA values were determined to confirm that active MV-CEA virus had been administered and to elucidate the kinetic profile of viral gene expression Major organs were harvested and examined histologically for acute (day 4), subacute (day 21) and chronic (day 90) toxicity We found that intraperitoneal administration of Vero cell lysate is almost completely innocuous in the CD46 transgenic mice, and the toxicities seen (all minor) were due primarily to MV-CEA administration The toxicities that were documented included transient abnormalities in biochemical and hematological parameters of alkaline phosphatase, BUN, total protein, and WBC count, as well as a slowed growth rate seen only in high single and multiple dose groups There was no evidence of peritonitis due to intraperitoneal administration of vehicle or virus at any dose level All organs examined, except for the spleen, appeared normal at every time point in every group In the early time points for the high single and multiple dose groups, we saw splenomegaly of 2-3 times normal size with microscopic findings of follicular hyperplasia with a pronounced increase of immunoblasts at the borders of the B cell follicles and focally infiltrating into the red pulp Late time points and lower dose groups did not show any abnormalities We not anticipate significant toxicities associated with MV-CEA in our proposed clinical study 343 Comparative Efficacy of Fusogenic Measles, Mumps, and Vaccinia Vectors Against Ovarian Cancer Xenografts Rae Myers,1 Marie Frenzke,1 Suzanne Greiner,1 Mary Harvey,1 Diane Soeffker,1 Katalin Abraham,2 Alan Shaw,2 Shmuel Rozenblatt,3 Kah-Whye Peng,4 Stephen Russell.1,4 Toxicology Core, Mayo Foundation, Rochester, MN; 2Merck Research Laboratories, West Point, PA; 3Molecular Microbiology and Biotechnology, Tel Aviv University, Tel Aviv, Israel; Molecular Medicine, Mayo Foundation, Rochester, MN We directly compare several gene therapy vectors against an in vivo intraperitoneal model of ovarian SKOV3ip.1 tumor in order to look at the relative survival time and thus the efficacy of the various viruses, as well as the dose-responsiveness of MV-CEA-treated tumor bearing mice MV-CEA is an attenuated oncolytic measles virus engineered to express the soluble extracellular domain of human carcinoembryonic antigen Viral gene expression can be followed noninvasively in MV-CEA treated tumor bearing animals by monitoring the plasma concentrations of CEA MV-Moraten is the Moraten vaccine strain of measles virus produced by Merck MVA-H/F is a recombinant modified vaccinia virus Ankara (MVA), encoding the measles virus (MV) hemagglutinin (H) and fusion (F) glycoproteins The mumps virus is a Jeryl-Lynn vaccine strain paramyxovirus Prior to the start of the study, all mice were microchipped and ear-notched to ensure accurate identification Female week old nude mice were challenged intraperitoneally with 3.5E6 SKOV3ip.1 cells day 0, with treatment administered beginning day by the same route Groups of 10 female mice were treated with (1) 10E7 pfu x MVCEA, (2) 10E7 pfu x MV-CEA, (3) 1.75 x 10E5 pfu x MV-CEA, (4) 1.75 x 10E5 pfu x MV-Moraten, (5) 10E7 pfu x MVA-H/F, (6) x 10E8 pfu x (7) x 10E8 x MVA, (8) 10E7 pfu x Mumps, or (9) neat x Vero cleared cell lysate control, and were monitored for survival MV-CEA treated mice had blood drawn periodically to obtain a CEA profile At necropsy, total intraperitoneal tumor burden and location was recorded All measles virus and mumps virus treated groups (MV-CEA, MV-Moraten, and Mumps) showed prolonged survival as compared to the vehicle control Survival was not prolonged in vaccinia virus treated groups (MVA and MVA-H/ F) and significant acute treatment-related toxicity was observed in these animals Survival of measles and mumps treated animals was Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy ...CANCER TARGETED GENE THERAPY I should allow for new protocols to be developed and readily applied in the ever changing and more demanding field of cellular and gene therapy 341 Large Scale Manufacturing... human carcinoembryonic antigen Viral gene expression can be followed noninvasively in MV-CEA treated tumor bearing animals by monitoring the plasma concentrations of CEA Based on the promising antitumor... MVA-H/ F) and significant acute treatment-related toxicity was observed in these animals Survival of measles and mumps treated animals was Molecular Therapy Vol 7, No 5, May 2003, Part of Parts