760 Human CD20 Transduced Packaging Cell Lines Express hCD20Δ Alternative Transcripts but Produce Low Level of Retroviral Particles Carrying Spliced RNA Molecular Therapy Volume 20, Supplement 1, May[.]
CELL PROCESSING AND VECTOR MANUFACTURE media were supplemented with polyvalent human immunoglobulin (pIgG) as an alternative to FBS The serum-free concentrated viruses still kept the high infectivity to peripheral T lymphocytes although the virus titers of them were slightly lower than those of cases containing FBS Furthermore, use of the concentrated viruses made it possible to set up the culture condition that favored proliferation of human cord blood CD34+ cells while maintaining their stemness and resulted in increasing the number of gene transferred immature cells that were capable of forming HPP-CFC With further modifications, the method described would provide an effective procedure to prepare a large amount of the viral supernatants of PG13-based retrovirus vectors in the Good Manufacturing Practice (GMP) 758 A Novel Platform of Genetically-Modified Cell Transplantation Using 3D Spheroid Culture System on Micropatterned Substrates and Polyplex Nanomicelles Keiji Itaka,1 Taisuke Endo,2 Kazunori Kataoka.2 Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, University of Tokyo, Japan; 2Department of Materials Science and Engineering, Graduate School of Engineering, University of Tokyo, Japan Three-dimentional (3D) spheroid cell culture comprises the complexity of tissues and mimics physiological micro-environments such as cell-cell and cell-matrix interactions It has great advantages in controlling biological mechanisms such as proliferation, differentiation and secretion of proteins from the cells, being potentially a highly useful platform of cell transplantation for therapeutic purposes Cell culture on micro-patterned substrates in which micrometer-sized domains are arranged as an active area of cell attachment is a powerful technique to obtain sufficient amount of spheroids with identical size We had already established spheroid systems for primary hepatocytes and MSC [1,2] For hepatocytes, the cell viability and liver-specific functions such as albumin secretion were well preserved for at least one month In this study, we investigated the feasibility of this system for genetically-modified cell transplantation For gene introduction into spheroids formed by primary hepatocytes, we used our nonviral polycation-based carriers, polyplex nanomicelle [3] After transfection of Gaussia lusiferase-encoding pDNA using nanomicelle, the luciferase expressions were well detected in culture medium for more than weeks The albumin secretion was also maintained as the control cells without transfection, suggesting that the safe gene introduction was achieved without affecting the biological functions of the cells Otherwise, when using other commercially lipid-based reagent such as FuGENE6, the shape of spheroids was significantly altered, with a decrease in albumin secretion To confirm the feasibility for cell transplantation, the spheroids after gene introduction were detached from substrates and reseeded into Matrigel for further incubation From the Matrigel, the luciferase expressions as well as albumin secretion were well obtained for more than weeks, strongly suggested that the cells maintained innate functions with sustained transgene expressions after transplantation Thus, we believe this system is promising for cell therapy with genetic modification [1] ChemBioChem 2004, 5, 850-5 [2] Biomaterials 2009, 30, 2705-15 [3] Curr Gene Ther 2011, 11, 457-65 759 cGMP Plasmid Manufacturing and Release Criteria for Phase I/II Trials Nicolas Taquet,1 Chris M Jay,1 Li Chen,1 Connor Phalon,1 John Nemunaitis,1 Phillip B Maples.1 Gradalis, Inc., Dallas Gradalis has gained FDA/RAC approvals for five DNA plasmidbased INDs in years The quality of plasmid DNA is critical for S292 successful and efficient gene transfer and outsourcing plasmid production can be costly We determined that for our advanced preclinical, Phase I and Phase II needs we could develop a costeffective cGMP plasmid production facility capable of producing up to 10g lots of plasmid during any given campaign A High-CellDensity Culture method for fed-batch fermentation was developed to produce high-quality plasmid DNA in Escherichia coli Fermentation conditions and feeding strategies, including the composition of start medium, the composition of feed medium, the time of start feeding, feeding flow rates, and dissolved oxygen level, were optimized for bacterial growth and plasmid yield Single use, disposable, and precertified materials were implemented The bacteria are collected by centrifugation on a Carr centrifuge into Teflon bags and stored frozen for pooling at the lysis step The fermentation and centrifugation are made in a negatively pressurized HEPA filtered Bioreactor room with bag in bag out filtration of the exhaust The plasmid is purified by alkaline lysis, clarified by filtration, anion exchange chromatography (AEX), hydrophobic interaction chromatography (HIC) and tangential flow filtration (TFF) within an ISO positively pressurized cleanroom Terminal filtration through reducing pore size filters is the last series of steps before filling The final fill occurs in a separate single pass air positively pressurized ISO cleanroom within an ISO biosafety cabinet The manufactured plasmid is released based on the following criteria: Optical density DNA concentration Appearance Bioburden DNA homogeneity Identity of uncut plasmid Restriction digest Sequencing Residual bacterial protein Residual bacterial DNA Residual bacterial RNA Residual Kanamycin by E coli growth inhibition test Endotoxin by LAL test USP sterility OD 260/280= 1.7-2.0 >1 mg/ml clear / colorless no growth on LB culture plates >80% supercoil co-migrates with standard on electrophoresis gel restriction bands on gel 100% DNA sequence match E coli