763 Peak Supply Implications of Serum and Consumable Availability for Clinicians, Researchers, and Commercial Translation Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American S[.]
CELL PROCESSING AND VECTOR MANUFACTURE mouse and human cells To obtain a high virus titer packaging cell line we performed a “ping-pong” transduction between amphotropic (GP86), pseudo-typed GALV (PG13) and dual tropic–10A1 (PT67) cells After CD20-immunomagnetic cell sorting we obtained, as assessed by cytometry, bulks at 98%, 95% and 98% purity of transduced cells, respectively for GP86, PG13 and PT67 1/ We detected, by RT-PCR, both transcripts (hCD20 and hCD20Δ) in all the cell lines 2/ Using our designed qPCR assay, we quantified the hCD20Δ at a percentage of 1.16%; 0.37% and 0.5% in cDNA cells and of 0.11%; 0.01% and 0.02% in viral supernatant (SN) for GP86, PG13 and PT67 respectively 3/ We never detected hCD20Δ sequence integrated within the DNA of transduced packaging cell lines, suggesting that the low level of hCD20Δ particles doesn’t allow to transduce cell lines 4/ By qRT-PCR, we detected both transcripts in GFP positive- P815 and -EL4-Luc transduced murine cell lines with GP86 SN Quantification of hCD20Δ transcripts on cDNA of these cell lines was 0.75% and 2.96% for P815 and EL4-Luc respectively As expected we didn’t detect hCD20Δ sequence on genomic DNA Our results show that murine packaging cell lines can produce alternatively spliced hCD20 transcripts and produce both types of viral particles: hCD20 and hCD20Δ Low level of hCD20Δ viral particles is not sufficient to affect the transduction and selection efficiencies However, our results obtained on cell bulks could be different in high producer clones that may have a high transcriptional/splicing activity Mismatching of murine splicing machinery on human gene could explain the low level of hCD20Δ transcripts However hCD20Δ transduced human primary T-cells could express higher level of hCD20Δ that may decrease RTX treatment efficiency Thus, the use of our splice site-corrected hCD20 or a codon optimized hCD20 sequence can prevent this phenomenon 761 Large-Scale cGMP Manufacture of a Plasmid Vector for Cystic Fibrosis Gene Therapy Clinical Trials Ying Cai,1 Stephen Rodriguez,1 Jared Nelson,1 Tara Batten,1 Henry Hebel,1 Stephen C Hyde,2 Deborah R Gill.2 VGXI, inc, The Woodlands, TX; 2Gene Medicine Group, NDCLS, University of Oxford, Oxford, United Kingdom Non-viral gene therapy employing a plasmid vector encoding the CFTR transgene has demonstrated its safety and efficacy As opposed to DNA vaccines, low CpG content in the plasmid preparation is required to minimize inflammatory responses after administration Small scale production of this CpG free plasmid to high purity has been demonstrated, however, a new set of obstacles arose during the cGMP manufacture at a large scale Constructed with the R6K replication of origin, plasmid replication is regulated by both π protein expression from the host cell and Kanamycin resistance protein expression from the vector, resulting in low plasmid yields during fermentation Pre-existing cell banks excluded the potential for improvements via strain or colony screening The high impurity profile originating from low plasmid copy number impeded the performance of membrane and column chromatography To address these challenges, process development was carried out to both upstream and downstream processing Media optimization was able to double the specific plasmid yield from 0.25 g/kg cells to 0.5 g/ kg cells in the simple batch mode Improving the homogeneity of the cell lysate ensured consistency and notably higher recovery at a 1000 L scale Greater than 70% RNA impurity was removed via salt precipitation and additional column guard pre-filtration, which enhanced column resolution and reduced processing time These process improvements were effectively implemented to cGMP manufacture In the end, 50 grams of plasmid DNA at clinical grade was successfully delivered on time for Cystic Fibrosis Gene Therapy Clinical Trials Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy 762 Characterisation of Viral Count by Nanoparticle Tracking and Analysis (NTA) Technique and Its Application to Gene Therapy Patrick Hole,1 Duncan Griffiths,2 Andrew Malloy,1 Bob Carr.1 Research and Development, NanoSight Ltd, Amesbury, Wiltshire, United Kingdom; 2NanoSight USA West, Costa Mesa, CA A new sizing and characterization technique, Nanoparticle Tracking and Analysis (NTA) is described allowing nanoparticles in a suspension to be individually but simultaneously detected and analysed in real time using a laser-based microscope system The size range that this technique optimally works for includes many of the viral types of particles used frequently in gene therapy The technique uniquely allows the user a simple and direct qualitative view of the sample under analysis and from which an independent quantitative estimation of sample size, but more crucially, concentration can immediately be obtained at a size range from 10nm up to 2000nm The ability to measure concentration of nanoparticles at this size range enables a rapid measurement of total viral particles Traditional methods such as qPCR and plaque assay are not able to measure all virus particles present (just those containing RNA/DNA or those which are infective, respectively) In addition, these techniques would typically take many hours to prepare and measure, whilst NTA can measure a sample in five minutes A good qualitative understanding of degree of aggregation is also obtained which is crucial to understanding delivered titre; both qPCR and plaque assay are unable to make any assessment of aggregation A range of viral types from adenovirus, influenza, herpes simplex etc (∼100nm) can readily be characteried rapidly by the technique, whereas a few much smaller vectors such as parvo virus (∼25nm) cannot be measured Whilst fluorescent labelling is not required, in cases where the buffer may contain a complex mix of material, labelling of the target structure can allow for improved measurements and/or investigation at an earlier stage of the production process This paper will present the operation of the technique along with its limitations The work of several user groups’ assessments of the method in its application to viral counting will be highlighted and discussed Several recent developments will also be highlighted and their applicability to this field explained 763 Peak Supply: Implications of Serum and Consumable Availability for Clinicians, Researchers, and Commercial Translation Natasha L Davie,1,2,3 David A Brindley,1,2 Emily J CulmeSeymour,4 Chris Mason,4 David W Smith,5 Jon A Rowley.5 Biochemical Engineering, University College London, London, United Kingdom; 2Harvard Stem Cell Institute, Cambridge, MA; Centre for Excellence in Vascular Biology, Harvard Medical School, Boston, MA; 4London Regenerative Medicine Network, London, United Kingdom; 5Lonza Biologics, Walkersville, MD The cell therapy industry (CTI) is emerging as a distinct and competitive component of global healthcare, creating value for investors and providing life-changing therapies to patients Industry growth has necessitated an increased focus on largescale manufacturing strategies to meet future demands One major challenge is the limited availability of some crucial raw materials used in cell therapy manufacturing – including bovine serum Without a sustainable supply or viable alternatives to these components, the commercial-scale production of cell therapies will be impossible, halting the momentum of the industry We propose that solutions to these challenges are achievable, and can be expedited by industrywide collaboration Bovine serum is currently used in the majority of cell therapy manufacturing processes Current stocks and production rates of serum suitable for GMP manufacture may only be sufficient to support the production of one blockbuster cell therapy Limitations in the availability of bovine serum thus act as a major cost driver S293 CELL PROCESSING AND VECTOR MANUFACTURE and significant barrier to the commercial success of the industry as a whole Thus, without an increase in serum production, or at least a significant increase in the development and implementation of serum-free production strategies, the growth and sustainability of the CTI will be severely constrained Furthermore, disposable cell culture vessels have been widely adopted throughout the industry We discuss the cost-benefit ratio of using such systems as well as the impact of supply on research, clinicians and the commercial translation of cellular therapies 764 Enhancing the Expression of Foreign Genes with Caffeine in Transfected and Infected Cells that that CTI has grown strongly over the five year period studied, by an average of 141.89% compared with a an -8.13% loss experienced by the NASDAQ While some volatility remains, we suggest that a distinct industry is maturing to a point at which the volatility should subside, providing a more attractive environment for future growth In particular, we highlight the vital role of CTI translation centres and industry organisations including the Bio Industry Association (BIA), the Alliance for Regenerative Medicine (ARM), International Society for Cellular Therapy (ISCT), the International Society for Stem Cell Research (ISSCR) and the American Society for Cell and Gene Therapy (ASCGT) Zhibing Liang,1 Jie Luo,1 Nana Pei,1 Yingying Mao,1 Yuming Jing,1 Yi Feng,2 Yangling Zhang,1 Weiwang Gu,2 Hongwei Li.1 School of Biotechnology, Southern Medical University, Guangzhou, China; 2Institute of Comparative Medicine and Center of Laboratory Animals, Southern Medical University, Guangzhou, China Studies have shown that caffeine can increase the lentiviral vector titre, however, the mechanism remains to be elucidated To understand the mechanism, we tested whether caffeine can enhance expression of foreign genes in transfected and transduced cells In this study, 293T cells were transfected by calcium phosphate with the plasmid pTYF-CMV-eGFP, which contains eGFP gene controlled by CMV promoter Prostate cancer cells (DU145) were transduced with the recombinant adenoviral vector Ad5-CMV-eGFP Caffeine was then added to the cell culture media to a final concentration of 2-4mM The data showed that the addition of caffeine could significantly enhance the fluorescence intensity or eGFP mRNA level of pTYF-CMV-eGFP transfected cells by to 6-fold and Ad5-CMV-eGFP transduced cells by to 4-fold There was no difference between the levels of eGFP DNA in both transfected and transduced cells with or without the addition of caffeine In summary, caffeine can increase the expression of foreign gene in transfected or transduced cells but not the efficiency of either plasmid transfection or adenoviral trandsuction Based on this data, we will continue to explore the mechanism by which caffeineThis may help us understand the mechanism by which caffeine increases the lentiviral vector titre, and indicate its application in gene therapy by enhancing foreign gene expression 765 Cell Therapy Industry Market Characteristics: Implications for Clinicians, Investors, Patients and Translational Researchers David A Brindley,1,2,3,4 Natasha L Davie,1,2,4,5 Emily J CulmeSeymour,4 Brock C Reeve,2 William A Sahlman,3 Chris Mason.1,4 Advanced Centre for Biochemical Engineering, University College London, London, United Kingdom; 2Harvard Stem Cell Institute, Cambridge, MA; 3Harvard Business School, Boston, MA; 4London Regenerative Medicine Network, London, United Kingdom; 5Centre for Excellence in Vascular Biology, Harvard Medical School, Boston, MA The global cell therapy industry (CTI) is emerging as the fourth and final pillar of global healthcare, providing exciting opportunities for investors and the potential for life changing therapies to patients However, in part due to it immaturity, the commercial characteristics of the CTI, including market volatility and the translational pathway, are poorly understood This situation has greatly hindered the investment(s) necessary to fund the translation of life saving therapies from lab bench to marketplace Here, provide a quantitative review of the financial performance of leading CTI companies, utilizing the Cell Therapy Industry Cumulative Growth Index, between the period September 2006–September 2010 and propose strategies to expedite the future translation of stem cells into therapy The index suggests S294 Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy ... cost-benefit ratio of using such systems as well as the impact of supply on research, clinicians and the commercial translation of cellular therapies 764 Enhancing the Expression of Foreign Genes... titre, and indicate its application in gene therapy by enhancing foreign gene expression 765 Cell Therapy Industry Market Characteristics: Implications for Clinicians, Investors, Patients and Translational... for investors and the potential for life changing therapies to patients However, in part due to it immaturity, the commercial characteristics of the CTI, including market volatility and the translational