308 Titer and Long Term Stability of Retroviral Titers Produced in Murine Based Producer Cell Lines gate into immobile clusters The results suggest that the epitopes are useful to assay the quality of[.]
gate into immobile clusters The results suggest that the epitopes are useful to assay the quality ofhMSCs cultures for use in animal models and clinical trials CELL PROCESSI NG VECTOR PRODUCTION 306 Testing Large-Scale Lentiviral Vector Preparations for Replication Competent Lentivirus Lisa Duffy,' Sue Koop ,' ling Yao,' Robert Getty,' Scott Cross,' Lakshmi Sastry, Kenneth Cornctta.' 'Medical and Molecular Genetics/National Gene Vector Laborato/yo Indiana University School ofMedicine, Indianapolis IN As lentiviral vectors move into the clinical arena, the need to screen for replication competent lentivirus (RCL) will be an im- portant step in certifying vector supernatant We have previously reportedan analysis ofRCL testing methods (Sastryet al Molecular Therapy 8:830-9, 2003) and now report our initial experience with RCL screening In brief, the two-phase assay utilizes an amplification phase where the T cell line C8166 is exposed to the test article After hour incubation the cells are passaged for weeks and cell free media is used to inoculate naive C8166 cells which are then passagedfor I week (indicator phase), RCL is presentwhen p24-gag I-IIV-I antigen (by ELISA)and/or psi-gag recombinant(by PCR)are detected in indicator phase cells.An attenuated HIV-I virus (R8.71) is used as a positive control The assay was validated by performing infection of C8166 cells according to the protocol using R8.71 at the TCID50 (0.5 infectious unit/culture).Three cultures were tested per assay and three independent assays were performed Seven of nine cultures tested positive at the indicator phase for both p24 antigen and psi-gag recombinants, which validated the assay and confirmed the limits of detection was approximately I infectious unit Since validation, the assay has been uscd to test large scale vectorpreparationsand cells in independent assays For each assay, three positive controls were inoculated at the estimated TCID50 at the start of the amplification phase and were subsequently carried through the indicatorphase In addition, fiveculturesof naive C8166 were inoculated with virus at the TCID50 at the start ofthe indicator phase In reviewing the positive control portion ofthe amplification phase, 24 of 27 cultures at thc end of the amplification phase had elevatedp24 antigen levelsand after passageintothe indicatorphase, 27 of27 had detectable virus by p24 and by psi-gag recombination PCR For the positive controls set up at the start of the indicator phase, 41 of 45 were positive for p24 and 36 of 45 were positive for psi-gag recombinants at the end of the week of culture These findings suggest the week amplification does increase sensitivity compared to a week amplification In terms of test articles, of the RCLassays were used to screen large scale vector productions that had a combined pre-concentration volume of over 100 liters, of which 5% of the total final product was tested In addition, 10' post-productioncells were also screened from each of the productions and assays were performed on cells transduced with clinical grade vector To date, no RCL has been detected in any of the test articles submitted for analysis This analysis provides encouraging news for lentiviral vector development, although continued refinement of the RCL assay is warranted to maximize sensitivity while streamlining the methodology S1I6 307 Generation of Lentivirus Vectors Using Recombinant Baculoviruses Hanna P Lesch.!' Sanna Turpeinen,':' Einari A Niskanen,' Anssi Mahonen,':' Kari Airenne, I Seppo Yla-Herttuala.v-' I Department ofBiotechnology and Molecular Medicine A.I Vlrtanen Institute University ofKuopio, Kuopio, Finland; 2Department ofMedicine and Gene Therapy Unit A./ Virtanen Institute University ofKuopio, Kuopio, Finland; j Kuopio University Hospital Kuopio University Hospital Kuopto, Finland; "Department ofBiological and Environmental Science NanoScience Center; University of'Jyvaskyla Jyviiskyld, Finland; sArk Therapeutics qg Ark Therap eutics qg Kuopio, Finland The production ofreplication defective lentiviral vectors in clinical scale is challenging The four plasmid method by conventional transienttransfectionto producethird generation lentiviralvectors is tedious, time consuming and suffers from batch-to-batch variation Some inducible stable production cell lines are available but the toxicity of several viral proteins prohibits constitutive expression Baculovirus technology offers an attractive possibility to a scalable virus production as a result of ease production and concentration of baculoviruses, efficient transduction of suspension mammalian cells in serum free conditions and safety of the baculoviruses, As a firststep towards scalable lentiviralproductionsystem we have constructed four recombinant baculoviruscs, BAC-transfer, BAC-gagpol, BAC-VSVg and BAC-rcv,expressing all c1cmcnts required for safe lentivirus vectorgeneration After 293T cell transduction with recombinant baculoviruses functional Ientiviruses were produced Different baculovirus concentrationswere used to findoptimal baculovirusconcentrationfor lentivirusproduction The un-concentrated lentiviral titers in cell culture mediums were on avarege 1,21 x 106 TUlml which are comparable to titers ofthe lentivirus produced by the conventional four plasmid method Lentivirus transduced Hela cells were grown for three months without loosing the GFP exprcssion Our results show for the first time that baculoviruses can be used for the production of lcntiviruscs in mammalian cells 308 Titer and Long Term Stability of Retroviral Titers Produced in Murine Based Producer Cell Lines Lisa Duffy,' Sue Koop,' ling Yao,' Kenneth Cornctta.' 'Medical and Molecular Genetics/National Gene l-ector Laboratory Indiana University School ofMedicine Indianapolis IN While the spectrum of integratingvectors under considerationfor clinical use continues to expand, vectors based on gamma retroviruses continue to be utilized and improved To better understand factors involved with vector production, we studied the impact of genome size, temperature, harvest timing, and long-term storage on vector titer.Tostudy genomesize we prospectively designed a series of deletions in the transgene portion of the GcSAM vector (kindly provided by Richard Morgan, NIH) that contains non-expressing neomycin phosphotransferase (neo) and beta galactosidases genes Eight constructs were evaluated with genome sizes ranging from 3258 to 6970 bascpairs Titer was assessed using real-time PCR for detection of vector RNA using a probe and primer sct designed within the packaging sequence Titer for vector genornes between 4559 and 6346 basepairs had similar titers, with a decrease outside this range The effect of temperature and harvest intervals was also evaluated Data from 27 PG13and GP+envAM12derived Master Cell Banks(MCB)generatedin our facilitywascompiled, evaluating titer of vector produced at 32 or 37°C and harvest intervals of 8, 12 or 24 hours Thc majority of PG 13 based ccll Iincs (13/27) had the highest titers whcn harvested at 24 hours and 32°C, although 6/27 wcre optimal at 37°C, 12 hour harvest; 4/27 wcrc optimal at 37°C, 24 hour harvest; and 4/27 were optimal at 32 DC, 12 hour Molecular The 4lpy Volume 15.Supplement tã ã\ br 2007 Co pyright â "111e American SOI;ic;ty o f Gene Thcrapj- harvest All GP+envAMI2 cell lines were optimal at 32 °C, but optimal harvest time varied The data suggests harvest conditions should be optimized for each MCB generated Also, those MCBs with an optimal titer at 24 hours often have a 12 hour titer that exceeds 50% of the 24 hour titer Therefore, more frequent harvests at shorter intervals can increase the total yield of vector particles and may be preferred when the material can be concentrated Finally, the stability of vector stored was measured over a year period Aliquots of the LNL6 vector (expressing neo) and the Ampho/GFP and GALVIGFP vectors (expressing enhance green f1 uorescent protein, MGF-eGFP) were maintained between -70 and -80 °C and the number oftransducing units was periodically measured using G418 selection and flow cytometry, respectively In all three vectors the titer appeared to be stable during the observation period suggesting an expiration date of5 years would be appropriate for unconcentrated vectors The information obtained serves as a baseline for assessing new packaging cell lines, including those designed to generate novel integrating vector systems such as HIV and foamy viruses 309 Plasmid DNA Production in a Wave Bioreactor under cGMP Conditions Kenneth A Laderman ,' Jonathan M Anderson ,' Ivy V Derecho ,' Nina T Dunphy,' Ross J Mclvlahon,' Valerie R Quezada,' David Hsu,' Larry A Couture.' 'Center for Biomedicine and Genetics Centerfor Applied Technology Development Beckman Research Institute ofCity of Hope Duarte CA Plasmid DNA production is an essential step in the generation of genetic therapies, either as the therapeutic agent or as reagents for the production of viral vectors A robust process for the generation of plasmid bearing E coli biomass is necessary as it is the rate limiting step for plasmid production and it is most variable as there are construct specific factors that affect the plasmid yield There are a number of factors that make the transition ofthe fermentation process from a traditional stirred tank reactor to a Wave bioreactor desirable Foremost among these are the disposable culture vessel in the form of the wave bag The disposable culture vessel minimizes the possibility of prior product contamination in a multi-product facility and decreased production cost while increasing throughput due to the ease of set up and tear down Two lentivirus helper plasmids, which had previously been produced in the facility using a cGMP stirred tank procedure, were used to develop the wave fermentation process Fermentations were completed using media and fermentation parameters adapted from the stirred tank fermentation process To mimic the aeration and agitation of the stirred tank the wave angle and rate were set to their practical maxima The mean specific yield (mg of plasmid I g wet weight) obtained from the wave bioreactor was found to range from equivalent to fold higher than that obtained from the same bacterial master cell bank in the stirred tank fermentor while the bacterial yield (g wet weight I liter of culture) from the wave bioreactor was approximately equivalent to that obtained from the stirred tank The wave fermentation has proven to be scalable with minor increases in specific yield observed as the culture volume is increased to 25 Iitcrs Limited optimization of temperature and pH conditions have established conditions that increase the specific yield approximately 100% from the default fermemation conditions used for the original feasibility assessment The resulting improvement in specific yield ofthe optimized process is approximately 600% greater than that obtained with the stirred tank system Molecular Therapy Volume15 Supplement I ~ br 2007 Copyright © The American Soci ety o r Gene "1l1f:r:lpy 310 A Rapid, Accurate and Precise Assay System To Analyze the Quality and the Quantity of Clinical Grade HSV Vector Stocks Ali Ozucr,' Steve K Wendell,2 Darren Wolfe; William F Goins; David M Krisky," Mohammad M Ataai,' Joseph C Glorioso.' f Molecular Genetics and Biochemistry University ofPittsburgh Pittsburgh PA; ]Dental Medicine, University ofPittsburgh Pittsburgh, PA; 'Chemical and Petroleum Engineering University of Pittsburgh Pittsburgh, PA; "Department ofPathology; University ofPittsburgh Pittsburgh PA As the number of clinical and research applications of herpesvirus-based vectors increase, it becomes critical to develop rapid, accurate and precise assay systems to analyze the quality and the quantity of clinical grade vector stocks Our assay system contains two independent and complementary DNA and protein quantification methods: a real-time quantitative PCR system and two micro-plate assays using PicoGreen and NanoOrange reagents for the quantification of total DNA and protein respectively This assay system is being optimized to quantify the amount of infectious versus defective HSV particles, and the purity ofHSV vector stocks Our real-time quantitative PCR assay employs two independent primer sets The first set, speeific for ICP47 sequences present in all HSV genomes, allows for the quantification of total viral particles and for defective DNA containing particles when compared with the number of plaque forming units generated by standard virological plaque assay A second primer set, specific for the human 18S gene, enables the estimation of purity ofgene therapy vector stocks in combination with NanoOrange protein assay system Our real time PCR assays are linear from to 4x 103 HSV genome copies per reaction, and 0.005 to 50 ng host cell genomic DNA per reaction In contrast to our PCR method that quantifies the viral and host cellular DNA concentration, we developed an independent microplate assay measuring the total DNA and protein concentrations of vector stocks Our micro-plate assays are fast and accurate, with detection limit as low as 0.5 ng of DNA and 0.4 mg/mL protein The resultant combination of real-time PCR and micro-plate DNA and protein quantitation assays represents a standard in the field of HSV vector quality assessment 311 Development of a High Cell Density Process for cGMP Production of VRX496 Modified HIV Infected CD4+ T Lymphocytes Andrew Worden,' Tony Encinas.' Yclena Skripchcnko.! James Rogers,' Reuben Cohen,' Vladimir Slepushkin,' Laurent Hu- meau.' f Research and Development VIRxSYS Corporation Gaithersburg AID; lOperations VIRxSYS Corporation Gaithersburg, MD VIIb:SYS Corporation has developed an autologous CD4+ T cell therapy for the treatment ofI-IlV infection, which uses a patient's own CD4+ T cells that have been genetically modified by a lentiviral vector, VRX496, carrying a 937-base antisense targeting the HIV envelope After a patient's white blood cells are harvested, the cells are then purified to obtain the CD4+ T cells, transduced with VRX496 , expanded, and then rcinfused into the patient We reported the successful completion of our Phase I clinical trial where we demonstrated the safety and tolerability ofa single dose consisting ofapproximately 10 billion autologous I-IIV infected CD4+ T cells transduced with VRX496, (Levine and Humeau et al., PNAS 2006) We immediately initiated a Phase II clinical trial testing the safety and tolerability ofsingle and repeated infusions ofVRX496-modifed CD4+ T cells with the aim to determine the optimal therapeutic dose A large scale cGMP cell process was developed and has been presented (Humeau et aI., ASGT 2006) Briefly, the positively seSI17 ... VRX496, (Levine and Humeau et al., PNAS 2006) We immediately initiated a Phase II clinical trial testing the safety and tolerability ofsingle and repeated infusions ofVRX496-modifed CD4+ T cells with... baseline for assessing new packaging cell lines, including those designed to generate novel integrating vector systems such as HIV and foamy viruses 309 Plasmid DNA Production in a Wave Bioreactor... uorescent protein, MGF-eGFP) were maintained between -70 and -80 °C and the number oftransducing units was periodically measured using G418 selection and flow cytometry, respectively In all three