381 A New Cell Line for Clinical Grade Production of E1 Deleted Adenovirus Encoding Toxic Proteins Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell T[.]
CELL PROCESSING AND VECTOR PRODUCTION 379 GMP Production of a SIN Lentiviral Vector for the Treatment of SCID-X1 Using a Stable Producer Cell Line Tim Lockey, Susan Sleep, Robert Throm, Mike Greene, Paolo Fagone,2 Harry Malech,3 Brian Sorrentino,2 John T Gray.2 Children’s GMP, LLC, St Jude Children’s Research Hospital, Memphis, TN; 2Hematology, St Jude Children’s Research Hospital, Memphis, TN; 3Laboratory of Host Defenses, NIAID, NIH, Bethesda, MD 1 2 We describe here our process and large scale production results for GMP grade production of a clinical lentiviral vector for the treatment of SCID-X1 using a stable, HIV based producer cell line (described in Throm, et al Blood, v113 n21 p5104) Process optimization was performed with cells from our master cell bank at and liter scale prior to pre-clinical production at liter scale and clinical production at 25 liter scale The final process consists of cell expansion in flasks prior to seeding into a WAVE bioreactor bag containing Fibracel disks for expansion to final production scale When daily glucose consumption exceeds – 1.5 g/L, the media (DMEM+10%FCS+1 ng/ml Doxycycline) is replaced with Doxycycline free media to induce vector production On Days through post induction (p.i.) supernatant is harvested and held at 4C On days 4, 6, and two harvests are pooled, adjusted to pH 8.0 and 300 mM NaCl, and flowed through Mustang Q cartridges to capture vector particles Following elution with 1.5 M NaCl, vector is diluted with buffer to reduce salt concentration and held at 4C until the final harvest has been similarly processed The three anion exchange eluates are then pooled, desalted, and concentrated by using tangential flow filtration with a 500 kDa cutoff membrane The final concentrated vector is formulated in PBS containing 0.5% human serum albumin Production yield and QC assay results for the pre-clinical and clinical batches were similar and results for the clinical product are presented here Infectious titer of the vector product was determined on ED7R cells, a T-cell line that does not significantly express the γc gene Peak supernatant titer occurred on day p.i at 1.25x107 tu/ml, and the average of all harvests was 5.5x106 tu/ml, giving a total transducing unit yield of 8x1011 tu p24 similarly peaked on day p.i at 5.2 µg/ ml, and averaged µg/ml for all harvests After purification, yield was 2.3x1011 total tu, or ∼30%, predominantly due to losses at the Mustang Q step Final volume was 500 mls and titer was 4.5x108 tu/ml, with an infectivity of ∼5000 tu/ng p24, which is comparable to that described for the transient transfection product used for the successful lentiviral treatment of adrenoleukodystrophy (Cartier et al, Science, v326 n5954 p818) The product has been extensively tested for adventitious agents including replication competent lentivirus and confirmed negative Critical potency measurements were performed by transducing mobilized adult human peripheral blood CD34+ cells at 108 tu/ml CFU-C derived from those transductions were typically >20% positive by PCR depending on the donor Final FDA review of this clinical grade product is pending, with anticipated initiation of clinical trial immediately following approval This represents to our knowledge the first clinical grade lentiviral vector produced from a stable cell line system S148 380 Generation and Characterization of AAV Producer Cell Lines John Martin,1 Amy Frederick,1 Yuxia Luo,1 Robert Jackson,1 Dianna Ambach,2 Christopher Renzi,2 Lisa Budzinski,2 Lois Horton,2 Angela Johnsen,2 Simon Godwin,2 Donna Armentano,1 Sam Wadsworth,1 Karen Vincent.1 Molecular Biology, Genzyme Corporation, Framingham, MA; Gene Therapy Development, Genzyme Corporation, Framingham, MA AAV producer cell lines represent an effective method for largescale production of AAV vectors for clinical applications In the system described here, a single plasmid containing three components: the vector sequence, the AAV rep and cap genes and a selectable marker gene is stably transfected into HeLa S3 cells Following transfection, candidates (referred to as masterwells or MW) are initially selected on the basis of a relative production screen where vector yields are estimated and used to rank MW relative to each other High-producing MW are then subjected to a specific production screen in which yield (of DNase-resistant particles; DRP) is determined on a per cell basis A transfection to generate an AAV2/SEAP producer cell line has been performed The relative production screen revealed that 6.4% of the MW yielded greater than x1010 DRP/ml Several of these showed vector yields in the x 104 to 2.0 x 105 DRP/cell range when assessed for specific production Analysis of genomic DNA in the high-producing MW showed plasmid copies integrated in a headto-tail configuration Upon infection with adenovirus, rep/cap copy number was amplified approximately 100-fold Rep expression in the producer cell line was first detected a few hours post-infection and was followed closely by cap; expression of both genes reached a peak at 48 hr This pattern of gene expression correlated with the appearance of packaged vector at the 24 hr time point Vector production reached a maximum at 48 hr and was maintained through the 96 hr (final) time point AAV2/SEAP was produced in a small-scale (1L) shaker culture and purified via affinity chromatography to provide vector for assessment of vector potency Results of experiments comparing AAV2/SEAP generated by the producer cell line with vector produced via the triple transfection method will be presented 381 A New Cell Line for Clinical Grade Production of E1-Deleted Adenovirus Encoding Toxic Proteins Rénald Gilbert,1,2 Claire Guilbault,1 David Gagnon,1 Danielle Jacob,3 Lucie Bourget,1 Amine Kamen,3 Bernard Massie.1,4 Genomics & Gene Therapy Vectors, Biotechnology Research Institute, Montreal, QC, Canada; 2Neuromuscular Research Group, Montreal Neurological Institute, McGill University, Montreal, QC, Canada; 3Animal Cell Technology, Biotechnology Research Institute, Montreal, QC, Canada; 4Département de Microbiologie et Immunologie, Université de Montréal, Montreal, QC, Canada First generation (E1-deleted) adenovirus vectors (FGAd) are important vectors for therapeutic applications such as cancer and vaccination An ideal cell line for clinical grade production of FGAd should produce high viral titers in suspension culture for scale-up and in serum-free medium for regulatory compliance Most importantly, it should not produce replication competent adenovirus (RCA) Some gene products carried by FGAd, such as those employed for cancer therapy, are cytotoxic For this reason, they should not be synthesized during the propagation of FGAd, because their toxicity reduces the vector yield The present study describes the construction of a cell line (Sf-BMAd-R) that possesses such properties The cell line was generated by stable transfection into A549 cells of the E1A and E1B regions of adenovirus and of the repressor of the cumateswitch (CymR) A clone producing elevated amounts of FGAd was Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy RNA VIRUS VECTORS II isolated and adapted to serum-free suspension culture CymR binds to the cumate operator (CuO) and prevents transcription when CuO is inserted downstream of a strong enhancer/promoter such as CMV Expression of a transgene regulated by the CMVCuO promoter is repressed when the FGAd carrying the transgene are propagated on Sf-BMAd-R cells The yield of FGAd expressing GFP regulated by CMVCuO (Ad-CMVCuOGFP) was 1000 transducing units (TU) per cell, a value comparable to the yield obtained with our 293SF cell line previously characterized Production of Ad-CMVCuOGFP and repression of GFP by CymR were stable for at least three months of culture in the absence of selective agent Production of Ad-CMVCuOGFP to a level of 1000 TU/cell was also validated in a 2.5 liter bioreactor in protein-free chemically defined medium We also demonstrated that the yield of FGAd encoding a suicide gene (Cytosine deaminase::uracil phosphoribosyltransferase) regulated by CMVCuO was times higher with our Sf-BMAd-R in comparison to our standard 293SF cells In summary, because of its interesting characteristics, the Sf-BMAd-R cell line should become a useful tool to produce clinical grade preparations of FGAd for gene therapy and vaccination RNA Virus Vectors II 382 Rapid Titration of Lentiviral and Gamma Retroviral Vectors Using a Beta-Lactamase Protein Fragment Complementation Assay Wu Ou,1 Connie Lu,2 Jakob Reiser.1 Division of Cellular and Gene Therapies, FDA/CBER, Bethesda, MD; 2University of Maryland, College Park, MD We have developed a rapid assay to titrate lentiviral and gamma retroviral vectors The assay is based on protein fragment complementation involving the N-terminal (Bla1) and C-terminal (Bla2) fragments of β-lactamase (BLAK), which were fused to the yeast FRB (the FKBP-rapamycin binding domain of TOR) and FKBP (FK506 binding protein) domains, respectively In addition, a 15-amino acid membrane anchoring sequence (S15) derived from c-Src was fused to the N-terminus of FRB-Bla1 For vector production a plasmid encoding S15-FRB-Bla1 was co-transfected with lentiviral packaging and vector plasmids into HEK 293T cells, resulting in the incorporation of S15-FRB-Bla1 in the vector’s envelope Transduction of HEK 293 cells stably expressing FKBP-Bla2 using vector particles containing S15-FRB-Bla1 resulted in BLAK fragment complementation, mediated by FRB and FKBP and rapamycin Enzymatically active BLAK could be detected by the conversion of a green fluorescent substrate CCF2/AM into a blue fluorescent product and BLAK activity could be easily quantified in that way using FACS The enzymatic conversion of CCF2/AM was found to be directly related to vector entry since a neutralizing antibody completely blocked the conversion The titers obtained using this rapid assay correlated well with titers measured by functional transduction assays The whole assay can be finished within hours This time frame is considerably shorter compared to other transduction-based titration assays for lentiviral and gamma retroviral vectors This assay may also be useful for rapid titration of other enveloped viruses and for studying virus entry processes Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy 383 Insulated Lentiviral Vectors towards Safer Gene Transfer to Stem Cells Caroline Duros,1 Alexandre Artus,1 Armelle Gaussin,2 Scholtz Simone,3 Daniela Cesana,4 Eugenio Montini,4 Manfred Schmidt,3 Christof von Kalle,3 Nicolas Mermod,2 Odile Y D CohenHaguenauer,1 Odile Y D Cohen-Haguenauer.5 LBPA and CLINIGENE, Ecole Normale Superieure, Cachan, France; 2Laboratory of Molecular Biotechnology, University of Lausanne (UNIL), Lausanne, Switzerland; 33Laboratory of Translational Oncology, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany; 4FCSR-TIGET, San Rafffaele, Milan, Italy; 5Medical Oncology and Oncogenetics, Hôpital SaintLouis and University Paris-Diderot, Paris, France The need for better gene transfer systems towards improved risk/benefit balance for patients remains a major challenge in the clinical translation of gene therapy (GT) We have investigated the improvement of integrating vectors safety in designing new short synthetic genetic insulator elements (GIE) In otherwise successful GT-trials in SCID, CGD and WASp patients, insertional mutagenesis has resulted in leukemia from transduced cells The identification of new GIEs which would prevent such activation effects is a main challenge since attempts e.g with the chicken β-globin HS4, have met with poor efficacy and genetic instability We have constructed SIN-insulated retrovectors with two candidate GIEs and compared them to native and SIN-LTRs With each constructs two internal promoters have been tested: either the strong Fr-MuLV-U3 or the housekeeping hPGK We could identify a specific combination of insulator repeats which translates into best functional activity, high titers and boundary effect in both gammaretro (p20) and lentivectors (DCaro4) In target cells a dramatic shift of expression is observed with an homogenous profile the level of which is strictly conditioned by the promoter strength These data remain stable in both HeLa cells over three months and cord blood HSCs for two months, irrespective of the multiplicity of infection (MOI) In comparison, control vectors show heterogeneous expression profiles with levels which depend on the MOI and prove unstable We have undertaken genotoxicity assessment in comparing integration patterns ingenuity in human target cells sampled over three months using high-throughput pyro-sequencing Data indicate reduced clonality developed over time In addition, utmost recent data in cancer-prone mice not show statistically different from mock-untransduced mice We have developed new lentivectors (and gammaretrovirus) which might provide improved versatile tools toward improvement of integrating gene therapy vectors 384 Generation of Transgene-Free Induced Pluripotent Stem Cells Using Polycistronic Foamy Virus Vectors Iram F Khan,1 Jordan Kho,3 David R Deyle,1 Yi Li,1 David W Russell.1,2 Medicine, University of Washington, Seattle, WA; 2Biochemistry, University of Washington, Seattle, WA; 3Program in Developmental Biology, Baylor College of Medicine, Houston, TX Induced pluripotent stem cells (iPSCs) generated by ectopic expression of transcription factors are an ideal source of patientspecific cells given their capacity to differentiate into different cell types However, multiple insertions of retroviral vectors at random sites within the genome can alter endogenous gene expression and render these cells unsuitable for clinical applications Expressing all transcription factors from a single vector can minimize the number of viral integrations Here we describe excisable, polycistronic Foamy Virus (FV) vectors for reprogramming human somatic cells into pluripotent stem cells Wild type FV is non-pathogenic and FV vectors exhibit no preference for integration within genes, S149 ... preparations of FGAd for gene therapy and vaccination RNA Virus Vectors II 382 Rapid Titration of Lentiviral and Gamma Retroviral Vectors Using a Beta-Lactamase Protein Fragment Complementation Assay Wu... Cohen-Haguenauer.5 LBPA and CLINIGENE, Ecole Normale Superieure, Cachan, France; 2Laboratory of Molecular Biotechnology, University of Lausanne (UNIL), Lausanne, Switzerland; 33Laboratory of Translational... Lu,2 Jakob Reiser.1 Division of Cellular and Gene Therapies, FDA/CBER, Bethesda, MD; 2University of Maryland, College Park, MD We have developed a rapid assay to titrate lentiviral and gamma retroviral