45 implementation of a reproducible and effective process for the GMP production of lentiviral vectors

45 Implementation of a Reproducible and Effective Process for the GMP Production of Lentiviral Vectors Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Ther[.]

CELL PROCESSING AND VECTOR PRODUCTION 43 A Novel Inducible System for Highly Efficient Production of Recombinant AdenoAssociated Virus (rAAV) Vectors in Insect Sf9 Cells George Aslanidi,1 Kenneth Lamb,1 Sergei Zolotukhin.1 Pediatrics, University of Florida, Gainesville, Fl Production of clinical grade gene therapy vectors for human trials remains a major hurdle in advancing cures for a number of otherwise incurable diseases In the current study, a novel simple and efficient system of rAAV production in insect cells is described The system takes advantage of DNA regulatory elements from two unrelated viruses - Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and AAV2 The endpoint design consists of only two components: 1) stable Sf9-based cell line incorporating integrated copies of rep and cap genes, and 2) Bac-GOI (gene of interest flanked by AAV inverted terminal repeats) Rep and cap genes are designed to remain silent until the cell is infected with Bac-GOI helper which provides both rAAV transgene cassette and immediate-early (IE-1) transcriptional transregulator Infection with Bac-GOI initiates the rescue/amplification of the integrated AAV helper genes resulting in dramatic induction of the expression and assembly of rAAV The integration cassette incorporates an IE-1 binding target sequence from wild type AcMNPV, a homologous region (hr2) In addition, the cassette includes Rep-binding element (RBE), a target site for AAV2 Rep78/68 proteins By binding to RBE, Rep proteins initiate a feed forward loop mediating the rescue/amplification of the integrated cassette All four Rep proteins (78, 68, 52, and 40) are expressed from one uninterrupted AAV2 rep sequence thus utilizing Sf9 cells splicing machinery Furthermore, the system had been also utilized to modulate the stoichiometry of VP1/VP2 proteins thus increasing phospholipase A2 content of the particle and improving infectivity of alternative serotypes such as AAV8 produced in insect Sf9 cells The arrangement provides high levels of Rep and Cap proteins in every cell thus improving rAAV yields by 10-fold The described vectors are modular in design and could be utilized for the production of other multiprotein complexes 44 Efficient Construction of Producer Cell Lines for SIN, Clinical Lentiviral Vectors by Concatemer Array Transfection Robert E Throm,1 Annastasia A Ouma,1 Anantharaman Chandrasekaran,1 Timothy Lockey,1 Michael Greene,1 Sheng Zhou,1 Derek A Persons,1 Harry Malech,2 Brian P Sorrentino,1 John T Gray.1 St Jude Children’s Research Hospital, Memphis, TN; 2NIAID, NIH, Bethesda, MD We describe a transfection and selection method for introducing vector genome expression cassettes into packaging cells which efficiently generates high titer producer cell lines for vectors containing self-inactivating (SIN) LTRs and chromatin insulators These vector design features have been shown to reduce genotoxic side effects, and yet clinical production of such vectors frequently utilizes cumbersome transient transfection methodology, as stable producer cell derivation is hampered by the fact that SIN vector genomes cannot be introduced into packaging cells by viral transduction methods We optimized transfection and selection methods of stable producer cell derivation by constructing in vitro ligated concatemeric arrays containing a 25:1 ratio of a SIN, GFP vector genome expression cassette and an antibiotic resistance cassette, which were subsequently transfected into our newly developed doxycycline regulated HIV-VSVG stable packaging cell line After selection with drug, individual clones were isolated, expanded, and screened for productive titer When compared to clones isolated after transfection of supercoiled plasmids mixed at identical ratios, clones derived from concatemeric array transfection yielded 10-fold higher titers (mean titer=1.76x107 tu/ml vs 1.8x106 S18 tu/ml, p=0.0002) We additionally showed by Southern blot analysis of producer cells that engineering of the concatemeric array to ensure the directional assembly of monomers facilitated the stable maintenance of the array in vivo We have successfully applied this new methodology to generate stable producer cell lines for separate SIN, insulated clinical vectors, all of which produce titers comparable to or exceeding those generated by transient transfection The furthest progressed vector expresses the human common γ-chain for SCID-X1 gene therapy, and our top clone reliably generates unconcentrated titers of 3-10 x 107 tu/ml on ED7R cells (a γ-chain deficient human T-cell line) This producer line has been expanded under GMP and vialed as a Master Cell Bank for clinical production Cells derived from this bank were passaged without selection for one month prior to seeding in a small scale WAVE bioreactor, which after induction generated high titer supernatants (>3x107 tu/ml) for consecutive daily harvests Another line developed for SCID-X1 gene therapy generates the IL2RG-Revgen vector, which contains the entire genomic locus of the human common γ-chain gene in the reverse orientation Titers from this line also exceed 107 tu/ml The third line produces an EF1α driven gp91phox vector for treatment of chronic granulomatous disease, and the fourth contains a complex γ-globin vector for treatment of beta-thalassemia and sickle cell anemia Both of these lines generate titers greater than 106 tu/ml, and yet still await production optimization In summary, the concatemeric array transfection method allows reliable generation of high-titer stable producer cell lines for safety modified, clinically relevant vectors 45 Implementation of a Reproducible and Effective Process for the GMP Production of Lentiviral Vectors Patricia Noguiez-Hellin,1 Otto-Wilhelm Merten,1 Sylvain Fauchille,1 Nicolas Laroudie,1 Céline Dugué,1 Sabine Charrier,1 Maria-Antonietta Zanta-Boussif,1 Helene Chautard,1 Didier Caizergues,1 Marina Radrizzani,2 Alessandro Aiuti,3 Luigi Naldini,3 Anne Galy.1 Genethon, Evry, France; 2Molmed, Milan, Italy; 3Tiget, San Raffaele Hospital, Milan, Italy We have obtained encouraging preclinical data supporting the development of a lentiviral vector (LV) for clinical gene therapy application in Wiskott Aldrich Syndrome (WAS) To implement the highest standards of quality and safety, the clinical studies will be performed with highly-purified vector particles For this purpose, we developed a process for the production of LV in cGMP This process is based on the transient transfection of a selected subclone of HEK293T cells followed by the downstream purification and concentration of the VSVg pseudotyped viral particles The process has been carefully validated jointly both at Genethon and Molmed/ Tiget We report here the manufacture of clinical grade vectors which was successfully implemented in Genethon’s manufacturing facility Two batches of the WAS lentiviral vector were produced in GMP The manufacturing operations started by cultivating cells from the master cell bank in 24 ten-stack CF-10 (CellFactories) After amplification, the cells were transfected with GMP-grade plasmids bringing in the functions necessary for the production of the vector (rev, gag-pol, VSV-g, vector with the therapeutic transgene) The supernatant fluid containing the LV was harvested, filtered, purified and concentrated by a multi-step downstream processing protocol including ion exchange chromatography, concentration/diafiltration, and gel filtration Such vector preparation was purified by a factor of about 1000 fold from protein and DNA contaminants and concentrated about 200 fold In the final product, specific host cell contaminants such as SV40large T antigen or E1A sequences were shown to be reduced Process byproducts such as residual benzonase were no longer detectable High titers of at least 1x 109 i.g./ml were obtained reproducibly in both batches, yielding a total amount of at least 2.5 x 1011 i.g produced Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy CELL PROCESSING AND VECTOR PRODUCTION per batch The vector integrity was confirmed by analysis of the integrated proviral sequences and RCLs were not detectable The vector was functional as demonstrated by gene transfer or transduction of immortalized WASp-deficient B cell lines or CD34+ cells, and there was no evidence of cellular toxicity induced by the vector These functionality assays revealed that the vector introduces about vector copy per cell in hematopoietic progenitor cells when the vector concentration was 5-10 x 107 ig/ml Under these conditions the transduction efficiency was about 40-50% The WAS LV batch produced at Genethon was therefore conform and released for clinical use In conclusion, the WAS project has permitted the implementation of large-scale GMP production, purification, and control of advanced HIV1-derived lentiviral technology at Genethon which should be useful for other applications 46 Production of Lentivirus by Transient Transfection of HEK 293T Grown on Spherical, Polystyrene Microcarriers Scott R Witting,1 Aparna Jasti,1 Sarah Dolan,1 Kenneth Cornetta.1 Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN Lentiviral vectors have evolved into an effective means of obtaining long-term transgene expression and a valuable tool for human gene therapy Therefore, techniques to efficiently produce large amounts of clinical grade vector are needed Transient transfection of mammalian cells with a plasmid cocktail containing the necessary vector components and transgene is a proven method of obtaining high titer lentiviral vector Advantages include time/cost savings by eliminating the tedious process of developing packaging cell lines and the ability to quickly utilize newly characterized pseudotypes Because transient lentivirus production on a large scale requires the cumbersome task of manipulating numerous cell culture vessels (i.e cell factories), we explored methods to simplify the process One possibility is the use of microcarriers as a cell growth substrate to replace the surface area of multiple culture vessels and downsize to a single vessel or bioreactor To investigate this possibility, Hillex II microcarriers were added to small shaker flasks such that 100 cm2 of growth surface was present Initial cell seeding experiments of HEK 293T cells were performed to determine the cell count and seeding time required to obtain 70-90% microcarrier surface coverage with less than 10% unbound cells The optimum conditions were found to be 6.25 x 106 cells in mL total volume, a seeding time of three hours, and gentle mixing every 45-60 minutes Cells were transfected with a plasmid cocktail, including vector plasmids and a GFP transgene plasmid, using jetPEI in concentrations consistent with those successfully used in static culture Infectious titer, as measured by GFP expression, averaged 5.7 x 106 IU/mL When scaled up to a 500 mL wave bioreactor, Hillex II microcarriers tended to clump without strong agitation - which in turn caused cell detachment We next compared lentivirus production with Hillex II (specific gravity = 1.11) to a lower density microcarrier, ProNectin F (specific gravity = 1.02), in shaker flasks Infectious titers were similar at 2.3 x 106 and 2.9 x 106 , respectively Additionally, the inclusion of 5% ficoll in the media was evaluated to increase the suspension efficiency of ProNectin F microcarriers This would allow for less harsh agitation conditions in wave bioreactor productions Infectious titers from cells in 5% ficoll were comparable to those without ficoll (3.2 x 106 and 2.9 x 106 IU/mL, respectively) Current work involves scaling up harvest volumes in a wave bioreactor using ProNectin F microcarriers and media containing 5% ficoll Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy 47 Rapid Generation of Genetically-Modified Primary T Cells and Antigen-Specific CTL for Preclinical and Clinical Applications Using a Novel Cell Bioreactor Juan F Vera,1 Lara Brenner,1 Ann M Leen,1 Minhtran C Ngo,1 John Wilson,2 Helen E Heslop,1 Gianpietro Dotti,1 Cliona M Rooney.1 Baylor College of Medicine, Texas Children’s Hospital, The Methodist Hospital, Houston; 2Wilson Wolf Manufacturing, New Brighton, MN The generation of genetically-modified primary T cells and antigenspecific cytotoxic T lymphocytes (CTL) for adoptive transfer is time-consuming, requiring 4-12wks to produce sufficient cells for therapeutic purposes, and expensive (media + plastics + cytokines + man hours), impeding the broader clinical application of T cell therapy T cell growth is limited by gas exchange, nutrients, and waste accumulation Bioreactors developed to overcome these limitations tend to be complex, involving mechanical rocking or stirring and continuous perfusion, which increases the expense and limits the number of products to the number of mechanical devices that can be housed and maintained We have now explored the use of a new static gas permeable (GP) Cell Bioreactor for T cell expansion This device is essentially a flask with a gas permeable membrane supported by a plastic lattice as its base The O2/CO2 exchange from the base allows large volumes of media to be added thereby reducing nutrient limitations and waste build-up, and consequently the manipulation required to sustain cell expansion We tested sizes, 10cm2 (GP40) and 100cm2 (GP2000) that hold a maximum of 40mL and 2000mL of media, respectively We first evaluated the expansion of geneticallymodified OKT3-stimulated T cells engrafted with a chimeric T cell receptor targeting the Kappa light chain of the B cell The cultured cells required minimal manipulation, with a single media change per week, while those cultured in 24-well plates required manipulation every 2-3 days (media replenishment and reseeding) to sustain optimal expansion Using the GP bioreactor we observed a 54.6 fold T cell expansion within days (range 37.8-64.6) vs 15.3 fold (range 11.8-29.2) Evaluation of the cells cultured using both systems revealed less apoptosis in the bioreactor-expanded cells, suggesting that the increased cell numbers resulted not from an increased rate of proliferation but from a cumulative increase in viable cells Importantly T cell phenotype and function was preserved Cytolytic activity evaluated by Cr51 release showed strong killing of a Kappa+ B cell tumor (Daudi) (99±5%vs89±2%, Bioreactor vs plate) with low killing of an irrelevant target, K562 (15±15%vs10±6%) Using a single GP2000 we could produce up to 8E+08 genetically-modified T cells, which would have required approximately 320 wells (>13 plates) We also evaluated the expansion of Epstein-Barr virus (EBV) CTL by coculturing APCs with established EBV-CTL at an optimized cell density These culture conditions enhanced CTL expansion (42.5 fold ±14.8 vs 3.4 fold ±1.2 within days) without requiring media change Their phenotype and antigen specificity, as evaluated by tetramer and IFN-γ ELIspot, was similar to conventional CTLs In summary, we have successfully utilized GP Cell Bioreactor technology to induce optimal in vitro T cell expansion with minimal handling We have also demonstrated that this system is suited to the clinical grade expansion of other cell types S19 ... was about 40-50% The WAS LV batch produced at Genethon was therefore conform and released for clinical use In conclusion, the WAS project has permitted the implementation of large-scale GMP production, ... of Gene Therapy 47 Rapid Generation of Genetically-Modified Primary T Cells and Antigen-Specific CTL for Preclinical and Clinical Applications Using a Novel Cell Bioreactor Juan F Vera,1 Lara... amounts of clinical grade vector are needed Transient transfection of mammalian cells with a plasmid cocktail containing the necessary vector components and transgene is a proven method of obtaining