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• W2991496875 abstract "Lentiviral vectors are increasingly utilized in cell and gene therapy applications because they efficiently transduce target cells such as hematopoietic stem cells and T cells. Large-scale production of current Good Manufacturing Practices-grade lentiviral vectors is limited because of the adherent, serum-dependent nature of HEK293T cells used in the manufacturing process. To optimize large-scale clinical-grade lentiviral vector production, we developed an improved production scheme by adapting HEK293T cells to grow in suspension using commercially available and chemically defined serum-free media. Lentiviral vectors with titers equivalent to those of HEK293T cells were produced from SJ293TS cells using optimized transfection conditions that reduced the required amount of plasmid DNA by 50%. Furthermore, purification of SJ293TS-derived lentiviral vectors at 1 L yielded a recovery of 55% ± 14% (n = 138) of transducing units in the starting material, more than a 2-fold increase over historical yields from adherent HEK293T serum-dependent lentiviral vector preparations. SJ293TS cells were stable to produce lentiviral vectors over 4 months of continuous culture. SJ293TS-derived lentiviral vectors efficiently transduced primary hematopoietic stem cells and T cells from healthy donors. Overall, our SJ293TS cell line enables high-titer vector production in serum-free conditions while reducing the amount of input DNA required, resulting in a highly efficient manufacturing option. Lentiviral vectors are increasingly utilized in cell and gene therapy applications because they efficiently transduce target cells such as hematopoietic stem cells and T cells. Large-scale production of current Good Manufacturing Practices-grade lentiviral vectors is limited because of the adherent, serum-dependent nature of HEK293T cells used in the manufacturing process. To optimize large-scale clinical-grade lentiviral vector production, we developed an improved production scheme by adapting HEK293T cells to grow in suspension using commercially available and chemically defined serum-free media. Lentiviral vectors with titers equivalent to those of HEK293T cells were produced from SJ293TS cells using optimized transfection conditions that reduced the required amount of plasmid DNA by 50%. Furthermore, purification of SJ293TS-derived lentiviral vectors at 1 L yielded a recovery of 55% ± 14% (n = 138) of transducing units in the starting material, more than a 2-fold increase over historical yields from adherent HEK293T serum-dependent lentiviral vector preparations. SJ293TS cells were stable to produce lentiviral vectors over 4 months of continuous culture. SJ293TS-derived lentiviral vectors efficiently transduced primary hematopoietic stem cells and T cells from healthy donors. Overall, our SJ293TS cell line enables high-titer vector production in serum-free conditions while reducing the amount of input DNA required, resulting in a highly efficient manufacturing option. HIV-1-derived lentiviral vectors (LVs) are efficient gene transfer vehicles used in both basic and clinical research settings. The ability of LVs to efficiently shuttle DNA into mammalian cells enables researchers to explore the function of various genes of interest.1Zheng Y. Yu F. Wu Y. Si L. Xu H. Zhang C. Xia Q. Xiao S. Wang Q. He Q. et al.Broadening the versatility of lentiviral vectors as a tool in nucleic acid research via genetic code expansion.Nucleic Acids Res. 2015; 43: e73Crossref PubMed Scopus (18) Google Scholar, 2Ranzani M. Annunziato S. Calabria A. Brasca S. Benedicenti F. Gallina P. Naldini L. Montini E. Lentiviral vector-based insertional mutagenesis identifies genes involved in the resistance to targeted anticancer therapies.Mol. Ther. 2014; 22: 2056-2068Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 3Ranzani M. Cesana D. Bartholomae C.C. Sanvito F. Pala M. Benedicenti F. Gallina P. Sergi L.S. Merella S. Bulfone A. et al.Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer.Nat. Methods. 2013; 10: 155-161Crossref PubMed Scopus (57) Google Scholar, 4Mak A.B. Moffat J. A versatile lentiviral expression system to identify mammalian protein-protein interactions.Methods. 2012; 57: 409-416Crossref PubMed Scopus (7) Google Scholar, 5Moffat J. Grueneberg D.A. Yang X. Kim S.Y. Kloepfer A.M. Hinkle G. Piqani B. Eisenhaure T.M. Luo B. Grenier J.K. et al.A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen.Cell. 2006; 124: 1283-1298Abstract Full Text Full Text PDF PubMed Scopus (1278) Google Scholar Clinically, LVs are used ex vivo to deliver therapeutic genes or expression cassettes to primary target cells, such as hematopoietic stem cells (HSCs), to treat genetic disorders or infectious diseases.6Naldini L. Genetic engineering of hematopoiesis: current stage of clinical translation and future perspectives.EMBO Mol. Med. 2019; 11: e9958Crossref PubMed Scopus (51) Google Scholar, 7Kumar S.R. Markusic D.M. Biswas M. High K.A. Herzog R.W. Clinical development of gene therapy: results and lessons from recent successes.Mol. Ther. Methods Clin. Dev. 2016; 3: 16034Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 8Levine B.L. Miskin J. Wonnacott K. Keir C. Global Manufacturing of CAR T Cell Therapy.Mol. Ther. Methods Clin. Dev. 2016; 4: 92-101Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 9Hu B. Tai A. Wang P. Immunization delivered by lentiviral vectors for cancer and infectious diseases.Immunol. Rev. 2011; 239: 45-61Crossref PubMed Scopus (61) Google Scholar LVs are well suited for use in ex vivo gene therapy because they can deliver a relatively large payload (>9 kb, including intron-containing genomes), have the ability to transduce dividing and non-dividing cells, and stably integrate into the genome of a target cell to provide lifelong correction to that cell and its progeny.10Naldini L. Blömer U. Gallay P. Ory D. Mulligan R. Gage F.H. Verma I.M. Trono D. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.Science. 1996; 272: 263-267Crossref PubMed Scopus (3868) Google Scholar, 11Kumar M. Keller B. Makalou N. Sutton R.E. Systematic determination of the packaging limit of lentiviral vectors.Hum. 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Andre K. Stein D. Blick G. Greenberg R.N. Kinder C. Zolopa A. Binder-Scholl G. et al.Patient monitoring and follow-up in lentiviral clinical trials.J. Gene Med. 2013; 15: 78-82Crossref PubMed Scopus (67) Google Scholar, 16Cesani M. Plati T. Lorioli L. Benedicenti F. Redaelli D. Dionisio F. Biasco L. Montini E. Naldini L. Biffi A. Shedding of clinical-grade lentiviral vectors is not detected in a gene therapy setting.Gene Ther. 2015; 22: 496-502Crossref PubMed Scopus (11) Google Scholar The promise of LV cell and gene therapy has led to the initialization of nearly 200 clinical trials, with notable successes in the treatment of hemoglobinopathies, primary immunodeficiencies, and leukemias.15McGarrity G.J. Hoyah G. Winemiller A. Andre K. Stein D. Blick G. Greenberg R.N. Kinder C. Zolopa A. Binder-Scholl G. et al.Patient monitoring and follow-up in lentiviral clinical trials.J. Gene Med. 2013; 15: 78-82Crossref PubMed Scopus (67) Google Scholar,17Maude S.L. Frey N. Shaw P.A. Aplenc R. Barrett D.M. Bunin N.J. Chew A. Gonzalez V.E. Zheng Z. Lacey S.F. et al.Chimeric antigen receptor T cells for sustained remissions in leukemia.N. Engl. J. Med. 2014; 371: 1507-1517Crossref PubMed Scopus (3079) Google Scholar, 18Porter D.L. Hwang W.T. Frey N.V. Lacey S.F. Shaw P.A. Loren A.W. Bagg A. Marcucci K.T. Shen A. Gonzalez V. et al.Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia.Sci. Transl. Med. 2015; 7: 303ra139Crossref PubMed Scopus (1012) Google Scholar, 19Cavazzana-Calvo M. Payen E. Negre O. Wang G. Hehir K. Fusil F. Down J. Denaro M. Brady T. Westerman K. et al.Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia.Nature. 2010; 467: 318-322Crossref PubMed Scopus (953) Google Scholar, 20De Ravin S.S. Wu X. Moir S. Anaya-O’Brien S. Kwatemaa N. Littel P. Theobald N. Choi U. Su L. 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Castiello M.C. et al.Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome.Science. 2013; 341: 1233151Crossref PubMed Scopus (746) Google Scholar, 24Ginn S.L. Amaya A.K. Alexander I.E. Edelstein M. Abedi M.R. Gene therapy clinical trials worldwide to 2017: An update.J. Gene Med. 2018; 20: e3015Crossref PubMed Scopus (373) Google Scholar, 25Mamcarz E. Zhou S. Lockey T. Abdelsamed H. Cross S.J. Kang G. Ma Z. Condori J. Dowdy J. Triplett B. et al.Lentiviral Gene Therapy Combined with Low-Dose Busulfan in Infants with SCID-X1.N. Engl. J. Med. 2019; 380: 1525-1534Crossref PubMed Scopus (98) Google Scholar In fact, LV-based cell and gene therapies have already been approved by the US Food and Drug Administration, Kymriah, and by the European Medicines Agency, Zynteglo. Furthermore, cancer immunotherapy, which currently includes the use of LV to modify T cell function, is an area of intense research. Thus, additional LV-based cellular therapies are likely to be approved in the future. However, the manufacturing of current Good Manufacturing Practices (cGMP)-grade LVs has been a challenge for early-phase clinical trials and is even more difficult for commercialization.26Milone M.C. O’Doherty U. Clinical use of lentiviral vectors.Leukemia. 2018; 32: 1529-1541Crossref PubMed Scopus (242) Google Scholar, 27Merten O.W. Hebben M. Bovolenta C. Production of lentiviral vectors.Mol. Ther. Methods Clin. Dev. 2016; 3: 16017Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 28McCarron A. Donnelley M. McIntyre C. Parsons D. Challenges of up-scaling lentivirus production and processing.J. Biotechnol. 2016; 240: 23-30Crossref PubMed Scopus (60) Google Scholar Thus, advances in industrial-scale vector manufacturing are required to meet current and future clinical needs for lentiviral-based cell and gene therapies. Research into the production and design of LVs for gene therapy has been ongoing for over 20 years, and although incremental advancements have been made, the current methodologies need optimization.29Zufferey R. Nagy D. Mandel R.J. Naldini L. Trono D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo.Nat. Biotechnol. 1997; 15: 871-875Crossref PubMed Scopus (1507) Google Scholar, 30Kim V.N. Mitrophanous K. Kingsman S.M. Kingsman A.J. Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1.J. Virol. 1998; 72: 811-816Crossref PubMed Google Scholar, 31Reiser J. Harmison G. Kluepfel-Stahl S. Brady R.O. Karlsson S. Schubert M. Transduction of nondividing cells using pseudotyped defective high-titer HIV type 1 particles.Proc. Natl. Acad. Sci. USA. 1996; 93: 15266-15271Crossref PubMed Scopus (288) Google Scholar, 32Kuroda H. Kutner R.H. Bazan N.G. Reiser J. Simplified lentivirus vector production in protein-free media using polyethylenimine-mediated transfection.J. Virol. Methods. 2009; 157: 113-121Crossref PubMed Scopus (73) Google Scholar Today, standard LVs are made using a third generation production system that relies on the transient transfection of HEK293T cells.33Dull T. Zufferey R. Kelly M. Mandel R.J. Nguyen M. Trono D. Naldini L. A third-generation lentivirus vector with a conditional packaging system.J. Virol. 1998; 72: 8463-8471Crossref PubMed Google Scholar This transient transfection method can generate high-titer LVs free of replication-competent lentivirus. However, because of the adherent and serum-dependent nature of the HEK293T cell line, cGMP LV production is limited due to the need to use multi-stack plastic tissue culture vessels, for example, 10-stack Cell Factory systems, which increases the amount of handling, personnel and consumables costs, and production time. Furthermore, the use of animal-derived serum to maintain the HEK293T cell line increases the risk for contamination by adventitious viruses. In addition, obtaining large enough lots of serum can often be challenging and costly to acquire. To address these challenges of industrial-scale cGMP-LV manufacturing, researchers have reported alternative means to produce LVs.27Merten O.W. Hebben M. Bovolenta C. Production of lentiviral vectors.Mol. Ther. Methods Clin. Dev. 2016; 3: 16017Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar,34Segura M.M. Mangion M. Gaillet B. Garnier A. New developments in lentiviral vector design, production and purification.Expert Opin. Biol. Ther. 2013; 13: 987-1011Crossref PubMed Scopus (73) Google Scholar Many strategies use HEK293-derived cells that are adapted to grow in suspension with serum-free media (SFM), fixed-bed bioreactors, and stable producer cell lines.35Segura M.M. Garnier A. Durocher Y. Coelho H. Kamen A. 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Mavilio F. Bordignon C. Rizzardi G.P. Bovolenta C. RD2-MolPack-Chim3, a packaging cell line for stable production of lentiviral vectors for anti-HIV gene therapy.Hum. Gene Ther. Methods. 2013; 24: 228-240Crossref PubMed Scopus (28) Google Scholar, 50Farson D. Witt R. McGuinness R. Dull T. Kelly M. Song J. Radeke R. Bukovsky A. Consiglio A. Naldini L. A new-generation stable inducible packaging cell line for lentiviral vectors.Hum. Gene Ther. 2001; 12: 981-997Crossref PubMed Scopus (117) Google Scholar, 51Ni Y. Sun S. Oparaocha I. Humeau L. Davis B. Cohen R. Binder G. Chang Y.N. Slepushkin V. Dropulic B. Generation of a packaging cell line for prolonged large-scale production of high-titer HIV-1-based lentiviral vector.J. Gene Med. 2005; 7: 818-834Crossref PubMed Scopus (68) Google Scholar, 52Klages N. Zufferey R. Trono D. A stable system for the high-titer production of multiply attenuated lentiviral vectors.Mol. Ther. 2000; 2: 170-176Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar However, these novel production schemes have been problematic for large-scale cGMP production for several reasons, including multiple handling steps, reduced titers, a limited production scale, continued use of tissue culture flasks, and reliance on animal-derived serum. To address these concerns, we adapted a HEK293T cell line to grow in suspension using a commercially available SFM. This newly derived cell line, SJ293TS, maintains the growth characteristics and LV productivity of the parental HEK293T cell line. We further improved the system’s fitness for cGMP-manufacture of LVs by simplifying the cell manipulation steps in the manufacturing protocol and introduced an antibiotic-free (AF) selection marker on all transfected plasmids. We show that this new system produces equivalent amounts of vesicular stomatitis virus envelope glycoprotein (VSV-G)-pseudotyped LVs as are produced by conventional methods using adherent, serum-dependent HEK293T cells. Moreover, the SJ293TS system produces at least 10-fold higher LV titers than that provided by other suspension-adapted HEK293 cells, specifically Viral Production Cells and Expi293F cells. Importantly, we achieve a more than 2-fold increase in the recovery of LV transducing units (TUs) after downstream purification compared with historical data from HEK293T producer cells.53Greene M.R. Lockey T. Mehta P.K. Kim Y.S. Eldridge P.W. Gray J.T. Sorrentino B.P. Transduction of human CD34+ repopulating cells with a self-inactivating lentiviral vector for SCID-X1 produced at clinical scale by a stable cell line.Hum. Gene Ther. Methods. 2012; 23: 297-308Crossref PubMed Scopus (33) Google Scholar, 54Merten O.W. Charrier S. Laroudie N. Fauchille S. Dugué C. Jenny C. Audit M. Zanta-Boussif M.A. Chautard H. Radrizzani M. et al.Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application.Hum. Gene Ther. 2011; 22: 343-356Crossref PubMed Scopus (119) Google Scholar, 55Bandeira V. Peixoto C. Rodrigues A.F. Cruz P.E. Alves P.M. Coroadinha A.S. Carrondo M.J. Downstream processing of lentiviral vectors: releasing bottlenecks.Hum. Gene Ther. Methods. 2012; 23: 255-263Crossref PubMed Scopus (42) Google Scholar Finally, we demonstrate that LVs produced from SJ293TS cells can efficiently transduce human cells including T cells and CD34+ cells. In order to convert the adherent HEK293T cell line to grow in suspension, we employed a commercially available SFM, Freestyle 293 Expression media (FS293). The cells were adapted by replacing the standard growth media, DMEM plus 10% fetal bovine serum (FBS) (D10), with FS293 supplemented with 10% FBS and seeded onto both non-tissue culture-treated and tissue culture-treated plates. Gradually, we reduced the amount of serum in the media at 1-week time intervals, and cells were passaged three times per week. After 4 weeks, the cells were growing solely in the presence of FS293. Cells that were successfully adapted directly using non-tissue culture-treated plates appeared to be less aggregated than those grown on tissue culture-treated plates; however, both approaches were ultimately successful in adapting HEK293T cells to grow in suspension without serum. The final adapted cell line, designated as SJ293TS, was then cultured in a shaking incubator to make a master cell bank. The cells were thawed, and their growth rate was determined by seeding the cells at 5 × 105 cells/mL followed by counting every 24 h for 2 days (Figure S1). SJ293TS cells doubled every 23.4 ± 2.7 h and reached a cell density of up to 3 × 106 cells/mL without compromising cell viability. For regular maintenance of the SJ293TS cell line, cultures were kept between 3 × 105 and 3 × 106 cells/mL and passaged every 2–3 days. We assessed vector production using SJ293TS cells via our previously established transient transfection method.42Throm R.E. Ouma A.A. Zhou S. Chandrasekaran A. Lockey T. Greene M. De Ravin S.S. Moayeri M. Malech H.L. Sorrentino B.P. Gray J.T. Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection.Blood. 2009; 113: 5104-5110Crossref PubMed Scopus (92) Google Scholar,56Hanawa H. Persons D.A. Nienhuis A.W. Mobilization and mechanism of transcription of integrated self-inactivating lentiviral vectors.J. Virol. 2005; 79: 8410-8421Crossref PubMed Scopus (48) Google Scholar Initially, we aimed to optimize transfection conditions using a simple GFP-expressing LV (vector CL40-MND-GFP) in 20-mL cultures in 125-mL shaker flasks for quick and easy readout. First, we tested the effect of cell density at the time of transfection. Based on our previous experience with adherent HEK293T cells as a starting point, where cell density is approximately 1 × 106 cells/mL, we transfected SJ293TS cells at various densities ranging from 0.5 to 3 × 106 cells/mL at two different concentrations of plasmid DNA, 0.55 and 1.1 μg DNA per 106 cells. Twenty-four hours post transfection, the cells were pelleted by centrifugation and suspended in 20 mL of fresh media. Supernatants were collected 48 h post transfection and titered on HOS cells. Although LV production did not differ significantly among cells transfected at 1.1 μg DNA per 106 cells, LV output using SJ293TS cells transfected with 0.55 μg DNA per 106 cells was significantly impacted by seeding density, with the 2 × 106 cells/mL density achieving the highest titers (Figure 1). Using a cell density fixed at 2 × 106 cells/mL, we found that 0.55 μg DNA/mL was optimal for achieving the highest titer yields (Figure S2). Next, we examined the timing for optimal polyplex formation between polyethylenimine (PEI) and plasmid DNA under the conditions described in Materials and Methods. A single master transfection mixture was made and incubated for up to 30 min. At various time points, an aliquot was taken from the transfection master mixture and used to transfect SJ293TS cells in a 20-mL culture. Although no significant differences were observed, peak vector production occurred when the incubation continued for 5 min, whereas longer incubation periods tended to decrease titer (Figure S3). Thus, all subsequent transfections of SJ293TS cells were performed after a 5-min incubation of PEI and plasmid DNA. To evaluate LV production in the suspension-adapted SJ293TS cell line, we compared 1-L productions of SJ293TS cells in 5-L shaker flasks against the parent HEK293T cells in 10-stack Cell Factory systems. For this comparison, three different clinically relevant LVs were produced: two containing shRNAmiR against human BCL11A with fluorophores (vectors SJL118 and SJL121) and the other with an expression cassette for a second generation anti-CD19 chimeric antigen receptor (vector αCD19-CAR). Media were replaced 24 h post transfection as described above, and vector was harvested 24 h later. HEK293T titers were 1.8-, 1.4-, and 2-fold higher than SJ293TS cells (Table 1).Table 1Titers of Either HEK293T or SJ293TS Cell-Derived Lentiviral Vectors Produced at 1-L ScaleProducer Cell Line in Culture VesselVectorTUs/mLaOne liter total volume, unprocessed supernatant titer.HEK293T cells in Cell FactorySJL1181.5 × 108SJ293TS cells in 5-L shaker flaskSJL1188.2 × 107HEK293T cells in Cell FactorySJL1215.3 × 108SJ293TS cells in 5-L shaker flaskSJL1213.7 × 108HEK293T cells in Cell Factory Prep 1αCD19-CAR2.00 × 108HEK293T cells in Cell Factory Prep 2αCD19-CAR2.00 × 108SJ293TS cells in 5-L shaker flaskαCD19-CAR1.00 × 108a One liter total volume, unprocessed supernatant titer. Open table in a new tab There are few commercially available suspension HEK293 cell lines. We compared the SJ293TS cell line against two different HEK293-based suspension cell lines commercially available, namely, Expi293F cells and Viral Production Cells. All three cell lines were transfected, at the 20-mL scale, to produce CL40-MND-GFP. A master polyplex was formed, and an equal amount was distributed to each flask. In addition to GFP-LV, a more complex LV production, V5m3-Sardinia expressing the human γ-globin gene, was compared in a similar setting. The SJ293TS cell line produced 10- to 25-fold more TUs than either Viral Production Cells or Expi293F cells, regardless of vector. Statistical significance was reached only with the GFP-expressing vector, with p values of 0.0252 and 0.0239 in comparison with Viral Production Cells and Expi293F cells, respectively (Figure 2). In order to improve the productivity and suitability of the SJ293TS cell line for cGMP manufacturing, we made modifications to the LV production process. Because there is growing concern using antibiotics for plasmid manufacturing, we looked for an alternative. An AF selection strategy, RNA-OUT, has been developed in which an approximately 150-bp cassette expresses a small antisense RNA against a counter-selectable marker expressed by the specialized bacterial host cell.57Luke J. Carnes A.E. Hodgson C.P. Williams J.A. Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system.Vaccine. 2009; 27: 6454-6459Crossref PubMed Scopus (69) Google Scholar We replaced the β-lactamase expression cassette on our helper and transfer plasmids with RNA-OUT, which has the added benefit of reducing the size of the plasmid backbone. We also reduced the posttransfection cell culture handling by diluting transfected cells with an equal volume of fresh media rather than performing a full media exchange. In the context of these changes, we also improved vector production by optimizing plasmid ratios used to transiently transfect SJ293TS cells. We found that a ratio of 14:4:2:0.25 of transfer vector:HIV-1 gagpol:Env:HIV-1 rev, compared with our previously used standard plasmid ratio of 12:6:2:0.25, led to a 1.7-fold increase in the titer of a pre-clinical LV (vector SJL644), which expresses a second generation anti-CD123 chimeric antigen receptor (p > 0.05) and a reduction in the amount of HIV-1 p24 present in harvested supernatants (p > 0.05) (Figures S4A and S4B). Inte" @default.
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