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1
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Chen H, Charles PD, Gu Q, Liberatori S, Robertson DL, Palmarini M, Wilson SJ, Mohammed S, Castello A. Omics Analyses Uncover Host Networks Defining Virus-Permissive and -Hostile Cellular States. Mol Cell Proteomics 2025; 24:100966. [PMID: 40204275 DOI: 10.1016/j.mcpro.2025.100966] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2024] [Revised: 03/24/2025] [Accepted: 04/04/2025] [Indexed: 04/11/2025] Open
Abstract
The capacity of host cells to sustain or restrict virus infection is influenced by their proteome. Understanding the compendium of proteins defining cellular permissiveness is key to many questions in fundamental virology. Here, we apply a multi-omic approach to determine the proteins that are associated with highly permissive, intermediate, and hostile cellular states. We observed two groups of differentially regulated genes: (i) with robust changes in mRNA and protein levels and (ii) with protein/RNA discordances. While many of the latter are classified as interferon-stimulated genes (ISGs), most exhibit no antiviral effects in overexpression screens. This suggests that IFN-dependent protein changes can be better indicators of antiviral function than mRNA levels. Phosphoproteomics revealed an additional regulatory layer involving non-signaling proteins with altered phosphorylation. Indeed, we confirmed that several permissiveness-associated proteins with changes in abundance or phosphorylation regulate infection fitness. Altogether, our study provides a comprehensive and systematic map of the cellular alterations driving virus susceptibility.
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Affiliation(s)
- Honglin Chen
- MRC-University of Glasgow Centre for Virus Research, Glasgow, UK; Department of Biochemistry, University of Oxford, Oxford, UK
| | | | - Quan Gu
- MRC-University of Glasgow Centre for Virus Research, Glasgow, UK
| | | | | | | | - Sam J Wilson
- Cambridge Institute of Therapeutic Immunol & Infect Disease, Jeffrey Cheah Biomedical Centre, Cambridge, UK
| | - Shabaz Mohammed
- Department of Biochemistry, University of Oxford, Oxford, UK; The Rosalind Franklin Institute, Oxfordshire, UK; Department of Chemistry, University of Oxford, Oxford, UK.
| | - Alfredo Castello
- MRC-University of Glasgow Centre for Virus Research, Glasgow, UK.
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2
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Mormin M, Rigonnot L, Chalumeau A, Miccio A, Fournier C, Pajanissamy S, Dewannieux M, Galy A. Cyclosporin H Improves the Transduction of CD34 + Cells with an Anti-Sickling Globin Vector, a Possible Therapeutic Approach for Sickle Cell Disease. Hum Gene Ther 2024; 35:896-903. [PMID: 39504955 DOI: 10.1089/hum.2024.098] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2024] Open
Abstract
Sickle cell disease (SCD) is a debilitating monogenic disease originating from mutations in the hemoglobin beta chain gene producing an abnormal hemoglobin HbS. The polymerization of HbS is responsible for the sickling of erythrocytes leading to anemia and vaso-occlusive events. Gene therapy is a promising treatment of SCD, and two different gene therapy drugs, using gene editing or gene transfer, have already reached the marketing stage. There is still a need to improve the efficacy of gene therapy in SCD, particularly when using anti-sickling beta-globin gene transfer strategies, which must outcompete the pathological HbS. One possibility is to increase transduction by inhibiting lentiviral restriction factors such as interferon-induced transmembrane proteins (IFITMs). This can be achieved by the addition of cyclosporin H (CsH) during the transduction process. This strategy was applied here in CD34+ hematopoietic progenitor and stem cells obtained from cord blood (CB). A first series of experiments with lentiviral vector coding for a green fluorescent protein (GFP) gene confirmed that the addition of CsH enhanced transgene expression levels and vector copy number per cell (VCN), while CD34+ cells remained viable and functional. Notably, the production of colony-forming cells (CFC) remained unaffected unless very high VCN values were reached. In a second step, CD34+ cells obtained from the CB of newborns with homozygous (n = 2) or heterozygous (n = 1) SCD mutations were transduced with the GLOBE-AS3 lentiviral vector coding for the HbAS3 anti-sickling beta globin. As with GFP, GLOBE-AS3 lentiviral transduction was clearly enhanced by CsH, leading to VCN > 2 and therapeutic levels of expression of the HbAS3. Moreover, the process did not affect the viability or functions of CFC. The combination of CB progenitors, the GLOBE-AS3 vector, and CsH is thus shown here to be a promising approach for the treatment of SCD.
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Affiliation(s)
| | - Luc Rigonnot
- Maternity/Obstetrics Unit, Centre Hospitalier Sud-Francilien, Corbeil-Essonnes, France
| | - Anne Chalumeau
- Institute Imagine, Inserm, Hopital Necker-Enfants Malades, Paris, France
| | - Annarita Miccio
- Institute Imagine, Inserm, Hopital Necker-Enfants Malades, Paris, France
| | | | | | | | - Anne Galy
- ART-TG, Inserm US35, Corbeil-Essonnes, France
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3
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Kontogiannis T, Braybrook J, McElroy C, Foy C, Whale AS, Quaglia M, Smales CM. Characterization of AAV vectors: A review of analytical techniques and critical quality attributes. Mol Ther Methods Clin Dev 2024; 32:101309. [PMID: 39234444 PMCID: PMC11372808 DOI: 10.1016/j.omtm.2024.101309] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/06/2024]
Abstract
Standardized evaluation of adeno-associated virus (AAV) vector products for biotherapeutic application is essential to ensure the safety and efficacy of gene therapies. This includes analyzing the critical quality attributes of the product. However, many of the current analytical techniques used to assess these attributes have limitations, including low throughput, large sample requirements, poorly understood measurement variability, and lack of comparability between methods. To address these challenges, it is essential to establish higher-order reference methods that can be used for comparability measurements, optimization of current assays, and development of reference materials. Highly precise methods are necessary for measuring the empty/partial/full capsid ratios and the titer of AAV vectors. Additionally, it is important to develop methods for the measurement of less-established critical quality attributes, including post-translational modifications, capsid stoichiometry, and methylation profiles. By doing so, we can gain a better understanding of the influence of these attributes on the quality of the product. Moreover, quantification of impurities, such as host-cell proteins and DNA contaminants, is crucial for obtaining regulatory approval. The development and application of refined methodologies will be essential to thoroughly characterize AAV vectors by informing process development and facilitating the generation of reference materials for assay validation and calibration.
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Affiliation(s)
- Theodoros Kontogiannis
- School of Biosciences, Division of Natural Sciences, University of Kent, Canterbury, Kent CT2 7NJ, UK
- National Measurement Laboratory at LGC, Teddington, Middlesex TW11 0LY, UK
| | - Julian Braybrook
- National Measurement Laboratory at LGC, Teddington, Middlesex TW11 0LY, UK
| | | | - Carole Foy
- National Measurement Laboratory at LGC, Teddington, Middlesex TW11 0LY, UK
| | - Alexandra S Whale
- National Measurement Laboratory at LGC, Teddington, Middlesex TW11 0LY, UK
| | - Milena Quaglia
- Reading Scientific Services Ltd, Reading Science Centre, Whiteknights Campus, Pepper Lane, Reading Berkshire RG6 6LA, UK
| | - C Mark Smales
- School of Biosciences, Division of Natural Sciences, University of Kent, Canterbury, Kent CT2 7NJ, UK
- National Institute for Bioprocessing Research and Training, Blackrock, Co, Foster Avenue, A94 X099 Mount Merrion, Dublin, Ireland
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4
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Luostarinen A, Kailaanmäki A, Turkki V, Köylijärvi M, Käyhty P, Leinonen H, Albers-Skirdenko V, Lipponen E, Ylä-Herttuala S, Kaartinen T, Lesch HP, Kekarainen T. Optimizing lentiviral vector formulation conditions for efficient ex vivo transduction of primary human T cells in chimeric antigen receptor T-cell manufacturing. Cytotherapy 2024; 26:1084-1094. [PMID: 38661611 DOI: 10.1016/j.jcyt.2024.04.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2023] [Revised: 03/10/2024] [Accepted: 04/03/2024] [Indexed: 04/26/2024]
Abstract
BACKGROUND AIMS Chimeric antigen receptor (CAR) T-cell products are commonly generated using lentiviral vector (LV) transduction. Optimal final formulation buffer (FFB) supporting LV stability during cryostorage is crucial for cost-effective manufacturing. METHODS To identify the ideal LV FFB composition for ex vivo CAR-T production, primary human T cells were transduced with vesicular stomatitis virus G-protein (VSV-G) -pseudotyped LVs (encoding a reporter gene or an anti-CD19-CAR). The formulations included phosphate-buffered saline (PBS), HEPES, or X-VIVOTM 15, and stabilizing excipients. The functional and viral particle titers and vector copy number were measured after LV cryopreservation and up to 24 h post-thaw incubation. CAR-Ts were produced with LVs in selected FFBs, and the resulting cells were characterized. RESULTS Post-cryopreservation, HEPES-based FFBs provided higher LV functional titers than PBS and X-VIVOTM 15, and 10% trehalose-20 mM MgCl2 improved LV transduction efficiency in PBS and 50 mM HEPES. Thawed vectors remained stable at +4°C, while a ≤ 25% median decrease in the functional titer occurred during 24 h at room temperature. Tested excipients did not enhance LV post-thaw stability. CAR-Ts produced using LVs cryopreserved in 10% trehalose- or sucrose-20 mM MgCl2 in 50 mM HEPES showed comparable transduction rates, cell yield, viability, phenotype, and in vitro functionality. CONCLUSION A buffer consisting of 10% trehalose-20 mM MgCl2 in 50 mM HEPES provided a feasible FFB to cryopreserve a VSV-G -pseudotyped LV for CAR-T-cell production. The LVs remained relatively stable for at least 24 h post-thaw, even at ambient temperatures. This study provides insights into process development, showing LV formulation data generated using the relevant target cell type for CAR-T-cell manufacturing.
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Affiliation(s)
- Annu Luostarinen
- Advanced Cell Therapy Centre, Finnish Red Cross Blood Service, Helsinki, Finland.
| | | | - Vesa Turkki
- Kuopio Center for Gene and Cell Therapy, Kuopio, Finland
| | | | - Piia Käyhty
- Kuopio Center for Gene and Cell Therapy, Kuopio, Finland
| | - Hanna Leinonen
- Kuopio Center for Gene and Cell Therapy, Kuopio, Finland
| | | | - Eevi Lipponen
- Kuopio Center for Gene and Cell Therapy, Kuopio, Finland
| | - Seppo Ylä-Herttuala
- Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland
| | - Tanja Kaartinen
- Advanced Cell Therapy Centre, Finnish Red Cross Blood Service, Helsinki, Finland
| | - Hanna P Lesch
- Kuopio Center for Gene and Cell Therapy, Kuopio, Finland
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5
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Hengelbrock A, Probst F, Baukmann S, Uhl A, Tschorn N, Stitz J, Schmidt A, Strube J. Digital Twin for Continuous Production of Virus-like Particles toward Autonomous Operation. ACS OMEGA 2024; 9:34990-35013. [PMID: 39157157 PMCID: PMC11325504 DOI: 10.1021/acsomega.4c04985] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 05/27/2024] [Revised: 07/05/2024] [Accepted: 07/12/2024] [Indexed: 08/20/2024]
Abstract
Lentiviral vector and virus-like particle (VLP) manufacturing have been published in fed-batch upstream and batch downstream modes before. Batch downstream and continuous upstream in perfusion mode were reported as well. This study exemplifies development and validation steps for a digital twin combining a physical-chemical-based mechanistic model for all unit operations with a process analytical technology strategy in order to show the efforts and benefits of autonomous operation approaches for manufacturing scale. As the general models are available from various other biologic manufacturing studies, the main step is model calibration for the human embryo kidney cell-based VLPs with experimental quantitative validation within the Quality-by-Design (QbD) approach, including risk assessment to define design and control space. For continuous operation in perfusion mode, the main challenge is the efficient separation of large particle manifolds for VLPs and cells, including cell debris, which is of similar size. Here, innovative tangential flow filtration operations are needed to avoid fast blocking with low mechanical stress pumps. A twofold increase of productivity was achieved using simulation case studies. This increase is similar to improvements previously described for other entities like plasmid DNAs, monoclonal antibodies (mAbs), and single-chain fragments of variability (scFv) fragments. The advantages of applying a digital twin for an advanced process control strategy have proven additional productivity gains of 20% at 99.9% reliability.
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Affiliation(s)
- Alina Hengelbrock
- Institute
for Separation and Process Technology, Clausthal
University of Technology, Clausthal 38678, Zellerfeld, Germany
| | - Finja Probst
- Institute
for Separation and Process Technology, Clausthal
University of Technology, Clausthal 38678, Zellerfeld, Germany
| | - Simon Baukmann
- Institute
for Separation and Process Technology, Clausthal
University of Technology, Clausthal 38678, Zellerfeld, Germany
| | - Alexander Uhl
- Institute
for Separation and Process Technology, Clausthal
University of Technology, Clausthal 38678, Zellerfeld, Germany
| | - Natalie Tschorn
- Faculty
of Applied Natural Sciences, Technische
Hochschule Köln, Leverkusen 51379, Germany
| | - Jörn Stitz
- Faculty
of Applied Natural Sciences, Technische
Hochschule Köln, Leverkusen 51379, Germany
| | - Axel Schmidt
- Institute
for Separation and Process Technology, Clausthal
University of Technology, Clausthal 38678, Zellerfeld, Germany
| | - Jochen Strube
- Institute
for Separation and Process Technology, Clausthal
University of Technology, Clausthal 38678, Zellerfeld, Germany
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6
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Stibbs DJ, Silva Couto P, Takeuchi Y, Rafiq QA, Jackson NB, Rayat AC. Quasi-perfusion studies for intensified lentiviral vector production using a continuous stable producer cell line. Mol Ther Methods Clin Dev 2024; 32:101264. [PMID: 38827249 PMCID: PMC11141457 DOI: 10.1016/j.omtm.2024.101264] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2024] [Accepted: 05/03/2024] [Indexed: 06/04/2024]
Abstract
Quasi-perfusion culture was employed to intensify lentiviral vector (LV) manufacturing using a continuous stable producer cell line in an 8-day process. Initial studies aimed to identify a scalable seeding density, with 3, 4, and 5 × 104 cells cm-2 providing similar specific productivities of infectious LV. Seeding at 3 × 104 cells cm-2 was selected, and the quasi-perfusion was modulated to minimize inhibitory metabolite accumulation and vector exposure at 37°C. Similar specific productivities of infectious LV and physical LV were achieved at 1, 2, and 3 vessel volumes per day (VVD), with 1 VVD selected to minimize downstream processing volumes. The optimized process was scaled 50-fold to 1,264 cm2 flasks, achieving similar LV titers. However, scaling up beyond this to a 6,320 cm2 multilayer flask reduced titers, possibly from suboptimal gas exchange. Across three independent processes in 25 cm2 to 6,320 cm2 flasks, reproducibility was high with a coefficient of variation of 7.7% ± 2.9% and 11.9% ± 3.0% for infectious and physical LV titers, respectively. The optimized flask process was successfully transferred to the iCELLis Nano (Cytiva) fixed-bed bioreactor, with quasi-perfusion at 1 VVD yielding 1.62 × 108 TU.
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Affiliation(s)
- Dale J. Stibbs
- Department of Biochemical Engineering, University College London, Bernard Katz Building, Gower Street, London WC1E 6BT, UK
| | - Pedro Silva Couto
- Department of Biochemical Engineering, University College London, Bernard Katz Building, Gower Street, London WC1E 6BT, UK
| | - Yasuhiro Takeuchi
- Division of Infection and Immunity, University College London, Cruciform Building, Gower Street, London WC1E 6BT, UK
- Biotherapeutics and Advanced Therapies, Scientific Research and Innovation, Medicines and Healthcare products Regulatory Agency, South Mimms EN6 3QC, Potters Bar, UK
| | - Qasim A. Rafiq
- Department of Biochemical Engineering, University College London, Bernard Katz Building, Gower Street, London WC1E 6BT, UK
| | - Nigel B. Jackson
- Cytiva, 5 Harbourgate Business Park, Southampton Road, Portsmouth PO6 4BQ, UK
| | - Andrea C.M.E. Rayat
- Department of Biochemical Engineering, University College London, Bernard Katz Building, Gower Street, London WC1E 6BT, UK
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7
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Stibbs DJ, Silva Couto P, Takeuchi Y, Rafiq QA, Jackson NB, Rayat AC. Continuous manufacturing of lentiviral vectors using a stable producer cell line in a fixed-bed bioreactor. Mol Ther Methods Clin Dev 2024; 32:101209. [PMID: 38435128 PMCID: PMC10907162 DOI: 10.1016/j.omtm.2024.101209] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2023] [Accepted: 02/07/2024] [Indexed: 03/05/2024]
Abstract
Continuous manufacturing of lentiviral vectors (LVs) using stable producer cell lines could extend production periods, improve batch-to-batch reproducibility, and eliminate costly plasmid DNA and transfection reagents. A continuous process was established by expanding cells constitutively expressing third-generation LVs in the iCELLis Nano fixed-bed bioreactor. Fixed-bed bioreactors provide scalable expansion of adherent cells and enable a straightforward transition from traditional surface-based culture vessels. At 0.5 vessel volume per day (VVD), the short half-life of LVs resulted in a low total infectious titer at 1.36 × 104 TU cm-2. Higher perfusion rates increased titers, peaking at 7.87 × 104 TU cm-2 at 1.5 VVD. The supernatant at 0.5 VVD had a physical-to-infectious particle ratio of 659, whereas this was 166 ± 15 at 1, 1.5, and 2 VVD. Reducing the pH from 7.20 to 6.85 at 1.5 VVD improved the total infectious yield to 9.10 × 104 TU cm-2. Three independent runs at 1.5 VVD and a culture pH of 6.85 showed low batch-to-batch variability, with a coefficient of variation of 6.4% and 10.0% for total infectious and physical LV yield, respectively. This study demonstrated the manufacture of high-quality LV supernatant using a stable producer cell line that does not require induction.
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Affiliation(s)
- Dale J. Stibbs
- Department of Biochemical Engineering, University College London, Bernard Katz Building, Gower Street, London WC1E 6BT, UK
| | - Pedro Silva Couto
- Department of Biochemical Engineering, University College London, Bernard Katz Building, Gower Street, London WC1E 6BT, UK
| | - Yasuhiro Takeuchi
- Division of Infection and Immunity, University College London, Cruciform Building, Gower Street, London WC1E 6BT, UK
- Biotherapeutics and Advanced Therapies, Scientific Research and Innovation, Medicines and Healthcare Products Regulatory Agency, South Mimms, Potters Bar EN6 3QC, UK
| | - Qasim A. Rafiq
- Department of Biochemical Engineering, University College London, Bernard Katz Building, Gower Street, London WC1E 6BT, UK
| | - Nigel B. Jackson
- Cytiva, 5 Harbourgate Business Park, Southampton Road, Portsmouth PO6 4BQ, UK
| | - Andrea C.M.E. Rayat
- Department of Biochemical Engineering, University College London, Bernard Katz Building, Gower Street, London WC1E 6BT, UK
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8
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Howe G, Wasmuth M, Emanuelle P, Massaro G, Rahim AA, Ali S, Rivera M, Ward J, Keshavarz-Moore E, Mason C, Nesbeth DN. Engineering an Autonucleolytic Mammalian Suspension Host Cell Line to Reduce DNA Impurity Levels in Serum-Free Lentiviral Process Streams. ACS Synth Biol 2024; 13:466-473. [PMID: 38266181 PMCID: PMC10877604 DOI: 10.1021/acssynbio.3c00682] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2023] [Revised: 01/17/2024] [Accepted: 01/18/2024] [Indexed: 01/26/2024]
Abstract
We engineered HEK293T cells with a transgene encoding tetracycline-inducible expression of a Staphylococcus aureus nuclease incorporating a translocation signal. We adapted the unmodified and nuclease-engineered cell lines to grow in suspension in serum-free media, generating the HEK293TS and NuPro-2S cell lines, respectively. Transient transfection yielded 1.19 × 106 lentiviral transducing units per milliliter (TU/mL) from NuPro-2S cells and 1.45 × 106 TU/mL from HEK293TS cells. DNA ladder disappearance revealed medium-resident nuclease activity arising from NuPro-2S cells in a tetracycline-inducible manner. DNA impurity levels in lentiviral material arising from NuPro-2S and HEK293TS cells were undetectable by SYBR Safe agarose gel staining. Direct measurement by PicoGreen reagent revealed DNA to be present at 636 ng/mL in lentiviral material from HEK293TS cells, an impurity level reduced by 89% to 70 ng/mL in lentiviral material from NuPro-2S cells. This reduction was comparable to the 23 ng/mL achieved by treating HEK293TS-derived lentiviral material with 50 units/mL Benzonase.
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Affiliation(s)
- Geoffrey Howe
- Department
of Biochemical Engineering, University College
London, Bernard Katz
Building, London WC1E 6BT, United Kingdom
| | - Matthew Wasmuth
- Department
of Biochemical Engineering, University College
London, Bernard Katz
Building, London WC1E 6BT, United Kingdom
| | - Pamela Emanuelle
- Department
of Biochemical Engineering, University College
London, Bernard Katz
Building, London WC1E 6BT, United Kingdom
| | - Giulia Massaro
- UCL
School of Pharmacy, University College London, London WC1N 1AX, U.K.
| | - Ahad A. Rahim
- UCL
School of Pharmacy, University College London, London WC1N 1AX, U.K.
| | - Sadfer Ali
- Department
of Biochemical Engineering, University College
London, Bernard Katz
Building, London WC1E 6BT, United Kingdom
| | - Milena Rivera
- Department
of Biochemical Engineering, University College
London, Bernard Katz
Building, London WC1E 6BT, United Kingdom
| | - John Ward
- Department
of Biochemical Engineering, University College
London, Bernard Katz
Building, London WC1E 6BT, United Kingdom
| | - Eli Keshavarz-Moore
- Department
of Biochemical Engineering, University College
London, Bernard Katz
Building, London WC1E 6BT, United Kingdom
| | - Chris Mason
- Department
of Biochemical Engineering, University College
London, Bernard Katz
Building, London WC1E 6BT, United Kingdom
| | - Darren N. Nesbeth
- Department
of Biochemical Engineering, University College
London, Bernard Katz
Building, London WC1E 6BT, United Kingdom
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9
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Kaygisiz K, Rauch‐Wirth L, Iscen A, Hartenfels J, Kremer K, Münch J, Synatschke CV, Weil T. Peptide Amphiphiles as Biodegradable Adjuvants for Efficient Retroviral Gene Delivery. Adv Healthc Mater 2024; 13:e2301364. [PMID: 37947246 PMCID: PMC11468294 DOI: 10.1002/adhm.202301364] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2023] [Revised: 10/20/2023] [Indexed: 11/12/2023]
Abstract
Retroviral gene delivery is the key technique for in vitro and ex vivo gene therapy. However, inefficient virion-cell attachment resulting in low gene transduction efficacy remains a major challenge in clinical applications. Adjuvants for ex vivo therapy settings need to increase transduction efficiency while being easily removed or degraded post-transduction to prevent the risk of venous embolism after infusing the transduced cells back to the bloodstream of patients, yet no such peptide system have been reported thus far. In this study, peptide amphiphiles (PAs) with a hydrophobic fatty acid and a hydrophilic peptide moiety that reveal enhanced viral transduction efficiency are introduced. The PAs form β-sheet-rich fibrils that assemble into positively charged aggregates, promoting virus adhesion to the cell membrane. The block-type amphiphilic sequence arrangement in the PAs ensures efficient cell-virus interaction and biodegradability. Good biodegradability is observed for fibrils forming small aggregates and it is shown that via molecular dynamics simulations, the fibril-fibril interactions of PAs are governed by fibril surface hydrophobicity. These findings establish PAs as additives in retroviral gene transfer, rivalling commercially available transduction enhancers in efficiency and degradability with promising translational options in clinical gene therapy applications.
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Affiliation(s)
- Kübra Kaygisiz
- Department Synthesis of MacromoleculesMax Planck Institute for Polymer ResearchAckermannweg 1055128MainzGermany
| | - Lena Rauch‐Wirth
- Institute of Molecular VirologyUlm University Medical CenterMeyerhofstraße 189081UlmGermany
| | - Aysenur Iscen
- Polymer Theory DepartmentMax Planck Institute for Polymer ResearchAckermannweg 1055128MainzGermany
| | - Jan Hartenfels
- Department Synthesis of MacromoleculesMax Planck Institute for Polymer ResearchAckermannweg 1055128MainzGermany
| | - Kurt Kremer
- Polymer Theory DepartmentMax Planck Institute for Polymer ResearchAckermannweg 1055128MainzGermany
| | - Jan Münch
- Institute of Molecular VirologyUlm University Medical CenterMeyerhofstraße 189081UlmGermany
| | - Christopher V. Synatschke
- Department Synthesis of MacromoleculesMax Planck Institute for Polymer ResearchAckermannweg 1055128MainzGermany
| | - Tanja Weil
- Department Synthesis of MacromoleculesMax Planck Institute for Polymer ResearchAckermannweg 1055128MainzGermany
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10
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Triantafyllou N, Sarkis M, Krassakopoulou A, Shah N, Papathanasiou MM, Kontoravdi C. Uncertainty quantification for gene delivery methods: A roadmap for pDNA manufacturing from phase I clinical trials to commercialization. Biotechnol J 2024; 19:e2300103. [PMID: 37797343 DOI: 10.1002/biot.202300103] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2023] [Revised: 07/01/2023] [Accepted: 09/28/2023] [Indexed: 10/07/2023]
Abstract
The fast-growing interest in cell and gene therapy (C>) products has led to a growing demand for the production of plasmid DNA (pDNA) and viral vectors for clinical and commercial use. Manufacturers, regulators, and suppliers need to develop strategies for establishing robust and agile supply chains in the otherwise empirical field of C>. A model-based methodology that has great potential to support the wider adoption of C> is presented, by ensuring efficient timelines, scalability, and cost-effectiveness in the production of key raw materials. Specifically, key process and economic parameters are identified for (1) the production of pDNA for the forward-looking scenario of non-viral-based Chimeric Antigen Receptor (CAR) T-cell therapies from clinical (200 doses) to commercial (40,000 doses) scale and (2) the commercial (40,000 doses) production of pDNA and lentiviral vectors for the current state-of-the-art viral vector-based CAR T-cell therapies. By applying a systematic global sensitivity analysis, we quantify uncertainty in the manufacturing process and apportion it to key process and economic parameters, highlighting cost drivers and limitations that steer decision-making. The results underline the cost-efficiency and operational flexibility of non-viral-based therapies in the overall C> supply chain, as well as the importance of economies-of-scale in the production of pDNA.
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Affiliation(s)
- Niki Triantafyllou
- The Sargent Centre for Process Systems Engineering, Imperial College London, London, UK
- Department of Chemical Engineering, Imperial College London, London, UK
| | - Miriam Sarkis
- The Sargent Centre for Process Systems Engineering, Imperial College London, London, UK
- Department of Chemical Engineering, Imperial College London, London, UK
| | | | - Nilay Shah
- The Sargent Centre for Process Systems Engineering, Imperial College London, London, UK
- Department of Chemical Engineering, Imperial College London, London, UK
| | - Maria M Papathanasiou
- The Sargent Centre for Process Systems Engineering, Imperial College London, London, UK
- Department of Chemical Engineering, Imperial College London, London, UK
| | - Cleo Kontoravdi
- The Sargent Centre for Process Systems Engineering, Imperial College London, London, UK
- Department of Chemical Engineering, Imperial College London, London, UK
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11
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Kilgore R, Minzoni A, Shastry S, Smith W, Barbieri E, Wu Y, LeBarre JP, Chu W, O'Brien J, Menegatti S. The downstream bioprocess toolbox for therapeutic viral vectors. J Chromatogr A 2023; 1709:464337. [PMID: 37722177 DOI: 10.1016/j.chroma.2023.464337] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2023] [Revised: 08/24/2023] [Accepted: 08/27/2023] [Indexed: 09/20/2023]
Abstract
Viral vectors are poised to acquire a prominent position in modern medicine and biotechnology owing to their role as delivery agents for gene therapies, oncolytic agents, vaccine platforms, and a gateway to engineer cell therapies as well as plants and animals for sustainable agriculture. The success of viral vectors will critically depend on the availability of flexible and affordable biomanufacturing strategies that can meet the growing demand by clinics and biotech companies worldwide. In this context, a key role will be played by downstream process technology: while initially adapted from protein purification media, the purification toolbox for viral vectors is currently undergoing a rapid expansion to fit the unique biomolecular characteristics of these products. Innovation efforts are articulated on two fronts, namely (i) the discovery of affinity ligands that target adeno-associated virus, lentivirus, adenovirus, etc.; (ii) the development of adsorbents with innovative morphologies, such as membranes and 3D printed monoliths, that fit the size of viral vectors. Complementing these efforts are the design of novel process layouts that capitalize on novel ligands and adsorbents to ensure high yield and purity of the product while safeguarding its therapeutic efficacy and safety; and a growing panel of analytical methods that monitor the complex array of critical quality attributes of viral vectors and correlate them to the purification strategies. To help explore this complex and evolving environment, this study presents a comprehensive overview of the downstream bioprocess toolbox for viral vectors established in the last decade, and discusses present efforts and future directions contributing to the success of this promising class of biological medicines.
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Affiliation(s)
- Ryan Kilgore
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States.
| | - Arianna Minzoni
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States
| | - Shriarjun Shastry
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States; Biomanufacturing Training and Education Center (BTEC), North Carolina State University, Raleigh, NC 27695, United States
| | - Will Smith
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States
| | - Eduardo Barbieri
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States
| | - Yuxuan Wu
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States
| | - Jacob P LeBarre
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States
| | - Wenning Chu
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States
| | - Juliana O'Brien
- Department of Chemistry, North Carolina State University, Raleigh, NC 27695, United States
| | - Stefano Menegatti
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States; Biomanufacturing Training and Education Center (BTEC), North Carolina State University, Raleigh, NC 27695, United States; North Carolina Viral Vector Initiative in Research and Learning, North Carolina State University, Raleigh, NC 27695, United States
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12
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Broussau S, Lytvyn V, Simoneau M, Guilbault C, Leclerc M, Nazemi-Moghaddam N, Coulombe N, Elahi SM, McComb S, Gilbert R. Packaging cells for lentiviral vectors generated using the cumate and coumermycin gene induction systems and nanowell single-cell cloning. Mol Ther Methods Clin Dev 2023; 29:40-57. [PMID: 36936448 PMCID: PMC10018046 DOI: 10.1016/j.omtm.2023.02.013] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2022] [Accepted: 02/22/2023] [Indexed: 02/27/2023]
Abstract
Lentiviral vectors (LVs) are important for cell therapy because of their capacity to stably modify the genome after integration. This study describes a novel and relatively simple approach to generate packaging cells and producer clones for self-inactivating (SIN) LVs pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). A novel gene regulation system, based on the combination of the cumate and coumermycin induction systems, was developed to ensure tight control for the expression of cytotoxic packaging elements. To accelerate clone isolation and ensure monoclonality, the packaging genes were transfected simultaneously into human embryonic kidney cells (293SF-3F6) previously engineered with the induction system, and clones were isolated after limiting dilution into nanowell arrays using a robotic cell picking instrument with scanning capability. The method's effectiveness to isolate colonies derived from single cells was demonstrated using mixed populations of cells labeled with two different fluorescent markers. Because the recipient cell line grew in suspension culture, and all the procedures were performed without serum, the resulting clones were readily adaptable to serum-free suspension culture. The best producer clone produced LVs expressing GFP at a titer of 2.3 × 108 transduction units (TU)/mL in the culture medium under batch mode without concentration.
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Affiliation(s)
- Sophie Broussau
- Department of Production Platforms & Analytics, Human Health Therapeutics Research Centre, National Research Council Canada, Montreal, QC H4P 2R2, Canada
| | - Viktoria Lytvyn
- Department of Production Platforms & Analytics, Human Health Therapeutics Research Centre, National Research Council Canada, Montreal, QC H4P 2R2, Canada
| | - Mélanie Simoneau
- Department of Production Platforms & Analytics, Human Health Therapeutics Research Centre, National Research Council Canada, Montreal, QC H4P 2R2, Canada
| | - Claire Guilbault
- Department of Production Platforms & Analytics, Human Health Therapeutics Research Centre, National Research Council Canada, Montreal, QC H4P 2R2, Canada
| | - Mélanie Leclerc
- Department of Production Platforms & Analytics, Human Health Therapeutics Research Centre, National Research Council Canada, Montreal, QC H4P 2R2, Canada
| | - Nazila Nazemi-Moghaddam
- Department of Production Platforms & Analytics, Human Health Therapeutics Research Centre, National Research Council Canada, Montreal, QC H4P 2R2, Canada
| | - Nathalie Coulombe
- Department of Production Platforms & Analytics, Human Health Therapeutics Research Centre, National Research Council Canada, Montreal, QC H4P 2R2, Canada
| | - Seyyed Mehdy Elahi
- Department of Production Platforms & Analytics, Human Health Therapeutics Research Centre, National Research Council Canada, Montreal, QC H4P 2R2, Canada
| | - Scott McComb
- Department of Immunology, Human Health Therapeutics Research Centre, National Research Council, Canada, Ottawa, ON K1A 0R6, Canada
- Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Rénald Gilbert
- Department of Production Platforms & Analytics, Human Health Therapeutics Research Centre, National Research Council Canada, Montreal, QC H4P 2R2, Canada
- Department of Bioengineering, McGill University, Montreal, QC H3A 0E9, Canada
- Département de Génie chimique, Université Laval, Québec, QC G1V 0A6, Canada
- Corresponding author: Rénald Gilbert, National Research Council Canada, Building Montreal, 6100 Avenue Royalmount, Montreal, QC H4P 2R2, Canada.
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13
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Hillebrandt N, Hubbuch J. Size-selective downstream processing of virus particles and non-enveloped virus-like particles. Front Bioeng Biotechnol 2023; 11:1192050. [PMID: 37304136 PMCID: PMC10248422 DOI: 10.3389/fbioe.2023.1192050] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2023] [Accepted: 05/08/2023] [Indexed: 06/13/2023] Open
Abstract
Non-enveloped virus-like particles (VLPs) are versatile protein nanoparticles with great potential for biopharmaceutical applications. However, conventional protein downstream processing (DSP) and platform processes are often not easily applicable due to the large size of VLPs and virus particles (VPs) in general. The application of size-selective separation techniques offers to exploit the size difference between VPs and common host-cell impurities. Moreover, size-selective separation techniques offer the potential for wide applicability across different VPs. In this work, basic principles and applications of size-selective separation techniques are reviewed to highlight their potential in DSP of VPs. Finally, specific DSP steps for non-enveloped VLPs and their subunits are reviewed as well as the potential applications and benefits of size-selective separation techniques are shown.
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Affiliation(s)
| | - Jürgen Hubbuch
- Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany
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14
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Moreira AS, Bezemer S, Faria TQ, Detmers F, Hermans P, Sierkstra L, Coroadinha AS, Peixoto C. Implementation of Novel Affinity Ligand for Lentiviral Vector Purification. Int J Mol Sci 2023; 24:3354. [PMID: 36834764 PMCID: PMC9966744 DOI: 10.3390/ijms24043354] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2023] [Revised: 01/27/2023] [Accepted: 02/01/2023] [Indexed: 02/10/2023] Open
Abstract
The use of viral vectors as therapeutic products for multiple applications such as vaccines, cancer treatment, or gene therapies, has been growing exponentially. Therefore, improved manufacturing processes are needed to cope with the high number of functional particles required for clinical trials and, eventually, commercialization. Affinity chromatography (AC) can be used to simplify purification processes and generate clinical-grade products with high titer and purity. However, one of the major challenges in the purification of Lentiviral vectors (LVs) using AC is to combine a highly specific ligand with a gentle elution condition assuring the preservation of vector biological activity. In this work, we report for the first time the implementation of an AC resin to specifically purify VSV-G pseudotyped LVs. After ligand screening, different critical process parameters were assessed and optimized. A dynamic capacity of 1 × 1011 total particles per mL of resin was determined and an average recovery yield of 45% was found for the small-scale purification process. The established AC robustness was confirmed by the performance of an intermediate scale providing an infectious particles yield of 54%, which demonstrates the scalability and reproducibility of the AC matrix. Overall, this work contributes to increasing downstream process efficiency by delivering a purification technology that enables high purity, scalability, and process intensification in a single step, contributing to time-to-market reduction.
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Affiliation(s)
- Ana Sofia Moreira
- IBET Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal
- ITQB Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal
| | - Sandra Bezemer
- Thermo Fisher Scientific, 2333 CH Leiden, The Netherlands
| | - Tiago Q. Faria
- IBET Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal
| | - Frank Detmers
- Thermo Fisher Scientific, 2333 CH Leiden, The Netherlands
| | - Pim Hermans
- Thermo Fisher Scientific, 2333 CH Leiden, The Netherlands
| | | | - Ana Sofia Coroadinha
- IBET Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal
| | - Cristina Peixoto
- IBET Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal
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15
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Kearney AM. Chromatographic Purification of Viral Vectors for Gene Therapy Applications. Methods Mol Biol 2023; 2699:51-60. [PMID: 37646993 DOI: 10.1007/978-1-0716-3362-5_4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/01/2023]
Abstract
Chromatography has been a mainstay in the downstream processing and purification of biopharmaceutical medicines. Until now, this has largely involved the purification of protein products such as recombinant enzymes and monoclonal antibodies using large-scale column chromatography methods. The development of advanced therapeutic medicinal products (ATMP) is heralding in a new era of therapeutics for a range of indications. These new therapeutics use diverse substances ranging from live stem cell preparations to fragments of nucleic acid enclosed in a viral delivery system. With these new technologies come new challenges in their purification. In this chapter, the challenges faced in producing and purifying viral vectors capable of delivering life-altering gene therapy to the patient will be discussed. Current methods of chromatography capable of adaptation to meet these new challenges and advancements that may be needed to increase the purification capabilities for these new products will also be discussed.
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16
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Celebi Torabfam G, Yetisgin AA, Erdem C, Cayli A, Kutlu O, Cetinel S. A feasibility study of different commercially available serum-free mediums to enhance lentivirus and adeno-associated virus production in HEK 293 suspension cells. Cytotechnology 2022; 74:635-655. [PMID: 36389283 PMCID: PMC9652196 DOI: 10.1007/s10616-022-00551-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2022] [Accepted: 09/30/2022] [Indexed: 02/02/2023] Open
Abstract
Lentivirus and adeno-associated viruses are invaluable tools for biotechnology applications due to their genetic material delivery abilities both in vitro and in vivo. However, their large-scale productions with Good Manufacturing Practices yield low efficiency when adherent and serum dependent HEK293 (Human Embryonic Kidney) cells are used as the host. To increase production efficiency, HEK293 cells are adapted to grow in suspension using commercially available and chemically defined serum-free mediums. Suspended cells can be transiently transfected for viral vector production; however, significant improvements are still needed to increase yield and thereby cost effectiveness. Here, we evaluated four most preferred commercially available mediums that are IVY, FreeStyle293, LV-MAX, and BalanCD HEK293 for the transient transfection feasibility of lentiviral (LV) and adeno-associated virus serotype 2 (AAV2) production in FlorabioHEK293 suspension cells. The highest transfection efficiency was over 90% and obtained by using polyethyleneimine (PEI) 25 K and by media adaptation in IVY without using any transfection enhancer. For the first time the feasibility of HEK293 cells, which were adapted to grow in suspension culture by Florabio and IVY media, were tested for virus production. This study demonstrates the best transfection medium for scalable and optimized production of Lentivirus and Adeno-Associated Virus in suspended HEK293 cell culture. Supplementary Information The online version contains supplementary material available at 10.1007/s10616-022-00551-1.
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Affiliation(s)
- Gizem Celebi Torabfam
- Sabanci University Nanotechnology Research and Application Center (SUNUM), Istanbul, 34956 Turkey
- Faculty of Engineering and Natural Sciences, Molecular Biology, Genetics, and Bioengineering Program, Sabanci University, Istanbul, 34956 Turkey
| | - Abuzer Alp Yetisgin
- Sabanci University Nanotechnology Research and Application Center (SUNUM), Istanbul, 34956 Turkey
- Faculty of Engineering and Natural Sciences, Materials Science and Nano Engineering, Sabanci University, Istanbul, 34956 Turkey
| | - Cem Erdem
- FloraBio Technology, Urla, 35430 İzmir Turkey
| | - Aziz Cayli
- FloraBio Technology, Urla, 35430 İzmir Turkey
| | - Ozlem Kutlu
- Sabanci University Nanotechnology Research and Application Center (SUNUM), Istanbul, 34956 Turkey
- Faculty of Engineering and Natural Sciences, Molecular Biology, Genetics, and Bioengineering Program, Sabanci University, Istanbul, 34956 Turkey
| | - Sibel Cetinel
- Sabanci University Nanotechnology Research and Application Center (SUNUM), Istanbul, 34956 Turkey
- Faculty of Engineering and Natural Sciences, Molecular Biology, Genetics, and Bioengineering Program, Sabanci University, Istanbul, 34956 Turkey
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17
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Yew CHT, Gurumoorthy N, Nordin F, Tye GJ, Wan Kamarul Zaman WS, Tan JJ, Ng MH. Integrase deficient lentiviral vector: prospects for safe clinical applications. PeerJ 2022; 10:e13704. [PMID: 35979475 PMCID: PMC9377332 DOI: 10.7717/peerj.13704] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2022] [Accepted: 06/19/2022] [Indexed: 01/17/2023] Open
Abstract
HIV-1 derived lentiviral vector is an efficient transporter for delivering desired genetic materials into the targeted cells among many viral vectors. Genetic material transduced by lentiviral vector is integrated into the cell genome to introduce new functions, repair defective cell metabolism, and stimulate certain cell functions. Various measures have been administered in different generations of lentiviral vector systems to reduce the vector's replicating capabilities. Despite numerous demonstrations of an excellent safety profile of integrative lentiviral vectors, the precautionary approach has prompted the development of integrase-deficient versions of these vectors. The generation of integrase-deficient lentiviral vectors by abrogating integrase activity in lentiviral vector systems reduces the rate of transgenes integration into host genomes. With this feature, the integrase-deficient lentiviral vector is advantageous for therapeutic implementation and widens its clinical applications. This short review delineates the biology of HIV-1-erived lentiviral vector, generation of integrase-deficient lentiviral vector, recent studies involving integrase-deficient lentiviral vectors, limitations, and prospects for neoteric clinical use.
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Affiliation(s)
- Chee-Hong Takahiro Yew
- Centre for Tissue Engineering and Regenerative Medicine (CTERM), Universiti Kebangsaan Malaysia Medical Centre (UKMMC), Kuala Lumpur, Malaysia
| | - Narmatha Gurumoorthy
- Centre for Tissue Engineering and Regenerative Medicine (CTERM), Universiti Kebangsaan Malaysia Medical Centre (UKMMC), Kuala Lumpur, Malaysia
| | - Fazlina Nordin
- Centre for Tissue Engineering and Regenerative Medicine (CTERM), Universiti Kebangsaan Malaysia Medical Centre (UKMMC), Kuala Lumpur, Malaysia
| | - Gee Jun Tye
- Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Pulau Pinang, Malaysia
| | | | - Jun Jie Tan
- Advanced Medical and Dental Institute, Universiti Sains Malaysia (USM), Bertam, Kepala Batas, Pulau Pinang, Malaysia
| | - Min Hwei Ng
- Centre for Tissue Engineering and Regenerative Medicine (CTERM), Universiti Kebangsaan Malaysia Medical Centre (UKMMC), Kuala Lumpur, Malaysia
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18
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Arsenijevic Y, Berger A, Udry F, Kostic C. Lentiviral Vectors for Ocular Gene Therapy. Pharmaceutics 2022; 14:pharmaceutics14081605. [PMID: 36015231 PMCID: PMC9414879 DOI: 10.3390/pharmaceutics14081605] [Citation(s) in RCA: 24] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2022] [Revised: 07/14/2022] [Accepted: 07/22/2022] [Indexed: 12/10/2022] Open
Abstract
This review offers the basics of lentiviral vector technologies, their advantages and pitfalls, and an overview of their use in the field of ophthalmology. First, the description of the global challenges encountered to develop safe and efficient lentiviral recombinant vectors for clinical application is provided. The risks and the measures taken to minimize secondary effects as well as new strategies using these vectors are also discussed. This review then focuses on lentiviral vectors specifically designed for ocular therapy and goes over preclinical and clinical studies describing their safety and efficacy. A therapeutic approach using lentiviral vector-mediated gene therapy is currently being developed for many ocular diseases, e.g., aged-related macular degeneration, retinopathy of prematurity, inherited retinal dystrophies (Leber congenital amaurosis type 2, Stargardt disease, Usher syndrome), glaucoma, and corneal fibrosis or engraftment rejection. In summary, this review shows how lentiviral vectors offer an interesting alternative for gene therapy in all ocular compartments.
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Affiliation(s)
- Yvan Arsenijevic
- Unit Retinal Degeneration and Regeneration, Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, Fondation Asile des Aveugles, 1004 Lausanne, Switzerland;
- Correspondence: (Y.A.); (C.K.)
| | - Adeline Berger
- Group Epigenetics of ocular diseases, Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, Fondation Asile des Aveugles, 1004 Lausanne, Switzerland;
| | - Florian Udry
- Unit Retinal Degeneration and Regeneration, Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, Fondation Asile des Aveugles, 1004 Lausanne, Switzerland;
| | - Corinne Kostic
- Group for Retinal Disorder Research, Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, Fondation Asile des Aveugles, 1004 Lausanne, Switzerland
- Correspondence: (Y.A.); (C.K.)
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19
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Ghosh R, Koley S, Gopal S, Rodrigues AL, Dordick JS, Cramer SM. Evaluation of Lentiviral Vector Stability and Development of Ion Exchange Purification Processes. Biotechnol Prog 2022; 38:e3286. [PMID: 35808852 DOI: 10.1002/btpr.3286] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2022] [Revised: 06/20/2022] [Accepted: 06/28/2022] [Indexed: 11/05/2022]
Abstract
In this manuscript we employ parallel batch stability and chromatographic screens in concert with linear and step gradient experiments to develop a high yield, HCP clearance anion exchange capture process for lentiviral vector (LVV) purification. An initial broad resin screen is carried out to determine anion exchange-based resins that exhibit high recovery of LVV. LVV stability is then evaluated and conditions are established where the vector exhibits good stability, namely phosphate buffer at pH 6.5-7.5, with low to moderate salt concentrations. A subsequent high-throughput batch screen is then carried out with a subset of resins selected from the first screen under stable conditions to identify optimal wash and elution steps to further improve product yield and protein clearance. Linear gradient experiments are also conducted in mini-column format to refine the operating conditions and final step gradient processes are established that exhibit greater than 70% yield of infectious LVV while also achieving up to 2.89 log reduction values (LRV) of HCPs during the process. The large set of stability and chromatographic data provided in this work represent an important contribution to knowledge in the field about the chromatographic efficacy of a wide range of resins for LVV bioprocessing under stable conditions.
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Affiliation(s)
- Ronit Ghosh
- Department of Chemical and Biological Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, United States
| | - Sushmita Koley
- Department of Chemical and Biological Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, United States
| | - Sneha Gopal
- Department of Chemical and Biological Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, United States
| | - Andre L Rodrigues
- Department of Chemical and Biological Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, United States
| | - Jonathan S Dordick
- Department of Chemical and Biological Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, United States
| | - Steven M Cramer
- Department of Chemical and Biological Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, United States
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20
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Lavado-García J, Pérez-Rubio P, Cervera L, Gòdia F. The cell density effect in animal cell-based bioprocessing: Questions, insights and perspectives. Biotechnol Adv 2022; 60:108017. [PMID: 35809763 DOI: 10.1016/j.biotechadv.2022.108017] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2022] [Revised: 05/31/2022] [Accepted: 07/01/2022] [Indexed: 11/28/2022]
Abstract
One of the main challenges in the development of bioprocesses based on cell transient expression is the commonly reported reduction of cell specific productivity at increasing cell densities. This is generally known as the cell density effect (CDE). Many efforts have been devoted to understanding the cell metabolic implications to this phenomenon in an attempt to design operational strategies to overcome it. A comprehensive analysis of the main studies regarding the CDE is provided in this work to better define the elements comprising its cause and impact. Then, examples of methodologies and approaches employed to achieve successful transient expression at high cell densities (HCD) are thoroughly reviewed. A critical assessment of the limitations of the reported studies in the understanding of the CDE is presented, covering the leading hypothesis of the molecular implications. The overall analysis of previous work on CDE may offer useful insights for further research into manufacturing of biologics.
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Affiliation(s)
- Jesús Lavado-García
- Grup d'Enginyeria Cel·lular i Bioprocessos, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Campus de Bellaterra, Cerdanyola del Vallès, 08193 Barcelona, Spain; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
| | - Pol Pérez-Rubio
- Grup d'Enginyeria Cel·lular i Bioprocessos, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Campus de Bellaterra, Cerdanyola del Vallès, 08193 Barcelona, Spain
| | - Laura Cervera
- Grup d'Enginyeria Cel·lular i Bioprocessos, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Campus de Bellaterra, Cerdanyola del Vallès, 08193 Barcelona, Spain
| | - Francesc Gòdia
- Grup d'Enginyeria Cel·lular i Bioprocessos, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Campus de Bellaterra, Cerdanyola del Vallès, 08193 Barcelona, Spain
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21
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Labisch JJ, Kassar M, Bollmann F, Valentic A, Hubbuch J, Pflanz K. Steric exclusion chromatography of lentiviral vectors using hydrophilic cellulose membranes. J Chromatogr A 2022; 1674:463148. [DOI: 10.1016/j.chroma.2022.463148] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2022] [Revised: 05/11/2022] [Accepted: 05/12/2022] [Indexed: 11/29/2022]
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22
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Quach ABV, Little SR, Shih SCC. Viral Generation, Packaging, and Transduction on a Digital Microfluidic Platform. Anal Chem 2022; 94:4039-4047. [PMID: 35192339 DOI: 10.1021/acs.analchem.1c05227] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Viral-based systems are a popular delivery method for introducing exogenous genetic material into mammalian cells. Unfortunately, the preparation of lentiviruses containing the machinery to edit the cells is labor-intensive, with steps requiring optimization and sensitive handling. To mitigate these challenges, we introduce the first microfluidic method that integrates lentiviral generation, packaging, and transduction. The new method allows the production of viral titers between 106 and 107 (similar to macroscale production) and high transduction efficiency for hard-to-transfect cell lines. We extend the technique for gene editing applications and show how this technique can be used to knock out and knock down estrogen receptor gene─a gene prominently responsible for 70% of breast cancer cases. This new technique is automated with multiplexing capabilities, which have the potential to standardize the methods for viral-based genome engineering.
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Affiliation(s)
- Angela B V Quach
- Department of Biology, Concordia University, 7141 Sherbrooke Street West, Montréal, Québec H4B 1R6, Canada.,Centre for Applied Synthetic Biology, Concordia University, 7141 Sherbrooke Street West, Montréal, Québec H4B 1R6, Canada
| | - Samuel R Little
- Centre for Applied Synthetic Biology, Concordia University, 7141 Sherbrooke Street West, Montréal, Québec H4B 1R6, Canada.,Department of Electrical and Computer Engineering, Concordia University, 1455 de Maisonneuve Blvd. West, Montréal, Québec H3G 1M8, Canada
| | - Steve C C Shih
- Department of Biology, Concordia University, 7141 Sherbrooke Street West, Montréal, Québec H4B 1R6, Canada.,Centre for Applied Synthetic Biology, Concordia University, 7141 Sherbrooke Street West, Montréal, Québec H4B 1R6, Canada.,Department of Electrical and Computer Engineering, Concordia University, 1455 de Maisonneuve Blvd. West, Montréal, Québec H3G 1M8, Canada
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23
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Shi R, Jia S, Liu H, Nie H. Clinical grade lentiviral vector purification and quality control requirements. J Sep Sci 2022; 45:2093-2101. [PMID: 35247228 DOI: 10.1002/jssc.202100937] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2021] [Revised: 02/27/2022] [Accepted: 03/01/2022] [Indexed: 11/10/2022]
Abstract
Lentiviral vectors have been proven to be a powerful tool in gene therapies that includes the ability to perform long-term gene editing in both dividing and non-dividing cells. In order to meet the rising demand of clinical grade lentiviral vectors for future clinical trials and requirements by regulatory agencies, new methods and technologies were developed, including the rapid optimization of production and purification processes. However, gaps still exist in achieving ideal yields and recovery rates in large-scale manufacturing process steps. The downstream purification process is a critical step required to obtain sufficient quantity and high-quality lentiviral vectors products, which is challenged by the low stability of the LV particles and large production volumes associated with the manufacturing process. This review summarizes the most recent and promising technologies and enhancements used in the large-scale purification process step of LV manufacturing and aims to provide a significant contribution towards the achievement of providing sufficient quantity and quality of LVs in scalable processes. This article is protected by copyright. All rights reserved.
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Affiliation(s)
- Ruina Shi
- Immunochina Pharmaceutical Co., Ltd., Beijing, China
| | - Shenghua Jia
- Immunochina Pharmaceutical Co., Ltd., Beijing, China
| | - Huwei Liu
- College of Life Sciences, Wuchang University of Technology, Wuhan, China.,Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, China
| | - Honggang Nie
- Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, China.,Analytical Instrumental Center, Peking University, Beijing, China
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24
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Aghaamoo M, Chen Y, Li X, Garg N, Jiang R, Yun JT, Lee AP. High-Throughput and Dosage-Controlled Intracellular Delivery of Large Cargos by an Acoustic-Electric Micro-Vortices Platform. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2022; 9:e2102021. [PMID: 34716688 PMCID: PMC8728830 DOI: 10.1002/advs.202102021] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/16/2021] [Revised: 09/23/2021] [Indexed: 05/20/2023]
Abstract
A high-throughput non-viral intracellular delivery platform is introduced for the transfection of large cargos with dosage-control. This platform, termed Acoustic-Electric Shear Orbiting Poration (AESOP), optimizes the delivery of intended cargo sizes with poration of the cell membranes via mechanical shear followed by the modulated expansion of these nanopores via electric field. Furthermore, AESOP utilizes acoustic microstreaming vortices wherein up to millions of cells are trapped and mixed uniformly with exogenous cargos, enabling the delivery of cargos into cells with targeted dosages. Intracellular delivery of a wide range of molecule sizes (<1 kDa to 2 MDa) with high efficiency (>90%), cell viability (>80%), and uniform dosages (<60% coefficient of variation (CV)) simultaneously into 1 million cells min-1 per single chip is demonstrated. AESOP is successfully applied to two gene editing applications that require the delivery of large plasmids: i) enhanced green fluorescent protein (eGFP) plasmid (6.1 kbp) transfection, and ii) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated gene knockout using a 9.3 kbp plasmid DNA encoding Cas9 protein and single guide RNA (sgRNA). Compared to alternative platforms, this platform offers dosage-controlled intracellular delivery of large plasmids simultaneously to large populations of cells while maintaining cell viability at comparable delivery efficiencies.
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Affiliation(s)
- Mohammad Aghaamoo
- Department of Biomedical EngineeringUniversity of California IrvineIrvineCA92697USA
- Center for Advanced Design & Manufacturing of Integrated Microfluidics (CADMIM)University of California IrvineIrvineCA92697USA
| | - Yu‐Hsi Chen
- Department of Biomedical EngineeringUniversity of California IrvineIrvineCA92697USA
- Center for Advanced Design & Manufacturing of Integrated Microfluidics (CADMIM)University of California IrvineIrvineCA92697USA
| | - Xuan Li
- Department of Biomedical EngineeringUniversity of California IrvineIrvineCA92697USA
- Center for Advanced Design & Manufacturing of Integrated Microfluidics (CADMIM)University of California IrvineIrvineCA92697USA
| | - Neha Garg
- Department of Biomedical EngineeringUniversity of California IrvineIrvineCA92697USA
- Center for Advanced Design & Manufacturing of Integrated Microfluidics (CADMIM)University of California IrvineIrvineCA92697USA
| | - Ruoyu Jiang
- Department of Biomedical EngineeringUniversity of California IrvineIrvineCA92697USA
- Center for Advanced Design & Manufacturing of Integrated Microfluidics (CADMIM)University of California IrvineIrvineCA92697USA
| | - Jeremy Tian‐Hao Yun
- Department of Biomedical EngineeringUniversity of California IrvineIrvineCA92697USA
- Palo Alto Senior High SchoolPalo AltoCA94301USA
| | - Abraham Phillip Lee
- Department of Biomedical EngineeringUniversity of California IrvineIrvineCA92697USA
- Center for Advanced Design & Manufacturing of Integrated Microfluidics (CADMIM)University of California IrvineIrvineCA92697USA
- Department of Mechanical & Aerospace EngineeringUniversity of California IrvineIrvineCA92697USA
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25
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Moreira A, Faria T, Oliveira J, Kavara A, Schofield M, Sanderson T, Collins M, Gantier R, Alves P, Carrondo M, Peixoto C. Enhancing the purification of Lentiviral vectors for clinical applications. Sep Purif Technol 2021. [DOI: 10.1016/j.seppur.2021.118598] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
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26
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Leinonen HM, Lepola S, Lipponen EM, Heikura T, Koponen T, Parker N, Ylä-Herttuala S, Lesch HP. Benchmarking of Scale-X Bioreactor System in Lentiviral and Adenoviral Vector Production. Hum Gene Ther 2021; 31:376-384. [PMID: 32075423 PMCID: PMC7087403 DOI: 10.1089/hum.2019.247] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
We have previously produced viral vectors (lentiviral vector, adenoviral vector, and adeno-associated viral vector) in small and in commercial scale in adherent cells using Pall fixed-bed iCELLis® bioreactor. Recently, a company called Univercells has launched a new fixed-bed bioreactor with the same cell growth surface matrix material, but with different fixed-bed structure than is used in iCELLis bioreactor. We sought to compare the new scale-X™ hydro bioreactor (2.4 m2) and iCELLis Nano system (2.67 m2) to see if the difference has any effect on cell growth or lentiviral vector and adenoviral vector productivity. Runs were performed using parameters optimized for viral vector production in iCELLis Nano bioreactor. Cell growth was monitored by counting nuclei, as well as by following glucose consumption and lactate production. In both bioreactor systems, cells grew well, and the cell distribution was found quite homogeneous in scale-X bioreactor. Univercells scale-X bioreactor was proven to be at least equally efficient or even improved in both lentiviral vector and adenoviral vector production. Based on the results, the same protocol and parameters used in viral vector production in iCELLis bioreactor can also be successfully used for the production in scale-X bioreactor system.
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Affiliation(s)
- Hanna M Leinonen
- Kuopio Center for Gene and Cell Therapy, Kuopio, Finland.,FinVector, Kuopio, Finland; and
| | - Saana Lepola
- Kuopio Center for Gene and Cell Therapy, Kuopio, Finland.,FinVector, Kuopio, Finland; and
| | - Eevi M Lipponen
- Kuopio Center for Gene and Cell Therapy, Kuopio, Finland.,FinVector, Kuopio, Finland; and
| | - Tommi Heikura
- Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland
| | - Tiina Koponen
- Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland
| | - Nigel Parker
- Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland
| | - Seppo Ylä-Herttuala
- Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland
| | - Hanna P Lesch
- Kuopio Center for Gene and Cell Therapy, Kuopio, Finland.,FinVector, Kuopio, Finland; and
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Ferrara F, Del Rosario JMM, da Costa KAS, Kinsley R, Scott S, Fereidouni S, Thompson C, Kellam P, Gilbert S, Carnell G, Temperton N. Development of Lentiviral Vectors Pseudotyped With Influenza B Hemagglutinins: Application in Vaccine Immunogenicity, mAb Potency, and Sero-Surveillance Studies. Front Immunol 2021; 12:661379. [PMID: 34108964 PMCID: PMC8182064 DOI: 10.3389/fimmu.2021.661379] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2021] [Accepted: 05/05/2021] [Indexed: 12/16/2022] Open
Abstract
Influenza B viruses (IBV) cause respiratory disease epidemics in humans and are therefore components of seasonal influenza vaccines. Serological methods are employed to evaluate vaccine immunogenicity prior to licensure. However, classical methods to assess influenza vaccine immunogenicity such as the hemagglutination inhibition assay (HI) and the serial radial hemolysis assay (SRH), have been proven to have many limitations. As such, there is a need to develop innovative methods that can improve on these traditional assays and provide advantages such as ease of production and access, safety, reproducibility, and specificity. It has been previously demonstrated that the use of replication-defective viruses, such as lentiviral vectors pseudotyped with influenza A hemagglutinins in microneutralization assays (pMN) is a safe and sensitive alternative to study antibody responses elicited by natural influenza infection or vaccination. Consequently, we have produced Influenza B hemagglutinin-pseudotypes (IBV PV) using plasmid-directed transfection. To activate influenza B hemagglutinin, we have explored the use of proteases in increasing PV titers via their co-transfection during pseudotype virus production. When tested for their ability to transduce target cells, the influenza B pseudotypes produced exhibit tropism for different cell lines. The pseudotypes were evaluated as alternatives to live virus in microneutralization assays using reference sera standards, mouse and human sera collected during vaccine immunogenicity studies, surveillance sera from seals, and monoclonal antibodies (mAbs) against IBV. The influenza B pseudotype pMN was found to effectively detect neutralizing and cross-reactive responses in all assays and shows promise as an effective and versatile tool in influenza research.
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Affiliation(s)
- Francesca Ferrara
- Viral Pseudotype Unit, Medway School of Pharmacy, University of Kent & University of Greenwich, Chatham, United Kingdom
| | - Joanne Marie M Del Rosario
- Viral Pseudotype Unit, Medway School of Pharmacy, University of Kent & University of Greenwich, Chatham, United Kingdom.,Department of Physical Sciences & Mathematics, College of Arts and Sciences, University of the Philippines Manila, Manila, Philippines
| | - Kelly A S da Costa
- Viral Pseudotype Unit, Medway School of Pharmacy, University of Kent & University of Greenwich, Chatham, United Kingdom
| | - Rebecca Kinsley
- Viral Pseudotype Unit, Medway School of Pharmacy, University of Kent & University of Greenwich, Chatham, United Kingdom.,Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom
| | - Simon Scott
- Viral Pseudotype Unit, Medway School of Pharmacy, University of Kent & University of Greenwich, Chatham, United Kingdom
| | - Sasan Fereidouni
- Research Institute of Wildlife Ecology, Veterinary Medicine University, Vienna, Austria
| | - Craig Thompson
- Department of Zoology, University of Oxford, Oxford, United Kingdom
| | - Paul Kellam
- Faculty of Medicine, Imperial College London, London, United Kingdom
| | - Sarah Gilbert
- The Jenner Institute, University of Oxford, Oxford, United Kingdom
| | - George Carnell
- Viral Pseudotype Unit, Medway School of Pharmacy, University of Kent & University of Greenwich, Chatham, United Kingdom.,Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom
| | - Nigel Temperton
- Viral Pseudotype Unit, Medway School of Pharmacy, University of Kent & University of Greenwich, Chatham, United Kingdom
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Belete TM. The Current Status of Gene Therapy for the Treatment of Cancer. Biologics 2021; 15:67-77. [PMID: 33776419 PMCID: PMC7987258 DOI: 10.2147/btt.s302095] [Citation(s) in RCA: 51] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2021] [Accepted: 03/04/2021] [Indexed: 02/06/2023]
Abstract
Gene therapy is the administration of foreign genomic material into the host tissue to modify the expression of a gene product or to change the biological properties of cells for therapeutic use. Initially, the major objective of gene therapy was to manage genetic diseases, but now different disorders with several patterns of acquired and inherited disorders are targets of gene therapy. Over three decades, the advancement of Genome engineering technologies facilitated gene therapy for the prevention and management of intractable diseases. Researchers are advancing with cautious optimism that safe and effective treatment will give to patients with single-gene disorders and complex acquired disorders. To date, over 3000 genes associates with disease-causing mutations, and about 2600 gene therapy trials are undergoing for the management of various disorders. This review summarizes the principles of genome-editing approaches, such as zinc finger nucleases, transcription activator-like effector nucleases, meganucleases, and the CRISPR/Cas9 system with the underlying mechanisms. This review also explains the types of gene delivery systems as viral [adenoviral, adeno association, herpes simplex virus] and nonviral delivery systems (physical: DNA bombardment, electroporation) and (chemical: Cationic lipids, cationic polymers). Finally, this review summarizes gene therapy medicines approved to treat cancer in detail, including names, indications, vectors, and mode of gene therapy. Gene therapy becomes an alternative to an existing management for different diseases. Therefore, gene products with safe vectors and better biotechnologies play a significant role in the prophylaxis and management of various disorders in the future.
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Affiliation(s)
- Tafere Mulaw Belete
- Department of Pharmacology, College of Medicine and Health Sciences, University of Gondar, Gondar, Amhara Region, Ethiopia
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29
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Comisel RM, Kara B, Fiesser FH, Farid SS. Lentiviral vector bioprocess economics for cell and gene therapy commercialization. Biochem Eng J 2021. [DOI: 10.1016/j.bej.2020.107868] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
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30
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Perry C, Rayat ACME. Lentiviral Vector Bioprocessing. Viruses 2021; 13:268. [PMID: 33572347 PMCID: PMC7916122 DOI: 10.3390/v13020268] [Citation(s) in RCA: 86] [Impact Index Per Article: 21.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2021] [Revised: 02/02/2021] [Accepted: 02/03/2021] [Indexed: 12/11/2022] Open
Abstract
Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are now commonly utilised within the growing field of cell and gene therapy for the treatment of monogenic diseases and adoptive therapies such as chimeric antigen T-cell (CAR-T) therapy. This is a comprehensive review of the individual bioprocess operations employed in LV production. We highlight the role of envelope proteins in vector design as well as their impact on the bioprocessing of lentiviral vectors. An overview of the current state of these operations provides opportunities for bioprocess discovery and improvement with emphasis on the considerations for optimal and scalable processing of LV during development and clinical production. Upstream culture for LV generation is described with comparisons on the different transfection methods and various bioreactors for suspension and adherent producer cell cultivation. The purification of LV is examined, evaluating different sequences of downstream process operations for both small- and large-scale production requirements. For scalable operations, a key focus is the development in chromatographic purification in addition to an in-depth examination of the application of tangential flow filtration. A summary of vector quantification and characterisation assays is also presented. Finally, the assessment of the whole bioprocess for LV production is discussed to benefit from the broader understanding of potential interactions of the different process options. This review is aimed to assist in the achievement of high quality, high concentration lentiviral vectors from robust and scalable processes.
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Affiliation(s)
- Christopher Perry
- The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Gower St, London WC1E 6BT, UK;
- Division of Advanced Therapies, National Institute for Biological Standards and Control, South Mimms EN6 3QG, UK
| | - Andrea C. M. E. Rayat
- The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Gower St, London WC1E 6BT, UK;
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31
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Bulcha JT, Wang Y, Ma H, Tai PWL, Gao G. Viral vector platforms within the gene therapy landscape. Signal Transduct Target Ther 2021; 6:53. [PMID: 33558455 PMCID: PMC7868676 DOI: 10.1038/s41392-021-00487-6] [Citation(s) in RCA: 718] [Impact Index Per Article: 179.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2020] [Revised: 10/05/2020] [Accepted: 10/23/2020] [Indexed: 01/30/2023] Open
Abstract
Throughout its 40-year history, the field of gene therapy has been marked by many transitions. It has seen great strides in combating human disease, has given hope to patients and families with limited treatment options, but has also been subject to many setbacks. Treatment of patients with this class of investigational drugs has resulted in severe adverse effects and, even in rare cases, death. At the heart of this dichotomous field are the viral-based vectors, the delivery vehicles that have allowed researchers and clinicians to develop powerful drug platforms, and have radically changed the face of medicine. Within the past 5 years, the gene therapy field has seen a wave of drugs based on viral vectors that have gained regulatory approval that come in a variety of designs and purposes. These modalities range from vector-based cancer therapies, to treating monogenic diseases with life-altering outcomes. At present, the three key vector strategies are based on adenoviruses, adeno-associated viruses, and lentiviruses. They have led the way in preclinical and clinical successes in the past two decades. However, despite these successes, many challenges still limit these approaches from attaining their full potential. To review the viral vector-based gene therapy landscape, we focus on these three highly regarded vector platforms and describe mechanisms of action and their roles in treating human disease.
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Affiliation(s)
- Jote T Bulcha
- Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, USA
- Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, USA
| | - Yi Wang
- Department of Pathophysiology, West China College of Basic medical sciences & Forensic Medicine, Sichuan University, Chengdu, China
| | - Hong Ma
- Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, USA
| | - Phillip W L Tai
- Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, USA.
- Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, USA.
- VIDE Program, University of Massachusetts Medical School, Worcester, MA, USA.
| | - Guangping Gao
- Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, USA.
- Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, USA.
- Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, USA.
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Kawka K, Wilton AN, Madadkar P, Medina MFC, Lichty BD, Ghosh R, Latulippe DR. Integrated development of enzymatic DNA digestion and membrane chromatography processes for the purification of therapeutic adenoviruses. Sep Purif Technol 2021. [DOI: 10.1016/j.seppur.2020.117503] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
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Abstract
Advances in the use of lentiviral vectors for gene therapy applications have created a need for large-scale manufacture of clinical-grade viral vectors for transfer of genetic materials. Lentiviral vectors can transduce a wide range of cell types and integrate into the host genome of dividing and nondividing cells, resulting in long-term expression of the transgene both in vitro and in vivo. In this chapter, we present a method to transfect human cells, creating an easy platform to produce lentiviral vectors for CAR-T cell application.
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Moreira AS, Cavaco DG, Faria TQ, Alves PM, Carrondo MJT, Peixoto C. Advances in Lentivirus Purification. Biotechnol J 2020; 16:e2000019. [PMID: 33089626 DOI: 10.1002/biot.202000019] [Citation(s) in RCA: 34] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2020] [Revised: 10/15/2020] [Indexed: 12/20/2022]
Abstract
Lentiviral vectors (LVs) have been increasingly used as a tool for gene and cell therapies since they can stably integrate the genome in dividing and nondividing cells. LV production and purification processes have evolved substantially over the last decades. However, the increasing demands for higher quantities with more restrictive purity requirements are stimulating the development of novel materials and strategies to supply the market with LV in a cost-effective manner. A detailed review of each downstream process unit operation is performed, limitations, strengths, and potential outcomes being covered. Currently, the majority of large-scale LV manufacturing processes are still based on adherent cell culture, although it is known that the industry is migrating fast to suspension cultures. Regarding the purification strategy, it consists of batch chromatography and membrane technology. Nevertheless, new solutions are being created to improve the current production schemes and expand its clinical use.
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Affiliation(s)
- Ana Sofia Moreira
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, Portugal.,Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, Oeiras, Portugal
| | - David Guia Cavaco
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, Portugal.,Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, Oeiras, Portugal
| | - Tiago Q Faria
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, Portugal.,Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, Oeiras, Portugal
| | - Paula M Alves
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, Portugal.,Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, Oeiras, Portugal
| | - Manuel J T Carrondo
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, Portugal
| | - Cristina Peixoto
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, Portugal
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Taştan C, Kançağı DD, Turan RD, Yurtsever B, Çakırsoy D, Abanuz S, Yılancı M, Seyis U, Özer S, Mert S, Kayhan CK, Tokat F, Açıkel Elmas M, Birdoğan S, Arbak S, Yalçın K, Sezgin A, Kızılkılıç E, Hemşinlioğlu C, İnce Ü, Ratip S, Ovalı E. Preclinical Assessment of Efficacy and Safety Analysis of CAR-T Cells (ISIKOK-19) Targeting CD19-Expressing B-Cells for the First Turkish Academic Clinical Trial with Relapsed/Refractory ALL and NHL Patients. Turk J Haematol 2020; 37:234-247. [PMID: 32755128 PMCID: PMC7702660 DOI: 10.4274/tjh.galenos.2020.2020.0070] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2020] [Accepted: 07/31/2020] [Indexed: 12/19/2022] Open
Abstract
Objective Relapsed and refractory CD19-positive B-cell acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL) are the focus of studies on hematological cancers. Treatment of these malignancies has undergone recent transformation with the development of new gene therapy and molecular biology techniques, which are safer and well-tolerated therapeutic approaches. The CD19 antigen is the most studied therapeutic target in these hematological cancers. This study reports the results of clinical-grade production, quality control, and in vivo efficacy processes of ISIKOK-19 cells as the first academic clinical trial of CAR-T cells targeting CD19-expressing B cells in relapsed/refractory ALL and NHL patients in Turkey. Materials and Methods We used a lentiviral vector encoding the CD19 antigen-specific antibody head (FMC63) conjugated with the CD8-CD28-CD3ζ sequence as a chimeric antigen receptor (CAR) along with a truncated form of EGFR (EGFRt) on human T-lymphocytes (CAR-T). We preclinically assessed the efficacy and safety of the manufactured CAR-T cells, namely ISIKOK-19, from both healthy donors’ and ALL/NHL patients’ peripheral blood mononuclear cells. Results We showed significant enhancement of CAR lentivirus transduction efficacy in T-cells using BX-795, an inhibitor of the signaling molecule TBK1/IKKƐ, in order to cut the cost of CAR-T cell production. In addition, ISIKOK-19 cells demonstrated a significantly high level of cytotoxicity specifically against a CD19+ B-lymphocyte cancer model, RAJI cells, in NOD/SCID mice. Conclusion This is the first report of preclinical assessment of efficacy and safety analysis of CAR-T cells (ISIKOK-19) targeting CD19-expressing B cells in relapsed/refractory ALL and NHL patients in Turkey.
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MESH Headings
- Animals
- Antigens, CD19/genetics
- Antigens, CD19/immunology
- Cytotoxicity, Immunologic/genetics
- Disease Models, Animal
- Gene Expression
- Genetic Vectors/genetics
- Humans
- Immunotherapy, Adoptive/methods
- Lentivirus/genetics
- Lymphocyte Activation
- Lymphoma, Non-Hodgkin/etiology
- Lymphoma, Non-Hodgkin/therapy
- Mice
- Mice, Inbred NOD
- Mice, SCID
- Precursor Cell Lymphoblastic Leukemia-Lymphoma/etiology
- Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy
- Receptors, Antigen, T-Cell/immunology
- Receptors, Chimeric Antigen/immunology
- T-Lymphocytes/immunology
- T-Lymphocytes/metabolism
- Transduction, Genetic
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Affiliation(s)
- Cihan Taştan
- Acıbadem Labcell Cellular Therapy Laboratory, İstanbul, Turkey
| | | | | | - Bulut Yurtsever
- Acıbadem Labcell Cellular Therapy Laboratory, İstanbul, Turkey
| | - Didem Çakırsoy
- Acıbadem Labcell Cellular Therapy Laboratory, İstanbul, Turkey
| | - Selen Abanuz
- Acıbadem Labcell Cellular Therapy Laboratory, İstanbul, Turkey
| | | | - Utku Seyis
- Acıbadem Labcell Cellular Therapy Laboratory, İstanbul, Turkey
| | - Samed Özer
- Acıbadem Mehmet Ali Aydınlar University, Animal Application and Research Center, İstanbul, Turkey
| | - Selin Mert
- Boğaziçi University, Center of Life Sciences and Technologies, İstanbul, Turkey
| | | | - Fatma Tokat
- Acıbadem Mehmet Ali Aydınlar University Faculty of Medicine, Department of Pathology, İstanbul, Turkey
| | - Merve Açıkel Elmas
- Acıbadem Mehmet Ali Aydınlar University Faculty of Medicine, Department of Histology and Embryology, İstanbul, Turkey
| | - Selçuk Birdoğan
- Acıbadem Mehmet Ali Aydınlar University, Electron Microscopy Laboratory, İstanbul, Turkey
| | - Serap Arbak
- Acıbadem Mehmet Ali Aydınlar University Faculty of Medicine, Department of Histology and Embryology, İstanbul, Turkey
| | - Koray Yalçın
- Acıbadem Labcell Cellular Therapy Laboratory, İstanbul, Turkey
- Medical Park Göztepe Hospital, Pediatric Hematopoetic Stem Cell Transplantation Unit, İstanbul, Turkey
| | | | | | | | - Ümit İnce
- Acıbadem Mehmet Ali Aydınlar University Faculty of Medicine, Department of Pathology, İstanbul, Turkey
| | - Siret Ratip
- Acıbadem Mehmet Ali Aydınlar University Faculty of Medicine, Department of Hematology, İstanbul, Turkey
| | - Ercüment Ovalı
- Acıbadem Labcell Cellular Therapy Laboratory, İstanbul, Turkey
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Martínez-Molina E, Chocarro-Wrona C, Martínez-Moreno D, Marchal JA, Boulaiz H. Large-Scale Production of Lentiviral Vectors: Current Perspectives and Challenges. Pharmaceutics 2020; 12:pharmaceutics12111051. [PMID: 33153183 PMCID: PMC7693937 DOI: 10.3390/pharmaceutics12111051] [Citation(s) in RCA: 39] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2020] [Revised: 10/20/2020] [Accepted: 10/31/2020] [Indexed: 02/07/2023] Open
Abstract
Lentiviral vectors (LVs) have gained value over recent years as gene carriers in gene therapy. These viral vectors are safer than what was previously being used for gene transfer and are capable of infecting both dividing and nondividing cells with a long-term expression. This characteristic makes LVs ideal for clinical research, as has been demonstrated with the approval of lentivirus-based gene therapies from the Food and Drug Administration and the European Agency for Medicine. A large number of functional lentiviral particles are required for clinical trials, and large-scale production has been challenging. Therefore, efforts are focused on solving the drawbacks associated with the production and purification of LVsunder current good manufacturing practice. In recent years, we have witnessed the development and optimization of new protocols, packaging cell lines, and culture devices that are very close to reaching the target production level. Here, we review the most recent, efficient, and promising methods for the clinical-scale production ofLVs.
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Affiliation(s)
- Eduardo Martínez-Molina
- Biopathology and Medicine Regenerative Institute (IBIMER), University of Granada (D.M.), 18016 Granada, Spain; (E.M.-M.); (C.C.-W.); (D.M.-M.); (J.A.M.)
- Department of Human Anatomy and Embryology, University of Granada, 18016 Granada, Spain
| | - Carlos Chocarro-Wrona
- Biopathology and Medicine Regenerative Institute (IBIMER), University of Granada (D.M.), 18016 Granada, Spain; (E.M.-M.); (C.C.-W.); (D.M.-M.); (J.A.M.)
- Department of Human Anatomy and Embryology, University of Granada, 18016 Granada, Spain
- Excellence Research Unit “Modeling Nature” (MNat), University of Granada, 18016 Granada, Spain
- Biosanitary Institute of Granada (ibs.GRANADA), SAS-Universidad de Granada, 18016 Granada, Spain
| | - Daniel Martínez-Moreno
- Biopathology and Medicine Regenerative Institute (IBIMER), University of Granada (D.M.), 18016 Granada, Spain; (E.M.-M.); (C.C.-W.); (D.M.-M.); (J.A.M.)
- Department of Human Anatomy and Embryology, University of Granada, 18016 Granada, Spain
- Excellence Research Unit “Modeling Nature” (MNat), University of Granada, 18016 Granada, Spain
- Biosanitary Institute of Granada (ibs.GRANADA), SAS-Universidad de Granada, 18016 Granada, Spain
| | - Juan A. Marchal
- Biopathology and Medicine Regenerative Institute (IBIMER), University of Granada (D.M.), 18016 Granada, Spain; (E.M.-M.); (C.C.-W.); (D.M.-M.); (J.A.M.)
- Department of Human Anatomy and Embryology, University of Granada, 18016 Granada, Spain
- Excellence Research Unit “Modeling Nature” (MNat), University of Granada, 18016 Granada, Spain
- Biosanitary Institute of Granada (ibs.GRANADA), SAS-Universidad de Granada, 18016 Granada, Spain
| | - Houria Boulaiz
- Biopathology and Medicine Regenerative Institute (IBIMER), University of Granada (D.M.), 18016 Granada, Spain; (E.M.-M.); (C.C.-W.); (D.M.-M.); (J.A.M.)
- Department of Human Anatomy and Embryology, University of Granada, 18016 Granada, Spain
- Excellence Research Unit “Modeling Nature” (MNat), University of Granada, 18016 Granada, Spain
- Biosanitary Institute of Granada (ibs.GRANADA), SAS-Universidad de Granada, 18016 Granada, Spain
- Correspondence: ; Tel.: +34-958-241-271
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Soldi M, Sergi Sergi L, Unali G, Kerzel T, Cuccovillo I, Capasso P, Annoni A, Biffi M, Rancoita PMV, Cantore A, Lombardo A, Naldini L, Squadrito ML, Kajaste-Rudnitski A. Laboratory-Scale Lentiviral Vector Production and Purification for Enhanced Ex Vivo and In Vivo Genetic Engineering. MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 2020; 19:411-425. [PMID: 33294490 PMCID: PMC7683235 DOI: 10.1016/j.omtm.2020.10.009] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/30/2020] [Accepted: 10/13/2020] [Indexed: 12/31/2022]
Abstract
Lentiviral vectors (LVs) are increasingly employed in gene and cell therapy. Standard laboratory production of LVs is not easily scalable, and research-grade LVs often contain contaminants that can interfere with downstream applications. Moreover, purified LV production pipelines have been developed mainly for costly, large-scale, clinical-grade settings. Therefore, a standardized and cost-effective process is still needed to obtain efficient, reproducible, and properly executed experimental studies and preclinical development of ex vivo and in vivo gene therapies, as high infectivity and limited adverse reactions are important factors potentially influencing experimental outcomes also in preclinical settings. We describe here an optimized laboratory-scale workflow whereby an LV-containing supernatant is purified and concentrated by sequential chromatographic steps, obtaining biologically active LVs with an infectious titer and specific activity in the order of 109 transducing unit (TU)/mL and 5 × 104 TU/ng of HIV Gag p24, respectively. The purification workflow removes >99% of the starting plasmid, DNA, and protein impurities, resulting in higher gene transfer and editing efficiency in severe combined immunodeficiency (SCID)-repopulating hematopoietic stem and progenitor cells (HSPCs) ex vivo, as well as reduced activation of inflammatory responses ex vivo and in vivo as compared to TU-matched, laboratory-grade vectors. Our results highlight the value of accessible purified LV production for experimental studies and preclinical testing.
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Affiliation(s)
- Monica Soldi
- San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCSS Ospedale San Raffaele, 20132 Milan, Italy
| | - Lucia Sergi Sergi
- San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCSS Ospedale San Raffaele, 20132 Milan, Italy
| | - Giulia Unali
- San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCSS Ospedale San Raffaele, 20132 Milan, Italy.,Vita-Salute San Raffaele University, School of Medicine, 20132 Milan, Italy
| | - Thomas Kerzel
- San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCSS Ospedale San Raffaele, 20132 Milan, Italy.,Vita-Salute San Raffaele University, School of Medicine, 20132 Milan, Italy
| | - Ivan Cuccovillo
- San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCSS Ospedale San Raffaele, 20132 Milan, Italy
| | - Paola Capasso
- San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCSS Ospedale San Raffaele, 20132 Milan, Italy
| | - Andrea Annoni
- San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCSS Ospedale San Raffaele, 20132 Milan, Italy
| | - Mauro Biffi
- San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCSS Ospedale San Raffaele, 20132 Milan, Italy
| | - Paola Maria Vittoria Rancoita
- CUSSB-University Center for Statistics and the Biomedical Statistics, Vita-Salute San Raffaele University, 20132 Milan, Italy
| | - Alessio Cantore
- San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCSS Ospedale San Raffaele, 20132 Milan, Italy.,Vita-Salute San Raffaele University, School of Medicine, 20132 Milan, Italy
| | - Angelo Lombardo
- San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCSS Ospedale San Raffaele, 20132 Milan, Italy.,Vita-Salute San Raffaele University, School of Medicine, 20132 Milan, Italy
| | - Luigi Naldini
- San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCSS Ospedale San Raffaele, 20132 Milan, Italy.,Vita-Salute San Raffaele University, School of Medicine, 20132 Milan, Italy
| | - Mario Leonardo Squadrito
- San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCSS Ospedale San Raffaele, 20132 Milan, Italy
| | - Anna Kajaste-Rudnitski
- San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCSS Ospedale San Raffaele, 20132 Milan, Italy
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38
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Abstract
Therapeutic viral gene delivery is an emerging technology which aims to correct genetic mutations by introducing new genetic information to cells either to correct a faulty gene or to initiate cell death in oncolytic treatments. In recent years, significant scientific progress has led to several clinical trials resulting in the approval of gene therapies for human treatment. However, successful therapies remain limited due to a number of challenges such as inefficient cell uptake, low transduction efficiency (TE), limited tropism, liver toxicity and immune response. To adress these issues and increase the number of available therapies, additives from a broad range of materials like polymers, peptides, lipids, nanoparticles, and small molecules have been applied so far. The scope of this review is to highlight these selected delivery systems from a materials perspective.
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Affiliation(s)
- Kübra Kaygisiz
- Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany.
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39
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Emerson J, Kara B, Glassey J. Multivariate data analysis in cell gene therapy manufacturing. Biotechnol Adv 2020; 45:107637. [PMID: 32980438 DOI: 10.1016/j.biotechadv.2020.107637] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2020] [Revised: 07/27/2020] [Accepted: 09/22/2020] [Indexed: 01/26/2023]
Abstract
The emergence of cell gene therapy (CGT) as a safe and efficacious treatment for numerous severe inherited and acquired human diseases has led to growing interest and investment in new CGT products. The most successful of these have been autologous viral vector-based treatments. The development of viral vector manufacturing processes and ex vivo patient cell processing capabilities is a pressing issue in the advancement of autologous viral vector-based CGT treatments. In viral vector production, scale-up is a critical task due to the limited scalability of traditional laboratory systems and the demand for high volumes of viral vector manufactured in accordance with current good manufacturing practice. Ex vivo cell processing methods require optimisation and automation before they can be scaled out, and several other manufacturing challenges are prevalent such as high levels of raw material and process variability, difficulty characterising complex materials, and a lack of knowledge of critical process parameters and their effect on critical quality attributes of the viral vector and cell drug products. Multivariate data analysis (MVDA) has been leveraged successfully in a variety of applications in the chemical and biochemical industries, including for tasks such as bioprocess monitoring, identification of critical process parameters and assessment of process variability and comparability during process development, scale-up and technology transfer. Henceforth, MVDA is reviewed here as a suitable tool for tackling some of the challenges faced in the development of CGT manufacturing processes. A summary of some key CGT manufacturing challenges is provided along with a review of MVDA applications to mammalian and microbial processes, and an exploration of the potential benefits, requirements and pre-requisites of MVDA applications in the development of CGT manufacturing processes.
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Affiliation(s)
- Joseph Emerson
- School of Engineering, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK.
| | - Bo Kara
- Currently, Evox Therapeutics, Medawar Centre, Oxford OX4 4HG, UK.
| | - Jarka Glassey
- School of Engineering, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK.
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Bae DH, Marino M, Iaffaldano B, Fenstermaker S, Afione S, Argaw T, McCright J, Kwilas A, Chiorini JA, Timmons AE, Reiser J. Design and Testing of Vector-Producing HEK293T Cells Bearing a Genomic Deletion of the SV40 T Antigen Coding Region. Mol Ther Methods Clin Dev 2020; 18:631-638. [PMID: 32775497 PMCID: PMC7397404 DOI: 10.1016/j.omtm.2020.07.006] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2020] [Accepted: 07/06/2020] [Indexed: 12/18/2022]
Abstract
The use of the human embryonic kidney (HEK) 293T cell line to manufacture vectors for in vivo applications raises safety concerns due to the presence of SV40 T antigen-encoding sequences. We used CRISPR-Cas9 genome editing to remove the SV40 T antigen-encoding sequences from HEK293T cells by transfecting them with a recombinant plasmid expressing Cas9 and two distinct single guide RNAs (sgRNAs) corresponding to the beginning and end of the T antigen coding region. Cell clones lacking T antigen-encoding sequences were identified using PCR. Whole-genome (WG) and targeted locus amplification (TLA) sequencing of the parental HEK293T cell line revealed multiple SV40 T antigen-encoding sequences replacing cellular sequences on chromosome 3. The putative T antigen null clones demonstrated a loss of sequence reads mapping to T antigen-encoding sequences. Western blot analysis of cell extracts prepared from the T antigen null clones confirmed that the SV40 large and small T antigen proteins were absent. Lentiviral vectors produced using the T antigen null clones exhibited titers up to 1.5 × 107 transducing units (TU)/mL, while the titers obtained from the parent HEK293T cell line were up to 4 × 107 TU/mL. The capacity of the T antigen-negative cells to produce high titer adeno-associated virus (AAV) vectors was also evaluated. The results obtained revealed that the lack of T antigen sequences did not impact AAV vector titers.
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Affiliation(s)
- Dahae Hailey Bae
- Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration (FDA), Silver Spring, MD, USA
| | - Michael Marino
- Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration (FDA), Silver Spring, MD, USA
| | - Brian Iaffaldano
- Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration (FDA), Silver Spring, MD, USA
| | - Sydney Fenstermaker
- Office of Biostatistics and Epidemiology, Center for Biologics Evaluation and Research, FDA, Silver Spring, MD, USA
| | - Sandra Afione
- AAV Biology Section, National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD, USA
| | - Takele Argaw
- Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration (FDA), Silver Spring, MD, USA
| | - Jacob McCright
- Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration (FDA), Silver Spring, MD, USA
| | - Anna Kwilas
- Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration (FDA), Silver Spring, MD, USA
| | - John A. Chiorini
- AAV Biology Section, National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD, USA
| | - Andrew E. Timmons
- Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration (FDA), Silver Spring, MD, USA
| | - Jakob Reiser
- Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration (FDA), Silver Spring, MD, USA
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41
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Formas‐Oliveira AS, Basílio JS, Rodrigues AF, Coroadinha AS. Overexpression of ER Protein Processing and Apoptosis Regulator Genes in Human Embryonic Kidney 293 Cells Improves Gene Therapy Vectors Production. Biotechnol J 2020; 15:e1900562. [DOI: 10.1002/biot.201900562] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2019] [Revised: 05/22/2020] [Indexed: 12/18/2022]
Affiliation(s)
- Ana S. Formas‐Oliveira
- iBET Instituto de Biologia Experimental e Tecnológica Apartado 12 2781‐901 Oeiras Portugal
- Instituto de Tecnologia Química e Biológica António Xavier Universidade Nova de Lisboa Av. da República 2780‐157 Oeiras Portugal
| | - João S. Basílio
- iBET Instituto de Biologia Experimental e Tecnológica Apartado 12 2781‐901 Oeiras Portugal
- Instituto de Tecnologia Química e Biológica António Xavier Universidade Nova de Lisboa Av. da República 2780‐157 Oeiras Portugal
| | - Ana F. Rodrigues
- iBET Instituto de Biologia Experimental e Tecnológica Apartado 12 2781‐901 Oeiras Portugal
- Instituto de Tecnologia Química e Biológica António Xavier Universidade Nova de Lisboa Av. da República 2780‐157 Oeiras Portugal
| | - Ana S. Coroadinha
- iBET Instituto de Biologia Experimental e Tecnológica Apartado 12 2781‐901 Oeiras Portugal
- Instituto de Tecnologia Química e Biológica António Xavier Universidade Nova de Lisboa Av. da República 2780‐157 Oeiras Portugal
- The Discoveries centre for Regenerative and Precision Medicine Nova University Lisbon Oeiras Campus, Av. da República 2780‐157 Oeiras Portugal
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42
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CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy. Cancers (Basel) 2020; 12:cancers12061704. [PMID: 32604839 PMCID: PMC7352666 DOI: 10.3390/cancers12061704] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2020] [Revised: 06/22/2020] [Accepted: 06/24/2020] [Indexed: 01/03/2023] Open
Abstract
T cells engineered with chimeric antigen receptors (CARs) show great promise in the treatment of some cancers. Modifying T cells to express CARs generally relies on T-cell transduction using viral vectors carrying a transgene, resulting in semi-random DNA integration within the T-cell genome. While this approach has proven successful and is used in generating the Food and Drug Administration (FDA, USA) approved B-lymphocyte antigen CD19-specific CAR T cells, it is possible the transgene could integrate into a locus that would lead to malignant transformation of the engineered T cells. In addition, manufacturing viral vectors is time-consuming and expensive. One way to overcome these challenges is site-specific gene integration, which can be achieved through clustered regularly interspaced short palindromic repeat (CRISPR) mediated editing and non-viral DNA, which serves as a template for homology-directed repair (HDR). This non-viral gene editing approach provides a rapid, highly specific, and inexpensive way to engineer T cells. Here, we describe an optimized protocol for the site-specific knock-in of a large transgene in primary human T cells using non-viral double stranded DNA as a repair template. As proof-of-principle, we targeted the T-cell receptor alpha constant (TRAC) locus for insertion of a large transgene containing green fluorescence protein (GFP) and interleukin-15 (IL-15). To optimize the knock-in conditions we tested template DNA concentration, homology arm length, cell number, and knock-in efficiency over time. We then applied these established guidelines to target the TRAC or interleukin-13 (IL-13) locus for the knock-in of synthetic molecules, such as a CAR, bispecific T-cell engager (BiTE), and other transgenes. While integration efficiency depends on the targeted gene locus and selected transgene, this optimized protocol reliably generates the desired insertion at rates upwards of 20%. Thus, it should serve as a good starting point for investigators who are interested in knocking in transgenes into specific loci.
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43
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Valkama AJ, Oruetxebarria I, Lipponen EM, Leinonen HM, Käyhty P, Hynynen H, Turkki V, Malinen J, Miinalainen T, Heikura T, Parker NR, Ylä-Herttuala S, Lesch HP. Development of Large-Scale Downstream Processing for Lentiviral Vectors. MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 2020; 17:717-730. [PMID: 32346549 PMCID: PMC7177191 DOI: 10.1016/j.omtm.2020.03.025] [Citation(s) in RCA: 40] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/04/2020] [Accepted: 03/25/2020] [Indexed: 02/06/2023]
Abstract
The interest in lentiviral vectors (LVs) has increased prominently for gene therapy applications, but few have reached the later stages of clinical trials. The main challenge has remained in scaling up the manufacturing process for the fragile vector to obtain high titers for in vivo usage. We have previously scaled up the LV production to iCELLis 500, being able to produce up to 180 L of harvest material in one run with perfusion. The following challenge considers the purification and concentration of the product to meet titer and purity requirements for clinical use. We have developed a downstream process, beginning with clarification, buffer exchange, and concentration, by tangential flow filtration. This is followed by a purification step using single membrane-based anion exchange chromatography and final formulation with tangential flow filtration. Different materials and conditions were compared to optimize the process, especially for the chromatography step that has been the bottleneck in lentiviral vector purification scale-up. The final infectious titer of the lentiviral vector product manufactured using the optimized scale-up process was determined to be 1.97 × 109 transducing units (TU)/mL, which can be considered as a high titer for lentiviral vectors.
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Affiliation(s)
- Anniina J Valkama
- Kuopio Center for Gene and Cell Therapy, 70210 Kuopio, Finland
- FinVector, 70210 Kuopio, Finland
- Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland
| | - Igor Oruetxebarria
- Kuopio Center for Gene and Cell Therapy, 70210 Kuopio, Finland
- FinVector, 70210 Kuopio, Finland
| | - Eevi M Lipponen
- Kuopio Center for Gene and Cell Therapy, 70210 Kuopio, Finland
- FinVector, 70210 Kuopio, Finland
| | - Hanna M Leinonen
- Kuopio Center for Gene and Cell Therapy, 70210 Kuopio, Finland
- FinVector, 70210 Kuopio, Finland
| | - Piia Käyhty
- Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland
| | - Heidi Hynynen
- Kuopio Center for Gene and Cell Therapy, 70210 Kuopio, Finland
- FinVector, 70210 Kuopio, Finland
| | - Vesa Turkki
- Kuopio Center for Gene and Cell Therapy, 70210 Kuopio, Finland
- FinVector, 70210 Kuopio, Finland
| | - Joonas Malinen
- Kuopio Center for Gene and Cell Therapy, 70210 Kuopio, Finland
- FinVector, 70210 Kuopio, Finland
| | - Tuukka Miinalainen
- Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland
| | - Tommi Heikura
- Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland
| | - Nigel R Parker
- Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland
| | - Seppo Ylä-Herttuala
- Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland
| | - Hanna P Lesch
- Kuopio Center for Gene and Cell Therapy, 70210 Kuopio, Finland
- FinVector, 70210 Kuopio, Finland
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44
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Boudeffa D, Bertin B, Biek A, Mormin M, Leseigneur F, Galy A, Merten OW. Toward a Scalable Purification Protocol of GaLV-TR-Pseudotyped Lentiviral Vectors. Hum Gene Ther Methods 2020; 30:153-171. [PMID: 31516018 DOI: 10.1089/hgtb.2019.076] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Lentiviral vectors (LV) that are used in research and development as well as in clinical trials are in majority vesicular stomatitis virus G glycoprotein (VSVg) pseudotyped. The predominance of this pseudotype choice for clinical gene therapy studies is largely due to a lack of purification schemes for pseudotypes other than VSVg. In this study, we report for the first time the development of a new downstream process protocol allowing high-yield production of stable and infectious gibbon ape leukemia virus (GaLV)-TR-LV particles. We identified critical conditions in tangential flow filtration (TFF) and chromatographic steps for preserving the infectivity/functionality of LV during purification. This was carried out by identifying for each step, the critical parameters affecting LV infectivity, including pH, salinity, presence of stabilizers, temperature, and by defining the optimal order of these steps. A three-step process was developed for GaLV-TR-LV purification consisting of one TFF and two chromatographic steps (ion-exchange chromatography and size exclusion chromatography) permitting recoveries of >27% of infectious particles. With this process, purified GaLV-pseudotyped LV enabled the transduction of 70% human CD34+ cells in the presence of the Vectofusin-1 peptide, whereas in the same conditions nonpurified vector transduced only 9% of the cells (multiplicity of infection 20). Our protocol will allow for the first time the purification of GaLV-TR-LV that are biologically active, stable, and with sufficient recovery in the perspective of preclinical studies and clinical applications. Obviously, further optimizations are required to improve final vector yields.
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Affiliation(s)
| | | | | | - Mirella Mormin
- Généthon, Evry, France.,Integrare Research Unit (UMR_S951), Généthon, Inserm, Université Evry Val-d'Essonne, Université Paris Saclay, EPHE, Evry, France
| | | | - Anne Galy
- Généthon, Evry, France.,Integrare Research Unit (UMR_S951), Généthon, Inserm, Université Evry Val-d'Essonne, Université Paris Saclay, EPHE, Evry, France
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45
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Kohn DB, Booth C, Kang EM, Pai SY, Shaw KL, Santilli G, Armant M, Buckland KF, Choi U, De Ravin SS, Dorsey MJ, Kuo CY, Leon-Rico D, Rivat C, Izotova N, Gilmour K, Snell K, Dip JXB, Darwish J, Morris EC, Terrazas D, Wang LD, Bauser CA, Paprotka T, Kuhns DB, Gregg J, Raymond HE, Everett JK, Honnet G, Biasco L, Newburger PE, Bushman FD, Grez M, Gaspar HB, Williams DA, Malech HL, Galy A, Thrasher AJ. Lentiviral gene therapy for X-linked chronic granulomatous disease. Nat Med 2020; 26:200-206. [PMID: 31988463 PMCID: PMC7115833 DOI: 10.1038/s41591-019-0735-5] [Citation(s) in RCA: 170] [Impact Index Per Article: 34.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2019] [Accepted: 12/10/2019] [Indexed: 12/24/2022]
Abstract
Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytic cells1,2. We report the initial results of nine severely affected X-linked CGD (X-CGD) patients who received ex vivo autologous CD34+ hematopoietic stem and progenitor cell-based lentiviral gene therapy following myeloablative conditioning in first-in-human studies (trial registry nos. NCT02234934 and NCT01855685). The primary objectives were to assess the safety and evaluate the efficacy and stability of biochemical and functional reconstitution in the progeny of engrafted cells at 12 months. The secondary objectives included the evaluation of augmented immunity against bacterial and fungal infection, as well as assessment of hematopoietic stem cell transduction and engraftment. Two enrolled patients died within 3 months of treatment from pre-existing comorbidities. At 12 months, six of the seven surviving patients demonstrated stable vector copy numbers (0.4-1.8 copies per neutrophil) and the persistence of 16-46% oxidase-positive neutrophils. There was no molecular evidence of either clonal dysregulation or transgene silencing. Surviving patients have had no new CGD-related infections, and six have been able to discontinue CGD-related antibiotic prophylaxis. The primary objective was met in six of the nine patients at 12 months follow-up, suggesting that autologous gene therapy is a promising approach for CGD patients.
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Affiliation(s)
| | - Claire Booth
- Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Foundation Trust, London, UK
| | - Elizabeth M Kang
- Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Sung-Yun Pai
- Boston Children's Hospital, Harvard Medical School, Boston, MA, USA
| | - Kit L Shaw
- University of California, Los Angeles, CA, USA
| | - Giorgia Santilli
- Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Foundation Trust, London, UK
| | - Myriam Armant
- Boston Children's Hospital, Harvard Medical School, Boston, MA, USA
| | - Karen F Buckland
- Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Foundation Trust, London, UK
| | - Uimook Choi
- Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Suk See De Ravin
- Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | | | | | - Diego Leon-Rico
- Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Foundation Trust, London, UK
| | - Christine Rivat
- Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Foundation Trust, London, UK
| | - Natalia Izotova
- Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Foundation Trust, London, UK
| | - Kimberly Gilmour
- Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Foundation Trust, London, UK
| | - Katie Snell
- Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Foundation Trust, London, UK
| | - Jinhua Xu-Bayford Dip
- Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Foundation Trust, London, UK
| | - Jinan Darwish
- Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Foundation Trust, London, UK
| | - Emma C Morris
- University College London Hospitals NHS Foundation Trust, London, UK
| | | | - Leo D Wang
- Boston Children's Hospital, Harvard Medical School, Boston, MA, USA
- City of Hope, Beckman Research Institute, Duarte, CA, USA
| | | | | | - Douglas B Kuhns
- Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA
| | - John Gregg
- University of Pennsylvania, Philadelphia, PA, USA
| | | | | | | | - Luca Biasco
- Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Foundation Trust, London, UK
| | | | | | | | - H Bobby Gaspar
- Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Foundation Trust, London, UK
- Orchard Therapeutics, London, UK
| | - David A Williams
- Boston Children's Hospital, Harvard Medical School, Boston, MA, USA
| | - Harry L Malech
- Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Anne Galy
- Genethon, Evry, France
- Inserm, University of Evry, Université Paris Saclay Genethon, Evry, France
| | - Adrian J Thrasher
- Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Foundation Trust, London, UK.
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Lavado-García J, Jorge I, Cervera L, Vázquez J, Gòdia F. Multiplexed Quantitative Proteomic Analysis of HEK293 Provides Insights into Molecular Changes Associated with the Cell Density Effect, Transient Transfection, and Virus-Like Particle Production. J Proteome Res 2020; 19:1085-1099. [DOI: 10.1021/acs.jproteome.9b00601] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Affiliation(s)
- Jesús Lavado-García
- Grup d’Enginyeria Cellular i Bioprocés, Departament d’Enginyeria Química, Biològica i Ambiental, Escola d’Enginyeria, Universitat Autònoma de Barcelona, Campus de Bellaterra, Cerdanyola del Vallès, 08193 Barcelona, Spain
| | - Inmaculada Jorge
- Laboratory of Cardiovascular Proteomics, Centro Nacional Investigaciones Cardiovasculares (CNIC), C/Melchor Fernández Almagro 3, Madrid 28029, Spain
- Centro de Investigación Biomédica en Red Enfermedades Cardiovasculares (CIBERCV), Madrid 28029, Spain
| | - Laura Cervera
- Grup d’Enginyeria Cellular i Bioprocés, Departament d’Enginyeria Química, Biològica i Ambiental, Escola d’Enginyeria, Universitat Autònoma de Barcelona, Campus de Bellaterra, Cerdanyola del Vallès, 08193 Barcelona, Spain
| | - Jesús Vázquez
- Laboratory of Cardiovascular Proteomics, Centro Nacional Investigaciones Cardiovasculares (CNIC), C/Melchor Fernández Almagro 3, Madrid 28029, Spain
- Centro de Investigación Biomédica en Red Enfermedades Cardiovasculares (CIBERCV), Madrid 28029, Spain
| | - Francesc Gòdia
- Grup d’Enginyeria Cellular i Bioprocés, Departament d’Enginyeria Química, Biològica i Ambiental, Escola d’Enginyeria, Universitat Autònoma de Barcelona, Campus de Bellaterra, Cerdanyola del Vallès, 08193 Barcelona, Spain
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Abstract
One of the most versatile gene transfer methods involves the use of recombinant lentiviral vectors since they can transduce both dividing and nondividing cells, are considered to be safe and provide long-term transgene expression since the integrated viral genome, the provirus, is passed on to daughter cells. These characteristics are highly desirable when a modified cell must continue to express the transgene even after multiple cell divisions. Lentiviral vectors are often used to introduce protein encoding cDNAs, such as reporter genes, or for noncoding sequences, such as mediators of RNA interference or genome editing, including shRNA or gRNA, respectively. In the gene therapy setting, lentiviral vectors have been used successfully for the modification of hematopoietic stem cells, resulting in restored immune function or correction of defects in hemoglobin, to name but a few examples. The success of chimeric antigen receptor (CAR) T cells for the treatment of B cell leukemias and lymphomas has been particularly striking and this approach has relied heavily on lentivirus-mediated gene transfer. Here we present a typical protocol for the production of lentivirus, concentration by ultracentrifugation and determination of virus titer. The resulting virus can then be used in laboratory assays of gene transfer, including the establishment of CAR T cells.
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48
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Lewandowicz-Uszyńska A, Pasternak G, Świerkot J, Bogunia-Kubik K. Primary Immunodeficiencies: Diseases of Children and Adults - A Review. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2020; 1289:37-54. [PMID: 32803731 DOI: 10.1007/5584_2020_556] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Primary immunodeficiencies (PIDs) belong to a group of rare congenital diseases occurring all over the world that may be seen in both children and adults. In most cases, genetic predispositions are already known. As shown in this review, genetic abnormalities may be related to dysfunction of the immune system, which manifests itself as recurrent infections, increased risk of cancer, and autoimmune diseases. This article reviews the various forms of PIDs, including their characterization, management strategies, and complications. Novel aspects of the diagnostics and monitoring of PIDs are presented.
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Affiliation(s)
- Aleksandra Lewandowicz-Uszyńska
- Third Department and Clinic of Pediatrics, Immunology and Rheumatology of Developmental Age, Wroclaw Medical University, Wroclaw, Poland. .,Department of Immunology and Pediatrics, The J. Gromkowski Provincial Hospital, Wroclaw, Poland.
| | - Gerard Pasternak
- Third Department and Clinic of Pediatrics, Immunology and Rheumatology of Developmental Age, Wroclaw Medical University, Wroclaw, Poland
| | - Jerzy Świerkot
- Department and Clinic of Rheumatology and Internal Medicine, Wroclaw Medical University, Wroclaw, Poland
| | - Katarzyna Bogunia-Kubik
- Laboratory of Clinical Immunogenetics and Pharmacogenetics, The Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland
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49
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Radek C, Bernadin O, Drechsel K, Cordes N, Pfeifer R, Sträßer P, Mormin M, Gutierrez-Guerrero A, Cosset FL, Kaiser AD, Schaser T, Galy A, Verhoeyen E, Johnston IC. Vectofusin-1 Improves Transduction of Primary Human Cells with Diverse Retroviral and Lentiviral Pseudotypes, Enabling Robust, Automated Closed-System Manufacturing. Hum Gene Ther 2019; 30:1477-1493. [PMID: 31578886 PMCID: PMC6919281 DOI: 10.1089/hum.2019.157] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2019] [Accepted: 09/21/2019] [Indexed: 01/07/2023] Open
Abstract
Cell and gene therapies are finally becoming viable patient treatment options, with both T cell- and hematopoietic stem cell (HSC)-based therapies being approved to market in Europe. However, these therapies, which involve the use of viral vector to modify the target cells, are expensive and there is an urgent need to reduce manufacturing costs. One major cost factor is the viral vector production itself, therefore improving the gene modification efficiency could significantly reduce the amount of vector required per patient. This study describes the use of a transduction enhancing peptide, Vectofusin-1®, to improve the transduction efficiency of primary target cells using lentiviral and gammaretroviral vectors (LV and RV) pseudotyped with a variety of envelope proteins. Using Vectofusin-1 in combination with LV pseudotyped with viral glycoproteins derived from baboon endogenous retrovirus, feline endogenous virus (RD114), and measles virus (MV), a strongly improved transduction of HSCs, B cells and T cells, even when cultivated under low stimulation conditions, could be observed. The formation of Vectofusin-1 complexes with MV-LV retargeted to CD20 did not alter the selectivity in mixed cell culture populations, emphasizing the precision of this targeting technology. Functional, ErbB2-specific chimeric antigen receptor-expressing T cells could be generated using a gibbon ape leukemia virus (GALV)-pseudotyped RV. Using a variety of viral vectors and target cells, Vectofusin-1 performed in a comparable manner to the traditionally used surface-bound recombinant fibronectin. As Vectofusin-1 is a soluble peptide, it was possible to easily transfer the T cell transduction method to an automated closed manufacturing platform, where proof of concept studies demonstrated efficient genetic modification of T cells with GALV-RV and RD114-RV and the subsequent expansion of mainly central memory T cells to a clinically relevant dose.
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Affiliation(s)
| | - Ornellie Bernadin
- CIRI—International Center for Infectiology Research, Team EVIR, Université de Lyon, Lyon, France
- Inserm, U1111, Ecole Normale Supérieure de Lyon, Lyon, France
- Université Lyon 1, CNRS, UMR5308, Lyon, France
| | | | - Nicole Cordes
- Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany
- Faculty of Biology, University of Freiburg, Freiburg, Germany
| | - Rita Pfeifer
- Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany
| | - Pia Sträßer
- Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany
| | - Mirella Mormin
- Integrare Research Unit UMR_S951, Genethon, INSERM, University Evry, EPHE, Evry, France
| | - Alejandra Gutierrez-Guerrero
- CIRI—International Center for Infectiology Research, Team EVIR, Université de Lyon, Lyon, France
- Inserm, U1111, Ecole Normale Supérieure de Lyon, Lyon, France
- Université Lyon 1, CNRS, UMR5308, Lyon, France
| | - François-loïc Cosset
- CIRI—International Center for Infectiology Research, Team EVIR, Université de Lyon, Lyon, France
- Inserm, U1111, Ecole Normale Supérieure de Lyon, Lyon, France
- Université Lyon 1, CNRS, UMR5308, Lyon, France
| | | | - Thomas Schaser
- Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany
| | - Anne Galy
- Integrare Research Unit UMR_S951, Genethon, INSERM, University Evry, EPHE, Evry, France
| | - Els Verhoeyen
- CIRI—International Center for Infectiology Research, Team EVIR, Université de Lyon, Lyon, France
- Inserm, U1111, Ecole Normale Supérieure de Lyon, Lyon, France
- Université Lyon 1, CNRS, UMR5308, Lyon, France
- Université Côte d'Azur, INSERM, Centre Méditerranéen de Médecine Moléculaire (C3M), Nice, France
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50
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Bauler M, Roberts JK, Wu CC, Fan B, Ferrara F, Yip BH, Diao S, Kim YI, Moore J, Zhou S, Wielgosz MM, Ryu B, Throm RE. Production of Lentiviral Vectors Using Suspension Cells Grown in Serum-free Media. MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 2019; 17:58-68. [PMID: 31890741 PMCID: PMC6931067 DOI: 10.1016/j.omtm.2019.11.011] [Citation(s) in RCA: 62] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/31/2019] [Accepted: 11/15/2019] [Indexed: 02/09/2023]
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.
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Affiliation(s)
- Matthew Bauler
- Vector Development and Production Laboratory, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Jessica K Roberts
- Vector Development and Production Laboratory, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Chang-Chih Wu
- Vector Development and Production Laboratory, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Baochang Fan
- Therapeutics Production and Quality, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Francesca Ferrara
- Vector Development and Production Laboratory, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Bon Ham Yip
- Vector Development and Production Laboratory, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Shiyong Diao
- Vector Development and Production Laboratory, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Young-In Kim
- Experimental Cell Therapeutics Lab, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Jennifer Moore
- Experimental Cell Therapeutics Lab, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Sheng Zhou
- Experimental Cell Therapeutics Lab, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Matthew M Wielgosz
- Vector Development and Production Laboratory, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Byoung Ryu
- Vector Development and Production Laboratory, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Robert E Throm
- Vector Development and Production Laboratory, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
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