Systematic improvements in lentiviral transduction of primary human natural killer cells undergoing ex vivo expansion
| Autor: | Richard W. Childs, Giacomo C. Waller, Mala Chakraborty, Michael J. Hochman, David S.J. Allan, Akkapon Poolcharoen, Robert Reger |
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| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
lcsh:QH426-470 Transgene Cell Green fluorescent protein Viral vector 03 medical and health sciences Transduction (genetics) 0302 clinical medicine Genetics medicine lcsh:QH573-671 Molecular Biology biology lcsh:Cytology Lymphoblast Interleukin biology.organism_classification Cell biology lcsh:Genetics 030104 developmental biology medicine.anatomical_structure Vesicular stomatitis virus 030220 oncology & carcinogenesis Molecular Medicine Original Article |
| Zdroj: | Molecular Therapy. Methods & Clinical Development Molecular Therapy: Methods & Clinical Development, Vol 20, Iss, Pp 559-571 (2021) |
| ISSN: | 2329-0501 |
| DOI: | 10.1016/j.omtm.2021.01.008 |
| Popis: | Transduction of primary human natural killer (NK) cells with lentiviral vectors has historically been challenging. We sought to evaluate multiple parameters to optimize lentiviral transduction of human peripheral blood NK cells being expanded to large numbers using a good manufacturing practice (GMP)-compliant protocol that utilizes irradiated lymphoblastoid (LCL) feeder cells. Although prestimulation of NK cells with interleukin (IL)-2 for 2 or more days facilitated transduction with vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped lentivirus, there was a subsequent impairment in the capacity of transduced NK cells to proliferate when stimulated with LCL feeder cells. In contrast, incubation of human NK cells with LCL feeder cells plus IL-2 before transduction in the presence of the TBK1 inhibitor BX795 resulted in efficient lentiviral integration (mean of 23% transgene+ NK cells) and successful subsequent proliferation of the transduced cells. Investigation of multiple internal promoter sequences within the same lentiviral vector revealed differences in percentage and level of transgene expression per NK cell. Bicistronic lentiviral vectors encoding both GFP and proteins suitable for the isolation of transduced cells with magnetic beads led to efficient transgene expression in NK cells. The optimized approaches described herein provide a template for protocols that generate large numbers of fully functional and highly purified lentivirus-transduced NK cells for clinical trials. Graphical Abstract By systematically evaluating multiple potential influencing factors, Childs and colleagues merge protocols to optimize lentiviral transduction and ex vivo expansion of human NK cells via co-culture with irradiated LCL feeder cells to produce large numbers of genetically modified NK cells for cancer immunotherapy. |
| Databáze: | OpenAIRE |
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