Transient blockade of TBK1/IKK epsilon allows efficient transduction of primary human NK cells with VSV-G-pseudotyped lentiviral vectors
In: Cytotherapy, 2021
academicJournal
Zugriff:
BACKGROUND AIMS: VSV-G pseudotyped lentiviral vectors (LVs) are widely used to reliably generate genetically modified, clinical grade T-cell products. However, the results of genetically modifying NK cells with VSV-G LVs have been variable. We explored if inhibition of the IKK-related protein kinases, TBK1/IKKε, key signaling molecules of the endosomal TLR4 pathway, which is activated by VSV-G, would enable the reliable transduction of NK cells by VSV-G LVs. METHODS: We activated NK cells from PBMCs using standard procedures and transduced them with VSV-G LVs encoding a marker gene (YFP) or functional genes (CARs, costimulatory molecules) in the presence of three TBK1/IKKε inhibitors (MRT67307, BX-795, Amlexanox). NK cell transduction was evaluated by flowcytometry and/or western blot and the functionality of expressed CARs was evaluated in vitro. RESULTS: Blocking TBK1/IKKε during transduction of NK cells enabled their efficient transduction by VSV-G LVs as judged by YFP expression of 40 to 50% with EC50s of 1.1 μM (MRT67307), 5 μM (BX-795), and 24.8 μM (Amlexanox). Focusing on MRT67307, we successfully generated NK cells expressing CD19-CARs or HER2-CARs with an inducible costimulatory molecule. CAR NK cells exhibited increased cytolytic activity and ability to produce cytokines in comparison to untreated controls, confirming CAR functionality. CONCLUSIONS: We demonstrate that inhibition of TBK1/IKKε enables the reliable generation of genetically modified NK cells using VSV-G LVs. Our protocol can be readily adapted to generate clinical grade NK cells and thus, has the potential to facilitate the clinical evaluation of genetically modified NK cell-based therapeutics in the future.
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Transient blockade of TBK1/IKK epsilon allows efficient transduction of primary human NK cells with VSV-G-pseudotyped lentiviral vectors
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Autor/in / Beteiligte Person: | Chockley, Peter ; Patil, Sagar L ; Gottschalk, Stephen |
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Zeitschrift: | Cytotherapy, 2021 |
Veröffentlichung: | 2021 |
Medientyp: | academicJournal |
DOI: | 10.1016/j.jcyt.2021.04.010 |
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