Dataset Information

Transient blockade of TBK1/IKKε allows efficient transduction of primary human natural killer cells with vesicular stomatitis virus G-pseudotyped lentiviral vectors.

ABSTRACT:

Background aims

Vesicular stomatitis virus G (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 natural killer (NK) cells with VSV-G LVs have been variable. The authors explored whether inhibition of the IKK-related protein kinases TBK1 and 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

The authors activated NK cells from peripheral blood mononuclear cells using standard procedures and transduced them with VSV-G LVs encoding a marker gene (yellow fluorescent protein [YFP]) or functional genes (chimeric antigen receptors [CARs], co-stimulatory molecules) in the presence of three TBK1/IKKε inhibitors (MRT67307, BX-795, amlexanox). NK cell transduction was evaluated by flow cytometry 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 YFPexpression of 40-50%, with half maximal effective concentrations of 1.1 µM (MRT67307), 5 µM (BX-795) and 24.8 µM (amlexanox). Focusing on MRT67307, the authors successfully generated NK cells expressing CD19-CARs or HER2-CARs with an inducible co-stimulatory molecule. CAR NK cells exhibited increased cytolytic activity and ability to produce cytokines in comparison to untreated controls, confirming CAR functionality.

Conclusions

The authors demonstrate that inhibition of TBK1/IKKε enables the reliable generation of genetically modified NK cells using VSV-G LVs. The authors' 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.

SUBMITTER: Chockley P

PROVIDER: S-EPMC8425283 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Publications

Transient blockade of TBK1/IKKε allows efficient transduction of primary human natural killer cells with vesicular stomatitis virus G-pseudotyped lentiviral vectors.

Chockley Peter P Patil Sagar L SL Gottschalk Stephen S

Cytotherapy 20210609 9

<h4>Background aims</h4>Vesicular stomatitis virus G (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 natural killer (NK) cells with VSV-G LVs have been variable. The authors explored whether inhibition of the IKK-related protein kinases TBK1 and IKKε, key signaling molecules of the endosomal TLR4 pathway, which is activated by VSV-G, would enable the reliable tran ...[more]

PMID: 34119434

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Project description:UnlabelledTo investigate the potential benefits which may arise from pseudotyping the HIV-1 lentiviral vector with its homologous gp41 envelope glycoprotein (GP) cytoplasmic tail (CT), we created chimeric RVG/HIV-1gp41 GPs composed of the extracellular and transmembrane sequences of RVG and either the full-length gp41 CT or C terminus gp41 truncations sequentially removing existing conserved motifs. Lentiviruses (LVs) pseudotyped with the chimeric GPs were evaluated in terms of particle release (physical titer), biological titers, infectivity, and in vivo central nervous system (CNS) transduction. We report here that LVs carrying shorter CTs expressed higher levels of envelope GP and showed a higher average infectivity than those bearing full-length GPs. Interestingly, complete removal of GP CT led to vectors with the highest transduction efficiency. Removal of all C-terminal gp41 CT conserved motifs, leaving just 17 amino acids (aa), appeared to preserve infectivity and resulted in a significantly increased physical titer. Furthermore, incorporation of these 17 aa in the RVG CT notably enhanced the physical titer. In vivo stereotaxic delivery of LV vectors exhibiting the best in vitro titers into rodent striatum facilitated efficient transduction of the CNS at the site of injection. A particular observation was the improved retrograde transduction of neurons in connected distal sites that resulted from the chimeric envelope R5 which included the "Kennedy" sequence (Ken) and lentivirus lytic peptide 2 (LLP2) conserved motifs in the CT, and although it did not exhibit a comparable high titer upon pseudotyping, it led to a significant increase in distal retrograde transduction of neurons.ImportanceIn this study, we have produced novel chimeric envelopes bearing the extracellular domain of rabies fused to the cytoplasmic tail (CT) of gp41 and pseudotyped lentiviral vectors with them. Here we report novel effects on the transduction efficiency and physical titer of these vectors, depending on CT length and context. We also managed to achieve increased neuronal transduction in vivo in the rodent CNS, thus demonstrating that the efficiency of these vectors can be enhanced following merely CT manipulation. We believe that this paper is a novel contribution to the field and opens the way for further attempts to surface engineer lentiviral vectors and make them more amenable for applications in human disease.

Dataset Information

Transient blockade of TBK1/IKKε allows efficient transduction of primary human natural killer cells with vesicular stomatitis virus G-pseudotyped lentiviral vectors.

Background aims

Methods

Results

Conclusions

Publications

Transient blockade of TBK1/IKKε allows efficient transduction of primary human natural killer cells with vesicular stomatitis virus G-pseudotyped lentiviral vectors.

Similar Datasets

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