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. 2020 Sep 23;18(1):363.
doi: 10.1186/s12967-020-02526-2.

Lentiviral delivery of combinatorial CAR/CRISPRi circuit into human primary T cells is enhanced by TBK1/IKKɛ complex inhibitor BX795

Lingyu Li  1 , Yuan Gao  2 , Richa Srivastava  2 , Wei Wang  3 , Qinghui Xiong  3 , Zhiming Fang  3 , Alejandra Pelayo  2 , Carolyn Denson  2 , Angshumala Goswami  2 , Rona Harari-Steinfeld  2 , Zhifen Yang  2 , Lihong Weng  3 , Lei Stanley Qi  4 , Francesco M Marincola  5
Affiliations

Lentiviral delivery of combinatorial CAR/CRISPRi circuit into human primary T cells is enhanced by TBK1/IKKɛ complex inhibitor BX795

Lingyu Li et al. J Transl Med. .

Abstract

Background: Adoptive transfer of engineered immune cells is a promising strategy for cancer treatment. However, low transduction efficiency particularly when large payload lentiviral vectors are used on primary T cells is a limitation for the development of cell therapy platforms that include multiple constructs bearing long DNA sequences. RB-340-1 is a new CAR T cell that combines two strategies in one product through a CRISPR interference (CRISPRi) circuit. Because multiple regulatory components are included in the circuit, RB-340-1 production needs delivery of two lentiviral vectors into human primary T cells, both containing long DNA sequences. To improve lentiviral transduction efficiency, we looked for inhibitors of receptors involved in antiviral response. BX795 is a pharmacological inhibitor of the TBK1/IKKɛ complex, which has been reported to augment lentiviral transduction of human NK cells and some cell lines, but it has not been tested with human primary T cells. The purpose of this study was to test if BX795 treatment promotes large payload RB-340-1 lentiviral transduction of human primary T cells.

Methods: To make the detection of gene delivery more convenient, we constructed another set of RB-340-1 constructs containing fluorescent labels named RB-340-1F. We incorporated BX795 treatment into the human primary T cell transduction procedure that was optimized for RB-340-1F. We tested BX795 with T cells collected from multiple donors, and detected the effect of BX795 on T cell transduction, phenotype, cell growth and cell function.

Results: We found that BX795 promotes RB-340-1F lentiviral transduction of human primary T cells, without dramatic change in cell growth and T cell functions. Meanwhile, BX795 treatment increased CD8+ T cell ratios in transduced T cells.

Conclusions: These results indicate that BX795 treatment is effective, and might be a safe approach to promote RB-340-1F lentiviral transduction of human primary T cells. This approach might also be helpful for other T cell therapy products that need delivery of complicated platform via large payload lentiviral vectors.

Keywords: BX795; CAR-T; CRISPRi; Lentiviral transduction; T cell engineering.

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Conflict of interest statement

The following patent US#9,856,497 relates to this work. Lei S. Qi is a co-founder and scientific advisor of Refuge Biotechnologies.

Figures

Fig. 1
Fig. 1
RB-340-1F two-vector platform and T cell transduction workflow. a Schematic representation of the first lentiviral vector LdCK-GFP and the second lentiviral vector CAR-TEV-mCherry. b The percentages of cells expressing the activation markers CD69, CD25, and cell surface protein LDLR. T cells were stimulated in anti-CD3/anti-CD28 pre-coated plate (CD3/CD28 activation). T cells cultured in non-coated plate were used as control (no activation). Results are the averages of two donors. c T cell transduction workflow and the time window to add BX795. The final concentration of BX795 in cell culture is 4 µM
Fig. 2
Fig. 2
Effect of BX795 treatment on RB-340-1F lentiviral vectors transduction efficiency. a Transgene expression was assessed by flow cytometry on day 6. b T cell transduction rate with the absence of BX795 (BX795-) or presence of BX795 (BX795+). GFP+/mCherry-: GFP single positive population. GFP+/mCherry + : GFP and mCherry double positive population. GFP-/mCherry+ : mCherry single positive population. c MFI of mCherry in CAR-TEV-mCherry transduced cells. d MFI of FITC in LdCK-GFP transduced cells. All results are the averages of four donors, except for figure A, which is flow cytometry plot from a representative donor. *p < 0.05; **p < 0.01
Fig. 3
Fig. 3
Effect of BX795 treatment on RB-340-1F transduction of CD4+ T cells and CD8+ T cells. a Percentage of CD4+ cells and percentage of CD8+ cells in the bulk population on day 6 before cell sorting. b Percentage of double transduced cells in CD4+ or CD8+ cells on day 6. BX795-: no BX795 treatment. BX795+ : with BX795 treatment. The above results are the average of four donors. c Percentage of CD4+ cells in double transduced cells on day 6 and day 13. d Percentage of CD8+ cells in double transduced cells on day 6 and day 13. BX795-: no BX795 treatment. BX795+ : with BX795 treatment. The above results are the average of three donors. *p < 0.05; **p < 0.01
Fig. 4
Fig. 4
Cell growth rate and CD27/CD28 expression of transduced T cells. a T cell expansion rate on day 3, day 6, and day 10, with the absence or presence of BX795. RB-340-1F transduction was performed on day 1 and day 2 as described in method, including non-transduced (NT) group as a control. Cell sorting is not carried out in this experiment. Fold of expansion is calculated by dividing the actual cell number on designated day with the cell number counted on day 1. b Expression of CD27 and CD28 in RB-340-1F double transduced cells on day 6 and day 10. Experiments were done with more than three donors. Data presented is from one representative donor
Fig. 5
Fig. 5
Functional assay of RB-340-1F double transduced cells. a Numbers of CAR + T cells existing in co-culture during 3 days killing assay against Fadu/PD-L1. b Numbers of Fadu/PD-L1 cells existing in co-culture during 3 days killing assay with non-transduced T cells (NT), RB-340-1F double transduced cells treated with or without BX795. c PD-1 positive percentage in double transduced T cells during 3 days killing assay against Fadu/PD-L1. Nosg groups are T cells transduced with LdCK-GFP vector and CAR-TEV-mCherry control vector without carrying any sgRNA sequence. PD1sg groups are RB-340-1F that contains LdCK-GFP and CAR-TEV-mCherry carrying PD-1 sgRNA sequence. d The concentration of secreted IL2, TNFα, and IFNγ in killing assay supernatant collected on day 1. All above results are the averages of triplicate wells. They are representative of five experiments done with T cells from two donors. e The average results of cytokine release from five experiments. To emphasize the fold change, cytokine concentrations in BX795 non-treated group are all normalized to 1. *p < 0.05; ns, not significant
Fig. 6
Fig. 6
Viral dose dependent transduction effect with BX795 treatment. a The percentage of double transduced cells with LdCK-GFP MOI 10, and CAR-TEV-mCherry MOI 5, 10, and 20, in the absence or presence of BX795. Data are the average of four experiments done with cells from two donors. b The percentage of double transduced cells with LdCK MOI 10, and MSLN CAR-TEV MOI 2.5, 5, 10, and 20, in the absence or presence of BX795. Data from one representative donor. c, d Virus dose-escalation for MSLN CAR-TEV transduction in human primary T cells in the absence or presence of BX795. Data from one representative donor
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