Efficient and Non-genotoxic RNA-Based Engineering of Human T Cells Using Tumor-Specific T Cell Receptors With Minimal TCR Mispairing

Abstract

Genetic engineering of T cells with tumor specific T-cell receptors (TCR) is a promising strategy to redirect their specificity against cancer cells in adoptive T cell therapy protocols. Most studies are exploiting integrating retro- or lentiviral vectors to permanently introduce the therapeutic TCR, which can pose serious safety issues when treatment-related toxicities would occur. Therefore, we developed a versatile, non-genotoxic transfection method for human unstimulated CD8⁺ T cells. We describe an optimized double sequential electroporation platform whereby Dicer-substrate small interfering RNAs (DsiRNA) are first introduced to suppress endogenous TCR α and β expression, followed by electroporation with DsiRNA-resistant tumor-specific TCR mRNA. We demonstrate that double sequential electroporation of human primary unstimulated T cells with DsiRNA and TCR mRNA leads to unprecedented levels of transgene TCR expression due to a strongly reduced degree of TCR mispairing. Importantly, superior transgenic TCR expression boosts epitope-specific CD8⁺ T cell activation and killing activity. Altogether, DsiRNA and TCR mRNA double sequential electroporation is a rapid, non-integrating and highly efficient approach with an enhanced biosafety profile to engineer T cells with antigen-specific TCRs for use in early phase clinical trials.

Keywords: DsiRNA; RNA transfection; TCR-gene transfer; adoptive cell therapy (ACT); electroporation.

Figure 1

Isolation and characterization of WT1₁₂₆-specific CTL clone. (A) WT1_126−134/HLA-A*02:01 tetramer staining and WT1_126−134 peptide-specific interferon (IFN)-γ and tumor necrosis factor (TNF)-α production of the WT1_126−134-reactive CTL clone. (B) Schematic representation of pST1 plasmid vectors containing the WT1_126−134-specific wild-type (WT1₁₂₆ TCR-wt) and WT1_126−134-specific codon-optimized (WT1₁₂₆ TCR-co) TCR cassettes. WT1, Wilms' tumor 1; wt, wild-type; co, codon-optimized; T7, T7 promoter; P2A, picornaviral 2A-like sequence; A120, 120-mer poly-A tail.

Figure 2

Silencing effect of DsiRNA against TRAC and TRBC upon simultaneous DsiRNA and TCR mRNA electroporation. (A) Analysis of TRAC and TRBC gene expression using RT-qPCR in Jurkat E6-1 cells after single electroporation with a control DsiRNA against EGFP (DsiRNA_EGFP), with DsiRNA targeting TRAC and TRBC (DsiRNA) or no DsiRNA (mock). Expression levels were normalized to the reference genes importin-8 and ribosomal protein L13A and analyzed relative to mock electroporation. (B) TCR-deficient 2D3 cells were electroporated simultaneously with wild-type (-wt) or codon-optimized (-co) WT1₁₂₆ TCR mRNA and DsiRNA against TRAC and TRBC or electroporated with WT1₁₂₆ TCR mRNA only. TCR surface expression was analyzed 24 h after transfection (mean ± SEM of 3 replicate experiments). Primary unstimulated CD8⁺ T cells were electroporated simultaneously with WT1₁₂₆ TCR-co mRNA and DsiRNA against TRAC and TRBC or with TCR mRNA only. The percentage of total TCR expression (C) and percentage of transgenic TCR expression (D) was measured in primary unstimulated CD8⁺ T cells at different time points after electroporation (n = 3; mean ± SEM). *P < 0.05; **P < 0.01; ***P < 0.001; TRAC, T-cell receptor alpha constant region; TRBC, T-cell receptor beta constant region; Mock, mock electroporation; DsiRNA_EGFP, Dicer-substrate small interfering RNA directed against EGFP gene; DsiRNA, Dicer-substrate small interfering RNAs directed against TRAC and TRBC genes; WT1, Wilms' tumor 1; wt, wild-type; co, codon-optimized.

Figure 3

Optimization of double sequential electroporation with DsiRNA and TCR mRNA in 2D3 cells. (A) Influence of different time spans between first and second sequential electroporation on transgenic TCR expression in TCRαβ-deficient 2D3 cells. DsiRNA electroporation was performed 6 or 24 h prior to WT1₁₂₆ TCR mRNA electroporation. (B) Kinetics of transgenic TCR expression in double sequentially-electroporated 2D3 cells. DsiRNA electroporation was performed 24 h prior to WT1₁₂₆ TCR mRNA electroporation. (C) Effect of mispairing on transgenic TCR expression. 2D3 cells were electroporated with a DsiRNA specific for EGFP (DsiRNA_EGFP) or DsiRNA for wild-type TRAC and TRBC genes (DsiRNA) 24 h before electroporation with WT1₃₇ TCR-co mRNA or a combination of WT1₃₇ TCR-co and WT1₁₂₆ TCR-wt mRNAs. Transgenic TCR expression was analyzed 24 h after mRNA transfection with WT1₃₇₋₄₅/HLA-A*02:01 tetramers (upper panel) and WT1_126−134/HLA-A*02:01 tetramers (lower panel). All graphs show the results for 3 independent experiments (mean ± SEM). ***P < 0.001; Mock, mock electroporated; WT1, Wilms' tumor 1; wt, wild-type; co, codon-optimized; DsiRNA, Dicer-substrate small interfering RNAs directed against TRAC and TRBC genes; DsiRNA_EGFP, Dicer-substrate small interfering RNA directed against EGFP gene.

Figure 4

Analysis of transgene WT1₁₂₆ TCR expression in human primary resting CD8⁺ T cells after double sequential electroporation with DsiRNA transfection performed 24 h prior to WT1₁₂₆ TCR mRNA transfection. (A) Representative flow cytometric analysis by WT1_126−134/HLA-A*02:01 tetramer staining 24 h after the second electroporation showing transgenic TCR expression from one out of 15 donors. The percentage of tetramer-positive CD8⁺ T cells is indicated in the upper right corner. (B) Transgenic TCR expression of double sequentially-electroporated resting CD8⁺ T cells was evaluated 24 h after TCR mRNA electroporation by WT1_126−134/HLA-A*02:01 tetramer analysis (n = 15, mean ± SEM). (C) Kinetics of transgenic TCR expression after second electroporation of resting CD8⁺ T cells (n = 3, mean ± SEM). *P < 0.05; **P < 0.01; ***P < 0.001; Mock, mock electroporated; WT1, Wilms' tumor 1; wt, wild-type; co, codon-optimized; DsiRNA, Dicer-substrate small interference RNAs directed against TRAC and TRBC genes.

Figure 5

Effect of DsiRNA-mediated silencing of endogenous TCR on WT1₁₂₆ TCR avidity and antigen-specific activation in resting CD8⁺ T cells after double sequential electroporation with DsiRNA transfection performed 24 h prior to WT1₁₂₆ TCR mRNA transfection. (A) Release of IFN-γ was measured by IFN-γ ELISpot after co-culture of double sequentially-electroporated CD8⁺ T cells and T2 cells that were pulsed with decreasing concentrations of WT1_126−134 peptide (n = 2, mean ± SEM). Within the graph, representative wells of co-cultures with non-peptide-pulsed T2 cells (a) or peptide-pulsed T2 cells (b, 1μM peptide). (B–D) Primary unstimulated CD8⁺ T cells were double sequentially-electroporated with WT1₁₂₆ TCR mRNA after DsiRNA or mock (no RNA) electroporation. Transfected CD8⁺ T cells were co-cultured with peptide-pulsed T2 cells in an effector:target ratio of 4:1. After 24 h, cells were pelleted by centrifugation and supernatants were collected. (B) Secretion of granzyme B was analyzed in supernatants using a human granzyme B ELISA kit (n = 4, mean ± SEM). Flow cytometric analysis of antigen-specific T cell activation was analyzed by activation-induced upregulation of surface markers CD69 (C) and CD137 (D) in CD8⁺ T cells (n = 5, mean ± SEM). *P < 0.05; **P < 0.01; ***P < 0.001; IFNγ, interferon-γ; Mock, mock electroporated; WT1, Wilms' tumor 1; co, codon-optimized; DsiRNA, Dicer-substrate small interference RNAs directed against TRAC and TRBC genes.

Figure 6

Antigen-specific cytotoxicity of primary resting CD8⁺ T cells is boosted after double sequential electroporation with DsiRNA and WT1₁₂₆ TCR-co mRNA. (A) Cytotoxic activity of double sequentially-electroporated CD8⁺ T cells after 6 h of co-culture with peptide-pulsed T2 cells (E:T ratio = 20:1, n = 8, mean ± SEM). WT1₃₇₋₄₅ peptide-pulsed T2 cells served as negative control target. (B) Representative example of WT1_126−134 peptide-pulsed T2 cell cytotoxicity mediated by double sequentially-electroporated CD8⁺ T cells after 6 h of co-culture. The percentage of cells is indicated in each quadrant. ***P < 0.001; Mock, mock electroporated; WT1, Wilms' tumor 1; wt, wild-type; co, codon-optimized; DsiRNA, Dicer-substrate small interfering RNAs directed against TRAC and TRBC genes.