Caspase Inhibition Improves Electrotransfer Efficiency
Comparison version between v1 by Fan Yuan and v3 by Dean Liu

Chimeric antigen receptor (CAR) T cell therapy has been approved to treat patients with various B cell-related tumors, including B-cell precursor acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma (PMBCL), and high-grade B-cell lymphoma. T cell receptor (TCR) knockout is a critical step in producing universal CAR T cells. A promising approach to achieving the knockout is to deliver the CRISPR/Cas9 system into T cells using electrotransfer technology.

1. Introduction

CAR T cells can be generated from the patient’s own T cells or those from healthy donors. The autologous CAR T cells are patient-specific, but it is a challenge to produce them for a subpopulation of cancer patients, and the production platform is currently inefficient for large-scale clinical applications[1] [1]. To avoid these problems, multiplexed genome editing strategies have been used to knock out certain endogenous genes, such as αβ T-cell receptor (TCR), in donor T cells to generate allogeneic universal CAR T cells[2][3] [2,3], which can be produced massively and applied to treat a large number of patients[2] [2]. The elimination of the αβ TCR is critical for avoiding the graft-versus-host-disease (GVHD) risk in cancer patients[4] [4]. Previous studies have shown that the elimination can happen in cells with T cell receptor-α constant (TRAC) mutation or be achieved through TRAC knockout[3][5] [3,5]. One of the promising approaches to gene knockout is to deliver the CRISPR/Cas9 system into cells using electrotransfer technology[6] [6].

The technology has been used for the delivery of various molecular cargo into cells, such as DNA, RNA, protein, and ribonucleoprotein (RNP)[7][8] [7,8]. It can be applied to all cell types and has few restrictions on the type of molecular cargo being delivered[7] [7]. Moreover, electrotransfer is easy to operate and can be readily scaled up for producing a large amount of cells needed for cell therapy in the clinic[7] [7]. Recently, electrotransfer has been employed in the production of CAR T cells in a clinical trial[9] [9]. Despite these advantages, electrotransfer may result in severe cell death[9][10] [9,10]. The viability of human primary T cells in gene electrotransfer experiments is highly dependent on the donors, varying from 20% to 40% under optimized experimental conditions for achieving adequate electrotransfer efficiency (e.g., 40%)[10][11][12] [10,11,12]. The low viability is a major issue that needs to be tackled before the technology can be applied successfully to manufacturing CAR-T cells.

2. Inhibition of Caspases in Human Primary T Cells Improves Gene Editing Efficiency

To demonstrate the capability of caspase inhibition for improving CAR T cell production, we treated human primary T cells with a pan-caspase inhibitor, z-vad-fmk, after pDNA or RNP electrotransfer. Our data showed that although the compound was nontoxic to T cells, it became highly toxic when the treatment is combined with electrotransfer. At the concentrations that worked for improving electrotransfer in Jurkat cells, the treatment with z-vad-fmk killed the majority of human T cells. Even at 0.5 µM, which was 100 times lower than the optimal concentration for Jurkat cells, the inhibitor treatment still killed approximately half of the T cell population, compared to the untreated controls. To reduce the toxicity caused presumably by non-specific inhibition of all caspases, we tested inhibitors more specific to caspase 3, such as z-devd-fmk and Ac-devd-cho. We observed that both inhibitors could increase the T cell viability by ~30% at the optimal treatment concentrations, compared to the matched controls (Figure 1A). Inhibition of caspase 3 in human T cells with z-devd-fmk treatment at 20 µM could effectively enhance the electrotransfer efficiency (Figure 1B–D). The treatment of T cells with Ac-devd-cho resulted in insignificant or minor changes in the electrotransfer efficiency (Figure 1B–D).

Cancers 12 02603 g008

Cancers 12 02603 g009Figure 2. Effects of apoptosis inhibition on cell viability and indel frequency in human primary T cells. The gene-editing was achieved through electrotransfer of an RNP targeting the TRAC gene. (AC) Apoptosis was inhibited with z-devd-fmk treatment; (DF) apoptosis was inhibited with Ac-devd-cho treatment. (A,D) ICE analysis, showing the outcome of TRAC editing in T cells; (B,E) cell viability measured at 24 h post pulsing; (C,F) Indel within TRAC gene determined at 48 h post pulsing using TIDE analysis. Pulsing condition: 650 V/0.2 cm, 300 µs, 2 pulses, 10 Hz. ns, non-significant. Error bars, SEM; * p < 0.05, Student’s t-test, N = 4.

The auhtors' data show that specific inhibitors for caspase 3, such as z-devd-fmk and Ac-devd-cho, are non-toxic to human primary T cells even when combined with electrotransfer. Thus, they can be used to improve the efficiency of gene-editing in T cells. Results from the study suggest that inhibition of caspases is a promising strategy for improving CAR T cell production, and more generally, electrotransfer of molecular cargo in cell engineering applications.

Reference

  1. Zhao, J.; Lin, Q.; Song, Y.; Liu, D. Universal CARs, universal T cells, and universal CAR T cells. J. Hematol. Oncol. 2018, 11, 132. [Google Scholar] [CrossRef]

  2. Liu, X.; Zhang, Y.; Cheng, C.; Cheng, A.W.; Zhang, X.; Li, N.; Xia, C.; Wei, X.; Liu, X.; Wang, H. CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells. Cell Res. 2017, 27, 154–157. [Google Scholar] [CrossRef]

  3. Torikai, H.; Reik, A.; Liu, P.-Q.; Zhou, Y.; Zhang, L.; Maiti, S.; Huls, H.; Miller, J.C.; Kebriaei, P.; Rabinovitch, B. A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR. Blood J. Am. Soc. Hematol. 2012, 119, 5697–5705. [Google Scholar] [CrossRef]

  4. Liu, J.; Zhou, G.; Zhang, L.; Zhao, Q. Building potent chimeric antigen receptor T cells with CRISPR genome editing. Front. Immunol. 2019, 10, 456. [Google Scholar] [CrossRef]

  5. Morgan, N.V.; Goddard, S.; Cardno, T.S.; McDonald, D.; Rahman, F.; Barge, D.; Ciupek, A.; Straatman-Iwanowska, A.; Pasha, S.; Guckian, M. Mutation in the TCRα subunit constant gene (TRAC) leads to a human immunodeficiency disorder characterized by a lack of TCRαβ+ T cells. J. Clin. Investig. 2011, 121, 695–702. [Google Scholar] [CrossRef]

  6. Chen, S.; Lee, B.; Lee, A.Y.-F.; Modzelewski, A.J.; He, L. Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. J. Biol. Chem. 2016, 291, 14457–14467. [Google Scholar] [CrossRef]

  7. Cervia, L.D.; Yuan, F. Current progress in electrotransfection as a nonviral method for gene delivery. Mol. Pharm. 2018, 15, 3617–3624. [Google Scholar] [CrossRef]

  8. Kebriaei, P.; Singh, H.; Huls, M.H.; Figliola, M.J.; Bassett, R.; Olivares, S.; Jena, B.; Dawson, M.J.; Kumaresan, P.R.; Su, S. Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells. J. Clin. Investig. 2016, 126, 3363–3376. [Google Scholar] [CrossRef] [PubMed]

  9. Jordan, E.T.; Collins, M.; Terefe, J.; Ugozzoli, L.; Rubio, T. Optimizing electroporation conditions in primary and other difficult-to-transfect cells. J. Biomol. Tech. JBT 2008, 19, 328. [Google Scholar] [PubMed]

  10. Zhang, Z.; Qiu, S.; Zhang, X.; Chen, W. Optimized DNA electroporation for primary human T cell engineering. BMC Biotechnol. 2018, 18, 4. [Google Scholar] [CrossRef] [PubMed]

  11. Bell, M.P.; Huntoon, C.J.; Graham, D.; McKean, D.J. The analysis of costimulatory receptor signaling cascades in normal T lymphocytes using in vitro gene transfer and reporter gene analysis. Nat. Med. 2001, 7, 1155–1158. [Google Scholar] [CrossRef] [PubMed]

  12. Aksoy, P.; Aksoy, B.A.; Czech, E.; Hammerbacher, J. Viable and efficient electroporation-based genetic manipulation of unstimulated human T cells. BioRxiv 2019, 466243.

References

  1. 1. Zhao, J.; Lin, Q.; Song, Y.; Liu, D. Universal CARs, universal T cells, and universal CAR T cells. J. Hematol. Oncol. 2018, 11, 132. [Google Scholar] [CrossRef]
  2. 2. Liu, X.; Zhang, Y.; Cheng, C.; Cheng, A.W.; Zhang, X.; Li, N.; Xia, C.; Wei, X.; Liu, X.; Wang, H. CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells. Cell Res. 2017, 27, 154–157. [Google Scholar] [CrossRef]
  3. 3. Torikai, H.; Reik, A.; Liu, P.-Q.; Zhou, Y.; Zhang, L.; Maiti, S.; Huls, H.; Miller, J.C.; Kebriaei, P.; Rabinovitch, B. A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR. Blood J. Am. Soc. Hematol. 2012, 119, 5697–5705. [Google Scholar] [CrossRef]
  4. 4. Liu, J.; Zhou, G.; Zhang, L.; Zhao, Q. Building potent chimeric antigen receptor T cells with CRISPR genome editing. Front. Immunol. 2019, 10, 456. [Google Scholar] [CrossRef]
  5. 5. Morgan, N.V.; Goddard, S.; Cardno, T.S.; McDonald, D.; Rahman, F.; Barge, D.; Ciupek, A.; Straatman-Iwanowska, A.; Pasha, S.; Guckian, M. Mutation in the TCRα subunit constant gene (TRAC) leads to a human immunodeficiency disorder characterized by a lack of TCRαβ+ T cells. J. Clin. Investig. 2011, 121, 695–702. [Google Scholar] [CrossRef]
  6. 6. Chen, S.; Lee, B.; Lee, A.Y.-F.; Modzelewski, A.J.; He, L. Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. J. Biol. Chem. 2016, 291, 14457–14467. [Google Scholar] [CrossRef]
  7. 7. Cervia, L.D.; Yuan, F. Current progress in electrotransfection as a nonviral method for gene delivery. Mol. Pharm. 2018, 15, 3617–3624. [Google Scholar] [CrossRef]
  8. 8. Kebriaei, P.; Singh, H.; Huls, M.H.; Figliola, M.J.; Bassett, R.; Olivares, S.; Jena, B.; Dawson, M.J.; Kumaresan, P.R.; Su, S. Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells. J. Clin. Investig. 2016, 126, 3363–3376. [Google Scholar] [CrossRef] [PubMed]
  9. 9. Jordan, E.T.; Collins, M.; Terefe, J.; Ugozzoli, L.; Rubio, T. Optimizing electroporation conditions in primary and other difficult-to-transfect cells. J. Biomol. Tech. JBT 2008, 19, 328. [Google Scholar] [PubMed]
  10. 10. Zhang, Z.; Qiu, S.; Zhang, X.; Chen, W. Optimized DNA electroporation for primary human T cell engineering. BMC Biotechnol. 2018, 18, 4. [Google Scholar] [CrossRef] [PubMed]
  11. 11. Bell, M.P.; Huntoon, C.J.; Graham, D.; McKean, D.J. The analysis of costimulatory receptor signaling cascades in normal T lymphocytes using in vitro gene transfer and reporter gene analysis. Nat. Med. 2001, 7, 1155–1158. [Google Scholar] [CrossRef] [PubMed]
  12. 12. Aksoy, P.; Aksoy, B.A.; Czech, E.; Hammerbacher, J. Viable and efficient electroporation-based genetic manipulation of unstimulated human T cells. BioRxiv 2019, 466243.

Keywords

CAR-T;Electrotransfer;Caspase-3;Apoptosis;Cell Viability