citations

                    Universal allogeneic CAR T cells engineered with Sleeping Beauty transposons and CRISPR-CAS9 for cancer immunotherapy
                

Authors:

Tipanee J, Samara-Kuko E, Gevaert T, Chuah MK, Vanden Driessche T

In:

Source: Mol Ther
Publication Date: (2022)
Issue: 30(10): 3155-3175

Research Area:

Cancer Research/Cell Biology

Immunotherapy / Hematology

Basic Research

Molecular Biology

Regenerative medicine

Platform:

4D-Nucleofector® X-Unit

Experiment

Delivery of CRISPR-Cas9 RNP into human CD8+ T cells by nucleofection
The P3 Primary Cell 4D-Nucleofector X Kit was used in all nucleofection experiments. 5 x 10^5 cells were washed with 1x Dulbecco’s PBS and resuspended in 20 µL of kit P3 Primary Cell 4D-Nucleofector X (Lonza). The prepared 70 pmol of CRISPR-Cas9 RNP particles was added to the cell-buffer mixture and mixed well. Nucleofection was
carried out in a 16-well strip using the EH-115 program. Immediately after nucleofection, 80 µL of pre-warmed complete T cell medium containing 50 U/mL of human rIL-2 was immediately added to cells, and the cells were rested for 30 min in 5% CO2 at 37°C. The transfected cells were transferred to 1 mL of complete T cell medium and culture for 48 h at 5% CO2 and 37°C.

Generation of CD19-28z.CAR/TCR KO T cells and SB-CD19- 28z.CAR/TCR KO T cells
Sequential nucleofection of TRAC-targeting CRISPR-Cas9 RNP and mcDNA vector encoding CD19-28z.CAR was used to engineer CAR/TCRneg T cells for transient and sustained CAR expression. To generate CD19-28z.CAR/TCR KO T cells for transient CAR expression, 700 pmol of TCR-1 RNP particles was transfected into 5 x 10^6 CD8+ T cells using the EH-115 program to obtain TCRneg T cells. TCR-1 RNP was substituted with scrambled gRNA RNP to generate the scrambled control T cells. Twenty-four hours later, 10 µg of mcCD19-28z.CAR was transfected into 5 x 10^6 TCRneg or scrambled control T cells using the EO-115 program. Alternatively, 10 µg of mcCD19-28z.CAR and 5 µg of mcSB100x were transfected into 5 x 10^6 TCRneg or scrambled control T cells using the EO-115 program to produce SB-CD19-28z.CAR/TCR KO and SB-CD19- 28z.CAR/scrambled T cells for stable CAR expression, respectively. As a control devoid of CAR expression, mock transfection of TCRneg and scrambled control T cells was carried out to generate mock/TCR KO and mock/scrambled control T cells, respectively. After 24 h of transfection, the surface CAR and TCR expression was examined by flow cytometry analysis, and the cells were harvested for functionality assays or CAR T cell culture.

Abstract

Allogeneic CD19-specific chimeric antigen receptor (CAR) T cells with inactivated donor T cell receptor (TCR) expression can be used as an "off-the-shelf" therapeutic modality for lymphoid malignancies, thus offering an attractive alternative to autologous, patient-derived T cells. Current approaches for T cell engineering mainly rely on the use of viral vectors. Here, we optimized and validated a non-viral genetic modification platform based on Sleeping Beauty (SB) transposons delivered with minicircles to express CD19-28z.CAR and CRISPR-Cas9 ribonucleoparticles to inactivate allogeneic TCRs. Efficient TCR gene disruption was achieved with minimal cytotoxicity and with attainment of robust and stable CD19-28z.CAR expression. The CAR T cells were responsive to CD19+ tumor cells with antitumor activities that induced complete tumor remission in NALM6 tumor-bearing mice while significantly reducing TCR alloreactivity and GvHD development. Single CAR signaling induced the similar T cell signaling signatures in TCR-disrupted CAR T cells and control CAR T cells. In contrast, TCR disruption inhibited T cell signaling/protein phosphorylation compared with the control CAR T cells during dual CAR/TCR signaling. This non-viral SB transposon-CRISPR-Cas9 combination strategy serves as an alternative for generating next-generation CD19-specific CAR T while reducing GvHD risk and easing potential manufacturing constraints intrinsic to viral vectors.

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