Nucleofection with Plasmid DNA for CRISPR/Cas9-Mediated Inactivation of Programmed Cell Death Protein 1 in CD133-Specific CAR T Cells

Hu B, Zou Y, Zhang L, Tang J, Niedermann G, Firat E, Huang X, Zhu X. 
Source: Hum Gene Ther
Publication Date: (2018)
Issue: :
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
PBMC, human
Species: human
Tissue Origin: blood
Nucleofector® I/II/2b

PBMCs were thawed and rested for 1.5–2 h.
Then, 2 x 107 cells were re-suspended in 100 µL of
buffer from the Human T cell Nucleofector kit
(Lonza; VPA-1002) and used for one nucleofection
reaction; 5 µg of pST1374-Cas9-GFP, 5 lg of pGL3-
U6-hPD-1-sgRNA(1 + 2), 5 µg of CD133-CAR piggy-
Bac transposon vector, and 5 µg of Super piggyBac
transposase plasmid were added. For preparation of
the conventional CAR T cells, only the piggyBac
transposon and transposase were used. Electroporation
was performed with a NucleofectorII/2b
device (Lonza) using the U-014 program


CRISPR/Cas9-mediated programmed cell death protein 1 (PD-1) disruption in chimeric antigen receptor (CAR) T cells could be an appealing choice to improve the therapeutic efficacy of CAR T cells in an immunosuppressive tumor microenvironment. In most of the reported cases, Cas9 was delivered into T cells by way of electroporation with RNA or protein. However, transient expression of Cas9 by transfection with a plasmid encoding its gene is apparently simpler, as it avoids the steps of in vitro transcription of DNA or protein production. This study tried nucleofection into human primary T cells of plasmids encoding both CRISPR/Cas9 for disrupting the PD-1 gene and the piggyBac transposon system for expressing CD133-specific CAR in one reaction. Based on drug selection, CD133-specific CAR T cells were obtained in which, on average, 91.5% of the PD-1 gene sites were disrupted, but almost no Cas9 gene expression was found in the final engineered CAR T cells. The PD-1-deficient CD133-specific CAR T cells showed similar levels of cytokine secretion and improved proliferation and cytotoxicity in vitro, and enhanced inhibition of tumor growth in an orthotopic mouse model of glioma, compared to conventional CD133-CAR T cells. The described method could be useful for the production of PD-1-deficient CAR T cells for cancer immunotherapy.