Nucleofection with Plasmid DNA for CRISPR/Cas9-Mediated Inactivation of Programmed Cell Death Protein 1 in CD133-Specific CAR T Cells.
Hum Gene Ther
; 30(4): 446-458, 2019 04.
Article
in En
| MEDLINE
| ID: mdl-29706119
ABSTRACT
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Plasmids
/
T-Lymphocytes
/
Gene Silencing
/
Programmed Cell Death 1 Receptor
/
CRISPR-Cas Systems
/
AC133 Antigen
/
Receptors, Chimeric Antigen
Limits:
Animals
/
Humans
Language:
En
Journal:
Hum Gene Ther
Journal subject:
GENETICA MEDICA
/
TERAPEUTICA
Year:
2019
Document type:
Article
Affiliation country:
China