Table 1.

Comparison of technical ease, elements needed, procedures, and efficacies between retroviral vector and lentiviral vector (RV/LV) gene delivery systems with CRISPR-Cas gene editing for production of chimeric antigen receptor (CAR)-T cells.

	RV/LV	CRISPR-Cas RNP
Generation of Gene transfer system	Viral packaging and purification, customized,complex, costly	Highly adaptable and modular, RNA/ DNA synthesis and recombinant protein, simple
QC of gene transfer system	Complex molecular biology and virology, biochemical, biological tests	Simple biochemical synthesis and biochemical tests
PBMC/T cell activation	1-2 days	1-3 days
T cell modification	Virus plus adjuvant, overnight incubation	Several reagents, electroporation and resting
T cell expansion	>1000 fold relative to input	Up to 200 fold relative to input
Insertion in genome	Mostly random and in pro­ oncogenic hotspots	Targeted to specific loci but off­ sites possible
Multicistronic gene transfer	Feasible within gene cargo capacity	Remains to be optimized
Production of HLA-KO Allogenic CAR-T cells	Feasible with shRNAs or gRNAs expressed in viral vector, and with electroporation of mRNAs expressing TALENs	Feasible with gRNAs included in gene editing procedure