Allogeneic t-cells and methods for production thereof

A method of producing a genetically modified T-cell line, comprising:

• a. introducing a nucleic acid molecule encoding a CRISPR-associated nuclease under the control of a controllable promoter into a T-cell line;
• b. integrating the nucleic acid molecule into the genome of the T-cell line;
• c. expanding the T-cell line;
• d. inducing expression of the CRISPR-associated nuclease by activating the controllable promoter;
• e. introducing a guide-RNA and a gene of interest into the expanded T-cell line;
• f. knocking out expression of a T-cell receptor and introducing the gene of interest into the genome of the T-cell line;
• and
• g. recovering the genetically modified T-cell line.

A method of producing a chimeric antigen receptor (CAR) T-cell line, comprising:

• a. introducing a nucleic acid molecule encoding a Cas9 nuclease or a Cas12 nuclease under the control of a controllable promoter into a T-cell line;
• b. integrating the nucleic acid molecule into the genome of the T-cell line;
• c. expanding the T-cell line;
• d. inducing expression of the Cas9 nuclease or Cas12 nuclease by activating the controllable promoter;
• e. introducing a guide-RNA and a nucleic acid encoding a chimeric antigen receptor (CAR) into the expanded T-cell line;
• f. knocking out expression of a T-cell receptor and introducing the nucleic acid encoding the CAR into the genome of the T-cell line;
• and
• g. recovering the CAR T-cell line.