Methods for manufacturing genetically engineered CAR-T cells

A method for manufacturing genetically engineered T cells, the method comprising:

• (i) providing a population of T cells;
• (ii) activating the population of T cells in step (i) to produce a population of activated T cells;
• (iii) performing a first electroporation to the activated T cells to introduce a first Cas9 enzyme, a first guide RNA (gRNA) targeting a Regnase 1 (Reg1) gene, and a second gRNA targeting a Transforming Growth Factor Beta Receptor II (TGFBRII) gene to produce a first population of genetically engineered T cells;
• (iv) culturing the first population of genetically engineered T cells in a medium for T cell recovery,
• (v) performing a second electroporation to the recovered cells from step (iv) to introduce a second Cas9 enzyme, a third Cas9 enzyme, a third gRNA targeting a T cell receptor alpha chain constant region (TRAC) gene, and a fourth gRNA targeting a beta-2 microglobulin (β2M) gene to produce a second population of genetically engineered T cells;
• (vi) incubating the second population of genetically engineered T cells with recombinant AAV particles, which comprise a donor template, wherein the donor template comprises a nucleic acid sequence encoding a chimeric antigen receptor that binds human CD19 (anti-CD19 CAR) flanked by a left homology arm and a right homology arm, wherein the left homology arm and the right homology arm are homologous to a locus in the TRAC gene;
• (vii) expanding the second genetically engineered T cells to produce an expanded T cell population;
• (viii) removing TCRαβ+ T cells from the expanded T cell population;
• and
• (ix) harvesting the genetically engineered T cells produced in step (viii),
• wherein the genetically engineered T cells harvested in step (viii) comprise a disrupted TRAC gene, a disrupted β2M gene, a disrupted Reg1 gene, a disrupted TGFBRII gene, and the nucleic acid sequence encoding the anti-CD19 CAR, which is inserted into the disrupted TRAC gene.

A method for manufacturing genetically engineered T cells, the method comprising:

• (i) providing a population of T cells;
• (ii) activating the population of T cells in step (i) to produce a population of activated T cells;
• (iii) performing a first electroporation to the activated T cells to introduce at least a first Cas9 enzyme, a first guide RNA (gRNA) targeting a Regnase 1 (Reg1) gene, a second gRNA targeting a Transforming Growth Factor Beta Receptor II (TGFBRII) gene, and a fourth gRNA targeting a β2M gene to produce a first population of genetically engineered T cells;
• (iv) culturing the first population of genetically engineered T cells in a medium for T cell recovery,
• (v) performing a second electroporation to the recovered cells from step (iv) to introduce at least a second Cas9 enzyme, a third gRNA targeting a T cell receptor alpha chain constant region (TRAC) gene, and a fifth gRNA targeting a CD70 gene to produce a second population of genetically engineered T cells;
• (vi) incubating the second population of genetically engineered T cells with recombinant AAV particles, which comprise a donor template, wherein the donor template comprises a nucleic acid sequence encoding a chimeric antigen receptor that binds human CD70 (anti-CD70 CAR) flanked by a left homology arm and a right homology arm, wherein the left homology arm and the right homology arm are homologous to a locus in the TRAC gene;
• (vii) expanding the second genetically engineered T cells to produce an expanded T cell population;
• (viii) removing TCRαβ+ T cells from the expanded T cell population;
• and
• (ix) harvesting the genetically engineered T cells produced in step (viii),
• wherein the genetically engineered T cells harvested in step (viii) comprise a disrupted TRAC gene, a disrupted β2M gene, a disrupted Reg1 gene, a disrupted TGFBRII gene, a disrupted CD70 gene, and the nucleic acid sequence encoding the anti-CD70 CAR, which is inserted into the disrupted TRAC gene.