Methods of manufacturing CAR-T cells

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

• (i) providing a first population of T cells;
• (ii) introducing into the first population of T cells by electroporation a first ribonucleoprotein (RNP) complex comprising a first Cas9 enzyme and a first guide RNA (gRNA) targeting a CD70 gene to produce a second population of T cells, wherein the second population of T cells comprises T cells having a disrupted CD70 gene;
• (iii) introducing into the second population of T cells a second RNP complex comprising a second Cas9 enzyme and a second gRNA targeting a T cell receptor alpha chain constant region (TRAC) gene, and a third RNP complex comprising a third Cas9 enzyme and a third gRNA targeting a beta-2 microglobulin (β2M) gene to produce a third population of T cells, wherein the third population of T cells comprises T cells having the disrupted CD70 gene disrupted, a disrupted TRAC gene, and a disrupted β2M gene, wherein the second RNP complex and the third RNP complex are introduced into the second population of T cells in one electroporation event and wherein the second RNP complex and the third RNP complex are formed prior to the electroporation event;
• (iv) incubating the third population of T cells with an adeno-associated viral (AAV) vector to produce a fourth population of T cells, wherein the AAV vector comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and wherein the nucleic acid sequence is flanked by homologous sequences to the TRAC gene, and wherein the fourth population of T cells comprises T cells expressing the CAR and having the disrupted CD70 gene, the disrupted TRAC gene, and the disrupted β2M gene;
• (v) expanding the fourth population of T cells thereby producing an expanded T cell population;
• (vi) removing TCRαβ+ T cells from the expanded T cell population to produce a population of genetically engineered T cells, wherein the population of genetically engineered T cells comprises T cells expressing the CAR and having the disrupted CD70 gene, the disrupted TRAC gene, and the disrupted β2M gene;
• and
• (vii) harvesting the population of genetically engineered T cells.