Genetically engineered T cells having improved persistence in culture
A T cell bank comprising genetically engineered T cells having one or more of the following features as compared to the non-engineered T cell counterparts: (a) enhanced expansion capacity in culture, (b) enhanced proliferation capacity, (c) reduced apoptosis, and (d) enhanced activation frequencies. Such genetically engineered T cells may comprise (i) a mutated gene involved in cell self-renewal; (ii) a disrupted gene involved in apoptosis; (iii) a disrupted gene involved in regulation of T cell exhaustion; or (iv) a combination of any one of (i)-(iii).
1. A population of genetically engineered T cells, comprising:
(i) a disrupted Ten-Eleven Translocation-2 (TET2) gene;
(ii) a disrupted T cell receptor alpha chain constant region (TRAC) gene;
(iii) a disrupted beta-2-microglobulin (β2M) gene;
(iv) a disrupted CD70 gene; and
(v) a disrupted FAS Cell Surface Death Receptor (FAS) gene;
wherein the population of genetically engineered T cells, as compared to non-engineered T cell counterparts, have the following features: (a) enhanced expansion capacity in culture, (b) enhanced proliferation capacity in vivo, (c) a reduced apoptosis level in vivo, and (d) an enhanced frequency of activation.
2. The population of genetically engineered T cells of claim 1 , wherein the disrupted TET2 gene is genetically edited in an exon selected from the group consisting of exon 1, exon 3, exon 4, exon 5, and exon 6, or a combination thereof.
3. The population of genetically engineered T cells of claim 1 , wherein the disrupted TET2 gene is genetically edited by CRISPR/Cas-mediated gene editing.
4. The population of genetically engineered T cells of claim 3 , wherein the disrupted TET2 gene is genetically edited by CRISPR/Cas-mediated gene editing with a guide RNA (gRNA) comprising a nucleotide sequence of SEQ ID NO: 14, 18, 22, 26, 112, 116, or 120.
5. The population of genetically engineered T cells of claim 1 , wherein the disrupted FAS and/or CD70 gene is genetically edited by CRISPR/Cas-mediated gene editing.
6. The population of genetically engineered T cells of claim 5 , wherein the disrupted FAS gene is genetically edited by CRISPR/Cas-mediated gene editing with a guide RNA (gRNA) comprising the nucleotide sequence of SEQ ID NO: 69, 73, 77, 81, or 85, and/or wherein the disrupted CD70 gene is genetically edited by CRISPR/Cas-mediated gene editing with a gRNA comprising the nucleotide sequence of SEQ ID NO: 34, 38, 42, 46, 50, 54, or 58.
7. The population of genetically engineered T cells of claim 1 , wherein the T cells are further engineered to express a chimeric antigen receptor (CAR).
8. The population of genetically engineered T cells of claim 7 , wherein the CAR targets a tumor antigen.
9. The population of genetically engineered T cells of claim 8 , wherein the tumor antigen is CD19, B cell maturation antigen (BCMA), or CD70.
10. The population of genetically engineered T cells of claim 7 , wherein the T cells comprise a nucleic acid encoding the CAR, and wherein the nucleic acid is inserted in the genome of the T cells.
11. The population of genetically engineered T cells of claim 10 , wherein the disrupted TRAC gene has an insertion of the nucleotide acid encoding the chimeric antigen receptor.
12. The population of genetically engineered T cells of claim 1 , wherein the T cells are derived from primary T cells of one or more human donors.
13. The population of genetically engineered T cells of claim 1 , wherein the T cells show cytokine-dependent growth.