Targeted replacement of endogenous T cell receptors
Provided herein are methods and compositions for editing the genome of a human T cell. In some embodiments, a heterologous T cell receptor (TCR)-β chain and a heterologous TCR-α chain are inserted into exon 1 of a TCR subunit constant gene in the genome of the T cell.
1. A modified primary human T cell comprising: at least one nucleic acid sequence comprising at least one heterologous gene non-virally inserted into one or both of:
an endogenous T cell receptor alpha subunit constant gene (TRAC); and
an endogenous T cell receptor beta subunit constant gene (TRBC), wherein the at least one heterologous gene encodes at least one antigen-specific receptor that specifically binds the target antigen expressed by a cancer, and wherein the at least one nucleic acid sequence is at least 500 bp in size.
2. The T cell of claim 1 , wherein the modified human T cell does not comprise a viral vector.
3. The T cell of claim 1 , wherein the at least one nucleic acid sequence is at least 1.5 kb in size.
4. The T cell of claim 1 , wherein the at least one nucleic acid sequence is non-virally inserted into an exon of the T cell receptor alpha subunit constant gene (TRAC) or into an exon of the T cell receptor beta subunit constant gene (TRBC).
5. The T cell of claim 1 , wherein the at least one heterologous gene comprises at least one of:
(a) a variable region of a heterologous T cell receptor alpha (TCR-α) chain and
(b) a variable region of a heterologous T cell receptor beta (TCR-β) chain.
6. The T cell of claim 1 , wherein the at least one heterologous gene comprises at least one of:
(1) a) a variable region of a heterologous T cell receptor alpha (TCR-α) chain or b) a variable region and constant region of the heterologous TCR-α chain; and
(2) a) a variable region of a heterologous T cell receptor beta (TCR-β) chain or b) a variable region and constant region of the heterologous TCR-β chain.
7. The T cell of claim 6 , wherein the at least one heterologous gene comprises each of:
(1) the a) variable region of the heterologous TCR-α chain orb) variable region and constant region of the heterologous TCR-α chain; and
(2) the a) variable region of the heterologous TCR-β chain orb) variable region and constant region of the heterologous TCR-β chain.
8. The T cell of claim 7 , wherein one or more coding sequences for the heterologous TCR-α chain and the heterologous TCR-β chain are linked by a linker sequence or a multicistronic element.
9. The T cell of claim 8 , wherein the linker sequence is a cleavable linker sequence that is cleaved to generate the heterologous TCR-α chain gene and the heterologous TCR-β chain gene.
10. The T cell of claim 8 , wherein the heterologous gene encodes the TCR-α chain and the heterologous TCR-β chain and wherein the heterologous gene is inserted into TRAC.
11. The T cell of claim 1 , wherein the at least one heterologous gene is non-virally inserted into an endogenous TRAC, wherein the at least one heterologous gene comprises each of:
(1) a) a variable region of the heterologous TCR-α chain orb) a variable region and constant region of the heterologous TCR-α chain; and
(2) a) a variable region of the heterologous TCR-β chain orb) a variable region and constant region of the heterologous TCR-β chain,
wherein the heterologous TCR-α chain and the heterologous TCR-β chain are operably linked by a cleavable linker sequence that is cleaved to generate the heterologous TCR-α chain and the heterologous TCR-β chain, and wherein the heterologous TCR-α chain and the heterologous TCR-β form an antigen-specific T cell receptor (TCR) that recognizes the target antigen expressed by the cancer.
12. The T cell of claim 1 , wherein expression of the at least one heterologous gene is under the control of an endogenous promoter.
13. The T cell of claim 1 , wherein the modified human T cell is a CD8+ T cell or a CD4+ T cell.
14. The T cell of claim 13 , wherein the CD8+ T cell or CD4+ T cell is an effector T cell or a naïve T cell.
15. The T cell of claim 1 , wherein the primary human T cell is obtained from a subject having cancer and modified to comprise the at least one nucleic acid sequence comprising at least one heterologous gene.
16. A population of cells comprising a plurality of the primary human T cell of claim 1 .