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. An isolated 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 comprises at least one of: (1) a variable region of a heterologous human T cell receptor alpha (TCR-α) chain gene and (2) a variable region of a heterologous human T cell receptor beta (TCR-β) chain gene.
2. The T cell of claim 1 , wherein the T cell does not comprise a viral vector for introducing the at least one nucleic acid sequence to the T cell.
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 at least 500 bp in size.
5. The T cell of claim 1 , wherein the at least one nucleic acid sequence is inserted into exon 1, 2, or 3 of TRAC.
6. The T cell of claim 1 , wherein the at least one nucleic acid sequence is inserted into exon 1, 2, or 3 of TRBC.
7. The T cell of claim 1 , wherein the T cell is a CD8+ T cell or a CD4+ T cell.
8. The T cell of claim 1 , wherein the at least one heterologous gene comprises at least one of: (1) a) variable region orb) variable region and constant region of the heterologous T cell receptor alpha (TCR-α) chain gene and (2) a) variable region or b) variable region and constant region of the heterologous T cell receptor beta (TCR-β) chain gene.
9. The T cell of claim 8 , wherein the at least one heterologous gene comprises each of: (1) the a) variable region orb) variable region and constant region of the heterologous T cell receptor alpha (TCR-α) chain gene and (2) the a) variable region or b) variable region and constant region of the heterologous T cell receptor beta (TCR-β) chain gene.
10. The T cell of claim 1 , wherein the T cell comprises each of (1) the a) variable region or b) variable region and constant region of the heterologous TCR-α chain gene and (2) the a) variable region orb) variable region and constant region of the heterologous TCR-β chain gene.
11. The T cell of claim 10 , wherein, upon expression, the heterologous TCR-α chain and the heterologous TCR-β form an antigen-specific T cell receptor (TCR).
12. The T cell of claim 10 , wherein the heterologous TCR-α chain gene and the heterologous TCR-β chain gene are operably linked by a linker sequence, optionally the linker sequence is a cleavable linker sequence or a multicistronic element.
13. The T cell of claim 12 , wherein the heterologous TCR-α chain gene and the heterologous TCR-β chain gene are inserted into TRAC.
14. The T cell of claim 1 , wherein expression of the at least one heterologous gene is driven by an endogenous promoter.
15. The T cell of claim 1 , wherein expression of one or both of endogenous TRAC and endogenous TRBC is reduced in the cell relative to a control T cell, wherein the control T cell is a primary human T cell that lacks the non-viral insertion.
16. An isolated population of primary human T cells of claim 1 , wherein the genome of at least 20% of primary human T cells in the population comprises the at least one nucleic acid sequence.
17. An isolated primary human T cell comprising:
at least one nucleic acid sequence comprising at least one heterologous gene 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 comprises at least one of: (1) a variable region of a heterologous human T cell receptor alpha (TCR-α) chain gene and (2) a variable region of a heterologous human T cell receptor beta (TCR-β) chain gene, and
wherein the T cell does not comprise a viral vector for introducing the at least one nucleic acid sequence to the T cell.
18. The T cell of claim 17 , wherein the at least one nucleic acid sequence is at least 1.5 kb in size.
19. The T cell of claim 17 , wherein the at least one nucleic acid sequence is at least 500 bp in size.
20. The T cell of claim 17 , wherein the at least one nucleic acid sequence is inserted into exon 1, 2, or 3 of TRAC.
21. The T cell of claim 17 , wherein the at least one nucleic acid sequence is inserted into exon 1, 2, or 3 of TRBC.
22. The T cell of claim 17 , wherein the T cell is a CD8+ T cell or a CD4+ T cell.
23. The T cell of claim 17 , wherein the at least one heterologous gene comprises at least one of: (1) a) variable region orb) variable region and constant region of the heterologous T cell receptor alpha (TCR-α) chain gene and (2) a) variable region or b) variable region and constant region of the heterologous T cell receptor beta (TCR-β) chain gene.
24. The T cell of claim 23 , wherein the at least one heterologous gene comprises each of: (1) the a) variable region or b) variable region and constant region of the heterologous T cell receptor alpha (TCR-α) chain gene and (2) the a) variable region or b) variable region and constant region of the heterologous T cell receptor beta (TCR-β) chain gene.
25. The T cell of claim 17 , wherein the T cell comprises each of (1) the a) variable region or b) variable region and constant region of the heterologous TCR-α chain gene and (2) the a) variable region or b) variable region and constant region of the heterologous TCR-β chain gene.
26. The T cell of claim 25 , wherein, upon expression, the heterologous TCR-α chain and the heterologous TCR-β form an antigen-specific T cell receptor (TCR).
27. The T cell of claim 25 , wherein the heterologous TCR-α chain gene and the heterologous TCR-β chain gene are operably linked by a linker sequence, optionally the linker sequence is a cleavable linker sequence or a multicistronic element.
28. The T cell of claim 27 , wherein the heterologous TCR-α chain gene and the heterologous TCR-β chain gene are inserted into TRAC.
29. The T cell of claim 17 , wherein expression of the at least one heterologous gene is driven by an endogenous promoter.
30. The T cell of claim 17 , wherein expression of one or both of endogenous TRAC and endogenous TRBC is reduced in the cell relative to a control T cell, wherein the control T cell is a primary human T cell that lacks the non-viral insertion.
31. An isolated population of primary human T cells of claim 17 , wherein the genome of at least 20% of primary human T cells in the population comprises the at least one nucleic acid sequence.