Targeted disruption of T cell receptor genes using talens
Disclosed herein are methods and compositions for modifying TCR genes, using nucleases (zinc finger nucleases or TAL nucleases) to modify TCR genes.
1. An isolated T-lymphocyte comprising a stably integrated exogenous sequence encoding a T-cell receptor (TCR), wherein at least one endogenous TCR gene within the cell is partially or completely inactivated by a nuclease, wherein the nuclease is a TALEN that binds to a target sequence site in the endogenous TCR gene, wherein said target sequence site is selected from SEQ ID NO: 147-153, the TALEN comprising a TAL-effector DNA-binding domain and a cleavage domain, and further wherein the TAL-effector DNA-binding domain comprises a C-terminal truncation as compared to a wild-type TAL-effector DNA-domain.
2. The isolated T-lymphocyte of claim 1 , wherein the endogenous TCR gene is a TCR α or TCR β gene.
3. The isolated T-lymphocyte of claim 1 , wherein a polynucleotide encoding the TALEN that binds to a target sequence site selected from SEQ ID NO: 147-153 is introduced into the cell using an integrase-defective lentiviral vector (IDLV), AAV, a plasmid or mRNA, the TALEN comprising a TAL-effector DNA-binding domain and a cleavage domain, and further wherein the TAL-effector DNA-binding domain comprises a C-terminal truncation as compared to a wild-type TAL-effector DNA-domain.
4. The isolated T-lymphocyte of claim 1 , wherein the exogenous sequence is introduced into an endogenous TCR gene, a CCR5 gene or an AAVS1 gene.
5. The isolated T-lymphocyte of claim 1 , wherein the exogenous sequence is selected from the group consisting of a tumor antigen specific TCR transgene wherein the TCR transgene is a TCR α transgene, a TCR β transgene and combinations thereof.
6. The isolated T-lymphocyte of claim 5 , wherein the tumor antigen comprises NY-ESO1.
7. A pharmaceutical composition comprising the isolated T-lymphocyte of claim 1 .
8. A method of generating a T-lymphocyte according to claim 1 , the method comprising:
inactivating an endogenous TCR gene in the T-lymphocyte using one or more polynucleotides encoding the one or more TALENs, each TALEN comprising a TAL-effector DNA-binding domain and a cleavage domain, and further wherein the TAL-effector DNA-binding domain comprises a C-terminal truncation as compared to a wild-type TAL-effector DNA-domain, wherein the TALEN cleave the endogenous TCR gene; and
stably integrating the exogenous sequence into the genome of the T-lymphocyte.
9. The method of claim 8 , wherein the endogenous TCR gene is a TCR α and/or TCR β gene.
10. The method of claim 8 , wherein the exogenous sequence is introduced into the cell using an integrase-defective lentiviral vector (IDLV), retroviral vector (RV) or lentiviral Vector (LV).
11. The method of claim 10 , wherein the exogenous sequence is introduced by IDLV into an endogenous TCR gene, a CCR5 gene or an AAVS1 gene.
12. The method of claim 8 , wherein the exogenous sequence is selected from the group consisting of a tumor antigen specific TCR transgene wherein the TCR transgene is a TCR α transgene, a TCR β transgene and combinations thereof.
13. The method of claim 12 , wherein the tumor antigen comprises NY-ESO1.