Targeted disruption of T cell receptor genes using engineered zinc finger protein nucleases
Disclosed herein are methods and compositions for inactivating TCR genes, using zinc finger nucleases (ZFNs) comprising a zinc finger protein and a cleavage domain or cleavage half-domain in conditions able to preserve cell viability. Polynucleotides encoding ZFNs, vectors comprising polynucleotides encoding ZFNs and cells comprising polynucleotides encoding ZFNs and/or cells comprising ZFNs are also provided. Disclosed herein are also methods and compositions for expressing a functional exogenous TCR in the absence of endogenous TCR expression in T lymphocytes, including lymphocytes with a central memory phenotype. Polynucleotides encoding exogenous TCR, vectors comprising polynucleotides encoding exogenous TCR and cells comprising polynucleotides encoding exogenous TCR and/or cells comprising exogenous TCR are also provided.
1. An isolated genetically modified cell comprising a genomic modification that modifies the endogenous T-cell receptor (TCR) gene, wherein the genomic modification is selected from the group consisting of insertions, deletions and combinations thereof, wherein the genomic modification is produced by a zinc finger nuclease comprising a zinc finger protein that binds to a target site in the TCR gene, wherein said target site is any of SEQ ID NO: 1, 7, 13, 18, 23, 29, 34, 37, 41, 45, 49, 54, 58 or 63, wherein the binding of the zinc finger protein at the target site in the TCR gene results in cleavage of the TCR gene such that the TCR gene is modified within any of SEQ ID NO: 1, 7, 13, 18, 23, 29, 34, 37, 41, 45, 49, 54, 58 and 63, thereby resulting in a genetically modified cell comprising the genomic modification at the endogenous TCR gene.
2. The genetically modified cell of claim 1 , wherein the genomic modification is within an endogenous TCRα or TCRβ gene.
3. The genetically modified cell of claim 2 , wherein the genomic modification is within exon 1 or exon 3 of an endogenous TCRα gene.
4. The genetically modified cell of claim 1 , wherein the endogenous TCR gene is inactivated.
5. The genetically modified cell of claim 1 , wherein the cell is a T-cell.
6. The genetically modified cell of claim 1 , wherein the cell is a stem cell.
7. The genetically modified cell of claim 6 , wherein the stem cell is a hematopoietic stem cell.
8. A genetically modified differentiated cell descended from the stem cell of claim 6 .
9. The isolated genetically modified cell of claim 1 , wherein the zinc finger nuclease is introduced into the cell as a polynucleotide.
10. The genetically modified cell of claim 1 , further comprising a donor polynucleotide encoding a transgene.
11. The genetically modified cell of claim 10 , wherein the transgene encodes an engineered T cell receptor subunit.
12. The genetically modified cell of claim 10 , wherein the transgene encodes a chimeric antigen receptor (CAR).
13. A pharmaceutical composition comprising the genetically modified cell of claim 1 .