LAGLIDADG homing endonuclease cleaving the T cell receptor alpha gene and uses thereof
View Patent ↗Disclosed herein are compositions for inactivating the human TCR-alpha gene comprising engineered LAGLIDADG homing endonucleases (LHEs) and their derivatives, particularly derived from members of the \-Onul subfamily of LHEs. Polynucleotides encoding such endonucleases, vectors comprising said polynucleotides, cells comprising or having been treated with such endonucleases, and therapeutic compositions deriving therefore are also provided.
1. A polypeptide comprising an I-Onul variant having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10,
wherein said I-Onul variant cleaves a target nucleic acid sequence within the T cell receptor alpha constant gene (TRAC),
wherein said I-Onul variant is fused to a TALE DNA binding domain.
2. The polypeptide of claim 1 comprising an I-Onul variant having at least 10 amino acid substitutions in positions selected from the group consisting of: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72, 75, 76, 77, 78, 80, 82, 138, 159, 168, 178, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 207, 223, 225, 227, 229, 231, 232, 234, 236, 238, 240, in reference to SEQ ID NO: 2.
3. The polypeptide of claim 1 comprising an I-Onul variant having at least 15 amino acid substitutions in positions selected from the group consisting of: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72, 75, 76, 77, 78, 80, 82, 138, 159, 168, 178, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 207, 223, 225, 227, 229, 231, 232, 234, 236, 238, 240, in reference to SEQ ID NO: 2.
4. The polypeptide of claim 1 comprising an I-Onul variant having at least 20 amino acid substitutions in positions selected from the group consisting of: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72, 75, 76, 77, 78, 80, 82, 138, 159, 168, 178, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 207, 223, 225, 227, 229, 231, 232, 234, 236, 238, 240, in reference to SEQ ID NO: 2.
5. The polypeptide of claim 1 comprising an I-Onul variant having at least 25 amino acid substitutions in positions selected from the group consisting of positions: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72, 75, 76, 77, 78, 80, 82, 138, 159, 168, 178, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 207, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240, in reference to SEQ ID NO: 2.
6. The polypeptide of claim 1 comprising an I-Onul variant having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.
7. The polypeptide of claim 1 comprising an I-Onul variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.
8. The polypeptide of claim 1 comprising an I-Onul variant having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.
9. The polypeptide of claim 1 comprising an I-Onul variant having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.
10. The polypeptide of claim 1 comprising an I-Onul variant having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.
11. The polypeptide of claim 1 comprising an I-Onul variant comprising the amino acid sequence of SEQ ID NO: 6.
12. The polypeptide of claim 1 comprising an I-Onul variant comprising the amino acid sequence of SEQ ID NO: 8.
13. The polypeptide of claim 1 comprising an I-Onul variant comprising the amino acid sequence of SEQ ID NO: 10.
14. The polypeptide of claim 1 , comprising the MegaTAL TCRA_S02 amino acid sequence of SEQ ID NO: 14.
15. The polypeptide of claim 1 , comprising the MegaTAL TCRA_S02 amino acid sequence of SEQ ID NO: 16.
16. The polypeptide of claim 1 , further comprising an additional protein domain that has catalytic activity selected from the group consisting of: nuclease activity, polymerase activity, kinase activity, phosphatase activity, methylase activity, topoisomerase activity, integrase activity, transposase activity, ligase activity, helicase activity, and recombinase activity.
17. The polypeptide of claim 16 , wherein said catalytic domain is a 5′-3′ exonuclease.
18. The polypeptide of claim 17 , wherein said catalytic domain is Trex2.
19. The polypeptide of claim 17 , wherein said catalytic domain is a single chain Trex2.
20. The polypeptide of claim 16 , wherein said additional protein domain is fused to the polypeptide by a peptide linker.
21. The polypeptide of claim 1 , wherein it cleaves a target nucleic acid sequence of SEQ ID NO: 3.