Laglidadg homing endonuclease cleaving the C-C chemokine receptor type-5 (CCR5) gene and uses thereof
View Patent ↗Disclosed herein are compositions for inactivating the human CCR5 gene comprising engineered LAGLIDADG homing endonucleases (LHEs) and their derivatives, particularly derived from members of the \-Onul subfamily of LHE. Polynucleotides encoding such endonucleases, vectors comprising said polynucleotides, cells comprising or having been treated with such endonucleases, and therapeutic compositions deriving therefrom are also provided.
1. A chimeric endonuclease comprising:
an endonuclease domain having at least 90% identity with the amino acid sequence of SEQ ID NO:19 that cleaves a target nucleic acid sequence within the C-C chemokine receptor type 5 gene (CCR5), wherein the target nucleic acid sequence is SEQ ID NO:5; and
at least an additional protein domain selected from the group consisting of: nucleic acid binding domain, catalytic domain, terminal epitope tags and fluorescent proteins.
2. A chimeric endonuclease comprising:
an endonuclease domain having at least 70% identity with the amino acid sequence of SEQ ID NO:19 and having at least 95% identity with the DNA recognition interface of SEQ ID NO:19 that cleaves a target nucleic acid sequence within the C-C chemokine receptor type 5 gene (CCR5),
wherein the target nucleic acid sequence is SEQ ID NO:5 and,
wherein the endonuclease domain comprises at least 10 amino acid substitutions at positions selected from the group consisting of: amino acid position 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, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240 in reference to SEQ ID NO:2,
and at least an additional protein domain selected from the group consisting of: nucleic acid binding domain, catalytic domain, terminal epitope tags and fluorescent proteins.
3. The chimeric endonuclease of claim 2 , comprising an endonuclease domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:19.
4. The chimeric endonuclease of claim 1 , wherein the chimeric endonuclease comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO17, SEQ ID NO:19 and SEQ ID NO:31.
5. The chimeric endonuclease of claim 2 , wherein the chimeric endonuclease comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 and SEQ ID NO.31.
6. The chimeric endonuclease of claim 1 , wherein the additional protein domain is a nucleic acid binding domain selected from the group consisting of TALE and Zinc Finger domain.
7. The chimeric endonuclease of claim 6 , wherein the chimeric endonuclease is the MegaTAL CCR5_S08 protein sequence selected from the group consisting of SEQ ID NO:25 and SEQ ID NO:33.
8. The chimeric endonuclease of claim 1 , wherein additional protein domain 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, recombinase activity.
9. The chimeric endonuclease of claim 1 , wherein the catalytic domain is a 5′-3′ exonuclease, more preferably Trex2 and more preferably single chain Trex2.
10. The chimeric endonuclease of claim 1 , wherein the additional protein domain is fused to the endonuclease domain by a peptide linker.
11. The chimeric endonuclease of claim 1 , fused to a nucleic acid binding domain and a catalytic domain.
12. The chimeric endonuclease of claim 11 , wherein the additional protein domain is a nucleic acid binding domain selected from the group consisting of TALE and Zinc Finger domain.
13. The chimeric endonuclease of claim 11 , wherein the catalytic domain is a 5′-3′ exonuclease, more preferably Trex2 and more preferably single chain Trex2.
14. The chimeric endonuclease of claim 11 , wherein the chimeric endonuclease comprises the amino acid sequence set forth in SEQ ID NO:33.