PDCD-1 HOMING ENDONUCLEASE VARIANTS
The present disclosure provides improved homing endonuclease variants and megaTALs reprogrammed to bind and cleave the PDCD-1 gene. The present disclosure relates to genome editing compositions with improved stability and activity. More particularly, the disclosure relates to improved nuclease variants, compositions, and methods of using the same for editing the human program cell death (PDCD-1) gene.
1 . A polypeptide comprising an I-OnuI homing endonuclease (HE) variant that cleaves a target site in the human programmed cell death 1 (PDCD-1) gene, the I-OnuI HE variant comprising the following amino acid substitutions: I14T, L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46T, T48M, V68S, A70L, S72N, N75H, A76Y, K80V, T82Y, R83A, L138M, T143N, N153V, K156R, S159P, F168G, E178D, C180S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223H, K225Y, K227G, F232R, D236Q, V238R, T240E, V261M, and G300R in an I-OnuI HE amino acid sequence set forth in any one of SEQ ID NOs: 1-5, or a biologically active fragment thereof.
2 . The polypeptide of claim 1 , wherein the I-OnuI HE variant comprises an amino acid sequence that is at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 6, or a biologically active fragment thereof.
3 . The polypeptide of claim 1 or claim 2 , wherein the I-OnuI HE variant comprises the amino acid sequence set forth in SEQ ID NO: 6, or a biologically active fragment thereof.
4 . The polypeptide of any one of claims 1 to 3 , wherein the I-OnuI HE variant binds the polynucleotide sequence set forth in SEQ ID NO: 8.
5 . The polypeptide of any one of claims 1 to 4 , wherein the I-OnuI HE variant binds the polynucleotide sequence set forth in SEQ ID NO: 10.
6 . The polypeptide of any one of claims 1 to 5 , further comprising a DNA binding domain.
7 . The polypeptide of claim 6 , wherein the DNA binding domain is selected from the group consisting of: a TALE DNA binding domain and a zinc finger DNA binding domain.
8 . The polypeptide of claim 7 , wherein the TALE DNA binding domain comprises about 9.5 TALE repeat units to about 15.5 TALE repeat units.
9 . The polypeptide of claim 7 or claim 8 , wherein the TALE DNA binding domain binds the polynucleotide sequence set forth in SEQ ID NO: 9.
10 . The polypeptide of claim 9 , wherein the polypeptide binds and cleaves the polynucleotide sequence set forth in SEQ ID NO: 10.
11 . The polypeptide of any one of claims 1 to 10 , further comprising a peptide linker and an end-processing enzyme or biologically active fragment thereof.
12 . The polypeptide of any one of claims 1 to 10 , further comprising a viral self-cleaving 2A peptide and an end-processing enzyme or biologically active fragment thereof.
13 . The polypeptide of claim 11 or claim 12 , wherein the end-processing enzyme or biologically active fragment thereof has 5′-3′ exonuclease, 5′-3′ alkaline exonuclease, 3′-5′ exonuclease, 5′ flap endonuclease, helicase or template-independent DNA polymerase activity.
14 . The polypeptide of any one of claims 11 to 13 , wherein the end-processing enzyme comprises Trex2 or a biologically active fragment thereof.
15 . The polypeptide of any one of claims 1 to 14 , wherein the polypeptide comprises an amino acid sequence at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 7 or a biologically active fragment thereof.
16 . The polypeptide of any one of claims 1 to 15 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7 or a biologically active fragment thereof.
17 . A polynucleotide encoding the polypeptide of any one of claims 1 to 16 .
18 . An mRNA encoding the polypeptide of any one of claims 1 to 16 .
19 . The mRNA of claim 18 , wherein the mRNA comprises the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12.
20 . The mRNA of claim 18 , wherein the mRNA comprises the sequence set forth in SEQ ID NO: 11.
21 . The mRNA of claim 18 , wherein the mRNA comprises the sequence set forth in SEQ ID NO: 12.
22 . A cDNA encoding the I-OnuI HE variant of any one of claims 1 to 16 .
23 . The cDNA of claim 22 , wherein an mRNA transcribed from the cDNA comprises the sequence set forth in SEQ ID NO: 11.
24 . The cDNA of claim 22 , wherein an mRNA transcribed from the cDNA comprises the sequence set forth in SEQ ID NO: 12.
25 . A vector comprising a polynucleotide encoding the polypeptide of any one of claims 1 to 16 ; a polynucleotide encoding the mRNA of any one of claims 18 to 21 ; or a polynucleotide encoding the cDNA of any one of claims 22 to 24 .
26 . The vector of claim 25 , when the vector is an expression vector, an episomal vector, or a viral vector.
27 . The vector of claim 26 , when the vector is an adeno-associated viral (AAV) vector.
28 . A cell comprising the polypeptide of any one of claims 1 to 16 , the polynucleotide of claim 17 , the mRNA of any one of claims 18 to 21 , the cDNA of any one of claims 22 to 24 , or the vector of any one of claims 25 to 27 .
29 . The cell of claim 28 , wherein the cell is a hematopoietic cell.
30 . The cell of claim 28 or claim 29 , wherein the cell is an immune effector cell.
31 . The cell of any one of claims 28 to 30 , wherein the cell is a T cell.
32 . The cell of any one of claims 28 to 31 , wherein the cell is a CD3*, CD4+, and/or CD8 + cell.
33 . The cell of any one of claims 28 to 32 , wherein the cell is a cytotoxic T lymphocytes (CTLs), a tumor infiltrating lymphocytes (TILs), or a helper T cells.
34 . The cell of any one of claims 28 to 30 , wherein the cell is a natural killer (NK) cell or natural killer T (NKT) cell.
35 . The cell of any one of claims 28 to 34 , wherein the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors.
36 . The cell of any one of claims 28 to 35 , wherein the cell comprises one or more modified PDCD-1 alleles.
37 . A method of editing a human PDCD-1 gene in a cell comprising: introducing a polynucleotide encoding the polypeptide of any one of claims 1 to 16 into the cell, wherein expression of the polypeptide creates a double strand break at a target site in a human PDCD-1 gene.
38 . A method of editing a human PDCD-1 gene in cell comprising: introducing a polynucleotide encoding the polypeptide of any one of claims 1 to 16 into the cell, wherein expression of the polypeptide creates a double strand break at a target site in a human PDCD-1 gene, wherein the break is repaired by non-homologous end joining (NHEJ).
39 . A method of editing a human PDCD-1 gene in a cell comprising: introducing a polynucleotide encoding the polypeptide of any one of claims 1 to 16 and a donor repair template into the cell, wherein expression of the polypeptide creates a double strand break at a target site in a human PDCD-1 gene and the donor repair template is incorporated into the human PDCD-1 gene by homology directed repair (HDR) at the site of the double-strand break (DSB).
40 . The method of any one of claims 37 to 39 , wherein the cell is a hematopoietic cell.
41 . The method of any one of claims 37 to 40 , wherein the cell is an immune effector cell.
42 . The method of any one of claims 37 to 41 , wherein the cell is a T cell.
43 . The method of any one of claims 37 to 42 , wherein the cell is a CD3 + , CD4 + , and/or CD8 + cell.
44 . The method of any one of claims 37 to 43 , wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.
45 . The method of any one of claims 37 to 41 , wherein the cell is a natural killer (NK) cell or natural killer T (NKT) cell.
46 . The method of any one of claims 37 to 45 , wherein the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors.
47 . The method of any one of claims 37 to 46 , wherein the polynucleotide encoding the polypeptide is an mRNA.
48 . The method of any one of claims 37 to 47 , wherein a polynucleotide encoding a 3′-5′ exonuclease is introduced into the cell.
49 . The method of any one of claims 37 to 48 , wherein a polynucleotide encoding Trex2 or a biologically active fragment thereof is introduced into the cell.
50 . The method of any one of claims 39 to 49 , wherein the donor repair template encodes a PDCD-1 gene or portion thereof comprising one or more mutations compared to the wild type PDCD-1 gene.
51 . The method of any one of claims 39 to 49 , wherein the donor repair template encodes an engineered antigen receptor.
52 . The method of claim 51 , wherein the engineered antigen receptor an engineered antigen receptor.
53 . The method of claim 52 , wherein the engineered antigen receptor is an αβTCR, a γδTCR, one or more components of a DARIC, a chimeric antigen receptor, or a zetakine.
54 . The method of any one of claims 37 to 53 , wherein the I-OnuI HE variant is more thermostable than an I-Onu HE variant that has not been refined to increase thermostability.
55 . The method of any one of claims 37 to 53 , wherein the I-OnuI HE variant is more thermostable than an I-Onu HE variant comprising an amino acid sequence set forth in any one of SEQ ID NOs: 15-20.