IMMUNOLOGICALLY DISCERNIBLE CELL SURFACE VARIANTS FOR USE IN CELL THERAPY

Abstract

The invention relates to a mammalian cell, particularly a human cell, expressing a first isoform of a surface protein, wherein the first isoform is functionally indistinguishable, but immunologically distinguishable from a second isoform, for use in a medical treatment of a patient having cells expressing the second isoform form of the surface protein. The invention further relates to an agent selected from 1) a compound comprising, or consisting of, an antibody or antibody-like molecule and 2) an immune effector cell bearing an antibody-like molecule or an immune effector cell bearing a chimeric antigen receptor, for use in a method of treatment of a medical condition, wherein the agent is specifically reactive to either a first or a second isoform of a surface protein, wherein the first isoform is functionally indistinguishable, but immunologically distinguishable from the second isoform, and wherein the agent is administered to ablate a cell bearing the isoform that the agent is reactive to.

Claims

1-37. (canceled)

38. A method for treating a human patient in need thereof, said method comprising administering a human cell expressing a first isoform of a surface protein, wherein said first isoform of said surface protein is functionally indistinguishable, but immunologically distinguishable from a second isoform of said surface protein, and wherein said second isoform of said surface protein is the native isoform of said human patient and said first isoform is a genetically engineered isoform of said surface protein.

39. The method according to claim 38, wherein said surface protein comprises an extracellular polypeptide sequence and said first isoform comprises an insertion, deletion and/or substitution of 1, 2, 3, 4 or 5 amino acids in comparison to said second isoform.

40. The method according to claim 38, wherein said first isoform can be distinguished from said second isoform by antibody-like molecule binding, antibody binding or by reaction of an immune effector cell bearing an antibody or an antibody-like molecule or an immune effector cell.

41. The method according to claim 38, wherein said first isoform can be distinguished by reaction of a T cell bearing a chimeric antigen receptor (CAR).

42. The method according to claim 38, wherein said surface protein is selected from CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDwl2, CD13, CD14, CD15, CD15u, CD15s, CD15su, CD16, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85a, CD85d, CD85j, CD85k, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b, CD122, CD123, CD124, CD125, CD126, CD127, CD129, CD130, CD131, CD132, CD133, CD134, CD135, CD136, CD137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CDw145, CD146, CD147, CD148, CDw149, CD150, CD151, CD152, CD153, CD154, CD155, CD156a, CD156b, CD156c, CD157, CD158e, CD158i, CD158k, CD159a, CD159c, CD160, CD161, CD162, CD163, CD164, CD165, CD166, CD167a, CD167b, CD168, CD169, CD170, CD171, CD172a, CD172b, CD172g, CD173, CD174, CD175, CD175s, CD176, CD177, CD178, CD179a, CD179b, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CD191, CD192, CD193, CD194, CD195, CD196, CD197, CDw198, CD199, CD200, CD201, CD202b, CD203c, CD204, CD205, CD206, CD207, CD208, CD209, CD210, CDw210b, CD212, CD213a1, CD213a2, CD215, CD217a, CD218a, CD218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235b, CD236, CD236R, CD238, CD239, CD240CE, CD240DCE, CD240D, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD252, CD253, CD254, CD256, CD266, CD267, CD268, CD269, CD270, CD271, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD286, CD289, CD290, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a, CD300c, CD300e, CD301, CD302, CD303, CD304, CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD308, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, CD371, BCMA, an Immunoglobulin light chain (lambda or kappa), a HLA protein and ?2-microglobulin.

43. The method according to claim 42, wherein said surface protein is selected from CD2, CD3, CD4, CD5, CD8, CD19, CD20, CD22, CD23, CD33, CD34, CD90, CD45, CD123, BCMA, an Immunoglobulin light chain (lambda or kappa), a HLA protein and ?2-microglobulin.

44. The method according to claim 38, wherein said first isoform is not encoded in the patient's native genomic DNA.

45. The method according to claim 38, wherein said first isoform is obtained by changing a sequence encoding said surface protein gene in the patient's native genomic DNA by gene editing or by changing mRNA encoding said surface protein by RNA editing.

46. The method according to claim 38, wherein said first isoform is obtained by insertion, deletion and/or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15 or 20 amino acids in the amino acid sequence of said second isoform of said surface protein.

47. The method according to claim 46, wherein said insertion, deletion and/or substitution is located in the extracellular portion of said first surface protein, particularly in an extracellular loop.

48. The method according to claim 38, wherein said cell is administered prior to, concomitant with or after specific ablation of cells expressing said second isoform of said surface protein.

49. The method according to claim 48, wherein ablation of cells expressing said second isoform of said surface protein is performed by administration to said patient of an agent selected from an antibody-like molecule, an antibody, an immune effector cell bearing an antibody or an antibody-like molecule and an immune effector cell, wherein said agent is specifically reactive to said second isoform but not to said first isoform of said cell surface protein.

50. The method according to claim 49, wherein ablation of cells expressing said second isoform of said surface protein is performed by administration of a T cell bearing a chimeric antigen receptor, wherein said chimeric antigen receptor is specifically reactive to said second isoform but not to said first isoform of said cell surface protein.

51. The method according to claim 38, wherein said cell expresses an antibody or an antibody-like molecule, particularly a chimeric antigen receptor, reactive against the second isoform of said surface protein.

52. The method according to claim 38, wherein said surface protein is selected from the group consisting of CD33, CD34, CD45, CD123, CD19, CD8 and CD4.

53. The method according to claim 38, wherein said surface protein is selected from CD45.

54. The method according to claim 38, wherein said human cell is a hematopoietic cell.

55. The method according to claim 38, wherein said human cell is selected from the group consisting of a hematopoietic stem cell (hemocytoblast), a CD4+ T cell, a CD8+ T cell, a memory T cell, a regulatory T cell (T reg), a natural killer cell (NK), an innate lymphoid cell (ILC), a dendritic cell (DC), a B-lymphocyte, a mucosal-associated invariant T cell (MAIT) and a gamma delta T cell (?? T).

56. The method according to claim 38, wherein said human cell is a human T cell.

57. The method according to claim 38, wherein said human cell is an immune effector cell bearing a chimeric antigen receptor and wherein said immune effector cell is specifically reactive to either a first or a second isoform of the surface protein.

58. The method according to claim 38, for treating a medical condition selected from the group consisting of (i) a hematopoietic disorder, (ii) a malignant hematopoietic disease (iii) a non-malignant hematopoietic disease, (iv) Graft-versus-host disease.

59. The method according to claim 38, for treating a medical condition selected from the group of graft-versus host disease caused by hematopoietic stem cell transplantation or adoptive transfer or organ transplantation.

60. The method according to claim 58, for treating a malignant hematopoietic disease refractive to treatment with anti-CD19 CAR T-cells.

61. The method according to claim 58, for treating graft-versus host disease caused by hematopoietic stem cell transplantation or adoptive transfer or organ transplantation.

62. A human hematopoietic stem cell, expressing a first isoform of a surface protein, wherein said first isoform of said surface protein is functionally indistinguishable, but immunologically distinguishable from a second isoform of said surface protein, and wherein said second isoform of said surface protein is the native isoform and said first isoform is a genetically engineered isoform of said surface protein, wherein said surface protein is CD45, CD33, CD34, CD19, CD4 or CD123.

63. A method for treating a medical condition of a subject in need thereof, comprising administering to said subject, a therapeutically efficient amount of an agent selected from a. a compound comprising, or consisting of, an antibody or antibody-like molecule, and; b. an immune effector cell bearing an antibody or an antibody-like molecule or an immune effector cell bearing a chimeric antigen receptor, wherein said agent is specifically reactive to either a first or a second isoform of a surface protein, wherein said first isoform of said surface protein is functionally indistinguishable, but immunologically distinguishable from said second isoform of said surface protein, and wherein said agent is administered to ablate a cell bearing the isoform that the agent is reactive to.

64. The method according to claim 63, wherein said antibody or antibody-like molecule is coupled to a toxin.

65. The method of claim 64, wherein said toxin is saporin.

66. The method according to claim 63, wherein said agent is a bispecific antibody or bispecific antibody-like molecule.

67. The method according to claim 66, wherein said agent is an immune effector cell bearing a bispecific antibody or bispecific antibody-like molecule.

68. The method according to claim 63, wherein said agent is an immune effector cell bearing a. a first antibody or antibody-like molecule specifically reactive to either a first or a second isofolin of a first surface protein, wherein said first and said second isoform of said first surface protein are functionally indistinguishable, but immunologically distinguishable, and b. a second antibody or antibody-like molecule specifically reactive to either a first or a second isoform of a second surface protein, wherein said first and said second isoform of said second surface protein are functionally indistinguishable, but immunologically distinguishable.

69. The method according to claim 63, wherein said agent is an immune effector cell bearing a chimeric antigen receptor, and said medical condition is a hematopoietic disease.

70. The method according to claim 69, wherein said immune effector cell is a T cell, and said hematopoietic disease is a malignant hematopoietic disease.

71. The method according to claim 63, for treating graft-versus-host disease.

72. A combination medicament, comprising: a. a first agent selected from i) a compound comprising, or consisting of, an antibody or antibody-like molecule, and; ii) an immune effector cell bearing an antibody or an antibody-like molecule or an immune effector cell bearing a chimeric antigen receptor, wherein said first agent is specifically reactive to a first isoform of a first surface protein but not a second isoform of said first surface protein, wherein said first isoform of said first surface protein is functionally indistinguishable, but immunologically distinguishable from said second isoform of said first surface protein, and b. a second agent selected from i) a compound comprising, or consisting of, an antibody or antibody-like molecule, and; ii) an immune effector cell bearing an antibody or an antibody-like molecule or an immune effector cell bearing a chimeric antigen receptor, wherein said second agent is specifically reactive to a first isoform of a second surface protein but not a second isoform of said second surface protein, wherein said first isoform of said second surface protein is functionally indistinguishable, but immunologically distinguishable from said second isoform of said second surface protein.

73. The combination medicament according to claim 72, wherein said first and said second agent are T cells bearing a chimeric antigen receptor.

74. The combination medicament according to claim 72, wherein either, (i) said first surface protein is CD19 and said second surface protein is CD45, or (ii) said first surface protein is CD34 and said second surface protein is CD45.

75. A method for in vivo tracking of a cell expressing a first isoform of a surface protein in a patient, wherein said first isoform of said surface protein is functionally indistinguishable, but immunologically distinguishable from a second isoform of said surface protein, said method comprising administrating to said patient a ligand specifically reactive to said first isoform.

76. A method for selectively depleting or enriching a cell in vivo, comprising the steps of a. providing a cell, wherein said cell expresses a first isoform of a surface protein, which is different from a second isoform of said surface protein with regard to an amino acid marker, wherein said first isoform comprises amino acid marker A encoded by nucleic acid sequence A, and said second isoform comprises amino acid marker B encoded by nucleic acid sequence B; b. inducing a mutation from said nucleic acid sequence A to said nucleic acid sequence B in the genomic DNA of said cell; c. selectively enriching/depleting said cell based on the expression of said first or said second isofolut of said surface protein.

77. A kit comprising the following components: a. a base editor guide RNA targeting a genomic location of a gene encoding a cell surface protein, wherein i. said gene exists in two isoforms that differ with regard to a nucleic acid marker sequence, wherein isoform 1 comprises a first marker sequence and isoform 2 comprises a second marker sequence; and ii. said genomic location comprises a base editor PAM sequence and said first or second marker sequence; and b. optionally, a first and a second antibody that bind specifically to the gene products of isoform 1 and isoform 2, respectively.

78. The kit according to claim 77, wherein said cell surface protein is murine Thy1 or murine CD45.

79. The kit according to claim 78, wherein said cell surface protein is murine CD45 and said base editor guide RNA comprises a nucleic acid sequence selected from SEO ID NO 004, SEO ID NO 005 and SEO ID NO 006.

80. A kit comprising the following components: a. a guide RNA targeting a genomic location of a gene encoding a cell surface protein, wherein i. said gene exists in two isoforms that differ with regard to a nucleic acid marker sequence, wherein isoform 1 comprises a first marker sequence and isoform 2 comprises a second marker sequence; and ii. said genomic location comprises a PAM sequence and said first or second marker sequence; and b. a DNA construct comprising i. said first marker sequence or said second marker sequence; ii. said PAM sequence, wherein in particular said PAM sequence is mutated and non-functional; iii. a pair of homology arms homologous to the genomic DNA sequences 5 and 3 of said genomic location of the gene encoding said cell surface protein; and c. optionally a first and a second antibody that bind specifically to the gene products of isoform 1 and isoform 2, respectively.

81. The kit according to claim 80, wherein said homology arms comprise at least 85 base pairs (bp) each.

82. The kit according to claim 81, said homology arms comprise at least 450 bp each.

83. The kit according to claim 81, said homology arms comprise approx. 2000 bp each.

84. The kit according to claim 80, wherein said cell surface protein is murine Thy1 or murine CD45.

85. The kit according to claim 80, wherein said cell surface protein is murine Thy1 and a. said guide RNA is SEQ ID NO 001 and said DNA construct is selected from SEQ ID NO 007 (no mut), SEQ ID NO 008 (mut), SEQ ID NO 009 (4x mut), SEQ ID NO 010 (2 kb), SEQ ID NO 011 (4 kb), SEQ ID NO 012 (1 kb) and SEQ ID NO 013 (160 bp); or b. said guide RNA is SEQ ID NO 002 and said DNA construct is selected from SEQ ID NO 014 (120 bp) and SEQ ID NO 015 (180 bp).

86. The kit according to claim 80, wherein said cell surface protein is murine CD45, said guide RNA is SEQ ID NO 003 and said DNA construct is selected from SEQ ID NO 016, SEQ ID NO 017 (1 kb), SEQ ID NO 018 (2 kb) and SEQ ID NO 019 (4 kb).

87. The kit according to claim 80, comprising murine T cells that have been genetically engineered for stable Cas9 expression.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0187] FIG. 1 illustrates the principle of engineering immunologically discernible variants of cell surface proteins. Isoform A and B are capable of performing the same function (are functionally comparable). In this example isoform A represents a naturally occurring surface protein that is engineered into variant isoform B.

[0188] FIG. 2 illustrates how immunologically discernible cell surface variants increase the safety of human cell therapy.

[0189] FIG. 3 shows that two isoforms of a surface protein can be distinguished by monoclonal antibodies (mAb). Sequences: upper row: SEQ ID NO 020, lower row: SEQ ID NO 021.

[0190] FIG. 4 shows a proof-of-concept experiment: Conversion of CD90.2 (allele A) to CD90.1 (allele B) in primary cells. The left flow cytometry panel shows a pure starting population of CD90.2+ primary T cells. Cells are then engineered in vitro to convert CD90.2 to CD90.1. Post editing 4 distinct populations can be discriminated: CD90.2+ homozygous cells (unedited starting population, upper left), CD90.1+/CD90.2+ heterozygous cells (upper right), CD90.1+ homozygous cells (lower right) and cells which lost CD90 (lower left). The allele engineered cells in the bottom right square no longer express the starting/original endogenous allele (CD90.2). A pure population of CD90.1+CD90.2? cells can be used to isolate correctly edited cells in vitro before transfer to a patient.

[0191] FIG. 5 shows a second proof-of-concept experiment: Conversion of CD45.2 (allele A) to CD45.1 (allele B) in primary cells. Sequences: upper row: SEQ ID NO 022, lower row: SEQ ID NO 023. As shown for CD90.2 to CD90.1 allele conversion (FIG. 4), the allele engineered cells in the bottom right square no longer express the starting/original endogenous allele (CD45.2). This can be used to isolate a pure population of correctly edited cells before transfer to a patient.

[0192] FIG. 6 shows gene editing in EL4 cells using Cas9 ribonucleoprotein particles (RNPs). EL4 cells were transfected with crRNA:tracrRNA/Cas9 complex and +/?HDR 2 kb template in the same way as for the plasmid based approach, except for electroporation conditions (described in methods section). This demonstrates that allele editing can be achieved by different approaches, e.g. plasmid-based or RNPs, i.e. is platform independent.

[0193] FIG. 7 shows gene editing in primary mouse T cells using Cas9 ribonucleoprotein particles (RNPs). Primary mouse T cells were transfected with crRNA:tracrRNA/Cas9 complex and +/?HDR 2 kb template in the same way as for the plasmid based approach. Like FIG. 6 this demonstrates that allele editing to convert allele A to allele B is platform independent.

[0194] FIG. 8 shows highly pure ex vivo selection of allele engineered cells. Left panel: After engineering CD45.2 cells into CD45.1 cells 4 population arise (comparable to FIG. 4). Flow cytometry was then used to purify all 4 populations to very high purity. The right panel displays the post-sort purity for each of the 4 populations. Conclusion: All 4 possible cell populations can be purified ex vivo to high purity based on the engineered allele. Correct gene editing was validated by Sanger DNA sequencing (not shown).

[0195] FIG. 9 shows the design of a base editor to convert CD45.1 to CD45.2 (A) and CD45.2 to CD45.1 (B). Sequences (A): upper row: SEQ ID NO 024, lower row: SEQ ID NO 025 (B): upper row (option 1 and 2): SEQ ID NO 026, lower row (option 1 and 2): SEQ ID NO 027. A) A guide RNA was designed to be used with the NNNRRT PAM restricted SaKKH-BE3 base editor. The chosen PAM sequence is highlighted by a dotted black bar (ATTTGT). The guide RNA is highlighted by a gray bar, the base editing window of guide RNA nucleotides 4-7 is highlighted. The G at position 5 will be converted to an A with this base editor. Thus, the CD45.1 allele will be converted to the CD45.2 allele. B) Engineered adenine base editors can convert A to G. To convert CD45.2 to CD45.1 two options were identified: Option 1: a sgRNA (gray bar) was designed to be used with Cas9 SaKKH (PAM highlighted by dotted black bar). This will convert the A at position 5 of the base editing window to a G. Option 2: a sgRNA (gray bar) to be used with Cas9 VQR. PAM highlighted by dashed black bar. This will convert the A at position 6 of the base editing window to G. Options 1 and 2 will convert the CD45.2 allele to the CD45.1 allele.

[0196] FIG. 10 shows the concept behind the optional ablation of the transferred cells. Cells are taken from a patient, then edited (ex vivo) to convert one allele to another one. Succssfully edited cells are selected to infuse a pure population of cells carrying the epitope necessary for selective depletion. Allele engineering has therefore added the option to ablate the transferred cells if needed.

[0197] FIG. 11 shows applications for selective depletion of transferred or host cells in vivo.

[0198] FIG. 12 shows a proof-of-concept experiment for selective ablation in vivo. Model for in vivo ablation: 1) Reconstitution of immunodeficient mice lacking all T cells (Rag KO) with a 1:1 mix of purified CD45.2+ and CD45.1+ T cells (congenic cells, not edited cells). 2) Antibody-mediated depletion (non-selective (anti-CD4) compared to selective (anti-CD45.2)). Anti-CD45.2 without toxin or coupled to toxin (Saporin). 3) Analysis 1 week later.

[0199] FIG. 13 shows selective depletion of CD45.2+ cells in vivo: Quantification of T cells in blood.

[0200] FIG. 14 shows selective depletion of CD45.2+ cells in vivo: Quantification of relative numbers of T cells in lymphoid organs. Same setup as in FIG. 12 but analysis of lymph nodes (LN) and mesenteric lymph nodes (mesLN).

[0201] FIG. 15 shows selective depletion of CD45.2+ cells in vivo: Quantification of relative numbers of T cells in lymphoid organs. Same setup as in FIG. 12 but analysis of spleen (SP).

[0202] FIG. 16 shows selective depletion of CD45.2+ cells in vivo: Quantification of absolute numbers of T cells in lymphoid organs. Same setup as in FIG. 12 but analysis of lymph nodes (LN) and mesenteric lymph nodes (mesLN).

[0203] FIG. 17 shows selective depletion of CD45.2+ cells in vivo: Quantification of absolute numbers of T cells in lymphoid organs. Same setup as in FIG. 12 but analysis of spleen (SP).

[0204] FIG. 18 shows a second proof-of-concept experiment for selective in vivo ablation of allele edited cells. Experimental setup: 1) Electroporation of CD45.2 T cells ex vivo to engineer to CD45.1 2) Adoptive transfer to T cell deficient mice (Rag KO); (no purity selection before transfer) 3) Weeks later quality control to demonstrate allele editing 4) Antibody-mediated depletion of CD45.2 cells leaving only edited cells. Conclusion: PoC demonstrating in vivo depletion of CD45.2+ host and unedited cells while sparing allele engineered CD45.1+ cells

[0205] FIG. 19 shows an application of the invention: Allele engineering increases safety, e.g. for CAR-T cell Therapy. Engineered cells express a first isoform of a cell surface protein (e.g. CD45) and a chimeric antigen receptor reactive to a target of interest, e.g. CD19 (CAR-19). While the CAR19 is engineered into the T cells the second CD45 allele is converted into the first CD45 allele to enable discrimination of the transferred CAR T cells from the non-engineered host cells. In the case of treatment related toxicity the pathogenic CAR-T cells can be selectively ablated to stop CAR-T related toxicity.

[0206] FIG. 20 shows the gene correction of scurfy cells and cells bearing the human Foxp3K276X mutation as well as enrichment of the relative frequency of gene-repaired cells when gating on an isoform-switched surrogate surface marker.

[0207] A) Alignment of genomic DNA sequences of wildtype foxp3 (C57BL/6) (SEQ ID NO 028), the Foxp3 locus with a targeted mutation Foxp3K276X (SEQ ID NO 029) which introduces a premature stop codon and the Foxp3 locus of scurfy mice (B6.Cg-Foxp3sf/J) which harbor a spontaneous 2 bp insertion leading to a frame-shift (SEQ ID NO 030). sgRNA binding sites (green line) and PAM sequences (black line). B) Protocol for gene editing of total CD4+ T cells from Foxp3K276X C57BL/6 mice. In vitro activation and electroporation (step 1) with plasmids encoding sgRNA targeting the Foxp3K276X mutation and a circular plasmid containing a 1 kb wildtype (wt) Foxp3 repair template. Successfully transfected cells are isolated based on GFP expression (step 2). Cell expansion in vitro for gene editing in presence of rhIL-2, TGF-6 alone or in combination with retinoic acid (RA) and cytokine neutralizing antibodies (anti-IL-4 and anti-IFN? for 7 days (step 3). C) Experimental setup as in B with total CD4+ T cells from control mice (WT) or Foxp3K276X mice. Flow cytometry of CD25 and Foxp3 expression (gated on live CD4+ T cells). Wildtype cells electroporated with empty px458 plasmid differentiate into CD4+Foxp3+CD25+ T cells (left panel), absence of Foxp3 differentiation in Foxp3K276X cells electroporated with sgRNA Foxp3K276X alone (middle panel) and restoration of Foxp3 protein expression in Foxp3K276X cells electroporated with sgRNA Foxp3K276X and 1 kb Foxp3 dsDNA repair template (right panel). Top row: Foxp3 induction with TGF-f3 alone, bottom row: Foxp3 induction with TGF-? combined with RA. Compared to TGF-? alone the combination of TGF-? and RA leads to a higher frequency of Foxp3 expressing cells in those cells which have an intact Foxp3 locus (i.e. wildtype and repaired cells). Representative data from 2 experiments with Foxp3.sup.K276X cells and one experiment with Foxp3.sup.sf/J cells. D) Enrichment of gene-repaired Foxp3 expressing cells using multiplexed CD45 isoform switching as a surrogate marker. Experimental setup as in b but simultaneous electroporation of plasmids encoding 2 sgRNAs (sgRNA Foxp3K276X and sgRNACD45.2_R1) and two 1 kb dsDNA templates (Foxp3 wildtype and CD45.1). Seven days later flow cytometry of CD45.2, CD45.1, CD25 and Foxp3 (gated on live CD4+ cells). Top panel: Pre-gating on CD45.1? cells (green line) and CD45.1+ cells (red line). Bottom panel: Enrichment of CD25+Foxp3+ cells in isoform switched CD45.1+ cells. Representative data from 2 experiments with Foxp3.sup.K276X cells and one experiment with Foxp3.sup.sf/J cells.

[0208] FIG. 21 shows repair of the Foxp3 gene using the plasmid based approach and the RNP based approach. A: CD4 T cells from Foxp3 KO mice were transfected with sgRNA plasmid alone or together with a Foxp3 wildtype HDR template. GFP+ and GFP? cells were sorted 24 h post transfection (plasmid transfection) and immediately after cell sorting expanded until the end of the experiment in the presence of Foxp3 differentiation cocktail. B: CD4 T cells from Foxp3 KO mice were transfected with crRNA:tracrRNA/Cas9 RNP complex alone or +/?HDR templates (180 bp ssDNA or 2 kb plasmid). Total pool of RNPs transfected cells were expanded until the end of the experiment in the presence of Foxp3 differentiation cocktail.

[0209] FIG. 22 shows successful Foxp3 gene repair in T cells in vitro. Upper row: Alignment of genomic DNA sequences of wt Foxp3 (C57BL/6) SEQ ID NO 031 and the Foxp3 locus with a targeted mutation Foxp3K276X SEQ ID NO 032 that introduces a premature stop codon. sgRNA binding sites (green line) and PAM sequences (black line). Total CD4+ T cells from wt control or Foxp3K276X mice were transfected. Flow cytometry of CD25 and Foxp3 expression (gated on live CD4+ T cells). Differentiation of wt cells electroporated with empty px458 plasmid into CD4+Foxp3+CD25+ T cells (upper panel), absence of Foxp3 differentiation in Foxp3K276X cells electroporated with sgRNAFoxp3K276X alone (middle panel) and restoration of Foxp3 protein expression in Foxp3K276X cells electroporated with sgRNAFoxp3K276X and 1 kb Foxp3 dsDNA repair template (lower panel). Foxp3 induction with TGFbeta combined with RA.

[0210] FIG. 23 shows a protocol for gene editing of total CD4+ T cells from Foxp3K276X C57BL/6 mice. In vitro activation and electroporation with plasmids encoding sgRNA targeting the Foxp3K276X mutation and a circular plasmid containing a 1 kb wt Foxp3 repair template. Successfully transfected cells are isolated based on GFP expression. Cells are the transferred in vivo for Foxp3 differentiation followed by IL-2 regimen.

[0211] FIG. 24 shows the clinical phenotype of mice 14 weeks after AT of cells. WT and repair mice are disease free, transfer of KO cells results in skin inflammation and alopecia. Experimental setup as in FIG. 23.

[0212] FIG. 25 shows the clinical phenotype (T cell infiltration) in mice that received KO cells correlates with infiltration of CD4/CD3+ T cells in ear and tail skin. No or very low numbers of CD4/CD3+ T cells are found in ear and tail skin of mice that received WT or Repaired T cells. Data are depicted as % and absolute numbers.

[0213] FIG. 26 shows the presence of CD25/Foxp3+ Treg cells in mice that received WT and repaired cells. No CD25/Foxp3+ T cells are found in mice that received KO cells. Data are displayed as % and MFI of Foxp3. Experimental setup as in FIG. 23.

[0214] FIG. 27 shows that repaired Treg respond to IL-2. Additional IL-2 treatment during the last week of experiment results in increased presence of CD25/Foxp3+ Treg cells in mice that received WT and Repaired cells. No CD25/Foxp3+ T cells are found in mice that received KO cells. These data demonstrate that WT and Repaired Treg cells respond to IL-2 to similar extent.

[0215] FIG. 28 shows that WT and repaired CD25/Foxp3 Treg cells express similar levels of different cell surface markers (GITR, ICOS, TIGIT, PD1 and KLRG1). The combination of CD25 and additional markers (e.g. GITR) can be used to enrich for these populations. Foxp3-deficient T cells are phenotypically different, e.g. they lack GITR and KLRG1 expression.

[0216] FIG. 29 shows that AT of CD45.1+ WT cells rescues Foxp3 deficient mice and expands their life span up to 8 weeks (latest time point examined, no indications of disease). High proportion of WT Treg cells (50%) found in a Foxp3 KO mice suppress host T cells activation (lower % of CD44high cells). This demonstrates that WT T cells have the capacity to fully restore immune regulation and control scurfy disease. Furthermore, the Foxp3-deficient microenvironment boosts Foxp3-suficient T cells to expand massively.

[0217] FIG. 30 shows a CD4 depletion experiment in Foxp3 KO mice (lymphnode, LN). Combined gene repair in T cells with selective antibody depletion: Deplete pathogenic cells, then transfer gene-corrected T cells. CD4 depletion largely prevents scurfy disease and extends life expectancy significantly. The observation is supported by FACS data showing low or non-existent CD4 T cells (KO depleted compared to KO or WT mice without treatment).

[0218] FIG. 31 shows a CD4 depletion experiment in Foxp3 KO mice (spleen, SP). Combined gene repair in T cells with selective antibody depletion: Deplete pathogenic cells, then transfer gene-corrected T cells. CD4 depletion largely prevents scurfy disease and extends life expectancy significantly. The observation is supported by FACS data showing low or non-existent CD4 T cells (KO depleted compared to KO or WT mice without treatment).

[0219] FIG. 32A shows that CD4 depletion prevents development of scurfy disease in Foxp3-deficient mice.

[0220] FIG. 32B shows that CD4 depletion largely prevents development of dermatitis in tail skin of Foxp3-deficient mice.

EXAMPLES

[0221] A description of efficient plasmid-based gene ablation in primary T cells, targeted introduction of point mutations in primary T cells, enrichment of HDR-edited cells through monitoring of isoform switching of a surrogate cell surface marker and a description of gene correction of murine scurfy cells can be found in EP16196860.7, EP16196858.1 and PCT/EP2017/059799.

[0222] General Considerations for the Design of Engineered Mutations (Allele Engineering):

[0223] Engineering an allele of a gene by introducing a targeted small mutation, possibly single nucleotide or single amino acid mutation, with the purpose of changing the specific binding of a ligand and allowing the binding of a second, specific ligand can be useful as a general principle for therapeutic use. The mutation is designed in a way to preserve the function of the engineered protein as much as possible. The immunogenicity needs to be changed to be able to raise specific binding ligands such as monoclonal antibodies (mAb) or antibody-like molecules such as affimers, DARPINS, nanobodies etc. Such ligands need to be screened for specific binding and the nature of the immunogen needs careful design. At the same time, the mutation constitutes a minor antigenic change and therefore is unlikely to cause a strong immunologic reaction in vivo. Congenic markers in mice fulfill exactly these criteria. The difference between CD90.1 and CD90.2 as well as between CD45.1 and CD45.2 is a single nucleotide leading to a single amino acid difference. In both cases, this difference can be detected by specific mAbs resulting in a pair of mAbs for each gene. Although mAbs could be raised against those differences when congenic cells are transferred into matching congenic host mice, i.e. CD90.1+ cells into CD90.2+ host mice or vice versa and CD45.1+ cells into CD45.2+ hosts and vice versa the cells are not immunologically rejected. This means that this minor antigenic difference is tolerated by the immune system in vivo. Collectively, these properties have made congenically marked cells a very useful tool for immunologic research which has been used for decades. Immunologically these cells are used as surrogate cells for autologous transfers (because the cells are genetically identical except for this one mutation) but the congenic minor difference allows to discriminate transferred from host cells.

[0224] Choice of a Protein to be Engineered:

[0225] To design a similar system for therapeutic use in humans several considerations need to be taken into account. The mutation can in principle be introduced into any gene encoding a protein. The expression pattern of the protein of interest will be relevant, i.e. ubiquitous or cell- or tissue-specific expression if desired. Proteins expressed on the surface can be most directly targeted and will therefore in most cases be the proteins of choice. Examples include but are not limited to the proteins characterized by the cluster of differentiation system CD1 to CD371) (Engel, J Immunol Nov. 15, 2015, 195 (10) 4555-4563 and http://www.hcdm.org/). Other proteins of interest can be ubiquitously expressed proteins such as beta-2-microglobulin or constant parts of HLA-class I which are expressed on all cells, including non-immunologic cells. Alternatively, proteins expressed in specific cell types can be of interest, e.g. human CD45 for hematopoietic cells, human CD3 for T cells, constant regions of human T cell receptor components, constant regions of B cell immunoglobulins such as the kappa and lambda light chain or constant regions of the heavy chains, human CD4 or CD8 coreceptors or B cell markers such as CD19, CD20, CD21, CD22, CD23, costimulatory molecules such as CD28 or CD40, CD34 on hematopoietic stem cells or even specific isoforms expressed on subsets of cells such as CD45RA or CD45RO. In this latter case the mutation would be designed into the variable region (alternatively spliced exons) which differs between CD45RA and CD45RO. Engineering a mutation into a ubiquitously expressed molecule such as beta-2-microglobulin or HLA-I could serve as a unique system which can be used in virtually any mammalian cell, more specifically any human cell. This could be used to track and ablate the engineered cells while sparing the non-engineered host cells. Such a feature could e.g. be useful as a kill switch for cell therapy, including various types of stem cells (e.g. muscle stem cells), hepatic cells or cells derived from induced pluripotent stem cells (iPS). Engineering a mutation into a cell type-specific protein such as CD45 could be useful to target all cells from that specific tissue. In the case of the hematopoietic system, engineering human CD45 could serve to mark hematopoietic stem cells (HSC) which are used to replace the endogenous hematopoietic system. All progeny of those HSCs will harbor the same mutation engineered into the genome of the original HSCs. If the previous host hematopoietic system is removed by any means (e.g. irradiation, chemotherapy, depleting ligands such as mAb or antibody-like molecules, mAb coupled with toxins, cell-mediated ablation, e.g. CAR-T cell mediated ablation etc.) then the replacing hematopoietic system, even if autologous, could be discriminated by the engineered mutation. Alternatively, it would allow the ablation of host cells while sparing the engineered cells.

[0226] Design Considerations: [0227] Expression pattern of the protein (ubiquitous versus cell- or tissue specific) [0228] Extracellular proteins: In most cases mutations will be engineered into the extracellular parts of proteins of interest which are accessible to specific ligands. The mutations can be engineered into constantly expressed exons or alternatively spliced exons if subset specific expression is desired. [0229] Conservation across species: conserved amino acids (aa) or structures are more likely to be functionally relevant and should therefore rather remain untouched. Mutations should rather be incorporated into less conserved regions. [0230] Chemical properties of the amino acids, e.g. polarity, electrical charge, hydrophobicity [0231] Similarity of amino acids: To preserve function the mutation should convert a given aa into a related aa but the changes still need to be detectable by specific ligands. [0232] Naturally occourring variations: mutations which correspond to amino acid variations observed at a give position in a multiple sequence alignememnts of members of a protein family (homologous sequences of different organisms) are likely to not affect the function and the structure of the protein, and can be slected to design mutations. [0233] Structural considerations: Structural data can assist in the rationale design of potential mutations. The amino acid to be engineered has to be accessible to ligand binding. Mutations in loops are functionally relatively well tolerated and are therefore of interest. [0234] Avoid known disease-causing mutations. [0235] Consider known human genetic variations: functionally tolerated single nucleotide polymorphisms (SNP)s are good candidate mutations. [0236] Sites important for secondary modifications such as glycosylations should not be mutated, [0237] Sites important for disulfide bonds should not be mutated. [0238] Sites important for reported interactions (e.g. hydrogen bonding, salt bridges, hydrophobic stacking interactions) should not be mutated, both protein internally but also protein-protein interactions for multiprotein complexes or receptor-ligand interactions. [0239] Consider previous successful systems such as murine CD90.1/CD90.2 (Q to R) [0240] Consider binding sites of known binding ligands such as known mAbs and computational modelling of complementarity determining regions (CDRs). [0241] Exclude known active sites, e.g. catalytic sites of enzymes [0242] Avoid structurally very similar domains present in other human proteins, since they are likely to increase the risk for cross-specific ligands [0243] Consider immunogenicity: choose a site that is likely to result in a suitable peptide immunogen.

[0244] Allele Engineering as a Generally Applicable, Platform-Independent Principle:

[0245] In EP16196860.7, EP16196858.1 and PCT/EP2017/059799, the inventors demonstrate and characterize how the CRISPR/Cas platform can be used to engineer a targeted point mutation for allele engineering followed by selective ablation. The inventors engineered two separate point mutations into two distinct genes and demonstrate that the engineered mutation can be used to track and/or selectively ablate engineered cells in vivo. The inventors relied on the CRISPR/Cas9 system and a dsDNA template to achieve homology directed repair (HDR). However, the example used is just one way of introducing the mutation. Alternatives include different types of nucleases such as zinc finger proteins, TALENs or other naturally occurring or engineered CRISPR/Cas systems such as Cas9, Cpf-1, high fidelity nucleases or nucleases with engineered PAM dependencies (Komor, Cell, 2017). Moreover, the described principle is independent of the form of delivery of the nucleases. They can be delivered as a plasmid, mRNA, recombinant protein, recombinant protein in complex with a guide RNA (i.e. ribonuclear complex, RNP), split recombinase or as an integrating or non-integrating virus, e.g. retrovirus, lentivirus, baculovirus or other viral delivery platform. The delivery modality can encompass electroporation or other forms such as lipofection, nanoparticle delivery, cell squeezing or physical piercing. In addition, the HDR template can be a ssDNA or dsDNA in the form of short ssDNA, long ssDNA or a circular or linear minicircle DNA, plasmid DNA or a viral DNA template, e.g. adeno-associated virus (AAV). Depending on the target cell, specific AAV serotypes are used to deliver cargo to the cells. For human T cells and human hematopoietic stem cells HDR templates are often provided as AAV6 but endonuclease RNP along with short ssDNA HDR templates can also successfully be used. Thus, the mode how the mutation is introduced can be flexible.

[0246] Allele Engineering Employing Alternative Approaches, e.g. Base Converters or Base Editors

[0247] The inventors demonstrated how a targeted dsDNA break followed by HDR can be exploited to introduce small, targeted, i.e. precise mutations for alleleic engineering. In addition, there are alternative approaches how designed point mutations could be engineered into the genome of a living cell. As an example, newly designed chimeric fusion proteins allow direct conversion of a target DNA base into another (Komor, Nature 2016; Nishida, Science 2016; Yang, Nat Comm, 2016; Ma, Nat Meth 2016). Enzymes such as deaminases can be fused to DNA binding modules such as (but not limited to) zinc finger proteins, TALENS or CRISPR/Cas systems. Naturally occurring cytidine deaminases (APOBEC1, APOBEC3F, APOBEC3G) and activation induced deaminase (AID) or the AID ortholog PmCDA1 (from sea lamprey) can convert cytidines (C) to uracils (U) in DNA. The DNA replication machinery will treat the U as T if DNA replication occurs before U repair. This leads to a conversion of a C:G to a T:A base pair (Yang, Nat Comm, 2016; Komor, Nature 2016). Therefore, several groups have developed engineered chimeric proteins with deaminases fused to DNA binding modules which are used to bring the deaminase to a specific genomic locus. For instance the CRISPR/Cas system can be used as a delivery system when an engineered, catalytically dead (dCas9) version of the Cas9 nuclease is used. This approach has successfully been used to target fused effector molecules to specific genomic loci. Applications include targeting fluorescent proteins to specific loci or bringing transcriptional transactivators or repressors to specific genomic loci to control specific gene expression (Wang, Ann Rev Biochem, 2016). Fusing a cytidine deaminase or AID to a nickase Cas9 and additional engineering to improve the base editing efficiency allows direct targeted base conversion (Komor, Nature 2016; Nishida, Science 2016). Under certain circumstances this could have advantages over the HDR approach since base editing neither induces a dsDNA break nor requires the delivery of a DNA HDR template. Therefore this alternative approach could lead to fewer indels and thus might be a safe or even safer alternative to HDR based genome engineering. In addition, delivery of the base editor as mRNA or RNP could be sufficient and could be less toxic for certain cell types. For human T cells Cas9 RNPs offer a successful genome editing approach (Schumann, PNAS, 2015), therefore it can be anticipated that base converters in the form of RNPs might be particularly well-suited for hematopoietic cells including hematopoietic stem cells (HSCs) and T cells but in principle base conversion might be a suitable approach for allele engineering applicable to any cell, including mammalian cells. The introduced designed point mutation can then be used for downstream applications such as cell marking, cell tracking and selective ablation. Base editors delivered as RNPs successfully edited target nucleotides in mammalian cells, mouse and zebrafish embryos and the inner ear of live mice (Rees, Nat Comm, 2017; Kim, Nat Biotech, 2017 doi:10.1038/nbt.3816). Thus, these studies demonstrate the feasibility of specific, DNA-free base editing. However, the number of nucleotides in the (human) genome which are amenable to base conversion is more restricted than the HDR approach since it not only depends on the PAM sequence (for CRISPR/Cas-based base converters) but underlies additional restrictions. Although cytidine deaminase base editors can only convert C to T (or G to A), newly engineered adenine base editors can convert A to G (or T to C (Gaudelli, Nature 2017)). However, in addition to the PAM requirement, base conversion only occurs within a certain window, specified by the specific design and/or engineering of the fusion protein. Thus, compared to HDR-mediated introduction of a point mutation the number of editable nucleotides is much more restricted if base editors are used (Komor, Nature 2016). In addition, the window of base conversion encompasses several nucleotides. For instance, for the so-called base editor 3 (BE3) that uses S. pyogenes Cas9 (SpBE3, see Komor et al., Nature 2016) this window encompasses about 5 nucleotides. Thus, editing a single C to T within a stretch of multiple adjacent C nucleotides will not be possible and will lead to additional unwanted mutations which may change the amino acid of the resulting protein. Similar editing windows apply to adenine base editors. Furthermore, a different BE3 that is based on S. aureus Cas9, SaBE3, can result in detectable base editing at target Cs outside the canonical BE3 editing window. To address these limitations, newer base converter versions were engineered to have narrower editing windows and different PAM specificities than NGG (e.g. NGA, NGAG, NGCG, NNGRRT and NNNRRT) but the design of a specific base editor for a given single nucleotide conversion remains challenging (Kim, Nat Biotech 2017, doi:10.1038/nbt.3803). Although the new base converters expand the range of targetable nucleotides in the mammalian genome, not every nucleotide can be edited at will. Nevertheless, in order to take advantage of some of the benefits of base converters the inventors attempted to design a base editor for allele engineering of CD90.1 to CD90.2, CD90.2 to CD90.1, CD45.1 to CD45.2 or CD 45.2 to CD45.1. However, with the original NGG restricted base editors none of these conversions could be achieved with the available base editors. This is despite the fact that for both genes, CD90 and CD45, the allelic difference is encoded by a G to A substitution which in principle should be amenable to deaminase conversion. The only solution was to use a BE3 version based on SaCas9 containing 3 mutations that relax this Cas9 variant's PAM requirement to NNNRRT (Kleinstiver, Nature 2015; Kim, Nat Biotech, 2016 (doi:10.1038/nbt.3803). Choosing this base editor made it possible to design a base editor with a matching sgRNA that will convert the CD45.1 allele to the CD45.2 allele (FIG. 9A). However, this base editor will only work to convert CD45.1 to CD45.2 but not CD45.2 to CD45.1 conversion we designed hypothetical adenine base editors (adenine base editor fusions with Cas9 SaKKH or Cas9 VQR (FIG. 9B). In contrast, none of the CD90 allele conversions are possible, even with the currently existing base editor version.

[0248] Methods

[0249] Detailed methods describig gene editing in primary murine CD4+ T cells, gene editing in EL-4 cells and a protocol for Foxp3 repair can be found in EP16196860.7, EP16196858.1 and PCT/EP2017/059799.

[0250] Human T-Cell Isolation and Antibodies

[0251] Human primary T cells were isolated from buffy coats (Blutspendezentrum, Basel) of healthy donors using Lymphoprep? (Stemcell Technologies) density gradient. Na?ve CD4.sup.+ T cells were pre-enriched with an Easysep Human na?ve CD4.sup.+ T-cell enrichment kit (Stemcell Technologies) according to the manufacturer's protocol. Alternatively, cord blood was used as source for PBMCs, without using na?ve T cells isolation step, given the high frequencies of na?ve T cells. Pre and post na?ve CD4.sup.+ T cells enrichment samples were stained with following antibodies in order to assess the purity: ?CD4-FITC (OKT-4), ?CD25-APC (BC96), ?CD45RA-BV711 (HI100), ?CD45RO-BV450 (UCHL1), ?CD62L-BV605 (DREG-56), ?CD3-PerCP (HIT3a) and Zombie-UV viability dye, all purchased at Biolegend.

[0252] In brief, for 1 buffy coat of 50 ml: prepare 2?50 ml Falcon tubes with filter and add 16 ml of Lymphoprep to each tube, spin ? 300 g for 1 min. Distribute the blood equally to both 50 ml filter tubes and top up with PBS to 50 ml. Spin ? 2000 rpm (acc 4, decc 1) for 15 min. Remove some of the serum and discard it. Carefully pool the white buffy coats to a fresh 50 ml Falcon tube. Add sterile PBS to the enriched PBMC fraction to approximately 50 ml and spin ? 300 g for 5 min. Discard the supernatant and resuspend pellet with 10 ml PBS and top up to 50 ml and spin ? 300 g for 5 min. Lyse the red blood cells, if needed, with red blood cell lysis buffer, before purification step.

[0253] Human T-Cell Transfection Protocol

[0254] Na?ve CD4.sup.+ T cells or total PBMCs from blood or cord blood were used for transfection. For T cell activation, 2?10.sup.6 cells were plated in a 24-well plate (Corning) coated with monoclonal antibodies (mAbs) a-CD3 (hybridoma clone OKT3, 5 (high), 2.5 (medium), 1 (low) ?g/ml) and a-CD28 (hybridoma clone CD28. 2.5 (high), 1 (medium), 0.5 (low) ?g/ml, both from Biolegend) for 24 h at 37? C. with 5% CO.sub.2 in the presence of 50 IU/ml recombinant human Interleukin-2 (rhIL-2) (RD systems). 24 h later T cells were harvested and washed with PBS. 2?10.sup.6 activated T cells were electroporated with the Amaxa Transfection System, T-020 program (for plasmid) or using Neon? Transfection System (ThermoFisher) at the following conditions: voltage (1600V), width (10 ms), pulses (3) 100 ?l tip, buffer R (for RNPs). Cells were transfected with 6.5 ?g of empty plasmid px458 (Addgene plasmid number: 48138) or crRNA:tracerRNA-Atto 550 (IDT) and Cas9 (Berkeley) complex. After electroporation cells were plated in 24-well plate in 650 ?l complete media with 50 IU rhIL-2/ml in the presence of plate-bound mAbs at half the concentrations used for the initial activation, i.e. anti-CD3 (2.5, 1.25, 0.5 ?g/ml) and anti-CD28 (1.25, 0.5, 0.25 ?g/ml). The expression of GFP.sup.+ or Atto550.sup.+ cells were assessed 24 h later by using Fortessa analyzer (BD Biosciences).

[0255] CD45.2Depletion Experiment

[0256] CD4.sup.+ T cells were isolated from C57BL6 (CD45.2) mice and C57BL6 congenic (CD45.1) mice using EasySep Mouse CD4.sup.+ T Cell Isolation Kit (Stem cell Technologies). RAG KO mice were reconstituted with 1:1 ration of 10?10.sup.6 CD45.2 and CD45.1 donor CD4.sup.+ T cells. Same day as T cells transfer, mice also received intraperitoneal injections of PBS (non treated group) or a depleting a-CD4 Ab (clone GK1.5, 250 ?g) for 3 consecutive days. CD45.2-ZAP immunotoxins were prepared by combining CD45.2 biotinylated antibody (160 kDa MW, Biolegend) with streptavidin-SAP conjugate (2.8 saporin molecules per streptavidin, 135 kDa MW, Advanced Targeting Systems) in a 1:1 molar ratio and subsequently diluted in PBS immediately before use, same as described in the initial publication: (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5179034/). In vivo administration of immunotoxin or the control with non-conjugated CD45.2 antibody was performed by intravenous injections. One week later, blood, peripheral lymph nodes (LN), mesenteric LN (mesLN) and spleen (SP) were collected and cells were stained with the following fluorochrome-conjugated mAbs: anti-CD45.2 (104), anti-CD45.1 (A20), anti-CD4 (RM4-5), anti-CD3 (145-2C11) all from Biolegend. Samples were acquired on a BD Fortessa (BD Biosciences) and analyzed with FlowJo software (Tree Star).

[0257] Items [0258] 1. A method to determine a first homology directed repair (HDR) event at a first genomic location within a eukaryotic cell, wherein [0259] said cell expresses a first isoform of a first surface protein, which is different from a second isoform of said first surface protein with regard to an amino acid marker, wherein said first isoform comprises amino acid marker A encoded by nucleic acid sequence A, and said second isoform comprises amino acid marker B encoded by nucleic acid sequence B; [0260] said first genomic location comprises said nucleic acid sequence A; [0261] and said method comprises the steps of [0262] a. inducing a first DNA double strand break at said first genomic location; [0263] b. providing a first DNA repair construct comprising said nucleic acid sequence B and a first pair of homology arms which are homologous to the DNA sequences 5 and 3 of said first genomic location; [0264] c. determining the expression of said first and/or said second isoform of said first surface protein on said cell and optionally purifying said cell based on the expression of said first and/or said second isoform of said surface protein; and [0265] d. determining the occurrence of said first HDR event, wherein expression of said second isoform of said first surface protein on said cell is equivalent to occurrence of said first HDR event. [0266] 2. The method according to item 1, wherein the occurrence of said first HDR event is determined at at least two different experimental conditions, and an increased ratio of expression of said second isoform to said first isoform at a first experimental condition compared to a second experimental condition indicates an increased HDR efficiency at said first experimental condition. [0267] 3. The method according to any one of the above items, wherein step a and b are performed in cell culture medium comprising vanillin and/or of rucaparib, particularly at a concentration of 50 ?M to 500 ?M vanillin and/or 0.5 ?M to 2.5 ?M of rucaparib, more particularly approx. 300 ?M vanillin and/or approx. 1 ?M of rucaparib. [0268] 4. The method according to any one of the above items, wherein said first and said second isoform of said first surface protein can be distinguished from each other by a ligand, particularly an antibody, wherein said ligand is capable of discriminatively binding to said amino acid marker A (and not to B) or to said amino acid marker B (and not to A), respectively. [0269] 5. The method according to any one of the above items, wherein said first surface protein is a native protein. [0270] 6. The method according to any one of the above items, wherein said first surface protein is a transgenic protein. [0271] 7. The method according to any one of the above items, wherein said purifying is effected by fluorescent activated cell sorting (FACS). [0272] 8. The method according to any one of items 1 to 6, wherein said purifying comprises magnetic-bead based enrichment of a cell expressing said first or said second isoform of said first surface protein. [0273] 9. The method according to any one of the above items, wherein said first surface protein is Thy1 or CD45. [0274] 10. The method according to any one of the above items, wherein said first double strand break is induced in said first genomic location by transfecting said cell with a DNA expression construct encoding a CRISPR-associated endonuclease (Cas9), and a guide RNA, wherein said guide RNA is capable of annealing to said first genomic location. [0275] 11. The method according to any one of the above items, wherein said homology arms comprise approximately 2000 basepairs (bp) each. [0276] 12. A method for selectively depleting or enriching an edited cell in a composition of non-edited and edited cells, wherein [0277] a. said non-edited cells express a first isoform of a surface protein and said edited cell has been edited by the method of any one of items 1 to 11 to express a second isoform of said surface protein, which is different from said first isoform with regard to an amino acid marker, wherein said first isoform comprises amino acid marker A encoded by nucleic acid sequence A, and said second isoform comprises amino acid marker B encoded by nucleic acid sequence B; and [0278] b. said edited cell is selectively enriched or depleted based on the expression of said first or said second isoform of said surface protein. [0279] 13. A method for selectively depleting or enriching a cell in a composition of cells, comprising the steps of [0280] c. providing a cell, wherein said cell expresses a first isoform of a surface protein, which is different from a second isoform of said surface protein with regard to an amino acid marker, wherein said first isoform comprises amino acid marker A encoded by nucleic acid sequence A, and said second isoform comprises amino acid marker B encoded by nucleic acid sequence B; [0281] d. inducing a DNA double strand break at a genomic location comprising said nucleic acid sequence A; [0282] e. providing a DNA repair construct comprising said nucleic acid sequence B and a pair of homology arms which are homologous to the DNA sequences 5 and 3 of said genomic location; [0283] f. selectively enriching/depleting said cell based on the expression of said first or said second isoform of said surface protein.