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WO2024219474A1 - Cellule présentant une délétion dans une ou plusieurs régions du groupe de gènes kir, du groupe de gènes lilr et du groupe de gènes klr, et son procédé de fabrication - Google Patents

Cellule présentant une délétion dans une ou plusieurs régions du groupe de gènes kir, du groupe de gènes lilr et du groupe de gènes klr, et son procédé de fabrication Download PDF

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WO2024219474A1
WO2024219474A1 PCT/JP2024/015500 JP2024015500W WO2024219474A1 WO 2024219474 A1 WO2024219474 A1 WO 2024219474A1 JP 2024015500 W JP2024015500 W JP 2024015500W WO 2024219474 A1 WO2024219474 A1 WO 2024219474A1
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gene
cell
cells
sequence
region
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康則 相澤
知幸 大野
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Logomix Inc
Logomix Inc Japan
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Logomix Inc
Logomix Inc Japan
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present disclosure relates to cells having deletions in one or more of the KIR gene cluster region, the LILR gene cluster region, and the KLR gene cluster region, and methods for producing the same.
  • KIR and LILR are receptors that are expressed on the membrane surface of natural killer (NK) cells and activate or inactivate the cytotoxic function of NK cells depending on the HLA protein of the target cell, and act depending on the type of HLA or allele type, but their mutual correspondence has often not been verified.
  • KIR, LILR, and HLA all have more frequent polymorphisms than normal genomic regions, and some KIR and LILR polymorphisms and HLA polymorphisms are known to be associated with disease, and it has been suggested that this is due to HLA allele type-dependent activation or inactivation of NK cells, but the causal relationship has not been verified.
  • KLR killer cell lectin-like receptor
  • Patent Document 1 discloses a technology for creating large-scale deletions in the genome.
  • Patent Document 3 cells have been obtained that are deleted in the region containing MLH1, the region containing p53, the region containing CD44, the region containing MET, and the region containing APP.
  • the present disclosure provides cells having deletions in one or more of the KIR gene cluster region, the LILR gene cluster region, and the KLR gene cluster region, and a method for producing the same.
  • the present inventors it was possible to induce deletion of the KIR gene group located within the killer cell immunoglobulin-like receptor (KIR) gene cluster region, deletion of the LILR gene group located within the LILR gene cluster region, and deletion of the KLR gene group located within the killer cell lectin-like receptor (KLR) gene cluster region in genomic DNA.
  • a method for suitably producing cells having such a genome is provided. According to the present inventors, it has been revealed that silencing can occur for negative selection markers during deletion cell selection. A method for producing cells that takes silencing into consideration is also provided.
  • a vertebrate cell comprising genomic DNA having a deletion, the deletion comprising: (i) A deletion of a part or the whole of a KIR gene cluster located within a killer cell immunoglobulin-like receptor (KIR) gene cluster region in one or two alleles, the part of the KIR gene cluster comprising at least one continuous region, the continuous region comprising 50% or more of the genes encoding the KIR genes contained in each of the regions; (ii) a cell comprising a deletion, in one or two alleles, of a portion or the entirety of the LILR gene group located within a leukocyte immunoglobulin-like receptor (LILR) gene cluster region, the portion of the KIR gene group comprising at least a contiguous region, and/or (iii) a cell comprising a deletion, in one or two alleles, of a portion or the entirety of the KLR gene group located within a killer cell lectin-like receptor (KLR) gene cluster
  • deletion comprises deletion of the entire KIR gene group.
  • deletion comprises a deletion of the entire leukocyte immunoglobulin-like receptor (LILR) gene cluster.
  • LILR leukocyte immunoglobulin-like receptor
  • the deletion comprises at least a portion of the KIR gene group and at least a portion of the LILR gene group.
  • the deletion includes a complete deletion of the KIR gene group and a complete deletion of the LILR gene group.
  • the deletion comprises a deletion of the entire KIR gene group in one allele.
  • the deletion comprises at least a portion of the KIR gene group and at least a portion of the LILR gene group in one allele.
  • the deletion comprises a deletion of the entire KIR gene group in one allele and a deletion of the entire LILR gene group in one allele.
  • the cell according to (1) above, wherein the deletion comprises a deletion of the entire KIR gene group in two alleles.
  • deletion includes at least a portion of the KIR gene group and at least a portion of the LILR gene group in two alleles.
  • deletion comprises a deletion of the entire KIR gene group in two alleles and a deletion of the entire LILR gene group in one allele.
  • a method for producing an isolated cell in which two or more alleles of a chromosomal genome are modified comprising: (a) introducing the following (i) and (ii) into an isolated cell (excluding a fertilized egg) containing two or more alleles to introduce a selection marker gene into each of the two or more alleles; (i) a sequence-specific nucleic acid cleaving molecule capable of targeting and cleaving a target region in two or more alleles of the chromosomal genome, or a genome modification system comprising a polynucleotide encoding the sequence-specific nucleic acid cleaving molecule; (ii) Two or more types of donor DNA for selection markers, each of which has an upstream homology arm having a base sequence capable of homologous recombination with a base sequence on the upstream side of the target region, and a downstream homology arm having a base sequence capable of homologous recombination with a base sequence on the downstream side of the target
  • a cell population comprising a plurality of cells, each cell comprises a set of positive and negative markers at two alleles (e.g., at a locus, e.g., at a locus that includes a gene cluster);
  • the positive and negative marker set includes a positive selection marker and a negative selection marker, the positive selection markers contained in the same cell being distinguishable from each other, and the negative selection markers contained in the same cell being distinguishable from each other;
  • a cell population, in which the percentage of cells in which a negative selection marker is silenced is 0.00001% or more and 10% or less.
  • a cell population comprising a vertebrate cell, Each vertebrate cell comprises a set of positive and negative markers on two alleles within a region of 500 kbp or less that includes a killer cell immunoglobulin-like receptor (KIR) gene cluster region, and/or a set of positive and negative markers on two alleles within a region of 500 kbp or less that includes a leukocyte immunoglobulin-like receptor (LILR) gene cluster region,
  • the positive and negative marker set includes a positive selection marker and a negative selection marker, the positive selection markers contained in the same cell being distinguishable from each other, and the negative selection markers contained in the same cell being distinguishable from each other;
  • a cell population, in which the percentage of cells in the cell population in which a negative selection marker is silenced is 10% or less.
  • the present invention includes, by way of example, the following aspects:
  • a method for modifying two or more alleles of a chromosomal genome at an MHC locus comprising: (a) introducing the following (i) and (ii) into a cell containing the chromosome: (i) a sequence-specific nucleic acid cleavage molecule that targets a target region of the chromosomal genome, or a genome modification system comprising a polynucleotide encoding the sequence-specific nucleic acid cleavage molecule; (ii) Two or more types of donor DNA for selection markers, comprising a base sequence of a selection marker gene between an upstream homology arm having a base sequence homologous to a base sequence adjacent to the upstream side of the target region and
  • step (b) after the step (a), selecting the cells based on all of the selection marker genes contained in the two or more types of selection marker donor DNAs.
  • the selection marker gene is a positive selection marker gene
  • the step (b) is a step of selecting cells expressing the positive selection marker gene in a number equal to the number of alleles.
  • the donor DNA for the selection marker further has a negative selection marker gene between the upstream homology arm and the downstream homology arm.
  • a genome modification kit for modifying two or more alleles of a chromosomal genome comprising: (i) a nucleic acid sequence for modifying a chromosomal genome; (i) a sequence-specific nucleic acid cleavage molecule that targets a target region of the chromosomal genome, or a genome modification system comprising a polynucleotide encoding the sequence-specific nucleic acid cleavage molecule; (ii) Two or more types of donor DNA for selection markers, comprising a base sequence of a selection marker gene between an upstream homology arm having a base sequence homologous to a base sequence adjacent to the upstream side of the target region and a downstream homology arm having a base sequence homologous to a base sequence adjacent to the downstream side of the target region, wherein the two or more types of donor DNA for selection markers have mutually different selection marker genes, and the number of types of donor DNA for selection markers is equal to or greater than the number of alleles to be subject to genome
  • the donor DNA for the selection marker further has a negative selection marker gene between the upstream homology arm and the downstream homology arm.
  • the sequence-specific nucleic acid cleaving molecule is a sequence-specific endonuclease.
  • the genome modification system includes a Cas protein and a guide RNA having a base sequence homologous to a base sequence in the target region.
  • a method for producing a cell in which two or more alleles of a chromosomal genome are modified comprising the steps of: (a) introducing the following (i) and (ii) into a cell containing two or more alleles to introduce a selection marker gene into each of the two or more alleles; (i) a sequence-specific nucleic acid cleaving molecule capable of targeting and cleaving a target region in two or more alleles of the chromosomal genome, or a genome modification system comprising a polynucleotide encoding the sequence-specific nucleic acid cleaving molecule; (ii) Two or more types of donor DNA for selection markers, each of which has an upstream homology arm having a base sequence capable of homologous recombination with
  • Two or more types of donor DNA for selection markers each have a selection marker gene for positive selection, a marker gene for negative selection, and a target sequence between the upstream homology arm and the downstream homology arm, and when the selection marker gene is used for both positive selection and negative selection, it is not necessary to have a separate selection marker gene for negative selection; (c) after the step (b), introducing the following (iii) and (iv) into the selected cells to introduce donor DNA for recombination into the two or more alleles: (iii) a sequence-specific nucleic acid cleaving molecule capable of targeting and cleaving the target sequence, or a genome modification system comprising a polynucleotide encoding the sequence-specific nucleic acid cleaving molecule; (iv) a donor DNA for recombination comprising a desired nucleotide sequence, the donor DNA
  • a genome modification kit for modifying two or more alleles of a chromosomal genome comprising the following (i) and (ii): (i) a sequence-specific nucleic acid cleaving molecule capable of targeting and cleaving a target region of the chromosomal genome, or a genome modification system comprising a polynucleotide encoding the sequence-specific nucleic acid cleaving molecule; (ii) Two or more types of donor DNA for selection markers, the donor DNA having an upstream homology arm having a base sequence capable of homologous recombination with a base sequence upstream of the target region, and a downstream homology arm having a base sequence capable of homologous recombination with a base sequence downstream of the target region, and comprising a base sequence of a selection marker gene between the upstream homology arm and the downstream homology arm, the two or more types of donor DNA for selection markers having mutually distinguishably different selection marker genes, the selection marker genes being unique for each type
  • step (b) The method according to any one of [1] to [11] and [20] to [25] above, wherein in step (b), single cell cloning is not performed in the process up to the selection of cells in which two or more alleles have been modified.
  • step (b) A cell having two or more alleles on a chromosomal genome with respect to a target region, wherein the target region of each of the two or more alleles is deleted, and the sequences upstream and downstream of the target region are seamlessly linked without any insertion, substitution, or deletion of bases.
  • the target region has a length of 5 kbp or more.
  • a method for producing an isolated cell in which two or more alleles of a chromosomal genome are modified comprising: (a) introducing the following (i) and (ii) into an isolated cell (preferably excluding a fertilized egg) containing two or more alleles to introduce a selection marker gene into each of the two or more alleles; (i) a sequence-specific nucleic acid cleaving molecule capable of targeting and cleaving a target region in two or more alleles of the chromosomal genome, or a genome modification system comprising a polynucleotide encoding the sequence-specific nucleic acid cleaving molecule; (ii) Two or more types of donor DNA for selection markers, each of which has an upstream homology arm having a base sequence capable of homolog
  • the selection marker gene for positive selection is preferably a drug resistance gene, and each of the two or more donor DNAs for selection markers has a selection marker gene for positive selection, a marker gene for negative selection, and a target sequence between the upstream homology arm and the downstream homology arm, (b) after the step (a), a step of selecting isolated cells expressing all of the introduced selectable marker genes for positive selection by homologous recombination of different types of selectable marker donor DNAs for the two or more alleles, respectively, and distinguishably different unique selectable marker genes being introduced into the two or more alleles (a step for positive selection); (c) after the step (b), introducing the following (iii) and (iv) into the selected cells to introduce donor DNA for recombination into the two or more alleles: (iii) a sequence-specific nucleic acid cleaving molecule capable of targeting and cleaving the target sequence, or a genome modification system comprising a polynucleotide encoding the sequence-specific
  • a method for modifying two or more alleles of a chromosomal genome comprising: (a) introducing the following (i) and (ii) into an isolated cell (preferably excluding a fertilized egg) containing two or more alleles to introduce a selection marker gene into each of the two or more alleles; (i) a sequence-specific nucleic acid cleaving molecule capable of targeting and cleaving a target region in two or more alleles of the chromosomal genome, or a genome modification system comprising a polynucleotide encoding the sequence-specific nucleic acid cleaving molecule; (ii) Two or more types of donor DNA for selection markers, each of which has an upstream homology arm having a base sequence capable of homologous recombination with the base sequence upstream of the target region, and a downstream homology arm having a base sequence capable of homologous recombination with the base sequence downstream of the target region, and between the upstream homo
  • ⁇ 3> The method according to ⁇ 1> or ⁇ 2> above, wherein the target region has a length of 10 kbp or more and 40 kbp or less.
  • ⁇ 4> The method according to ⁇ 3> above, wherein the target region has a length of 10 kbp or more and 20 kbp or less.
  • ⁇ 5> The method according to any one of ⁇ 1> to ⁇ 4> above, wherein the upstream homology arm and the downstream homology arm are each 500 bp to 3,000 bp.
  • ⁇ 6> The method according to ⁇ 3> above, wherein the upstream homology arm and the downstream homology arm are each 500 bp to 3,000 bp.
  • ⁇ 7> The method according to ⁇ 4> above, wherein the upstream homology arm and the downstream homology arm are each 500 bp to 3,000 bp.
  • ⁇ 8> The method according to any one of ⁇ 1> to ⁇ 7> above, wherein the region between the upstream homology arm and the downstream homology arm of the donor DNA for recombination has a length of 5 kbp or more.
  • ⁇ 9> The method according to ⁇ 3> or ⁇ 4> above, wherein the region between the upstream homology arm and the downstream homology arm of the donor DNA for recombination has a length of 5 kbp or more.
  • ⁇ 10> The method according to ⁇ 4> above, wherein the region between the upstream homology arm and the downstream homology arm of the donor DNA for recombination has a length of 5 kbp or more.
  • ⁇ 11> The method according to ⁇ 8> above, wherein the region between the upstream homology arm and the downstream homology arm of the donor DNA for recombination has a length of 8 kbp or more.
  • a genome modification kit for modifying two or more alleles of a chromosomal genome comprising the following (i) and (ii), the kit being for use in the method according to ⁇ 1> or ⁇ 2> above: (i) a sequence-specific nucleic acid cleaving molecule capable of targeting and cleaving a target region of the chromosomal genome, or a genome modification system comprising a polynucleotide encoding the sequence-specific nucleic acid cleaving molecule; (ii) Two or more types of donor DNA for selection markers, the donor DNA having an upstream homology arm having a base sequence capable of homologous recombination with a base sequence on the upstream side of the target region, and a downstream homology arm having a base sequence capable of homologous recombination with a base sequence on the downstream side of the target region, and a base sequence of a selection marker gene for positive selection between the upstream homology arm and the downstream homology arm, the two
  • ⁇ 13> The kit described in ⁇ 12> above, wherein the target region has a length of 10 kbp or more and 40 kbp or less.
  • ⁇ 14> The kit described in ⁇ 13> above, wherein the target region has a length of 10 kbp or more and 20 kbp or less.
  • kit according to any one of ⁇ 12> to ⁇ 14> above, further comprising a donor DNA for recombination,
  • the donor DNA for recombination has an upstream homology arm having a base sequence capable of homologous recombination with a base sequence on the upstream side of the target region, and a downstream homology arm having a base sequence capable of homologous recombination with a base sequence on the downstream side of the target region.
  • kit. ⁇ 16> The kit described in ⁇ 15> above, wherein the region between the upstream homology arm and the downstream homology arm of the donor DNA for recombination has a length of 5 kbp or more.
  • ⁇ 17> The kit according to ⁇ 15> or ⁇ 16> above, wherein the region between the upstream homology arm and the downstream homology arm of the donor DNA for recombination has a length of 8 kbp or more.
  • ⁇ 18> The kit described in ⁇ 15> or ⁇ 16> above, wherein the target region has a length of 5 kbp or more, and the region between the upstream homology arm and the downstream homology arm of the recombination donor DNA has a length of 5 kbp or more.
  • ⁇ 19> The kit described in ⁇ 18> above, wherein the target region has a length of 8 kbp or more, and the region between the upstream homology arm and the downstream homology arm of the recombination donor DNA has a length of 8 kbp or more.
  • ⁇ 20> The method according to any one of ⁇ 1> to ⁇ 4> above, wherein the donor DNA for recombination has no base sequence in a region between an upstream homology arm and a downstream homology arm of the donor DNA for recombination, and in the two or more alleles of the chromosomal genome obtained after the modification, the sequences upstream and downstream of the target region are seamlessly linked without any insertion, substitution or deletion of bases.
  • ⁇ 21> The method according to ⁇ 3> or ⁇ 4> above, wherein the donor DNA for recombination has no base sequence in the region between the upstream homology arm and the downstream homology arm of the donor DNA for recombination, and in the two or more alleles of the chromosomal genome obtained after modification, the sequences upstream and downstream of the target region are seamlessly linked without any insertion, substitution or deletion of bases.
  • ⁇ 22> The method according to ⁇ 3> above, wherein a target sequence for a site-specific recombinase is not present in the two or more alleles of the chromosomal genome obtained after the modification.
  • ⁇ 23> The method according to ⁇ 4> above, wherein a target sequence for a site-specific recombinase is not present in the two or more alleles of the chromosomal genome obtained after the modification.
  • ⁇ 24> The method according to any one of ⁇ 1> to ⁇ 4> above, wherein a target sequence for a site-specific recombinase is not present in the two or more alleles of the chromosomal genome obtained after the modification.
  • ⁇ 25> The method according to ⁇ 3> or ⁇ 4> above, wherein a target sequence for a site-specific recombinase is not present in the two or more alleles of the chromosomal genome obtained after the modification.
  • ⁇ 26> The method according to any one of ⁇ 1> to ⁇ 11> and ⁇ 20> to ⁇ 25> above, wherein in the step (b), single-cell cloning is not performed in the process up to the selection of a cell in which two or more alleles have been modified.
  • ⁇ 27> The method according to ⁇ 3> or ⁇ 4> above, wherein the region between the upstream homology arm and the downstream homology arm of the donor DNA for recombination has a length of 8 kbp or more.
  • ⁇ 28> The kit described in ⁇ 15> above, wherein the donor DNA for recombination does not have a base sequence in the region between the upstream homology arm and the downstream homology arm of the donor DNA for recombination.
  • This figure illustrates the KLR gene cluster region in the human reference genome (hg38), a map of the region, and the location of the deleted region on the map.
  • the specific flow of the scheme for creating a deletion in the KLR gene cluster region and the results of confirming the introduction of the deletion in the obtained cells are shown.
  • 1 shows a map of the KIR gene cluster region in the human reference genome (hg38) and the location of the deleted region on the map.
  • the introduction positions of the markers of the present disclosure (positive and negative marker set) in the KIR gene cluster region (region 0 to region 7) are illustrated. Illustrates silencing of a negative selection marker by the location of introduction of the disclosed markers (positive-negative marker set) in the KIR gene cluster region.
  • the specific flow of the scheme for creating a deletion in the KIR gene cluster region and the results of confirming the introduction of the deletion in the obtained cells are shown.
  • the map showing the region including the KIR gene cluster region and its surrounding areas and the position of the deleted region on the map are shown.
  • the specific flow of the construction scheme for a region including the KIR gene cluster region and its surrounding regions and the results of confirming the introduction of deletions in the obtained cells are shown.
  • the term "cell” refers to a cell of an individual organism, including a cell of an animal having an immune system (i.e., a vertebrate).
  • the cell may be, for example, a mammalian cell, e.g., a cell of a primate such as a human, a rodent such as a mouse and a rat, a livestock animal such as a cow, a horse, a sheep, a llama, a camel, a goat, a pig, a pet animal such as a dog, a cat, and an avian cell such as a chicken.
  • a mammalian cell e.g., a cell of a primate such as a human, a rodent such as a mouse and a rat, a livestock animal such as a cow, a horse, a sheep, a llama, a camel, a goat, a pig, a pet animal such as a dog, a cat,
  • allele refers to a set of base sequences present at the same locus on a chromosomal genome.
  • a diploid cell has two alleles at the same locus, and a triploid cell has three alleles at the same locus.
  • additional alleles may be formed by abnormal copies of a chromosome or abnormal additional copies at the locus.
  • Mammalian cells, except for specialized cells such as germ cells, are usually diploid and have two alleles at each locus.
  • Genome modification or “genome editing” are used interchangeably and refer to the introduction of a mutation at a desired location (target region) on a genome.
  • Genome modification may include the use of a sequence-specific nucleic acid cleavage molecule designed to cleave the target region DNA.
  • genome modification may include the use of a nuclease engineered to cleave the target region DNA.
  • genome modification may include the use of a nuclease (e.g., TALEN or ZFN) engineered to cleave a target sequence having a specific base sequence in the target region.
  • genome modification may involve the use of sequence-specific endonucleases such as meganucleases that have only one cleavage site in the genome (e.g., restriction enzymes with 16-base sequence specificity (theoretically present at a ratio of 1 out of 4 16 bases), restriction enzymes with 17-base sequence specificity (theoretically present at a ratio of 1 out of 4 17 bases), and restriction enzymes with 18-base sequence specificity (theoretically present at a ratio of 1 out of 4 18 bases)) to cleave target sequences having a specific base sequence in the target region.
  • sequence-specific endonucleases such as meganucleases that have only one cleavage site in the genome (e.g., restriction enzymes with 16-base sequence specificity (theoretically present at a ratio of 1 out of 4 16 bases), restriction enzymes with 17-base sequence specificity (theoretically present at a ratio of 1 out of 4 17 bases), and restriction enzymes with 18-base sequence
  • a double-stranded break is induced in the DNA of the target region by the use of a site-specific nuclease, and then the genome is repaired by endogenous processes of the cell, such as Homologous Directed Repair (HDR) and Non-Homologous End-Joining Repair (NHEJ).
  • NHEJ is a repair method that joins the ends of double-stranded breaks without using donor DNA, and insertions and/or deletions (indels) are frequently induced during repair.
  • HDR is a repair mechanism that uses donor DNA, and it is also possible to introduce desired mutations into the target region.
  • a preferred example of a genome modification technique is the CRISPR/Cas system.
  • meganucleases include I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-CeuI, I-CeuAIIP, I-CreI, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIIP, I-CrepsbIVP, I-TliI, I-PpoI, PI-PspI, F-SceI, F-SceII, F-SuvI, F-TevI, F-TevII, I-AmaI, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CsmI, I-CvuI, I-CvuAIP, I-DdiI, I-DdiII, I-DirI
  • target region refers to a genomic region that is subject to genome modification.
  • “Deletion” includes deletions of one or more bases and deletions of one or more genes relative to a reference genome.
  • the deletions can be deletions of 100 bp or more, deletions of 200 bp or more, deletions of 300 bp or more, deletions of 400 bp or more, deletions of 500 bp or more, deletions of 600 bp or more, deletions of 700 bp or more, deletions of 800 bp or more, deletions of 900 bp or more, deletions of 1 kbp or more, deletions of 10 kbp or more, deletions of 50 kbp or more, deletions of 100 kbp or more, deletions of 200 kbp or more, deletions of 300 kbp or more, deletions of 400 kbp or more, deletions of 500 kbp or more, or deletions of 1 Mbp or more or less.
  • the deletions can be deletions of 1 Mbp or less.
  • the deletion may be a deletion of 700 kbp or less.
  • the deletion may be a deletion of 600 kbp or less.
  • the deletion may be a deletion of 500 kbp or less.
  • the deletion may be a deletion of 10 kbp to 600 kbp or less.
  • the deletion may be a deletion of 100 kbp to 600 kbp or less.
  • the deletion may be a deletion of 100 kbp to 500 kbp or less.
  • donor DNA refers to DNA used to repair double-stranded DNA breaks and capable of homologous recombination with DNA surrounding a target region.
  • the donor DNA contains a base sequence upstream and a base sequence downstream of the target region (e.g., a base sequence adjacent to the target region) as homology arms.
  • a homology arm consisting of a base sequence upstream of a target region e.g., a base sequence adjacent to the upstream side
  • upstream homology arm a homology arm consisting of a base sequence downstream of a target region
  • downstream homology arm consisting of a base sequence downstream of a target region
  • the donor DNA may contain a desired base sequence between the upstream homology arm and the downstream homology arm.
  • the length of each homology arm is preferably 300 bp or more, and is usually about 500 to 3000 bp.
  • the lengths of the upstream homology arm and the downstream homology arm may be the same or different from each other. If the target region is successfully induced to undergo homologous recombination with the donor DNA after sequence-dependent cleavage, the sequence between the upstream and downstream base sequences of the target region will be replaced with the sequence of the donor DNA.
  • Upstream of a target region means the DNA region located 5' of the reference nucleotide strand in the double-stranded DNA of the target region.
  • Downstream of a target region means the DNA located 3' of the reference nucleotide strand.
  • the reference nucleotide strand is usually the sense strand.
  • a promoter is located upstream of a protein coding sequence.
  • a terminator is located downstream of a protein coding sequence.
  • sequence-specific nucleic acid cleaving molecule refers to a molecule that can recognize a specific nucleic acid sequence and cleave a nucleic acid at said specific nucleic acid sequence.
  • a sequence-specific nucleic acid cleaving molecule is a molecule that has the activity of cleaving a nucleic acid in a sequence-specific manner (sequence-specific nucleic acid cleaving activity).
  • target sequence refers to a DNA sequence in a genome that is to be cleaved by a sequence-specific nucleic acid cleaving molecule.
  • sequence-specific nucleic acid cleaving molecule is a Cas protein
  • the target sequence refers to a DNA sequence in a genome that is to be cleaved by the Cas protein.
  • Cas9 protein is used as the Cas protein
  • the target sequence must be adjacent to the 5' side of a protospacer adjacent motif (PAM).
  • the target sequence is usually selected as a sequence of 17 to 30 bases (preferably 18 to 25 bases, more preferably 19 to 22 bases, and even more preferably 20 bases) adjacent to and immediately preceding the 5' side of the PAM.
  • a known design tool such as CRISPR DESIGN (crispr.mit.edu/) can be used to design the target sequence.
  • Cas protein refers to a CRISPR-associated protein.
  • the Cas protein forms a complex with a guide RNA and exhibits endonuclease activity or nickase activity.
  • Examples of Cas proteins include, but are not limited to, Cas9 protein, Cpf1 protein, C2c1 protein, C2c2 protein, and C2c3 protein.
  • Cas proteins include wild-type Cas proteins and their homologs (paralogs and orthologs), as well as mutants thereof, so long as they cooperate with a guide RNA to exhibit endonuclease activity or nickase activity.
  • the Cas protein is involved in the class 2 CRISPR/Cas system, more preferably in the type II CRISPR/Cas system.
  • a preferred example of the Cas protein is the Cas9 protein.
  • a preferred example of the Cas protein is the Cas3 protein.
  • Cas9 protein refers to a Cas protein involved in the type II CRISPR/Cas system.
  • the Cas9 protein forms a complex with a guide RNA and exhibits the activity of cleaving DNA in a target region in cooperation with the guide RNA.
  • the Cas9 protein includes wild-type Cas9 protein and its homologs (paralogs and orthologs), as well as mutants thereof, so long as it has the above-mentioned activity.
  • the wild-type Cas9 protein has a RuvC domain and an HNH domain as nuclease domains, but the Cas9 protein in this specification may have either the RuvC domain or the HNH domain inactivated.
  • Cas9 in which either the RuvC domain or the HNH domain is inactivated introduces a single-stranded break (nick) into double-stranded DNA. Therefore, when Cas9 in which either the RuvC domain or the HNH domain has been inactivated is used to cleave double-stranded DNA, a modified system can be constructed in which Cas9 target sequences are set for each of the sense and antisense strands, and nicks in the sense and antisense strands are generated at positions sufficiently close to each other, thereby inducing double-stranded cleavage.
  • the species of organism from which the Cas9 protein is derived is not particularly limited, and preferred examples include bacteria belonging to the genus Streptococcus, Staphylococcus, Neisseria, or Treponema. More specifically, preferred examples include Cas9 proteins derived from S. pyogenes, S. thermophilus, S. aureus, N. meningitidis, or T. denticola. In a preferred embodiment, the Cas9 protein is a Cas9 protein derived from S. pyogenes.
  • the amino acid sequences of various Cas proteins and information on their coding sequences can be obtained from various databases such as GenBank, UniProt, and Addgene.
  • the amino acid sequence of the Cas9 protein of S. pyogenes can be that registered in Addgene as plasmid number 42230.
  • An example of the amino acid sequence of the Cas9 protein of S. pyogenes is shown in SEQ ID NO:1.
  • guide RNA and "gRNA” are used interchangeably and refer to an RNA that can form a complex with Cas protein and guide Cas protein to a target region.
  • the guide RNA comprises CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA).
  • the crRNA is involved in binding to a target region on the genome, and the tracrRNA is involved in binding to Cas protein.
  • the crRNA comprises a spacer sequence and a repeat sequence, and the spacer sequence binds to the complementary strand of the target sequence in the target region.
  • the tracrRNA comprises an anti-repeat sequence and a 3' tail sequence.
  • the anti-repeat sequence has a sequence complementary to the repeat sequence of the crRNA and forms a base pair with the repeat sequence, and the 3' tail sequence usually forms three stem loops.
  • the guide RNA may be a single guide RNA (sgRNA) in which the 5' end of the tracrRNA is linked to the 3' end of the crRNA, or the crRNA and the tracrRNA may be separate RNA molecules in which the repeat sequence and the anti-repeat sequence form base pairs.
  • the guide RNA is an sgRNA.
  • the crRNA repeat sequence and tracrRNA sequence can be selected appropriately depending on the type of Cas protein, and those derived from the same bacterial species as the Cas protein can be used.
  • the length of the sgRNA can be about 50 to 220 nucleotides (nt), preferably about 60 to 180 nt, and more preferably about 80 to 120 nt.
  • the length of the crRNA can be about 25 to 70 bases including the spacer sequence, preferably about 25 to 50 nt.
  • the length of the tracrRNA can be about 10 to 130 nt, preferably about 30 to 80 nt.
  • the repeat sequence of the crRNA may be the same as that in the bacterial species from which the Cas protein is derived, or may be one in which a part of the 3' end has been deleted.
  • the tracrRNA may have the same sequence as the mature tracrRNA in the bacterial species from which the Cas protein is derived, or may be a truncated type in which the 5' end and/or the 3' end of the mature tracrRNA has been truncated.
  • the tracrRNA may be a truncated type in which about 1 to 40 nucleotide residues have been removed from the 3' end of the mature tracrRNA.
  • the tracrRNA may also be a truncated type in which about 1 to 80 nucleotide residues have been removed from the 5' end of the mature tracrRNA.
  • the tracrRNA may also be a truncated type in which, for example, about 1 to 20 nucleotide residues have been removed from the 5' end and about 1 to 40 nucleotide residues have been removed from the 3' end.
  • Various crRNA repeat sequences and tracrRNA sequences for sgRNA design have been proposed, and those skilled in the art can design sgRNA based on known techniques (e.g., Jinek et al. (2012) Science, 337, 816-21; Mali et al.
  • protospacer adjacent motif and “PAM” are used interchangeably and refer to a sequence recognized by the Cas protein during DNA cleavage by the Cas protein.
  • the sequence and position of the PAM vary depending on the type of Cas protein. For example, in the case of the Cas9 protein, the PAM must be immediately adjacent to the 3' side of the target sequence.
  • the sequence of the PAM corresponding to the Cas9 protein varies depending on the bacterial species from which the Cas9 protein is derived. For example, the PAM corresponding to the Cas9 protein of S. pyogenes is "NGG", the PAM corresponding to the Cas9 protein of S. thermophilus is "NNAGAA”, the PAM corresponding to the Cas9 protein of S.
  • NGRRT or "NNGRR(N)
  • NNNNGATT the PAM corresponding to the Cas9 protein of T.
  • NAAAAC corresponds to the Cas9 protein of B. denticola (where "R” is A or G; “N” is A, T, G, or C).
  • spacer sequence and "guide sequence” are used interchangeably and refer to a sequence contained in a guide RNA that can bind to a complementary strand of a target sequence.
  • the spacer sequence is the same sequence as the target sequence (with the exception that T in the target sequence becomes U in the spacer sequence).
  • the spacer sequence may contain one or more base mismatches with the target sequence. When multiple base mismatches are contained, the mismatches may be located adjacent to each other or may be located distant from each other.
  • the spacer sequence may contain 1 to 5 base mismatches with the target sequence. In a particularly preferred embodiment, the spacer sequence may contain one base mismatch with the target sequence.
  • the spacer sequence is positioned 5' to the crRNA.
  • operably linked when used with respect to a polynucleotide means that a first base sequence is positioned sufficiently close to a second base sequence that the first base sequence can affect the second base sequence or a region under the control of the second base sequence.
  • a polynucleotide is operably linked to a promoter means that the polynucleotide is linked such that it is expressed under the control of the promoter.
  • expressible refers to a state in which a polynucleotide can be transcribed in a cell into which it has been introduced.
  • expression vector refers to a vector containing a target polynucleotide and equipped with a system that allows the target polynucleotide to be expressed in a cell into which the vector is introduced.
  • Cas protein expression vector refers to a vector that can express Cas protein in a cell into which the vector is introduced.
  • guide RNA expression vector refers to a vector that can express guide RNA in a cell into which the vector is introduced.
  • unique sequence means a unique sequence with no other similar sequences, specifically a sequence with an identity value of 80% or more for its entire length as determined by BLAT search, which exists only once on the genome having the unique sequence. More preferably, the unique sequence is a sequence with an identity value of 75% or more as determined by BLAT search, which exists only once on the genome having the unique sequence, even more preferably, a sequence with an identity value of 70% or more as determined by BLAT search, which exists only once on the genome having the unique sequence, even more preferably, a sequence with an identity value of 65% or more as determined by BLAT search, which exists only once on the genome having the unique sequence, and even more preferably, a sequence with an identity value of 60% or more as determined by BLAT search, which exists only once on the genome having the unique sequence. "Unique sequence” may be used interchangeably with “unique sequence”.
  • KIRs iller cell immunoglobulin-like receptors
  • NK natural killer cells and T cells.
  • the human KIR gene group is clustered on chromosome 19q13.4 and is said to contain 15 KIR genes and two pseudogenes.
  • the KIR gene group is present in the cluster at hg38:chr19:54,724,497-54,866,731.
  • KIRs can interact with major histocompatibility complex (MHC) class I on other cells to control the cytotoxic activity of the other cells.
  • MHC major histocompatibility complex
  • KIRs are polymorphic and their sequences are known to vary between individuals.
  • KIRs are named based on the number of extracellular immunoglobulin (Ig)-like domains and the length of the cytoplasmic tail (long: L, short: S, or pseudogene: P). For example, if there are two Ig-like domains and a long cytoplasmic tail, it is named KIR2DL1. The last 1 is an identification number to distinguish it from similar KIRs. Since KIRs have polymorphisms, information on the polymorphism can be added after the name.
  • Ig extracellular immunoglobulin
  • P pseudogene
  • KIR2DL1*0010101 is a seven-digit number with an asterisk between them to distinguish alleles. Most KIRs are inhibitory, but a few have the effect of enhancing immune activity.
  • the KIR gene group can include, for example, KIR3DL3, KIR2DL3, KIR2DL1, KIR3DL1, KIR2DL4, KIR3DS4, and KIR2DL2.
  • KLRs iller cell lectin-like receptors
  • NK natural killer cells and T cells.
  • Human KLR genes form a cluster on chromosome 12p13.1. The cluster is present, for example, at hg38:chr12:10,308,078-10,451,156.
  • KLRs can interact with major histocompatibility complex (MHC) class I on other cells to control the cytotoxic activity of the other cells.
  • MHC major histocompatibility complex
  • the control of NK cell cytotoxic activity by KLRs is difficult to verify by individual analysis due to the diversity of expression of KIRs, LILRs, and KLRs on NK cells in vivo. Therefore, many aspects of the functions of KLR genes and polymorphisms remain unclear.
  • the KLR gene group may include, for example, KLRD1, KLRK1, KLRC4, KLRC3, KLRC2, and KLRC1.
  • LILRs Leukocyte immunoglobulin-like receptors
  • NK natural killer
  • monocytes monocytes
  • macrophages macrophages
  • dendritic cells B cells and T cells.
  • Human LILR genes are clustered on chromosome 19q13.4. The cluster is present, for example, at hg38:chr19:54,217,096-54,668,016.
  • LILRs can interact with major histocompatibility complex (MHC) class I on other cells to control the cytotoxic activity of the other cells.
  • MHC major histocompatibility complex
  • LILRs are polymorphic and their sequences are known to vary between individuals.
  • the LILR includes a centromeric LILR cluster and a telomeric LILR.
  • the centromeric LILR includes, from the centromeric side, LILRB3, LILRB5, LILRB2, LILRA3, and LILRA4, and the telomeric LILR includes, from the centromeric side, LILRA2, LILRA1, LILRB1, and LILRB4.
  • a KIR cluster region is present further to the telomeric side of the LILR.
  • a "chimeric antigen receptor” is a chimeric molecule having an antigen-binding fragment of an antibody (particularly, scFv) and an activation domain of an immune cell.
  • a CAR is generally a molecule formed by linking an scFv, an extracellular hinge domain, a transmembrane domain (e.g., CD8 ⁇ or CD28), and an activation signaling domain (e.g., CD3 ⁇ ).
  • a CAR can be introduced into a cell and expressed on the cell surface.
  • a cell expressing a CAR can target a specific antigen.
  • a CAR is introduced into an immune cell, such as, for example, a T cell or an NK cell, and targets the immune cell, such as a T cell or an NK cell, to a cancer.
  • an scFv, an extracellular hinge domain, a transmembrane domain (e.g., CD8 ⁇ or CD28), and an activation signaling domain (e.g., CD3 ⁇ ) are linked, but a second generation CAR further includes a costimulatory molecule signaling domain for activating the immune cell into which the CAR is introduced.
  • Costimulatory factors such as CD28, 4-1BB, OX40, CD27, and ICOS are used as costimulatory molecule signaling domains.
  • CARs In third generation CARs, multiple costimulatory factors are incorporated. Thus, CARs have been improved to allow immune cells into which the CAR has been introduced to proliferate continuously in vivo. All domains other than the scFv portion are preferably derived from human proteins. When CARs bind to cells expressing a target antigen, they can activate CAR-expressing cells.
  • sequence identity (or homology) between base sequences or amino acid sequences is determined by juxtaposing two base sequences or amino acid sequences with gaps at the insertion and deletion sites so that the corresponding bases or amino acids are most commonly identical, and calculating the ratio of matching bases or amino acids to the entire base sequence or entire amino acid sequence excluding gaps in the resulting alignment.
  • Sequence identity between base sequences or amino acid sequences can be determined using various homology search software known in the art.
  • the sequence identity value (identity value) of base sequences is not particularly limited, and can be obtained, for example, by a BLAT search installed in the known homology search software UCSC Genome Browser.
  • hg38 is a reference genome released by the University of California, Santa Cruz (UCSC) in December 2013.
  • the reference genome is a reference genome created by combining various genomes, and it does not mean that there is a human having this genome.
  • the decoded fragmentary sequence information is linked to construct a continuous sequence on a computer, and the sequence of the genomic DNA of the human individual can be estimated.
  • the genomic DNA of an individual such as a human individual is usually decoded by matching the sequence of the genomic DNA of the human individual to the reference genome.
  • a position or region corresponding to a specific position or specific region of the hg38 genome sequence means a position or region linked to the specific position or specific region in the genome of another individual having a different specific sequence.
  • a position or region having a sequence characteristic of the position or region based on sequence identity corresponds to a specific position or region of the hg38 genome sequence.
  • the corresponding position can be determined by aligning the partial sequences of two genomic DNAs. Even if there is a difference in the specific sequence, the correspondence between the two genomic DNAs can be determined by aligning them if they have an orthologous relationship or sequence identity.
  • the cell may include cells at various stages of development into various tissues.
  • the cell may be a pluripotent cell (e.g., a pluripotent stem cell such as an embryonic stem cell or an induced pluripotent stem cell), a tissue stem cell (e.g., a stem cell such as a hematopoietic stem cell or a mesenchymal stem cell), a tissue progenitor cell, or a terminally differentiated cell.
  • the cell may be a normal cell (e.g., a non-tumor cell) or a tumor cell.
  • the cell may be an immune cell or a non-immune cell.
  • the immune cell may be, for example, an antigen-specific immune cell (e.g., a T cell, a B cell, a NK cell, a NKT cell, or a macrophage).
  • the antigen-specific immune cell may have, for example, a nucleic acid encoding an antigen-specific receptor (e.g., a T cell receptor and a chimeric antigen receptor), or may express the receptor on the cell surface.
  • the composition containing the cells may be unfrozen, typically at a temperature between 0° C. and 37° C., preferably between 2° C. and 8° C., or may be frozen. Freezing may be accomplished, for example, by chilling a tube containing the composition in liquid nitrogen. Thawing may be accomplished in a warm bath (e.g., at about 37° C.).
  • the cell of the present disclosure may be an iPS cell.
  • the cell of the present disclosure may be an iPS cell derived from a human cell (human iPS cell).
  • the cell of the present disclosure may be used after being differentiated into an immune cell, for example. Therefore, the cell of the present disclosure may be an immune cell (particularly an immune cell derived from an iPS cell).
  • the immune cell may be, for example, one or more immune cells selected from the group consisting of T cells (e.g., CD4 single positive T cells and CD8 single positive T cells), natural killer T cells (NKT cells), natural killer cells (NK cells), regulatory T cells (Treg), ⁇ T cells, ⁇ T cells, and macrophages, and may preferably be NK cells.
  • the immune cell may have a T cell receptor (TCR) having antigen specificity.
  • TCR T cell receptor
  • the immune cell may not express a T cell receptor having antigen specificity (e.g., may have a TCR ⁇ chain deficiency).
  • the immune cell may express a chimeric antigen receptor (CAR).
  • the immune cell can be a T cell, NKT cell, NK cell, or ⁇ T cell that expresses a CAR but may not express a TCR with antigen specificity.
  • the cell can be, for example, a primary cell (e.g., a primary immune cell).
  • the cell can be, for example, a cell line (e.g., an immune cell line).
  • the cell can be a non-cancer cell.
  • a cell expressing a CAR can express, for example, either or both of endogenous or exogenous interleukin-7 (IL-7) and endogenous or exogenous CCL19.
  • the endogenous or exogenous IL-7 and endogenous or exogenous CCL19 are operably linked to a regulatory sequence, and the cell expressing a CAR has either or preferably both of said IL-7 and CCL19.
  • a cell expressing a CAR can express endogenous or exogenous endo- ⁇ -D-glucuronidase (HPSE), or both of endogenous or exogenous endo- ⁇ -D-glucuronidase (HPE ... ).
  • HPSE endogenous or exogenous endo- ⁇ -D-glucuronidase
  • HPE ... endogenous or exogenous endo- ⁇ -D-glucuronidase
  • the immune cells can also express endogenous or exogenous CD3.
  • the cells may or may not express one or more endogenous or exogenous factors selected from the group consisting of HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A2AR, CAR, and TCR.
  • the cells have a nucleic acid encoding one or more endogenous or exogenous factors selected from the group consisting of HLA-E, HLA-G, CD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A2AR, CAR, and TCR, the nucleic acid being operably linked to a regulatory sequence.
  • the cells expressing CAR can be used in combination with antibody therapy.
  • the target molecule (tumor antigen) of the antibody may be knocked out in the cells expressing CAR.
  • tumor antigens include CD38 and CD52.
  • anti-CD38 antibodies include daratumumab and isatuximab (the disease to be treated may be, for example, multiple myeloma).
  • anti-CD52 antibodies include alemtuzumab (the disease to be treated may be, for example, leukemia, lymphocytic leukemia, or chronic lymphocytic leukemia).
  • the cells may also have NLRC5 knocked out, which may suppress the onset of, for example, graft-versus-host disease (GVHD).
  • a cell of an embodiment e.g., an immune cell or a non-immune cell
  • a cell of the disclosure has functional ⁇ 2 microglobulin and/or functional CIITA.
  • a cell of the disclosure may express, for example, an IL-15:IL15R ⁇ fusion protein.
  • the cell comprises genomic DNA having a deletion.
  • the deletion is (i) A deletion of a portion or the entirety of a killer cell immunoglobulin-like receptor (KIR) gene cluster located within a KIR gene cluster region in one or two alleles, the portion of the KIR gene cluster comprising at least one continuous region; (ii) a deletion of part or the entirety of the LILR gene cluster located within the leukocyte immunoglobulin-like receptor (LILR) gene cluster region, in one or two alleles, said part of the KIR gene cluster comprising at least one contiguous region, and/or (iii) a deletion of part or the entirety of the KLR gene cluster located within the killer cell lectin-like receptor (KLR) gene cluster region, in one or two alleles, said part of the KLR gene cluster comprising at least one contiguous region.
  • KIR killer cell immunoglobulin-like receptor
  • the portion of the KIR gene group comprises 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the genes encoded by the KIR genes contained in each of the above regions.
  • the portion of the LILR gene group comprises 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the genes encoded by the LILR genes contained in each of the above regions.
  • the portion of the KLR gene group comprises 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the genes encoded by the KLR genes contained in each of the above regions.
  • the deletion includes at least a portion of the KIR gene group and at least a portion of the KLR gene group in the region on the genome. At least a portion of the KIR gene group and at least a portion of the KLR gene group in the region on the genome may each be a contiguous region on the genome. In a preferred embodiment, the deletion includes the entire KIR gene group and the entire KLR gene group in the region on the genome. All of the KIR gene group and all of the KLR gene group in the region on the genome may each be a contiguous region on the genome.
  • the deletion comprises at least a portion of the KIR gene group and at least a portion of the LILR gene group in the region on the genome. At least a portion of the KIR gene group and at least a portion of the LILR gene group in the region on the genome may each be a contiguous region, or the two regions as a whole may be a contiguous region. In a preferred embodiment, the deletion comprises the entire LILR gene group and the entire KLR gene group in the region on the genome. In a preferred embodiment, the deletion comprises the centromeric LILR. In a preferred embodiment, the deletion comprises the telomeric LILR. In a more preferred embodiment, the deletion comprises the centromeric LILR and the telomeric LILR.
  • the deletion comprises at least a portion of the KIR gene group, at least a portion of the LILR gene group, and at least a portion of the KLR gene group in the region on the genome. At least a portion of the KIR gene group, at least a portion of the LILR gene group, and at least a portion of the KLR gene group in the region on the genome may each be a continuous region. At least a portion of the KIR gene group and at least a portion of the LILR gene group in the region on the genome may be a continuous region as a whole. In a preferred embodiment, the deletion comprises the entire KIR gene group, the entire LILR gene group, and the entire KLR gene group in the region on the genome.
  • the deletion comprises a region of the genome corresponding to hg38:chr19:54,217,096-54,941,711. This region encompasses both the KIR gene cluster region and the LILR gene cluster region.
  • the cells having the deletion do not exhibit a decrease in cell proliferation rate of 50% or more, 40% or more, 30% or more, 25% or more, 20% or more, 15% or more, 10% or more, or 5% or more compared to unmodified cells having no deletion under normal culture conditions (e.g., in an environment suitable for culturing unmodified cells having no deletion).
  • the cells having the deletion do not exhibit a decrease in viability of 25% or more, 20% or more, 15% or more, 10% or more, or 5% or more under normal culture conditions (e.g., in an environment suitable for culturing unmodified cells having no deletion). If the cells have a drug resistance gene, a drug (particularly a drug at an appropriate concentration used for normal selection) may be present.
  • the cell may include an insertion of a nucleic acid at the site of the deletion or at another site.
  • the nucleic acid may include a gene expression cassette, the gene expression cassette including a regulatory sequence and a gene operably linked to the regulatory sequence.
  • the gene may be any gene of interest.
  • the gene may be, for example, a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
  • the chimeric antigen receptor may, for example, comprise a single chain antibody (e.g., scFv) comprising a heavy chain and a light chain, an extracellular hinge domain (e.g., CD8), a transmembrane domain (e.g., CD8 ⁇ and CD28), a co-stimulatory molecule signaling domain (4-1BB, CD28 and CD137), and an activation signaling domain (CD3 ⁇ ).
  • the chimeric antigen receptor thereby generates an activation signal to the cell upon binding to the target.
  • the chimeric antigen receptor may bind to an antigen expressed on a cancer cell.
  • Such antigens include, for example, CD16, CD19, CD20, CD22, CD123, CD171, epidermal growth factor receptor (EGFR), particularly EGFRvIII, type 3 EGFR, de2-7EGFR, and HER2. 2, carcinoembryonic antigen (CEA), prostate stem cell antigen (PSCA), B cell maturation antigen (BCMA), CS1, NKG2D, NKp30, B7H6, MUC-16 (CA125), orphan receptor for receptor tyrosine kinase 1 (ROR-1), GD3, GM2, glypican-3 (GPC3), mesothelin, IL13R, c-KIT, c-MET, NY-ESO-1, WT1, MAGE-A3, MAGE-A4, MAGE-A10, HPV E6, HPV E7, CMV, AFP, PRAME, SSX2, KRAS, HER2, and PD-L1.
  • CEA carcinoembryonic antigen
  • PSCA prostate
  • the chimeric antigen receptor may also be capable of binding to, for example, a proteinaceous tag.
  • the antigen of the scFv can be, for example, a fluorescent protein such as fluorescein isothiocyanate (FITC), because cancer cells can also be killed by binding an antibody labeled with a proteinaceous tag to a cancer antigen on the surface of the cancer cells and further administering cells expressing a chimeric antigen receptor that targets the tag.
  • FITC fluorescein isothiocyanate
  • TCRs may have antigen specificity.
  • TCRs with antigen specificity may bind to, for example, cancer antigens.
  • TCRs with antigen specificity may bind to, for example, CD16, CD19, CD20, CD22, CD123, CD171, epidermal growth factor receptor (EGFR), in particular EGFRvIII, EGFR type 3, de2-7EGFR and HER2, carcinoembryonic antigen (CEA), prostate stem cell antigen (PSCA), B cell maturation antigen (BCMA), CS1, NKG2D, NKp30, B7H6, MUC-16 (CA125), receptor tyrosine kinase (RKT), and NKp30.
  • EGFR epidermal growth factor receptor
  • CEA carcinoembryonic antigen
  • PSCA prostate stem cell antigen
  • CS1 NKG2D
  • NKp30 B7H6, MUC-16
  • RKT receptor tyrosine kinase
  • It can bind to one or more selected from the group consisting of orphan receptor for rosine kinase 1 (ROR-1), GD3, GM2, glypican-3 (GPC3), mesothelin, IL13R, c-KIT, c-MET, NY-ESO-1, WT1, MAGE-A3, MAGE-A4, MAGE-A10, HPV E6, HPV E7, CMV, AFP, PRAME, SSX2, KRAS, HER2, and PD-L1.
  • the TCR can be selected to match the HLA.
  • target genes include genes encoding one or more immunosuppressive factors, such as CD47, CD24, CD200, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, FASL, Serpmb9, CC121, and Mfge8.
  • Other target genes may be, for example, genes that produce a therapeutic benefit.
  • Therapeutic genes may be proinflammatory proteins, such as cytokines.
  • Therapeutic genes may be anti-inflammatory proteins, such as cytokines.
  • Other target genes may be, for example, suicide genes.
  • suicide genes include thymidine kinase genes, particularly herpes virus-derived thymidine kinase genes (HSVtk), cytotoxic signal receptors (e.g., diphtheria toxin receptors), and iCas9. Thymidine kinase genes phosphorylate ganciclovir to generate cytotoxic ganciclovir triphosphate.
  • iCas9 inducible caspase-9 is a protein whose CARD has been replaced by FKBP12, and induces cell death in iCas9-expressing cells in the presence of a tacrolimus derivative (e.g., AP1903).
  • Such suicide genes are useful when it is desired to remove cells (e.g., therapeutic cells) that evade the immune system from the body.
  • the positive-negative marker set When inserting a gene expression cassette into DNA, the positive-negative marker set can be removed in the presence of recombination donor DNA containing the gene expression cassette. In this way, the gene expression cassette can be inserted in place of the region where a large-scale deletion has occurred.
  • the control sequence is, for example, a promoter.
  • the promoter is not particularly limited, and for example, various pol II promoters can be used. Examples of pol II promoters include, but are not particularly limited to, CMV promoter, EF1 promoter (EF1 ⁇ promoter), SV40 promoter, MSCV promoter, hTERT promoter, ⁇ -actin promoter, CAG promoter, CBh promoter, etc.
  • the promoter may be an inducible promoter.
  • An inducible promoter is a promoter that can induce the expression of a polynucleotide functionally linked to the promoter only in the presence of an inducer that drives the promoter.
  • Inducible promoters include promoters that induce gene expression by heating, such as heat shock promoters. Inducible promoters also include promoters in which the inducer that drives the promoter is a drug. Examples of such drug-inducible promoters include, for example, a cumate operator sequence, a lambda operator sequence (e.g., 12 ⁇ Op), and a tetracycline-based inducible promoter. Examples of tetracycline-inducible promoters include promoters that drive gene expression in the presence of tetracycline or its derivatives (e.g., doxycycline), or reverse tetracycline-controlled transactivators (rtTA). Examples of tetracycline-inducible promoters include the TRE3G promoter.
  • the present invention provides a method for modifying two or more alleles of a chromosomal genome, the method comprising the steps of: (a) introducing the following (i) and (ii) into a cell containing the chromosome; and (i) a genome modification system comprising a sequence-specific nucleic acid cleavage molecule that targets a target region of the chromosome genome, or a polynucleotide encoding the sequence-specific nucleic acid cleavage molecule, (ii) Two or more types of donor DNA for selection markers, comprising a base sequence of a selection marker gene between an upstream homology arm having a base sequence homologous to a base sequence adjacent to the upstream side of the target region and a downstream homology arm having a base sequence homologous to a base sequence adjacent to the downstream side of the target region, wherein the two or more donor DNAs for selection markers have mutually different selection marker genes, and the number of types of the donor
  • the selection marker gene may be unique for each type of selection marker donor DNA.
  • the step (b) may be a step (a) of selecting cells that express all of the introduced, distinguishably different, unique selection marker genes by homologous recombination of different types of selection marker donor DNAs with respect to the two or more alleles (a step for positive selection).
  • the above method may be a method of producing a cell in which two or more alleles of a chromosomal genome are modified.
  • the method described below is useful as a method for efficiently introducing the same modification simultaneously into two or more alleles of a chromosomal genome, and is suitable, for example, for creating a deletion of about 100 kb to 500 kb in a sequence-specific manner in a target chromosomal region, and can be preferably used to create the above-mentioned cells.
  • This method can also be applied to the modification of haploid cells.
  • This method can also be applied to cells in which there is only one allele of the HLA gene region on the genomic DNA.
  • the invention provides a method for producing a cell in which two or more alleles of a chromosomal genome have been modified, comprising the steps of: (a) introducing the following (i) and (ii) into a cell containing two or more alleles to introduce a selection marker gene into each of the two or more alleles; (i) a sequence-specific nucleic acid cleaving molecule capable of targeting and cleaving a target region in two or more alleles of the chromosomal genome, or a genome modification system comprising a polynucleotide encoding the sequence-specific nucleic acid cleaving molecule; (ii) Two or more types of donor DNA for selection markers, each of which has an upstream homology arm having a base sequence capable of homologous recombination with a base sequence on the upstream side of the target region and a downstream homology arm having a base sequence capable of homologous recombination with a base sequence on the downstream side of the
  • step (a) In step (a), (i) and (ii) are introduced into a cell containing the chromosome.
  • the cells used in the genome modification method of this embodiment are not particularly limited, and may be cells having a haploid or diploid or higher chromosomal genome.
  • the cells may be diploid, triploid, or quadruploid or higher.
  • the cells include, but are not particularly limited to, eukaryotic cells.
  • the cells may be plant cells, animal cells, or fungal cells.
  • the animal cells include, but are not particularly limited to, cells of humans, non-human mammals (e.g., non-human primates such as monkeys, non-human mammals such as dogs, cats, cows, horses, sheep, goats, llamas, and rodents), birds, reptiles, amphibians, fish, and other vertebrates.
  • the target region to be subjected to genome modification can be any region on the genome having one or more alleles.
  • the size of the target region is not particularly limited.
  • the genome modification method of this embodiment can modify a region of a larger size than conventional methods.
  • the target region may be, for example, 10 kbp or more.
  • the target region may be, for example, 100 bp or more, 200 bp or more, 400 bp or more, 800 bp or more, 1 kbp or more, 2 kbp or more, 3 kbp or more, 4 kbp or more, 5 kbp or more, 8 kbp or more, 10 kbp or more, 20 kbp or more, 40 kbp or more, 80 kbp or more, 100 kbp or more, 200 kbp or more, 300 kbp, 400 kbp or more, 500 kbp or more, 600 kbp or more, 700 kbp or more, 800 kbp or more, 900 kbp or more, or 1 Mbp or more, or any number less than or equal to the above.
  • the target region is deleted in the modified cell.
  • Genome modification system refers to a molecular mechanism capable of modifying a desired target region.
  • the genome modification system includes a sequence-specific nucleic acid cleavage molecule that targets a target region of a chromosomal genome, or a polynucleotide that encodes the sequence-specific nucleic acid cleavage molecule.
  • sequence-specific nucleic acid cleaving molecule is not particularly limited as long as it is a molecule that has sequence-specific nucleic acid cleaving activity, and may be a synthetic organic compound or a biopolymer compound such as a protein.
  • synthetic organic compounds that have sequence-specific nucleic acid cleaving activity include pyrrole-imidazole polyamides.
  • proteins that have sequence-specific site cleaving activity include sequence-specific endonucleases.
  • a sequence-specific endonuclease is an enzyme that can cleave nucleic acids at a specific sequence.
  • a sequence-specific endonuclease can cleave double-stranded DNA at a specific sequence.
  • Sequence-specific endonucleases are not particularly limited, but examples include zinc finger nucleases (ZFNs), TALENs (transcription activator-like effector nucleases), Cas proteins, etc., but are not limited to these.
  • ZFNs are artificial nucleases that contain a nucleic acid cleavage domain conjugated to a binding domain that contains a zinc finger array.
  • cleavage domains include the cleavage domain of the type II restriction enzyme FokI.
  • Zinc finger nucleases capable of cleaving a target sequence can be designed by known methods.
  • TALENs are artificial nucleases that contain a DNA-binding domain of a transcription activator-like (TAL) effector in addition to a DNA-cleavage domain (e.g., a FokI domain).
  • TALE constructs capable of cleaving a target sequence can be designed by known methods (e.g., Zhang, Feng et. al. (2011) Nature Biotechnology 29 (2)).
  • the genome modification system includes a CRISPR/Cas system. That is, the genome modification system preferably includes a Cas protein and a guide RNA having a base sequence homologous to a base sequence in the target region.
  • the guide RNA may include a sequence homologous to a sequence in the target region (target sequence) as a spacer sequence.
  • the guide RNA may be capable of binding to DNA in the target region, and does not need to have a completely identical sequence to the target sequence. This binding may be formed under physiological conditions in the cell nucleus.
  • the guide RNA may include, for example, 0 to 3 base mismatches with respect to the target sequence.
  • the number of mismatches is preferably 0 to 2 bases, more preferably 0 to 1, and even more preferably no mismatches.
  • the guide RNA may be designed based on a known method.
  • the genome modification system is preferably a CRISPR/Cas system, and preferably includes a Cas protein and a guide RNA.
  • the Cas protein is preferably a Cas9 protein.
  • sequence-specific endonuclease may be introduced into the cell as a protein, or may be introduced into the cell as a polynucleotide encoding the sequence-specific endonuclease.
  • the mRNA of the sequence-specific endonuclease may be introduced, or an expression vector of the sequence-specific endonuclease may be introduced.
  • the coding sequence of the sequence-specific endonuclease (sequence-specific endonuclease gene) is functionally linked to a promoter.
  • the promoter is not particularly limited, and for example, various pol II promoters can be used.
  • pol II promoters include, but are not limited to, the CMV promoter, the EF1 promoter (EF1 ⁇ promoter), the SV40 promoter, the MSCV promoter, the hTERT promoter, the ⁇ -actin promoter, the CAG promoter, and the CBh promoter.
  • the promoter may be an inducible promoter.
  • An inducible promoter is a promoter that can induce expression of a polynucleotide functionally linked to the promoter only in the presence of an inducer that drives the promoter.
  • Inducible promoters include promoters that induce gene expression by heating, such as heat shock promoters.
  • Inducible promoters also include promoters in which the inducer that drives the promoter is a drug.
  • drug-inducible promoters include, for example, cumate operator sequences, lambda operator sequences (e.g., 12 ⁇ Op), tetracycline-based inducible promoters, and the like.
  • Tetracycline-based inducible promoters include, for example, promoters that drive gene expression in the presence of tetracycline or a derivative thereof (e.g., doxycycline), or reverse tetracycline-controlled transactivator (rtTA). Tetracycline-based inducible promoters include, for example, the TRE3G promoter.
  • any known expression vector can be used without any particular restrictions.
  • expression vectors include plasmid vectors and viral vectors.
  • the expression vector may contain a guide RNA coding sequence (guide RNA gene) and in addition to the coding sequence of the Cas protein (Cas protein gene).
  • guide RNA coding sequence (guide RNA gene) is functionalized in a pol III promoter.
  • pol III promoters include mouse and human U6-snRNA promoters, human H1-RNase P RNA promoters, and human valine-tRNA promoters.
  • the donor DNA for a selection marker is a donor DNA for knocking in a selection marker into a target region.
  • the donor DNA for a selection marker contains the base sequence of one or more selection marker genes between an upstream homology arm having a base sequence homologous to a base sequence adjacent to the upstream side of the target region and a downstream homology arm having a base sequence homologous to a base sequence adjacent to the downstream side of the target region.
  • the donor DNA for the selection marker may have a length of, but is not limited to, 1 kb or more, 2 kb or more, 3 kb or more, 4 kb or more, 5 kb or more, 6 kb or more, 7 kb or more, 8 kb or more, 9 kb or more, 9.5 kb or more, or 10 kb or more.
  • the donor DNA for the selection marker may have a length of, but is not limited to, 50 kb or less, 45 kb or less, 40 kb or less, 35 kb or less, 30 kb or less, 25 kb or less, 20 kb or less, 15 kb or less, 14 kb or less, 13 kb or less, 12 kb or less, 11 kb or less, 10 kb or less, 9 kb or less, 8 kb or less, 7 kb or less, 6 kb or less, 5 kb or less, or 4 kb or less.
  • a “selection marker” refers to a protein that can select cells based on the presence or absence of its expression.
  • a selection marker gene is a gene that codes for a selection marker. When selecting cells that express a selection marker in a cell population in which selection marker-expressing cells and non-expressing cells are mixed, the selection marker is called a "positive selection marker” or a “selection marker for positive selection”. When selecting cells that do not express a selection marker in a cell population in which selection marker-expressing cells and non-expressing cells are mixed, the selection marker is called a "negative selection marker” or a “selection marker for negative selection”.
  • selection markers When selection markers are different from each other, it means that they can be distinguished from each other (e.g., they are distinguishably different), and for example, they can be distinguished from each other at least in physiological properties such as the property of drug resistance that they confer on cells into which the selection marker is introduced or in other physicochemical properties. In other words, when selection markers are different from each other, it means that different selection markers can be detected in a distinguishable manner from other selection markers, or that they can be selected for drugs in a distinguishable manner from other selection markers.
  • the selective marker gene being unique to each type of donor DNA for selective markers means that the selective marker gene possessed by one type of donor DNA for selective markers is not contained in other types of donor DNA for selective markers, or, when contained in multiple types of donor DNA, is configured so that it is not expressed from two or more types of donor DNA at the same time.
  • the two or more types of donor DNA may be identical except for the selective marker, or may differ in the sequence and/or structure other than the selective marker.
  • positive selection marker genes include drug resistance genes, fluorescent protein genes, luminescent enzyme genes, and chromogenic enzyme genes.
  • the negative selection marker is not particularly limited as long as it is capable of selecting cells that do not express it.
  • negative selection marker genes include suicide genes (such as thymidine kinase), fluorescent protein genes, luminescent enzyme genes, and chromogenic enzyme genes.
  • suicide genes such as thymidine kinase
  • fluorescent protein genes such as a suicide gene
  • luminescent enzyme genes such as a suicide gene
  • chromogenic enzyme genes such as thymidine kinase
  • the negative selection marker gene can be functionally linked to an inducible promoter.
  • the negative selection marker gene can be expressed only when it is desired to remove cells that have the negative selection marker gene.
  • the negative selection marker gene has little negative effect on cell survival, such as when it is an optically detectable marker gene (visible marker gene) that is fluorescent, luminescent, or chromogenic, it may be expressed constitutively.
  • Examples of drug resistance genes include, but are not limited to, a puromycin resistance gene, a blasticidin resistance gene, a geneticin resistance gene, a neomycin resistance gene, a tetracycline resistance gene, a kanamycin resistance gene, a zeocin resistance gene, a hygromycin resistance gene, and a chloramphenicol resistance gene.
  • Examples of fluorescent protein genes include, but are not limited to, green fluorescent protein (GFP) gene, yellow fluorescent protein (YFP) gene, red fluorescent protein (RFP) gene, and the like.
  • Examples of the luminescent enzyme gene include, but are not limited to, the luciferase gene.
  • chromogenic enzyme genes include, but are not limited to, ⁇ -galactosidase gene, ⁇ -glucuronidase gene, alkaline phosphatase gene, and the like.
  • suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (HSV-TK), inducible caspase 9, and the like.
  • the selection marker gene contained in the selection marker donor DNA is preferably a positive selection marker gene.
  • cells expressing the selection marker can be selected as cells in which the selection marker gene has been knocked in.
  • the upstream homology arm has a base sequence capable of homologous recombination with the base sequence upstream of the target region in the genome to be modified, for example, a base sequence homologous to the base sequence adjacent to the upstream side of the target sequence.
  • the downstream homology arm has a base sequence capable of homologous recombination with the base sequence upstream of the target region in the genome to be modified, for example, a base sequence homologous to the base sequence adjacent to the downstream side of the target sequence.
  • the length and sequence of the upstream homology arm and the downstream homology arm are not particularly limited as long as they are capable of homologous recombination with the surrounding region of the target region.
  • the upstream homology arm and the downstream homology arm do not necessarily have to completely match the upstream or downstream sequence of the target region as long as they can perform homologous recombination.
  • the upstream homology arm can be a sequence having 90% or more sequence identity (homology) with the base sequence adjacent to the upstream side of the target region, and it is preferable that the sequence identity is 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
  • the downstream homology arm can be a sequence having 90% or more sequence identity (homology) with the base sequence adjacent to the downstream side of the target region, and preferably has 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity.
  • the efficiency of allele modification can be increased.
  • “close” can mean that the distance between the two sequences is 100 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, or 10 bp or less.
  • the selection marker gene is located between the upstream homology arm and the downstream homology arm.
  • the selection marker gene is introduced into the target region by HDR (if a gene is destroyed by this, it is called gene knockout, and if a desired gene is introduced by this, it is called gene knockin, in which a gene can be knocked out while another gene is knocked in).
  • the selection marker gene is preferably functionally linked to a promoter so that it is expressed under the control of an appropriate promoter.
  • the promoter can be appropriately selected depending on the type of cell into which the donor DNA is introduced. Examples of promoters include SR ⁇ promoter, SV40 early promoter, retroviral LTR, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, HSV-TK (herpes simplex virus thymidine kinase) promoter, EF1 ⁇ promoter, metallothionein promoter, and heat shock promoter.
  • the donor DNA for the selection marker may have any control sequence such as an enhancer, polyA addition signal, or terminator.
  • the donor DNA for the selection marker may have an insulator sequence.
  • An "insulator” refers to a sequence that blocks or alleviates the influence of the adjacent chromosomal environment and ensures or enhances the independence of the transcriptional regulation of the DNA sandwiched between the regions.
  • An insulator is defined by its enhancer blocking effect (the effect of blocking the effect of the enhancer on promoter activity by inserting it between an enhancer and a promoter) and its suppression effect on position effect (the effect of preventing the expression of the introduced gene from being affected by the position on the genome where it is inserted by sandwiching both sides of the introduced gene with insulators).
  • the donor DNA for the selection marker may have an insulator sequence between the upstream arm and the selection marker gene (or between the upstream arm and the promoter that controls the selection marker gene).
  • the donor DNA for the selection marker may have an insulator sequence between the downstream arm and the selection marker gene.
  • the donor DNA for the selection marker may be linear or circular, but is preferably circular.
  • the donor DNA for the selection marker is a plasmid.
  • the donor DNA for the selection marker may contain any sequence in addition to the above sequences. For example, it may contain a spacer sequence in all or part of the sequences between the upstream homology arm, the insulator, the selection marker gene, and the downstream homology arm.
  • donor DNA for selection markers is introduced into cells in a number equal to or greater than the number of alleles to be modified in the genome.
  • Different types of donor DNA for selection markers have mutually different (distinguishable) types of selection marker genes.
  • different types of donor DNA for selection markers do not have completely identical selection marker genes or sets thereof. That is, a first type of donor DNA for selection markers has a first type of selection marker gene, a second type of donor DNA for selection markers has a second type of selection marker gene, a third type of donor DNA for selection markers has a third type of selection marker gene, and so on for subsequent types of donor DNA for selection markers.
  • there are two alleles to be modified in the genome there are two or more types of donor DNA for selection markers.
  • one donor DNA for a selection marker may have two or more mutually different (distinguishable) selection markers (even in this case, the different types of donor DNA for a selection marker must have mutually different (distinguishable) types (e.g., unique) of selection marker genes).
  • the donor DNA for a selection marker does not have a recombination sequence of a site-specific recombinase (e.g., a loxP sequence recombined by Cre recombinase and its variants).
  • the method of the present invention does not use a site-specific recombinase and its recombination sequence (e.g., a loxP sequence recombined by Cre recombinase and its variants).
  • a site-specific recombinase usually one recombination sequence of the site-specific recombinase remains in the genome after editing.
  • the modified genome of the cell obtained by the method of the present invention does not have a recombination sequence (which is foreign) of a site-specific recombinase.
  • the number of types of donor DNA for selection markers may be equal to or greater than the number of alleles to be targeted for genome modification, with no particular upper limit.
  • donor DNA for selection markers of a number equal to or greater than the number of alleles to be targeted for genome modification, two or more alleles can be stably modified.
  • the number of types of donor DNA for selection markers is preferably equal to the number of alleles to be targeted for genome modification or about 1 to 2 more, and more preferably equal to the number of alleles to be targeted for genome modification.
  • the method of introducing (i) and (ii) into cells is not particularly limited, and known methods can be used without particular limitation.
  • Examples of methods of introducing (i) and (ii) into cells include, but are not limited to, viral infection, lipofection, microinjection, calcium phosphate, DEAE-dextran, electroporation, and particle gun.
  • two or more types of donor DNA for selection marker can be knocked into two or more alleles of the target region randomly when the upstream homology arm and downstream homology arm of each are identical.
  • two or more types of donor DNA for selection markers can modify each of the two or more alleles as long as the donor DNA for selection markers has a base sequence of a homology arm that can undergo homologous recombination with the upstream and downstream sequences of the target region of each of the two or more alleles, and therefore does not need to have completely identical base sequences of the homology arms.
  • the donor DNA for selection markers may have base sequences of the upstream and downstream homology arms that are more identical to the upstream and downstream sequences of the target region of each allele (e.g., may be optimized in this way).
  • the donor DNA for the selection marker has an upstream homology arm and a downstream homology arm, and has a selection marker gene between the upstream homology arm and the downstream homology arm, and preferably may further have a target sequence for an endonuclease (a base sequence-specific nucleic acid cleavage molecule) such as a cleavage site for a meganuclease.
  • the selection marker includes a selection marker gene for positive selection and a marker gene for negative selection.
  • the selection marker includes a selection marker for positive selection, but may not include a negative selection marker gene separately.
  • the selection marker gene for positive selection can also be used for negative selection, and such a marker gene may include a visualization marker gene.
  • a set of two or more donor DNAs for selection markers is a combination of the above donor DNAs for selection markers, and each of them has a selectable marker gene for positive selection that can be distinguished from the others.
  • the above set may further have a target sequence for an endonuclease (base sequence-specific nucleic acid cutting molecule) such as a cleavage site of a meganuclease, and the target sequences may be different from each other, but are preferably the same (or can be cut by the same base sequence-specific nucleic acid cutting molecule).
  • the length of the donor DNA for selection markers is as described above, but may be, for example, 5 kbp or more, 8 kbp or more, or 10 kbp or more.
  • step (b) After the step (a), step (b) is performed.
  • step (b) cells into which two or more alleles have been introduced with selectable marker genes or a combination thereof that are distinct from each other are selected based on the expression of the distinctly different selectable marker genes. More specifically, in step (b), cells that express all of the distinctly different selectable marker genes introduced into the two or more alleles by homologous recombination of different types of selectable marker donor DNAs with respect to the two or more alleles are selected.
  • step (b) cells whose alleles have been modified by the introduction of different selectable marker donor DNAs are selected based on the expression of all selectable marker genes contained in the two or more selectable marker donor DNAs and integrated into the chromosomal genome. In one aspect, in step (b), cells are selected based on all selectable marker genes contained in the two or more selectable marker donor DNAs. In one aspect, in step (b), cells in which each allele has been modified by the introduction of a distinguishable donor DNA for selection marker are selected based on the expression of all the selection marker genes (marker genes for positive selection) that are contained in the two or more types of donor DNA for selection marker and that have been integrated into the chromosomal genome.
  • the cells obtained in step (b) have different marker genes for positive selection in each allele. In one aspect, the cells obtained in step (b) have a common marker gene for positive selection in each allele.
  • single cell cloning is not performed in step (b) ⁇ however, it may or may not include single cell cloning after selecting cells in which two or more alleles have been modified in step (b) ⁇ . In one aspect, in step (b), the selection of cells is performed based on the expression of multiple distinguishable marker genes for positive selection introduced into each allele.
  • step (b) is not performed by a method of estimating the number of modified alleles based on the expression intensity of a single selection marker gene (e.g., expression intensity or fluorescence intensity of a fluorescent protein).
  • a single selection marker gene e.g., expression intensity or fluorescence intensity of a fluorescent protein.
  • step (b) cells may be appropriately selected depending on the type of selection marker gene used in step (a). In this case, cells are selected based on the expression of all of the selection marker genes used in step (a).
  • the selection marker gene when the selection marker gene is a positive selection marker gene, cells expressing all the selection marker genes that are incorporated (or have been incorporated) into the chromosomal genome to be modified can be selected, for example, cells expressing the same number of positive selection markers as the number of alleles to be modified can be selected.
  • the positive selection marker gene is a drug resistance gene, cells expressing the positive selection marker can be selected by culturing the cells in a medium containing the drug.
  • the positive selection marker gene is a fluorescent protein gene, a luminescent enzyme gene, or a chromogenic enzyme gene
  • cells expressing the positive selection marker can be selected by selecting cells that exhibit fluorescence, luminescence, or color development due to the fluorescent protein, luminescent enzyme, or chromogenic enzyme.
  • the number of alleles to be modified is n or less, and when the number of selection marker donor DNAs of types greater than or equal to n are incorporated into the genome, at least the alleles to be modified (which are two or more alleles) are modified.
  • the number of alleles to be modified is n, and the corresponding number of types of donor DNA for the selection marker are incorporated into the chromosomal genome, and all alleles are modified.
  • the same number or more types of donor DNA for the selection marker are used as the number of alleles to be modified, so that the number of positive selection markers expressed by the cells means that the corresponding number of alleles have been reliably modified. From the viewpoint of increasing the efficiency of cell selection in step (b), it is preferable that the number of alleles to be modified is the same as the number of types of donor DNA for the selection marker.
  • the genome modification method of this embodiment by inducing HDR using n types of donor DNA for selection markers to modify n alleles in an n-ploid cell, it is possible to efficiently obtain cells in which all alleles possessed by the cell have been modified. Furthermore, because it is possible to reliably obtain cells in which all alleles have been modified, it is possible to efficiently obtain cells in which the target region has been modified even if the target region is large in size (e.g., 10 kbp or more). This makes large-scale genome modification possible.
  • step (b) allows for the selection of modified cells from a pool containing cells obtained by step (a) without cloning the cells. By eliminating the cloning step, the time required for the process can be reduced.
  • the pool may contain 10 or more, 10 or more, 10 or more, or 10 or more cells.
  • the genome modification method of the present embodiment may have any step in addition to the above steps (a) and (b).
  • Examples of the optional step include the following steps (c) and (d): (c) after step (b), introducing into the cells a donor DNA for recombination comprising a desired base sequence between an upstream homology arm having a base sequence homologous to a base sequence adjacent to the upstream side of the target region and a downstream homology arm having a base sequence homologous to a base sequence adjacent to the downstream side of the target region; and (d) after step (c), selecting cells that do not express the negative selection marker.
  • the genome modification method of this embodiment may have any step in addition to the above steps (a) and (b).
  • two or more types of donor DNA for selection markers each have a selection marker gene for positive selection and a separate marker gene for negative selection and a target sequence between the upstream homology arm and the downstream homology arm, and when the selection marker gene is used for both positive selection and negative selection, the separate selection marker gene for negative selection may not be present, and may have, for example, the following steps (c) and (d): (c) after the step (b), introducing the following (iii) and (iv) into the selected cells to introduce donor DNA for recombination into the two or more alleles: (iii) a sequence-specific nucleic acid cleaving molecule capable of targeting and cleaving the additional target sequence, or a genome modification system comprising a polynucleotide encoding the sequence-specific nucleic
  • Step (c) may be performed after step (b).
  • a donor DNA for recombination containing or not containing a desired base sequence between an upstream homology arm and a downstream homology arm is introduced into the cell selected in step (b).
  • a donor DNA for recombination containing a desired base sequence between an upstream homology arm having a base sequence homologous to a base sequence adjacent to the upstream side of the target region and a downstream homology arm having a base sequence homologous to a base sequence adjacent to the downstream side of the target region is introduced into the cell selected in step (b).
  • the donor DNA for recombination may contain a desired base sequence to be knocked in.
  • the desired base sequence is not particularly limited.
  • a base sequence in which a part or all of the base sequence of the target region is deleted can be used as the desired base sequence.
  • a base sequence containing the gene can be used as the desired base sequence.
  • the size of the desired base sequence is not particularly limited and can be any size.
  • the desired base sequence can be, for example, 10 bp or more, 20 bp or more, 40 bp or more, 80 bp or more, 200 bp or more, 400 bp or more, 800 bp or more, 1 kbp or more, 2 kbp or more, 3 kbp or more, 4 kbp or more, 5 kbp or more, 6 kbp or more, 7 kbp or more, 8 kbp or more, 9 kbp or more, 10 kbp or more, 15 kbp or more, 20 kbp or more, 40 kbp or more, 80 kbp or more, 100 kbp or more, or 200 kbp or more.
  • cells in which the desired base sequence is knocked into two or more alleles can be efficiently selected. Therefore, even DNA of a large size, for example, 5 kbp or more, 8 kbp or more, or 10 kbp or more, can be knocked in.
  • the donor DNA for recombination may be shorter in length than the donor DNA for selection marker, for example.
  • the upstream homology arm and downstream homology arm of the donor DNA for recombination may be the same as or different from those of the donor DNA for the selection marker.
  • the upstream homology arm and downstream homology arm of the donor DNA for the selection marker may be referred to as the "first upstream homology arm” and the "first downstream homology arm”
  • the upstream homology arm and downstream homology arm of the donor DNA for recombination may be referred to as the "second upstream homology arm” and the "second downstream homology arm”.
  • the length and sequence of the second upstream homology arm and the second downstream homology arm are not particularly limited, for example, as long as they are capable of homologous recombination with the first upstream homology arm or a region upstream thereof, and are capable of homologous recombination with the first downstream homology arm or a region downstream thereof (in one aspect, the length and sequence are not particularly limited, as long as they are capable of homologous recombination with a region surrounding the target region).
  • the base sequence of the donor DNA for selection marker is completely removed from the genome by recombination with the donor DNA for recombination.
  • Various genes carried by the donor DNA for selection marker are removed by recombination with the donor DNA for recombination.
  • two or more alleles of a cell can be replaced by the donor DNA for recombination.
  • the donor DNA for recombination may have a desired base sequence, and as a result, a cell in which two or more alleles have been modified will have the desired base sequence in the modified allele.
  • the desired base sequence is located between the second upstream homology arm and the second downstream homology arm.
  • the foreign gene is preferably functionally linked to a promoter.
  • the recombinant donor DNA may have any control sequence such as an enhancer, a polyA addition signal, a terminator, etc.
  • the recombinant donor DNA may have an insulator sequence upstream and downstream of the foreign gene.
  • the recombinant donor DNA contains a spacer sequence between the second upstream homology arm and the second downstream homology arm.
  • the donor DNA for recombination is configured so that when cells having a negative selection marker gene contained in the donor DNA for selection marker are removed, the donor DNA for recombination does not express a gene that is the same as (or indistinguishable from) the gene under conditions that exert its toxicity.
  • the donor DNA for recombination does not have a negative selection marker gene and a second target sequence between the second upstream homology arm and the second downstream homology arm.
  • the donor DNA for recombination is preferably introduced into the cell together with the above (i).
  • the DNA in the target region is cleaved by the sequence-specific nucleic acid cleaving molecule of the above (i), and then the desired base sequence in the donor DNA for recombination is knocked into the target region by HDR.
  • the cell into which the donor DNA for recombination is introduced in this step is the cell selected in step (b), so the base sequence of the donor DNA for selection marker is knocked into the target region. Therefore, the target sequence of the genome modification system in (i) is the base sequence contained in the target region after the donor DNA for selection marker is knocked in.
  • the target sequence of the genome modification system in step (a) may be referred to as the "first target sequence”
  • the target sequence of the genome modification system in step (c) may be referred to as the "second target sequence”.
  • the second target sequence may be any sequence contained in the target region of the cell after step (b).
  • the second target sequence in the donor DNA for selection marker may be a sequence that does not exist in the genome of the cell.
  • the second target sequence in the donor DNA for the selection marker is a sequence that is not present in the genome of the cell, and is a sequence that is different from other sequences to the extent that it does not cleave other sequences in the genome due to off-targets.
  • the second target sequence in the donor DNA for the selection marker can be a cleavage site of the meganuclease that is not present in the genome.
  • the second target sequence is a region other than the negative selection marker gene in step (d).
  • the donor DNA for recombination is configured so that homologous recombination by the donor DNA for recombination is not significantly inhibited. If the first target sequence remains in the target region of the cell after step (b) or if the first target sequence is reintroduced by the donor DNA for the selection marker, the second target sequence may be the same as or different from the first target sequence.
  • the donor DNA for recombination does not have to contain a base sequence between the upstream homology arm and the downstream homology arm, but may contain a base sequence between the upstream homology arm and the downstream homology arm of 10 bp or less, 20 bp or less, 30 bp or less, 40 bp or less, 50 bp or less, 60 bp or less, 70 bp or less, 80 bp or less, 90 bp or less, 100 bp or less, 200 bp or less, 300 bp or less, 400 bp or less, 500 bp or less, 600 bp or less, 700 bp or less, 800 bp or less, 900 bp or less, or 1 kbp or less.
  • the donor DNA for recombination may contain a base sequence of 1 kbp or more, 2 kbp or more, 3 kbp or more, 4 kbp or more, 5 kbp or more, 6 kbp or more, 7 kbp or more, 8 kbp or more, 9 kbp or more, or 10 kbp or more between the upstream homology arm and the downstream homology arm.
  • the donor DNA for recombination contains, between the upstream homology arm and the downstream homology arm, one or more or all selected from the group consisting of a selection marker gene, a target sequence for a site-specific recombinase, a gene encoding a physiologically active factor, a gene encoding a cytotoxic factor, and a promoter sequence, or does not contain one or more or all selected from the above group.
  • step (c) a donor DNA for recombination is introduced into the cells selected in step (b).
  • the cells selected in step (b) have a selection marker gene knocked in to the target region.
  • Step (c) can also be said to be a step of removing the selection marker gene knocked in to the target region or replacing it with a desired base sequence.
  • Step (d) Step (c) may be followed by step (d), in which cells that do not express the negative selection marker are selected.
  • the donor DNA for the selection marker in step (a) may contain a positive selection marker gene and a negative selection marker gene. That is, the donor DNA for the selection marker used in step (a) may contain a positive selection marker gene and a negative selection marker gene between the upstream homology arm and the downstream homology arm.
  • the positional relationship between the positive selection marker gene and the negative selection marker gene is not particularly limited, and the positive selection marker gene may be upstream of the negative selection marker gene, or vice versa.
  • the donor DNA for the selection marker has a positive selection marker gene and a negative selection marker gene
  • a base sequence encoding a self-cleaving peptide or an IRES (internal ribozyme entry site) sequence or the like may be interposed between the positive selection marker gene and the negative selection marker gene.
  • IRES internal ribozyme entry site
  • 2A peptides examples include 2A peptide (F2A) derived from foot-and-mouth disease virus (FMDV), 2A peptide (E2A) derived from equine rhinitis A virus (ERAV), 2A peptide (P2A) derived from porcine teschovirus (PTV-1), and 2A peptide (T2A) derived from Thosea asigna virus (TaV).
  • FMDV foot-and-mouth disease virus
  • E2A peptide
  • E2A derived from equine rhinitis A virus
  • P2A porcine teschovirus
  • T2A 2A peptide derived from Thosea asigna virus
  • the same selection marker gene may be used as a positive selection marker in step (a) and as a negative selection marker in step (d).
  • the selection marker gene is a marker gene (visualization marker gene) related to fluorescence or color development such as a fluorescent protein gene, a luminescent enzyme gene, or a chromogenic enzyme gene
  • cells that exhibit fluorescence, luminescence, or color development due to expression of the fluorescent protein, luminescent enzyme, or chromogenic enzyme may be selected in step (a), and cells that have lost this fluorescence, luminescence, or color development may be selected in step (c).
  • Cases in which the same selection marker gene serves as both a positive selection marker and a negative selection marker are also included when the donor DNA for the selection marker further has a negative selection marker in addition to a positive selection marker.
  • the negative selection marker gene may be different or the same for each type of selection marker donor DNA. By using a common negative selection marker gene, the cell selection process in step (d) is simplified.
  • step (d) cells may be appropriately selected depending on the type of negative selection marker gene used in step (a). In this case, cells that do not express any of the negative selection marker genes used in step (a) are selected.
  • the negative selection marker gene is a visualization marker gene such as a fluorescent protein gene, a luminescent enzyme gene, or a chromogenic enzyme gene
  • the negative selection marker gene is a suicide gene
  • cells that do not express the negative selection marker can be selected by culturing the cells in a medium containing a drug that expresses toxicity due to the expression of the suicide gene.
  • a thymidine kinase gene is used as the suicide gene, cells can be cultured in a medium containing ganciclovir.
  • the disappearance of the expression of the negative selection marker gene means that the negative selection marker gene incorporated into the target region in step (a) has been replaced with a polynucleotide containing the desired base sequence of the donor DNA for recombination. At this time, it is considered that the replacement of the polynucleotide has occurred in the entire base sequence knocked in in step (a). Therefore, by selecting cells in which the expression of the negative selection marker gene has disappeared, cells in which the base sequence knocked in in step (a) has been replaced with the desired base sequence of the donor DNA for recombination can be efficiently selected.
  • a negative selection marker gene such as a suicide gene, may be operably linked to an inducible promoter, and cells that do not express the negative selection marker may be selected by culturing the cells in the presence of a drug that drives the inducible promoter so that the negative selection marker gene is expressed under conditions that exert its toxicity.
  • the negative selection marker gene may be a gene that encodes a cytotoxin (e.g., ricin and diphtheria toxin) that is toxic to cells when expressed alone.
  • cells in which two or more alleles have been modified can be selected from a pool containing the cells obtained by step (c) without cloning the cells.
  • the pool may contain 10 or more, 10 or more, 10 or more, or 10 or more cells.
  • cells in which all alleles in the cells have been modified to the desired sequence can be efficiently obtained.
  • cells in which all alleles have been modified can be reliably obtained, even if the desired base sequence is large in size (e.g., 10 kbp or more), cells in which the base sequence has been knocked into the target region can be efficiently obtained.
  • the target region is deleted in all alleles in the cells, and the sequences before and after it (i.e., the sequences in which the upstream homology arm and the downstream homology arm each undergo homologous recombination) are seamlessly linked without one or more selected from the group consisting of base insertion, substitution, and deletion (e.g., without base insertion, substitution, and deletion).
  • the base sequences on the upstream and downstream sides of the deleted region are seamlessly linked.
  • step (e) can be performed after step (b).
  • step (e) when the number of surviving cells is low or no surviving cells are obtained in step (b), the target region is narrowed and the number of genes to be eliminated from the genome is reduced to identify genes that affect cell proliferation or survival.
  • the gene is a gene that affects cell proliferation or survival.
  • step (f) can be performed.
  • Step (f) includes knocking in the identified gene that affects cell proliferation or survival into another region of the genome to be modified (e.g., a safe harbor region, etc.) ⁇ recombination donor DNA may be used for knocking in ⁇ .
  • step (a) can exclude a region that eliminates cell proliferation or survival from the target region.
  • the present invention includes, for example, knocking in a desired gene into the chromosomal region (i) or a region other than (i) (e.g., a safe harbor region, etc.) of the genomic DNA of a cell having a deletion in the chromosomal region (i).
  • Such a cell has a genome having a deletion in the chromosomal region (i) and a gene encoding the desired gene in the chromosomal region (i) or a region other than (i) (e.g., the region (ii) above having a deletion and a safe harbor region, etc.).
  • a cell having a genome with a deletion (e.g., a deletion of a region of up to 1 Mb, or 500 kb or less, 450 kb or less, 400 kb or less, 350 kb or less, or 300 kb or less) in all alleles of the genome (two alleles in the case of diploidy), the deletion including a region including some or preferably all of the gene clusters encoding KIR and/or KLR (sometimes referred to as the KIR gene cluster and the KLR gene cluster, respectively).
  • the deletion of repetitive sequences with similar sequences can improve the readability of the genome and/or increase the targeting efficiency of genes in the genome (e.g., KIR genes or KLR genes).
  • the cell having the deletion may have an insertion of an endogenous or exogenous gene.
  • the inserted gene is preferably operably linked to a regulatory sequence.
  • the region including the group of genes encoding KIR may further include the surrounding region.
  • the surrounding region may include, for example, LILRB1 and/or LILRB4.
  • the surrounding region may include, for example, any one, two, or three selected from the group consisting of FCAR, NCR1, and NRRP7.
  • the deletion is in a region that includes the KIR gene cluster, and the size of the deletion is, but is not limited to, for example, 140 kbp to 500 kbp, 200 kbp to 500 kbp, 300 kbp to 500 kbp, 140 kbp to 400 kbp, 200 kbp to 400 kbp, or 300 kbp to 400 kbp.
  • the deletion is in a region that includes the KLR gene cluster, and the size of the deletion is, but is not limited to, for example, 140 kbp to 500 kbp, 200 kbp to 500 kbp, 300 kbp to 500 kbp, 140 kbp to 400 kbp, 200 kbp to 400 kbp, or 300 kbp to 400 kbp.
  • the cell has a deletion, the deletion including all or part of a region that includes the KLR gene cluster (e.g., the region corresponding to hg38:chr12:10,308,078-10,451,156). In one embodiment, the cell has a deletion, the deletion including all or part of a region that includes the KIR gene cluster (e.g., the region corresponding to hg38:chr19:54,724,497-54,866,731). In one embodiment, the cell has a deletion, the deletion including all or part of a region that includes the KIR gene cluster (e.g., the region corresponding to hg38:chr19:54,630,354-54,944,284).
  • the deletion including all or part of a region that includes the KLR gene cluster e.g., the region corresponding to hg38:chr12:10,308,078-10,451,156. In one embodiment, the cell has a deletion, the deletion including all or part of a region that includes the KIR
  • the genes encoding KIR, LILR, and KLR can be targeted by their own unique sequences.
  • the clusters containing these genes are present within a region of approximately 150 kbp to 450 kbp. Therefore, it is necessary to find at least one unique sequence in that region. This is because targeting and cutting a unique sequence makes it possible to modify the genome of that region and introduce a positive-negative marker set.
  • one unique sequence is specifically cut on each side of the region to be deleted. Therefore, one unique sequence is required on each side of the region to be deleted.
  • a positive and negative marker set includes a set of multiple nucleic acids including a combination of a positive selection marker and a negative selection marker, and the positive selection marker and the negative selection marker in a certain nucleic acid are distinguishable from the positive selection marker and the negative selection marker in another nucleic acid during selection.
  • a first combination includes a green fluorescent protein (GFP) and a puromycin resistance gene (Puro)
  • a second combination includes a red fluorescent protein (RFP) and a blasticidin resistance gene (Blst), where GFP and RFP are negative selection markers that are distinguishable during selection by flow cytometry, and Puro and Blst are positive selection markers that are distinguishable during drug selection.
  • the positive selection marker is preferably a drug resistance gene
  • the negative selection marker is preferably a gene encoding a fluorescent protein.
  • the positive and negative marker set is usually expected to be introduced into the corresponding positions (particularly the same positions) of the two alleles of genomic DNA, respectively.
  • specific cleavage is induced at the site to be modified in the cells to be edited, and then drug selection is performed to obtain clones in which different drug resistance genes are inserted into each allele.
  • a large-scale deletion of 1 kbp or more, 2 kbp or more, 3 kbp or more, 4 kbp or more, 5 kbp or more, 6 kbp or more, 7 kbp or more, 8 kbp or more, 9 kbp or more, or 10 kbp or more.
  • a large-scale DNA deletion can be induced by the second recombination by introducing specific cleavage at one or more sites in the vicinity of the region containing the positive-negative marker set, preferably at two sites so as to sandwich the positive-negative marker set.
  • the junction portion of the DNA after the deletion will have the sequence that the donor DNA for recombination had. In this way, deletion can be caused exactly as designed at the single base level without using a site recombination enzyme, and the sequence of the DNA after the deletion will also be as designed.
  • cells with deletions can be effectively selected by expression of the negative selection marker. If the cells are diploid, clones that express only one negative selection marker are clones with deletions in only one allele, and clones that do not express any negative selection marker are clones with deletions in both alleles.
  • a certain locus can induce silencing of the expression of a negative selection marker in a positive-negative marker set.
  • Silencing may occur uniformly in all cells, or may occur only in some cells. If it occurs only in some cells, the presence of cells in which silencing occurs, even if it is a small percentage, is a major obstacle to negative selection. Specifically, in negative selection, clones that should truly be selected (hit clones) are generated at a rate of about 1/10,000. In contrast, if expression is stopped even in a very small percentage of cells due to silencing, it becomes extremely difficult to obtain a clone that should truly be selected due to a large number of false negatives.
  • the present disclosure provides methods for detecting whether such silencing has occurred. For example, if silencing has occurred, a positive-negative marker set can be introduced at another site, and if silencing has not occurred, negative selection can be performed.
  • a method for detecting whether silencing of a negative selection marker in a positive negative marker set has occurred comprises: providing a cell having a positive and negative marker set at each allele of a particular locus, the positive and negative marker set including a positive and negative selection marker, wherein the positive and negative selection markers are distinguishably different for each allele and the negative and positive selection markers are distinguishably different for each allele; measuring the expression level of a negative selection marker in the cells to determine the proportion of cells in which expression of the negative selection marker is silenced; wherein the positive selection marker is preferably a drug resistance gene, and the negative selection marker is preferably a gene encoding a fluorescent protein.
  • a method for detecting whether silencing of a negative selection marker in a positive-negative marker set has occurred is The method may further comprise assessing that silencing has occurred when the proportion of cells in which silencing has occurred exceeds a reference value, and determining that silencing has not occurred or that the above-mentioned negative selection is possible when the proportion of cells in which silencing has occurred is equal to or less than the reference value.
  • the present disclosure also provides a method for determining whether negative selection is possible, comprising: The method includes assessing that silencing has occurred when the proportion of cells in which silencing has occurred exceeds a reference value, and determining that the negative selection is possible when the proportion of cells in which silencing has occurred is equal to or less than the reference value.
  • a method for producing an isolated cell having two or more modified alleles of a chromosomal genome comprising: (a) introducing the following (i) and (ii) into an isolated cell (excluding a fertilized egg) containing two or more alleles to introduce a selection marker gene into each of the two or more alleles; (i) a sequence-specific nucleic acid cleaving molecule capable of targeting and cleaving a target region in two or more alleles of the chromosomal genome, or a genome modification system comprising a polynucleotide encoding the sequence-specific nucleic acid cleaving molecule; (ii) Two or more types of donor DNA for selection markers, each of which has an upstream homology arm having a base sequence capable of homologous recombination with a base sequence on the upstream side of the target region, and a downstream homology arm having a base sequence capable of homologous recombination with a base sequence on
  • the above silencing can be observed, for example, in a region where selective expression control is performed in a gene cluster region (for example, a region where allelic exclusion is performed, such as immunoglobulin (Ig) genes, Ig ⁇ and ⁇ light chain genes, olfactory receptor genes, vomeronasal receptor V1R genes, T cell receptor genes, etc.), but is not particularly limited thereto.
  • a gene cluster region for example, a region where allelic exclusion is performed, such as immunoglobulin (Ig) genes, Ig ⁇ and ⁇ light chain genes, olfactory receptor genes, vomeronasal receptor V1R genes, T cell receptor genes, etc.
  • the isolated cells selected in the step for positive selection can be allowed to grow, and the proportion of cells that are silenced can be confirmed after growth. In a preferred embodiment, if the proportion exceeds a reference value, it can be shown that negative selection is not easy due to silencing.
  • a cell population including cells for example, vertebrate cells, for example, mammalian cells, for example, human cells
  • Each cell contains a positive and negative marker set for each of two alleles within a region of 500 kbp or less that includes the gene cluster region
  • the positive and negative marker set includes a positive selection marker and a negative selection marker, the positive selection markers contained in the same cell being distinguishable from each other, and the negative selection markers contained in the same cell being distinguishable from each other;
  • a cell population in which the percentage of cells in which a negative selection marker is silenced is 10% or less (or a reference value or less), the percentage of silenced cells in the cell population is 1x10-7 % or more, 1x10-6 % or more, 1x10-5 % or more, 0.00001% or more, 0.00002% or more, 0.00003% or more, 0.00004% or more, 0.00005%
  • the percentage of silenced cells may be 1 ⁇ 10 ⁇ 7 % to 5%. It may be 1 ⁇ 10 ⁇ 7 % to 1%. In some embodiments, the percentage of silenced cells may be 0.00001% or more to 5%, 4% or less, 3% or less, 2% or less, or 1% or less. In some embodiments, the percentage of silenced cells may be 0.0001% or more and 2% or less. In some embodiments, the percentage of silenced cells may be 0.001% or more and 2% or less. In some embodiments, the percentage of silenced cells may be 0.01% or more and 2% or less. In some embodiments, the percentage of silenced cells may be 0.1% or more and 2% or less.
  • the percentage of silenced cells may be 0.1% or more and 1% or less. Although it is considered that it is usually possible to confirm deletion in about 100 cells or more and about 1000 cells or less, in order to reduce the number of cells to be confirmed, it is preferable that the percentage of silenced cells is as small as possible.
  • the selected cells may be subjected to a step for negative selection ⁇ however, in order to remove false negatives from negative selection marker-negative cells in negative selection, it may be necessary to confirm whether deletion has occurred for more clones ⁇ , but, for example, the selected cells may not be subjected to a subsequent step for negative selection (or it may be decided not to subject them to the step).
  • n/m is 500 or less, 400 or less, 300 or less, 200 or less, 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1 or less, 0.9 or less, 0.8 or less , 0.7 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, 0.01 or less, 0.009 or less, 0.008 or less, 0.007 or less
  • n (under this premise, it can be assumed that there is one hit clone in n x 100) can be determined by comparing the effort of screening with the effort of identifying other points with low n.
  • the method of the present invention may further include selecting cells having a deletion of the above-mentioned gene group from cells that do not express the negative selection marker.
  • the deletion of the gene group can be determined by analyzing the DNA sequence. For example, it is possible to determine whether or not the deletion of the gene group has occurred in the DNA by determining the size of the amplified product obtained by the PCR method, the size of the restriction enzyme fragment, or the sequence by sequencing.
  • the cell has a positive-negative marker set in the KIR gene cluster region and/or in the KLR gene cluster region. In one preferred embodiment, the cell has no deletion in the KIR gene cluster region and has a positive-negative marker set in the KLR gene cluster region. In one preferred embodiment, the cell has a deletion of some or all of the KIR genes in the KIR gene cluster region and has a positive-negative marker set in the KLR gene cluster region. In one preferred embodiment, the cell has a positive-negative marker set in the KIR gene cluster region and has no deletion in the KLR gene cluster region. In one preferred embodiment, the cell has a positive-negative marker set in the KIR gene cluster region and has a deletion of some or all of the KLR genes in the KLR gene cluster region.
  • the cell has a positive-negative marker set in each of the KIR gene cluster region and the KLR gene cluster region.
  • the positive and negative marker set is present at one locus, the positive and negative selection markers introduced at each of the two alleles are distinguishably different from each other, but when the positive and negative markers are present at two or more loci, the positive and negative selection markers introduced at any allele at any locus are distinguishably different from any other positive and negative selection marker.
  • the marker is present between the KIR3DL1 and KIR2DL4 genes in one allele or preferably in the good allele.
  • the cells having the positive and negative marker set preferably have a silencing rate of the negative selection marker of 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.09% or less, 0.08% or less, 0.07% or less, 0.06% or less, 0.05% or less, 0.04% or less, 0.03% or less, 0.02% or less, 0.01% or less, 0.009% or less, 0.008% or less, 0.007% or less, 0.00 6% or less, 0.005% or less, 0.004% or less, 0.003% or less, 0.002% or less, 0.001% or less, 0.0009% or less, 0.0008% or less, 0.0007% or less, 0.0006% or less, 0.0005% or less, 0.0004% or less, 0.0003% or less, 0.0002% or less, 0.0001%
  • a cell population includes cells.
  • the cells have a positive-negative marker set in the KIR gene cluster region and/or the KLR gene cluster region, and the percentage of cells in which the negative selection marker of any allele is silenced is 1% or less.
  • the cells have a positive-negative marker set in the KIR gene cluster region, and the percentage of cells in which the negative selection marker of any allele is silenced is 1% or less.
  • the cells have a positive-negative marker set in the KLR gene cluster region, and the percentage of cells in which the negative selection marker of any allele is silenced is 1% or less.
  • a cell population includes cells, wherein in one aspect, each cell comprises a set of positive and negative markers on two alleles within a region of 500 kbp or less (or 450 kb or less, 400 kb or less, 350 kb or less, 300 kb or less, 250 kb or less, or 200 kbp or less) that includes a killer cell immunoglobulin-like receptor (KIR) gene cluster region, and/or a set of positive and negative markers on two alleles within a region of 500 kbp or less (or 450 kb or less, 400 kb or less, 350 kb or less, 300 kb or less, 250 kb or less, or 200 kbp or less) that includes a killer cell lectin-like receptor (KLR) gene cluster region;
  • the positive and negative marker set includes positive and negative selection markers, the positive selection markers being distinguishable from one another, and the negative selection markers being distinguishable from one another, The percentage of cells in the cell population
  • Cells containing a positive-negative marker set can be preferably used to induce deletion (and replacement) of a region containing the positive-negative marker set.
  • the size and position of the deletion can be determined by the cleavage position of the target DNA and the design of the recombination donor DNA. Whether the deletion is to be a replacement can also be determined by the design of the recombination donor DNA.
  • the sequence between the upstream homology arm and the downstream homology arm of the recombination donor DNA replaces the region to be deleted in the resulting cells.
  • the complete region is deleted, and if a sequence exists between the upstream homology arm and the downstream homology arm, the region to be deleted is replaced by the sequence between the upstream homology arm and the downstream homology arm.
  • cells containing a positive-negative marker set can be preferably used for further genetic engineering.
  • the proportion of cells in which the negative selection marker is silenced is below a standard value in order to facilitate the selection of deleted cells.
  • regions are regions where silencing can occur, it is important to keep the proportion of silenced cells small.
  • the cells are vertebrate cells, preferably human cells.
  • the modified cells of the present disclosure may be included, for example, in a composition (particularly an aqueous composition).
  • the composition may further include water, salts, and additives (e.g., pH adjusters, isotonicity agents, dispersants, etc.).
  • the pluripotent cells When the cells are pluripotent cells, the pluripotent cells and compositions comprising the pluripotent cells are provided.
  • the pluripotent cells can be differentiated into a desired cell and then used. For example, they can be differentiated into immune cells (e.g., NK cells or T cells) and then used. It may be easier to genetically modify pluripotent cells than differentiated cells, in which case the modifications desired to be made to the differentiated cells may be made in the pluripotent cells or at other differentiation stages.
  • the immune cells are NK cells, genetic modifications can be made at the hematopoietic stem cells, or common lymphoid progenitor cells, and NK, as well as at the differentiation stages between the two of these.
  • the cells of the present disclosure may further lack all or part of HLA class I and/or class II. Since silencing did not occur in the HLA region, it is believed that it is possible to obtain clones lacking all or part of the HLA region by negative selection using a negative selection marker inserted into that region.
  • KIR locus and LILR locus which have sequence polymorphisms, repeat polymorphisms, and copy number polymorphisms, as well as the KLR locus.
  • the KLR gene is expressed in natural killer cells (NK cells) and is involved in controlling the ability to damage target cells, so its expression can be problematic in NK cell transplantation therapy.
  • NK cells natural killer cells
  • the KIR gene is expressed in NK cells and some T cell subsets and is involved in controlling the activity of KIR-expressing cells by target cells. Many KIRs are inhibitory, so their expression can be problematic in NK cell or T cell transplantation therapy.
  • these genes have sequence polymorphisms and copy number polymorphisms, and therefore are difficult to decipher even with current sequence decoding technology.
  • sequence polymorphisms When similar sequences are accumulated in one region, it is also difficult to specifically edit one gene in this region. It is useful to create cells with deleted loci and haploid cells for these loci. It is possible to remove the entire region that is difficult to decipher or specifically target.
  • Example 1 Deletion of KLR gene cluster region
  • Figure 1 shows a map of the human KLR gene locus.
  • KLR genes form a cluster on chromosome 12 (p13.2) ( Figure 1).
  • the cluster region is located at hg38:chr12:10,308,078-10,451,156 and has a length of approximately 143 kb.
  • An attempt was made to delete a target region (hg38:chr12:10,301,563-10,454,767) of approximately 153 kb length that includes the entire cluster.
  • iPS cells were used as the cells. Large-scale deletions were created based on the method disclosed in WO2021/206054A. Specifically, first, different selectable markers (positive and negative marker sets) that were distinguishable were introduced into each of the two alleles in the deleted region. Specifically, the first marker included green fluorescent protein (GFP) and a puromycin resistance gene (Puro), and the second marker included red fluorescent protein (RFP) and a blasticidin resistance gene (Blst), with GFP and RFP being negative selectable markers and inserted into each allele. GFP and RFP are distinguishable during selection by flow cytometry, and Puro and Blst are positive selectable markers that are distinguishable during drug selection.
  • GFP green fluorescent protein
  • Puro puromycin resistance gene
  • RFP red fluorescent protein
  • Blst blasticidin resistance gene
  • iPS cells were seeded on a 24-well plate pre-coated with iMatrix-511 diluted 150-fold with DPBS.
  • StemFit AK02N (10 ⁇ M Y-27632) was used as the medium.
  • Marker insertion 300 ng of gRNA/Cas9 expression plasmid for cleaving the marker insertion sequence, 100 ng of donor plasmid (GFP-Puro), and 100 ng of donor plasmid (RFP-Blst) were introduced into cells seeded on a 24-well plate.
  • the donor plasmid (GFP-Puro) had a gene encoding green fluorescent protein (GFP) operably linked to the EF1 promoter and a drug resistance gene, puromycin resistance gene (Puro). GFP and Puro were linked via a T2A sequence.
  • the donor plasmid had a gene encoding red fluorescent protein (RFP) operably linked to the EF1 promoter and a drug resistance gene, blasticidin resistance gene (Blst). RFP and Blst were linked via a T2A sequence. Details were as shown in Example 1 and Figure 1 of WO2021/206054.
  • the markers carried by the donor plasmid are referred to as a positive-negative marker set, and cells into which the positive-negative marker set has been introduced are referred to as positive-negative marker set-introduced cells. Additionally, positive-negative marker set-introduced cells are also referred to as first-stage cells.
  • Drug selection and expansion The obtained cells were seeded on a 6-well plate and drug selection of the cells was performed in the presence of puromycin and blasticidin.
  • StemFitR AK02N (containing 1 ⁇ M puromycin and 10 ⁇ M blasticidin) was used as the drug selection medium.
  • the surviving cells after drug selection are resistant to two drugs, that is, cells that have both the puromycin resistance gene and the blasticidin resistance gene.
  • such cells are generated when a puromycin resistance gene is introduced into one allele of the region targeted by gRNA and a blasticidin resistance gene is introduced into the other allele.
  • the surviving cells were expanded to obtain cells with a positive and negative marker set.
  • the cells with a positive and negative marker set were cloned as necessary.
  • the cells were cloned by collecting one colony of the surviving cells after drug selection and expanding the cells.
  • iPS cells were seeded on a 24-well plate pre-coated with iMatrix-511 diluted 150-fold with DPBS.
  • StemFit AK02N (10 ⁇ M Y-27632) was used as the medium.
  • gRNA/Cas9 Two gRNAs were created to target both ends of the region to be deleted. 62.5 ng of gRNA (left side), 62.5 ng of gRNA (right side), and 750 ⁇ g of Cas9 protein were introduced into cells seeded in a 24-well plate. After 24 hours, the medium was changed.
  • Flow cytometry After introducing gRNA/Cas9, GFP-negative and RFP-negative cells were selected using a flow cytometer after 5-7 days of culture, and then each cell was seeded onto a 96-well plate and allowed to proliferate. In this way, cells in which the region containing the positive-negative marker set introduction region has been deleted in both alleles can be obtained.
  • Genome purification Approximately 2 x 105 mutagenized cells were collected and the genome was purified. The primers used for genotyping were those shown in Table 1 below. PCR was performed by a conventional method. The guide RNAs shown in Table 2 below were used for marker insertion. The guide RNAs shown in Table 3 below were used for large-scale deletion formation.
  • the KLR gene cluster region is located at chromosome 12, position 10,308,078-10,451,156 (hg38:chr12:10,308,078-10,451,156) in the gh38 reference genome (see Figure 1). In this case, a large-scale deletion of a wider region including the cluster region was attempted. A positive-negative marker set was inserted into the deleted region. Specifically, it was introduced into the KLRD1 gene (more specifically, between hg38:chr12:10,307,328 and 10,307,329) (see Figures 1 and 2).
  • This insertion was obtained by generating a specific cut in the KLRD1 gene in the presence of selectable marker donor DNAs carrying the positive-negative marker set, and then culturing the cells in the presence of puromycin and blasticidin.
  • the insertion was confirmed by junction PCR. That is, the amplification of Puro(L) and Puro(R) in FIG. 2, as well as the amplification of Blst(L) and Blst(R) were confirmed. If an insertion is present, amplification can be obtained. According to FIG. 2, an amplified band was confirmed at a predetermined position in each, and the insertion of the selection marker was confirmed.
  • Example 2 Induction of large-scale deletion of KIR gene cluster region An attempt was made to further delete the entire KIR gene cluster region from one of the clones obtained in Example 1 in which the entire KLR gene cluster region was deleted.
  • the KIR gene cluster is present at hg38:chr19:54,724,497-54,866,731.
  • the KIR gene cluster region was a region that induced silencing in gene expression.
  • Cells (GFP negative) that have been removed from both alleles from a cell group having a positive-negative marker set are obtained with a probability of approximately 1 in 10,000. If 1% of GFP negative cells occur in 10,000 cells that have the positive-negative marker set in both alleles, this alone will result in 100 GFP negative cells being mixed in as false negatives. The presence of 1% GFP negative cells makes it difficult to obtain one defective cell in 10,000 cells.
  • silencing makes it almost impossible to screen for defective cells.
  • silencing was 0% in cells into which a gene encoding GFP was introduced into the HLA region. Therefore, it is understood that the KIR region is a difficult-to-edit region that exhibits a silencing effect.
  • FIG. 7 it was found that the silencing effect was acceptable even when a positive-negative marker set was introduced between hg38:chr19:54,939,860 and 54,939,861.
  • Cells were obtained in which a positive-negative marker set was introduced.
  • the above region includes the group of genes shown in FIG. 8 (for example, LILRB1, LILRB4, FCAR, NCR, and NPRP7 in addition to KIR). Next, the entire region including these was deleted.
  • the positive-negative marker set was inserted into the NPRP7 gene. The insertion was confirmed by junction PCR for Puro and Blst (see FIG. 8).

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Abstract

La présente invention concerne une délétion (plus particulièrement une délétion à grande échelle) dans une ou plusieurs régions de la région du groupe de gènes KIR, de la région du groupe de gènes LILR et de la région du groupe de gènes KLR. La présente invention concerne une cellule présentant une délétion dans une ou plusieurs régions de la région du groupe de gènes KIR, de la région du groupe de gènes LILR et de la région du groupe de gènes KLR. La présente invention propose également une cellule comprenant un marqueur positif-négatif placé dans une région de 1000 kpb comprenant une ou plusieurs régions de la région du groupe de gènes KIR, de la région du groupe de gènes LILR et de la région du groupe de gènes KLR.
PCT/JP2024/015500 2023-04-21 2024-04-19 Cellule présentant une délétion dans une ou plusieurs régions du groupe de gènes kir, du groupe de gènes lilr et du groupe de gènes klr, et son procédé de fabrication Pending WO2024219474A1 (fr)

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