WO2026005012A1 - Procédé d'amélioration de l'efficacité de recombinaison homologue dans l'édition génomique in vitro et in vivo - Google Patents

Procédé d'amélioration de l'efficacité de recombinaison homologue dans l'édition génomique in vitro et in vivo

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WO2026005012A1
WO2026005012A1 PCT/JP2025/023203 JP2025023203W WO2026005012A1 WO 2026005012 A1 WO2026005012 A1 WO 2026005012A1 JP 2025023203 W JP2025023203 W JP 2025023203W WO 2026005012 A1 WO2026005012 A1 WO 2026005012A1
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cells
dna
sequence
nucleic acid
target
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弘真 原
史雄 中原
豊 花園
新 本多
史倫 谷原
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Jichi Medical University
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Jichi Medical University
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    • 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
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • 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

  • This disclosure relates to methods for increasing the efficiency of homologous recombination in genome editing in vitro and in vivo in the presence of donor DNA (recombinant template DNA).
  • This disclosure provides a method for culturing cells having a double-strand break in a target sequence in the presence of donor DNA and an ATM inhibitor.
  • Genome editing technology has been developed, and a homologous recombination repair method has been developed to replace target regions in a cell's genome with a desired sequence.
  • the world's first genome therapy drug (CASGEVYTM) using the CRISPR/Cas9 system was approved for the treatment of sickle cell disease and beta-thalassemia (https://www.fda.gov/news-events/press-announcements/fda-approves-first-gene-therapies-treat-patients-sickle-cell-disease).
  • CASGEVY treats the above diseases by disrupting the BCL11A gene, stopping the production of mutated adult hemoglobin and promoting the production of functional fetal hemoglobin.
  • Double-strand breaks are specifically introduced at target sites in the genome using various sequence-specific nucleic acid cleaving molecules. Double-strand break repair induces errors, and is therefore used for gene disruption as described above. However, in the presence of donor DNA (recombinant template DNA), the double-strand break induces homology-directed repair (HDR) at the break site using the donor DNA, resulting in the precise replacement of the region between the left and right homology arms of the donor DNA with the corresponding genomic sequence. Because of the precision of the editing, genome editing using homology-directed repair is desirable for many medical purposes, but low editing efficiency remains a challenge (Frangoul et al. NEJM, 2021). Homologous recombination occurs particularly between the S and G2 phases. However, in cells with slow cell cycles, such as hematopoietic stem cells, the efficiency of homologous recombination is low, making them prone to other inaccurate repair processes such as non-homologous end joining (NHEJ).
  • NHEJ non-homolog
  • the present disclosure provides a method for increasing the efficiency of homologous recombination in genome editing in the presence of donor DNA (recombinant template DNA).
  • the present disclosure provides a method for culturing cells having a double-strand break in a target sequence in the presence of donor DNA and an ATM inhibitor.
  • ATM inhibitors increase the efficiency of homologous recombination during genome editing in the presence of linear donor DNA (recombination template DNA). Specifically, ATM inhibitors increased the efficiency of homologous recombination during genome editing in the presence of donor DNA (recombination template DNA) in various cells, such as pluripotent stem cells, hematopoietic stem cells, and fertilized eggs.
  • a method for inducing homologous recombination in a target cell comprising: introducing a double-strand break into DNA at a target site in the subject cell; culturing the subject cells in the presence of donor DNA and an ATM inhibitor, wherein the donor DNA comprises an upstream homology arm homologous to an upstream portion of the target site and a downstream homology arm homologous to a downstream portion of the target site, and further comprises a sequence of interest or no sequence between the upstream homology arm and the downstream homology arm; and after culturing, obtaining cells having DNA in which the target sequence is inserted between the upstream and downstream of the target site or DNA in which the target sequence is deleted between the upstream and downstream of the target site.
  • the donor DNA is derived from a circular DNA containing the target sequence of the sequence-specific nucleic acid cleaving molecule and is a linear DNA generated by cleaving the target sequence with the sequence-specific nucleic acid cleaving molecule.
  • the donor DNA is single-stranded DNA and is introduced into cells by an adeno-associated virus vector having the sequence of the donor DNA.
  • the target cells include blood cells.
  • the target cells include hematopoietic stem cells.
  • a method for editing a genome in a subject comprising: administering to a subject effective amounts of a sequence-specific nucleic acid cleaving molecule or a sequence-specific nucleic acid cleaving molecule complex or a nucleic acid encoding the same, template DNA, and an ATM inhibitor, respectively, to induce homologous recombination repair in the subject, thereby inducing homologous recombination between the target site in the subject and the template DNA;
  • the template DNA comprises an upstream homology arm homologous to the upstream of the target site and a downstream homology arm homologous to the downstream of the target site, and may further comprise a target sequence between the upstream homology arm and the downstream homology arm, or may not comprise a sequence of interest.
  • a genome editing kit for use in the method according to (10) above, an effective amount (i) a sequence-specific nucleic acid cleaving molecule, or a sequence-specific nucleic acid cleaving molecule complex, or a nucleic acid encoding the same; (ii) template DNA; (iii) one or more components selected from the group consisting of ATM inhibitors; Genome editing kit.
  • This figure shows the homologous recombination repair efficiency of various ATM inhibitors in hematopoietic stem progenitor cells (HSPCs). Treatment with ATM inhibitors tends to increase knock-in efficiency, and knock-in efficiency is also increased in CD150 + CD201 + KSL cells, a hematopoietic stem cell fraction. Same as above. Shows the efficiency of homologous recombination repair by various ATM inhibitors in immortalized bone marrow stromal cells (non-blood cells).
  • the term "cell” refers to a basic unit of life that contains at least genomic DNA, cytoplasm, and a membrane structure that encases these. Examples of cells include, but are not limited to, prokaryotic cells and eukaryotic cells. Genomic DNA contains the cell's endogenous DNA, but is not necessarily composed solely of the cell's endogenous factors.
  • cell population refers to a composition containing multiple cells.
  • Isolation means separating a cell of interest from at least one other component. Isolation can be performed, for example, by separating and removing a cell in its natural state from other components with which it exists in its natural state. Isolation can be performed, for example, by separating and removing a portion of cells from a multicellular organism. Techniques that involve the handling of isolated cells are referred to herein as in vitro techniques.
  • purification refers to further separating isolated cells of interest from other components that coexist with them. Purification can be performed, for example, by separating the cells of interest from other components based on morphology or surface markers. Purification can be performed by limiting dilution and/or cloning of cells. Cloned cells are cells that have been replicated from a single cell. Purification can be performed by establishing a cell line of interest. If the cells of interest have a marker gene, such as a drug resistance gene or a gene encoding a fluorescent protein, purification can be performed based on the expression of the marker gene. As used herein, “enrichment” refers to increasing the density of the cells of interest.
  • HDR is a repair mechanism that uses donor DNA and can also introduce desired mutations into the target region.
  • a preferred example of a genome modification technique is the CRISPR/Cas system (more preferably the CRISPR/Cas9 system).
  • Other genome editing systems that can be used include CRISPR/Cas3, CRISPR/Cas12, CRISPR-StAR, and MAD7.
  • target sequence refers to a DNA sequence in a genome that is targeted for cleavage 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 targeted for cleavage 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 typically 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) immediately adjacent to the 5' side of the PAM.
  • Known design tools such as CRISPR DESIGN (crispr.mit.edu/) can be used to design target sequences.
  • 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, the Cas9 protein.
  • Cas proteins include wild-type Cas proteins and their homologs (paralogs and orthologs), as well as mutants thereof, as long as they exhibit endonuclease activity or nickase activity in cooperation with a guide RNA.
  • the Cas protein is involved in a class 2 CRISPR/Cas system, more preferably a type II CRISPR/Cas system.
  • a preferred example of the Cas protein is the Cas9 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 proteins and their homologs (paralogs and orthologs), as well as mutants thereof, as long as they have the above-mentioned activity.
  • the wild-type Cas9 protein has a RuvC domain and an HNH domain as nuclease domains, but the Cas9 protein herein 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-strand break (nick) into double-stranded DNA. Therefore, when using Cas9 in which either the RuvC domain or the HNH domain has been inactivated 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 in positions sufficiently close to each other, thereby inducing double-strand cleavage.
  • the biological species from which the Cas9 protein is derived is not particularly limited, but 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, T. denticola, etc. In a preferred embodiment, the Cas9 protein is derived from S. pyogenes.
  • guide RNA and "gRNA” are used interchangeably and refer to an RNA that can form a complex with a Cas protein and guide the Cas protein to a target region.
  • the guide RNA comprises a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA).
  • the crRNA is involved in binding to a target region on the genome, and the tracrRNA is involved in binding to the 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 base pairs 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 tracrRNA may be separate RNA molecules in which base pairs are formed at the repeat and anti-repeat sequences.
  • 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 can be derived from the same bacterial species as the Cas protein.
  • the Cas9 protein, crRNA, and tracrRNA (or sgRNA) derived from S. pyogenes can be used.
  • Various crRNA repeat sequences and tracrRNA sequences for sgRNA design have been proposed, and those skilled in the art can design sgRNAs based on known techniques (e.g., Jinek et al. (2012) Science, 337, 816-21; Mali et al. (2013) Science, 339: 6121, 823-6; Cong et al. (2013) Science, 339: 6121, 819-23; Hwang et al. (2013) Nat. Biotechnol. 31: 3, 227-9; Jinek et al. (2013) eLife, 2, e00471).
  • ATM Ataxia telangiectasia mutated refers to the gene that causes the hereditary disease ataxia-telangiectasia (AT).
  • ATM is known to function as a sensor for double-strand breaks in DNA. If ATM does not function normally for a long period of time, DNA damage will not be properly repaired, which may affect cell division and proliferation.
  • the amino acid sequence of human ATM may be, for example, the amino acid sequence registered under Genbank registration number AAI37170.1.
  • Many ATM inhibitors have been developed, and they are expected to be used as anticancer drugs that inhibit DNA repair, causing damage to accumulate in cancer cells and killing them, for example.
  • the present disclosure provides a method for inducing homologous recombination in a target cell.
  • the method of the present disclosure may be an in vitro or ex vivo method.
  • the method of the present disclosure is an industrially applicable method. Since the method of the present disclosure can increase the rate of homologous recombination in a target cell, the method of the present disclosure can be interpreted as a method for increasing the rate of homologous recombination in a target cell.
  • a cell with a double-strand break introduced into its DNA attempts to repair the DNA using its own DNA repair mechanisms. If donor DNA (e.g., double-stranded DNA or preferably single-stranded DNA) is present in the cell and/or nucleus, the broken DNA is repaired by homologous recombination using the donor DNA. This type of repair is called homologous recombination repair. Details of genome editing using the CRISPR/Cas9 system are disclosed, for example, in F. A. Ran et al., Nature Protocols, Vol. 8, No. 11, 2281-2301, 2013, which is incorporated herein by reference in its entirety.
  • the method of the present disclosure may further include the step of introducing donor DNA into the cell.
  • the donor DNA can be introduced into the cell by transfection or, if the donor DNA is integrated into a viral genome, by a viral vector.
  • the method of the present disclosure comprises: 1. A method for inducing homologous recombination in a subject cell, comprising: The method of the present disclosure may include introducing into a subject cell a sequence-specific nucleic acid cleaving molecule capable of cleaving a target sequence on a genome, or DNA encoding the molecule, and donor DNA.
  • a method of the present disclosure is a method of inducing homologous recombination in a subject cell, the method comprising: providing a cell in which a double-strand break has been introduced into a target site in DNA within the cell; culturing the subject cells in the presence of donor DNA and an ATM inhibitor, wherein the donor DNA comprises an upstream homology arm homologous to an upstream portion of the target site and a downstream homology arm homologous to a downstream portion of the target site, and further comprises a sequence of interest or no sequence between the upstream homology arm and the downstream homology arm; After culturing, cells having DNA in which the target sequence is inserted between the upstream and downstream of the target site or DNA in which the target sequence is deleted between the upstream and downstream of the target site can be obtained.
  • Donor DNA is a template that undergoes homologous recombination with genomic DNA and is incorporated into the genomic DNA.
  • the donor DNA contains an upstream homology arm homologous to the upstream of the target site and a downstream homology arm homologous to the downstream of the target site, and may further contain a target sequence between the upstream and downstream homology arms.
  • the DNA is repaired by homologous recombination so that the sequence between the upstream and downstream of the target site on the genome is replaced with the sequence between the upstream and downstream homology arms.
  • the donor DNA contains a target sequence between the upstream and downstream homology arms, the sequence between the upstream and downstream of the target site on the genome is replaced with the target sequence; if the donor DNA does not contain a target sequence, the sequence between the upstream and downstream of the target site on the genome is deleted. In this way, DNA is repaired so that the sequence between the upstream and downstream of the target site on the genome is replaced with the sequence between the upstream and downstream homology arms of the donor DNA.
  • the donor DNA is sometimes called a donor template.
  • the sequence between the upstream and downstream of the target site will be completely missing (deleted).
  • Seamless or scarless editing can be achieved if a marker sequence or an artificially introduced recombination sequence is not used as the target sequence. Seamless or scarless editing is utilized during the editing process and is a preferred embodiment in situations where it is required that the edited genomic DNA is not affected by residual sequences that are no longer needed after editing (see, e.g., Xi et al., Genome Biol., 16:231, 2015; Roger Askew et al., Mol. Cel. Biol., 4115-4124, 1993; WO2019/018534; and WO2021/206054, which are incorporated by reference in their entireties).
  • the upstream and downstream homology arms of the donor DNA can be designed appropriately by those skilled in the art, but may have a length of, for example, 300 to 2000 nucleotides, 300 to 1500 nucleotides, or 300 to 1000 nucleotides, e.g., 350 to 700 nucleotides.
  • the target sequence of the donor DNA may be 0 nucleotides in length (i.e., absent or deleted), or 1 to 10,000 nucleotides in length, e.g., 1 to 5000 nucleotides, 1 to 1000 nucleotides, or 1 to 300 nucleotides.
  • the target sequence of the donor DNA can also be designed appropriately by those skilled in the art depending on the DNA sequence obtained after repair.
  • Donor DNA can be designed near the double-strand break (DSB) introduction site.
  • DSB double-strand break
  • any homology arm can be designed within 200 base pairs, preferably within 100 base pairs, and more preferably within 50 base pairs of the DSB introduction site. Even more preferably, homology arms can be designed upstream and downstream of the DSB introduction site.
  • modifications can include, for example, one or more selected from the group consisting of addition, deletion, insertion, deletion, and substitution, and combinations thereof.
  • Modifications can include those that confer activity to a loss-of-function mutation (e.g., reverting to the wild type), those that reduce the activity of a gain-of-function mutation (e.g., reverting to the wild type), those that introduce a loss-of-function mutation into the wild type, or those that introduce a gain-of-function mutation into the wild type.
  • the efficiency of the above-mentioned homologous recombination repair can be increased by culturing cells in the presence of an effective amount of an ATM inhibitor (ATMi).
  • ATM inhibitor ATM inhibitor
  • Donor DNA is linear single-stranded DNA (ssDNA) or linear double-stranded DNA (dsDNA).
  • Donor DNA (particularly linear single-stranded DNA and linear double-stranded DNA) can be introduced into cells as appropriate, for example, together with a sequence-specific nucleic acid cleaving molecule for genome editing.
  • the homology arms of the donor DNA may be designed to be complementary to either strand of the genomic DNA. The efficiency of homologous recombination is increased when donor DNA has homology arms of at least 40 bases on both sides.
  • Donor DNA may also be circular DNA to which a recognition sequence for a sequence-specific nucleic acid cleaving molecule has been added. When the sequence-specific nucleic acid cleaving molecule cleaves the recognition sequence on the genome, the circular DNA is cleaved and converted into linear DNA, resulting in the same results as when linear DNA is used as donor DNA.
  • the donor DNA is integrated into the genome of a virus or viral vector having a single-stranded DNA genome. In another embodiment, the donor DNA is integrated into the genome of a virus or viral vector having a double-stranded DNA genome. In this embodiment, the donor DNA is incorporated into the cells along with the viral genome by culturing the cells in the presence of the virus or viral vector before, simultaneously with, or after the introduction of a sequence-specific nucleic acid cleaving molecule into the cells. This is advantageous in that the donor DNA can be introduced into the cells by utilizing the infectivity of the virus.
  • Viruses with single-stranded DNA genomes include Parvoviridae viruses (e.g., parvovirus B19), Circoviridae viruses, and adeno-associated viruses. Viral vectors derived from these viruses also retain linear single-stranded DNA in their genomic DNA and are suitable for introducing linear single-stranded DNA into cells.
  • donor DNA is integrated into the genome of an adeno-associated virus (AAV) vector.
  • AAV vectors can infect cells and supply them with genomes containing donor DNA. In this way, the use of AAV vectors makes it easy to introduce donor DNA into cells from outside the cell.
  • AAV serotypes There are various AAV serotypes, and vectors derived from any serotype may be used as long as they are capable of infecting target cells.
  • AAV include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.
  • AAV can typically introduce DNA up to about 4.7 kb.
  • the length of the target sequence can be up to about 3.2 kb to about 4.1 kb.
  • the donor DNA portion in the AAV functions as donor DNA for homologous recombination.
  • Viruses that have linear double-stranded DNA include adenoviruses, adenovirus vectors, herpes viruses, herpes virus vectors, pox viruses, and pox virus vectors.
  • the donor DNA which is linear double-stranded DNA, can be introduced into cells.
  • Adenovirus vectors lack the E1A, E1B, and E3 genes, and the E1A and E1B genes necessary for virus production are supplied by packaging cells.
  • the adenovirus vector can be, for example, an adenovirus serotype 5 (Ad5)-derived vector (see, e.g., Suzuki M, Saito I et al., Gene Ther, 1-9, 2015; Miyake S, Saito I et al., PNAS, 93:1320-1324, 1996; and Bett AJ et al., JVirol, 67:5911-5921, 1993; WO2020/067004A).
  • Ad5 adenovirus serotype 5
  • Herpes virus vectors are characterized by their high gene carrying capacity and the development of vector delivery technologies to targeted cells and tissues (e.g., Proc Natl Acad Sci U S A 2015, 112: E1632-E1641; Nat Med 2022, 28: 780-788; Mol Ther 2013, 21: 61-569; J Virol 2010, 84: 12200-12209; Gene Ther 2016; 23: 479-488).
  • poxvirus vectors include first-generation herpesvirus vectors deleted for the ICP4 or ICP27 gene, second-generation herpesvirus vectors deleted for the ICP22 gene in addition to the ICP4 and ICP27 genes, and third-generation herpesvirus vectors deleted for all immediate-early (IE) genes.
  • Poxvirus vectors including vaccinia virus vectors, are suitable for transferring relatively large genes (e.g., up to about 30 kbp).
  • the vaccinia virus strain used is not limited, but examples include the Lister strain, New York City Board of Health (NYBH) strain, Wyeth strain, Copenhagen strain, Western Reserve (WR) strain, Modified Vaccinia Ankara (MVA) strain, EM63 strain, Ikeda strain, Dalian strain, and Tian Tan strain.
  • the Lister strain and MVA strain are available from the American Type Culture Collection (ATCC VR-1549 and ATCC VR-1508, respectively).
  • Vaccinia virus strains that have been established from the Lister strain, such as the LC16 strain, LC16m8 strain, and LC16mO strain, can also be used.
  • the LC16mO strain was developed by low-temperature passage using the Lister strain as a parent strain, followed by the LC16 strain.
  • the LC16m8 strain was developed by further low-temperature passage of the LC16mO strain. It is an attenuated strain due to a frameshift mutation in the B5R gene, which encodes a viral membrane protein, resulting in the loss of expression and function of this protein (Protein Nucleic Acid Enzyme, 2003, Vol. 48, pp. 1693-1700).
  • the complete genome sequences of the Lister, LC16m8, and LC16mO strains are known, for example, as Accession No. AY678276.1, Accession No. AY678275.1, and Accession No. AY678277.1, respectively.
  • Viral vectors with linear double-stranded DNA genomes can be useful in that they can deliver donor DNA, nucleic acid encoding a sequence-specific nucleic acid cleavage molecule, and nucleic acid encoding an ATM inhibitor to cells. Therefore, such vectors are suitable for in vitro and in vivo applications.
  • Viruses and viral vectors can be selected taking into consideration their cell infectivity. Pseudotyped viruses that transiently express envelope proteins or specific proteins from other viruses to confer cell infectivity can also be preferably used.
  • circular double-stranded DNA examples include plasmid DNA and viral genomic DNA.
  • Circular double-stranded DNA can be cleaved within a cell to form linear double-stranded DNA, which includes donor DNA.
  • the circular double-stranded DNA preferably has a cleavage site (target sequence) for a sequence-specific nucleic acid cleaving molecule, for example.
  • Target sequence for a sequence-specific nucleic acid cleaving molecule
  • Linear double-stranded DNA can be used for HDR by including donor DNA.
  • viruses include human papillomavirus, baculovirus, and polyomavirus.
  • Human papillomavirus, baculovirus, and polyomavirus have each been converted into vectors, and human papillomavirus vectors, baculovirus vectors, and polyomavirus vectors can also be used.
  • Viruses and viral vectors can be selected taking into consideration their tropism for infecting cells. Pseudotyped viruses that transiently express envelope proteins or specific proteins from other viruses to confer cell infectivity can also be preferably used.
  • the circular double-stranded DNA may further contain DNA encoding a sequence-specific nucleic acid cleaving molecule and DNA encoding a guide RNA.
  • the circular double-stranded DNA may further contain a nucleic acid that inhibits ATM expression or DNA encoding such a nucleic acid.
  • one or more circular double-stranded DNAs can be used to introduce the sequence-specific nucleic acid cleaving molecule, guide RNA, and donor DNA (and optionally the nucleic acid that inhibits ATM expression) into cells.
  • Viral vectors having a genome containing circular double-stranded DNA containing the sequence-specific nucleic acid cleaving molecule, guide RNA, and donor DNA, as well as DNA encoding the nucleic acid that inhibits ATM expression, or viral vectors having a genome containing linear double-stranded DNA containing guide RNA, donor DNA, and DNA encoding the nucleic acid that inhibits ATM expression can supply all of the ATM inhibitor, donor DNA, and sequence-specific nucleic acid cleaving molecule to cells. For this reason, such vectors can be preferably used in vitro and in vivo.
  • the cleavage site (target sequence) for the sequence-specific nucleic acid cleaving molecule is inserted in a location that does not significantly interfere with the expression.
  • ATM inhibitors inhibit the ATM protein encoded by ataxia telangiectasia mutated (ATM), the gene responsible for ataxia telangiectasia.
  • ATM inhibitors can be selective inhibitors of ATM.
  • Various ATM inhibitors have been developed, and any of them can be used.
  • ATM inhibitors disclosed in the following publications may be used: WO 2017/046216, WO 2015/170081, WO 2018/167203, WO 2017/153578, WO 2017/162611, WO 2017/162605, WO 2017/174446, WO 2017/076895, WO 2017/076898, WO 2017/194632, WO 2019/057757, WO 2021/260580 (the entire specifications of which are incorporated herein by reference).
  • the ATM inhibitor is used at a concentration suitable for ATM inhibition.
  • ATM inhibitors do not exhibit unacceptable cytotoxicity at concentrations suitable for ATM inhibition.
  • ATM inhibitors may also include, for example, compounds selected from: AZD1390: 7-fluoro-1-isopropyl-3-methyl-8-[6-[3-(1-piperidyl)propoxy]-3-pyridyl]imidazo[4,5-c]quinolin-2-one, and its deuterated form: 4,6-dideutero-7-fluoro-1-isopropyl-3-methyl-8-[6-[3-(1-piperidyl)propoxy]-3-pyridyl]imidazo[4,5 -c]quinolin-2-one, and 4-deutero-7-fluoro-1-isopropyl-3-methyl-8-[6-[3-(1-piperidyl)propoxy]-3-pyridyl]imidazo[4,5-c]quinolin-2-one; AZD0156: 8-[6-(3-dimethylaminopropoxy)pyridin-3-yl]-3-methyl-1-(oxan-4-y
  • ATM inhibitors include, but are not limited to, dactolisib, wortmannin, KU55933, KU59403, KU60019, Torin2, AZD0156, Mirin, AZD1390, CP466722, CGK733, AZ31, AZ32, cinobufagin, raltesertib, SKLB197, NVP-BEZ235, and M3541.
  • oral administration of AZD1390 has been shown to deliver AZD1390 to the brain (Sci Adv. 2018;4(6):eaat1719).
  • An ATM inhibitor can be a nucleic acid that suppresses ATM gene expression.
  • nucleic acids that suppress ATM gene expression include siRNA, shRNA, antisense oligos, gapmers, and mixers directed against the ATM gene.
  • siRNA two RNA strands, typically about 21 to 23 nucleotides in length, bind complementarily to form a double-stranded complex.
  • shRNA is a single-stranded RNA, typically consisting of two complementary hybridizing RNA strands, each about 21 to 23 nucleotides in length, linked by a hairpin.
  • Gapmers have a structure in which modified nucleic acids (wing regions) are linked to both sides of antisense DNA (gap region).
  • RNA When gapmers bind to target RNA, they induce cleavage in the target RNA through the action of RNase H.
  • RNase H RNA sequence, and modifications to design nucleic acids suitable for suppressing ATM gene expression.
  • RNA may also be produced intracellularly by introducing DNA encoding it into cells.
  • Nucleic acids may contain modified nucleic acids for stabilization or improved target binding.
  • Various modified nucleic acids have been developed, and those skilled in the art can use them as appropriate.
  • knockdown nucleic acids consist solely of modified nucleic acids.
  • modified nucleic acids include fluorescent dye-modified nucleic acids, biotinylated nucleic acids, and nucleic acids with cholesteryl groups introduced.
  • the sugar moiety of the base may be modified with 2'-O-methyl, 2'-fluoro, or 2'-methoxyethyl (MOE).
  • U.S. Pat. No. 9,469,664B discloses modified nucleic acids in which the phosphorus of the intersubunit (internucleotide bond) is replaced with a tertiary amine.
  • the phosphodiester bond of the internucleotide bond may also be replaced with a phosphorothioate bond.
  • the phosphodiester bond of the internucleotide bond may also be replaced with a phosphorodiamidate bond.
  • the phosphodiester bond of the internucleotide bond may also be replaced with a peptide bond (e.g., peptide nucleic acid).
  • modified nucleic acids with modified sugars include nucleic acids in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged.
  • LNA locked nucleic acid
  • ENA in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via an ethylene
  • BNA COC in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via -CH 2 OCH 2 -
  • BNA NC in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via -NR-CH 2 - (where R is a methyl or hydrogen atom);
  • cMOE in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via -CH 2 (OCH 3 ) -
  • BNA in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via -CH
  • the treatment time with the ATM inhibitor can be set appropriately.
  • the treatment time is not particularly limited, but sufficient effects can be obtained, for example, even for about 24 hours.
  • the treatment time can be, for example, 16 to 48 hours.
  • the treatment can be performed immediately after the cleavage treatment, and can begin before, during, or after cleavage.
  • a treatment to cleave the target site in the genomic DNA can be performed, and then donor DNA and an ATM inhibitor can be supplied to the cells.
  • cells can be cultured in a first medium in the presence of a sequence-specific nucleic acid cleaving molecule, then cultured in a second medium in the presence of donor DNA and an ATM inhibitor, and then cultured in a third medium.
  • the second medium may contain an ATM inhibitor
  • the third medium may not contain donor DNA or an ATM inhibitor.
  • Donor DNA not incorporated into a vector capable of infecting cells can be introduced into cells, for example, simultaneously with the introduction of the sequence-specific nucleic acid cleaving molecule into the cells.
  • the donor DNA incorporated into a vector capable of infecting cells can be added to the second culture medium after, for example, treating the genomic DNA with double-strand breaks, thereby introducing the DNA into the cells.
  • Target cells are not particularly limited as long as they have functional ATM, and include eukaryotic cells, including, but not limited to, mammalian cells.
  • Mammals include vertebrates, mammals (human or non-human mammals), primates (e.g., humans, chimpanzees, gorillas, orangutans, monkeys, marmosets, and bonobos), non-primate mammals such as mice, rats, pigs, cows, sheep, goats, llamas, camels, horses, cats, and dogs, fish, amphibians, reptiles, crustaceans, birds, insects, and plants.
  • the cells are human cells.
  • the cells are mouse cells.
  • the cells are pig cells.
  • the target cells are not particularly limited, but may be, for example, pluripotent cells (embryonic stem cells, induced pluripotent stem cells, epiblast stem cells, and other pluripotent stem cells), tissue stem cells, progenitor cells, somatic cells, germ cells, and fertilized eggs or egg cells.
  • pluripotent cells embryonic stem cells, induced pluripotent stem cells, epiblast stem cells, and other pluripotent stem cells
  • tissue stem cells tissue stem cells
  • progenitor cells somatic cells
  • germ cells germ cells
  • fertilized eggs or egg cells fertilized eggs or egg cells.
  • the target cells may be, for example, cells with a long cell cycle or non-dividing cells.
  • examples of such cells include cells other than epidermal cells and intestinal epithelial cells, and more specifically, include hematopoietic stem cells, hematopoietic progenitor cells (collectively referred to as "hematopoietic stem and progenitor cells"), nerve cells, and muscle cells (e.g., skeletal muscle cells and cardiac muscle cells).
  • the target cells are non-dividing cells.
  • a long cell cycle is not particularly limited, but may be, for example, a cell cycle of 48 hours or more, 72 hours or more, 144 hours or more, one week or more, or one month or more, or may be 72 hours or less, 144 hours or less, one week or less, one month or less, or several months or less (e.g., 5 to 7 months or less), for example, 48 hours to several months. Although it depends on the conditions, it has been reported that hematopoietic stem cells divide once every 36 to 145 days (see E.M. Pietras, et al., J. Cell Biol., 195(5): 709-720, 2011).
  • the disclosed method is suitable for use with cells with long cell cycles, such as those dividing once every one to five months, and in principle, is also considered effective for cells with even longer cell cycles (e.g., cell cycles of 5 months to one year, one to three years, or three to ten years, or longer) and non-dividing cells.
  • the target cells do not necessarily have to have long cell cycles; they may also have cell cycles of 48 hours or less, 42 hours or less, 36 hours or less, or 30 hours or less.
  • the cell cycle of embryonic pluripotent stem cells is typically about 24 hours ⁇ 4 hours, while the cell cycle of stromal cells is typically about 36 hours ⁇ 6 hours.
  • the cell cycle can vary depending on culture conditions and cell state, but the method of the present disclosure can be effective regardless of the length of the cell cycle, and can also be effective for cells with long cell cycles and low HDR efficiency.
  • the target cell may be a fertilized egg, for example, a mammalian fertilized egg. Genome editing in a fertilized egg may lead to the editing of all cells that make up the body, which may be beneficial.
  • the target cell may be a pluripotent cell, for example, a mammalian pluripotent cell. Genome editing of an animal is possible by selecting, proliferating, and introducing the edited cell into an embryo. If the mammal is livestock, this may offer benefits in animal husbandry. If the mammal is human, this may offer benefits in the treatment of disease.
  • the target cells When administered to humans, the target cells can be autologous or allogeneic (allogeneic). In the case of allogeneic transplantation, the cells are low immunogenic. For example, cells in which HLA class I and II or their production have been disrupted are low immunogenic to adaptive immunity. As a more specific example, cells in which ⁇ 2-microglobulin has been disrupted no longer express HLA class I and do not activate adaptive immunity. Furthermore, cells in which CIITA has been disrupted no longer express HLA class II and do not activate adaptive immunity. Examples of such modifications include cell surface expression of immune checkpoint molecules and cell surface expression of molecules that emit "don't eat me" signals, such as CD47.
  • low immunogenic cells Cells that have been modified in this way and are suitable for allogeneic transplantation are called low immunogenic cells, and cells that can be administered to a variety of others are called universal donor cells.
  • the cells are preferably low immunogenic cells, and more preferably universal donor cells.
  • the blood disease may be sickle cell disease, beta-thalassemia, X-linked severe combined immunodeficiency (X-SCID), chronic granulomatous disease (CGD), or other blood disease caused by a genetic mutation.
  • the method of the present disclosure can be used to correct the cellular abnormality by modifying the genetic mutation to a normal sequence.
  • the method of the present disclosure may be an ex vivo method in which cells from within the body that have a genetic mutation are removed from the body, isolated cells are obtained, and the method can be applied to the isolated cells.
  • the method of the present disclosure may be an in vivo method in which effective amounts of donor DNA, a sequence-specific nucleic acid cleaving molecule or a nucleic acid encoding the molecule, and an ATM inhibitor or, if the ATM inhibitor is a nucleic acid, a nucleic acid encoding the ATM inhibitor are administered to a subject, thereby modifying the genetic mutation to a normal sequence within the subject's body.
  • the target cells may be hematopoietic cells.
  • Hematopoietic cells are hematopoietic stem cells and cells resulting from the differentiation of hematopoietic stem cells. Hematopoietic cells are broadly classified into hematopoietic stem cells, hematopoietic progenitor cells, and blood cells, depending on the differentiation stage. Hematopoietic stem cells differentiate into blood cells via hematopoietic progenitor cells. More specifically, hematopoietic stem cells can differentiate into lymphocytes (e.g., T cells, B cells, NK cells) via lymphoblasts.
  • lymphocytes e.g., T cells, B cells, NK cells
  • Hematopoietic stem cells can also differentiate into monocytes via hematopoietic progenitor cells and monoblasts. Hematopoietic stem cells can also differentiate into hematopoietic progenitor cells, common myeloid progenitor cells (CMP), granulocytic/monocytic progenitor cells (GMP), and then into granulocytic leukocytes such as neutrophils, eosinophils, or basophils, or macrophages.
  • CMP common myeloid progenitor cells
  • GMP granulocytic/monocytic progenitor cells
  • Hematopoietic stem cells can also differentiate into red blood cells via hematopoietic progenitor cells, common myeloid progenitor cells (CMP), megakaryocyte/erythroid progenitor cells (MEP), and megakaryocytes. Hematopoietic stem cells can also differentiate into platelets via hematopoietic progenitor cells, common myeloid progenitor cells (CMP), megakaryocyte/erythroid progenitor cells (MEP), and megakaryocytes. All cells derived from these hematopoietic stem cells are blood cells. Human hematopoietic stem cells are CD34 positive and CD38 negative. Whether they are positive or negative can be determined by flow cytometry by one skilled in the art.
  • the target cells may be primary cells, established cell lines, or immortalized cells.
  • the target cells are preferably isolated cells.
  • the target cells may be primary cells that have not been established or immortalized, in consideration of the safety of transplantation into humans.
  • the cells may also be pluripotent stem cells or cells derived from pluripotent stem cells.
  • the break is repaired by homology-directed DNA repair (HDR) within the cells, using the donor DNA as a template.
  • HDR homology-directed DNA repair
  • NHEJ nuclear-derived DNA repair
  • the presence of an effective amount of an ATM inhibitor promotes this repair by homology-directed repair.
  • inducing HDR in cells in the presence of an ATM inhibitor increases the efficiency of HDR (i.e., increases the percentage of cells in which HDR is induced) compared to inducing HDR in cells under identical conditions except in the absence of the ATM inhibitor. Therefore, the method of the present disclosure includes culturing cells with a double-strand break at a target site in DNA in the presence of donor DNA and an ATM inhibitor.
  • the ATM inhibitor is as described above.
  • the culture medium may be serum-free, albumin-free, and/or cytokine-free. Such media include chemically defined media.
  • Culture media include basal media. Basal media include S-clone SF-3 medium, F12 medium, StemSpan or StemSpan SFEM (Stem Cell technologies), STEM ⁇ (STEM ALPHA), StemPro-34 serum-free medium (Gibco Invitrogen), StemPro MSC serum-free medium (Invitrogen), and HSC-CFU medium (Mi Iltenyl Biotech), S-Clone serum-free medium (SF-02, SF-03, CM-B, SF-B) (Sanko Junyaku), HPGM medium (Sanko Junyaku), AIM V medium (Invitrogen), Marrow MAX bone marrow medium (Invitrogen), KnockOut DMEM/F-12 medium (Invitrogen), Stemline hematopoietic stem cell growth medium (S
  • culture media include basal medium.
  • the culture medium may contain one or more or all of insulin, apo-transferrin, sodium selenite, and ethanolamine.
  • the culture medium may contain HEPES, sodium pyruvate, vitamins, amino acids, heparin, heparan sulfate, chondroitin sulfate, and the like.
  • the culture medium may contain antibiotics (e.g., penicillin and streptomycin).
  • the culture medium may also contain glutamine.
  • the culture medium may contain, for example, insulin, transferrin (apo), sodium selenite, ethanolamine, and antibiotics, and may further contain HEPES.
  • the present disclosure may further include obtaining, after culturing, cells having DNA in which the target sequence is inserted between the upstream and downstream of the target site, or DNA in which the sequence is deleted between the upstream and downstream of the target site.
  • Such cells can be obtained by nucleic acid amplification (such as polymerase chain reaction (PCR)) and sequencing of the target site.
  • PCR polymerase chain reaction
  • a person skilled in the art can appropriately introduce a double-strand break at a target site, induce homologous recombination repair by culturing cells having the double-strand break in the presence of donor DNA, and obtain cells having DNA in which the target sequence is inserted between the upstream and downstream of the target site or DNA in which the sequence is deleted between the upstream and downstream of the target site.
  • the obtained cells or cell population can be administered to a subject in need of such cells or cell population.
  • the administered cells or cell population may be cloned or uncloned; for example, a cell population that has been treated to induce HDR may be administered as is.
  • the genetic mutation that causes the disease can be corrected to a normal sequence using the method of the present disclosure, or cells whose sequence has been corrected to alleviate the abnormality, the disease can be treated in the subject.
  • performing the methods of the present disclosure in a subject may induce genome modification through homologous recombination or treat a disease in the subject.
  • the present disclosure provides an in vivo method comprising administering to a subject effective amounts of a sequence-specific nucleic acid cleaving molecule (or a sequence-specific nucleic acid cleaving molecule complex) or a nucleic acid encoding the same, template DNA (e.g., single-stranded DNA or double-stranded DNA), and an ATM inhibitor (or, if the ATM inhibitor is a nucleic acid, a nucleic acid encoding the ATM inhibitor), thereby inducing homologous recombination repair in the subject and inducing homologous recombination between the target region of the subject and the template DNA.
  • template DNA e.g., single-stranded DNA or double-stranded DNA
  • ATM inhibitor or, if the ATM inhibitor is a nucleic acid, a nucleic acid encoding the ATM inhibitor
  • the template DNA is preferably linear.
  • the in vivo method of the present disclosure may include administering to a subject having a genetic mutation (e.g., a disease-causing one) effective amounts of a sequence-specific nucleic acid cleaving molecule (or a sequence-specific nucleic acid cleaving molecule complex) or a nucleic acid encoding the same, template DNA (e.g., single-stranded DNA or double-stranded DNA), and an ATM inhibitor (if the ATM inhibitor is a nucleic acid, a nucleic acid encoding the ATM inhibitor) to induce homologous recombination repair in the subject, thereby repairing the genetic mutation to a sequence on the template DNA (particularly a sequence encoding a functional protein or a portion thereof).
  • the subject has a genetic mutation and a disease caused by the genetic mutation, and the method of the present disclosure may repair the genetic mutation and treat or prevent the disease.
  • a sequence-specific nucleic acid cleaving molecule (or sequence-specific nucleic acid cleaving molecule complex) or a nucleic acid encoding the same, template DNA (e.g., single-stranded DNA or double-stranded DNA), and an ATM inhibitor (if the ATM inhibitor is a nucleic acid, a nucleic acid encoding the ATM inhibitor) can be administered by being encapsulated in a carrier such as, but not limited to, a micelle, liposome, lipid nanoparticle, or polyion complex.
  • a carrier such as, but not limited to, a micelle, liposome, lipid nanoparticle, or polyion complex.
  • the present disclosure provides a carrier (e.g., lipid nanoparticle) containing one or more or all selected from the group consisting of a sequence-specific nucleic acid cleaving molecule (or sequence-specific nucleic acid cleaving molecule complex) or a nucleic acid encoding the same, template DNA (e.g., single-stranded DNA or double-stranded DNA), and an ATM inhibitor (if the ATM inhibitor is a nucleic acid, a nucleic acid encoding the ATM inhibitor), a composition or pharmaceutical composition containing the carrier, and these compositions for use in editing applications.
  • a carrier e.g., lipid nanoparticle
  • the sequence-specific nucleic acid cleaving molecule or sequence-specific nucleic acid cleaving molecule complex
  • the nucleic acid encoding it and the ATM inhibitor can be administered by being encapsulated in a carrier such as, but not limited to, a micelle, liposome, lipid nanoparticle, or polyion complex
  • the template DNA can be administered by being carried on a vector such as a virus.
  • the carrier and vector such as a virus may be administered simultaneously or sequentially.
  • the ATM inhibitor does not necessarily need to be encapsulated in a carrier; the carrier may contain the sequence-specific nucleic acid cleaving molecule (or sequence-specific nucleic acid cleaving molecule complex) or the nucleic acid encoding it, and may be used in combination with the ATM inhibitor and template DNA.
  • the order of administration is not particularly limited, and they may be administered simultaneously or sequentially.
  • the template DNA and the sequence-specific nucleic acid cleaving molecule (or sequence-specific nucleic acid cleaving molecule complex) or the nucleic acid encoding it may be administered after the ATM inhibitor is administered.
  • lipid nanoparticles are not particularly limited, but examples of lipid nanoparticles that can be used include those described in US Pat. Nos. 9,364,435B, 8,822,668B, 8,802,644B, and 8,058,069B2.
  • mRNA may be encapsulated in a polyion complex micelle or a polyion complex polymersome (Miyata et al., Chem. Soc. Rev., 2012, 41, 2562-2574).
  • Lipid nanoparticles can deliver encapsulated nucleic acids to, for example, B cells (e.g., Loomis et al., Exp. Mol. Pahol., 88(2):238-249, 2010).
  • the lipid nanoparticles are not particularly limited, but include, for example: ionizable lipids (e.g., DLin-MC3-MDA, ALC-0315, and SM-102); PEG lipids (e.g., DMG-PEG (2000), and ALC-0159);
  • ionizable lipids e.g., DLin-MC3-MDA, ALC-0315, and SM-102
  • PEG lipids e.g., DMG-PEG (2000), and ALC-0159
  • the present invention relates to a method for treating a fibrous ...
  • 1,2-DSPC represents 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DMG-PEG(2000) represents alpha-(3'- ⁇ [1,2-di(myristyloxy)propanoxy]carbonylamino ⁇ propyl)- ⁇ -methoxy
  • polyoxyethylene and
  • ALC-0159 represents 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide. Ionized lipids are neutral at physiological pH but cationic in acidic environments.
  • the lipid nanoparticles preferably comprise: ionizable lipids (e.g., DLin-MC3-MDA, ALC-0315, and SM-102); PEG lipids (e.g., DMG-PEG (2000), and ALC-0159);
  • the lipid nanoparticles comprise a phospholipid (e.g., 1,2-DSPC) and cholesterol, and the molar ratio of the lipids (ionized lipid/PEG lipid/phospholipid/cholesterol) may be 45-50/5-15 (preferably 8-10)/35-45 (preferably 37-42)/1-2 (provided that the total is 100 or less).
  • lipid nanoparticles see, for example, Int. J. Pharm., 601:120586, 2021.
  • patisiran contains siRNA-containing lipid nanovesicles as an active ingredient, and is composed of Lin-MC3-DMA/1,2-DSPC/DMG-PEG(2000)/cholesterol in a molar ratio of 50/10/38.5/1.5. Lipid nanoparticles containing lipids in this molar ratio may also be used in the present disclosure.
  • lipid nanoparticles comprising Lin-MC3-DMA, 1,2-DSPC, DMG-PEG(2000), and cholesterol are provided, which comprise a knockdown nucleic acid of the present disclosure, and the lipid nanoparticles preferably contain Lin-MC3-DMA/1,2-DSPC/DMG-PEG(2000)/cholesterol in a molar ratio of 45-50/5-15 (preferably 8-10)/35-45 (preferably 37-42)/1-2 (provided that the total is 100 or less).
  • lipid nanoparticles comprising Lin-MC3-DMA, 1,2-DSPC, DMG-PEG(2000), and cholesterol are provided, which comprise a knockdown nucleic acid of the present disclosure, and the lipid nanoparticles contain Lin-MC3-DMA/1,2-DSPC/DMG-PEG(2000)/cholesterol, preferably in a molar ratio of 50/10/38.5/1.5.
  • Lipid nanoparticles can be obtained, for example, by preparing an alcohol solution containing a lipid mixture and an aqueous solution containing nucleic acid, and then mixing the two solutions using a microfluidic device or the like.
  • Lipid nanoparticles can include, for example, micelles (e.g., reverse micelles) in which nucleic acid is encapsulated by ionized lipids and cholesterol inside an outer shell formed from PEG lipids and cholesterol, but are not limited to these.
  • the present disclosure provides an ATM inhibitor or a composition comprising an ATM inhibitor for use in the methods of the present disclosure.
  • the present disclosure also provides the use of an ATM inhibitor in the manufacture of a composition for use in the methods of the present disclosure.
  • kits for use in the methods of the present disclosure
  • kits comprising: (A) (i) a sequence-specific nucleic acid cleaving molecule, or a sequence-specific nucleic acid cleaving molecule complex, or a nucleic acid encoding the same; (ii) template DNA; (iii) one or more members selected from the group consisting of ATM inhibitors; (B) (i) and (ii) above, (C) (ii) and (iii) above, (D) (i) and (iii) above, or (E) (i), (ii), and (iii)
  • the kit may include an ATM inhibitor.
  • the kit may include a viral vector having a single-stranded DNA genome (preferably an AAV vector), a plasmid for constructing the viral vector containing the genome of a viral vector having a linear double-stranded DNA genome (e.g., an adenovirus vector, a herpesvirus vector, or a poxvirus vector), a plasmid for constructing the viral vector containing the genome of a viral vector having a circular double-stranded genome containing double-stranded donor DNA (e.g., a human papillomavirus vector, a polyomavirus vector, or a baculovirus vector), and/or a helper plasmid.
  • a viral vector having a single-stranded DNA genome preferably an AAV vector
  • a plasmid for constructing the viral vector containing the genome of a viral vector having a linear double-stranded DNA genome e.g., an adenovirus vector, a herpesvirus vector,
  • the viral vector genome may have, for example, two inverted repeat sequences (ITRs), preferably a cloning site (preferably a multiple cloning site) between them, allowing donor DNA to be integrated. Therefore, the resulting viral vector contains the sequence of the donor DNA between the two ITRs.
  • ITRs inverted repeat sequences
  • a helper plasmid is used when producing the viral vector. Therefore, the kit may further include such a helper plasmid.
  • the helper plasmid may contain, for example, a gene encoding a capsid (e.g., VP1, VP2, and VP3) and a gene region responsible for the adenovirus helper function (e.g., E1A, E1B, E2A, VA, and E4orf6).
  • a capsid e.g., VP1, VP2, and VP3
  • a gene region responsible for the adenovirus helper function e.g., E1A, E1B, E2A, VA, and E4orf6
  • E1A, E1B, E2A, VA, and E4orf6 e.g., E1A, E1B, E2A, VA, and E4orf6
  • the kit of the present disclosure may contain a helper plasmid in addition to a plasmid for constructing a viral vector.
  • the kit of the present disclosure may further contain one or more ATM inhibitors.
  • the present disclosure provides the use of a plasmid for constructing a viral vector, a helper plasmid, and/or an ATM inhibitor in the manufacture of the kit.
  • the present disclosure provides a composition comprising one or more of a sequence-specific nucleic acid cleaving molecule (or a sequence-specific nucleic acid cleaving molecule complex) or a nucleic acid encoding the same, template DNA (e.g., single-stranded DNA or double-stranded DNA), and an ATM inhibitor (a nucleic acid encoding the ATM inhibitor if the ATM inhibitor is a nucleic acid), for use in such a method.
  • template DNA e.g., single-stranded DNA or double-stranded DNA
  • an ATM inhibitor a nucleic acid encoding the ATM inhibitor if the ATM inhibitor is a nucleic acid
  • the present disclosure provides an ATM inhibitor or a composition comprising the ATM inhibitor for use in the above-described in vivo method.
  • the ATM inhibitor is RNA
  • the ATM inhibitor may be incorporated into a viral vector genome as DNA encoding the RNA operably linked to a promoter.
  • the kit may not separately include an ATM inhibitor, or may further include an ATM inhibitor.
  • compositions containing modified cells obtained by the methods of the present disclosure The present disclosure provides modified cells obtainable by the methods of the present disclosure, and compositions comprising the modified cells.
  • the modified cells and compositions obtainable by the methods of the present disclosure may comprise an ATM inhibitor.
  • the present disclosure provides a viral vector (preferably an AAV vector) comprising linear single-stranded donor DNA or a composition comprising the viral vector for use in the method of the present disclosure.
  • a viral vector e.g., an adenovirus vector, a herpesvirus vector, and a poxvirus vector such as a vaccinia virus vector
  • linear double-stranded donor DNA for use in the method of the present disclosure.
  • the present disclosure also provides a circular double-stranded viral vector (e.g., a human papillomavirus vector, a polyomavirus vector, and a baculovirus vector) comprising double-stranded donor DNA for use in the method of the present disclosure.
  • a viral vector preferably an AAV vector
  • AAV vectors are described above.
  • the ATM inhibitor is RNA
  • the ATM inhibitor may be incorporated into the viral vector genome as DNA encoding the RNA operably linked to a promoter.
  • Example 1 Effect of ATM inhibitors on genome editing efficiency by homology-directed repair (HDR)
  • HDR homology-directed repair
  • the present inventors screened 174 small molecule compounds using a reporter ES cell line that can distinguish between double-strand breaks (DSBs), non-homologous end joining (NHEJ), and homology-directed repair (HDR) as editing results in genome editing in the presence of donor nucleic acid.
  • An adeno-associated virus (AAV) vector was used as the donor nucleic acid, and the CRISPR/Cas9 system was used for genome editing.
  • AAV adeno-associated virus
  • ES cells were cultured on 0.1% (w/v) gelatin-coated tissue culture plates in 2i medium: Glasgow minimum essential medium (Sigma-Aldrich), 10% fetal bovine serum (FBS) (Moregate), 1000 U/mL leukemia inhibitory factor (Fujifilm), 3 ⁇ M CHIR99021 (StemCell Technologies), 1 ⁇ M PD032591 (StemCell Technologies), 0.1 mM ⁇ -mercaptoethanol (Thermo Fisher Scientific), 1 ⁇ GlutaMAX (Thermo Fisher Scientific), 1 mM sodium pyruvate (Thermo Fisher Scientific), 1 ⁇ non-essential amino acids (Thermo Fisher Scientific), and penicillin-streptomycin (Thermo Fisher Scientific).
  • 2i medium Glasgow minimum essential medium (Sigma-Aldrich), 10% fetal bovine serum (FBS) (Moregate), 1000 U/mL leukemia inhibitory factor (Fujifilm), 3 ⁇ M CHIR99021 (S
  • stromal cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% FBS, 1x GlutaMAX, and penicillin-streptomycin. Cells were cultured at 37°C in 5% CO2 and passaged every two days.
  • DMEM Dulbecco's modified Eagle's medium
  • tracrRNA IDTT
  • target-specific crRNA IDT
  • tracrRNA IDCT
  • target-specific crRNA IDT
  • AAV6 was produced using the AAVpro Helper-Free System (Takara Bio) according to the minimal purification method (17).
  • a mixture of pAAV-Reporter-donor or pAAV-Actb-donor was added to AAV6.
  • EGFP, pHelper, and pRC6 encoding the AAV2 rep and AAV6 capsid genes were prepared at a molecular ratio of 1:2:2.
  • a mixture of circular plasmid DNA and PEI Max (Polysciences) was transfected into HEK293T cells at a mass ratio of 1:6. After 24 hours, the medium was replaced with FBS-free DMEM, and the cells were cultured for 5 days. The medium was collected and filtered through a 0.22 ⁇ m filter (Merck Millipore). The AAV in the filtrate was concentrated using a Vivaspin 20 column (Sigma-Aldrich) with a 100k molecular weight cutoff at 2600 rpm for 3 hours. To measure the AAV concentration, DNA was extracted using the phenol-chloroform-isoamyl alcohol method. AAV vector genome (vg) was quantified using qPCR.
  • pActb-TagBFP The sequence of TagBFP was obtained from Integrated DNA Technologies (IDT) as gBlock. It was then amplified by PCR using a primer set that added a linker sequence immediately before TagBFP (see Table 1). The 5' and 3' arms were amplified by PCR from mouse genomic DNA. These two PCR fragments were cloned into linearized pUC19 (Takara Bio) using the NEBuilder cloning kit (NEB).
  • pActb-lacZ-mCherry To extend the length of intron 3 of Actb, the sequence shown in Supplementary Table S2 was ordered as a gBlock and amplified by PCR.
  • Oligonucleotides containing the splice acceptor and linker sequences and the gRNA recognition sequence for TagBFP were synthesized, and the sense and antisense strands were annealed. LacZ and mCherry were amplified by PCR. The 5' and 3' arms were obtained by PCR amplification of mouse genomic DNA. These five fragments were cloned into a linearized pUC19 backbone amplified using the primer set (see Table 1) and the NEBuilder cloning kit.
  • pReporter-Donor Oligonucleotides containing linker and 2A sequences were synthesized and annealed for both the sense and antisense strands.
  • pReporter-donor-HITI The sense and antisense strands of the homology-independent target insertion (HITI) sequences designed at both the 5' and 3' ends were synthesized, annealed, and then inserted into the pReporter-donor vector using the NEBuilder cloning kit.
  • the 5' end was inserted between the HindIII and NotI sites located upstream of the 5' STOP sequence of the pReporter-donor vector using the NEBuilder cloning kit.
  • the 3' end HITI fragment was inserted between the AflII and SalI sites located downstream of the 3' STOP sequence.
  • pAAV-Reporter-donor The plasmid was digested with AflII and HindIII to excise the pReporter-donor construct, which was then cloned into a backbone derived from pAAV-CMV-EGFP (Takara Bio).
  • EGFP amplified with the primer set (Table 1) was cloned into the SphI-XhoI digested backbone from pActb-TagBFP using the NEBuilder cloning kit.
  • pAAV-Actb-EGFP The plasmid was digested with HindIII and EcoRI to excise the pActb-EGFP construct. The excised fragment was cloned into a backbone derived from pAAV-CMV-EGFP (Takara Bio). This vector contained a gene encoding EGFP operably linked to a ⁇ -actin promoter.
  • the gene contained an upstream homology arm (5' arm) capable of homologous recombination with the target site upstream, a downstream homology arm (3' arm) capable of homologous recombination with the 5' side of the EGFP-encoding gene and its downstream LacZ gene, and a nonsense mutation (STOP sequence).
  • This vector was used as donor DNA for genome editing.
  • Cas9/gRNA expression vector To introduce the gRNA sequence into the Cas9 expression vector, pX330-U6-Chimeric_BB-CBh-hSpCas9 (a gift from Feng Zhang, Addgene #42230) was digested with BbsI (16). Annealed oligonucleotides (listed in Table 1) were then inserted into the digested vector using DNA ligase (Toyobo).
  • Mouse ES cells (1 ⁇ 10 cells) were suspended in 100 ⁇ L of Opti-MEM (Gibco) and either 3 ⁇ g of pX330-Actb-Exon6 and 10 ⁇ g of pActb-TagBFP or 1.5 ⁇ g of pX330-Actb-Intron3, 1.5 ⁇ g of pX330-Actb-Exon6, and 10 ⁇ g of pActb-lacZ-mCherry were added.
  • Each mixture was transferred to a 2 mm electroporation cuvette and electroporated using NEPA21 (Nepagene). The electroporation settings were five 2-ms polling pulses at 145 V, followed by five 50-ms transfer pulses at 20 V.
  • TagBFP-positive or mCherry-positive cells were single-cell sorted and cloned using an SH800 cell sorter (Sony Biotechnology). Heterozygous insertion clones were selected by PCR (see Table 2). The resulting reporter ES cells contained TagBFP in one allele and mCherry, a linker, lacZ, and mCherry in the other allele. The DSB detection ability of the reporter system (Actb-TagBFP) was verified using the Surveyor Mutation Detection Kit (IDT) and deep sequencing (see Table 2). Before cleavage, these reporter ES cells emitted blue and red fluorescence from BFP and mCherry.
  • IDTT Surveyor Mutation Detection Kit
  • indels When indels were generated and ligated in-frame, they emitted red fluorescence from mCherry. When the donor was integrated into the target by HDR, they emitted yellow fluorescence from mCherry and EGFP. When the donor was integrated into the target by NHEJ, they emitted green fluorescence from EGFP. Therefore, the difference in fluorescence color allows us to distinguish between pre-editing and post-editing edits.
  • pX330-TagBFP and 10 ⁇ g of pReporter-donor were electroporated with or without the HITI sequence under the same conditions as those used for reporter cell establishment. Cells were plated on gelatin-coated dishes and cultured for 4 days.
  • AAV AAV as the targeting vector
  • 1 ⁇ 10 6 reporter ES cells were suspended in 100 ⁇ L of Opti-MEM containing Cas9/RNP complex (final: 0.744 ⁇ M), electroporated, and plated on gelatin-coated dishes.
  • stromal cells were suspended in 100 ⁇ L of Opti-MEM containing 3 ⁇ g of pX330-Actb-Exon6 and 10 ⁇ g of pActb-EGFP. The mixture was transferred to a 2 mm cuvette for a NEPA21 electroporator. Electroporation settings included five 2-ms polling pulses at 125 V, followed by five 50-ms transfer pulses at 20 V. Electroporated cells were cultured in DMEM containing 20% FBS for 4 days.
  • stromal cell lines were suspended in 100 ⁇ L of Opti-MEM containing Cas9/RNP complex (final: 0.744 ⁇ M) and electroporated under the same conditions as for circular plasmid-mediated targeting.
  • the cells were then mixed with 1x106 vg/cell of AAV-Actb-EGFP and cultured in serum-free DMEM for 24 hours, followed by culture in DMEM containing 20% FBS for 3 days.
  • the efficiency of genome editing was assessed using flow cytometry (SH800) and DAPI staining to exclude dead cells.
  • electroporated reporter ES cells were treated with compounds from a DNA damage/DNA repair compound library (Selleck Chemicals) to assess the effect of reagents on genome editing efficiency. This analysis was performed using an LSRFortessa X-20 flow cytometer (BD Bioscience) equipped with a sample loader.
  • KU55933 is an ATM inhibitor.
  • ATM is known to be involved in the detection of double-strand breaks.
  • ATM inhibitors other than KU55933, such as KU60019, AZD1390, M4076, and AZ32, on the efficiency of HDR using the CRISPR/Cas9 system.
  • donor DNA for recombination was provided by an AAV vector (AAV6-Actb-EGFP-1kb arm).
  • FIGS 1A and 1B show that both ATM inhibitors improved the efficiency of genome editing-mediated HDR in whole stromal cells (second row from the top in Figures 1A and 1B) and hematopoietic stem/progenitor cells (KSL cells).
  • ATM inhibitors enhance HDR efficiency using donor DNA (single-stranded DNA) delivered by AAV vectors in stromal cells and hematopoietic stem/progenitor cells.
  • KU55933 and AZ32 also showed a tendency to enhance HDR efficiency using donor DNA delivered by AAV vectors.
  • ATM inhibitors reduced HDR efficiency using donor DNA delivered by circular plasmid DNA.
  • Double-stranded DNA excised from pReporter-donor with a restriction enzyme was used as the dsODN.
  • the lsODN was prepared by converting the dsODN described above to single-stranded DNA using a denaturing buffer. 1 x 10 6 reporter ES cells described above were seeded onto a gelatin-coated 10 cm culture dish and cultured in 2i medium with the above composition. After 24 hours, the medium was replaced with 10 mL of fresh medium containing 10 ⁇ M KU55933 as an ATM inhibitor.
  • Recombinant AAV6 was purchased from VectorBuilder after purification by cesium salt density gradient method.
  • scAAV6 was produced using the AAVpro Helper Free System (Takara Bio) with minimal purification.
  • a plasmid mixture was prepared by mixing pscAAV-Reporter-donor, pHelper, and pRC6 encoding the AAV2 rep gene and the AAV6 capsid gene at a mass ratio of 1:2:2.
  • HEK293T cells were transfected with a mixture of plasmid DNA and PEI Max (Polysciences) at a mass ratio of 1:6. 24 hours after transfection, the medium was replaced with FBS-free DMEM, and the cells were cultured for 5 days.
  • the culture supernatant was collected and filtered through a 0.22 ⁇ m filter (Merck Millipore).
  • the AAV in the filtrate was concentrated by centrifugation at 2,600 rpm for 3 hours using a Vivaspin 20 column (Sigma-Aldrich) with a molecular weight cutoff of 100 kJ.
  • the resulting AAV was washed twice in PBS using Vivaspin 20.
  • DNA was extracted using the phenol-chloroform-isoamyl alcohol method. Quantification of the AAV vector genome (vg) was performed using qPCR to examine self-complementary AAV (scAAV), a double-stranded AAV. scAAV responded similarly to AAV (see Figure 2C, upper right panel), suggesting that strand type is not a determining factor.
  • dsDNA linear double-stranded DNA
  • pReporter-donor linear double-stranded DNA
  • ssDNA was obtained by digesting the plsODN-Reporter-donor plasmid with Nb.BbvCI and EcoRI (NEB). The digested DNA was separated by 1.2% agarose gel electrophoresis. DNA bands were visualized using crystal violet staining. The target band was excised and purified using the Long ssDNA Preparation Kit (BDL). Similar experiments were performed with the obtained ssDNA. As shown in the upper left panel of Figure 2C, ATMi significantly improved the knock-in efficiency.
  • Example 2 Genome editing of hematopoietic stem/progenitor cells
  • hematopoietic stem/progenitor cells were genome-edited using the above-mentioned method, and then administered to animals to observe engraftment.
  • the cells used were bone marrow cells derived from the tibia and femur of B6 mice.
  • Red blood cells were lysed, and bone marrow hematopoietic stem and progenitor cells were enriched using CD117-beads with the Direct Lineage Depletion Kit (DepleteS) and AutoMACS (PosselS).
  • DepleteS Direct Lineage Depletion Kit
  • AutoMACS Pieris Biodepletion Kit
  • 1 ⁇ 10 bone marrow cells were seeded in a single well of a fibronectin-coated 24-well plate in HemEx-Type 9A medium (10 ng/ml SCF, 100 ng/ml TPO). On day 2, 90% of the medium was replaced. This medium replacement was repeated every two days, and bone marrow cells were cultured for a total of 7 days.
  • the gRNA and targeting vector used in the mouse stromal cell experiments were introduced into the cells by electroporation. Electroporation: 2-4 x 10 cells were placed in 20 ⁇ l of nucleofection buffer (16.4 ⁇ l of P3 nucleofection solution + 3.6 ⁇ l of nucleofection supplement) and 2.5 ⁇ g/cuvette of Cas9/RNP was added. Cells were then transferred to a 16-well cuvette and electroporated using the CM137 program in a 4D Lonza nucleofector.
  • the cells were then seeded: after nucleofection, 80 ⁇ l of medium was added to the wells of the cuvette, and then all the cells were transferred to a 1.5 ml tube.
  • ATM inhibitor treatment 5 ⁇ 10 4 cells were diluted in 200 ⁇ l of culture medium (containing 10 nM AZD1390) and seeded into one well of a fibronectin-coated 96-well plate.
  • AAV transduction 1x104 vg/cell AAV (AAV6-Actb-EGFP-1kb arm) was added to the well ( 5x108 vg AAV for 5x104 cells). 24 hours after genome editing, 90% of the medium was replaced with fresh medium. In vitro evaluation was performed 3 days after genome editing by calculating HDR efficiency in the CD150+ CD201+ KSL fraction using flow cytometry.
  • Figures 3 and 4 compare the engraftment rates of genome-edited cells without ATM inhibitor treatment (GE) and genome-edited cells with ATM inhibitor treatment (GE+ATMi).
  • Figure 3 shows that GE+ATMi had a significantly higher cell percentage than GE in peripheral blood 8 weeks after transplantation.
  • GE+ATMi also had a significantly higher percentage of CD45.1-positive cells (i.e., transplanted cells) than GE.
  • GE+ATMi also had a significantly higher percentage of bone marrow cells, T cells, B cells, and NK cells than GE.
  • Figure 3 shows that ATM inhibitor treatment increased the percentage of cells with HDR-mediated knock-in in peripheral blood.
  • Figure 4 also shows that ATM inhibitor treatment increased the percentage of HDR-mediated knock-in cells in bone marrow KSL and HSC cells 12 weeks after transplantation.
  • Example 3 Genome editing of fertilized eggs
  • an ATM inhibitor on the induction of homologous recombination in fertilized eggs was examined.
  • IVM Egg retrieval, in vitro maturation (IVM), and in vitro fertilization (IVF) Oocyte collection, IVM, and IVF were performed as described in Reproduction in Domestic Animals 52:969-75, 2017. Swine ovaries were collected from prepubertal female pigs at a local slaughterhouse. Cumulus cell-oocyte complexes were collected and cultured in maturation medium for 44 hours.
  • Matured oocytes were cultured with frozen-thawed ejaculated sperm (Fuji Micra, Shizuoka, Japan) (2 ⁇ 10 /mL) from microminipigs in porcine fertilization medium (Functional Peptide Institute, Yamagata, Japan) for 5 hours, followed by 7 hours in porcine zygote medium (PZM-5; Functional Peptide Institute). Oocytes were cultured in a humidified incubator at 39°C and 5% CO .
  • Electroporation and in vitro culture Electroporation was performed as described in Sci Adv 2:e1600803, 2016. Fertilized zygotes were placed in the electrode gap of a chamber slide (LF501PT1-20; BEX, Tokyo, Japan) filled with Nuclease-Free Duplex Buffer (IDT) containing 100 ng/ ⁇ L gRNA targeting porcine Rosa26 and 100 ng/ ⁇ L Cas9 protein (Guide-it Recombinant Cas9; Takara Bio Inc., Shiga, Japan). The zygotes were then electroporated (five 1-ms pulses at 25 V) using a CUY21EDIT II electroporator (BEX).
  • IDT Nuclease-Free Duplex Buffer
  • the zygotes were then electroporated (five 1-ms pulses at 25 V) using a CUY21EDIT II electroporator (BEX).
  • the zygotes were cultured in PZM-5 containing 3 ⁇ 10 9 vg/mL of AAV6 vector (AAV6-Rosa26-KuO) in the presence or absence of various concentrations of KU55933 for 24 hours, and then cultured in PZM-5 for 6 days.
  • Zygotes and embryos were cultured in a humidified incubator at 39°C under 5% CO 2 , 5% O 2 , and 90% N 2 .
  • the guide RNA sequences were as follows: pROSA26 gRNA91: gtgagagttatctgaccgtaagg
  • the primers for knock-in detection were as follows: pROSA26_KI_EGFP_F1: ACGAGCTGTACAAGTAAGCG R2: ggcatgtgtggaaaattgtg
  • Example 4 Editing of Fertilized Eggs and Development to Blastocysts.
  • the relationship between AAV concentration and knock-in efficiency using the ATMi AZ32 was investigated. Specifically, pig fertilized eggs were infected with AAV at various concentrations using the same method as in Example 3 (except that AZ32 was used as the ATMi and mEGFP was knocked into the 3' end of the ⁇ -actin gene), knock-in was induced, and the embryos were cultured to the blastocyst stage. The knock-in efficiency was estimated based on the mEGFP positivity rate. As shown in Figure 6, ATMi significantly increased knock-in efficiency using AAV as template DNA over a wide range. Furthermore, 1 ⁇ M AZ32 did not have any significant adverse effects on blastocyst development.

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Abstract

La présente invention concerne un procédé d'amélioration de l'efficacité de recombinaison homologue dans l'édition génomique in vitro et in vivo en présence d'ADN donneur (ADN matrice recombinant). La présente invention concerne un procédé de culture de cellules ayant une cassure double brin dans une séquence cible en présence d'ADN donneur et d'un inhibiteur d'ATM.
PCT/JP2025/023203 2024-06-28 2025-06-27 Procédé d'amélioration de l'efficacité de recombinaison homologue dans l'édition génomique in vitro et in vivo Pending WO2026005012A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120244131A1 (en) * 2011-02-04 2012-09-27 Cellectis Sa Method for modulating the efficiency of double-strand break-induced mutagenesis
US20140304847A1 (en) * 2011-06-07 2014-10-09 Ralf Kühn Recombination efficiency by inhibition of nhej dna repair
JP2016509063A (ja) * 2013-02-25 2016-03-24 サンガモ バイオサイエンシーズ， インコーポレイテッド ヌクレアーゼ媒介性遺伝子破壊を増強するための方法および組成物
JP2017535271A (ja) * 2014-11-21 2017-11-30 リジェネロン・ファーマシューティカルズ・インコーポレイテッドＲｅｇｅｎｅｒｏｎ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． ガイドｒｎａのペアを使用したターゲティングによる遺伝子改変の方法及び組成物
JP2020516258A (ja) * 2017-04-10 2020-06-11 マックス−プランク ゲゼルシャフト ツァー フォルデルング デア ビッセンシャフテン エー．ファウ． ゲノム編集効率を増大させるための化合物
JP2021511038A (ja) * 2018-01-17 2021-05-06 バーテックス ファーマシューティカルズ インコーポレイテッドＶｅｒｔｅｘ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ Ｉｎｃｏｒｐｏｒａｔｅｄ Ｄｎａ−ｐｋ阻害剤

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120244131A1 (en) * 2011-02-04 2012-09-27 Cellectis Sa Method for modulating the efficiency of double-strand break-induced mutagenesis
US20140304847A1 (en) * 2011-06-07 2014-10-09 Ralf Kühn Recombination efficiency by inhibition of nhej dna repair
JP2016509063A (ja) * 2013-02-25 2016-03-24 サンガモ バイオサイエンシーズ， インコーポレイテッド ヌクレアーゼ媒介性遺伝子破壊を増強するための方法および組成物
JP2017535271A (ja) * 2014-11-21 2017-11-30 リジェネロン・ファーマシューティカルズ・インコーポレイテッドＲｅｇｅｎｅｒｏｎ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． ガイドｒｎａのペアを使用したターゲティングによる遺伝子改変の方法及び組成物
JP2020516258A (ja) * 2017-04-10 2020-06-11 マックス−プランク ゲゼルシャフト ツァー フォルデルング デア ビッセンシャフテン エー．ファウ． ゲノム編集効率を増大させるための化合物
JP2021511038A (ja) * 2018-01-17 2021-05-06 バーテックス ファーマシューティカルズ インコーポレイテッドＶｅｒｔｅｘ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ Ｉｎｃｏｒｐｏｒａｔｅｄ Ｄｎａ−ｐｋ阻害剤

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