Compositions and methods for treating hematopoietic malignancies

Abstract

[0003] Embodiments of the present disclosure provide methods and compositions for treating hematopoietic malignancies (e.g., acute myeloid leukemia). In some embodiments, the present disclosure provides methods of treatment using a population of genetically engineered CD33-deficient hematopoietic cells and a cytotoxic agent comprising an anti-CD33 antigen-binding domain.

Description

[Background technology]

[0001] Related applications This application claims the benefits under 35 U.S. SC 119(e) of U.S. Provisional Application No. 63 / 106,136 filed on 27 October 2020 and U.S. Provisional Application No. 63 / 165,950 filed on 25 March 2021, which are incorporated herein by reference in their entirety.

[0002] Hematopoietic stem cell transplantation (HCT) can be used to treat patients with acute myeloid leukemia (AML) who are in complete remission but at high risk of relapse, particularly those with moderate or adverse disease-associated genetic features. While transplant-related mortality has decreased over the past few decades, leukemia relapse after HCT remains an obstacle to overall improvement in prognosis. Several recent studies have shown that the presence of minimal residual disease (MRD) during morphological complete remission at HCT is independently associated with a significant increased risk of leukemia relapse and death. Furthermore, patients showing evidence of residual leukemic blasts in the bone marrow are at very high risk of early relapse after HCT. [Overview of the Initiative]

[0003] The methods described herein provide novel methods for reducing the risk of leukemia relapse and improving overall prognosis in patients with acute myeloid leukemia. In some embodiments, the disclosure provides a method comprising administering to a subject an effective amount of genetically engineered hematopoietic cells or a population of their offspring, which include a modified gene encoding CD33 that has been engineered to have reduced or eliminated expression of the CD33 antigen, and an effective amount of a cytotoxic agent including an anti-CD33 antigen-binding domain.

[0004] In some embodiments, the present disclosure provides a method comprising administering to a subject an effective amount of a cytotoxic agent comprising an anti-CD33 antigen-binding domain. In some embodiments, the subject has received or is receiving a population of genetically engineered hematopoietic cells, or progeny thereof, comprising a modified gene encoding CD33 that has been engineered to have reduced or eliminated expression of the CD33 antigen.

[0005] In some embodiments, the present disclosure provides a method comprising administering to a subject a population of genetically engineered hematopoietic cells, or progeny thereof, comprising a modified gene encoding CD33 that has been engineered to have reduced or eliminated expression of the CD33 agent. In some embodiments, the subject has received or is receiving an effective amount of a cytotoxic agent comprising an anti-CD33 antigen-binding domain.

[0006] In some embodiments, the cytotoxic agent is an antibody-drug conjugate (ADC). In some embodiments, the ADC is gemtuzumab ozogamicin (GO).

[0007] In some embodiments, the effective amount of the population of genetically engineered hematopoietic cells is from about 10 6 cells / kg of subject body weight to about 10 7 cells / kg of subject body weight. In some embodiments, the effective amount of the population of genetically engineered hematopoietic cells is about 3.0×10 6 cells / kg of subject body weight.

[0008] In some embodiments, the effective amount of the cytotoxic agent is from about 0.1 mg / m 2 of subject body surface area to about 2.0 mg / m 2 of subject body surface area. In some embodiments, the effective amount of the cytotoxic agent is about 0.1 mg / m 2 , about 0.25 mg / m 2 , about 0.5 mg / m 2 , about l.0 mg / m 2 , or about 2.0 mg / m 2In some embodiments, the effective dose of the cytotoxic agent is approximately 2.0 mg / m² of the target body surface area. 2 That is the case.

[0009] In some embodiments, the genetically modified hematopoietic cell population and the cytotoxic agent are administered in close proximity. In some embodiments, close-proximity administration includes administering the genetically modified hematopoietic cell population and the cytotoxic agent in a single-dose regimen. In some embodiments, close-proximity administration includes administering the genetically modified hematopoietic cell population and the cytotoxic agent simultaneously. In some embodiments, close-proximity administration includes administering the genetically modified hematopoietic cell population and the cytotoxic agent simultaneously. In some embodiments, close-proximity administration includes administering the genetically modified hematopoietic cell population and the cytotoxic agent sequentially. In some embodiments, close-proximity administration includes administering the genetically modified hematopoietic cell population within 120 days of the administration of the cytotoxic agent (e.g., within 90 days of the administration of the cytotoxic agent, or within 60 days of the administration of the cytotoxic agent).

[0010] In some embodiments, the genetically modified hematopoietic cell population is administered before a cytotoxic agent. In some embodiments, the genetically modified hematopoietic cell population is administered in a single-dose treatment regimen. In some embodiments, the genetically modified hematopoietic cell population is administered intravenously.

[0011] In some embodiments, the cytotoxic agent is administered in multiple doses of an effective amount every four weeks. In some embodiments, the cytotoxic agent is administered at approximately 2.0 mg / m² every four weeks. 2 It is administered in multiple doses.

[0012] In some embodiments, a population of genetically modified hematopoietic cells is thawed from a cryopreserved form before administration. In some embodiments, cytotoxic agents are reconstituted from a lyophilized form before administration.

[0013] In some embodiments, the subject is preconditioned before administration of a cytotoxic agent and / or a population of genetically modified hematopoietic cells. In some embodiments, the method further includes preconditioning the subject before administration of a cytotoxic agent and / or a population of genetically modified hematopoietic cells. In some embodiments, preconditioning includes administering one or more chemotherapeutic agents to the subject. In some embodiments, preconditioning includes whole-body irradiation of the subject. In some embodiments, the chemotherapeutic agent is selected from the group consisting of busulfan, melphalan, fludarabine, cyclophosphamide, and thiotepa. In some embodiments, preconditioning includes administering an antibody that binds to human T cells (T lymphocytes), and optionally, the antibody includes rabbit anti-thymocyte globulin (rATG).

[0014] In some embodiments, the subject has or has been diagnosed with a hematopoietic malignancy or a pre-hematopoietic malignancy, where the hematopoietic malignancy is characterized by the presence of CD33-positive malignant cells, or the pre-hematopoietic malignancy is characterized by the presence of CD33-positive pre-malignant cells. In some embodiments, the subject has or has been diagnosed with CD33-positive acute myeloid leukemia. In some embodiments, the subject has or has been diagnosed with CD33-positive myelodysplastic syndrome. In some embodiments, the subject has or has been diagnosed with CD33-positive myelodysplastic syndrome and is at high risk of developing acute myeloid leukemia.

[0015] In some embodiments, the subjects are unsensitized to chemotherapy and / or radiotherapy. In some embodiments, the subjects are unsensitized to any treatment aimed at addressing hematopoietic malignancies or pre-hematopoietic malignancies.

[0016] In some embodiments, the subject has previously received chemotherapy. In some embodiments, the subject has previously received induction therapy. In some embodiments, the subject is already in complete hematological remission, and optionally, complete hematological remission is characterized by incomplete recovery of peripheral counts. In some embodiments, the subject has one or more risk factors associated with early leukemia relapse. In some embodiments, one or more risk factors associated with early leukemia relapse are selected from the group consisting of bone marrow in morphological complete remission with the presence of a moderate or high-risk disease-associated genetic trait, the presence of minimal residual disease (MRD) after cytoreductive therapy, bone marrow with residual leukemic blasts after cytoreductive therapy, and bone marrow blast counts of approximately 10% or less that do not contain circulating blasts.

[0017] In some embodiments, the subjects do not have a homozygous dominant genotype for the CD33 single nucleotide polymorphism (SNP) rs12459419. In some embodiments, the subjects do not have acute promyelocytic leukemia or chronic myeloid leukemia. In some embodiments, the subjects do not have a translocation associated with acute promyelocytic leukemia or chronic myeloid leukemia, and optionally, the translocation is t(15;17)(q22;q21) or t(9;22)(q34;q11). In some embodiments, the subjects have not previously received autologous or allogeneic stem cell transplantation. In some embodiments, the subjects have not previously received cytotoxic agents.

[0018] In some embodiments, the method further includes determining the percentage of donor chimerization and / or the level of CD33-negative myeloid hematopoiesis in a peripheral blood sample derived from the subject. In some embodiments, the subject has a CD33-negative absolute neutrophil count (ANC) of at least 1000 / dL before receiving a cytotoxic agent.

[0019] In some embodiments, the genetically modified population of hematopoietic cells is hematopoietic stem cells. In some embodiments, the hematopoietic stem cells are derived from bone marrow cells, umbilical cord blood cells, or peripheral blood mononuclear cells (PBMCs). In some embodiments, the hematopoietic stem cells are CD34 + / CD33- In some embodiments, the hematopoietic cells are autologous. In some embodiments, the method further comprises obtaining autologous hematopoietic stem cells from a subject, and optionally, the method further comprises genetically engineering the autologous stem cells to have reduced or eliminated CD33 antigen expression, and returning the genetically engineered hematopoietic stem cells to the subject.

[0020] In some embodiments, the hematopoietic cells are allogeneic. In some embodiments, the hematopoietic cells are allogeneic hematopoietic stem cells obtained from a donor having an HLA haplotype matching the target HLA haplotype. In some embodiments, the method further includes obtaining hematopoietic cells from a donor having an HLA haplotype matching the target HLA haplotype.

[0021] In some embodiments, the method further includes preparing a population of genetically engineered hematopoietic cells by modifying the endogenous gene of hematopoietic cells encoding the CD33 antigen. In some embodiments, all or part of the endogenous gene encoding the CD33 cell surface antigen is deleted. In some embodiments, all or part of the endogenous gene is deleted using genome editing. In some embodiments, genome editing requires a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a CRISPR-Cas system.

[0022] In some embodiments, the disclosure provides a composition comprising a population of genetically modified hematopoietic cells or their offspring, comprising a modified gene encoding CD33 that has been engineered to have reduced or eliminated expression of the CD33 antigen. In some embodiments, the hematopoietic cells are hematopoietic stem cells derived from bone marrow cells, umbilical cord blood cells, or peripheral blood mononuclear cells (PBMCs). In some embodiments, the hematopoietic stem cells are CD34+ / CD33-.

[0023] In some embodiments, all or part of the endogenous gene encoding the CD33 antigen is deleted. In some embodiments, all or part of the endogenous gene is deleted using genome editing. In some embodiments, the genome editing performed requires a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a CRISPR-Cas system. In some embodiments, the CRISPR-Cas system includes a nucleic acid encoding a gRNA and an RNA guide nuclease. In some embodiments, the gRNA includes a target domain containing one of the sequences SEQ ID NOs. 9–15.

[0024] In some embodiments, the disclosure provides a combination comprising any population of genetically modified hematopoietic cells described herein and a cytotoxic agent comprising an anti-CD33 antigen-binding domain. In some embodiments, the cytotoxic agent is an antibody-drug conjugate (ADC). In some embodiments, the ADC is gemtuzumab ozogamicin.

[0025] In some embodiments, the disclosure provides at least 1 × 10 per milliliter (mL) of culture medium. 6The present invention provides a composition comprising a population of cells, comprising genetically modified hematopoietic cells or their offspring, comprising a modified gene encoding CD33 which has been manipulated to have reduced or removed expression of the CD33 antigen. In some embodiments, the culture medium has a volume of about 40–50 mL. In some embodiments, the culture medium has a volume of about 5–150 mL. In some embodiments, the culture medium has a volume of about 10–100 mL. In some embodiments, the culture medium has a volume of about 25–75 mL. In some embodiments, the culture medium has a volume of about 30–70 mL. In some embodiments, the culture medium has a volume of about 40–60 mL. In some embodiments, the culture medium has a volume of about 45 mL. In some embodiments, the culture medium has a volume of about 30 mL. In some embodiments, the culture medium has a volume of about 35 mL. In some embodiments, the culture medium has a volume of about 40 mL. In some embodiments, the culture medium has a volume of about 50 mL. In some embodiments, the culture medium has a volume of about 55 mL. In some embodiments, the culture medium has a volume of about 60 mL. In some embodiments, the culture medium has a volume of approximately 70 mL.

[0026] In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the cells in the population are genetically modified hematopoietic cells or their offspring that have reduced or eliminated CD33 antigen expression. In some embodiments, the population contains at least 0.5 × 10¹⁶ cells per mL. 6 Each cell, at least 1 × 10⁶ cells per 1 mL 6 Each cell, at least 2 × 10⁶ cells per 1 mL 6 Each cell, at least 3 × 10⁶ cells per 1 mL 6 Each cell, at least 4 × 10⁶ cells per 1 mL 6 Each cell, at least 5 × 10⁶ cells per 1 mL 6 Each cell, at least 6 × 10⁶ cells per 1 mL 6 Each cell, at least 7 × 10⁶ cells per 1 mL 6 Each cell, at least 8 × 10⁶ cells per 1 mL 6Individual cells, or at least 9 × 10⁶ cells per 1 mL. 6 It contains individual cells. In some embodiments, the population is at least 0.5 × 10 per mL. 6 Each cell, at least 1 × 10⁶ cells per 1 mL 6 Each cell, at least 2 × 10⁶ cells per 1 mL 6 Each cell, at least 3 × 10⁶ cells per 1 mL 6 Each cell, at least 4 × 10⁶ cells per 1 mL 6 Each cell, at least 5 × 10⁶ cells per 1 mL 6 Each cell, at least 6 × 10⁶ cells per 1 mL 6 Each cell, at least 7 × 10⁶ cells per 1 mL 6 Each cell, at least 8 × 10⁶ cells per 1 mL 6 Individual cells, or at least 9 × 10⁶ cells per 1 mL. 6 It contains a number of cells. In some embodiments, the cell population is at least 1 × 10 9 A single living cell, at least 2 × 10⁶ 9 A single living cell, at least 3 × 10⁶ 9 A single living cell, at least 4 × 10⁶ 9 A single living cell, at least 5 × 10⁶ 9 A single living cell, at least 6 × 10⁶ 9 A single living cell, at least 7 × 10⁶ 9 A single living cell, at least 8 × 10⁶ 9 A single living cell, at least 9 × 10⁶ 9 A single living cell, at least 1 × 10⁶ 10 A single living cell, at least 2 × 10⁶ 10 A single living cell, at least 3 × 10⁶ 10 A single living cell, at least 4 × 10⁶ 10 A single living cell, at least 5 × 10⁶ 10 A single living cell, at least 6 × 10⁶ 10 A single living cell, at least 7 × 10⁶ 10 A single living cell, at least 8 × 10⁶ 10 A single living cell, at least 9 × 10⁶ 1 0 living cells, or at least 1 × 10⁶ 11The organism comprises living cells, and in some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the cells in the population are genetically modified hematopoietic cells or their offspring having reduced or eliminated CD33 antigen expression.

[0027] In some embodiments, the culture medium is a cryopreservation medium containing an antifreeze agent. In some embodiments, the antifreeze agent contains dimethyl sulfoxide (DMSO) in an amount of about 10% (v / v).

[0028] In some embodiments, hematopoietic cells are CD34+ / CD33-. In some embodiments, all or part of the endogenous gene encoding the CD33 antigen is deleted. In some embodiments, all or part of the endogenous gene is deleted using genome editing. In some embodiments, the genome editing performed requires a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a CRISPR-Cas system. In some embodiments, the CRISPR-Cas system includes a nucleic acid encoding a gRNA and an RNA guide nuclease. In some embodiments, the gRNA includes a target domain containing one of the sequences SEQ ID NOs: 9-15. In some embodiments, the composition does not contain detectable levels of RNA guide nuclease.

[0029] In some embodiments, the composition is in a frozen state.

[0030] In some embodiments, the composition is subjected to a cryopreservation process. In some embodiments, the cryopreservation process is a controlled-rate freezing process.

[0031] The above summary is intended in a non-limiting manner to illustrate some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the modes, drawings, examples, and claims for carrying out the invention. [Brief explanation of the drawing]

[0032] The accompanying drawings, which constitute part of this specification, illustrate several embodiments of the present invention and, together with the description, help to illustrate the principles of the present invention.

[0033] [Figure 1] Figure 1 is a schematic diagram showing an example of a drug regimen for a clinical trial. [Figure 2] Figure 2 is a schematic diagram showing an example of an experimental design for evaluating the effectiveness of the method described herein. Briefly, it involves CD34+ hematopoietic stem cells and progenitor cells (HSPCs) derived from recruited PMBCs (mPBMCs) obtained from human donors. The HSPCs are either mock-electroporated ("control") or, for example, CD33 Using gRNA and the CRISPR-Cas system, genome editing is performed to reduce or eliminate the expression of the CD33 antigen (CD33KO), and then administered to sublethally irradiated NOD / scid / IL2Rγnull((NOD.Cg-PrkdcscidIl2rgtm1Wjl, NSG®) mice. Blood samples are taken at 8 and 12 weeks after HSPC transplantation. Mice are administered at approximately 15 weeks to a cytotoxic agent containing an anti-CD33 antigen binding domain, such as gemtuzumab ozogamicin / Mylotarg®, or a vehicle control, at a dose of 0.33 mg / kg. Approximately 8 days after gemtuzumab ozogamicin / Mylotarg® treatment (16 weeks after HSPC transplantation), mice are sacrificed, and terminal bone marrow (BM), blood, and spleen samples are collected and subjected to analysis, for example, by flow cytometry. [Figure 3AB]Figures 3A-3E show plots of flow cytometry analysis results of cell populations after transplantation of human HSPCs derived from donor 1, according to the experimental design shown in Figure 2. Figure 3A shows human leukocyte chimerization determined by the percentage of human CD45+ cells. Figure 3B shows CD33+ bone marrow cells as the percentage of human CD45+ cells. [Figure 3CDE] Figure 3C shows CD14+ bone marrow cells as a percentage of human CD45+ cells. Figure 3D shows CD11b+ bone marrow cells as a percentage of human CD45+ cells. Figure 3E shows the presence of CD33 in CD14+ bone marrow cells, indicating that CD14+ cells edited for CD33 were protected from gemtuzumab ozogamicin / Mylotarg® cytotoxicity. For each plot, Mock EP refers to transplantation using mock electroporated human HSPCs, and CD33 gRNA refers to transplantation using human HSPCs edited with CD33 gRNA. "Mylotarg" refers to mice treated with gemtuzumab ozogamicin / Mylotarg®, and "Vehicle" refers to control mice treated with a vehicle control. Each data point represents an individual value for a mouse, along with the mean and standard deviation shown (n=9-10). *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001. [Figure 4ABC]Figures 4A-4C show plots illustrating the results of flow cytometry analysis of cell populations after transplantation of human HSPCs derived from donor 1, according to the experimental design shown in Figure 2. Figure 4A shows CD3+ T cells as a percentage of human CD45+ cells. Figure 4B shows CD19+ B cells as a percentage of human CD45+ cells. Figure 4C shows CD34+CD38- primitive HSPCs as a percentage of human CD45+ cells. For each plot, "Mock EP" refers to transplantation using mock electroporated human HSPCs, and "CD33 gRNA" refers to transplantation using human HSPCs edited with CD33 gRNA. "Mylotarg" refers to mice treated with gemtuzumab ozogamicin / Mylotarg®, and "Vehicle" refers to control mice treated with a vehicle control. Each data point represents the individual value for a mouse, along with the mean and standard deviation shown (n=9-10). *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001. [Figure 5AB] Figures 5A–5D show plots of flow cytometry analysis results of cell populations after transplantation of human HSPCs derived from donor 2, according to the experimental design shown in Figure 2. Figure 5A shows human leukocyte chimerization determined by the percentage of human CD45+ cells. Figure 5B shows CD33+ bone marrow cells as the percentage of human CD45+ cells. [Figure 5CD] Figure 5C shows CD14+ bone marrow cells as a percentage of human CD45+ cells. Figure 5D shows CD11b+ bone marrow cells as a percentage of human CD45+ cells. For each plot, Mock EP refers to transplantation using mock electroporated human HSPCs, and CD33 gRNA refers to transplantation using human HSPCs edited with CD33 gRNA. "Mylotarg" refers to mice treated with gemtuzumab ozogamicin / Mylotarg®, and "Vehicle" refers to control mice treated with the vehicle control. Each data point represents an individual value for a mouse, along with the mean and standard deviation shown (n=9-10). *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001. [Figure 6ABC] Figures 6A–6C show plots illustrating the results of flow cytometry analysis of cell populations after transplantation of human HSPCs derived from donor 2, according to the experimental design shown in Figure 2. Figure 6A shows CD3+ T cells as a percentage of human CD45+ cells. Figure 6B shows CD19+ B cells as a percentage of human CD45+ cells. Figure 6C shows CD34+CD38- primitive HSPCs as a percentage of human CD45+ cells. For each plot, "Mock EP" refers to transplantation using mock electroporated human HSPCs, and "CD33 gRNA" refers to transplantation using human HSPCs edited with CD33 gRNA. "Mylotarg" refers to mice treated with gemtuzumab ozogamicin / Mylotarg®, and "Vehicle" refers to control mice treated with a vehicle control. Each data point represents the individual value for a mouse, along with the mean and standard deviation shown (n=9–10). *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001. [Modes for carrying out the invention]

[0034] This disclosure provides a targeted therapeutic approach for use in the treatment of hematopoietic malignancies that overcomes the limitations of existing therapies. For example, current CD33-targeted therapies for acute myeloid leukemia (AML) are limited to "on-target, off-leukemia" cytotoxicity against normal, healthy myeloid cells expressing CD33. The loss of non-cancerous CD33+ cells can deplete the patient's hematopoietic system. To address this depletion, the subject may be administered rescue cells (e.g., hematopoietic cells) containing modifications to the CD33 gene. These CD33-modified cells may be resistant to anti-CD33 cancer therapies and thus allow for the rearrangement of the hematopoietic system during or after anti-CD33 therapy. In this way, normal bone marrow compartments are protected from the on-target effects of CD33-targeted agents, resulting in improved therapeutic metrics and improved patient prognosis for these agents.

[0035] cell Aspects of the present disclosure have concerned genetically engineered hematopoietic cells (also referred herein as eHSCs or eHSPCs) or their offspring, which include a modified gene encoding CD33 that has been engineered to have reduced or eliminated expression of the CD33 antigen. In some embodiments, the genetically engineered hematopoietic cells of the present disclosure having a modification of the gene encoding CD33 (e.g., hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs)) are genetically engineered using any gene editing method known in the art.

[0036] In some embodiments, genetically engineered hematopoietic cells of this disclosure (e.g., hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs)) having a modification of the gene encoding CD33 are genetically engineered using the nucleases and / or gRNAs described herein. In some embodiments, cells (e.g., HSCs or HPCs) having a modification of CD33 and a modification of a second lineage-specific cell surface antigen are produced using the nucleases and / or gRNAs described herein. It is understood that the cells may be produced by contacting the cells themselves with the nucleases and / or gRNAs, or that the cells may be daughter cells of cells that have been contacted with the nucleases and / or gRNAs. In some embodiments, the cells described herein (e.g., HSCs or HPCs) are capable of reconstituting a hematopoietic system of interest. In some embodiments, the cells described herein (e.g., HSCs or HPCs) are capable of engrafting in a human subject, producing myeloid cells, and producing lymphoid cells, one or more (e.g., all of them).

[0037] In some embodiments, the cells contain only one gene modification. In some embodiments, the cells are genetically modified only at the CD33 locus, such as in the sequence of exon 3 of CD33. In some embodiments, the cells are genetically modified at a second locus. In some embodiments, the cells do not contain transgenic proteins, such as chimeric antigen receptors (CARs).

[0038] The terms “CD33 antigen” and “CD33 protein” are used interchangeably herein and refer to the CD33 protein, or a portion or fragment thereof, such as a cytotoxic agent containing an anti-CD33 antigen-binding domain, that is targeted by an anti-CD33 agent.

[0039] In some embodiments, the genetically engineered hematopoietic cells described herein are substantially devoid of the CD33 protein (CD33 antigen). In some embodiments, the genetically engineered hematopoietic cells described herein are substantially devoid of the wild-type CD33 protein but contain the mutant CD33 protein. In some embodiments, the mutant CD33 protein is not conjugated by an agent that targets CD33 for therapeutic purposes. When used herein, genetically engineered hematopoietic cells that have or do not have reduced CD33 expression may be referred to as "CD33KO eHSC" or "CD33KO eHSPC".

[0040] In some embodiments, the cells are circulating blood cells, e.g., reticulocytes, megakaryocyte erythroblast precursor (MEP) cells, myeloid progenitor cells (CMP / GMP), lymphoid progenitor (LP) cells, hematopoietic stem cells (HSCs), or hematopoietic progenitor cells (HPCs), which may be referred to as hematopoietic stem and progenitor cells (HSPCs), or endothelial cells (ECs). In some embodiments, the cells are myeloid cells (e.g., reticulocytes, erythrocytes (e.g., erythroblasts), megakaryocyte-erythroblast precursor cells (MEP cells), myeloid progenitor cells (CMP / GMP), lymphocyte-dominant (LP) cells, erythroblast precursor (EP) cells, HSCs, pluripotent progenitor (MPP) cells, endothelial cells (ECs), hematopoietic endothelial cells (HE) cells, or pluripotent stem cells). In some embodiments, the cells are myeloid progenitor cells (e.g., common myeloid progenitor (CMP) cells or granulocyte-macrophage progenitor (GMP) cells). In some embodiments, the cells are lymphoid progenitor cells (e.g., common lymphoid progenitor (CLP) cells). In some embodiments, the cells are erythrocyte progenitor cells (e.g., MEP cells). In some embodiments, the cells are hematopoietic stem / progenitor cells (e.g., long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs), MPP cells, or lineage-limiting progenitor (LRP) cells). In some embodiments, the cells are CD34+ cells, CD34+CD90+ cells, CD34+CD38+ cells, CD34+CD90+CD49^CD38+CD45RA cells, CD105+ cells, CD31+, or CD133+ cells, or CD34+CD90+CD133+ cells. In some embodiments, the cells are umbilical cord blood CD34+ HSPCs, umbilical vein endothelial cells, umbilical artery endothelial cells, amniotic fluid CD34+ cells, amniotic fluid endothelial cells, placental endothelial cells, or placental hematopoietic CD34+ cells. In some embodiments, the cells are recruited peripheral blood hematopoietic CD34+ cells (after the patient has been treated with a recruiting agent, e.g., G-CSF and / or plerixafor). In some embodiments, the cells are peripheral blood endothelial cells, or a population of cells.

[0041] In some embodiments, the cells are hematopoietic cells, e.g., hematopoietic stem cells. Hematopoietic stem cells (HSCs) are typically stem cells capable of giving rise to both myeloid and lymphoid progenitor cells, which can further give rise to myeloid cells (e.g., monocytes, macrophages, neutrophils, basophils, dendritic cells, erythrocytes, platelets, etc.) and lymphoid cells (e.g., T cells, B cells, NK cells), respectively. HSCs are characterized by the expression of the cell surface marker CD34 (e.g., CD34+), which can be used to identify and / or isolate HSCs, and the absence of this cell surface marker is associated with a leaning towards a particular cell lineage.

[0042] In some embodiments, the genetically modified hematopoietic cell population described herein includes a plurality of hematopoietic stem cells. In some embodiments, the genetically modified hematopoietic cell population described herein includes a plurality of hematopoietic progenitor cells. In some embodiments, the genetically modified hematopoietic cell population described herein includes a plurality of hematopoietic stem cells or a plurality of hematopoietic progenitor cells.

[0043] In some embodiments, hematopoietic stem cells (HSCs) refer to cells of stem cell lineages that give rise to all blood cell types, including erythrocytes (red blood cells or red blood cells (RBCs)), myeloid cells (monocytes and macrophages, neutrophils, basophils, eosinophils, megakaryocytes / platelets, and dendritic cells), and lymphoid cells (T cells, B cells, NK cells). In some embodiments, the cells used herein are selected from the group consisting of circulating blood cells, recruited blood cells, myeloid cells, myeloid progenitor cells, lymphoid progenitor cells, pluripotent progenitor cells, lineage-limited progenitor cells, endothelial cells, or mesenchymal stromal cells. In some embodiments, HSCs are from a non-umbilical cord blood source, an umbilical cord source, or an umbilical cord blood source. In one embodiment, HSCs are CD34+ cells. In some embodiments, HSC cells can be differentiated in vivo after transplantation into a subject. In some embodiments, HSC cells can be differentiated into B cells, T cells, erythrocytes, and / or myeloid cells. In some embodiments, HSC cells can reconstitute hematopoiesis in a subject. In some embodiments, hematopoietic stem cells have at least one of the cell surface marker features of hematopoietic progenitor cells: CD34+, CD59+, Thyl / CD90+, CD38lo / -, and C-kit / CD117+. In some embodiments, the hematopoietic progenitor cells are CD34+.

[0044] In some embodiments, hematopoietic stem cells are peripheral blood stem cells obtained from a subject after the subject has been treated with granulocyte colony-stimulating factor (G-CSF) (optionally in combination with plerixafor). In some embodiments, CD34+ cells are enriched using the CliniMACS® cell sorting system (Miltenyi Biotec). In some embodiments, CD34+ cells are weakly stimulated in serum-free medium (e.g., CellGrow SCGM medium, CellGenix) containing cytokines (e.g., SCF, rhTPO, rhFLT3) before genome editing. In some embodiments, the addition of SR1 and dmPGE2 and / or other factors is intended to improve long-term engraftment.

[0045] In some embodiments, the population of genetically engineered hematopoietic cells for administration according to this disclosure may be allogeneic hematopoietic progenitor cells obtained from one or more donors. As used herein, “allogeneic” means hematopoietic progenitor cells or a biological sample comprising hematopoietic progenitor cells obtained from one or more different donors of the same species, where the genes at one or more loci are not identical. For example, the population of hematopoietic cells administered to a subject may be derived from umbilical cord blood obtained from one or more unrelated donor subjects or one or more non-identical siblings. In some embodiments, a syngeneic hematopoietic cell population may be used, such as one obtained from genetically identical animals or identical twins. In some embodiments, the hematopoietic cells are autologous cells, i.e., the hematopoietic progenitor cells are obtained from or isolated from a subject and administered to the same subject (i.e., the donor and recipient are the same).

[0046] In some embodiments, the genetically modified hematopoietic cell population described herein includes a plurality of genetically modified hematopoietic stem cells. In some embodiments, the genetically modified hematopoietic cell population described herein includes a plurality of genetically modified hematopoietic progenitor cells. In some embodiments, the genetically modified hematopoietic cell population described herein includes a plurality of genetically modified hematopoietic stem cells and a plurality of genetically modified hematopoietic progenitor cells.

[0047] In some embodiments, HSCs or HPCs are obtained from a subject, such as a human subject. A method for obtaining HSCs is described, for example, in PCT application US2016 / 057339, which is incorporated herein in its entirety by reference. In some embodiments, the HSCs are peripheral blood HSCs. In some embodiments, the mammalian subject is a non-human primate, a rodent (e.g., mouse or rat), a cattle, pig, horse, or livestock. In some embodiments, the HSCs are obtained from a human subject, such as a human subject with hematopoietic malignancies. In some embodiments, the HSCs or HPCs are obtained from a healthy donor. In some embodiments, the HSCs or HPCs are obtained from a subject to which a cytotoxic agent containing an anti-CD33 antigen-binding domain is subsequently administered. HSCs or HPCs administered to the same subject from which the cells were obtained are referred to as autologous cells, while HSCs or HPCs obtained from a subject other than the subject to which the cells were administered are referred to as allogeneic cells.

[0048] In some embodiments, the population of genetically engineered hematopoietic cells is a heterogeneous population of cells, e.g., a heterogeneous population of genetically engineered hematopoietic cells containing different CD33 mutations. In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the CD33 copies in the cell population have mutations affected by the genome editing approach described herein, e.g., a CRISPR / Cas system using the gRNA described herein. As an example, the population may contain multiple different CD33 mutations, each of which contributes to the proportion of CD33 copies in the population of cells having the mutation.

[0049] In some embodiments, the expression of CD33 on genetically modified hematopoietic cells is compared to the expression of CD33 on naturally occurring hematopoietic cells (e.g., wild-type counterparts). In some embodiments, the genetic modification results in a reduction of CD33 expression levels of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to the expression of CD33 on naturally occurring hematopoietic cells (e.g., wild-type counterparts). For example, in some embodiments, genetically engineered hematopoietic cells express less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of CD33 compared to naturally occurring hematopoietic cells (e.g., wild-type counterparts).

[0050] In some embodiments, the genetic engineering results in a reduction of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the expression level of wild-type CD33 compared to the expression of wild-type CD33 on naturally occurring hematopoietic cells (e.g., wild-type counterparts). For example, in some embodiments, genetically engineered hematopoietic cells express less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of CD33 compared to naturally occurring hematopoietic cells (e.g., wild-type counterparts).

[0051] In some embodiments, the genetic manipulation results in a reduction of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the expression level of wild-type lineage-specific cell surface antigen (e.g., CD33) compared to a preferred control (e.g., a cell or a group of cells). In some embodiments, a preferred control includes the measured or expected level of wild-type lineage-specific cell surface antigen in a group of unmanipulated cells from the same subject. In some embodiments, a preferred control includes the measured or expected level of wild-type lineage-specific cell surface antigen in a group of cells from a healthy subject. In some embodiments, a preferred control includes the measured or expected level of wild-type lineage-specific cell surface antigen in a population of cells from a pool of healthy individuals (e.g., 10, 20, 50, or 100 individuals). In some embodiments, preferred controls include subjects requiring the treatment described herein, e.g., anti-CD33 therapy, e.g., subjects having cancer and cancer cells expressing CD33, and levels of wild-type lineage-specific cell surface antigen measured or expected in the subjects. In some embodiments, preferred controls include levels of wild-type lineage-specific cell surface antigen measured in cells before being subjected to genetic manipulation to reduce or eliminate CD33 expression.

[0052] In some embodiments, the method for producing genetically modified hematopoietic cells described herein includes the step of preparing wild-type cells, e.g., wild-type hematopoietic stem or progenitor cells. In some embodiments, the wild-type cells are unedited cells containing (e.g., expressing) two functional copies of a lineage-specific cell surface antigen (e.g., CD33). In some embodiments, the cells contain the CD33 gene sequence according to SEQ ID NO: 16. In some embodiments, the cells contain the CD33 gene sequence encoding the CD33 protein encoded by SEQ ID NO: 16, and for example, the CD33 gene sequence may contain one or more silent mutations relative to SEQ ID NO: 16. In some embodiments, the wild-type cells express the lineage-specific cell surface antigen (e.g., CD33) or produce more differentiated cells that express the lineage-specific cell surface antigen at levels equivalent to HL60 or MOLM-13 cells (or within 90%-110%, 80%-120%, 70%-130%, 60%-140%, or 50%-150%). In some embodiments, wild-type cells either bind to an antibody that conjugates a lineage-specific cell surface antigen (e.g., an anti-CD33 antibody, e.g., P67.6), or produce more differentiated cells that bind to the antibody at levels equivalent to (or within 90%–110%, 80%–120%, 70%–130%, 60–140%, or 50%–150%) of the antibody binding to HL60 or MOLM-13 cells. Antibody binding can be measured, for example, by flow cytometry.

[0053] In some embodiments, genetically engineered hematopoietic stem or progenitor cells contain a gene mutation in exon 3 of the endogenous CD33 gene, the gene mutation being located at a site described herein (see Table 1). One embodiment of this disclosure provides genetically engineered hematopoietic stem and / or progenitor cells containing a gene mutation in exon 3 of the endogenous CD33 gene, the gene mutation being located at a site targeted by a gRNA, such as one of the gRNAs presented in Table 1.

[0054] In some embodiments, the manipulated cells described herein contain two mutations, the first mutation being in CD33 and the second mutation being in a second lineage-specific cell surface antigen. Such cells may, in some embodiments, be resistant to agents targeting both antigens: an anti-CD33 agent and a second lineage-specific cell surface antigen. In some embodiments, such cells can be produced using two or more gRNAs described herein, for example, the gRNAs in Table 3 and a second gRNA. In some embodiments, the cells can be produced using, for example, ZFNs or TALENs. This disclosure also provides populations comprising the cells described herein.

[0055] In some embodiments, the second mutation is located in a gene encoding a lineage-specific cell surface antigen, such as one of the lineage-specific cell surface antigens described herein.

[0056] Typically, mutations affected by the methods and compositions provided herein, such as mutations in a target gene like CD33, result in a loss of function of the gene product encoded by the target gene, for example, in the case of a mutation in the CD33 gene, resulting in a loss of function of the CD33 protein. In some embodiments, the loss of function is a reduction in the level of expression of the gene product, for example, a reduction to a lower level of expression, or a complete inactivation of the gene product's expression. In some embodiments, the mutation results in the expression of a non-functional variant of the gene product. For example, a gene product characterized by a modified amino acid sequence that renders the gene product non-functional, such as a mutation that produces an immature stop codon in a shortened gene product, or a mutation that produces a nonsense or missense mutation. In some embodiments, the function of the gene product is to bind to or recognize a binding partner. In some embodiments, the reduction in the expression of a gene product, such as CD33, a second lineage-specific cell surface antigen, or both, is 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, or 1% or less, compared to wild-type or unmanipulated corresponding cells.

[0057] In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the copies of CD33 in a population of genetically engineered hematopoietic cells produced by and / or using the methods provided herein are mutated. In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the copies of a second lineage-specific cell surface antigen in a population of genetically engineered hematopoietic cells are mutated. In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the copies of CD33 and the second lineage-specific cell surface antigen in a population of genetically engineered hematopoietic cells are mutated. In some embodiments, the population includes one or more wild-type cells. In some embodiments, the population includes one or more cells containing one wild-type copy of CD33. In some embodiments, the population includes one or more cells containing one wild-type copy of a second lineage-specific cell surface antigen.

[0058] Cytotoxic agents

[0059] Aspects of this disclosure relate to cytotoxic agents containing an anti-CD33 antigen-binding domain. Administration of cytotoxic agents containing an anti-CD33 antigen-binding domain interacts with CD33-expressing cells and induces cytotoxicity. As described herein, administration of such cytotoxic agents can induce cytotoxicity not only in cancer cells expressing CD33, but also in normal, healthy cells that also express CD33, e.g., "on-target, off-leukemia" effects.

[0060] In some embodiments, the cytotoxic agents of this disclosure are antibody-drug conjugates (ADCs). An ADC may be a molecule comprising an antibody or its antigen-binding fragment conjugated to a toxin or drug molecule. The binding of the antibody or its fragment to the corresponding antigen enables the delivery of the toxin or drug molecule to a cell that presents the antigen on its cell surface (e.g., a target cell), thereby causing the death of the target cell.

[0061] In some embodiments, the binding of ADCs to epitopes of cell surface lineage-specific proteins (e.g., CD33) induces internalization of ADCs, allowing drugs (or toxins) to be released into cells. In some embodiments, the binding of ADCs to epitopes of cell surface lineage-specific proteins induces the internalization of toxins or drugs, which allows the toxin or drug to kill cells expressing the lineage-specific protein. In some embodiments, the binding of ADCs to epitopes of cell surface lineage-specific proteins induces the internalization of toxins or drugs, which can modulate the activity of cells expressing the lineage-specific protein. The types of toxins or drugs used with ADCs described herein are not limited to any specific type.

[0062] Toxins or drugs suitable for use in ADCs are known in the art and will be obvious to those skilled in the art. See, for example, Peters et al. Biosci. Rep. (2015) 35(4):e00225; Beck et al. Nat Rev Drug Disc (2017) 16:315-337; Marin-Acevedo et al. J. Hematol. Oncol. (2018) 11:8; Elgundi et al. Advanced Drug Delivery Reviews (2017) 122:2-19.

[0063] In some embodiments, the ADC may further include a linker (e.g., a peptide linker such as a cleavable linker) for attaching the antibody and drug molecules.

[0064] In some embodiments, the cytotoxic agent of this disclosure is gemtuzumab ozogamicin. Gemtuzumab ozogamicin is a recombinant humanized anti-CD33 monoclonal antibody (IgG4 K antibody hP67.6) conjugated (covalently) to the cytotoxic antitumor antibiotic calitiamycin (N-acetyl-γ-calitiamycin) via a bifunctional linker (4-(4-acetylphenoxy)butanoic acid). Gemtuzumab ozogamicin is marketed as Mylotarg® (Wyeth Pharmaceuticals, Philadelphia, Pa.). The antibody portion of gemtuzumab ozogamicin, referred to as hP67.6, specifically binds to the CD33 antigen.

[0065] Gemtuzumab ozogamicin contains an amino acid sequence that is approximately 98.3% of human origin. The constant region and framework region contain human sequences, while the complementarity-determining region is derived from a mouse antibody (P67.6) that binds to CD33. This antibody is linked to N-acetyl-gamma-kalythiamycin via a bifunctional linker. Gemtuzumab ozogamicin contains approximately 50% of an antibody loaded with 4-6 moles of kalythiamycin per mole of antibody. The remaining 50% of the antibody is not linked to a kalythiamycin derivative. Gemtuzumab ozogamicin has a molecular weight of 151-153 kDa. Gemtuzumab ozogamicin and methods for preparing the same are described in U.S. Patents 5,733,001; 5,739,116; 5,767,285; 5,877,296; 5,606,040; 5,712,374; and 5,714,586, which are incorporated herein by reference in their entirety.

[0066] Amino acid sequence of the heavy chain of gemtuzumab ozogamicin / Mylotarg(registered trademark) EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 26)

[0067] Amino acid sequence of the light chain of gemtuzumab ozogamicin / Mylotarg(registered trademark) DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 27)

[0068] The anti-CD33 antibody-binding domains for use in the construction of CD33-targeting cytotoxic agents described herein may include the same heavy chain and / or light chain CDR regions as those in SEQ ID NO: 26 and SEQ ID NO: 27. Such antibodies may include amino acid residue variations in one or more of the framework regions. In some cases, the anti-CD33 antibody fragment may include a heavy chain variable region that shares at least 70% sequence identity (e.g., 75%, 80%, 85%, 90%, 95% or more) with the heavy chain variable region of gemtuzumab ozogamicin (provided in the heavy chain sequence of SEQ ID NO: 26), and / or a light chain variable region that shares at least 70% sequence identity (e.g., 75%, 80%, 85%, 90%, 95% or more) with the light chain variable region of gemtuzumab ozogamicin (provided in the light chain sequence of SEQ ID NO: 27).

[0069] CD33 is also a target of the anti-CD33 immunotoxin vadastuximab-tarilin (SGN-CD33A, also known as 33A) (Seattle Genetics). SGN-CD33A is an antibody-drug conjugate that can reduce multidrug resistance observed in response to treatment with gemtuzumab ozogamicin. In some embodiments, SGN-CD33A is used to treat the subject. In some embodiments, gemtuzumab ozogamicin and SGN-CD33A are used in combination to treat the subject, for example, concurrently or sequentially. In some embodiments, the subject is also treated with chemotherapy.

[0070] Further examples of ADCs include, but are not limited to, brentuximab vedotin, glenvatumumab vedotin / CDX-011, depatuxizumab mahodotin / ABT-414, and PSMA. ADC, Polatuzumab Vedotin / RG7596 / DCDS4501A, Denintuzumab Vedotin / SGN-CD19A, AGS-16C3F, CDX-014, RG7841 / DLYE5953A, RG7882 / DMUC406A, RG7986 / DCDS0780A, SGN-LIV1A, Enfortomab Vedotin / ASG-22ME, AG-15ME, AGS67E, Terisotuzumab Vedotin / ABBV-399, ABBV-221, ABBV-085, GSK-2857916, Tisotuzumab Vedotin / HuMax-TF-ADC, HuMax-Axl-ADC, Pinatuzumab Vedotin / RG7593 / DCDT2980S, Rifastuzumab Vedotin / RG7599 / DNIB0600A, Indusatuzumab Vedotin / MLN-0264 / TAK-264, Bundutuzumab Vedotin / RG7450 / DSTP3086S, Sofituzumab Vedotin / RG7458 / DMUC5754A, RG7600 / DMOT4039A, RG7336 / DEDN6526A, ME1547, PF-06263507 / ADC 5T4, Trastuzumab emtansine / T-DM1, Milbetuximab sorabutansine / IMGN853, Coltuximab labutansine / SAR3419, Naratuximab emtansine / IMGN529, Induximab labutansine / BT-062, Anetumab labutansine / BAY 94-9343, SAR408701, SAR428926, AMG224, PCA062, HKT288, LY3076226, SAR566658, lorbotuzumab meltansine / IMGN901, cantuzumab meltansine / SB-408075, cantuzumab meltansine / IMGN242, laprituximab emtansine / IMGN289, IMGN388, vibatuzumab meltansine, AVE9633, BIIB015, MLN2704, AMG 172, AMG 595, LOP 628. Vadastuximab-Tarilin / SGN-CD33A, SGN-CD70A, SGN-CD19B, SGN-CD123A, SGN-CD352A, Robalpituzumab-Tecillin / SC16LD6.5, SC-002, SC-003, ADCT-301 / HuMax-TAC-PBD, ADCT-402, MEDI3726 / ADC-401, IMGN779, IMGN632, Gemtuzumab ozogamicin, Inotuzumab ozogamicin / CMC-544, PF-06647263, CMD-193, CMB-401, Trastuzumab duocalmazine / SYD985, BMS-936561 / MDX-1203, Sacituzumab govitecan / IMMU-132, Rabetsuzumab govitecan / IMMU-130, DS-8201a, U Examples of toxins and drugs contained in 3-1402, milatuzumab doxorubicin / IMMU-110 / hLL1-DOX, BMS-986148, RC48-ADC / hertuzumab-vc-MMAE, PF-06647020, PF-06650808, PF-06664178 / RN927C, lupalzumab amadotin / BAY1129980, applezumab ixadotin / BAY1187982, ARX788, AGS62P1, XMT-1522, AbGn-107, MEDI4276, and DSTA4637S / RG7861 include.

[0071] Methods of treatment and administration Aspects of this disclosure provide a method comprising administering to a subject an effective amount of a population of genetically modified hematopoietic cells described herein (referred herein as eHSC or eHSPC) and an effective amount of a cytotoxic agent comprising an anti-CD33 antigen-binding domain. In some embodiments, a subject is diagnosed with a hematopoietic malignancy and receives combination therapy comprising the administration of a population of genetically modified hematopoietic cells described herein and an effective amount of a cytotoxic agent comprising an anti-CD33 antigen-binding domain. As will be understood by those skilled in the art, combination therapy encompasses more than one aspect of therapy that may be administered together (e.g., simultaneously or in a single composition), but encompasses more than one aspect of therapy within a treatment regimen aimed at treating a malignancy or any symptom or sign thereof. For example, some methods described herein include combination therapy for treating a hematopoietic malignancy (e.g., acute myeloid leukemia) comprising the administration of an effective amount of a population of genetically modified hematopoietic cells described herein and an effective amount of a cytotoxic agent comprising an anti-CD33 antigen-binding domain. In some embodiments, the methods described herein include combination therapy for treating pre-malignant stages of hematopoietic malignancies (e.g., myelodysplastic syndrome (MDS)), comprising administering an effective amount of a population of genetically modified hematopoietic cells described herein and an effective amount of a cytotoxic agent containing an anti-CD33 antigen-binding domain. Some combination therapy methods provided herein include, for example, the administration of a population of genetically modified hematopoietic cells (e.g., CD33KO eHSPC) and a cytotoxic agent (e.g., Mylotarg®) following the administration of the genetically modified hematopoietic cells, followed by the administration of the cytotoxic agent (e.g., Mylotarg®). The combination therapy provided herein, for example, the therapeutic modality of a population of genetically modified hematopoietic cells and a cytotoxic agent, may be administered as part of a combination therapy according to the same or different dosing regimens (including dosing frequency, amount, and route of administration), which may overlap in time or be consecutive.

[0072] In some embodiments, an effective number of genetically modified hematopoietic stem cells, such as CD33-modified hematopoietic stem cells as described herein, are administered in combination with a cytotoxic agent containing an anti-CD33 antigen-binding domain (e.g., an anti-CD33 cancer therapy such as gemtuzumab ozogamicin / Mylotarg®). In some embodiments, an effective number of cells containing modified CD33 and a modified second lineage-specific cell surface antigen are administered in combination with a cytotoxic agent. In some embodiments, the cytotoxic agent includes an antibody, an ADC, or immune cells expressing a chimeric antigen receptor (CAR). In some embodiments, the cytotoxic agent includes gemtuzumab ozogamicin.

[0073] In some embodiments, the effective dose of a population of genetically modified hematopoietic stem cells, such as the CD33-modified hematopoietic stem cells described herein, is about 10 2 Cells / Target body weight in kilograms ~ approximately 10 10 The cell / subject weight is in kilograms. In some embodiments, the effective dose of a population of genetically modified hematopoietic stem cells, e.g., CD33-modified hematopoietic stem cells as described herein, is about 10 4 Cells / Target body weight in kilograms ~ approximately 10 8 The cell / subject weight is in kilograms. In some embodiments, the effective dose of a population of genetically modified hematopoietic stem cells, e.g., CD33-modified hematopoietic stem cells as described herein, is about 10 6 Cells / Target body weight in kilograms ~ approximately 10 8 The cell / subject weight is in kilograms. In some embodiments, the effective dose of a population of genetically modified hematopoietic stem cells, e.g., CD33-modified hematopoietic stem cells as described herein, is about 10 5 Cells / Target body weight in kilograms ~ approximately 10 7 The cell / subject weight is in kilograms. In some embodiments, the effective dose of a population of genetically modified hematopoietic stem cells, e.g., CD33-modified hematopoietic stem cells as described herein, is about 10 6 Cells / Target body weight in kilograms ~ approximately 10 7Includes the weight in kilograms of the cells / subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic stem cells, such as the CD33-modified hematopoietic stem cells described herein, is about 10 5 cells / kilogram of the weight of the subject, about 10 6 cells / kilogram of the weight of the subject, about 10 7 cells / kilogram of the weight of the subject, or about 10 8 cells / kilogram of the weight of the subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic stem cells, such as the CD33-modified hematopoietic stem cells described herein, is at least 10 2 cells, at least 10 3 cells, at least 10 4 cells, at least 10 5 cells, at least 5×10 5 cells, at least 10 6 cells, at least 2×10 6 cells, at least 3×10 6 cells, at least 4×10 6 cells, at least 5×10 6 cells, at least 6×10 6 cells, at least 7×10 6 cells, at least 8×y10 6 cells, at least 9×10 6 [[ID=~36]]cells, at least 1×10 7 cells, or a plurality thereof. [[ID=~39]] [[ID=~40]]

[0074] [[ID=~41]] In some embodiments, an effective amount of a population of genetically engineered hematopoietic stem cells, such as the CD33-modified hematopoietic stem cells described herein, is about 1.0×10 5 , about 2.0×10 5 , about 3.0×10 5 , about 4.0×10 5 , about 5.0×10 5 , about 6.0×10 5 , about 7.0×10 5 , about 8. O×10 5 , about 9.0×10 5 , about 1.0×10 6 , about 2.0×10 6 , about 3.0×10 6 Please note that there seem to be some tags with "~" in the original which might be errors. I've translated them as they are but they might need to be corrected in the original source for a more accurate translation context., about 4.0×10 6 , about 5.0×10 6 , about 6.0×10 6 , about 7.0×10 6 , about 8.0×10 6 , about 9.0×10 6 , about 1.0×10 7 , about 2.0×10 7 , about 3.0×10 7 , about 4.0×10 7 , 5.0×10 7 , about 6.0×10 7 , about 7.0×10 7 , about 8.0×10 7 , about 9.0×10 7 , or about 1.0×10 8 cells / kilogram of the subject's body weight. In some embodiments, an effective amount of a population of genetically engineered hematopoietic stem cells, e.g., hematopoietic stem cells in which CD33 as described herein is modified, is about 3.0×10 6 cells / kilogram of the subject's body weight.

[0075] In at least some embodiments, hematopoietic stem cells that can act as starting materials for generating genetically engineered hematopoietic stem cells, e.g., hematopoietic stem cells in which CD33 as described herein is modified, e.g., CD34+ hematopoietic stem cells, can be derived from one or more donors or can be obtained from autologous sources. In some embodiments, genetically engineered hematopoietic stem cells, e.g., hematopoietic stem cells in which CD33 as described herein is modified, are expanded in culture prior to administration to a subject that needs them.

[0076] Typical numbers of cells, e.g., immune cells or hematopoietic cells, administered to a mammal (e.g., a human) can be, for example, in the range of 1 million to 100 billion cells, although amounts below or above this exemplary range are also within the scope of the present disclosure.

[0077] In some embodiments, a cytotoxic agent, such as gemtuzumab ozogamicin, is used in combination with, for example, a population of genetically modified hematopoietic stem cells, such as the CD33-modified hematopoietic stem cells described herein, in a therapeutically effective dose. In some embodiments, the effective dose of the cytotoxic agent (e.g., gemtuzumab ozogamicin) is approximately 0.01 mg / m² of the target body surface area. 2 ~Approximately 3.0 mg / target body surface area m² 2 In some embodiments, the effective dose of the cytotoxic agent is approximately 0.05 mg / m² of the target body surface area. 2 ~Approximately 2.5 mg / target body surface area m² 2 Approximately 0.1 mg / target body surface area m 2 ~Approximately 2.0 mg / target body surface area m² 2 Approximately 0.1 mg / target body surface area m 2 ~Approximately 1.0 mg / target body surface area m² 2 Approximately 1.0 mg / target body surface area m 2 ~Approximately 2.0 mg / target body surface area m² 2 , or approximately 1.5 mg / target body surface area m² 2 ~Approximately 2.5 mg / target body surface area m² 2 In some embodiments, the effective dose of the cytotoxic agent is approximately 0.05 mg / m² of the target body surface area. 2 Approximately 0.1 mg / target body surface area m 2 Approximately 0.25 mg / target body surface area m² 2 Approximately 0.5 mg / target body surface area m 2 Approximately 1.0 mg / target body surface area m 2 Approximately 1.5 mg / target body surface area m² 2 Approximately 2.0 mg / target body surface area m 2 , or approximately 2.5 mg / target body surface area m 2 In some embodiments, the effective dose of the cytotoxic agent is approximately 2.0 mg / m² of the target body surface area. 2 That is the case.

[0078] In some embodiments, gemtuzumab ozogamicin is used in combination with, for example, genetically modified hematopoietic stem cells, such as a population of CD33-modified hematopoietic stem cells as described herein, in a therapeutically effective dose. In some embodiments, the effective dose of gemtuzumab ozogamicin is approximately 0.01 mg / m² of the target body surface area. 2 ~Approximately 3.0 mg / target body surface area m² 2 In some embodiments, the effective dose of gemtuzumab ozogamicin is approximately 0.05 mg / m² of the target body surface area. 2 ~Approximately 2.5 mg / target body surface area m² 2 Approximately 0.1 mg / target body surface area m 2 ~Approximately 2.0 mg / target body surface area m² 2 Approximately 0.1 mg / target body surface area m 2 ~Approximately 1.0 mg / target body surface area m² 2 Approximately 1.0 mg / target body surface area m 2 ~Approximately 2.0 mg / target body surface area m² 2 , or approximately 1.5 mg / target body surface area m² 2 ~Approximately 2.5 mg / target body surface area m² 2 In some embodiments, the effective dose of gemtuzumab ozogamicin is approximately 0.05 mg / m² of the target body surface area. 2 Approximately 0.1 mg / target body surface area m 2 Approximately 0.25 mg / target body surface area m² 2 Approximately 0.5 mg / target body surface area m 2 Approximately 1.0 mg / target body surface area m 2 Approximately 1.5 mg / target body surface area m² 2 Approximately 2.0 mg / target body surface area m 2 , or approximately 2.5 mg / target body surface area m 2 In some embodiments, the effective dose of gemtuzumab ozogamicin is approximately 2.0 mg / m² of the target body surface area. 2 That is the case.

[0079] In some embodiments, the effective dose of a population of genetically modified hematopoietic cells, such as the CD33-modified hematopoietic stem cells described herein, is about 10 4 Cells / Target body weight in kilograms ~ approximately 10 8The effective dose of a cytotoxic agent (e.g., gemtuzumab ozogamicin) is approximately 0.01 mg / m² of the target's body surface area, where the effective dose is cell / kilogram of the target's body weight. 2 ~Approximately 3.0 mg / m² of the target body surface area 2 In some embodiments, the effective amount of a population of genetically modified hematopoietic cells, for example, CD33-modified hematopoietic stem cells as described herein, is about 10 6 Cells / Target body weight in kilograms ~ approximately 10 7 The effective dose of a cytotoxic agent is approximately 0.1 mg / m² of the target's body surface area, with the effective dose being approximately 0.1 mg / m² of the target's body surface area. 2 ~Approximately 2.0 mg / m² of the target body surface area 2 In some embodiments, the effective amount of a population of genetically modified hematopoietic cells, such as the CD33-modified hematopoietic stem cells described herein, is approximately 3.0 × 10⁻⁶. 6 The effective dose of a cytotoxic agent is approximately 0.1 mg / m² of the target's body surface area, with the effective dose being approximately 0.1 mg / m² of the target's body surface area. 2 Approximately 0.25 mg / m² of the target body surface area 2 Approximately 0.5 mg / m² of the target body surface area 2 Approximately 1.0 mg / m² of the target body surface area 2 , or approximately 2.0 mg / m² of the target body surface area 2 In some embodiments, the effective amount of a population of genetically modified hematopoietic cells, such as the CD33-modified hematopoietic stem cells described herein, is approximately 3.0 × 10⁻⁶. 6 The effective dose of a cytotoxic agent is approximately 2.0 mg / m² of the target's body surface area, with the effective dose being approximately 2.0 mg / m² of the target's body surface area. 2 That is the case.

[0080] In some embodiments, the effective dose of a population of genetically modified hematopoietic cells, such as the CD33-modified hematopoietic stem cells described herein, is about 10 4 Cells / Target body weight in kilograms ~ approximately 10 8 The cell-to-body weight in kilograms is the effective dose of gemtuzumab ozogamicin, which is approximately 0.01 mg per square meter of the target's body surface area. 2 ~Approximately 3.0 mg / m² of the target body surface area 2In some embodiments, the effective amount of a population of genetically modified hematopoietic cells, for example, CD33-modified hematopoietic stem cells as described herein, is about 10 6 Cells / Target body weight in kilograms ~ approximately 10 7 The cell-to-body weight in kilograms is the effective dose of gemtuzumab ozogamicin, which is approximately 0.1 mg per square meter of the target's body surface area. 2 ~Approximately 2.0 mg / m² of the target body surface area 2 In some embodiments, the effective amount of a population of genetically modified hematopoietic cells, such as the CD33-modified hematopoietic stem cells described herein, is approximately 3.0 × 10⁻⁶. 6 The cell-to-body weight in kilograms is the effective dose of gemtuzumab ozogamicin, which is approximately 0.1 mg per square meter of the target's body surface area. 2 Approximately 0.25 mg / m² of the target body surface area 2 Approximately 0.5 mg / m² of the target body surface area 2 Approximately 1.0 mg / m² of the target body surface area 2 , or approximately 2.0 mg / m² of the target body surface area 2 In some embodiments, the effective amount of a population of genetically modified hematopoietic cells, such as the CD33-modified hematopoietic stem cells described herein, is approximately 3.0 × 10⁻⁶. 6 The cell-to-body weight in kilograms is the effective dose of gemtuzumab ozogamicin, which is approximately 2.0 mg per square meter of the target's body surface area. 2 That is the case.

[0081] In some embodiments, a population of genetically modified hematopoietic cells and a cytotoxic agent (e.g., gemtuzumab ozogamicin) are administered at the same time or at different times close together. As used herein, in some embodiments, close together refers to the timing of the administration of the population of genetically modified hematopoietic cells relative to the administration of the cytotoxic agent. Naturally, unless the ordering is explicitly stated, no particular ordering is implicit in the use of this term. For example, the administration of a population of genetically modified hematopoietic cells and a cytotoxic agent at close together may include the administration of hematopoiesis before, after, or nearly simultaneously with the administration of the cytotoxic agent. Furthermore, the treatments may be mixed or in separate volumes. For example, in some embodiments, combined administration includes administration in the same therapeutic process, for example, in a therapeutic process using anti-CD33 therapy, where the subject may be administered a sufficient number of CD33-modified cells simultaneously with or consecutively with the cytotoxic agent, for example, before, during, or after the treatment.

[0082] In some embodiments, a near-time dose includes administering a population of genetically modified hematopoietic stem cells and a cytotoxic agent in a single treatment regimen. In some embodiments, a near-time dose includes administering a population of genetically modified hematopoietic stem cells and a cytotoxic agent simultaneously or concurrently. In some embodiments, a near-time dose includes administering a population of genetically modified hematopoietic stem cells and a cytotoxic agent sequentially (e.g., administering one treatment before the other). In some embodiments, the population of genetically modified hematopoietic stem cells is administered before the cytotoxic agent. In some embodiments, a near-time dose includes administering a population of genetically modified hematopoietic stem cells within 120 days of the administration of the cytotoxic agent (e.g., within 90 days, 60 days, 30 days, 20 days, 10 days, 7 days, or 1 day). In some embodiments, the near-time administration includes administering the genetically modified hematopoietic stem cell population within 120 days prior to administration of a cytotoxic agent to the subject (e.g., within 90 days, 60 days, 30 days, 20 days, 10 days, 7 days, or 1 day). In some embodiments, the near-time administration includes administering the genetically modified hematopoietic stem cell population within 120 days after administration of a cytotoxic agent to the subject (e.g., within 90 days, 60 days, 30 days, 20 days, 10 days, 7 days, or 1 day).

[0083] In some embodiments, subjects are evaluated based on one or more parameters, such as the level of engraftment, after administration of a population of genetically modified hematopoietic cells and before administration of a cytotoxic agent as described herein. In some embodiments, subjects have a CD33-negative absolute neutrophil count (ANC) above a threshold (e.g., at least 1000 / dL) before administration of the cytotoxic agent.

[0084] In some embodiments, the cytotoxic agent is administered in multiple doses at regular intervals (e.g., weekly, every two weeks, every three weeks, every four weeks, monthly, every two months, every three months, every four months, every five months, or every six months). In some embodiments, the cytotoxic agent is administered in multiple effective doses every four weeks. For example, in some embodiments, an effective amount of the cytotoxic agent is administered in a first dose, followed by one or more subsequent effective doses, with each dose spaced about four weeks apart (e.g., 28 days). In some embodiments, each dose is spaced about two to six weeks apart (e.g., about two weeks, about three weeks, about four weeks, about five weeks, about six weeks, about three to five weeks, or about four to six weeks). In some embodiments, an effective amount of the cytotoxic agent is administered to the subject in at least one dose, at least two doses, at least three doses, one to six doses, one to four doses, one to three doses, or four doses. In some embodiments, each dose of the cytotoxic agent is approximately 2.0 mg / m². 2 In some embodiments, the cytotoxic agent is administered at approximately 2.0 mg / m² every four weeks. 2 It is administered in multiple doses.

[0085] In some embodiments, the subjects requiring treatment according to this disclosure are identified as those with newly diagnosed novel CD33-positive AML. When given as part of a combination regimen for the treatment of newly diagnosed novel CD33-positive AML, the recommended course of treatment including gemtuzumab ozogamicin consists of one induction cycle and two consolidation cycles. For the induction cycle, the recommended dose of gemtuzumab ozogamicin is 3 mg / m² on days 1, 4, and 7. 2 (Up to one 4.5 mg vial) is administered in combination with daunorubicin and cytarabine. For patients requiring a second induction cycle, gemtuzumab ozogamicin is not introduced during the second induction cycle. For the consolidation cycle, the recommended dose of gemtuzumab ozogamicin is 3 mg / m² on day 1 in combination with daunorubicin and cytarabine. 2 (Maximum of one 4.5 mg vial.)

[0086] In some embodiments, the subjects requiring treatment according to this disclosure are identified as having newly diagnosed CD33-positive AML. When administered as monotherapy for the treatment of newly diagnosed CD33-positive AML, the recommended course of treatment of gemtuzumab ozogamicin consists of one induction cycle and up to eight cycles of continuation therapy. For the induction cycle, the recommended dose of gemtuzumab ozogamicin as monotherapy is 6 mg / m² on day 1. 2 On the 8th day, 3 mg / m² 2 It is administered. For continued use, the recommended dose of gemtuzumab ozogamicin is 2 mg / m² as monotherapy on day 1 every 4 weeks. 2 That is the case.

[0087] In some embodiments, subjects requiring treatment according to this disclosure are diagnosed with, or suspected of having, relapsed, or refractory CD33-positive AML. When administered as monotherapy for the treatment of relapsed or refractory CD33-positive AML, the recommended dose of gemtuzumab ozogamicin is 3 mg / m² on days 1, 4, and 7. 2 (Maximum of one 4.5 mg vial.)

[0088] In some embodiments, subjects are pre-treated with one or more of the following: corticosteroids, antihistamines, and acetaminophen, prior to the administration of gemtuzumab ozogamicin. In some embodiments, subjects are pre-treated about 1 hour before the administration of gemtuzumab ozogamicin (e.g., about 30 minutes to 1.5 hours, about 45 minutes to 1.5 hours, about 1 to 2 hours, or about 45 minutes to 1 hour). In some embodiments, subjects are pre-treated with about 650 mg of acetaminophen (e.g., orally) and about 50 mg of diphenhydramine (e.g., orally or intravenously) 1 hour before the administration of gemtuzumab ozogamicin. In some embodiments, subjects are pre-treated with about 1 mg / kg of methylprednisolone or an equivalent dose of an alternative corticosteroid within 30 minutes before the administration of gemtuzumab ozogamicin. Children may be pre-treated orally or intravenously with acetaminophen 15 mg / kg (maximum 650 mg), diphenhydramine 1 mg / kg (maximum 50 mg), and methylprednisolone 1 mg / kg, with additional doses of acetaminophen and diphenhydramine administered every 4 hours of the initial treatment dose. Pre-treatment may be repeated with the same dose of methylprednisolone or an equivalent corticosteroid for any signs of infusion reaction, such as fever, chills, hypotension, or dyspnea, during or within 4 hours after infusion.

[0089] In some embodiments, the subjects do not have a homozygous dominant genotype for the CD33 single nucleotide polymorphism (SNP) rs12459419. In some embodiments, the subjects do not have acute promyelocytic leukemia or chronic myeloid leukemia. In some embodiments, the subjects do not have a translocation associated with acute promyelocytic leukemia or chronic myeloid leukemia, and optionally, the translocation is t(15;17)(q22;q21) or t(9;22)(q34;q11). In some embodiments, the subjects have not previously received autologous or allogeneic stem cell transplantation. In some embodiments, the subjects have not previously received cytotoxic agents.

[0090] In some embodiments, the method further includes determining the percentage of donor chimerization and / or the level of CD33-negative myeloid hematopoiesis in a peripheral blood sample derived from the subject.

[0091] In some embodiments, gemtuzumab ozogamicin is reconstituted from a lyophilized form before administration. In some embodiments, the lyophilized form contains approximately 4.5 mg of lyophilized cake or powder. In some embodiments, the lyophilized form contains lyophilized cake or powder in a single-dose vial for reconstitution and / or dilution.

[0092] In some embodiments, the subject may be treated with one or more antibodies that selectively bind to CD33 or its antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment that selectively binds to CD33 is linked to a toxin to target CD33-expressing cancer cells in the subject. Any antibody that selectively binds to CD33 may be used.

[0093] In some embodiments, subjects are preconditioned before administration of cytotoxic agents and / or a population of genetically modified hematopoietic stem cells. In some embodiments, preconditioning of subjects includes administering one or more chemotepas to the subjects. Examples of chemotepas include, but are not limited to, busulfan, melphalan, fludarabine, cyclophosphamide, and thiotepa. In some embodiments, preconditioning includes whole-body irradiation of the subjects. In some embodiments, preconditioning includes administering antibodies that bind to human T cells (e.g., rabbit anti-thymocyte globulin (rATG)). In some embodiments, preconditioning occurs within two weeks prior to administration of cytotoxic agents and / or hematopoietic cells (e.g., within 14 days, 12 days, 10 days, 9 days, 7 days). In some embodiments, preconditioning occurs over a period of about 1 to 10 days. In some embodiments, preconditioning occurs over a period of about 9 days.

[0094] In some embodiments, the compositions of the present disclosure (e.g., populations of hematopoietic cells, cytotoxic agents) are not limited to enteral (intestinal), gastrointestinal, epidural (intradural), oral (oral), percutaneous, epidural, intracerebral (intracerebral), intraventricular (intraventricular), supercutaneous (applied to the skin), intradermal (intracutaneous), subcutaneous (under the skin), nasal administration (through the nose), intravenous (intravenous), intravenous bolus, intravenous infusion, intraarterial (intraarterial), intramuscular (intramuscular), intracardiac (intracardiac), intraosseous injection (intramuscular), subarachnoid (intraspinal canal), intraperitoneal (intraperitoneal injection or injection), intravesical injection, intravitreous (through the eye), intracavitary injection. Intracellular (pathological sinus), intracavitary (base of the penis), intravaginal administration, intrauterine, extraamniotic administration, transdermal (dispersion via untreated skin for systemic distribution), transmucosal (dispersion via mucosa), transvaginal, gas injection (nasal breath), sublingual, sublabial, enema, eye drops (supraconjunctival), intraocular ear drops, auricle (intra-ear or via), oral cavity (towards the cheek), conjunctiva, skin, teeth (into lobes or multiple teeth), electroosmosis, intracervical, internal sinuses, intratracheal, extracorporeal, hemodialysis, infiltration, intrastitium, intraperitoneal, intraamniotic fluid, intraarticular, intrabile, intrabronchial, sacral, intracartilaginous, intrasacral (cauda equina), intracisional (cisticerebellum, medulla oblongata), intracorneal, dental cornea Intracavitary, intracoronary (in the coronary arteries), intracavernosal (in the expandable space of the corpus cavernosum of the penis), intradiscal (in the papilla), intraductal (in the duct of a gland), intraduodenum (in the duodenum), intradural (in or below the dura mater), intraepidermal (in the epidermis), intraesophageal (in the esophagus), intrastomal (in the stomach), intragingival (in the gingiva), intraileum (in the distal part of the small intestine), intrafocal (in or directly into a local lesion), intraluminal (in the lumen), intralymphatic (in the lymphatic fluid), intramedullary (in the medullary cavity of bone), intrameningeal (in the meningeal), intramyocardium (in the myocardium), intraocular (in the eye), intraovarian (in the ovary), intrapericardial (in the pericardial), intrapleural (in the pleura), intraprostatic (in the prostate), intrapulmonary (lung or (Inside the bronchi), inside the nose (inside the nose or perioboribular sinus), inside the spinal cord (inside the spinal column), inside the synovial membrane (inside the synovial cavity of a joint), inside the tendon (inside the tendon), inside the testis (inside the testis), inside the subarachnoid space (inside the cerebrospinal fluid at any level of the cerebrospinal axis), inside the thoracic cavity (inside the chest), inside the tubule (inside the renal tubule of an organ), inside the tumor (inside the tumor), inside the tympanic cavity (inside the middle ear), inside the blood vessel (inside a blood vessel or multiple blood vessels), inside the ventricle (inside the ventricle), iontophoresis (soluble salt ions enter the body's tissues by current density), irrigation (to immerse or flow into an open wound or body cavity), larynx (directly into the larynx), nasogastric (through the nose and into the stomach), occlusive dressing techniques (local route administration,Subsequently, the drug may be administered via routes such as (sealing the area covered by bandage), eye drops (outside the eye), oropharyngeal (directly into the mouth and pharynx), parenteral, transdermal, periarticular, epidural, perineurial, periodontal, rectal, respiratory (intra-airway by oral or nasal inhalation for local or systemic effects), retroocular (behind the pons or behind the eye), intramyocardial (intramyocardial), soft tissue, subarachnoid, subconjunctival, submucosal, local, transplacental (through or beyond the placenta), transtracheal (through the tracheal wall), middle ear (through or beyond the tympanic cavity), ureter (into the ureter), urethra (into the urethra), vagina, sacral block, diagnostic methods, nerve block, biliary perfusion, cardiac perfusion, photopheresis, and spinal cord. As will be understood by those skilled in the art, the administration of a population of genetically modified hematopoietic cells and cytotoxic agents may be carried out by the same route of administration (e.g., intravenous infusion) or by different routes of administration.

[0095] Modes of administration include injection, intravenous infusion, and / or ingestion. “Injection” includes, but is not limited to, intravenous, intramuscular, intra-arterial, subarachnoid, intraventricular, intraleninal, intraorbital, intradermal, intraperitoneal, transtracheal, subcutaneous, intra-articular, sublental, subarachnoid, intraspinal, intraventricular, and intrasternal injections and infusions. In some examples, the route is intravenous. For cell delivery, administration may be by injection or infusion. In some embodiments, a population of genetically engineered hematopoietic stem cells may be administered systemically. The terms “systemic administration,” “systemically administered,” “peripherally administered,” and “peripherally administered” refer to the administration of a population of progenitor cells other than directly to a target site, tissue, or organ, as a result of which it enters the circulatory system of the target and is therefore subject to metabolism and other similar processes.

[0096] The effectiveness of a treatment having a composition for the treatment of hematopoietic malignancies (e.g., AML) can be determined by those skilled in the art. However, a treatment is considered “effective” if any or all of the signs or symptoms of a hematopoietic malignancy are altered in a beneficial manner, or if other clinically acceptable symptoms or markers are improved or ameliorated. Effectiveness can also be measured by whether the individual does not worsen, as assessed by the need for hospitalization or medical intervention (e.g., whether the progression of the disease is stopped or at least delayed). Methods for measuring these indicators are known to those skilled in the art and / or are described herein. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or mammals) and includes (1) inhibiting the disease, e.g., stopping or delaying the progression of symptoms, or (2) alleviating the disease, e.g., causing regression of symptoms, and (3) preventing or reducing the likelihood of the onset of symptoms.

[0097] Clinical preparations comprising a population of cells, including either the genetically modified hematopoietic cells described herein or their offspring, are also provided herein. In some embodiments, the composition contains at least 1 × 10⁶ cells per milliliter (mL) of culture medium. 6 The cell population includes genetically modified hematopoietic cells or their offspring, which include a modified gene encoding CD33 that has been engineered to have reduced or eliminated CD33 antigen expression. In some embodiments, the population contains at least 2 × 10¹⁶ cells per mL. 6 Cells, at least 3 × 10⁶ per 1 mL 6 Cells, at least 4 × 10⁶ per 1 mL 6 Cells, at least 5 × 10 per 1 mL 6 Cells, at least 6 × 10⁶ per 1 mL 6 Cells, at least 7 × 10 per 1 mL 6 Cells, at least 8 × 10 per 1 mL 6 Cells, or at least 9 × 10 per 1 mL 6 Contains cells.

[0098] In some embodiments, the culture medium has a volume of approximately 5 to 150 mL. In some embodiments, the culture medium has a volume of approximately 10 to 100 mL. In some embodiments, the culture medium has a volume of approximately 25 to 75 mL. In some embodiments, the culture medium has a volume of approximately 30 to 70 mL. In some embodiments, the culture medium has a volume of approximately 40 to 60 mL. In some embodiments, the culture medium has a volume of approximately 45 mL. In some embodiments, the culture medium has a volume of approximately 30 mL. In some embodiments, the culture medium has a volume of approximately 35 mL. In some embodiments, the culture medium has a volume of approximately 40 mL. In some embodiments, the culture medium has a volume of approximately 50 mL. In some embodiments, the culture medium has a volume of approximately 55 mL. In some embodiments, the culture medium has a volume of approximately 60 mL. In some embodiments, the culture medium has a volume of approximately 70 mL. In some embodiments, the culture medium has a volume of approximately 40 to 50 mL. In some embodiments, the culture medium has a volume of approximately 40 mL, 41 mL, 42 mL, 43 mL, 44 mL, 45 mL, 46 mL, 47 mL, 48 mL, 49 mL, or approximately 50 mL. In some embodiments, the culture medium has a volume of approximately 45 mL.

[0099] In some embodiments, the composition is approximately 1 × 10 in total in the culture medium. 6 ~1 × 10 8 It contains a population of cells. In some embodiments, the composition contains a total of about 1 × 10⁶ cells in the culture medium. 7 , 2×10 7 , 3 x 10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , or 1 × 10 8 It contains a population of cells. In some embodiments, the population is at least 0.5 × 10⁶ per mL. 6 Each cell, at least 1 × 10⁶ cells per 1 mL 6 Each cell, at least 2 × 10⁶ cells per 1 mL 6 Each cell, at least 3 × 10⁶ cells per 1 mL 6 Each cell, at least 4 × 10⁶ cells per 1 mL6 Each cell, at least 5 × 10⁶ cells per 1 mL 6 Each cell, at least 6 × 10⁶ cells per 1 mL 6 Each cell, at least 7 × 10⁶ cells per 1 mL 6 Each cell, at least 8 × 10⁶ cells per 1 mL 6 Individual cells, or at least 9 × 10⁶ cells per 1 mL. 6 It contains individual cells. In some embodiments, the population is at least 0.5 × 10 per mL. 6 Each cell, at least 1 × 10⁶ cells per 1 mL 6 Each cell, at least 2 × 10⁶ cells per 1 mL 6 Each cell, at least 3 × 10⁶ cells per 1 mL 6 Each cell, at least 4 × 10⁶ cells per 1 mL 6 Each cell, at least 5 × 10⁶ cells per 1 mL 6 Each cell, at least 6 × 10⁶ cells per 1 mL 6 Each cell, at least 7 × 10⁶ cells per 1 mL 6 Each cell, at least 8 × 10⁶ cells per 1 mL 6 Individual cells, or at least 9 × 10⁶ cells per 1 mL. 6 It contains a number of cells. In some embodiments, the cell population is at least 1 × 10 9 A single living cell, at least 2 × 10⁶ 9 A single living cell, at least 3 × 10⁶ 9 A single living cell, at least 4 × 10⁶ 9 A single living cell, at least 5 × 10⁶ 9 A single living cell, at least 6 × 10⁶ 9 A single living cell, at least 7 × 10⁶ 9 A single living cell, at least 8 × 10⁶ 9 A single living cell, at least 9 × 10⁶ 9 A single living cell, at least 1 × 10⁶ 10 A single living cell, at least 2 × 10⁶ 10 A single living cell, at least 3 × 10⁶ 10 A single living cell, at least 4 × 10⁶ 10 A single living cell, at least 5 × 10⁶ 10 A single living cell, at least 6 × 10⁶ 10 A single living cell, at least 7 × 10⁶10 A single living cell, at least 8 × 10⁶ 10 A single living cell, at least 9 × 10⁶ 1 0 living cells, or at least 1 × 10⁶ 11 The organism comprises living cells, and in some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the cells in the population are genetically modified hematopoietic cells or their offspring having reduced or eliminated CD33 antigen expression.

[0100] In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in a population are genetically modified hematopoietic cells or their offspring that have reduced or eliminated CD33 antigen expression.

[0101] In some embodiments, the culture medium is a cryopreservation medium containing an antifreeze agent. Non-limiting examples of antifreeze agents include cetamide, agarose, alginate, 1-analine, albumin, ammonium acetate, butanediol, chondroitin sulfate, chloroform, choline, diethylene glycol, dimethylacetamide, dimethylformamide, dimethyl sulfoxide (DMSO), erythritol, ethanol, ethylene glycol, formamide, glucose, glycerol, α-glycerol phosphate, glycerol monoacetate, glycine, hydroxyethyl starch, inositol, lactose, and magnesium chloride. Examples include magnesium sulfate, maltose, mannitol, mannose, methanol, methylacetamide, methylformamide, methylurea, phenol, pluronic polyol, polyethylene glycol, polyvinylpyrrolidone, proline, propylene glycol, pyridine N-oxide, ribose, serine, sodium bromide, sodium chloride, sodium iodide, sodium nitrate, sodium sulfate, sorbitol, sucrose, trehalose, triethylene glycol, trimethylamine acetate, urea, valine, and xylose. In some embodiments, the antifreeze agent contains DMSO in an amount of about 10% (v / v).

[0102] In some embodiments, the composition is in a frozen state. In some embodiments, the composition is subjected to a cryopreservation process. As will be apparent to those skilled in the art, a cryopreservation process is a method aimed at preserving (viable) cells by, for example, cooling a sample containing cells to a low temperature (e.g., below -80°C) and storing it. In some embodiments, the cryopreservation process is a rate-controlled freezing.

[0103] Genome editing Aspects of this disclosure relate to a population of genetically engineered hematopoietic cells or their offspring, comprising a modified gene encoding CD33 which has been engineered to have reduced or eliminated expression of the CD33 antigen. The gene encoding CD33 may be engineered by any means known in the art so that the cell has reduced or eliminated expression of the CD33 antigen.

[0104] As used herein with respect to gRNA interactions with a target domain, the term “binding” refers to the gRNA molecule and the target domain that forms a complex. The complex may include two strands forming a double-stranded structure, or three or more strands forming a multi-stranded complex. Binding may constitute a step in a broader process, such as cleavage of the target domain by a Cas endonuclease. In some embodiments, the gRNA binds to the target domain in complete complementarity, while in other embodiments, the gRNA binds to the target domain in partial complementarity, for example, with one or more mismatches. In some embodiments, when the gRNA binds to the target domain, the entire target domain of gRNA bases pairs with the target domain. In other embodiments, only portions of the target domain and / or only portions of the target domain bases pair with each other. In one embodiment, the interaction is sufficient to mediate a target domain-mediated cleavage phenomenon.

[0105] As used herein, the term “Cas9 molecule” refers to a molecule or polypeptide that interacts with and, in cooperation with gRNA, can migrate to or localize to a site containing a target domain. Cas9 molecules include naturally occurring Cas9 molecules and manipulated, altered, or modified Cas9 molecules that differ from, for example, naturally occurring Cas9 molecules by at least one amino acid residue.

[0106] The terms “gRNA” and “guide RNA” are used interchangeably throughout this text and refer to nucleic acids that facilitate the specific targeting or homing of the gRNA / Cas9 molecular complex to a target nucleic acid. gRNA may be monomolecule (having a single RNA molecule) or modular (containing more than one, typically two distinct RNA molecules), and may be referred to herein as sgRNA. The gRNA may bind to a target domain in the host cell’s genome. The gRNA (e.g., its targeting domain) may be partially or fully complementary to the target domain. The gRNA may also contain a “scaffolding sequence” (e.g., a tracrRNA sequence) that recruits the Cas9 molecule to the target domain bound to the gRNA sequence (e.g., by the targeting domain of the gRNA sequence). The scaffolding sequence may contain at least one stem-loop structure and recruit an endonuclease. Exemplary scaffolding arrangements can be found, for example, in Jinek, et al. Science (2012) 337(6096):816-821, Ran, et al. Nature Protocols (2013) 8:2281-2308, PCT Publication WO2014 / 093694, and PCT Publication WO2013 / 176772.

[0107] The term “mutation” is used herein to refer to a genetic alteration (e.g., insertion, deletion, or substitution) of a nucleic acid compared to a reference sequence, e.g., the corresponding wild-type nucleic acid. In some embodiments, a mutation in a gene detargets the protein produced by that gene. In some embodiments, the detargeted CD33 protein is not bound, or is bound, to CD33-targeting drugs.

[0108] The “target domain” of a gRNA is complementary to the “target domain” on the target nucleic acid. The chain of target nucleic acid containing a nucleotide sequence complementary to the core domain of the gRNA is referred to herein as the “complementary chain” of the target nucleic acid. Guidance on the selection of target domains can be found, for example, in Fu Y et al, Nat Biotechnol (2014) 32:279-284 (doi:10.1038 / nbt.2808) and Sternberg SH et al., Nature (2014) 507(7490):62-7 (doi:10.1038 / naturel3011).

[0109] Nuclease In some embodiments, the cells described herein (e.g., HSCs or HPCs) are prepared using the nucleases described herein. Exemplary nucleases include Cas molecules (e.g., Cas9 or Cas12a), TALENs, ZFNs, and meganucleases. In some embodiments, the nucleases are used in combination with the CD33 gRNAs described herein (e.g., according to Table 3).

[0110] Cas9 molecule In some embodiments, the CD33 gRNA described herein is complexed with a Cas9 molecule. Various Cas9 molecules may be used. In some embodiments, a Cas9 molecule with desired PAM specificity is selected to target the gRNA / Cas9 molecule complex against a target domain in CD33. In some embodiments, genetic engineering of cells involves introducing one or more (e.g., one, two, three or more) Cas9 molecules into the cells.

[0111] Various species of Cas9 molecules can be used in the methods and compositions described herein. In one embodiment, the Cas9 molecule is of or derived from Streptococcus pyogenes (SpCas9), Staphylococcus aureus (SaCas9), or Streptococcus thermophilus. Additional suitable Cas9 molecules include Staphylococcus aureus, Neisseria meningitidis (NmCas9), Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces, Cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni(CjCas9), Campylobacter lari, Candidatus puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, gamma proteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeriaivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum spp., Simonsiella muelleri, Sphingomonas spp., Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcus, Subdoligranulum, Tistrella mobilis, Treponema, or Verminephrobacter This includes things from or derived from eiseniae.

[0112] In some embodiments, the Cas9 molecule is a naturally occurring Cas9 molecule. In some embodiments, the Cas9 molecule is an engineered, modified, or altered Cas9 molecule that differs, for example, by at least one amino acid residue from a reference sequence, such as the most similar naturally occurring Cas9 molecule, or the sequence in Table 50 of PCT Publication WO2015 / 157070, which is incorporated herein by reference in its entirety.

[0113] Naturally occurring Cas9 molecules typically comprise two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe, each of which further comprises domains described, for example, in PCT Publication WO2015 / 157070, for example, in Figures 9A-9B therein (the application is incorporated herein by reference in its entirety).

[0114] The REC lobe contains an arginine-rich bridge helix (BH), the REC1 domain, and the REC2 domain. The REC lobe is considered to be a Cas9-specific functional domain. The BH domain is a long alpha helix and arginine-rich region containing amino acids 60-93 of the S. pyogenes Cas9 sequence. The REC1 domain is involved in the recognition of repeat:anti-repeat double strands, for example, in gRNA or tracrRNA. The REC1 domain contains two REC1 motifs at amino acids 94-179 and 308-717 of the S. pyogenes Cas9 sequence. These two REC1 domains are separated by the REC2 domain in the linear primary structure, but assemble in the tertiary structure to form the REC1 domain. The REC2 domain, or a part thereof, may also play a role in the recognition of repeat:anti-repeat double strands. The REC2 domain contains amino acids 180-307 of the S. pyogenes Cas9 sequence.

[0115] The NUC lobe comprises a RuvC domain (also referred herein as a RuvC-like domain), an HNH domain (also referred herein as an HNH-like domain), and a PAM interaction (PI) domain. The RuvC domain shares structural similarities with members of the retroviral integrase superfamily and cleaves single strands, such as the non-complementary strand of a target nucleic acid molecule. The RuvC domain is assembled from three splintered RuvC motifs (commonly referred in the art as the RuvCI domain, or the N-terminal RuvC domain, RuvCII domain, and RuvCIII domain, respectively) at amino acids 1-59, 718-769, and 909-1098 of the S. pyogenes Cas9 sequence. Similar to the REC1 domain, the three RuvC motifs are linearly separated by other domains in the primary structure, but in the tertiary structure, the three RuvC motifs assemble to form the RuvC domain. The HNH domain shares structural similarities with HNH endonucleases and cleaves single-stranded proteins, such as the complementary strand of a target nucleic acid molecule. The HNH domain is located between the RuvC II-III motifs and contains amino acids 775-908 of the S. pyogenes Cas9 sequence. The PI domain interacts with the PAM of the target nucleic acid molecule and contains amino acids 1099-1368 of the S. pyogenes Cas9 sequence.

[0116] The crystal structure has been determined for naturally occurring bacterial Cas9 molecules (see, e.g., Jinek et al., Science, (2014) 343(6176):1247997) and S. pyogenes Cas9 with guide RNA (e.g., synthetic fusion of crRNA and tracrRNA) (see Nishimasu et al., Cell (2014) 156:935-949 and Anders et al., Nature (2014) doi:10.1038 / naturel3579).

[0117] In some embodiments, the Cas9 molecule described herein has nuclease activity, e.g., double-strand break activity. In some embodiments, the Cas9 molecule is modified to inactivate one of the catalytic residues of an endonuclease. In some embodiments, the Cas9 molecule is a nickase and produces single-strand breaks. See, for example, Dabrowska et al. Frontiers in Neuroscience (2018) 12(75). It has been shown that one or more mutations in the RuvC and HNH catalytic domains of the enzyme can improve Cas9 efficiency. See, for example, Sarai et al. Currently Pharma. Biotechnol. (2017) 18(13). In some embodiments, the Cas9 molecule is fused to a second domain, e.g., a domain that modifies DNA or chromatin, e.g., a deaminase or demethylase domain. In some such embodiments, the Cas9 molecule is modified to eliminate its endonuclease activity.

[0118] In some embodiments, the Cas9 molecule described herein is administered together with a template for homologous inducible repair (HDR). In some embodiments, the Cas9 molecule described herein is administered without an HDR template.

[0119] In some embodiments, Cas9 molecules are used that are modified to enhance enzyme specificity (e.g., to reduce off-target effects and maintain robust target cleavage). In some embodiments, the Cas9 molecule is an enhanced specificity Cas9 variant (e.g., eSPCas9). See, for example, Slaymaker et al. Science (2016) 351(6268):84-88. In some embodiments, the Cas9 molecule is a high fidelity Cas9 variant (e.g., SpCas9-HF1). See, for example, Kleinstiver et al. Nature (2016) 529:490-495.

[0120] Various Cas9 molecules are known in the art, available from various sources, and can be manipulated / modified to modulate one or more of the enzyme's activity or specificity. In some embodiments, Cas9 molecules are manipulated / modified to recognize one or more PAM sequences. In some embodiments, Cas9 molecules are manipulated / modified to recognize one or more PAM sequences different from those recognized by the Cas9 molecule without manipulation / modification. In some embodiments, Cas9 molecules are manipulated / modified to reduce the enzyme's off-target activity.

[0121] In some embodiments, the nucleotide sequence encoding the Cas9 molecule is further modified to alter the specificity of endonuclease activity (e.g., to reduce off-target cleavage, decrease intracellular endonuclease activity or survival time, increase homologous recombination, and reduce non-homologous end joining). See, for example, Komor et al. Cell (2017) 168:20-36. In some embodiments, the nucleotide sequence encoding the Cas9 molecule is modified to alter the PAM recognition of the endonuclease. For example, the Cas9 molecule SpCas9 recognizes the PAM sequence NGG, while incomplete variants of SpCas9, including one or more modifications of the endonuclease (e.g., VQR SpCas9, EQR SpCas9, VRER SpCas9), may recognize the PAM sequences NGA, NGAG, and NGCG. PAM recognition of a modified Cas9 molecule is considered "incomplete" if the Cas9 molecule recognizes more potential PAM sequences compared to an unmodified Cas9 molecule. For example, while the Cas9 molecule SaCas9 recognizes the PAM sequence NNGRRT, an incomplete variant of SaCas9 containing one or more modifications (e.g., KKH SaCas9) may recognize the PAM sequence NNNRRT. Another example is the Cas9 molecule FnCas9, which recognizes the PAM sequence NNG, while an incomplete variant of FnCas9 containing one or more modifications to the endonuclease (e.g., RHA FnCas9) may recognize the PAM sequence YG. Another example is a Cas9 molecule that is a Cpf1 endonuclease containing the substitution mutations S542R and K607R and recognizes the PAM sequence TYCV. Another example is a Cas9 molecule that is a Cpf1 endonuclease containing the substitution mutations S542R, K607R, and N552R and recognizes the PAM sequence TATV. For example, see Gao et al. Nat. Biotechnol. (2017) 35(8):789-792.

[0122] In some embodiments, more than one (e.g., two, three, or more) Cas molecules, such as Cas9 molecules, are used. In some embodiments, at least one of the Cas9 molecules is the Cas9 enzyme. In some embodiments, at least one of the Cas molecules is the Cpf1 enzyme. In some embodiments, at least one of the Cas9 molecules is derived from Streptococcus pyogenes. In some embodiments, at least one of the Cas9 molecules is derived from Streptococcus pyogenes, and at least one Cas9 molecule is derived from an organism other than Streptococcus pyogenes.

[0123] In some embodiments, the Cas9 molecule is a base editor. Base editor endonucleases generally contain a catalytically inactive Cas9 molecule fused to a functional domain. See, for example, Eid et al. Biochem. J. (2018) 475(11):1955-1964 and Rees et al. Nature Reviews Genetics (2018) 19:770-788. In some embodiments, the catalytically inactive Cas9 molecule is dCas9. In some embodiments, the catalytically inactive Cas9 molecule (dCas9) is fused to one or more uracilglycosylase inhibitor (UGI) domains. In some embodiments, the endonuclease contains dCas9 fused to an adenine base editor (ABE), e.g., an ABE evolved from RNA adenine deaminase TadA. In some embodiments, the endonuclease comprises dCas9 fused to a cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDA1, activation-induced cytidine deaminase (AID)). In some embodiments, the catalytically inactive Cas9 molecule is nCas9, having reduced activity. In some embodiments, the Cas9 molecule comprises nCas9 fused to one or more uracilglycosylase inhibitor (UGI) domains. In some embodiments, the Cas9 molecule comprises nCas9 fused to an adenine base editor (ABE), e.g., an ABE evolved from RNA adenine deaminase TadA. In some embodiments, the Cas9 molecule comprises nCas9 fused to a cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDA1, activation-induced cytidine deaminase (AID)).

[0124] Examples of base editors, though not limited to them, include BE1, BE2, BE3, HF-BE3, BE4, BE4max, BE4-Gam, YE1-BE3, EE-BE3, YE2-BE3, YEE-CE3, VQR-BE3, VRER-BE3, SaBE3, SaBE4, SaBE4-Gam, Sa(KKH)-BE3, Target-AID, Target-AID-NG, xBE3, eA3A-BE3, BE-PLUS, TAM, CRISPR-X, ABE7.9, ABE7.10, ABE7.10*, xABE, ABESa, VQR-ABE, VRER-ABE, Sa(KKH)-ABE, and CRISPR-SKIP. Examples of additional base editors can be found, for example, in U.S. Publication No. 2018 / 0312825A1, U.S. Publication No. 2018 / 0312828A1, and PCT Publication No. 2018 / 165629A1, which are incorporated herein by reference in their entirety.

[0125] In some embodiments, the base editor has been further modified to inhibit base excision repair at a target site and induce cellular mismatch repair. Any of the Cas9 molecules described herein can be fused to a Gam domain (bacteriophage Mu protein) to protect the Cas9 molecule from degradation and exonuclease activity. See, for example, Eid et al. Biochem. J. (2018) 475(11):1955-1964.

[0126] In some embodiments, the Cas9 molecule belongs to class 2V Cas endonucleases. Class 2V Cas endonucleases can be further classified as VA, VB, VC, and VU types. See, for example, Stella et al. Nature Structural & Molecular Biology (2017). In some embodiments, the Cas molecule is a VA type Cas endonuclease, such as Cpf1 nuclease. In some embodiments, the Cas9 molecule is a VB type Cas endonuclease, such as C2c1 endonuclease. See, for example, Shmakov et al. Mol Cell (2015) 60:385-397. In some embodiments, the Cas molecule is Mad7 (Inscripta). Alternatively, or in addition, the Cas9 molecule is a Cpf1 nuclease or a variant thereof. As will be understood by those skilled in the art, the Cpf1 nuclease may also be called Cas12a. See, for example, Strohkendl et al. Mol. Cell (2018) 71:1-9. In some embodiments, the compositions or methods described herein include a Cpf1 nuclease derived from the genera Prevotella or Francisella, the genera Acidaminococcus (AsCpf1), the bacterium Lachnospira (LpCpf1), or Eubacterium rectore, or a host cell expressing it. In some embodiments, the nucleotide sequence encoding the Cpf1 nuclease may be a codon optimized for expression in the host cell. In some embodiments, the nucleotide sequence encoding the Cpf1 endonuclease is further modified to alter the activity of the protein.

[0127] In some embodiments, catalytically inactive variants of Cas molecules (e.g., Cas9 or Cas12a) are used according to the methods described herein. A catalytically inactive variant of Cpf1(Cas12a) may be called dCas12a. As described herein, a catalytically inactive variant of Cpf1 may be fused to a functional domain to form a base editor. See, for example, Rees et al. Nature Reviews Genetics (2018) 19:770-788. In some embodiments, the catalytically inactive Cas9 molecule is dCas9. In some embodiments, the endonuclease comprises dCas12a fused to one or more uracilglycosylase inhibitor (UGI) domains. In some embodiments, the Cas9 molecule comprises dCas12a fused to an adenine base editor (ABE), for example, an ABE evolved from RNA adenine deaminase TadA. In some embodiments, the Cas molecule includes dCas12a fused to a cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDA1, activation-induced cytidine deaminase (AID)).

[0128] Alternatively, or in addition, the Cas9 molecule is a Cas14 endonuclease or a variant thereof. Cas14 endonucleases originate from archaea and tend to be small in size (e.g., 400-700 amino acids). Furthermore, Cas14 endonucleases do not require a PAM sequence. See, for example, Harrington et al. Science (2018) 362(6416).

[0129] Any of the Cas9 molecules described herein may be regulated to control the level of Cas9 expression and / or activity at a desired time. For example, it may be advantageous to increase the level of Cas9 expression and / or activity during a specific phase(s) of the cell cycle. It has been demonstrated that the level of homology-designated repair is reduced during the G1 phase of the cell cycle, and therefore, increasing the level of Cas9 expression and / or activity during the S, G2, and / or M phases may increase homology-designated repair after Cas endonuclease editing. In some embodiments, the level of Cas9 expression and / or activity is increased during the S, G2, and / or M phases of the cell cycle. In one example, the Cas9 molecule is fused to the N-terminal region of human geminin. See, for example, Gutschner et al. Cell Rep. (2016) 14(6):1555-1566. In some embodiments, the level of Cas9 expression and / or activity is reduced during the G1 phase of the cell cycle. In one example, the Cas9 molecule is modified so that its activity decreases during the G1 phase. See, for example, Lomova et al. Stem Cells (2018) 37(2):284-294.

[0130] Alternatively, or in addition, any of the Cas9 molecules described herein may be fused to an epigenetic modifier (e.g., a chromatin modifying enzyme, e.g., DNA methylase, histone deacetylase). See, for example, Kungulovski et al. Trends Genet. (2016) 32(2):101-113. A Cas9 molecule fused to an epigenetic modifier is called an “epieffector” and may enable transient and / or transient endonuclease activity. In some embodiments, the Cas9 molecule is dCas9 fused to a chromatin modifying enzyme.

[0131] Zinc finger nuclease In some embodiments, the cells or cell populations described herein are produced using zinc finger (ZFN) technology. In some embodiments, the ZFN recognizes target domains as described herein, for example, in Table 1. Generally, zinc finger-mediated genome editing involves the use of a zinc finger nuclease, which typically comprises a zinc finger DNA-binding domain and a nuclease domain. The zinc finger-binding domain may be engineered to recognize and bind to any target domain of interest, and may be designed to recognize, for example, DNA sequences in the range of about 3 to about 21 nucleotides in length, or about 8 to about 19 nucleotides in length. The zinc finger-binding domain typically comprises at least three zinc finger-recognition regions (e.g., zinc fingers).

[0132] Restriction endonucleases (restriction enzymes) capable of sequence-specific binding to DNA (at the recognition site) and cleaving DNA at or near the binding site are known in the art and can be used to form ZFNs for use in genome editing. For example, IIS-type restriction endonucleases cleave DNA at the site removed from the recognition site and have separable binding and cleavage domains. In one example, the DNA cleavage domain may be derived from a FokI endonuclease.

[0133] TALEN In some embodiments, the cells or cell populations described herein are produced using TALEN technology. In some embodiments, TALEN recognizes target domains as described herein, for example, in Table 1. Generally, TALEN is an engineered restriction enzyme capable of specifically binding to and cleaving a desired target DNA molecule. TALEN typically contains a transcription activator-like effector (TALE) DNA-binding domain fused to a DNA-cleaving domain. The DNA-binding domain may contain a highly conserved 33-34 amino acid sequence with a branched 2-amino acid RVD (repeating variable dipeptide motif) at positions 12 and 13. The RVD motif can be engineered to determine binding specificity to nucleic acid sequences and to specifically bind to a desired DNA sequence. In one example, the DNA-cleaving domain may be derived from a FokI endonuclease. In some embodiments, the FokI domain functions as a dimer using two constructs, each having a DNA-binding domain specific to a site in the target genome at an appropriate orientation and spacing.

[0134] Double-strand breaks (DSBs) can be produced in cells using TALENs specific to the target gene. If the repair mechanism improperly repairs the damage via non-homologous end joining, mutations can be introduced at the break site. For example, improper repair can result in frameshift mutations. Alternatively, a foreign DNA molecule with a desired sequence can be introduced into the cell along with the TALEN. Depending on the sequence of the foreign DNA and the chromosomal sequence, this process can be used to correct defects, introduce DNA fragments into the target gene, or introduce such defects into endogenous genes, thereby reducing the expression of the target gene.

[0135] Some exemplary, non-limiting embodiments of endonucleases and nuclease variants suitable for use in connection with the guide RNA and genetic manipulation methods provided herein are described above. Additional suitable nucleases and nuclease variants will be apparent to those skilled in the art based on this disclosure and knowledge of the art. This disclosure is not limited in this respect.

[0136] gRNA sequence and arrangement gRNAs can contain multiple domains. In one embodiment, a single molecule, sgRNA, or chimeric gRNA may include, for example, a target domain (complementary to the target nucleic acid in the CD33 gene), a first complementarity domain, a ligation domain, a second complementarity domain (complementary to the first complementarity domain), a proximal domain, and optionally, a tail domain, from 5' to 3'. Each of these domains is described in more detail here.

[0137] The target domain may include a nucleotide sequence that is, for example, 80, 85, 90, or 95% complementary to the target sequence on the target nucleic acid, e.g., complete complementarity. The target domain is part of an RNA molecule and therefore contains the base uracil (U), while any DNA encoding a gRNA molecule contains the base thymine (T). Although we do not wish to be constrained by theory, in one embodiment, the complementarity of the target domain to the target sequence is thought to contribute to the specificity of the interaction of the gRNA / Cas9 molecular complex with the target nucleic acid. In the target domain and target sequence pair, it is understood that the uracil bases in the target domain pair with the adenine bases in the target sequence. In one embodiment, the target domain itself includes an optional secondary domain and a core domain in the 5' to 3' direction. In one embodiment, the core domain is completely complementary to the target sequence. In one embodiment, the target domain is 5 to 50 nucleotides long. The target domain may be 15 to 25 nucleotides long, 18 to 22 nucleotides long, or 19 to 21 nucleotides long. In some embodiments, the target domain is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. In some embodiments, the target domain is 10–30, or 15–25 nucleotides long.

[0138] In some embodiments, the targeting domain includes a core domain and a secondary targeting domain, as described in PCT Publication WO2015 / 157070, which is incorporated in its entirety, for example, by reference. In one embodiment, the core domain includes about 8 to about 13 nucleotides from the 3' end of the targeting domain (e.g., the 8 to 13 nucleotides at the very 3' position of the targeting domain). In one embodiment, the secondary domain is positioned 5' relative to the core domain. In many embodiments, the core domain has exact complementarity with the corresponding region of the target sequence. In other embodiments, the core domain may include one or more nucleotides that are not complementary to the corresponding nucleotides of the target sequence.

[0139] The first complementary domain is complementary to the second complementary domain and, in one embodiment, is sufficiently complementary to the second complementary domain to form a double-stranded region under at least some physiological conditions. In one embodiment, the first complementary domain is 5 to 30 nucleotides in length. In one embodiment, the first complementary domain includes three subdomains, which are a 5' subdomain, a central subdomain, and a 3' subdomain, in the 5' to 3' direction. In one embodiment, the 5' subdomain is 4 to 9 nucleotides in length, for example, 4, 5, 6, 7, 8, or 9 nucleotides. In one embodiment, the central subdomain is 1, 2, or 3 nucleotides in length, for example, 1 nucleotide. In one embodiment, the 3' subdomain is 3 to 25 nucleotides in length, for example, 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. The first complementarity domain may share homology with or derive from a naturally occurring first complementarity domain. In one embodiment, it has at least 50% homology with the first complementarity domain of S. pyogenes, S. aureus, or S. thermophilus.

[0140] The arrangement and configuration of the domains described above are described in detail in PCT Publication WO2015 / 157070, which is incorporated herein by reference in its entirety, including pages 88–112.

[0141] The binding domain plays a role in binding the first complementarity domain of a single gRNA molecule to the second complementarity domain. The binding domain can bind the first and second complementarity domains covalently or non-covalently. In one embodiment, the binding is covalent. In one embodiment, the binding domain is or includes a covalent bond interposed between the first and second complementarity domains. In some embodiments, the binding domain contains one or more nucleotides, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the binding domain contains at least one non-nucleotide bond, for example, disclosed in PCT Publication WO2018 / 126176, the entire contents of which are incorporated herein by reference.

[0142] The second complementary domain is at least partially complementary to the first complementary domain and, in one embodiment, is sufficiently complementary to the second complementary domain to form a double-stranded region under at least some physiological conditions. In one embodiment, the second complementary domain may include sequences that are not complementary to the first complementary domain, such as sequences that form a loop from the double-stranded region. In one embodiment, the second complementary domain is 5 to 27 nucleotides long. In one embodiment, the second complementary domain is longer than the first complementary region. In one embodiment, the complementary domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. In one embodiment, the second complementary domain includes three subdomains, which are the 5' subdomain, the central subdomain, and the 3' subdomain, in the 5' to 3' direction. In one embodiment, the 5' subdomain is 3 to 25 nucleotides long, for example, 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In one embodiment, the central subdomain is 1, 2, 3, 4, or 5 nucleotides long, for example, 3 nucleotides. In one embodiment, the 3' subdomain is 4 to 9 nucleotides long, for example, 4, 5, 6, 7, 8, or 9 nucleotides. In one embodiment, the 5' and 3' subdomains of the first complementarity domain are complementary, for example, perfectly complementary, to the 3' and 5' subdomains of the second complementarity domain, respectively.

[0143] In one embodiment, the proximal domain is 5 to 20 nucleotides in length. In one embodiment, the proximal domain may share homology with or be derived from a naturally occurring proximal domain. In one embodiment, it has at least 50% homology with the proximal domain of S. pyogenes, S. aureus, or S. thermophilus.

[0144] A wide range of tail domains are suitable for use with gRNA. In one embodiment, the tail domain is 0 (absent), 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, the tail domain nucleotides are derived from or share homology with sequences from the 5' end of naturally occurring tail domains. In one embodiment, the tail domains are complementary to each other and contain sequences that form a double-stranded region under at least some physiological conditions. In one embodiment, the tail domain is absent or has a length of 1 to 50 nucleotides. In one embodiment, the tail domain may share homology with or be derived from naturally occurring proximal tail domains. In one embodiment, this has at least 50% homology with the S. pyogenes, S. aureus, or S. thermophilus tail domains. In one embodiment, the tail domain contains nucleotides at its 3' end that are relevant to in vitro or in vivo transcription methods.

[0145] In some embodiments, the modular gRNA comprises, for example, a first strand comprising a target domain (complementary to the target nucleic acid in the CD33 gene) and a first complementarity domain from 5' to 3', and preferably a second strand comprising an optional 5' extension domain, a second complementarity domain, a proximal domain, and optionally a tail domain from 5' to 3'.

[0146] In some embodiments, the gRNA is chemically modified. For example, the gRNA may include one or more modifications selected from phosphorothioate backbone modifications, 2'-O-Me modified sugars (e.g., one or both of the 3' and 5' ends), 2'-F modified sugars, substitution of a ribose sugar with a bicyclic nucleotide-cEt, 3' thioPACE (MSP), or any combination thereof. Suitable gRNA modifications are described, for example, in Rahdar et al. PNAS (2015) 112(51) E7110-E7117 and Hendel et al., Nat Biotechnol. 2015) Sep;33(9):985-989, which are incorporated herein by reference in their entirety. In some embodiments, the gRNA described herein includes one or more 2'-O-methyl-3'-phosphorothioate nucleotides, for example, at least 2, 3, 4, 5, or 6 2'-O-methyl-3'-phosphorothioate nucleotides. In some embodiments, the gRNAs described herein include modified nucleotides (e.g., 2'-O-methyl-3'-phosphorothioate nucleotides) at three terminal positions and the 5' end, and / or at three terminal positions and the 3' end. In some embodiments, the gRNAs may include one or more modified nucleotides, as described in PCT Publications WO2017 / 214460, WO2016 / 089433, and WO2016 / 164356, which are incorporated in their entirety by reference, for example.

[0147] In some embodiments, the gRNAs described herein are chemically modified. For example, a gRNA may contain one or more 2'-O modified nucleotides, such as 2'-O-methylnucleotides. In some embodiments, the gRNA contains a 2'-O modified nucleotide, such as 2'-O-methylnucleotide, at its 5' end. In some embodiments, the gRNA contains a 2'-O modified nucleotide, such as 2'-O-methylnucleotide, at its 3' end. In some embodiments, the gRNA contains a 2'-O modified nucleotide, such as 2'-O-methylnucleotide, at both its 5' and 3' ends. In some embodiments, the gRNA is 2'-O modified and 2'-O-methyl modified, for example, at a nucleotide at the 5' end of the gRNA, a second nucleotide from the 5' end of the gRNA, and a third nucleotide from the 5' end of the gRNA. In some embodiments, the gRNA is 2'-O modified, for example, by 2'-O-methyl modification of the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA. In some embodiments, the gRNA is 2'-O modified, for example, by 2'-O-methyl modification of the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA. In some embodiments, the gRNA is 2'-O modified, for example, by 2'-O-methyl modification of the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA. In some embodiments, the nucleotide at the 3' end of the gRNA is not chemically modified. In some embodiments, the nucleotide at the 3' end of the gRNA does not have a chemically modified sugar.In some embodiments, the gRNA is 2'-O modified and 2'-O-methyl modified, for example, at the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA. In some embodiments, the 2'-O-methyl nucleotide includes a phosphate bond to an adjacent nucleotide. In some embodiments, the 2'-O-methyl nucleotide includes a phosphorothioate bond to an adjacent nucleotide. In some embodiments, the 2'-O-methyl nucleotide includes a thioPACE bond to an adjacent nucleotide.

[0148] In some embodiments, the gRNA may contain one or more 2'-O modified and 3'-phosphorus modified nucleotides, for example, 2'-O-methyl3'-phosphorothioate nucleotide. In some embodiments, the gRNA contains a 2'-O modified and 3'-phosphorus modified nucleotide at the 5' end of the gRNA, for example, 2'-O-methyl3'-phosphorothioate nucleotide. In some embodiments, the gRNA contains a 2'-O modified and 3'-phosphorus modified nucleotide at the 3' end of the gRNA, for example, 2'-O-methyl3'-phosphorothioate nucleotide. In some embodiments, the gRNA contains a backbone in which one or more unbridged oxygen atoms are replaced by sulfur atoms. In some embodiments, the gRNA is 2'-O modified and 3'-phosphorus modified, for example, by 2'-O-methyl3'-phosphorothioate modification of the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA. In some embodiments, the gRNA is 2'-O modified and 3'-phosphorus modified, for example, by 2'-O-methyl3'-phosphorothioate modification of the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA. In some embodiments, the gRNA is 2'-O modified and 3'-phosphorus modified, for example, by 2'-O-methyl3'-phosphorothioate modification of the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA. In some embodiments, the gRNA is 2'-O modified and 3'-phosphorus modified, for example, by 2'-O-methyl3'phosphorothioate modification at the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA.In some embodiments, the nucleotide at the 3' end of the gRNA is not chemically modified. In some embodiments, the nucleotide at the 3' end of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2'-O modified and 3' phosphorus modified, for example, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA are 2'-O-methyl 3' phosphorothioate modified.

[0149] In some embodiments, the gRNA may contain one or more 2'-O modifications and 3'-phosphorus modifications, for example, a 2'-O-methyl3'thioPACE nucleotide. In some embodiments, the gRNA contains a 2'-O modification and a 3'-phosphorus modification, for example, a 2'-O-methyl3'thioPACE nucleotide, at the 5' end of the gRNA. In some embodiments, the gRNA contains a 2'-O modification and a 3'-phosphorus modification, for example, a 2'-O-methyl3'thioPACE nucleotide, at the 3' end of the gRNA. In some embodiments, the gRNA contains a backbone in which one or more unbridged oxygen atoms are replaced with sulfur atoms and one or more unbridged oxygen atoms are replaced with acetate groups. In some embodiments, the gRNA is 2'-O modified and 3'-phosphorus modified, for example, by 2'-O-methyl3'thioPACE modification at the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA. In some embodiments, the gRNA is 2'-O modified and 3'-phosphorus modified, for example, by 2'-O-methyl3'thioPACE modification at the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA. In some embodiments, the gRNA is 2'-O modified and 3'-phosphorus modified, for example, by 2'-O-methyl3'thioPACE modification at the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA. In some embodiments, the gRNA is 2'-O modified and 3'-phosphorus modified, and for example, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA are 2'-O-methyl3'thioPACE modified. In some embodiments, the nucleotide at the 3' end of the gRNA is not chemically modified.In some embodiments, the nucleotide at the 3' end of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2'-O modified and 3'-phosphorus modified, for example, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA are 2'-O-methyl3'thioPACE modified.

[0150] In some embodiments, the gRNA contains a chemically modified backbone. In some embodiments, the gRNA contains a phosphorothioate bond. In some embodiments, one or more non-crosslinked oxygen atoms are replaced with sulfur atoms. In some embodiments, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA each contain a phosphorothioate bond. In some embodiments, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each contain a phosphorothioate bond. In some embodiments, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each contain a phosphorothioate bond. In some embodiments, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA each contain a phosphorothioate bond. In some embodiments, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA each contain a phosphorothioate bond.

[0151] In some embodiments, the gRNA contains a thioPACE bond. In some embodiments, the gRNA contains a skeleton in which one or more non-crosslinked oxygen atoms are replaced by sulfur atoms and one or more non-crosslinked oxygen atoms are replaced by acetate groups. In some embodiments, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA each contain a thioPACE bond. In some embodiments, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each contain a thioPACE bond. In some embodiments, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each contain a thioPACE bond. In some embodiments, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA each contain a thioPACE bond. In some embodiments, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA each contain a thioPACE bond.

[0152] Some exemplary non-limiting embodiments of modifications suitable for use in connection with the guide RNAs and gene manipulation methods provided herein, such as chemical modifications, are described above. Additional suitable modifications, such as chemical modifications, will be apparent to those skilled in the art based on the knowledge in the art of the present disclosure and, without limitation, Hendel, A. et al., Nature Biotech. (2015) Vol 33, No. 9; PCT Publication No. 2017 / 214460; No. 2016 / 089433; and / or No. 2016 / 164356, each of which is incorporated herein by reference in its entirety.

[0153] gRNA targeting CD33

[0154] The present disclosure provides a number of useful gRNAs that can target an endonuclease to human CD33. In some embodiments, the gRNAs used in the methods described herein target sequences in exon 3 of CD33. Table 1 below illustrates target domains within the human endogenous CD33 gene that can be bound by the gRNAs described herein.

[0155] Table 1. Exemplary Cas9 target site sequences of human CD33 are provided, as are exemplary targeting domain sequences useful for targeting such sites. For each target site, the first sequence represents the DNA target domain sequence, the second sequence represents its reverse complement, and the third sequence represents an exemplary targeting domain sequence of a gRNA that can be used to target the respective target site. [Table 1]

[0156] The CD33 (CCDS33084.1) cDNA sequence is provided below as SEQ ID NO: 16. Exon 3 is underlined. [Chemical formula]

[0157] Exon 3 of CD33 is provided below as Sequence ID No. 17. The underlined region indicates the region complementary to gRNA A, gRNA B, gRNA C, gRNA D (or their reverse complement). Note that the target regions of gRNA A, gRNA B, and gRNA D partially overlap. [ka]

[0158] Dual gRNA composition and its use In some embodiments, the gRNAs described herein (e.g., the gRNAs in Table 1) can be used in combination with a second gRNA to target, for example, a CRISPR / Cas nuclease to two sites in the genome. For example, in some embodiments, it is desirable to produce hematopoietic cells lacking CD33 and a second lineage-specific cell surface antigen, etc., so that the cells can exhibit resistance to two drugs: an anti-CD33 agent and a drug targeting a second lineage-specific cell surface antigen. In some embodiments, it is desirable to contact the cells with two different gRNAs targeting different regions of CD33 to perform two cleavages and create a deletion between the two cleavage sites. Thus, this disclosure provides various combinations of gRNAs.

[0159] In some embodiments, two or more (e.g., two, three, or four or more) gRNAs described herein are mixed. In some embodiments, each gRNA is in a separate container. In some embodiments, the kit described herein (e.g., a kit containing one or more gRNAs according to Table 1) also contains a Cas9 molecule, or a nucleic acid encoding a Cas9 molecule.

[0160] In some embodiments, the first and second gRNAs are the gRNAs or their variants shown in Table 1.

[0161] In some embodiments, the first gRNA is CD33 as described herein. The second gRNA is a gRNA (e.g., a gRNA or its variant in Table 1), and targets a lineage-specific cell surface antigen selected from the following: CD5, CD6, CD7, BCMA, CD19, CD20, CD30, ROR1, B7H6, B7H3, CD23, CD38, C-type lectin-like molecule-1, CS1, IL-5, L1-CAM, PSCA, PSMA, CD138, CD133, CD70, CD7, CD13, NKG2D, NKG2D ligand, CLEC12A, CD11, CD123, CD56, CD34, CD14, CD66b, CD41, CD61, CD62, CD235a, CD146, CD326, LMP2, CD22, CD52, CD10, CD3 / TCR, CD79 / BCR, and CD26.

[0162] In some embodiments, the first gRNA is a CD33 gRNA as described herein (e.g., a gRNA or variant thereof as shown in Table 1), and the second gRNA is, but is not limited to, CD20, CD22 (non-Hodgkin lymphoma, B-cell lymphoma, chronic lymphocytic leukemia (CLL)), CD52 (B-cell CLL), CD33 (acute myeloid leukemia (AML)), CD10 (gp100) (common (pre-B) acute lymphoblastic leukemia and malignant melanoma), CD3 / T-cell receptor ( It targets lineage-specific cell surface antigens associated with specific types of cancer, such as TCR (T-cell lymphoma and leukemia), CD79 / B-cell receptor (BCR) (B-cell lymphoma and leukemia), CD26 (epithelial and lymphoid malignancies), human leukocyte antigen (HLA)-DR, HLA-DP, HLA-DQ (lymphoid malignancies), RCAS1 (gynecologic cancers, cholangiocarcinomas, and pancreatic ductal adenocarcinomas), and prostate-specific membrane antigens.

[0163] In some embodiments, the first gRNA is a CD33 gRNA as described herein (e.g., a gRNA or variant thereof as shown in Table 1), and the second gRNA targets a lineage-specific cell surface antigen selected from: CD5, CD6, CD7, CD13, CD19, CD22, CD20, CD25, CD30, CD32, CD38, CD44, CD45, CD47, CD56, 96, CD117, CD123, CD135, CD174, CLL-1, BCMA, folate receptor β, IL1RAP, MUC1, NKG2D / NKG2DL, TIM-3, or WT1.

[0164] In some embodiments, the first gRNA is a CD33 gRNA as described herein (e.g., a gRNA or variant thereof as shown in Table 1), and the second gRNA targets a lineage-specific cell surface antigen selected from the following: CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD16, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD2 3, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32a, CD32b, CD32c, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD 42d, CD43, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD5 4, CD55, CD56, CD57, CD58, CD59, CD60a, CD61, CD62E, CD62L, CD62P, CD63, CD64a, CD65, CD65s, CD66a, CD66b, CD66c, CD66F, CD68, CD69, CD70, CD7 1, CD72, CD73, CD74, CD75, CD75S, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85A, CD85C, CD85D, CD85E, CD85F, CD85G, CD85H, CD85I, CD8 5J, CD85K, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD104, CD105 , CD106, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b,CD121a、CD121b、CD122、CD123、CD124、CD125、CD126、CD127、CD129、CD130、 CD131、CD132、CD133、CD134、CD135、CD136、CD137、CD138、CD139、CD140a、C D140b、CD141、CD142、CD143、CD14、CDw145、CD146、CD147、CD148、CD150、CD 152、CD152、CD153、CD154、CD155、CD156a、CD156b、CD156c、CD157、CD158b1、 CD158b2, CD158d, CD158e1 / e2, CD158f, CD158g, CD158h, CD158i, CD158j, CD158k, CD159a, CD159c, CD160, CD161, CD163, CD164, CD165, CD166, CD167a 、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172g、CD173、CD174、CD17 5、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD181、CD182、CD18 3、CD184、CD185、CD186、CD191、CD192、CD193、CD194、CD195、CD196、CD197、 CDw198、CDw199、CD200、CD201、CD202b、CD203c、CD204、CD205、CD206、CD20 7, CD208, CD209, CD210a, CDw210b, CD212, CD213a1, CD213a2, CD215, CD217, CD218a, CD218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227 CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235b, CD236, CD236R, CD238, CD239, CD240, CD241, CD242, CD243, CD244, CD245, CD246, C D247, CD248, CD249, CD252, CD253, CD254, CD256, CD257, CD258, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD272, CD272CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD286, CD288, CD289, CD290, CD292, CDw293, CD294, CD295, C D296, CD297, CD298, CD299, CD300a, CD300c, CD300e, CD301, CD302, CD303, CD304, CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD309, CD 312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD359, CD360, CD361, CD362 or CD363,

[0165] In some embodiments, the first gRNA is a CD33 gRNA as described herein (e.g., a gRNA or its variant as shown in Table 1), and the second gRNA targets a lineage-specific cell surface antigen selected from: CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); type C lectin-like molecule-1 (CLECL1); epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (CD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlep(1-1)Cer); TNF receptor family member B cell maturation (BCMA), Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) Antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine protein kinase ERBB2 (Her2 / neu); Mucin 1, cell surface-related (MUC1); Epidermal growth factor receptor (EGFR); Neuronal adhesion molecule (NCAM); Prostase; Prostatic acid phosphatase (PAP); Elongation factor 2 mutation (ELF2M); Ephrin B2; Fibroblast-activating protein alpha (FAP); Insulin-like growth factor I receptor (IGF-I receptor), Carbonic anhydrase IX (CAIX), Proteasome (Macropain) subunit, beta-type 9 (LMP2); Glycoprotein 100 (gp100);Oncogene fusion protein consisting of a critical cluster region (BCR) and Ebelson mouse leukemia virus oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor beta ; Tumor vascular endothelial marker 1 (TEM1 / CD248); Tumor vascular endothelial marker 7-related (TEM7R); Claudin 6 (CLDN6); Thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); Chromosome X open reading frame 61 (CXORF61); CD97; CD179a; Anaplastic lymphoma kinase (ALK); Polysialic acid; Placenta-specific 1 (PLAC1); Hexasaccharide portion of globoH glycoceramide (GloboH); Mammary gland differentiation antigen (NY-BR-1); Uroplakin 2 (UPK2); Hepatitis A virus cell receptor 1 (HAVCR1); Adrenergic receptor beta-3 (ADRB3); Panexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex; Gene locus K9 (LY6K); Taste receptor 51E2 (OR51E2); TCR gamma surrogate leading frame protein (TARP); Wilms tumor protein (WT1); Cancer / testicular antigen 1 (NY-ESO-1); Cancer / testicular antigen 2 (LAGE-1a); Melanoma-associated antigen 1 (MAGE-A1), ETS translocation located on chromosome 12p Variant gene 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); melanoma cancer testicular antigen-1 (MAD-CT-1); melanoma cancer testicular antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 variant; prostain; survivor; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cell 1 (MelanA or MART1);Rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation limit; apoptotic melanoma inhibitor (ML-1AP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); pair-forming box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelomatosis virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC binding factor (zinc finger protein)-like (BORIS or similar, regulatory factors of imprinted sites), squamous cell carcinoma antigen recognized by T cell 3 (SART3); pair-forming box protein Pax-5 (PAX5); pro-acrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X limit point 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous protein 1 (RU1); renal ubiquitous protein 2 (RU2); regmine; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxyesterase; heat shock protein 70-2 mutation (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1);

[0166] In some embodiments, the first gRNA is a CD33 gRNA as described herein (e.g., a gRNA or variant thereof as shown in Table 1), and the second gRNA targets a lineage-specific cell surface antigen selected from: CD11a, CD18, CD19, CD20, CD31, CD34, CD44, CD45, CD47, CD51, CD58, CD59, CD63, CD97, CD99, CD100, CD102, CD123, CD127, CD133, CD135, CD157, CD172b, CD217, CD300a, CD305, CD317, CD321, and CLL1.

[0167] In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA or variant thereof as shown in Table 1), and the second gRNA targets a lineage-specific cell surface antigen selected from: CD123, CLL1, CD38, CD135 (FLT3), CD56 (NCAM1), CD117 (c-KIT), FRβ (FOLR2), CD47, CD82, TNFRSF1B (CD120B), CD191, CD96, PTPRJ (CD148), CD70, LILRB2 (CD85D), CD25 (IL2Ralpha), CD44, CD96, NKG2D ligand, CD45, CD7, CD15, CD19, CD20, CD22, CD37, and CD82.

[0168] In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., the gRNA according to Table 1 or a variant thereof), and the second gRNA targets a lineage-specific cell surface antigen selected from: CD7, CD11a, CD15, CD18, CD19, CD20, CD22, CD25, CD31, CD34, CD37, CD38, CD44, CD45, CD47, CD51, CD56, CD58, CD59, CD63, CD70, CD82, CD85D, CD96, CD97, CD99, CD100, CD102, CD117, CD120B, CD123, CD127, CD133, CD135, CD148, CD157, CD172b, CD191, CD217, CD300a, CD305, CD317, CD321, CLL1, FRβ (FOLR2), or NKG2D ligand.

[0169] In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., the gRNA according to Table 1 or a variant thereof), and the second gRNA targets CLL-1. In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., the gRNA according to Table 1 or a variant thereof), and the second gRNA targets CD123. [Table 2]

[0170] Some of the embodiments, advantages, features, and uses of the technology disclosed herein will be more fully understood from the following examples. The examples are intended to illustrate some of the benefits of the present disclosure and to describe specific embodiments, but are not intended to illustrate the full scope of the present disclosure and, accordingly, do not limit the scope of the present disclosure. [Examples]

[0171] Example 1. Generation of Genetically Engineered Hematopoietic Cells Containing a Modified Gene Encoding CD33 The Cas9 sgRNAs shown in Table 1 were designed based on SpCas9 PAM(5′-NGG-3′) located close to the target region, and evaluated according to predicted specificity by minimizing potential target external locations within the human genome using online search algorithms (e.g., Benchling algorithm, Doench et al 2016, Hsu et al 2013).

[0172] Cas9 sgRNA is synthesized using the gRNA target domain and Cas9 sgRNA scaffold sequence provided below. 5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC UUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3' (Sequence ID 18).

[0173] For example, the nucleotide sequence of sgRNA A is:

number

[0174] For example, the nucleotide sequence of sgRNA B is:

number

[0175] For example, the nucleotide sequence of sgRNA C is:

number

[0176] For example, the nucleotide sequence of sgRNA D is:

number

[0177] For example, the nucleotide sequence of sgRNA E is:

number

[0178] All designed synthetic sgRNAs were produced using chemically modified nucleotides at three terminal positions in both the 5' and 3' ends. The modified nucleotides included 2'-O-methyl-3'-phosphorothioate (abbreviated as "ms"), and the ms-sgRNA was purified by HPLC. The Cas9 protein was purchased from Synthego.

[0179] For example, the nucleotide sequence of sgRNA A showing modified nucleotides is:

number

[0180] For example, the nucleotide sequence of sgRNA E showing modified nucleotides is:

number

[0181] Peripheral blood mononuclear cells are collected from healthy donor subjects by apheresis after hematopoietic stem cell recruitment. Donor CD34+ cells are electroporated with the Cas9 protein and the CD33-targeted Cas9 gRNAs disclosed herein, such as gRNA A, gRNA B, gRNA C, gRNA D, or gRNA E, which have the targeting domain sequence provided in Tables 1 and 3. [Table 3]

[0182] The edited cells are cultured for less than 48 hours. After harvesting, the cells are washed, resuspended in the final formulation, and cryopreserved.

[0183] Representative samples of edited HSCs are stained for CD33 using an anti-CD33 antibody (e.g., P67.7) and analyzed by flow cytometry to evaluate CD33 viability and expression, or absence thereof. Edited CD33KO eHSPC populations exhibiting at least 70% cell viability and at least 45% CD33 editing efficiency (i.e., absence of CD33 expression in at least 45% of cells within the cell population) 48 hours after electroporation are used for HCT.

[0184] Example 2: Combination therapy using CD33KO eHSPC and CD33-targeted ADC (Mylotarg) CD33 is a transmembrane receptor expressed in normal bone marrow cells as well as many leukemic myeloblasts (e.g., Larson et al. Cancer (2005) 104(7):1442-1452; Kenderian et al. Leukemia (2015) 29(8):1637-47; Wang et al. Mol.Ther. (2015) 23(1):184-91; Pollard et al. J.Clin.Oncol. (2016) 34(7:747-55). Hematopoietic stem cells genetically engineered to reduce or eliminate CD33 expression ("CD33KO eHSC" or "CD33KO eHSPC") are not sensitive to on-target, off-cancer cell damage reported to be associated with CD33-targeted therapy, thus potentially improving the safety and efficacy of CD33-targeted therapy and thus enabling the administration of CD33-targeted therapy without optimal dose and schedule, e.g., treatment delay or dose optimization.

[0185] The treatment regimens provided herein relate to subjects with acute myeloid leukemia or its pre-malignant stage, such as myelodysplastic syndrome. Currently, CD33-targeted therapy is limited by on-target cytotoxicity targeting normal myeloid cells. The approach provided herein eliminates this on-target toxicity by administering genetically modified HSPCs that lack expression of the CD33 epitope recognized by CD33-targeted therapy. Subsequently, normal bone marrow compartments are protected from the on-target effects of CD33-targeted therapy, resulting in improved treatment index for these drugs and potentially a better prognosis for subjects with AML.

[0186] This embodiment provides a therapeutic regimen using allogeneic or autologous CRISPR / Cas9 genome-edited CD33KO eHSPCs that lack CD33 expression. Allogeneic eHSPCs are obtained by processing CD34-positive (CD34+) enriched stem cells obtained from a healthy donor with HLA matching that of the recipient, i.e., a subject receiving CD33KO eHSPCs. Autologous eHSPCs are obtained by processing CD34-positive (CD34+) enriched stem cells obtained from the same subject being treated, i.e., the HSPC donor and the subject receiving CD33KO eHSPCs are the same. CD33KO eHSPCs are injected into the recipient subject after they have received a conditioning regimen as part of hematopoietic stem cell transplantation (HCT).

[0187] Gemtuzumab ozogamicin Gemtuzumab ozogamicin / Mylotarg® is a U.S. Food and Drug Administration (FDA) approved CD33-targeted antibody-drug conjugate (ADC) for the treatment of newly diagnosed AML in CD33-positive (CD33+) adults, as well as in infants aged 1 month or older with relapsed or refractory AML (R / R AML).

[0188] For patients with relapsed or refractory AML, gemtuzumab ozogamicin / Mylotarg® is currently the only CD33-targeted therapy approved by the U.S. FDA. Analysis provided in the "Prescription Information Highlights" section of the Mylotarg® U.S. Prescription Information (Mylotarg 2020) and in the U.S. FDA publication regarding the approval summary is based on a dose of 2 mg / m². 2 (Norsworthy 2018) suggests that available CD33 is saturated in subjects with AML. Furthermore, the risk of sinusoidal occlusive syndrome / venous occlusive disease (SOS / VOD), a severe and sometimes fatal toxicity associated with gemtuzumab ozogamicin / Mylotarg® administration, is significantly reduced at lower doses. This potentially greater safety limit supports dose reduction in post-HCT settings, where subjects are known to be at higher risk of SOS / VOD. The current gemtuzumab ozogamicin / Mylotarg® US FDA-approved product label recommends administering 2 mg / m² on day 1 every 4 weeks for up to 8 cycles. 2 The “continued” dose and schedule are listed for subjects with AML who have no evidence of disease progression.

[0189] The clinical treatment regimens provided herein use similar “continued” doses and schedules in subjects who received CD33KO eHSPC as part of their HCT, approximately 60 days after the HCT. This is based on the argument that administering gemtuzumab ozogamicin / Mylotarg® in a post-HCT setting to suppress early leukemia relapses may have greater safety limits, allowing subjects to undergo more robust immunological reconstitution and thereby resulting in a longer-lasting “graft-versus-leukemia” effect.

[0190] subject Clinical regimens provided herein for treating subjects with AML or its pre-malignant forms using stem cell transplantation comprising CD33KO eHSPC and ADC gemtuzumab ozogamicin / Mylotarg® are useful for treating subjects with AML characterized by CD33 expression, or pre-malignant forms of AML characterized by CD33 expression, such as MDS, or those diagnosed with such conditions. This includes subjects who have not received AML therapy; subjects who have received some form of AML therapy, including induction therapy; subjects who have experienced complete hematological remission (including complete remission with incomplete recovery of peripheral counts [CRi], CR1 or CR2) in response to AML therapy; and subjects with residual disease or progressive disease, including subjects with residual disease, including subjects with myeloblast count ≤10% and no clinical evidence of circulating blasts. The regimens provided herein are also useful for treating subjects with myelodysplastic syndrome (MDS) characterized by CD33 expression, including subjects at high risk of progression from MDS to AML.

[0191] Clinical regimens for treating subjects with AML or pre-malignant tumors, including hematopoietic stem cell transplantation containing CD33KO eHSPC and ADC gemtuzumab ozogamicin / Mylotarg®, are provided herein and are more useful for treating subjects exhibiting one or more of the following adverse risk characteristics: 1) a presentation with evidence of MRD+ at the time of HCT and a genetic predisposition to a moderate or high risk of the disease in the bone marrow; or 2) persistent leukemic blasts (≤10%) in the bone marrow alone at the time of HCT (with a genetic predisposition to the disease in any risk category in the presentation).

[0192] HCT pre-conditioning Typically, a clinical treatment regimen, including hematopoietic stem cell transplantation including CD33KO eHSPCs, including HLA-matched allogeneic HCTs, as provided in some of the examples herein, includes a complete conditioning regimen. The conditioning regimen may include, for example, busulfan / melphalan / fludarabine / rabbit anti-thymocyte globulin (rATG); or total body irradiation / cyclophosphamide / thiotepa / rATG. An appropriate conditioning regimen is selected for a given subject in accordance with clinical guidelines, taking into account the subject's health and medical history.

[0193] Clinical monitoring Subjects undergoing clinical regimens including hematopoietic stem cell transplantation, which include CD33KO eHSPC and ADC gemtuzumab ozogamicin / Mylotarg®, as provided herein, will be monitored for treatment-related adverse events during the course of the treatment regimen and any possible conditioning or induction regimens, and their disease status and the status of the HCT graft and hematopoietic system will be evaluated during and / or after the completion of the treatment regimen.

[0194] Example 3: Treatment of patients with AML using CD33KO eHSPC generated using the gRNA A and CD33-targeted ADC gemtuzumab ozogamicin / Mylotarg®. Patients with CD33-positive AML are treated with allogeneic HCT including CD33KO eHSPC and ADC gemtuzumab ozogamicin / Mylotarg®.

[0195] For HCT, a population of cells containing CD34+ hematopoietic stem cells is obtained from healthy donors whose HLA matches that of the target at 8 / 8 gene loci (HLA-A, -B, -C, DRB1).

[0196] After G-CSF / prelixafor mobilization, at least 10 × 10 units of G-CSF / prelixafor should be taken from the donor for processing and subsequent administration to the recipient. 6A maximum of two apheresis procedures are performed to obtain 1000 living cells / kg (kg refers to the weight of the recipient). From this apheresis product, at least 3.0 × 10⁶ cells are obtained. 6 The live cells / kg (recipient weight) undergo minimal processing and are cryopreserved to serve as a backup stem cell source, for example, for use as a rescue dose. The remaining apheresis products are used for processing and preparation of the CD33KO eHSPC population for HCT. The CD33KO eHSPC population for HCT is enriched with apheresis products for CD34+ cells, followed by nucleotide sequencing.

number

[0197] The edited cells are then cultured for <48 hours. Upon harvesting, after the culture period, the cells are washed, resuspended in the final formulation, and cryopreserved. Cell viability and editing efficiency are confirmed using representative samples as described in Example 1, and the CD33KO eHSPC population that meets the criteria described in Example 1 (at least 70% viability and at least 45% CD33 editing efficiency) is used for HCT. The population for administration to the subjects should be at least 3 × 10⁶ 6 The population includes CD33KO eHSPC populations that meet these viability and editing efficiency criteria per kg of body weight of the cell / recipient target, preferably with at least 4 × 10 6 Cells / recipient body weight in kg, 5 × 10 6 Cells / recipient body weight in kg, 6 x 10 6 Cells / recipient weight in kg, or 7 × 10 6 Includes the weight (kg) of the cells / recipient.

[0198] After completion of the conditioning regimen, the patient receives HCT containing thawed CD33KO eHSPC via intravenous (IV) infusion. The day of HCT is day 0 of the treatment regimen.

[0199] At day 28, the engraftment of CD33KO eHSPCs in the subjects will be evaluated by measuring the absolute peripheral neutrophil count (ANC) of CD33KO (CD33-) neutrophils. If a subject shows an absolute peripheral CD33KO neutrophil count of ≥1000 / dL CD33- ANC 28 days after CD33KO eHSPC HCT, the subject is considered to have neutrophil recovery (also referred to as good CD33KO neutrophil engraftment).

[0200] If a subject shows neutrophil recovery on day 28, a bone marrow biopsy is taken from the subject on day 60 to assess the disease status and hematopoietic recovery. Furthermore, the percentage of donor chimerization and CD33-negative (CD33-) myeloid hematopoiesis are determined from peripheral blood at this point. If a subject shows good CD33- HSC engraftment and CD33- hematopoiesis on day 60, the subject is subsequently administered gemtuzumab ozogamicin / Mylotarg®. CD33-ANC is monitored in the subject before administration of gemtuzumab ozogamicin / Mylotarg®, and the subject should preferably have ≥1000 / dL CD33-ANC before receiving gemtuzumab ozogamicin / Mylotarg®.

[0201] Gemtuzumab ozogamicin / Mylotarg® administration should preferably be initiated within 30 days of the bone marrow biopsy on day 60, i.e., preferably by day 90. However, if the clinical condition of the subject requires such a delay, for example, in light of comorbidities including HCT-related comorbidities, in order to enable the subject to achieve ≥1000 / dL CD33-ANC, the initiation of gemtuzumab ozogamicin / Mylotarg® may be delayed up to day 120. If gemtuzumab ozogamicin / Mylotarg® is initiated 30 days or more after the bone marrow biopsy on day 60, repeated bone marrow biopsies should be completed before the initiation of gemtuzumab ozogamicin / Mylotarg®.

[0202] Gemtuzumab ozogamicin / Mylotarg(registered trademark) 0.1 mg / m² 2 ~2mg / m 2 Doses within the range, for example, 0.1 mg / m² 2 , 0.25 mg / m² 2 , 0.5 mg / m² 2 , 1 mg / m² 2 , or 2 mg / m² 2 The following dose is administered to the target population. For most subjects, the dose is 2 mg / m². 2 Gemtuzumab ozogamicin / Mylotarg® is preferred. However, some subjects may be administered at lower doses, for example, in the case of treatment-related side effects, such as dose-limiting toxicity (DLT), or in consideration of the individual subject's health condition, comorbidities, or medical history.

[0203] Gemtuzumab ozogamicin / Mylotarg® is administered to subjects in a 4-week (28-day) treatment cycle regimen, for example, at a dose of 2 mg / m². 2 With gemtuzumab ozogamicin / Mylotarg®, the full dose for each treatment cycle is received on day 1 of each 4-week (28-day) treatment cycle.

[0204] For most subjects, four consecutive treatment cycles of gemtuzumab ozogamicin / Mylotarg®, and therefore four doses of gemtuzumab ozogamicin / Mylotarg® spaced four weeks apart, are preferred. However, in some subjects, additional gemtuzumab ozogamicin / Mylotarg® treatment cycles, e.g., up to four further “continued” treatment cycles, may be initiated, e.g., at the same dose as the initial four treatment cycles, or at a lower dose, if clinically desirable, e.g., based on the subject’s clinical condition.

[0205] At the completion of the final gemtuzumab ozogamicin / Mylotarg® treatment cycle, subjects will be monitored for disease status and hematopoietic chimerism, and these parameters will be monitored every six months for five years following the completion of the final treatment cycle.

[0206] Example 4: Treatment of subjects with AML using CD33KO eHSPC generated using the gRNA B and CD33-targeted ADC gemtuzumab ozogamicin / Mylotarg®. Patients with CD33-positive AML are treated with allogeneic HCT including CD33KO eHSPC and ADC gemtuzumab ozogamicin / Mylotarg®.

[0207] For HCT, a population of cells containing CD34+ hematopoietic stem cells is obtained from healthy donors whose HLA matches that of the target at 8 / 8 gene loci (HLA-A, -B, -C, DRB1).

[0208] After G-CSF / prelixafor mobilization, at least 10 × 10 units of G-CSF / prelixafor should be taken from the donor for processing and subsequent administration to the recipient. 6 A maximum of two apheresis procedures are performed to obtain 1000 living cells / kg (kg refers to the weight of the recipient). From this apheresis product, at least 3.0 × 10⁶ cells are obtained. 6The live cells / kg (recipient weight) undergo minimal processing and are cryopreserved to serve as a backup stem cell source, for example, for use as a rescue dose. The remaining apheresis products are used for processing and preparation of the CD33KO eHSPC population for HCT.

[0209] CD33KO eHSPC population for HCT enriched with apheresis products for CD34+ cells, followed by nucleotide sequencing

number

[0210] The edited cells are then cultured for <48 hours. Upon harvesting, after the culture period, the cells are washed, resuspended in the final formulation, and cryopreserved. Cell viability and editing efficiency are confirmed using representative samples as described in Example 1, and the CD33KO eHSPC population that meets the criteria described in Example 1 (at least 70% viability and at least 45% CD33 editing efficiency) is used for HCT. The population for administration to the subjects should be at least 3 × 10⁶ 6 The population includes CD33KO eHSPC populations that meet these viability and editing efficiency criteria per kg of body weight of the cell / recipient target, preferably with at least 4 × 10 6 Cells / recipient body weight in kg, 5 × 10 6 Cells / recipient body weight in kg, 6 x 10 6 Cells / recipient weight in kg, or 7 × 10 6 Includes the weight (kg) of the cells / recipient.

[0211] After completion of the conditioning regimen, the patient receives HCT containing thawed CD33KO eHSPC via intravenous (IV) infusion. The day of HCT is day 0 of the treatment regimen.

[0212] At day 28, the engraftment of CD33KO eHSPCs in the subjects will be evaluated by measuring the absolute peripheral neutrophil count (ANC) of CD33KO (CD33-) neutrophils. If a subject shows an absolute peripheral CD33KO neutrophil count of ≥1000 / dL CD33- ANC 28 days after CD33KO eHSPC HCT, the subject is considered to have neutrophil recovery (also referred to as good CD33KO neutrophil engraftment).

[0213] If a subject shows neutrophil recovery on day 28, a bone marrow biopsy is taken from the subject on day 60 to assess the disease status and hematopoietic recovery. Furthermore, the percentage of donor chimerization and CD33-negative (CD33-) myeloid hematopoiesis are determined from peripheral blood at this point. If a subject shows good CD33- HSC engraftment and CD33- hematopoiesis on day 60, the subject is subsequently administered gemtuzumab ozogamicin / Mylotarg®. CD33-ANC is monitored in the subject before administration of gemtuzumab ozogamicin / Mylotarg®, and the subject should preferably have ≥1000 / dL CD33-ANC before receiving gemtuzumab ozogamicin / Mylotarg®.

[0214] Gemtuzumab ozogamicin / Mylotarg administration is preferably initiated within 30 days of the bone marrow biopsy on day 60, i.e., preferably by day 90. However, if the clinical condition of the subject requires such a delay, for example, in light of comorbidities including HCT-related comorbidities, in order to enable the subject to achieve ≥1000 / dL CD33-ANC, the initiation of gemtuzumab ozogamicin / Mylotarg® may be delayed up to day 120. If gemtuzumab ozogamicin / Mylotarg® is initiated 30 days or more after the bone marrow biopsy on day 60, repeated bone marrow biopsies should be completed before initiating gemtuzumab ozogamicin / Mylotarg®.

[0215] Gemtuzumab ozogamicin / Mylotarg(registered trademark) 0.1 mg / m² 2 ~2mg / m 2 Doses within the range, for example, 0.1 mg / m² 2 , 0.25 mg / m² 2 , 0.5 mg / m² 2 , 1 mg / m² 2 , or 2 mg / m² 2 The following dose is administered to the target population. For most subjects, the dose is 2 mg / m². 2 Gemtuzumab ozogamicin / Mylotarg® is preferred. However, some subjects may be administered at lower doses, for example, in the case of treatment-related side effects, such as dose-limiting toxicity (DLT), or in consideration of the individual subject's health condition, comorbidities, or medical history.

[0216] Gemtuzumab ozogamicin / Mylotarg® is administered to subjects in a 4-week (28-day) treatment cycle regimen, for example, at a dose of 2 mg / m². 2 With gemtuzumab ozogamicin / Mylotarg®, the full dose for each treatment cycle is received on day 1 of each 4-week (28-day) treatment cycle.

[0217] For most subjects, four consecutive treatment cycles of gemtuzumab ozogamicin / Mylotarg®, and therefore four doses of gemtuzumab ozogamicin / Mylotarg® spaced four weeks apart, are preferred. However, in some subjects, additional gemtuzumab ozogamicin / Mylotarg® treatment cycles, e.g., up to four further “continued” treatment cycles, may be initiated, e.g., at the same dose as the initial four treatment cycles, or at a lower dose, if clinically desirable, e.g., based on the subject’s clinical condition.

[0218] At the completion of the final gemtuzumab ozogamicin / Mylotarg® treatment cycle, subjects will be monitored for disease status and hematopoietic chimerism, and these parameters will be monitored every six months for five years following the completion of the final treatment cycle.

[0219] Example 5: Treatment of subjects with AML using CD33KO eHSPC generated using gRNA C and CD33-targeted ADC gemtuzumab ozogamicin / Mylotarg®. Patients with CD33-positive AML are treated with allogeneic HCT including CD33KO eHSPC and ADC gemtuzumab ozogamicin / Mylotarg®.

[0220] For HCT, a population of cells containing CD34+ hematopoietic stem cells is obtained from healthy donors whose HLA matches that of the target at 8 / 8 gene loci (HLA-A, -B, -C, DRB1).

[0221] After G-CSF / prelixafor mobilization, at least 10 × 10 units of G-CSF / prelixafor should be taken from the donor for processing and subsequent administration to the recipient. 6 A maximum of two apheresis procedures are performed to obtain 1000 living cells / kg (kg refers to the weight of the recipient). From this apheresis product, at least 3.0 × 10⁶ cells are obtained. 6 The live cells / kg (recipient weight) undergo minimal processing and are cryopreserved to serve as a backup stem cell source, for example, for use as a rescue dose. The remaining apheresis products are used for processing and preparation of the CD33KO eHSPC population for HCT.

[0222] CD33KO eHSPC population for HCT enriched with apheresis products for CD34+ cells, followed by nucleotide sequencing

number

[0223] The edited cells are then cultured for <48 hours. Upon harvesting, after the culture period, the cells are washed, resuspended in the final formulation, and cryopreserved. Cell viability and editing efficiency are confirmed using representative samples as described in Example 1, and the CD33KO eHSPC population that meets the criteria described in Example 1 (at least 70% viability and at least 45% CD33 editing efficiency) is used for HCT. The population for administration to the subjects should be at least 3 × 10⁶ 6 The population includes CD33KO eHSPC populations that meet these viability and editing efficiency criteria per kg of body weight of the cell / recipient target, preferably with at least 4 × 10 6 Cells / recipient body weight in kg, 5 × 10 6 Cells / recipient body weight in kg, 6 x 10 6 Cells / recipient weight in kg, or 7 × 10 6 Includes the weight (kg) of the cells / recipient.

[0224] After completion of the conditioning regimen, the patient receives HCT containing thawed CD33KO eHSPC via intravenous (IV) infusion. The day of HCT is day 0 of the treatment regimen.

[0225] At day 28, the engraftment of CD33KO eHSPCs in the subjects will be evaluated by measuring the absolute peripheral neutrophil count (ANC) of CD33KO (CD33-) neutrophils. If a subject shows an absolute peripheral CD33KO neutrophil count of ≥1000 / dL CD33- ANC 28 days after CD33KO eHSPC HCT, the subject is considered to have neutrophil recovery (also referred to as good CD33KO neutrophil engraftment).

[0226] If a subject shows neutrophil recovery on day 28, a bone marrow biopsy is taken from the subject on day 60 to assess the disease status and hematopoietic recovery. Furthermore, the percentage of donor chimerization and CD33-negative (CD33-) myeloid hematopoiesis are determined from peripheral blood at this point. If a subject shows good CD33- HSC engraftment and CD33- hematopoiesis on day 60, the subject is subsequently administered gemtuzumab ozogamicin / Mylotarg®. CD33-ANC is monitored in the subject before administration of gemtuzumab ozogamicin / Mylotarg®, and the subject should preferably have ≥1000 / dL CD33-ANC before receiving gemtuzumab ozogamicin / Mylotarg®.

[0227] Gemtuzumab ozogamicin / Mylotarg administration is preferably initiated within 30 days of the bone marrow biopsy on day 60, i.e., preferably by day 90. However, if the clinical condition of the subject requires such a delay, for example, in light of comorbidities including HCT-related comorbidities, in order to enable the subject to achieve ≥1000 / dL CD33-ANC, the initiation of gemtuzumab ozogamicin / Mylotarg® may be delayed up to day 120. If gemtuzumab ozogamicin / Mylotarg® is initiated 30 days or more after the bone marrow biopsy on day 60, repeated bone marrow biopsies should be completed before initiating gemtuzumab ozogamicin / Mylotarg®.

[0228] Gemtuzumab ozogamicin / Mylotarg(registered trademark) 0.1 mg / m² 2 ~2mg / m 2 Doses within the range, for example, 0.1 mg / m² 2 , 0.25 mg / m² 2 , 0.5 mg / m² 2 , 1 mg / m² 2 , or 2 mg / m² 2 The following dose is administered to the target population. For most subjects, the dose is 2 mg / m². 2Gemtuzumab ozogamicin / Mylotarg® is preferred. However, some subjects may be administered at lower doses, for example, in the case of treatment-related side effects, such as dose-limiting toxicity (DLT), or in consideration of the individual subject's health condition, comorbidities, or medical history.

[0229] Gemtuzumab ozogamicin / Mylotarg® is administered to subjects in a 4-week (28-day) treatment cycle regimen, for example, at a dose of 2 mg / m². 2 With gemtuzumab ozogamicin / Mylotarg®, the full dose for each treatment cycle is received on day 1 of each 4-week (28-day) treatment cycle.

[0230] For most subjects, four consecutive treatment cycles of gemtuzumab ozogamicin / Mylotarg®, and therefore four doses of gemtuzumab ozogamicin / Mylotarg® spaced four weeks apart, are preferred. However, in some subjects, additional gemtuzumab ozogamicin / Mylotarg® treatment cycles, e.g., up to four further “continued” treatment cycles, may be initiated, e.g., at the same dose as the initial four treatment cycles, or at a lower dose, if clinically desirable, e.g., based on the subject’s clinical condition.

[0231] At the completion of the final gemtuzumab ozogamicin / Mylotarg® treatment cycle, subjects will be monitored for disease status and hematopoietic chimerism, and these parameters will be monitored every six months for five years following the completion of the final treatment cycle.

[0232] Example 6: Treatment of subjects with AML using CD33KO eHSPC generated using the gRNA D and CD33-targeted ADC gemtuzumab ozogamicin / Mylotarg®. Patients with CD33-positive AML are treated with allogeneic HCT including CD33KO eHSPC and ADC gemtuzumab ozogamicin / Mylotarg®.

[0233] For HCT, a population of cells containing CD34+ hematopoietic stem cells is obtained from healthy donors whose HLA matches that of the target at 8 / 8 gene loci (HLA-A, -B, -C, DRB1).

[0234] After G-CSF / prelixafor mobilization, at least 10 × 10 units of G-CSF / prelixafor should be taken from the donor for processing and subsequent administration to the recipient. 6 Perform apheresis procedures up to two times to obtain 10⁴ living cells / kg (kg refers to the weight of the recipient). From this apheresis product, obtain at least 3.0 × 10⁴ 6 The live cells / kg (recipient weight) undergo minimal processing and are cryopreserved to serve as a backup stem cell source, for example, for use as a rescue dose. The remaining apheresis products are used for processing and preparation of the CD33KO eHSPC population for HCT.

[0235] CD33KO eHSPC population for HCT enriched with apheresis products for CD34+ cells, followed by nucleotide sequencing

number

[0236] The edited cells are then cultured for <48 hours. Upon harvesting, the cells are washed after the culture period, resuspended in the final formulation, and cryopreserved. Cell viability and editing efficiency are confirmed using representative samples as described in Example 1, and CD33KO eHSPC populations that meet the criteria described in Example 1 (at least 70% viability and at least 45% CD33 editing efficiency) are used for HCT. The population for administration to the subjects should be at least 3 × 10⁶ 6The population includes CD33KO eHSPC populations that meet these viability and editing efficiency criteria per kg of body weight of the cell / recipient target, preferably with at least 4 × 10 6 Cells / recipient body weight in kg, 5 × 10 6 Cells / recipient body weight in kg, 6 x 10 6 Cells / recipient weight in kg, or 7 × 10 6 Includes the weight (kg) of the cells / recipient.

[0237] After completion of the conditioning regimen, the patient receives HCT containing thawed CD33KO eHSPC via intravenous (IV) infusion. The day of HCT is day 0 of the treatment regimen.

[0238] At day 28, the engraftment of CD33KO eHSPCs in the subjects will be evaluated by measuring the absolute peripheral neutrophil count (ANC) of CD33KO (CD33-) neutrophils. If a subject shows an absolute peripheral CD33KO neutrophil count of ≥1000 / dL CD33-ANC 28 days after CD33KO eHSPC HCT, the subject is considered to have neutrophil recovery (also referred to as good CD33KO neutrophil engraftment).

[0239] If a subject shows neutrophil recovery on day 28, a bone marrow biopsy is taken from the subject on day 60 to assess the disease state and hematopoietic recovery. Furthermore, the percentage of donor chimerization and CD33-negative (CD33-) myeloid hematopoiesis are determined from peripheral blood at this point. If a subject shows good CD33-HSC engraftment and CD33-hematopoiesis on day 60, the subject is subsequently administered gemtuzumab ozogamicin / Mylotarg®. CD33-ANC is monitored in the subject before administration of gemtuzumab ozogamicin / Mylotarg®, and the subject should preferably have ≥1000 / dL CD33-ANC before receiving gemtuzumab ozogamicin / Mylotarg®.

[0240] Gemtuzumab ozogamicin / Mylotarg® administration should preferably be initiated within 30 days of the bone marrow biopsy on day 60, i.e., preferably by day 90. However, if the clinical condition of the subject requires such a delay, for example, in light of comorbidities including HCT-related comorbidities, in order to enable the subject to achieve ≥1000 / dL CD33-ANC, the initiation of gemtuzumab ozogamicin / Mylotarg® may be delayed up to day 120. If gemtuzumab ozogamicin / Mylotarg® is initiated 30 days or more after the bone marrow biopsy on day 60, repeated bone marrow biopsies should be completed before the initiation of gemtuzumab ozogamicin / Mylotarg®.

[0241] Gemtuzumab ozogamicin / Mylotarg(registered trademark) 0.1 mg / m² 2 ~2mg / m 2 Doses within the range, for example, 0.1 mg / m² 2 , 0.25 mg / m² 2 , 0.5 mg / m² 2 , 1 mg / m² 2 , or 2 mg / m² 2 The following dose is administered to the target population. For most subjects, the dose is 2 mg / m². 2 Gemtuzumab ozogamicin / Mylotarg® is preferred. However, some subjects may be administered at lower doses, for example, in the case of treatment-related side effects, such as dose-limiting toxicity (DLT), or in consideration of the individual subject's health condition, comorbidities, or medical history.

[0242] Gemtuzumab ozogamicin / Mylotarg® is administered to subjects in a 4-week (28-day) treatment cycle regimen, for example, at a dose of 2 mg / m². 2 With gemtuzumab ozogamicin / Mylotarg®, the full dose for each treatment cycle is received on day 1 of each 4-week (28-day) treatment cycle.

[0243] For most subjects, four consecutive treatment cycles of gemtuzumab ozogamicin / Mylotarg®, and therefore four doses of gemtuzumab ozogamicin / Mylotarg® spaced four weeks apart, are preferred. However, in some subjects, additional gemtuzumab ozogamicin / Mylotarg® treatment cycles, e.g., up to four further “continued” treatment cycles, may be initiated, e.g., at the same dose as the initial four treatment cycles, or at a lower dose, if clinically desirable, e.g., based on the subject’s clinical condition.

[0244] At the completion of the final gemtuzumab ozogamicin / Mylotarg® treatment cycle, subjects will be monitored for disease status and hematopoietic chimerism, and these parameters will be monitored every six months for five years following the completion of the final treatment cycle.

[0245] Example 7: Treatment of subjects with AML using CD33KO eHSPC generated using the gRNA E and CD33-targeted ADC gemtuzumab ozogamicin / Mylotarg®. Patients with CD33-positive AML are treated with allogeneic HCT including CD33KO eHSPC and ADC gemtuzumab ozogamicin / Mylotarg®.

[0246] For HCT, a population of cells containing CD34+ hematopoietic stem cells is obtained from healthy donors whose HLA matches that of the target at 8 / 8 gene loci (HLA-A, -B, -C, DRB1).

[0247] After G-CSF / prelixafor mobilization, at least 10 × 10 units of G-CSF / prelixafor should be taken from the donor for processing and subsequent administration to the recipient. 6 A maximum of two apheresis procedures are performed to obtain 1000 living cells / kg (kg refers to the weight of the recipient). From this apheresis product, at least 3.0 × 10⁶ cells are obtained. 6The live cells / kg (recipient weight) undergo minimal processing and are cryopreserved to serve as a backup stem cell source, for example, for use as a rescue dose. The remaining apheresis products are used for processing and preparation of the CD33KO eHSPC population for HCT.

[0248] CD33KO eHSPC population for HCT enriched with apheresis products for CD34+ cells, followed by nucleotide sequencing

number

[0249] The edited cells are then cultured for <48 hours. Upon harvesting, after the culture period, the cells are washed, resuspended in the final formulation, and cryopreserved. Cell viability and editing efficiency are confirmed using representative samples as described in Example 1, and the CD33KO eHSPC population that meets the criteria described in Example 1 (at least 70% viability and at least 45% CD33 editing efficiency) is used for HCT. The population for administration to the subjects should be at least 3 × 10⁶ 6 The population includes CD33KO eHSPC populations that meet these viability and editing efficiency criteria per kg of body weight of the cell / recipient target, preferably with at least 4 × 10 6 Cells / recipient body weight in kg, 5 × 10 6 Cells / recipient body weight in kg, 6 x 10 6 Cells / recipient weight in kg, or 7 × 10 6 Includes the weight (kg) of the cells / recipient.

[0250] After completion of the conditioning regimen, the patient receives HCT containing thawed CD33KO eHSPC via intravenous (IV) infusion. The day of HCT is day 0 of the treatment regimen.

[0251] At day 28, the engraftment of CD33KO eHSPCs in the subjects will be evaluated by measuring the absolute peripheral neutrophil count (ANC) of CD33KO (CD33-) neutrophils. If a subject shows an absolute peripheral CD33KO neutrophil count of ≥1000 / dL CD33-ANC 28 days after CD33KO eHSPC HCT, the subject is considered to have neutrophil recovery (also referred to as good CD33KO neutrophil engraftment).

[0252] If a subject shows neutrophil recovery on day 28, a bone marrow biopsy is taken from the subject on day 60 to assess the disease state and hematopoietic recovery. Furthermore, the percentage of donor chimerization and CD33-negative (CD33-) myeloid hematopoiesis are determined from peripheral blood at this point. If a subject shows good CD33-HSC engraftment and CD33-hematopoiesis on day 60, the subject is subsequently administered gemtuzumab ozogamicin / Mylotarg®. CD33-ANC is monitored in the subject before administration of gemtuzumab ozogamicin / Mylotarg®, and the subject should preferably have ≥1000 / dL CD33-ANC before receiving gemtuzumab ozogamicin / Mylotarg®.

[0253] Gemtuzumab ozogamicin / Mylotarg® administration should preferably be initiated within 30 days of the bone marrow biopsy on day 60, i.e., preferably by day 90. However, if the clinical condition of the subject requires such a delay, for example, in light of comorbidities including HCT-related comorbidities, in order to enable the subject to achieve ≥1000 / dL CD33-ANC, the initiation of gemtuzumab ozogamicin / Mylotarg® may be delayed up to day 120. If gemtuzumab ozogamicin / Mylotarg® is initiated 30 days or more after the bone marrow biopsy on day 60, repeated bone marrow biopsies should be completed before the initiation of gemtuzumab ozogamicin / Mylotarg®.

[0254] Gemtuzumab ozogamicin / Mylotarg(registered trademark) 0.1 mg / m² 2 ~2mg / m 2 Doses within the range, for example, 0.1 mg / m² 2 , 0.25 mg / m² 2 , 0.5 mg / m² 2 , 1 mg / m² 2 , or 2 mg / m² 2 The following dose is administered to the target population. For most subjects, the dose is 2 mg / m². 2 Gemtuzumab ozogamicin / Mylotarg® is preferred. However, some subjects may be administered at lower doses, for example, in the case of treatment-related side effects, such as dose-limiting toxicity (DLT), or in consideration of the individual subject's health condition, comorbidities, or medical history.

[0255] Gemtuzumab ozogamicin / Mylotarg® is administered to subjects in a 4-week (28-day) treatment cycle regimen, for example, at a dose of 2 mg / m². 2 With gemtuzumab ozogamicin / Mylotarg®, the full dose for each treatment cycle is received on day 1 of each 4-week (28-day) treatment cycle.

[0256] For most subjects, four consecutive treatment cycles of gemtuzumab ozogamicin / Mylotarg®, and therefore four doses of gemtuzumab ozogamicin / Mylotarg® spaced four weeks apart, are preferred. However, in some subjects, additional gemtuzumab ozogamicin / Mylotarg® treatment cycles, e.g., up to four further “continued” treatment cycles, may be initiated, e.g., at the same dose as the initial four treatment cycles, or at a lower dose, if clinically desirable, e.g., based on the subject’s clinical condition.

[0257] At the completion of the final gemtuzumab ozogamicin / Mylotarg® treatment cycle, subjects will be monitored for disease status and hematopoietic chimerism, and these parameters will be monitored every six months for five years following the completion of the final treatment cycle.

[0258] Example 8: Combination Therapy Subjects with acute myeloid leukemia (AML) are matched to healthy stem cell donors based on 8 / 8 gene loci (HLA-A, -B, -C, DRB1). The minimum number of recipient subjects is 10 × 10⁶. 6 To obtain individual living cells per kilogram, up to two apheresis techniques are performed on the donor subject.

[0259] For recipients of CD34+ healthy donor cells, at least approximately 10 × 10 6 Using 3.0 × 10⁶ living cells / kg, the minimum required to produce the CD33KO eHSPC HCT product and backup graft (i.e., rescue dose) is 3.0 × 10⁶. 6 The process produces 10

[0260] Recipients may undergo a bone marrow ablation conditioning regimen (preconditioning) before administration of CD33-edited hematopoietic cells. After completion of the conditioning regimen, CD33KO eHSPC HCT is administered to the subject via intravenous (IV) infusion on day 0. The subject is monitored for neutrophil recovery, defined as the recovery of peripheral neutrophil counts 28 days after infusion. On day 60, if the subject has good neutrophil engraftment, the subject undergoes a bone marrow biopsy to assess disease status and hematopoietic recovery. The percentage of donor chimerization and CD33-negative (CD33-) myeloid hematopoiesis is determined from peripheral blood. The subject must have a neutrophil count above the threshold (e.g., ≥1000 / dL CD33-ANC) before administration of gemtuzumab ozogamicin / Mylotarg®. The subject is then administered a dose of approximately 0.1 mg / m².2 ~2.0 mg / m² 2 Then, administer gemtuzumab ozogamicin / Mylotarg(registered trademark).

[0261] The subjects may continue to be evaluated and may receive one or more additional doses of gemtuzumab ozogamicin / Mylotarg® at the same dose as before or at a different (increased or decreased) dose.

[0262] Example 9: Combination therapy using multiplex editing Subjects with acute myeloid leukemia (AML) are matched to healthy stem cell donors based on 8 / 8 gene loci (HLA-A, -B, -C, DRB1). The minimum number of recipient subjects is 10 × 10⁶. 6 To obtain individual living cells per kilogram, up to two apheresis techniques are performed on the donor subject.

[0263] For recipients of CD34+ healthy donor cells, at least approximately 10 × 10 6 Using 3.0 × 10⁶ living cells / kg, the minimum required to produce a double-edited hematopoietic cell product and backup graft (i.e., rescue dose) is 3.0 × 10⁶. 6 The process produces 10

[0264] During collection, after the culture period is complete, the cells are washed, resuspended in the final formulation, and then cryopreserved.

[0265] Recipients may undergo a bone marrow ablation conditioning regimen (preconditioning) before administration of double-edited hematopoietic cells. After completion of the conditioning regimen, double-edited hematopoietic cells are administered to the subject via intravenous (IV) infusion on day 0. The subject is monitored for neutrophil recovery, defined as the recovery of peripheral neutrophil counts 28 days after infusion. On day 60, if the subject has good neutrophil engraftment, the subject undergoes a bone marrow biopsy to assess disease status and hematopoietic recovery. The percentage of donor chimerization and CD33-negative (CD33-) myeloid hematopoiesis are determined from peripheral blood. The subject must have a neutrophil count above the threshold (e.g., ≥1000 / dL CD33-ANC) before administration of gemtuzumab ozogamicin / Mylotarg®. The subject is then administered a dose of approximately 0.1 mg / m². 2 ~2.0 mg / m² 2 Then, administer gemtuzumab ozogamicin / Mylotarg(registered trademark).

[0266] The subjects may continue to be evaluated and may receive one or more additional doses of gemtuzumab ozogamicin / Mylotarg® at the same dose as before or at a different (increased or decreased) dose.

[0267] Example 10: Xenotransplant model for use of human CD33 KO eHSPC generated using gRNA E and CD33-targeted gemtuzumab ozogamicin / Mylotarg® The objective of this study was to investigate, using a mouse model, whether human hematopoietic stem cells and progenitor cells (HSPCs), or their induced cells (and their offspring), genetically engineered to reduce or eliminate CD33 expression, are protected from gemtuzumab ozogamicin / Mylotarg®-mediated cytotoxicity.

[0268] The recruited PBMCs were obtained from two human donors (Donor 1 and Donor 2) and screened to confirm that the cells were not homozygous for a single nucleotide polymorphism (SNP) at rs12459419, which resulted in a substitutively spliced ​​transcript variant lacking exon 2, leading to reduced expression of the full-length CD33 isoform, and therefore not recognized or targeted by gemtuzumab ozogamicin / Mylotarg®.

[0269] In short, as shown in Figure 2, donor-derived CD34+ HSPCs were electroporated with the CD33 gRNA E Cas9 ribonucleoprotein complex as described in Example 1. Cells from the same donor were electroporated without RNP and used as a negative control ("false EP"). CD33 editing efficiency is shown in Table 4. Cell number and viability were also quantified before and after electroporation (Table 5). [Table 4] [Table 5]

[0270] CD33 edited HSPC (CD33KO) and sham EP control CD34+HSPC were sublethally irradiated with NOD / scid / IL2Rγ. null ((NOD.Cg-Prkdc scid Il2rg tm1WjlThe drug was injected into NSG® mice via the tail vein. Eight weeks after transplantation, posterior orbital blood was collected from each mouse for flow cytometry analysis to evaluate engraftment of CD34+ HSPCs. Postorbital hemorrhage was also performed 12 weeks after transplantation to collect plasma and cell pellets, which were saved for further analysis. Fifteen weeks after transplantation, following hematopoietic system rearrangement with transplanted human HSPCs, mice were intravenously administered either gemtuzumab ozogamicin / Mylotarg® (0.33 mg / kg) or DPBS (Dulbeccio phosphate-buffered saline) as a vehicle control ("vehicle"). Eight days after treatment with gemtuzumab ozogamicin / Mylotarg® or the vehicle (16 weeks post-transplantation), the mice were euthanized, and bone marrow, blood, and spleen were collected for flow cytometry analysis. The experimental groups of cells obtained from Donor 1 and Donor 2 are shown in Tables 6 and 7, respectively. [Table 6] [Table 7]

[0271] As shown in Figure 3A, in mice transplanted with pseudo-EP HSPCs derived from Donor 1, human leukocyte rearrangement (hCD45+ cells) was reduced by gemtuzumab ozogamicin / Mylotarg® treatment, while mice transplanted with CD33-edited HSPCs derived from Donor 1 were unaffected by gemtuzumab ozogamicin / Mylotarg®. Furthermore, CD33 gene editing did not result in significant changes in human leukocyte chimerism, as demonstrated by human CD45 staining. In mice transplanted with HSPCs derived from Donor 2, no statistically significant differences in human cell chimerism were observed between groups treated with gemtuzumab ozogamicin / Mylotarg® versus vehicle controls, or between mice transplanted with CD33-edited HSPCs versus pseudo-EP HSPCs (Figure 5A).

[0272] The proportion of CD33+ cells among all human leukocytes (hCD45+ cells) in the bone marrow of transplanted animals was analyzed by flow cytometry. For each donor-derived HSPC, mice transplanted with a sham EP HSPC and subsequently treated with gemtuzumab ozogamicin / Mylotarg® showed a significant reduction in CD33+ cells compared to treatment with vehicle alone (Figures 3B, 5B). Furthermore, mice transplanted with CD33-edited HSPCs showed almost complete loss of CD33+ cells compared to mice transplanted with sham EP HSPCs, regardless of treatment with gemtuzumab ozogamicin / Mylotarg® or a vehicle control (Figures 3B, 5B). While not intended to be tied to any particular theory, these results are thought to be due to the highly efficient editing of donor human HSPCs and demonstrate robust ablation of CD33+ cells by gemtuzumab ozogamicin / Mylotarg®. This suggests that CD33-edited cells exhibit long-term persistence in this xenograft model.

[0273] Cells expressing different human myeloid markers from human CD45+ (mouse CD45-) populations were also evaluated. The fraction of monocytes (CD14+ myeloid cells) within total human leukocytes (hCD45+) in the bone marrow of transplanted mice was analyzed. For both donors, gemtuzumab ozogamicin / Mylotarg® treatment led to almost complete removal of CD14+ myeloid cells in mice transplanted with sham EP HSPCs (Figure 3C, 5C). In contrast, gemtuzumab ozogamicin / Mylotarg® treatment had little effect on the proportion of CD14+ myeloid cells in mice transplanted with CD33-edited HSPCs (Figure 3C, 5C). These results indicate that CD33-edited cells were protected from gemtuzumab ozogamicin / Mylotarg®-induced cytotoxicity (Figure 3E). No significant difference in the percentage of CD14+ myeloid cells was observed between mice transplanted with pseudo-EP HSPCs and mice transplanted with CD33-edited HSPCs, which supports the idea that the loss of CD33 does not impair the long-term differentiation of HSPC-derived CD14+ myeloid cells.

[0274] CD11b+ myeloid cells within the hCD45+ cell population were also analyzed. In mice transplanted with pseudo-EP HSPCs derived from either donor, gemtuzumab ozogamicin / Mylotarg® treatment eliminated the majority of CD11b+ myeloid cells (Figures 3D, 5D). In contrast, gemtuzumab ozogamicin / Mylotarg® treatment had little effect on the proportion of CD11b+ myeloid cells in mice transplanted with CD33-edited HSPCs (Figures 3D, 5D). These results support the idea that reduction of CD33 protects CD11b+ myeloid cells from gemtuzumab ozogamicin / Mylotarg®. No significant difference in the proportion of CD11b+ myeloid cells was observed between mice transplanted with pseudo-EP HSPCs and those transplanted with CD33-edited HSPCs, which supports the idea that loss of CD33 does not impair the long-term differentiation of HSPC-derived CD11b+ myeloid cells.

[0275] In addition to bone marrow cells, the proportion of CD3+ T cells among all human CD45+ cells in the bone marrow of transplanted mice was analyzed. No statistically significant difference was observed in the proportion of CD3+ T cells in mice transplanted with pseudo-EP HSPCs compared to mice transplanted with CD33-edited HSPCs (Figures 4A, 6A). As expected, CD3+ T cells were not affected by gemtuzumab ozogamicin / Mylotarg® treatment.

[0276] The proportion of CD19+ B cells among all human CD45+ cells in the bone marrow of transplanted animals was analyzed. No statistically significant difference was observed in the proportion of CD19+ B cells in mice transplanted with pseudo-EP HSPCs compared to mice transplanted with CD33-edited HSPCs (Figures 4B, 6B). As expected, CD19+ B cells were not affected by gemtuzumab ozogamicin / Mylotarg® treatment.

[0277] Comparing vehicle-treated groups, mice transplanted with CD33-edited HSPCs and pseudo-EP HSPCs had comparable levels of B cells, suggesting that B cell differentiation from HSPCs is not inhibited by CD33 knockout. Comparing gemtuzumab ozogamicin / Mylotarg®-treated mice, mice transplanted with pseudo-EP HSPCs had a higher percentage of CD19+ B cells in total human leukocytes than mice transplanted with CD33-edited HSPCs (Figures 4B, 6B). This likely reflects the fact that, since the myeloid and lymphoid fractions constitute the majority of the total human leukocyte fraction in the bone marrow of this mouse model, reduced myeloid protection (due to gemtuzumab ozogamicin / Mylotarg® treatment) results in a clear proportional increase in the lymphoid fraction in mice treated with pseudo-EP HSPCs.

[0278] Furthermore, the proportion of CD34+CD38-human primitive HSPCs was evaluated in the bone marrow of transplanted mice. No statistically significant differences were observed in the proportion of CD34+CD38- cells between mice receiving sham EP HSPCs versus CD33-edited HSPCs, and between mice treated with gemtuzumab ozogamicin / Mylotarg® versus vehicle controls (Figure 4C, 6C). These results suggest that CD33 loss does not affect CD34+CD38- primitive HSPCs, and that primitive HSPCs are not targeted by gemtuzumab ozogamicin / Mylotarg® cytotoxicity.

[0279] In summary, these analyses demonstrate that CD33-deficient cells are protected from gemtuzumab ozogamicin / Mylotarg cytotoxicity. Upon engraftment, CD33-edited HSPCs reconstituted a multiseries hematopoietic system, including comparable levels of human leukocyte chimerism, lymphoid and myeloid systems, and primitive HSPCs as control HSPCs. Furthermore, significant loss of CD33+ cells was observed in mice transplanted with CD33-edited HSPCs, indicating highly efficient CD33 distribution and long-term persistence of CD33-deficient cells after 16 weeks. In mice transplanted with control (unedited) HSPCs, gemtuzumab ozogamicin / Mylotarg® treatment efficiently eliminated CD14+ and CD11b+ myeloid cells. In contrast, animals transplanted with CD33-edited HSPCs retained significantly higher levels of myeloid cells after gemtuzumab ozogamicin / Mylotarg® treatment. In summary, these findings indicate that CD33 depletion provides substantial protection to myeloid cells against gemtuzumab ozogamicin / Mylotarg® cytotoxicity in vivo.

[0280] Example 11: Clinical-scale production of human CD33KO eHSPC Two clinical-scale batches of allogeneic CRISPR / Cas9 genome-edited hematopoietic stem cells / progenitor cells (HSPCs) lacking the CD33 protein were manufactured for the treatment of human leukocyte antigen (HLA)-matched patients with high-risk CD33+ acute myeloid leukemia (AML). The resulting HSPC population is suitable for infusion into HLA-matched human AML patients undergoing hematopoietic stem cell transplantation, such as those known to be at high risk of leukemia relapse and post-transplant mortality.

[0281] The final HSPC population was formulated in 45 mL volumes in cryopreservation medium prepared for cryopreservation, stored in the gas phase of liquid nitrogen, then thawed and administered to recipient patients by intravenous (IV) infusion.

[0282] Each batch was manufactured from leukocyte apheresis starting material obtained from a single donor, generating HLA-matched products from one donor to one recipient, thereby enabling the production of products specifically matched for patients.

[0283] For each batch, healthy donors were subjected to leukocyte apheresis. Leukocyte apheresis starting material was collected and stored at 2–8°C before the start of cell manipulation. Cell number and viability of the leukocyte apheresis starting material were tested by flow cytometry. Cell viability was confirmed to be ≥80%. Samples were removed for cell analysis and other evaluations.

[0284] The leukocyte apheresis rescue dose is taken from the leukocyte apheresis material and divided into 3 × 10⁻⁶ units. 6 A volume containing individual CD34+ cells / patient kg was obtained. Rescue dose material was cryopreserved and stored in the gas phase of liquid nitrogen at -140°C.

[0285] After obtaining the rescue dose, the leukocyte apheresis starting material was processed to extract red blood cells, platelets, and plasma. The processed materials were then concentrated for CD34-positive cells and transferred to 250 mL conical tubes.

[0286] The Cas9 / gRNA ribonucleoprotein (RNP) complex was prepared prior to electroporation by mixing Cas9 protein and gRNA E under sterile conditions. Cells in a 250 mL conical tube were spun down at 200 × g, resuspended in electroporation buffer, mixed with the prepared RNP complex, and electroporated using a single-use sterile electroporation cassette.

[0287] After electroporation, the cells were removed from the cassette, transferred to culture medium, and suspended in culture at 37°C and 5% CO2. The cell culture was monitored for cell number and viability. After harvesting the cells from electroporation (where cell viability was determined to be ≥80%), the cells were washed to reduce cell residue and other debris, and resuspended in serum-free, animal-component-free, delimited cryopreservation medium containing 10% DMSO. The cells were formulated in 45 mL volumes and cryopreserved in a controlled-rate freezer (CRF). Cell number, viability, percentage of cells expressing specific markers (e.g., CD34, CD3, CD19, CD56), editing efficiency, and residue Cas9 were determined as shown in Table 8 below. [Table 8]

[0288] Listed embodiments 1. A population of genetically modified hematopoietic cells, or their offspring, that include a modified gene encoding CD33 that has been engineered to have an effective amount of reduced or eliminated CD33 antigen expression; and A method comprising administering an effective amount of a cytotoxic agent containing an anti-CD33 antigen-binding domain. 2. The method according to Embodiment 1, wherein the cytotoxic agent is an antibody-drug conjugate (ADC). 3. The method according to Embodiment 2, wherein the ADC is gemtuzumab ozogamicin. 4. The effective dose of a population of genetically modified hematopoietic cells is approximately 10 6 Cells / body weight in kilograms ~ approximately 10 7 The method according to any one of Embodiments 1 to 3, wherein the cell / body weight is in kilograms. 5. The effective dose of a population of genetically modified hematopoietic cells is approximately 3.0 × 10⁻⁶. 6 The method according to Embodiment 4, wherein the cell / subject weight is in kilograms. 6. The effective dose of the cytotoxic agent is approximately 0.1 mg / m² of the target body surface area. 2 ~Approximately 2.0 mg / m² of the target body surface area 2The method according to any one of Embodiments 1 to 5. 7. The effective dose of the cytotoxic agent is approximately 0.1 mg / m² of the target body surface area. 2 Approximately 0.25 mg / m² of the target body surface area 2 Approximately 0.5 mg / m² of the target body surface area 2 Approximately 1.0 mg / m² of the target body surface area 2 , or approximately 2.0 mg / m² of the target body surface area 2 The method according to Embodiment 6. 8. The effective dose of the cytotoxic agent is approximately 2.0 mg / m² of the target body surface area. 2 The method according to Embodiment 7. 9. The method according to any one of Embodiments 1 to 8, wherein a population of genetically modified hematopoietic cells and a cytotoxic agent are administered in close succession. 10. The method according to Embodiment 9, wherein a short-term administration involves administering a population of genetically modified hematopoietic cells and a cytotoxic agent in a single treatment regimen. 11. The method according to Embodiment 9, wherein the administration at close intervals includes the simultaneous administration of a population of genetically modified hematopoietic cells and a cytotoxic agent. 12. The method according to Embodiment 9, wherein the administration at close intervals includes the simultaneous administration of a population of genetically modified hematopoietic cells and a cytotoxic agent. 13. The method according to Embodiment 9, wherein the administration in close succession includes sequentially administering a population of genetically modified hematopoietic cells and a cytotoxic agent. 14. The method according to Embodiment 9, wherein the administration at a close time is the administration of a population of genetically modified hematopoietic cells within 120 days of the administration of the cytotoxic agent. 15. The method according to Embodiment 14, wherein the administration at a close time includes administering a population of genetically modified hematopoietic cells within 90 days of the administration of the cytotoxic agent. 16. The method of Embodiment 15, wherein the administration at a close time includes administering a population of genetically modified hematopoietic cells within 60 days of the administration of the cytotoxic agent. 17. The method according to any one of Embodiments 1-9 or 12-16, wherein a population of genetically modified hematopoietic cells is administered before a cytotoxic agent. 18. The method according to any one of Embodiments 1 to 17, wherein a population of genetically modified hematopoietic cells is administered in a single treatment regimen. 19. The method according to any one of Embodiments 1 to 18, wherein a population of genetically modified hematopoietic cells and / or a cytotoxic agent is administered intravenously. 20. The method according to any one of Embodiments 1 to 19, wherein a cytotoxic agent is administered in multiple effective doses every four weeks. 21. A cytotoxic agent is administered every four weeks at a dose of approximately 2.0 mg / m². 2 The method according to any one of Embodiments 1 to 20, administered in multiple doses. 22. The method according to any one of Embodiments 1 to 21, wherein a population of genetically modified hematopoietic cells is thawed from a cryopreserved form prior to administration. 23. The method according to any one of Embodiments 1 to 22, wherein the cytotoxic agent is reconstituted from a lyophilized form before administration. 24. The method according to any one of Embodiments 1 to 23, wherein the subject is preconditioned before administration of a cytotoxic agent and / or hematopoietic cells. 25. The method according to any one of Embodiments 1 to 24, further comprising preconditioning the subject before administration of a cytotoxic agent and / or hematopoietic cells. 26. The method according to Embodiment 24 or Embodiment 25, wherein preconditioning includes administering one or more chemotherapeutic agents to the target. 27. The method according to any one of embodiments 24 to 26, wherein preconditioning includes whole-body irradiation of the subject. 28. The method according to any one of embodiments 24 to 27, wherein the chemotherapeutic agent is selected from the group consisting of busulfan, melphalan, fludarabine, cyclophosphamide, and thiotepa. 29. The method according to any one of Embodiments 24 to 28, wherein preconditioning comprises administering an antibody that binds to human T cells, and optionally the antibody comprises rabbit anti-thymocyte globulin (rATG). 30. The method according to any one of Embodiments 1 to 29, wherein the subject has or has been diagnosed with a hematopoietic malignancy or a pre-hematopoietic malignancy, the hematopoietic malignancy being characterized by the presence of CD33-positive malignant cells, or the pre-hematopoietic malignancy being characterized by the presence of CD33-positive pre-malignant cells. 31. The method according to any one of Embodiments 1 to 30, wherein the subject has or has been diagnosed with CD33-positive acute myeloid leukemia. 32. The method according to any one of Embodiments 1 to 31, wherein the subject has or has been diagnosed with CD33-positive myelodysplastic syndrome. 33. The method according to any one of Embodiments 1 to 30, wherein the subject has or has been diagnosed with CD33-positive myelodysplastic syndrome, and the subject is at high risk of developing acute myeloid leukemia. 34. The method according to any one of Embodiments 1 to 33, wherein the subject is unsensitized to chemotherapy and / or radiotherapy, and optionally, the subject is unsensitized to any treatment aimed at addressing hematopoietic malignancies or pre-hematopoietic malignancies. 35. The method according to any one of Embodiments 1 to 34, wherein the subject has previously received chemotherapy. 36. The method according to any one of Embodiments 1 to 35, wherein the subject has previously received induction therapy. 37. The method according to any one of Embodiments 1 to 36, wherein the subject has previously entered complete hematological remission, and optionally, complete hematological remission is characterized by incomplete recovery of peripheral counts. 38. The method according to any one of Embodiments 1 to 37, wherein the subject has one or more risk factors associated with early relapse of leukemia. 39. The method according to Embodiment 38, wherein one or more risk factors associated with early leukemia relapse are selected from the group consisting of bone marrow in morphological complete remission with the presence of a genetic trait associated with a moderate or high-risk disease; the presence of minimal residual disease (MRD) after cytoreductive therapy; bone marrow with residual leukemic blasts after cytoreductive therapy; and bone marrow blast count of about 10% or less, excluding circulating blasts. 40. The method according to any one of Embodiments 1 to 39, wherein the subject does not have a homozygous dominant genotype for the CD33 single nucleotide polymorphism (SNP) rs12459419. 41. The method according to any one of Embodiments 1 to 40, wherein the subject does not have acute promyelocytic leukemia or chronic myeloid leukemia. 42. The method according to any one of Embodiments 1 to 41, wherein the subject does not have a gene translocation associated with acute promyelocytic leukemia or chronic myeloid leukemia, and the gene translocation is optionally t(15;17)(q22;q21) or t(9;22)(q34;q11). 43. The method according to any one of Embodiments 1 to 42, wherein the subject has not previously undergone stem cell transplantation. 44. The method according to any one of Embodiments 1 to 43, wherein the subject has not previously been treated with a cytotoxic agent. 45. The method according to any one of Embodiments 1 to 44, further comprising determining the percentage of donor chimerization and / or the level of CD33-negative myeloid hematopoiesis in a peripheral blood sample derived from a subject. 46. ​​The method according to any one of Embodiments 1 to 45, wherein the subject has a CD33-negative absolute neutrophil count (ANC) of at least 1000 / dL before receiving a cytotoxic agent. 47. The method according to any one of Embodiments 1 to 46, wherein the hematopoietic cells are hematopoietic stem cells. 48. The method according to Embodiment 47, wherein the hematopoietic stem cells are derived from bone marrow cells, umbilical cord blood cells, or peripheral blood mononuclear cells (PBMCs). 49. Hematopoietic stem cells, CD34 + / CD33 - The method according to Embodiment 47 or Embodiment 48. 50. The method according to any one of Embodiments 1 to 49, wherein the hematopoietic cells are autologous. 51. The method according to Embodiment 50, further comprising obtaining autologous hematopoietic stem cells from a subject, optionally genetically modifying the autologous stem cells to have reduced or eliminated CD33 antigen expression, and returning the genetically modified hematopoietic stem cells to the subject. 52. The method according to any one of Embodiments 1 to 51, wherein the hematopoietic cells are of the same species. 53. The method according to Embodiment 52, wherein the hematopoietic cells are allogeneic hematopoietic stem cells obtained from a donor having an HLA haplotype that matches the target HLA haplotype. 54. The method according to any one of Embodiments 1 to 53, further comprising obtaining hematopoietic cells from a donor having an HLA haplotype that matches the target HLA haplotype. 55. The method according to any one of Embodiments 1 to 54, further comprising preparing hematopoietic cells by modifying the endogenous gene of hematopoietic cells encoding the CD33 antigen. 56. The method according to Embodiment 55, wherein all or part of the endogenous gene encoding the CD33 cell surface antigen is deleted. 57. The method according to Embodiment 55 or Embodiment 56, wherein all or part of an endogenous gene is deleted using genome editing. 58. The method according to Embodiment 57, wherein genome editing comprises a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a CRISPR-Cas system. 59. A method comprising administering to a subject an effective amount of a cytotoxic agent comprising an anti-CD33 antigen-binding domain, wherein the subject has received or is receiving an effective amount of a population of genetically modified hematopoietic cells or their offspring, comprising a modified gene encoding CD33 which has been manipulated to reduce or decrease the expression of the CD33 agent. 60. The method according to Embodiment 59, wherein the cytotoxic agent is an antibody-drug conjugate (ADC). 61. The method according to Embodiment 60, wherein ADC is gemtuzumab ozogamicin. 62. The effective dose of a population of genetically modified hematopoietic cells is approximately 10 6 Cells / body weight in kilograms ~ approximately 10 7 The method according to any one of embodiments 59 to 61, wherein the cell / body weight in kilograms is the weight of the subject. 63. The effective dose of a population of genetically modified hematopoietic cells is approximately 3.0 × 10⁻⁶. 6The method according to Embodiment 62, wherein the cell / subject weight is in kilograms. 64. The effective dose of a cytotoxic agent is approximately 0.1 mg / m² of the target body surface area. 2 ~Approximately 2.0 mg / m² of the target body surface area 2 The method according to any one of embodiments 59 to 63. 65. The effective dose of a cytotoxic agent is approximately 0.1 mg / m² of the target body surface area. 2 Approximately 0.25 mg / m² of the target body surface area 2 Approximately 0.5 mg / m² of the target body surface area 2 Approximately 1.0 mg / m² of the target body surface area 2 , or approximately 2.0 mg / m² of the target body surface area 2 The method according to embodiment 64. 66. The effective dose of a cytotoxic agent is approximately 2.0 mg / m² of the target body surface area. 2 The method according to embodiment 65. 67. The method according to any one of embodiments 59 to 66, wherein an effective amount of cytotoxic agent is administered in close proximity to an effective amount of a population of genetically modified hematopoietic cells. 68. The method of Embodiment 67, wherein the administration of a population of genetically modified hematopoietic cells and a cytotoxic agent in a single treatment regimen over a short period of time. 69. The method according to Embodiment 67, wherein the administration at close intervals includes the simultaneous administration of a population of genetically modified hematopoietic cells and a cytotoxic agent. 70. The method of Embodiment 67, wherein the administration over a period of time includes the simultaneous administration of a population of genetically modified hematopoietic cells and a cytotoxic agent. 71. The method according to Embodiment 67, wherein the administration in close succession includes sequential administration of a population of genetically modified hematopoietic cells and a cytotoxic agent. 72. The method of Embodiment 67, wherein the administration at close range includes administering a cytotoxic agent within 120 days of administration of a population of genetically modified hematopoietic cells. 73. The method of Embodiment 67, wherein the administration at close range includes administering a cytotoxic agent within 90 days of administration of a population of genetically modified hematopoietic cells. 74. The method of Embodiment 67, wherein the administration at close range includes administering a cytotoxic agent within 60 days of administering a population of genetically modified hematopoietic cells. 75. The method according to any one of embodiments 59 to 74, wherein a population of genetically modified hematopoietic cells is administered before a cytotoxic agent. 76. The method according to any one of embodiments 59 to 75, wherein a population of genetically modified hematopoietic cells is administered in a single treatment regimen. 77. The method according to any one of embodiments 59 to 76, wherein a population of genetically modified hematopoietic cells and / or a cytotoxic agent is administered intravenously. 78. The method according to any one of embodiments 59 to 77, wherein a cytotoxic agent is administered in multiple effective doses every four weeks. 79. A cytotoxic agent is administered every four weeks at a dose of approximately 2.0 mg / m². 2 The method according to any one of embodiments 59 to 78, administered in multiple doses. 80. The method according to any one of Embodiments 59 to 79, wherein a population of genetically modified hematopoietic cells is thawed from a cryopreserved form before administration. 81. The method according to any one of embodiments 59 to 80, wherein the cytotoxic agent is reconstituted from a lyophilized form before administration. 82. The method according to any one of embodiments 59 to 81, wherein the subject is preconditioned before administration of a cytotoxic agent and / or hematopoietic cells. 83. The method according to any one of Embodiments 59 to 82, further comprising preconditioning the subject before administration of a cytotoxic agent and / or a population of genetically modified hematopoietic cells. 84. The method according to Embodiment 82 or Embodiment 83, wherein preconditioning includes administering one or more chemotherapeutic agents to the target. 85. The method according to any one of embodiments 82 to 84, wherein preconditioning includes whole-body irradiation of the subject. 86. The method according to any one of embodiments 82 to 85, wherein the chemotherapeutic agent is selected from the group consisting of busulfan, melphalan, fludarabine, cyclophosphamide, and thiotepa. 87. The method according to any one of Embodiments 82 to 86, wherein preconditioning comprises administering an antibody that binds to human T cells, and optionally the antibody comprises rabbit anti-thymocyte globulin (rATG). 88. The method according to any one of Embodiments 59 to 87, wherein the subject has or has been diagnosed with a hematopoietic malignancy or a pre-hematopoietic malignancy, the hematopoietic malignancy being characterized by the presence of CD33-positive malignant cells, or the pre-hematopoietic malignancy being characterized by the presence of CD33-positive pre-malignant cells. 89. The method according to any one of embodiments 59 to 88, wherein the subject has or has been diagnosed with CD33-positive acute myeloid leukemia. 90. The method according to any one of embodiments 59 to 88, wherein the subject has or has been diagnosed with CD33-positive myelodysplastic syndrome. 91. The method according to any one of Embodiments 59 to 88, wherein the subject has or has been diagnosed with CD33-positive myelodysplastic syndrome, and the subject is at high risk of developing acute myeloid leukemia. 92. The method according to any one of Embodiments 59 to 91, wherein the subject is unsensitized to chemotherapy and / or radiotherapy, and optionally, the subject is unsensitized to any treatment aimed at addressing hematopoietic malignancies or pre-hematopoietic malignancies. 93. The method according to any one of embodiments 59 to 91, wherein the subject has previously received chemotherapy. 94. The method according to any one of embodiments 59 to 93, wherein the subject has previously received induction therapy. 95. The method according to any one of embodiments 59 to 94, wherein the subject has previously entered complete hematological remission, and optionally, complete hematological remission is characterized by incomplete recovery of peripheral counts. 96. The method according to any one of embodiments 59 to 95, wherein the subject has one or more risk factors associated with early relapse of leukemia. 97. The method according to Embodiment 96, wherein one or more risk factors associated with early leukemia relapse are selected from the group consisting of bone marrow in morphological complete remission with the presence of a genetic predisposition to the disease at moderate or high risk; the presence of minimal residual disease (MRD) after cytoreductive therapy; bone marrow with residual leukemic blasts after cytoreductive therapy; and bone marrow with a bone marrow blast count of about 10% or less that does not contain circulating blasts. 98. The method according to any one of Embodiments 59 to 97, wherein the subject does not have a homozygous dominant genotype for the CD33 single nucleotide polymorphism (SNP) rs12459419. 99. The method according to any one of embodiments 59 to 98, wherein the subject does not have acute promyelocytic leukemia or chronic myeloid leukemia. 100. The method according to any one of Embodiments 59 to 99, wherein the subject does not have a gene translocation associated with acute promyelocytic leukemia or chronic myeloid leukemia, and the gene translocation is optionally t(15;17)(q22;q21) or t(9;22)(q34;q11). 101. The method according to any one of embodiments 59 to 100, wherein the subject has not previously undergone stem cell transplantation. 102. The method according to any one of embodiments 59 to 101, wherein the subject has not previously been treated with a cytotoxic agent. 103. The method according to any one of Embodiments 59 to 102, further comprising determining the percentage of donor chimerization and / or the level of CD33-negative myeloid hematopoiesis in a peripheral blood sample derived from a subject. 104. The method according to any one of embodiments 59 to 103, wherein the subject has a CD33-negative absolute neutrophil count (ANC) of at least 1000 / dL before receiving the cytotoxic agent. 105. The method according to any one of embodiments 59 to 104, wherein the hematopoietic cells are hematopoietic stem cells. 106. The method according to Embodiment 105, wherein the hematopoietic stem cells are derived from bone marrow cells, umbilical cord blood cells, or peripheral blood mononuclear cells (PBMCs). 107. Hematopoietic stem cells, CD34 + / CD33 - The method according to Embodiment 105 or Embodiment 106. 108. The method according to any one of embodiments 59 to 107, wherein the hematopoietic cells are autologous. 109. The method of Embodiment 108, further comprising obtaining autologous hematopoietic stem cells from a subject, optionally genetically modifying the autologous stem cells to have reduced or eliminated CD33 antigen expression, and returning the genetically modified hematopoietic stem cells to the subject. 110. The method according to any one of embodiments 59 to 107, wherein the hematopoietic cells are of the same species. 111. The method according to Embodiment 110, wherein the hematopoietic cells are allogeneic hematopoietic stem cells obtained from a donor having an HLA haplotype that matches the target HLA haplotype. 112. The method according to any one of embodiments 59 to 111, further comprising obtaining hematopoietic cells from a donor having an HLA haplotype that matches the target HLA haplotype. 113. The method according to any one of embodiments 59 to 112, further comprising preparing hematopoietic cells by modifying the endogenous gene of hematopoietic cells encoding the CD33 antigen. 114. The method according to Embodiment 113, wherein all or part of the endogenous gene encoding the CD33 cell surface antigen is deleted. 115. The method according to Embodiment 113 or Embodiment 114, wherein all or part of an endogenous gene is deleted using genome editing. 116. The method according to Embodiment 115, wherein genome editing comprises a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a CRISPR-Cas system. 117. A method comprising administering to a subject a population of genetically modified hematopoietic cells, or their offspring, which contains a modified gene encoding CD33 that has been engineered to have reduced or eliminated expression of the CD33 antigen, wherein the subject has received or is receiving an effective amount of a cytotoxic agent containing an anti-CD33 antigen-binding domain. 118. The method according to Embodiment 117, wherein the cytotoxic agent is an antibody-drug conjugate (ADC). 119. The method according to Embodiment 118, wherein ADC is gemtuzumab ozogamicin. 120. The effective dose of a population of genetically modified hematopoietic cells is approximately 10 6 Cells / body weight in kilograms ~ approximately 10 7 The method according to any one of embodiments 117 to 119, wherein the cell / body weight in kilograms is the weight of the subject. 121. The effective dose of a population of genetically modified hematopoietic cells is approximately 3.0 × 10⁻⁶. 6 The method according to Embodiment 120, wherein the cell / subject weight is in kilograms. 122. The effective dose of a cytotoxic agent is approximately 0.1 mg / m² of the target body surface area. 2 ~Approximately 2.0 mg / m² of the target body surface area 2 The method according to any one of embodiments 117 to 121. 123. The effective dose of a cytotoxic agent is approximately 0.1 mg / m² of the target body surface area. 2 Approximately 0.25 mg / m² of the target body surface area 2 Approximately 0.5 mg / m² of the target body surface area 2 Approximately 1.0 mg / m² of the target body surface area 2 , or approximately 2.0 mg / m² of the target body surface area 2 The method according to Embodiment 122. 124. The effective dose of a cytotoxic agent is approximately 2.0 mg / m² of the target body surface area. 2 The method according to Embodiment 123. 125. The method according to any one of Embodiments 117 to 124, wherein an effective amount of cytotoxic agent is administered in close proximity to an effective amount of a population of genetically modified hematopoietic cells. 126. The method according to Embodiment 125, wherein the administration of a population of genetically modified hematopoietic cells and a cytotoxic agent in a single treatment regimen over a short period of time. 127. The method according to Embodiment 125, wherein the administration of a population of genetically modified hematopoietic cells and a cytotoxic agent at close intervals is performed simultaneously. 128. The method according to Embodiment 125, wherein the administration over a short period of time includes the simultaneous administration of a population of genetically modified hematopoietic cells and a cytotoxic agent. 129. The method according to Embodiment 125, wherein the administration in close succession includes sequentially administering a population of genetically modified hematopoietic cells and a cytotoxic agent. 130. The method according to Embodiment 125, wherein the administration is within 120 days of the administration of a cytotoxic agent, comprising administering a population of genetically modified hematopoietic cells within a similar timeframe. 131. The method according to Embodiment 125, wherein the administration at a close time is the administration of a population of genetically modified hematopoietic cells within 90 days of the administration of the cytotoxic agent. 132. The method according to Embodiment 125, wherein the administration at a close time includes administering a population of genetically modified hematopoietic cells within 60 days of the administration of a cytotoxic agent. 133. The method according to any one of embodiments 117 to 132, wherein a population of genetically modified hematopoietic cells is administered before a cytotoxic agent. 134. The method according to any one of embodiments 117 to 133, wherein a population of genetically modified hematopoietic cells is administered in a single treatment regimen. 135. The method according to any one of embodiments 117 to 134, wherein a population of genetically modified hematopoietic cells and / or a cytotoxic agent is administered intravenously. 136. The method according to any one of embodiments 117 to 135, wherein a cytotoxic agent is administered in multiple effective doses every four weeks. 137. A cytotoxic agent is administered every four weeks at a dose of approximately 2.0 mg / m². 2 The method according to any one of embodiments 117 to 136, administered in multiple doses. 138. The method according to any one of Embodiments 117 to 137, wherein a population of genetically modified hematopoietic cells is thawed from a cryopreserved form prior to administration. 139. The method according to any one of embodiments 117 to 138, wherein the cytotoxic agent is reconstituted from a lyophilized form before administration. 140. The method according to any one of Embodiments 117 to 139, wherein the subject is preconditioned before administration of a cytotoxic agent and / or a population of genetically modified hematopoietic cells. 141. The method according to any one of Embodiments 117 to 140, further comprising preconditioning the subject before administration of a cytotoxic agent and / or a population of genetically modified hematopoietic cells. 142. The method according to Embodiment 140 or Embodiment 141, wherein preconditioning includes administering one or more chemotherapeutic agents to the target. 143. The method according to any one of embodiments 140 to 142, wherein preconditioning includes whole-body irradiation of the subject. 144. The method according to Embodiment 142 or Embodiment 143, wherein the chemotherapeutic agent is selected from the group consisting of busulfan, melphalan, fludarabine, cyclophosphamide, and thiotepa. 145. The method according to any one of Embodiments 140 to 144, comprising preconditioning by administering an antibody that binds to human T cells, wherein the antibody optionally comprises rabbit anti-thymocyte globulin (rATG). 146. The method according to any one of Embodiments 117 to 145, wherein the subject has or has been diagnosed with a hematopoietic malignancy or a pre-hematopoietic malignancy, the hematopoietic malignancy being characterized by the presence of CD33-positive malignant cells, or the pre-hematopoietic malignancy being characterized by the presence of CD33-positive pre-malignant cells. 147. The method according to any one of embodiments 117 to 146, wherein the subject has or has been diagnosed with CD33-positive acute myeloid leukemia. 148. The method according to any one of embodiments 117 to 146, wherein the subject has or has been diagnosed with CD33-positive myelodysplastic syndrome. 149. The method according to any one of embodiments 117 to 146, wherein the subject has or has been diagnosed with CD33-positive myelodysplastic syndrome, and the subject is at high risk of developing acute myeloid leukemia. 150. The method according to any one of Embodiments 117 to 149, wherein the subject is unsensitized to chemotherapy and / or radiotherapy, and optionally, the subject is unsensitized to any treatment aimed at addressing hematopoietic malignancies or pre-hematopoietic malignancies. 151. The method according to any one of embodiments 117 to 149, wherein the subject has previously received chemotherapy. 152. The method according to any one of embodiments 117 to 151, wherein the subject has previously received induction therapy. 153. The method according to any one of embodiments 117 to 152, wherein the subject has previously entered complete hematological remission, and optionally, complete hematological remission is characterized by incomplete recovery of peripheral counts. 154. The method according to any one of embodiments 117 to 153, wherein the subject has one or more risk factors associated with early relapse of leukemia. 155. The method according to Embodiment 154, wherein one or more risk factors associated with early leukemia relapse are selected from the group consisting of bone marrow in morphological complete remission with the presence of a moderate or high-risk disease-associated genetic feature; the presence of minimal residual disease (MRD) after cytoreductive therapy; bone marrow with residual leukemic blasts after cytoreductive therapy; and bone marrow with a bone marrow blast count of about 10% or less that does not contain circulating blasts. 156. The method according to any one of Embodiments 117 to 155, wherein the subject does not have a homozygous dominant genotype for the CD33 single nucleotide polymorphism (SNP) rs12459419. 157. The method according to any one of embodiments 117 to 156, wherein the subject does not have acute promyelocytic leukemia or chronic myeloid leukemia. 158. The method according to any one of Embodiments 117 to 157, wherein the subject does not have a gene translocation associated with acute promyelocytic leukemia or chronic myeloid leukemia, and the gene translocation is optionally t(15;17)(q22;q21) or t(9;22)(q34;q11). 159. The method according to any one of embodiments 117 to 158, wherein the subject has not previously undergone stem cell transplantation. 160. The method according to any one of embodiments 117 to 159, wherein the subject has not previously been treated with a cytotoxic agent. 161. The method according to any one of Embodiments 117 to 160, further comprising determining the percentage of donor chimerization and / or the level of CD33-negative myeloid hematopoiesis in a peripheral blood sample derived from a subject. 162. The method according to any one of Embodiments 117 to 161, wherein the subject has a CD33-negative absolute neutrophil count (ANC) of at least 1000 / dL before receiving a cytotoxic agent. 163. The method according to any one of embodiments 117 to 162, wherein the hematopoietic cells are hematopoietic stem cells. 164. The method according to Embodiment 163, wherein the hematopoietic stem cells are derived from bone marrow cells, umbilical cord blood cells, or peripheral blood mononuclear cells (PBMCs). 165. Hematopoietic stem cells, CD34 + / CD33 - The method according to embodiment 163 or embodiment 164. 166. The method according to any one of embodiments 117 to 165, wherein the hematopoietic cells are autologous. 167. The method according to Embodiment 166, further comprising obtaining autologous hematopoietic stem cells from a subject, optionally genetically modifying the autologous stem cells to have reduced or eliminated CD33 antigen expression, and returning the genetically modified hematopoietic stem cells to the subject. 168. The method according to any one of embodiments 117 to 165, wherein the hematopoietic cells are of the same species. 169. The method according to Embodiment 168, wherein the hematopoietic cells are allogeneic hematopoietic stem cells obtained from a donor having an HLA haplotype that matches the target HLA haplotype. 170. The method according to any one of embodiments 117 to 169, further comprising obtaining hematopoietic cells from a donor having an HLA haplotype that matches the target HLA haplotype. 171. The method according to any one of Embodiments 117 to 170, further comprising preparing hematopoietic cells by modifying the endogenous gene of hematopoietic cells encoding the CD33 antigen. 172. The method according to Embodiment 171, wherein all or part of the endogenous gene encoding the CD33 cell surface antigen is deleted. 173. The method according to Embodiment 171 or Embodiment 172, wherein all or part of an endogenous gene is deleted using genome editing. 174. The method according to Embodiment 173, wherein genome editing comprises a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a CRISPR-Cas system. 175. A composition comprising a population of genetically modified hematopoietic cells, or their offspring, which comprises a modified gene encoding CD33 that has been manipulated to have reduced or eliminated expression of the CD33 antigen. 176. The composition according to Embodiment 175, wherein the hematopoietic cells are hematopoietic stem cells derived from bone marrow cells, umbilical cord blood cells, or peripheral blood mononuclear cells (PBMCs). 177. The composition according to Embodiment 176, wherein the hematopoietic stem cells are CD34+ / CD33-. 178. A composition according to any one of embodiments 175 to 177, wherein all or part of the endogenous gene encoding the CD33 antigen is deleted. 179. The composition according to any one of embodiments 175 to 178, wherein all or part of an endogenous gene is deleted using genome editing. 180. The composition according to Embodiment 179, wherein the genome editing performed comprises a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a CRISPR-Cas system. 181. The composition according to Embodiment 180, wherein the CRISPR-Cas system comprises a nucleic acid encoding a gRNA and an RNA guide nuclease. 182. The composition according to Embodiment 181, wherein the gRNA comprises a target domain containing the sequence described in any one of Sequence IDs 9 to 15. 183. A combination comprising a population of genetically modified hematopoietic cells as described in any one of Embodiments 175 to 182, and a cytotoxic agent containing an anti-CD33 antigen-binding domain. 184. The combination according to Embodiment 183, wherein the cytotoxic agent is an antibody-drug conjugate (ADC). 185. The combination according to Embodiment 184, wherein ADC is gemtuzumab ozogamicin. 185. At least 1 × 10⁶ per milliliter (mL) of culture medium. 6 A composition comprising a population of cells, wherein the population of cells comprises genetically modified hematopoietic cells or their offspring, the population comprising a modified gene encoding CD33 which has been manipulated to have reduced or eliminated expression of the CD33 antigen. 186. The composition according to Embodiment 185, wherein the culture medium has a volume of approximately 45 mL. 187. The composition according to Embodiment 185 or Embodiment 186, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the cells in the population are genetically modified hematopoietic cells or their offspring having reduced or eliminated CD33 antigen expression. 188. The population contains at least 2 × 10 per 1 mL. 6 Cells, at least 3 × 10⁶ per 1 mL 6 Cells, at least 4 × 10⁶ per 1 mL 6 Cells, at least 5 × 10 per 1 mL 6 Cells, at least 6 × 10⁶ per 1 mL 6 Cells, at least 7 × 10 per 1 mL 6 Cells, at least 8 × 10 per 1 mL 6 Cells, or at least 9 × 10 per 1 mL 6A composition according to any one of embodiments 185 to 187, comprising cells. 189. The composition according to any one of Embodiments 185 to 188, wherein the culture medium is a cryopreservation medium containing an antifreeze agent. 190. The composition according to Embodiment 189, wherein the antifreeze agent comprises dimethyl sulfoxide (DMSO) in an amount of approximately 10% (v / v). 191. The composition according to any one of embodiments 185 to 190, wherein the hematopoietic cells are CD34+ / CD33-. 192. The composition according to any one of Embodiments 185 to 191, wherein all or part of the endogenous gene encoding the CD33 antigen is deleted. 193. The composition according to any one of Embodiments 185 to 192, wherein all or part of an endogenous gene is deleted using genome editing. 194. The composition according to Embodiment 193, wherein the genome editing performed comprises a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a CRISPR-Cas system. 195. The composition according to Embodiment 194, wherein the CRISPR-Cas system comprises nucleic acids encoding gRNA and RNA guide nuclease. 196. The composition according to Embodiment 195, wherein the gRNA comprises a target domain containing the sequence described in any one of Sequence IDs 9 to 15. 197. The composition according to Embodiment 195 or Embodiment 196, which does not contain detectable levels of RNA guide nuclease. 198. A composition according to any one of embodiments 185 to 197, in a frozen state. 199. A cryopreserved composition comprising the composition described in any one of Embodiments 185 to 198, which has been subjected to a cryopreservation process. 200. A cryopreserved composition according to Embodiment 199, wherein the cryopreservation process is a rate-controlled freezing.

[0289] Equivalents and range Those skilled in the art will recognize many equivalents of the exemplary embodiments described herein, or can verify them by routine experimentation alone. The scope of this disclosure is not intended to be limited to the above description.

[0290] Articles such as "a," "an," and "the" can mean one or more unless otherwise indicated or made clear from the context. A claim or specification containing "or" between two or more members of a group is deemed satisfied if one, more than one, or all of the members of the group are present, unless otherwise indicated or made clear from the context. Disclosures of groups containing "or" between two or more members of a group provide embodiments in which exactly one member of the group is present, embodiments in which more than one member of the group is present, and embodiments in which all members of the group are present. For brevity, these embodiments are not described in detail individually herein, but it will be understood that each of these embodiments is provided herein and may be specifically asserted or refuted.

[0291] It should be understood that the present invention includes all variations, combinations, and permutations of one or more limitations, elements, clauses, or descriptive terms from one or more of the claims or relevant parts of the specification that are introduced into other claims. For example, a claim dependent on another claim may be modified to include one or more limitations found in any other claim dependent on the same basic claim. Furthermore, if a claim lists compositions, it should be understood that unless otherwise indicated, or unless it is obvious to a person skilled in the art that this would result in a contradiction or inconsistency, it includes methods of preparing or using the compositions by any of the methods of preparation or use disclosed herein, or, if any, by methods known in the art.

[0292] Where elements are presented as a list, it should be understood that all possible individual elements or subgroups of the element are also disclosed, and any element or subgroup of an element may be removed from the group. It should also be noted that the term “includes” is intended to be open and allow for the inclusion of additional elements, features, or steps. Generally, where an embodiment is referred to as including a particular element, feature, or step, it should be understood that embodiments consisting of, or essentially derived from, such elements, features, or steps are also provided. For brevity, these embodiments are not individually detailed herein, but it should be understood that each of these embodiments is provided herein and may be specifically asserted or refuted.

[0293] Where a range is given, it includes an endpoint. Furthermore, unless otherwise indicated, or unless it is obvious from the context and / or from the understanding of those skilled in the art, values ​​expressed as a range may, in some embodiments, be assumed to be any specific value within the range described, up to 1 / 10 of the lower limit unit of the range, unless the context explicitly indicates otherwise. For brevity, each value within a range is not individually detailed herein, but it will be understood that each of these values ​​is provided herein and may be specifically asserted or denied. Also, unless otherwise indicated, or unless it is obvious from the context and / or from the understanding of those skilled in the art, values ​​expressed as a range may be assumed to be any subrange within a given range, and the endpoint of a subrange may be expressed with a precision equivalent to 1 / 10 of the lower limit unit of the range.

[0294] In addition, it should be understood that any particular embodiment of the present invention may be expressly excluded from one or more of the claims. Where a range is given, any value within that range may be expressly excluded from one or more of the claims. For brevity, not all embodiments in which one or more elements, features, purposes, or aspects are excluded are expressly described herein. This disclosure intends any one or all combinations of the embodiments described above, as well as any one or all combinations of the embodiments described in the Detailed Description and Examples.

[0295] Similar or equivalent methods and materials may be used in carrying out or testing the present invention, but preferred methods and materials are described herein. All publications, patent applications, patents, and other references (e.g., sequence database reference numbers) mentioned herein are incorporated in their entirety by reference. For example, all GenBank sequences, Unigene sequences, and Entrez sequences mentioned herein, for example, in any table herein, are incorporated by reference. Unless otherwise specified, sequence accession numbers expressed herein refer to database entries as of May 23, 2019, in any table herein. If a gene or protein references multiple sequence accession numbers, all sequence variants are included.

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