Method and composition for transducing hematopoietic stem cells and hematopoietic progenitor cells in vivo.

Abstract

This invention provides compositions and methods for improving the in vivo transduction of hematopoietic stem cells and hematopoietic progenitor cells. [Solution] A method for transducing a population of hematopoietic stem cells or hematopoietic progenitor cells mobilized from the bone marrow of a mammalian subject into the peripheral blood, wherein a CXCR2 agonist is used to mobilize the subject's hematopoietic stem cells or hematopoietic progenitor cells into the peripheral blood, and the method comprises (a) administering a nucleic acid containing a selection marker for transducing hematopoietic stem cells or hematopoietic progenitor cells in vivo to the subject, and (b) administering a selection agent for selecting hematopoietic stem cells or hematopoietic progenitor cells that have been transduced with the nucleic acid containing the selection marker, wherein hematopoietic stem cells or hematopoietic progenitor cells that have not been transduced will not survive.

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application is based on U.S. Provisional Application No. 63 / 016,212, filed on April 27, 2020, and on May 12, 2020. We claim priority rights to the filed U.S. Provisional Application No. 63 / 023,749. The entirety of this is incorporated by reference in this application.

[0002] [Array List] This application includes an array list submitted electronically in ASCII format, the entirety of which is referenced in this application. It is incorporated by [the specified method]. The aforementioned ASCII copy, created on April 22, 2021, is MGA-008WO_SL.t The file is named xt and has a size of 3,440 bytes.

[0003] [Field of Invention] This invention relates to hematopoietic stem cells and hematopoietic progenitor cells in subjects such as humans. In vivo transduction of stem and progenitor cells (HSPCs), and in particular, hematological diseases. This relates to the treatment of individuals suffering from various pathological conditions such as metabolic disorders, cancer, and autoimmune diseases. do. [Background technology]

[0004] [Background of the Invention] Despite advances in medical technology, hematopoietic disorders, in particular, such as those involving specific blood cells, remain a challenge. To treat diseases (e.g., sickle cell disease (SCD)), metabolic disorders, cancer, and autoimmune conditions. And there is still a need for this. Current apps for gene therapy for such diseases Roach examples include gene therapy for ex vivo hematopoietic stem cells and hematopoietic progenitor cells, This involves costly methods that require complex manufacturing processes involving cell culture, and toxic substances. It is a conditioning regimen.

[0005] Therefore, in vivo transduction of hematopoietic stem cells and hematopoietic progenitor cells is particularly important for ex vivo gene transfer. It may be desirable in geographically challenging areas where treatment is difficult. However, in vivo hematopoiesis Direct transduction of stem cells and hematopoietic progenitor cells is hindered by the physical barrier of the bone marrow stroma. It's not efficient.

[0006] Furthermore, using G-CSF as a mobilizing agent can lead to several abnormalities such as sickle cell disease. It is contraindicated in patients with hemoglobinopathy. Furthermore, G-CSF is non-selective in bone marrow cells. This leads to cell mobilization, which results in leukocytosis and an increase in cytokine-producing cells in the periphery. This leads to contact with the intravenously injected viral vector. The number of cytokine-producing cells in the periphery increases, and this, in turn, mobilizes Compared to animals that were not subjected to the procedure, animals that were mobilized had higher cytokine levels. It becomes more so. Peripheral mobilized (committed) bone marrow cells also become infected with the virus. Because it encloses the vector, the effective dose for immature HSPCs decreases. Furthermore, G-CSF / AMD3 Due to the 5-day treatment regimen and high cost associated with 100, alternative mobilization regimens Developing [this] is a reasonable thing to do.

[0007] Therefore, compositions for improving in vivo transduction of hematopoietic stem cells and hematopoietic progenitor cells and A method is needed now. [Overview of the project] [Problems that the invention aims to solve]

[0008] [Overview of the prefecture] The present invention relates to compositions and methods for in vivo transduction of hematopoietic stem cells and hematopoietic progenitor cells. To provide. Using such methods, for example, a gene defect that leads to a disease of blood cells. We can provide gene therapy to correct the condition.

[0009] The above method, as described in this application, involves CXC chemokine receptor type 2 (CXC R2) Agonist (e.g., Gro-β or its variant [e.g., cleavage of Gro-β (e.g., (Gro-β T)) is optionally used for CXC chemokine receptor type 4 (CXCR4) Gonists (for example, 1,1'-[1,4-phenylenebis(methylene)]-bis-1,4,8,11-tetra-aza) In combination with cyclotetradecane or its variants, hematopoietic stem cells and hematopoietic progenitor cells are extracted from bone marrow. This may include recruiting cells, such as hematopoietic stem cells and hematopoietic progenitor cells. Transduction can be performed using nucleic acids containing a selection marker. Selective drugs can be used to select a It is possible to select hematopoietic stem cells or hematopoietic progenitor cells that have been transduced with nucleic acids containing the maker. Therefore, hematopoietic stem cells that were not transduced with nucleic acids containing a selection marker or Hematopoietic progenitor cells do not survive. [Means for solving the problem]

[0010] Therefore, in one aspect, the present disclosure relates to hematopoietic stem cells mobilized from the bone marrow into the peripheral blood of mammalian subjects. A method for transducing a population of cells or hematopoietic progenitor cells, where approximately 0.001 mg / kg to approximately 0.1 m Gro-β, Gro-β T, and their respective doses are administered in doses of g / kg, or in fixed doses of approximately 1 mg to approximately 8 mg. Using a CXCR2 agonist selected from the group consisting of variants, the target hematopoietic stem The method relates to the recruitment of cells or hematopoietic progenitor cells into the peripheral blood. The method relates to hematopoietic stem cells or This involves a nucleic acid containing a selection marker for transducing hematopoietic progenitor cells in vivo, applied to the aforementioned target. The administration step, and hematopoietic stem cells transduced with nucleic acids containing a selection marker or A step of administering a selective agent to select hematopoietic progenitor cells, thereby selecting a selection marker. Hematopoietic stem cells or hematopoietic progenitor cells that were not transduced with nucleic acids containing do not survive. Sometimes it happens.

[0011] In a particular embodiment, the nucleic acid is a gene editing or genetic engineering system, for example, CRISPR-Cas9 system, Sleeping Beauty Transposase y Transposase) 100x (SB100x) system, and recombinase system (e.g., FLP-F Includes components such as the RT system.

[0012] In a particular embodiment, the nucleic acid includes a therapeutic gene such as the γ-globin gene. In a particular embodiment, the nucleic acid is FANC AF; factor VIII (F8); factor IX (F9); X Factor (F10); Wiscott-Aldrich syndrome protein (WASP); Cytochrome B-245 Elastase chain (CYBB); Elastase neutrophil expressed (ELANE); Hemoglobin subunit alpha (HBA); hemoglobin subunit beta (HBB) ); pyruvate kinase, liver and RBC (PKLR); ribosomal protein S19 (RPS19); ATP Conjugation cassette subfamily D member 1 (ABCD1); aryl sulfatase A (ARSA); g Glucosylceramidase beta (GBA); Iduronate 2-sulfatase (IDS); Iduronate -ase, alpha-L (IDUA); T-cell immunomodulatory factor 1 (TCIRG1); adenosine deaminase (AD A); Interleukin-2 receptor subunit gamma (IL2RG); Bruton's tyrosine • Kinase (Bruton's Tyrosine Kinase (BTK)); Adenosine Deaminase (ADA); IL2R G; CD40 ligand (CD40LG); Forkhead Box P3 (FOXP3)); I Interleukin 4, 10, 13 (IL-4, 10, 13); Perforin 1 (PRF1); Artificial T cell receptor ( TCR; Chimeric antigen receptor (CAR); or CC motif chemokine receptor 5 (CCR5) Therapeutic genes include at least a portion of the genes that encode the subject.

[0013] In a particular embodiment, the selection marker is human O(6)-methylguanine-DNA-methyl It is a transferase (MGMT) variant.

[0014] In certain embodiments, the selected agent includes a methylating agent. The methylating agent is O6-benzylguanine (O6BG), bis-chloroethylnitrosourea. (BCNU), temozolomide, and combinations thereof are selected from among.

[0015] In a particular embodiment, the nucleic acid is a vector (e.g., a lentiviral vector). It exists in the rAAV vector or HDAd5 / 35++ vector, etc.

[0016] In a particular embodiment, the nucleic acid is the CXCR2 agonist and / or the CXCR4 agonist. The drug should be administered approximately 10 minutes to 10 hours after the administration of the tagonist.

[0017] In a particular embodiment, the selected drug is administered approximately 4 weeks to approximately 24 weeks after the administration of the nucleic acid. Administer at a later time.

[0018] In certain embodiments, the dose of the CXCR2 agonist ranges from over approximately 0.015 mg / kg to approximately 0.05 mg / kg It was less than 0 kg. In a particular embodiment, the CXCR2 agonist was administered at approximately 0.03 mg / kg. Administer in doses. In certain embodiments, the CXCR2 agonist is administered in doses ranging from approximately 2.5 mg to approximately 5.5 mg. Administer in a fixed dose of g. In a particular embodiment, the CXCR2 agonist is administered in approximately 1.3 mg. It is administered in a fixed dose. In a particular embodiment, the CXCR2 agonist is Gro-β T Includes.

[0019] In a particular embodiment, the method further includes the step of administering a CXCR2 agonist. nothing.

[0020] In a particular embodiment, the CXCR2 agonist and the CXCR4 antagonist are used Then, the target hematopoietic stem cells or hematopoietic progenitor cells are mobilized into the peripheral blood. A specific implementation In one embodiment, the CXCR4 antagonist is prelixafor. The subject is administered prelixafor at a dose of approximately 240 μg / kg.

[0021] In a particular embodiment, the CXCR2 agonist is used simultaneously with the CXCR4 antagonist. Administer. In a particular embodiment, the CXCR2 agonist is administered to the CXCR4 antagonist. It is administered after the following. In a particular embodiment, the CXCR2 agonist is administered after the CXCR4 antagonist. The CXCR2 agonist is administered within approximately 4 hours of administering the nyst. In a particular embodiment, the CXCR2 agonist is administered within 4 hours of administering the nyst. The drug is administered approximately 2 hours after the CXCR4 antagonist. In a particular embodiment, the The CXCR2 agonist and the CXCR4 antagonist are each administered for two consecutive days. In a particular embodiment, the CXCR2 agonist and the CXCR4 antagonist are, respectively It is administered once a day for two consecutive days. [Brief explanation of the drawing]

[0022] [Figure 1] Figure 1A is an overview of the protocol used for in vivo transduction of CD46-transgenic mice in Example 1. Blood cells were recruited using GCSF + plerixafor (5 days) or Gro-β + plerixafor (simultaneous subcutaneous administration), and then the mice were injected with the indicated amounts of the HDAd5 / 35++ mgmt / GFP vector + HDAd-SB vector to be incorporated, 1 hour later. Figure 1B is a graph showing the number of LSK (series-cKit+Sca1+) cells measured by flow cytometry at various time points after MGTA-145 injection. Figure 1C is a graph showing the number of colony-forming cells present in peripheral blood, measured by methylcellulose assay. [Figure 2] Figure 2 is a graph showing the number of CFUs generated after plating blood from mice recruited with GCSF + prelixafor or Gro-β + prelixafor. [Figure 3]Figure 3A is a graph showing the cell / mL counts of various blood types collected at different time points after prelixafor administration and subjected to hemabet analysis. The bars, from left to right, represent leukocytes (WBC), neutrophils (NE), lymphocytes (LY), monocytes (MO), and eosinophils (EO). Figure 3B is a graph showing that using MGTA-145 + prelixafor recruits fewer mononuclear cells (MNCs) one hour after the last drug injection compared to using G-CSF + prelixafor. Figure 3C is a graph showing the percentage of reticulocytes detected by Brilliant Cresil Blue. [Figure 4] Figure 4 shows the in vivo transduction / selection scheme for Example 1. CD46-transgenic mice were transduced by intravenous injection of HDAd-mgmt / GFP + HDAd-SB. Four rounds of selection were performed at weeks 4, 6, 8, and 10 post-transduction by IP injection of O6BG / BCNU. Primary mice were euthanized at week 12 post-transduction. Strain-negative cells were isolated from primary mice and injected into lethally irradiated C57Bl / 6 mice. These secondary transplanted mice were followed up to week 16 for final analysis. [Figure 5] Figure 5A is a graph showing GFP labeling in peripheral blood mononuclear cells (PBMCs) at various time points after transduction according to the scheme of Example 1. Figure 5B is a graph showing GFP expression in CD3-, CD19-, and Gr-1-positive mononuclear cells (MNCs) from blood, spleen, and bone marrow at 16 weeks. LSK cells in bone marrow samples are also shown. Figure 5C is a graph showing the percentage of pooled colony cells expressing GFP+ after a methylcellulose assay of lineage-negative cells isolated from bone marrow 16 weeks after transduction. [Figure 6]Figure 6A shows the engraftment rate when human CD46+ cells in PBMCs were detected by flow cytometry. As shown, leukocytosis in the MGTA-145 + prelixafor group is much lower than in the G-CSF + prelixafor group. Figure 6B is a graph showing the percentage of GFP-expressing PBMCs at various time points up to 16 weeks post-transplant. [Figure 7] Figure 7A is a graph showing the cellular composition of MNCs in blood, spleen, and bone marrow 16 weeks post-secondary transplantation. Each dot represents one animal. Naive animals that had not undergone transduction were used as controls. Figure 7B shows the number of colony-forming units (CFUs) / 2500 Lin-cells of lineage-negative cells isolated from bone marrow 16 weeks post-transduction after a 10-day methylcellulose assay. [Figure 8] Figure 8 is a graph showing serum IL-6 (pg / ml) levels in mice 1 and 6 hours after transduction. Each dot represents one animal. Samples from unmobilized mice were used as a control. *, p<0.05. [Figure 9] Figure 9A is a graph showing the number of LSK (strain-cKit+Sca1+) cells measured by flow cytometry one hour after MGTA-145 injection in a thalassemia mouse model. Each dot represents one animal. Figure 9B is a graph showing the number of colony-forming cells present in peripheral blood as measured by a methylcellulose assay in a thalassemia mouse model. Each dot represents one animal. [Figure 10]Figure 10 shows the phenotypes of Hbbth3 / CD46tg (thalassemia) and Hbbtm2 / CD46tg (Townes or sickle cell disease model) cells before treatment. RBC morphology was measured by Giemsa / May-Grunwald staining of blood smears. The percentage of reticulocytes was measured by brilliant cresil blue staining. Samples from CD46 mice were used as a "healthy" control. [Modes for carrying out the invention]

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[0041] [Detailed explanation] The present invention relates to compositions and methods for in vivo transduction of hematopoietic stem cells and hematopoietic progenitor cells. To provide. Using such methods, for example, a gene defect that leads to a disease of blood cells. We can provide gene therapy to correct the condition.

[0042] The above method, as described in this application, involves CXC chemokine receptor type 2 (CXC R2) Agonist (e.g., Gro-β or its variant [e.g., cleavage of Gro-β (e.g., Gr o-β T)) is optionally used as an antagonist for the CXC chemokine receptor type 4 (CXCR4). Nist (for example, 1,1'-[1,4-phenylenebis(methylene)]-bis-1,4,8,11-tetra-azasi In combination with clotetradecane or its variants, hematopoietic stem cells and hematopoietic progenitor cells are extracted from bone marrow. This may include mobilizing cells. The mobilized hematopoietic stem cells and hematopoietic progenitor cells are Nucleic acids containing a selection marker can be used for transduction. A selection agent can be used to introduce a selection marker. Hematopoietic stem cells or hematopoietic progenitor cells transduced with nucleic acids containing Kerr can be selected. As a result, hematopoietic stem cells or cells that were not transduced with nucleic acids containing a selection marker Hematopoietic progenitor cells do not survive.

[0043] The present invention, in part, relates to CXCR2 agonists (e.g., Gro-β, Gro-β T, or their variants). (e.g., rianto) can be optionally used with a CXCR4 antagonist (e.g., prelixafor or In combination with the pharmaceutically acceptable salt (such as the salt), the mobilized hematopoietic stem cells and hematopoietic progenitor cells are in In vivo transduction is performed to correct defects in genes that lead to diseases of blood cells, for example. This is based on the discovery that this is possible. Furthermore, CD34 + CD90 + CD45RA - Cells (stem related to long-term engraftment) A population exhibiting a cellular phenotype is effectively mobilized by the administration method described in this application. Therefore, mobilized hematopoiesis produced using the compositions and methods described in this application. Stem cell and hematopoietic progenitor cell populations are used in combination with in vivo transduction (e.g., gene therapy). It is particularly suitable for this purpose.

[0044] As described in this application, hematopoietic stem cells differentiate into numerous cell types in the hematopoietic lineage. Therefore, in vivo transduction can be used to correct genetic defects in cell types and treat the condition. In individuals, it is possible to engraft or regenerate cell types that are defective or missing. Yes, it is possible. The patient may have, for example, an autoimmune disease, cancer, an abnormal hemoglobin disorder, or other hematopoietic conditions. Patients may have one or more blood disorders, and therefore, hematopoietic stem Cell gene therapy is required. Therefore, the present invention is particularly relevant to Fanconi anemia (Fanconi a Hemophilia, hemophilia A, hemophilia B, factor X deficiency, Wiskot-Aldrich syndrome t-Aldrich syndrome, X-linked chronic granulomatous disease, Kostmann's syndrome ome), alpha-thalassemia, beta-thalassemia, sickle cell disease (sickle cell anemia), pi Rubinate kinase deficiency, Diamond-Blackfan anemia ), X-linked adrenoleukodystrophy, metachromatic leukodystrophy Lophie's disease, Gaucher disease, Hunter syndrome, Mucopolysaccharidosis type 1, Osteopetrosis, Adenosine degeneration Aminase (ADA)-deficient severe combined immunodeficiency, X-linked severe combined immunodeficiency, X-linked agamma Globulinemia, X-linked hyper-IgM syndrome, IPEX syndrome, early-onset inflammatory disease, hemophagocytosis Lymphoid tissue disease (hemophagocytic lymphohistiocytosis), Schwakman-Diamond disease Schwachman-Diamond syndrome, human immunodeficiency virus infection, and acquired immunodeficiency virus infection. We provide methods to treat all syndromes, as well as various hematopoietic symptoms such as cancer and autoimmune diseases. ru.

[0045] The following sections describe the process of moving a population of hematopoietic stem cells or hematopoietic progenitor cells from the stem cell niche to the peripheral blood. In this location, the hematopoietic stem cells or hematopoietic progenitor cells undergo in vivo transduction, for example. , one or more stem cell disorders [e.g., cancer, autoimmune diseases], metabolic disorders as described in this application To induce mobilization for the treatment of the harm (correcting the defective gene), This section will explain CXCR4 antagonists and CXCR2 agonists that can be administered to this patient.

[0046] [Definition] As used in this application, the term "about" means a value that is 10% higher or lower than the stated value. For example, the term "approximately 5 nM" refers to a range from 4.5 nM to 5.5 nM.

[0047] As used in this application, the term “antibody” means an antibody that specifically binds to a particular antigen or is specific to a particular antigen. This refers to immunoglobulin molecules that are immunologically reactive with an antigen. Antibodies include... Liclonal antibodies, monoclonal antibodies, genetically modified antibodies, and antibody variants (e.g.) For example, although not limited to them, chimeric antibodies, humanized antibodies, heteroconjugate antibodies [For example, bi-, tri- and quad-specific antibodies, diabody, triabody] [i, and tetrabodies], as well as antigen-binding fragments of antibodies [e.g., Fab', F(ab')2] Examples include [Fab, Fv, rlgG, and scFv fragments]). Unless otherwise specified, In short, the term "monoclonal antibody" (mAb) refers to an antibody that specifically binds to a target protein. Intact molecules, as well as antibody fragments (e.g., Fab and F(ab')2) can be used. This means including both fragments of Fab and Fab. The F(ab')2 fragment is an antibody fragment lacking the Fc fragment of an intact antibody. This refers to [the following]. Examples of these antibody fragments are described in this application.

[0048] As used in this application, the term "antigen-binding fragment" is specific to the target antigen. This refers to a fragment of one or more antibodies that retains the ability to bind to an antigen. The combination function can be performed by a fragment of a full-length antibody. For example, Fab, F(ab')2, scFv, Diabody, Triabody, Affibody, Nanobody It may be a d-body, i-body, aptamer, or domain antibody. Antigen-binding of the antibody Examples of bound fragments that fall under the term "fragment" are limited to the following: However, the following can be mentioned: (i) Fab fragments, V L , V H , C L and C H From 1 domain a monovalent fragment; (ii) a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region; (iii) an Fd fragment consisting of a V and C H domains; (iv) an Fv fragment consisting of the V H and V domains of a single arm of an antibody; (v) a dAb comprising the V L and V H domains; (vi) a dAb fragment consisting of a V domain (see, for example, Ward et al., Nature 341:544-546, (1989)); (vii) a dAb consisting of a V H or V L domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more (e.g., two, three, four, five, or six) isolated H CDRs optionally linked by a synthetic linker. Further, the two domains (V and V H or V L ) of the Fv fragment are encoded by separate genes, but they can be linked by a linker using recombinant methods such that the resulting single protein chain (the V and V regions pair to form a monovalent molecule) (known as single-chain Fv (scFv); see, for example, Bird et al., Sc L ience 242:423-426, (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879 H -5883, (1988)). These antibody fragments are prepared by conventional techniques known to those of skill in the art . The two domains (V L and V H ) of the Fv fragment are encoded by separate genes, but they can be linked by a linker using recombinant methods such that the resulting single protein chain (the V and V regions pair to form a monovalent molecule) (known as single-chain Fv (scFv); see, for example, Bird et al., Sc It can be obtained using, and the fragment can be combined with an intact antibody. The same method can be used to screen for usefulness. Antigen-binding fragments This involves the enzymatic or chemical cleavage of intact immunoglobulins using recombinant DNA technology. By, or in some cases by chemical peptide synthesis procedures known in the art. It can be produced.

[0049] As used in this application, the term "bispecific antibody" means: At least two different antigens, or two different epitons, may be present on the same antigen. A monoclonal antibody, often a human antibody, can bind to a pyogenes molecule. This refers to a t-induced antibody.

[0050] As used in this application, the term "complementarity-determining region" (CDR) refers to the variable light and heavy chain regions of an antibody. This refers to a highly variable region found in both the main and subdomains. The skeletal region is called the skeletal region (FR). The amino acid positions representing the hypervariable region of an antibody depend on the context. This may change depending on various definitions known in the relevant technical field. Some positions can be considered hybrid hypervariable positions, and these positions are Under one set of criteria, it may be considered to be in the hypervariable region, while under another set of criteria... Under these conditions, it may be considered to be outside the hypervariable region. One or more of these positions are also They may also be found in the extended hypervariable region. The antibodies described in this application are these The hybrid hypervariable position may include modifications. The inherent variable domains of the heavy and light chains are Each of them mainly adopts a β-sheet structure and is connected by three CDRs (CDRs are β-sheets). Four skeletal regions that connect the structure, and in some cases form part of it, forming loops. The region is included. The CDRs in each chain are in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in the skeletal region. Therefore, they protrude closely and cluster together, and together with CDRs derived from other antibody chains, they target the antibody. Contributes to the formation of binding sites (Kabat et al., Sequences of Proteins of Immunological In See Terest, National Institute of Health, Bethesda, MD., 1987. When used, the numbering of amino acid residues of immunoglobulins should be Kaba unless otherwise specified. This is done according to the numbering system for amino acid residues of immunoglobulins developed by t et al.

[0051] When used in this application, the terms “conservative mutation,” “conservative substitution,” or “conservative amino acid” are used. "Substitution" indicates similarity in physicochemical properties such as polarity, static charge, and stereovolume. This refers to the substitution of one or more different amino acids for one or more other amino acids. These characteristics are... Table 1 below summarizes each of the 20 natural amino acids. [Table 1] + Based on the volume of A3: 50-100 is small, 100-150 is medium, 150-200 is large, and >200 is bulky.

[0052] From this table, examples of conserved amino acid families include (i) G, A, V, L, I, P, and M; (ii) D and E; (iii) C, S and T; (iv) H, K and R; (v) N and Q; and (vi) F, Y and W , for example. Therefore, a conservative mutation or substitution is the same amino acid formula as a given amino acid formula. This replaces the members of Millie (for example, replacing Ser with Thr, or Lys with Arg). .

[0053] When used in this application, "CRU (competitive repopulating unit)" is used. "))" refers to a unit of measurement for long-term engrafted stem cells that can be detected after transplantation in vivo.

[0054] As used in this application, the term "diabody" means a divalent antibody comprising two polypeptide chains. This refers to the body, where each polypeptide chain is on the same peptide chain. H and V L Domain portion Linkers that are too short for intramural association (for example, phosphorus composed of 5 amino acids) V connected by (car) H and V L Includes domains. This configuration allows each domain The in pairs with a complementary domain on another polypeptide chain to form a homodimer structure. Therefore, the term "tribody" refers to a trivalent antibody containing three peptide chains, and each of them These are V molecules within the same peptide chain. H and V L It is far too short to allow intramolecular association of domains. A single V linked by a linker (for example, a linker composed of 1 to 2 amino acids) H Domain and one V L It contains a domain. The peptide constructed in this way is, by nature In order to fold into this structure, the V of the adjacent peptide chain is usually H Domain and V L Domaine The molecules trimerize so that they are spatially close to each other (for example, Holliger et al., Proc. Na See tl. Acad. Sci. USA 90:6444-48, (1993).

[0055] As used in this application, the term "disrupt" with respect to genes means disrupting a functional gene product. This refers to inhibiting the formation of a gene. The gene product will only function if it satisfies its normal (wild-type) function. It is functional. Disrupting the aforementioned gene prevents the expression of the functional factor encoded by that gene. to express, and a gene, and / or a process necessary for the expression of the gene in an animal. Insertion of one or more bases in the sequence encoded by the motor and / or operator This includes insertion, deletion, or substitution. The disrupted gene is, for example, from the genome of an animal. To remove at least a portion of the offspring, or to prevent the expression of the functional factor encoded by the gene. Therefore, modifying the aforementioned gene, interfering RNA, or dominant-negative genes The expression of vital factors can cause destruction. Genetically modifying hematopoietic stem / progenitor cells. The materials and methods are described in U.S. Patent Application Publication No. 8,518,701; U.S. Patent Application Publication No. 2010 / 0251395 Each of these disclosures, as detailed in U.S. Patent Application Publication No. 2012 / 0222143, The entirety of this is incorporated by reference in this application (in case of any conflict, this specification shall prevail).

[0056] When used in this application, "dual variable domain immunoglobulin" Munoglobulin ("DVD-Ig") is a tetravalent, dual-targeting monotherapy. To generate a dual-targeting single agent (nt), two monocles are sent via the linker. This refers to an antibody to which the target-binding variable domain of a ronal antibody has been conjugated (for example, Gu et al. See ., Meth. Enzymol., 502:25-41, (2012).

[0057] As used in this application, the term “endogenous” means naturally occurring in a particular living organism, such as a human patient. Molecules, cells, tissues, or organs found (e.g., hematopoietic stem cells, or megakaryocytes, platelets (thro mbocytes, platelets, red blood cells, mast cells, myeloblasts, basophils Neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macules Lophages, dendritic cells, natural killer cells, T lymphocytes, or B lymphocytes, etc. Describe substances such as hematopoietic cells.

[0058] As used in this application, the term "engraftment potential" refers to hematopoietic stem cells and The term is used to refer to the ability of hematopoietic progenitor cells to regenerate and adhere to tissues, and such cells are naturally Whether circulating or provided by transplantation. The aforementioned terms refer to a group of cells. Surrounding engraftment, such as tissue homing and colonization of cells within the tissue of interest, Or it encompasses all events leading to successful engraftment. The efficiency or rate of engraftment is determined by the discretion of those skilled in the art. It can be evaluated or quantified using clinically acceptable parameters of intent, for example, competition. To evaluate competitive repopulating units (CRUs); stem cells In tissues that have grown, colonized, or engrafted, the marker is incorporated. To or to manifest; or the progression of the disease, the survival of hematopoietic stem cells and hematopoietic progenitor cells, Alternatively, this may include evaluating the progression of the disease based on the recipient's survival. Engraftment can also be determined by measuring the number of white blood cells in the peripheral blood after transplantation. In bone marrow aspiration samples, engraftment occurs when bone marrow cells are restored by transduced cells. It can also be evaluated by measuring what is done.

[0059] As used in this application, the term "exogenous" refers to a specific life, such as that of a human patient. Molecules, cells, tissues, or organs not found naturally in the body (e.g., hematopoietic stem cells, or megamolecule). nuclear cells, platelets, red blood cells, mast cells, myeloblasts ast), basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigens - Presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, or B-lymphocytes This section describes substances such as hematopoietic cells (like lymphocytes). Exogenous substances include those supplied from external sources. This includes substances provided to living organisms or cultures extracted therefrom.

[0060] As used in this application, the terms "skeletal region" or "FW region" refer to an antibody or its antigen-binding region. It contains amino acid residues adjacent to the CDR of the fragment. The residues in the FW region are, for example, human antibodies. Humanized antibodies, monoclonal antibodies, antibody fragments, Fab fragments, single-chain antibody fragments Present in fragments, scFv fragments, antibody domains, and bispecific antibodies, etc. There are things that need to be done.

[0061] As used in this application, the term "hematopoietic progenitor cell" refers to several cell types of the hematopoietic system (particularly (including, but not limited to, granulocytes, monocytes, erythrocytes, megakaryocytes, B-cells, and T-cells) This includes pluripotent cells that can differentiate into hematopoietic cells. Hematopoietic progenitor cells are hematopoietic cells. They are committed to a lineage and generally do not self-replicate. Hematopoietic progenitor cells are, for example, It can be identified by the expression pattern of cell surface antigens and has the following immunophenotypes Includes cells that do: Lin - KLS + Flk2 - CD34 + Hematopoietic progenitor cells include short-term hematopoietic stem cells and pluripotent stem cells. Multipotent progenitor cells, common myeloid progenitor cells, granulocytes - monopotent progenitor cells This includes bulbar progenitor cells and megakaryocyte-erythrocyte progenitor cells. The presence of hematopoietic progenitor cells is confirmed. Functionally, for example, colony-forming unit cells, for example, complete methylcellulose In a complete methylcellulose assay, detection is performed, or the output Flow cytometry and cell selection described in the application and known in the art. Phenotypic determination can be made through the detection of cell surface markers using a separate assay. ru.

[0062] As used in this application, the term "hematopoietic stem cell" ("HSC") means , the ability to self-replicate, and granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), red blood Spheres (e.g., reticulocytes, red blood cells), platelets (e.g., megakaryoblasts, platelet-producing megakaryoblasts) Nuclear cells, platelets, monocytes (e.g., monocytes, macrophages), dendritic cells, microg This includes, but is not limited to, rheumatoid arthritis, osteoclasts, and lymphocytes (e.g., NK cells, B- cells, and T- cells). Immature blood cells that have the ability to differentiate into mature blood cells, including a diverse range of lineages that are not yet defined. Yes. One such cell is CD34. + Cells are mentioned. CD34 + The cells have a CD34 cell surface marker. These are immature cells that express Kerr. In humans, CD34 + The cells are stem cells as defined above. It is thought to contain a subpopulation of cells with specific characteristics, but in mice, HSCs are CD34- Furthermore, HSC also refers to long-term regenerative HSC (LT-HSC) and short-term regenerative HSC (ST-HSC). LT-HS C and ST-HSC are related to the development of functional potential and cell surface markers. They are distinguished based on their current characteristics. For example, human HSCs are classified as CD34. + CD38 - CD45RA - CD90 + CD49F + , and lin - (Includes CD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, CD235A, etc.) (Negative for mature lineage markers). In mice, bone marrow LT-HSCs are CD34- SCA-1+, C-kit+, CD135-, Slamfl / CD150+, CD48-, lin-(Ter119, CD11b, Gr1, CD3, CD4 (Negative for mature series markers including CD8, B220, IL7ra, etc.) On the other hand, S T-HSC is CD34 + SCA-1 + C-kit + CD135 - Slamfl / CD150 + , and lin -(Ter119, CD11b, G (Negative for mature series markers including r1, CD3, CD4, CD8, B220, IL7ra, etc.) Furthermore, ST-HSCs have lower quiescence and proliferative capacity than LT-HSCs under homeostatic conditions. It is expensive. However, LT-HSCs have a high self-renewal capacity (i.e., LT-HSCs survive throughout adulthood). While ST-HSC can be transplanted sequentially through the recipient over generations, ST-HSC is self-replicating Its production capacity is limited (i.e., ST-HSCs survive for only a limited period and cannot be transplanted continuously). (This is not possible). Any of these HSCs may be used in the manner described in this application. ST -HSCs are highly proliferative and therefore can produce differentiated offspring more quickly. , it is particularly useful.

[0063] When used in this application, the term "hematopoietic stem cell functional potential" refers to the functional potential of hematopoietic stem cells. "Cell functional potential" refers to 1) pluripotency (multi-potency) (this is limited) It is not a granulocyte (for example, promyelocytes, neutrophils, eosinophils, basophils), red blood cells (for example) For example, reticulocytes, red blood cells, platelets (e.g., megakaryoblasts, platelet-producing megakaryocytes, blood cells) Platelets, monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, Multiple types of blood cells, including osteocytes and lymphocytes (e.g., NK cells, B- cells, and T- cells). 1) The ability to differentiate into lineages, 2) Self-replication (this refers to the ability to have the same potential as the parent cell). This refers to the ability of hematopoietic stem cells to produce daughter cells that possess (tial), and furthermore, this ability is depleted. (This can occur repeatedly throughout an individual's lifetime without any intervention), and 3) hematopoietic stem cells or their descendants They are reintroduced into transplant recipients, home to the hematopoietic stem cell niche, and become productive and sustainable. This refers to the functional properties of hematopoietic stem cells, including their ability to re-establish hematopoiesis.

[0064] As used in this application, the term "human antibody" means substantially all parts of a protein (for example) However, all CDRs, skeletal regions, C L , C H Domain [for example, C H 1, C H 2, C H 3], hinge, arrangement V L and V H The domain is substantially non-immunogenic in humans, and only slight sequence changes or This refers to antibodies that only contain mutations. Human antibodies are expressed in human cells (for example, by recombinant expression). (or functionally reconstituted human immunoglobulins (e.g., heavy chain and / or light chain)) It may be produced by non-human animals or prokaryotic or eukaryotic cells capable of expressing offspring. If a human antibody is a single-chain antibody, it is a linker pepto not found in innate human antibodies. It may contain glycine or other amino acid residues. For example, Fv may contain 2 to about 8 glycine or other amino acid residues. It may contain linker peptides, the linker peptides being variable regions of the heavy chain. and links the variable region of the light chain. Such linker peptides are of human origin. This is considered to be the case. Human antibodies are obtained using an antibody library derived from human immunoglobulin sequences. They are prepared by various methods known in the art, including phage display methods. Human antibodies can also not express functional endogenous immunoglobulins. However, transgenic mice capable of expressing human immunoglobulin genes are available. It can also be used to produce (for example, PCT publication numbers WO 1998 / 24893; WO 1992 / 01047; WO 1 996 / 34096; WO 1996 / 33735; US Patent No. 5,413,923; No. 5,625,126; No. 5,633,425; No. 5,5 No. 69,825; No. 5,661,016; No. 5,545,806; No. 5,814,318; No. 5,885,793; No. 5,916,771; See also issues 5,939,598).

[0065] As used in this application, the term "humanized" antibody is derived from non-human immunoglobulins. This refers to antibodies that contain the minimum amount of the sequence. Generally, humanized antibodies contain all, or substantially all, of the sequence. The CDR region corresponds to at least one CDR region of non-human immunoglobulin, and is typically This includes virtually all of the two variable domains. All or virtually all of the FW area is human immunodeficiency. It may also be the FW region of the immunoglobulin sequence. The humanized antibody also performs immunoglobulin assay. At least a portion of the normal region (Fc), typically a small amount of the human immunoglobulin consensus sequence. It may contain some of the above. Methods for humanizing antibodies are publicly known in the relevant art, And, for example, Riechmann et al., Nature 332:323-7, 1988; U.S. Patent No. 5,530,101; No. 5,585, It is described in issues 089; 5,693,761; 5,693,762; and 6,180,370.

[0066] When used in this application, patients who "need" in vivo transduction and / or gene therapy. This includes patients who have defects or deficiencies in one or more types of blood cells, as well as stem cell disorders, autologous Patients with immune disorders, cancer, or other conditions described in this application include hematopoietic stem cells. Cells are generally 1) multipotent, therefore not limited to, granules Spheres (e.g., promyelocytes, neutrophils, eosinophils, basophils), red blood cells (e.g., reticulocytes, red blood cells) Spheres), platelets (thrombocytes) (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets (pl Cells (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts , and multiple different blood series, including lymphocytes (e.g., NK cells, B- cells and T- cells) 1) It can differentiate, 2) it can self-replicate, and therefore it produces daughter cells that have the same potential as the parent cell. It can be made to do so, and 3) it has the ability to undergo in vivo transduction, and to undergo in vivo transduction. Afterward, the cells home to the hematopoietic stem cell niche and re-establish productive and sustainable hematopoiesis. For example, The aforementioned patient has sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and wi Abnormal hemoglobin disorders such as Scott-Aldrich syndrome (e.g., non-malignant abnormal hemoglobin disorders) The subject may have adenosine deaminase severe combined immune deficiency. ADA SCID, HIV / AIDS, metachromatic leukodystrophy, diamond-black phenotype The subject may have anemia or Schwakman-Diamond syndrome. The subjects mentioned above have hereditary blood disorders (e.g., sickle cell anemia) or autoimmune diseases, Or it may be affected by it. Furthermore, or instead, the subject is neuroblastoma. Alternatively, they may have or be affected by malignant tumors such as blood cancer. In this embodiment, the subject may have leukemia, lymphoma, or myeloma. In some embodiments, the target is acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia. Leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or It has non-Hodgkin's lymphoma. In some embodiments, The subject has myelodysplastic syndrome. In some embodiments, the subject has scleroderma, Autoimmune diseases such as multiple sclerosis, ulcerative colitis, Crohn's disease, and type 1 diabetes, or the present application It has another autoimmune pathological condition as described. In some embodiments, the subject is a chemoembolic. The subject requires antigen receptor T-cell (CART) therapy. In some embodiments, the subject is , has metabolic storage disorders or is affected by them. The subject is glycogen storage Sulfurous dysplasia, mucopolysaccharidosis, Gaucher disease, Haller's disease, sphingolipidosis, metachromatic leukodystrophy Globoid cell leukodystrophy, cerebral adrenal gland Metabolism selected from the group consisting of cerebral adrenoleukodystrophy. Disabilities, or any other diseases for which one may benefit from the treatments and therapies disclosed in this application. Or a disorder (not limited to, but including severe combined immunodeficiency, Wiscott-Aldone syndrome) Richie syndrome, hyperimmune globulin M syndrome, Chediak-Dong disease, hereditary lymphohistiocytosis Osteopetrosis, osteogenesis imperfecta, storage disease, thalassemia major, sickle cell anemia, systemic Sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis, and "Bone Ma rrow Transplantation for Non-Malignant disease", ASH Education Book, 1:319-338(2 000)(This disclosure may be treated by prescribing hematopoietic stem cell transplantation therapy (This relates to the pathological condition, and the entirety of which is incorporated by reference in this application.) You may have a disease or disorder, or if you do not, you may be affected. Sometimes it is.

[0067] As used in this application, the term "leukocyte" refers to a heterogeneous group of nucleated blood cell types, and red blood Spheres and platelets are excluded. White blood cells are divided into two general groups: neutrophils Polymorphonuclear cells, including globules, eosinophils, and basophils, and mononuclear cells, including lymphocytes and monocytes. Nuclear cells contain many cytoplasmic granules and multilobed nuclei, including: neutrophils (Generally, they are amoeba-like in form, phagocytic, and resistant to both basic and acidic pigments.) (stained), as well as eosinophils and basophils (stained with acidic dyes and basic dyes, respectively) (Contains cytoplasmic granules).

[0068] As used in this application, the term "lymphocyte" refers to mononuclear leukocytes involved in initiating an immune response. This refers to lymphocytes. Generally, lymphocytes include B lymphocytes, T lymphocytes, and NK cells. ru.

[0069] In use in this application, the terms "mobilize" and "mobilization" are used. This refers to a population of hematopoietic stem cells or hematopoietic progenitor cells that originates from the stem cell niche, for example, the bone marrow of the target area, and extends to the periphery. This refers to the process of release into the bloodstream. It involves the recruitment of hematopoietic stem cells and hematopoietic progenitor cells. For example, the amount or concentration of hematopoietic stem cells or hematopoietic progenitor cells in a peripheral blood sample isolated from the subject. The degree can be monitored by evaluating it. For example, for the above object, After prescribing a hematopoietic stem cell or hematopoietic progenitor cell recruitment regimen, peripheral blood samples are taken from the subject. The sample is collected, and the amount or concentration of hematopoietic stem cells or hematopoietic progenitor cells in the peripheral blood sample is evaluated. It may be worthwhile. The aforementioned mobilization regimen is, for example, the CXCR4 antagonist described in this application. CXCR4 antagonists such as st (e.g., prelixafor or its variants), and The CXCR2 agonist described in this application (for example, Gro-β or its variant, for example, Gro- This may include β-cleavages, for example, CXCR2 agonists such as Gro-β T). After prescribing the regimen, hematopoietic stem cells or hematopoiesis in peripheral blood samples isolated from the subject. The quantity or concentration of progenitor cells is determined by the amount of peripheral cells isolated from the subject before prescribing the mobilization regimen. The amount or concentration of hematopoietic stem cells or hematopoietic progenitor cells in the blood sample may be compared. After prescribing the member regimen, hematopoietic stem cells or hematopoietic progenitor cells were detected in the peripheral blood of the subject. If an increase in quantity or concentration is observed, the subject is responding to the mobilization regimen. This refers to the fact that hematopoietic stem cells and hematopoietic progenitor cells originate from one or more stem cell niches, such as bone marrow, and that This indicates that it was released into the peripheral blood circulation. In some embodiments, hematopoiesis The amount or concentration of stem cells or hematopoietic progenitor cells after the prescription of the mobilization regimen is 1%, 100%, 1,000%, or more (for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%) 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% An increase of 1,000% or more in the peripheral blood of the subject has been observed prior to The subjects described are responding to the aforementioned mobilization regimen, and hematopoietic stem cells and hematopoietic progenitor cells are also included. The cells were released into the peripheral blood circulation from one or more stem cell niches (e.g., bone marrow). This indicates that the amount or concentration of hematopoietic stem cells or hematopoietic progenitor cells is measured. The method described in this application and known in the art is, For example, hematopoietic stem cells or hematopoietic progenitor cells are identified based on their antigen expression profile. Examples include quantitative flow cytometry techniques, which are described in this application. Human HSCs are CD34 + CD38 - CD45RA - CD90 + CD49F + , and lin-(for example, CD2, CD3, Regarding mature lineage markers such as CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, and CD235A... (and negative). Hematopoietic stem cells or pre-hematopoietic cells in peripheral blood samples isolated from the subject. Further methods for measuring the quantity or concentration of progenitor cells include the colony-forming cells in the sample. One example is an assay to quantify the number of condensed functional units (CFUs), which uses an appropriate culture medium and ink. When ovulated, it produces a group of individual hematopoietic stem cells or hematopoietic progenitor cells, which are living hematopoiesis. It is a measure of the quantity of stem cells or hematopoietic progenitor cells.

[0070] As used in this application, the term "mobilizing amount" refers to mammals. When administered to subjects such as (for example, human subjects), it moves a population of hematopoietic stem cells or hematopoietic progenitor cells. The amount of one or more drugs (for example, the CXCR4 antagonist and / or as described in this application) The amount of CXCR2 agonist [in some embodiments, prelixafor, or its varian] Gro-β, and / or its variants (e.g., cleaved Gro-β, e.g., Gro-β) This refers to the amount of β(T). Examples of the amounts of these drugs that would mobilize the following populations include: A sufficient quantity to be dispensed is given, for example, about 20 to about 40 CD34 + Cells / 1 μL Peripheral blood, for example, approximately 21 to 39 CD34 + Approximately 22 to 38 CD34 cells per 1 μL of peripheral blood + Thin Approximately 23 to 37 CD34 cells per 1 μL of peripheral blood + Cells / 1 μL of peripheral blood, approximately 24 to 36 CD34 + Cells / 1 μL of peripheral blood, approximately 25 to 35 CD34 + Cells / 1 μL of peripheral blood, approximately 26 to 34 CDs 34 + Approximately 27 to 33 CD34 cells per 1 μL of peripheral blood + Approximately 28 to 32 cells per 1 μL of peripheral blood CD34 + Cells / 1 μL of peripheral blood, or approximately 29 to 31 CD34 + Cells / 1 μL of peripheral blood (for example) Approximately 20 CD34 + Cells / 1 μL of peripheral blood, 21 CD34 + Cells / 1 μL of peripheral blood, 22 CD34 + Cells / 1 μL of peripheral blood, 23 CD34 + Cells / 1 μL of peripheral blood, 24 CD34 + Peripheral cells / 1 μL Blood, 25 CD34 + Cells / 1 μL of peripheral blood, 26 CD34 + Cells / 1 μL of peripheral blood, 27 CD34 +cells / 1 μL of peripheral blood, 28 CD34 + cells / 1 μL of peripheral blood, 29 CD34 + cells / 1 μL of peripheral blood , 30 CD34 + cells / 1 μL of peripheral blood, 31 CD34 + cells / 1 μL of peripheral blood, 32 CD34 + cells / 1 μL of peripheral blood, 33 CD34 + cells / 1 μL of peripheral blood, 34 CD34 + cells / 1 μL of peripheral blood , 35 CD34 + cells / 1 μL of peripheral blood, 36 CD34 + cells / 1 μL of peripheral blood, 37 CD34 + cells / 1 μL of peripheral blood, 38 CD34 + cells / 1 μL of peripheral blood, 39 CD34 + cells / 1 μL of peripheral blood, 4 0 CD34 + cells / 1 μL of peripheral blood, or more). In certain embodiments, the amount that mobilizes these agents includes an amount sufficient to release the following population, e.g ., about 5 to about 20 CD34+CD90+CD45RA− cells / 1 μL of peripheral blood, e.g., about 5 to about 8 CD34+CD90+CD45RA− cells / 1 μL of peripheral blood, about 5 to about 10 CD34+CD90+CD45RA− cells / 1 μL of peripheral blood, about 5 to about 12 CD34+CD90+CD45RA− cells / 1 μL of peripheral blood, about 5 to about 15 CD34+CD90+CD45RA− cells / 1 μL of peripheral blood, about 5 to about 18 CD34+CD90+CD45RA− cells / 1 μL of peripheral blood, about 8 to about 10 CD34+CD90+CD45RA− cells / 1 μL of peripheral blood, about 8 to about 12 CD34+CD90+CD45RA- cells / 1 μL of peripheral blood, approximately 8 to 15 CD34+CD90+CD45RA- cells / 1 μL of peripheral blood, or approximately 8 to 18 CD34+CD90+CD45RA- cells / 1 μL of peripheral blood, approximately 8 Approximately 20 CD34+CD90+CD45RA- cells / 1 μL of peripheral blood, approximately 10 to 12 CD34+CD90+CD45RA - Cells / 1 μL of peripheral blood, approximately 10 to 15 CD34+CD90+CD45RA- cells / 1 μL of peripheral blood, approximately 10 Approximately 18 CD34+CD90+CD45RA- cells / 1 μL of peripheral blood, approximately 10 to approximately 20 CD34+CD90+CD45 RA- cells / 1 μL of peripheral blood, approximately 12 to approximately 15 CD34+CD90+CD45RA- cells / 1 μL of peripheral blood, approximately Approximately 10 to 18 CD34+CD90+CD45RA- cells / 1 μL of peripheral blood, approximately 10 to 20 CD34+CD90+CD 45RA- cells / 1 μL of peripheral blood, approximately 12 to approximately 15 CD34+CD90+CD45RA- cells / 1 μL of peripheral blood, Approximately 12 to 18 CD34+CD90+CD45RA- cells / 1 μL of peripheral blood, approximately 12 to 20 CD34+CD90+ cells CD45RA- cells / 1 μL of peripheral blood, approximately 15 to 18 CD34+CD90+CD45RA- cells / 1 μL of peripheral blood , or about 15 to about 20 CD34+CD90+CD45RA- cells / 1 μL of peripheral blood. In a particular embodiment Therefore, the amount of these drugs mobilized is at least twice the release of CD34+CD90+CD45RA- Cell population / 1 μL of peripheral blood, for example, at least 3 times release, at least 4 times release, less At least five times the amount released, at least six times the amount released, at least seven times the amount released, at least eight times the amount released, Population of CD34+CD90+CD45RA- cells with at least 9x or at least 10x release / 1 A sufficient amount to yield μL of peripheral blood is mentioned. In a particular embodiment, this The amount of these drugs mobilized ranges from 2 times to 10 times the normal release, for example, 2 to 4 times the normal release. Release, 2 to 6 times the amount, 2 to 8 times the amount, 4 to 6 times the amount, 4 to 8 times the amount, 4 times CDs with 10 times the release, 6 to 8 times the release, 6 to 10 times the release, or 8 to 10 times the release. A sufficient amount to yield a population of 34+CD90+CD45RA- cells per 1 μL of peripheral blood is given.

[0071] As used in this application, the term "monoclonal antibody" refers to any eukaryote, prokaryote, Alternatively, it refers to antibodies obtained from a single clone, including a phage clone, and the production of such antibodies. It's not about the method of making a living.

[0072] As used in this application, the term "monocyte" is defined as CD14 + And CD34 - Peripheral blood mononuclear cells This refers to a blood mononuclear cell (PBMC), which generally contains one or more microbial products. Upon activation by foreign substances, they can differentiate into macrophages and / or dendritic cells. In particular, monocytes may have elevated levels of CD14 surface antigen marker expression, and CD64, CD93, CD180, CD328 (also known as sialic acid-bound Ig-like lectin 7 or Siglec7) ), and CD329 (sialic acid-bound Ig-like lectin 9 or Siglec9), and peanut argyl At least one of the following selected types: tinine protein (peanut agglutinin protein (PNA)) may express a biomarker.

[0073] When used in this application, "peptide" refers to a single-chain polyamide containing a plurality of amino acid residues such as natural and / or non-natural amino acid residues, which are continuously linked by amide bonds. Examples of peptides include shorter fragments of full-length proteins (such as naturally occurring full-length proteins, etc.).

[0074] When used in this application, the term "sample" refers to a specimen collected from a subject (such as blood, blood components (such as serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (such as placenta or dermis), pancreatic juice, chorionic villus sample, and cells). The sample may be, for example, peripheral blood collected from a subject who has received or has received a mobilization regimen for hematopoietic stem cells or hematopoietic progenitor cells as described in this application.

[0075] When used in this application, the term "scFv" refers to a single-chain Fv antibody in which the variable domain of the heavy chain derived from an antibody and the variable domain of the light chain are combined to form a single chain. The scFv fragment is a single polypeptide chain containing the variable region (V L ) of the antibody light chain (such as CDR-L1, CDR-L2, and / or CDR-L3), as well as the variable region (V H ) of the antibody heavy chain (such as CDR-H1, CDR-H2, and / or CDR-H3). The linker connecting the V L region and the V H region of the scFv fragment may be a peptide linker composed of amino acids constituting a protein. An alternative linker may be the resistance of the scFv fragment to proteolysis. To increase the dissolution of scFv fragments (for example, a linker containing D-amino acids), To increase properties (for example, polyethylene glycol-containing linker or repeated Hydrophilic linkers such as polypeptides containing glycine and serine residues, and the biological properties of the molecule To improve physical stability (for example, by forming intramolecular or intermolecular disulfide bonds) A linker containing cysteine ​​residues, or a linker that weakens the immunogenicity of the scFv fragment. It may be used for purposes such as (for example, a linker containing a glycosylated site). The variable regions of the described scFv molecules are modified from the antibody molecules from which they originate by altering their amino acid sequences. Those skilled in the art will also understand that it can be modified in this way. For example, the function of scFv To preserve or enhance (the ability of the corresponding antibody to bind to the antigen it recognizes), amino acids Conservative substitutions or nucleotide or amino acid substitutions that result in changes at a residue (e.g., CDR and This may occur (at or in skeletal residues).

[0076] When used in this application, the term "stem cell disorder" refers to the in vivo phenotypic induction of hematopoietic stem cells in a patient. This refers to any disease, disorder, or symptom that can be treated or cured by treatment. In a broad sense, this refers to examples of conditions that can be treated by in vivo transduction of hematopoietic stem cells within a patient. Typical diseases include sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and WISC-14 anemia. Aldrich syndrome, ADA SCID, HIV / AIDS, metachromatic leukodystrophy, diamorphosis This application concerns Nd-Blackfan anemia and Schwakman-Diamond syndrome. Further diseases that may be treated by in vivo transduction of hematopoietic stem cells as described above include These include blood disorders (e.g., sickle cell anemia), as well as autoimmune diseases such as scleroderma, multiple sclerosis, ulcerative colitis , and Crohn's disease. Further diseases that may be treated by in vivo transduction of hematopoietic stem cells include cancer, such as the cancers described in the present application . Exemplary stem cell disorders are malignant tumors (e.g., neuroblastoma, or blood cancers ( e.g., leukemia, lymphoma, and myeloma)). In some embodiments, the cancer is , acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multi ple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma (non-Hodgkin's lymphoma). Further diseases treatable using in vivo transduction of hematopoietic stem cells include myelodysplastic syndromes. In some embodiments, the patient has or is affected by a metabolic storage disorder. For example, the patient has a metabolic disorder selected from the group consisting of glycogen storage disease, mucopolysaccharidosis, Gaucher's disease, Hurler's disease, sphingolipidosis , metachromatic leukodystrophy, globoid cell leukodystrophy , cerebral adrenoleukodystrophy, or any other disease or disorder (not limited to, but including severe combined immunodeficiency, Wiskott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chediak-Higashi disease , hereditary lymphohistiocytosis, marble bone disease, osteogenesis imperfecta, storage diseases, thalassemia major ), or may benefit from the treatments and therapies disclosed in the present application ). Selected from the group consisting of cerebral adrenoleukodystrophy, or any other disease or disorder that may benefit from the treatments and therapies disclosed in the present application (not limited to, but including severe combined immunodeficiency, Wiskott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chediak-Higashi disease ), hereditary lymphohistiocytosis, marble bone disease, osteogenesis imperfecta, storage diseases, thalassemia major ), or any other disease or disorder (not limited to, but including severe combined immunodeficiency, Wiskott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chediak-Higashi disease , hereditary lymphohistiocytosis, marble bone disease, osteogenesis imperfecta, storage diseases, thalassemia major ), or any other disease or disorder that may benefit from the treatments and therapies disclosed in the present application (not limited to, but including severe combined immunodeficiency, Wiskott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chediak-Higashi disease - One, sickle cell anemia, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis), and "Bone Marrow Transplantation for N on-Malignant disease", ASH Education Book, 1:319-338(2000)(The present disclosure relates to conditions that may be treated by prescribing hematopoietic stem cell transplantation therapy or hematopoietic progenitor cell transplantation therapy, the entire of which is incorporated herein by reference), diseases or disorders described therein, may be suffering from, or, if not, may be affected.

[0077] When used in the context of hematopoietic stem cell mobilization in the present application, the term "stem cell niche" refers to the microenvironment within a mammalian subject (e.g., a human subject) in which endogenous hematopoietic stem cells or hematopoietic progenitor cells are present. An example of a stem cell niche is bone marrow tissue.

[0078] When used in the present application, the terms "subject" and "patient" refer to a living being such as a human who is undergoing treatment for a particular disease or condition as described in the present application. In some embodiments, a patient such as a human patient who requires in vivo hematopoietic stem cell gene therapy may undergo treatment including transducing a population of hematopoietic stem cells to treat a stem cell disorder, e.g., a cancer, autoimmune disease, or metabolic disorder described in the present application. In some embodiments, the hematopoietic stem cells to be transduced in the patient can be mobilized within the patient by administering a CXCR4 antagonist and / or a CXCR2 agonist.

[0079] ​​​​​​​​​​​​​As used in this application, the terms “transfection” or “transfection” are used in the context of the electrified application. Poration, lipofection, calcium phosphate precipitate, DEAE-dextran tran Methods such as DNA transmission introduce exogenous DNA into prokaryotic or eukaryotic host cells. This refers to any and all the different techniques commonly used for in vivo transduction or Transfection is typically described in more detail in this application, This is performed using an Ilus vector.

[0080] As used in this application, the terms "to treat" or "treatment" mean therapeutic procedures. In that context, the purpose is to prevent undesirable physiological changes or other issues in patients receiving treatment. This involves preventing or slowing down (mitigating) disabilities, or promoting beneficial phenotypes. The beneficial results of the therapies described in this application also include hematopoietic stem cells and hematopoietic progenitor cells. After in vivo transduction, one or more hematopoietic cells (e.g., megakaryocytes, platelets (thrombocytosis)) te), platelets, red blood cells, mast cells, myeloblasts, basophils, neutrophils, eosinophils, Microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells An increase in the number of cells (such as natural killer cells, T lymphocytes, or B lymphocytes) One possible consequence is an increase in relative concentration. The beneficial effects of the therapies described in this application The results also include an increase in the activity or function of one or more hematopoietic cells. Further beneficial results include the removal of disease-causing cells such as cancer cells or autoimmune cells. One possible explanation is a decrease in the amount of the resulting cell population.

[0081] In use in this application, the terms “variant” and “derivative” are to be used interchangeably. The naturally occurring compounds, peptides, proteins, or other substrates described in this application This refers to synthetic and semi-synthetic analogs. The compounds, peptides, and other compounds described in this application are... Proteins, or variants or derivatives of other substances, retain the biological activity of the original substance or There is room for improvement.

[0082] As used in this application, the term "vector" refers to plasmids, DNA vectors, plasmids. nucleic acid vectors such as RNA vectors, viral vectors, or other suitable replicons. Includes. The expression vector described in this application is a polynucleotide sequence, and for example, These polynucleotides express proteins and / or are placed in the genome of mammalian cells. It may contain further sequence elements used to incorporate the sequence. Peptides and For expressing proteins, such as the peptides and proteins described in this application Certain vectors that can contain a promoter region that directs gene transcription and an enzyme Plasmids containing regulatory sequences such as Hansar are included. The peptides described in this application and Other useful vectors for expressing proteins enhance the translation rate of these genes. Polynucleotides either improve the stability or nuclear translocation of mRNA resulting from gene transcription. It contains Otid sequences. These sequence elements efficiently express genes on an expression vector, for example. It includes 5' and 3' untranslated regions and a polyadenylation signal site, which are transcribed to [the specified location]. There is. The expression vector described in this application also selects cells containing such vectors. It may also contain polynucleotides that encode markers for the appropriate markers. Examples include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, and nourseothricin (nourseothricin).

[0083] When used in this application, the term "alkyl" refers to, for example, a straight-chain or branched-chain alkyl group having 1 to 20 carbon atoms in the chain . Examples of alkyl groups include methyl, ethyl, n- propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and the like.

[0084] When used in this application, the term "alkylene" refers to a straight-chain or branched-chain divalent alkyl group . The divalent position may be on the same atom or different atoms within the alkyl chain . Examples of alkylene include methylene, ethylene, propylene, isopropylene, and the like .

[0085] When used in this application, the term "heteroalkyl" refers to, for example, a straight-chain or branched-chain alkyl group having 1 to 20 carbon atoms in the chain and further containing one or more heteroatoms (for example, especially oxygen, nitrogen, or sulfur ) in the chain.

[0086] When used in this application, the term "heteroalkylene" refers to a straight-chain or branched-chain divalent heteroalkyl group . The divalent position may be on the same atom or different atoms within the heteroalkyl chain . The divalent position may be one or more heteroatoms.

[0087] When used in this application, the term "alkenyl" refers to, for example, a straight-chain or branched-chain alkenyl group having 2 to 20 carbon atoms in the chain ​This refers to a linear or branched alkenyl group having [a specific characteristic]. Examples of alkenyl groups include vinyl, p This includes lopenyl, isopropenyl, butenyl, tert-butyrenyl, hexenyl, etc.

[0088] As used in this application, the term "alkenylene" refers to a linear or branched divalent alkenyl group. This refers to the position of the divalent nucleotide, whether on the same atom or a different atom within the alkenyl chain. This is also good. Examples of alkenylenes include etenylene, propenylene, isopropenylene, and b It contains tenirene, etc.

[0089] As used in this application, the term "heteroalkenyl" means, for example, 2 to 20 carbon atoms in a chain. It has elementary atoms, and further contains one or more heteroatoms in the chain (for example, oxygen, nitrogen, or sulfur, in particular). This refers to linear or branched alkenyl groups containing (yellow).

[0090] As used in this application, the term "heteroalkenylene" refers to a linear or branched divalent hetero This refers to an alkenyl group. The divalent position can be different even if it is on the same atom within a heteroalkenyl chain. It may be located on an atom. The divalent position may be one or more heteroatoms.

[0091] As used in this application, the term "alkynyl" means, for example, a chain containing 2 to 20 carbon atoms. This refers to a linear or branched alkynyl group having [a specific characteristic]. An example of an alkynyl group is propargyl This includes ru, butynyl, pentynyl, hexynyl, etc.

[0092] As used in this application, the term "alkynylene" refers to a linear or branched divalent alkynyl group. This refers to the position of the divalent nucleotide, whether on the same atom or a different atom within the alkynyl chain. That's good too.

[0093] As used in this application, the term "heteroalkynyl" means, for example, 2 to 20 carbon atoms in a chain. It has elementary atoms, and further contains one or more heteroatoms in the chain (for example, oxygen, nitrogen, or sulfur, in particular). This refers to linear or branched alkynyl groups containing (yellow).

[0094] As used in this application, the term "heteroalkylylene" refers to a linear or branched divalent heteroalkylene. This refers to an alkynyl group. The divalent position can be different even if it is on the same atom within a heteroalkynyl chain. It may be located on an atom. The divalent position may be one or more heteroatoms.

[0095] As used in this application, the term "cycloalkyl" means saturated and, for example, 3 From a monocyclic, condensed, bridged, or spiropolycyclic ring structure having 12 carbon ring atoms Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, and cyclopentyl. , cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[3.1.0]hexane, etc. Born.

[0096] As used in this application, the term "cycloalkylene" refers to a divalent cycloalkyl group. The divalent position may be on the same atom or on a different atom within the ring structure. Examples of roalkylenes include cyclopropylene, cyclobutylene, cyclopentylene, and cyclobutylene. It contains chlorohexylene, etc.

[0097] As used in this application, the term "heterocycloalkyl" means saturated and, for example, For example, from carbon atoms and, more particularly, nitrogen, oxygen, and sulfur, a more selected hete Monocyclic or condensed rings having 3 to 12 ring atoms per ring structure, selected from the ∫ atoms. This refers to a cross-linked or spiropolycyclic ring structure. The ring structure is, for example, made of carbon, nitrogen, or sulfur. The ring member may contain one or more oxo groups.

[0098] As used in this application, the term "heterocycloalkylene" means a divalent heterocycloalkylene. This refers to the Kill group. The divalent position can be on the same atom or on a different atom within the ring structure. That's good too.

[0099] As used in this application, the term "aryl" means, for example, a single atom containing 6 to 19 carbon atoms. This refers to a cyclic or polycyclic aromatic ring system. Examples of aryl groups include phenyl, fluorenyl, and naphth. This includes, but is not limited to, til. The divalent position is occupied by one or more heteroatoms. It can happen.

[0100] As used in this application, the term "arylene" refers to a divalent aryl group. These elements may be located on the same atom or on different atoms.

[0101] As used in this application, the term "heteroaryl" means monocyclic heteroaromatic or bicyclic heteroaromatic. Or, it refers to a tricyclic fused ring heteroaromatic group. Heteroaryl groups include pyridyl and pyrrolyl. , furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, i Sothiazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadi Azolyl, 1,2,4-Oxadiazolyl, 1,2,5-Oxadiazolyl, 1,3,4-Oxadiazolyl , 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuryl, [2,3-dihydro]benzofuryl Isobenzofuryl, benzothienyl, benzotriazolyl, isobenzothienyl, i Indolyl, isoindolyl, 3H-indolyl, benzimidazolyl, imidazo[1,2-a]pyri Dil, benzothiazolyl, benzoxazolyl, quinolidinil, quinazolinil, phthalazi Nyl, quinoxalinyl, sinnolinyl, naptilidinyl, pyrido[3,4-b]pyridyl, pyr do[3,2-b]pyridyl, pyrido[4,3-b]pyridyl, quinolyl, isoquinolyl, tetrazolyl, 5,6,7,8-tetrahydroquinolyl, 5,6,7,8-tetrahydroisoquinolyl, prynyl, ptery Examples include dinyl, carbazolyl, xanthenyl, and benzoquinolil.

[0102] As used in this application, the term "heteroarylene" refers to a divalent heteroaryl group. The divalent position can be on the same atom or on different atoms. It may consist of more than one heteroatom.

[0103] Unless otherwise restricted by the definition of each substituent, the aforementioned chemical substructures, e.g. For example, "alkyl", "alkylene", "heteroalkyl", "heteroalkylene", "Alkenyl", "Alkenylene", "Heteralkenyl", "Heteralkenylene", "Alkynyl", "Alkynylene", "Heteroalkynyl", "Heteroalkynylene", "Cycloalkyl", "Cycloalkylene", "Heterocycloalkyl", "Heterocyclo "Roalkylene", "aryl", "arylene", "heteroaryl", and "heteroaryl The "riylene" group, etc., may be optionally substituted. When used in this application, The phrase "to be arbitrarily replaced" means that one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, This refers to a compound or substructure containing substituents (or more), and the aforementioned compound or This is permissible depending on the substructure or the valency of that part, for example, alkyl, alkeni Alkynyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkyl Al-heteroaryl, alkyl-cycloalkyl, alkyl-heterocycloalkyl, A Mino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alcohol Xycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, Sulfonyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, shea Compounds containing substituents selected from the group consisting of no, hydroxy, mercapto, nitro, etc. Or it refers to a substructure. The substitution is such that adjacent substituents form a ring closure, for example, adjacent bonds Following the ring closure of potential substituents, for example, lactams, lactones, cyclic anhydrides, and acetals are formed. , forming hemiacetals, thioacetals, aminals, and hemiacetals, for example For example, this could include situations where a protecting group is provided.

[0104] Methods for mobilizing hematopoietic stem cells and hematopoietic precursor cells, and cells for proliferation and therapeutic use. How to release This disclosure, in part, describes CXCR2 agonists (e.g., Gro-β, Gro-β T, or their variants). Specific doses of (such as riant) are optionally used in combination with a CXCR4 antagonist for infant feeding. By administering it to animal subjects (e.g., human subjects), hematopoietic stem cells and hematopoietic progenitor cells are produced. It is based on the discovery that it is possible to mobilize people.

[0105] CXCR2 Agonist Gro-β, Gro-β T, and their variants An exemplary CXCR2 agonist that may be used in conjunction with the compositions and methods described in this application is Gro-β and its variants. Gro-β (growth regulatory protein β, chemokine (CXC) This is also known as the motif ligand 2 (CXCL2) and macrophage inflammatory protein 2-α (MIP2-α). (To be detected) For example, by stimulating the release of proteases from peripheral neutrophils. Therefore, it is a cytokine that can mobilize hematopoietic stem cells and hematopoietic progenitor cells.

[0106] In addition to Gro-β, exemplary CX may be used in conjunction with the compositions and methods described in this application. CR2 agonists are characterized by the deletion of one to eight amino acids at the N-terminus of Gro-β. (For example, 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids) Characterized by the deletion of the N-terminus of 1 amino acid, 6 amino acids, 7 amino acids, or 8 amino acids. It is a cleaved product of Gro-β of the peptide. In some embodiments, as described in this application CXCR2 agonists that can be used in conjunction with the composition and method include the first four segments from the N-terminus of Gro-β. It contains Gro-β T, which is characterized by the deletion of one amino acid. Gro-β T is particularly Advantageous biological properties, for example, hematopoietic stem cells and hematopoietic progenitor cells with several orders of magnitude better efficacy than Gro-β. It exhibits an effect that induces the mobilization of Gro-β and Gro-β T, for example, U.S. 6,08 It is described in Patent No. 0,398, and its disclosure is incorporated in its entirety by reference in this application. ru.

[0107] Furthermore, exemplary CXCR2 agonists that may be used in conjunction with the compositions and methods described in this application The asparagine residue at position 69 of sequence number (SEQ ID NO): 1 is replaced with an asparagine residue. This is a variant of Gro-β containing an acidic acid residue. This peptide is referred to as Gro-β N69D in this application. Similarly, CXCR2 agonists that may be used in conjunction with the compositions and methods described in this application In this case, the asparagine residue at position 65 of sequence number (SEQ ID NO): 2 is replaced with asparagine This peptide contains a variant of Gro-β T that includes acidic residues. It is called N65D and not only retains the ability to mobilize hematopoietic stem cells and hematopoietic progenitor cells, but also Gro -βT has a considerably greater potency than that of βT. For example, U.S. Patent No. 6,447,766 Gro-β N69D and Gro-β T N65D are described, and the entirety of their disclosure is published here. It is incorporated into the petition by reference.

[0108] The amino acid sequences of Gro-β, Gro-β T, Gro-β N69D, and Gro-β T N65D are shown in Table 2 below. show. [Table 2]

[0109] Additional CXCR2 agonists that may be used in conjunction with the compositions and methods described in this application include: Therefore, compared to other variants of Gro-β, for example, one or more amino acid substitutions, Examples include peptides having insertions and / or deletions. In some embodiments, CXCR2 agonists that may be used in conjunction with the compositions and methods described in the application include: SEQ ID NO: Peptides having at least 85% sequence identity with amino acid sequence 1 (For example, SEQ ID NO: 1, at least 85%, 90%, 95% of the amino acid sequence) Examples include peptides having 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity. In some embodiments, the amino acid sequence of the CXCR2 agonist consists of one or more conserved amino acids. The amino acid sequence differs from sequence number (SEQ ID NO): 1 only by substitution. Several implementations In this state, the amino acid sequences of CXCR2 agonists are 20 or less, 15 or less, 10 or less, and 5 or less. Alternatively, the amino acid sequence of sequence number (SEQ ID NO): 1 may be altered by one or fewer non-conservative amino acid substitutions. This differs from the above. In some embodiments, the CXCR2 agonist is Gro-β. In some embodiments, the Gro-β T is not covalently modified. Therefore, Gro-β is a polyalkylene glycoside such as polyethylene glycol. The substructure is not modified by covalent bonding.

[0110] Additional examples of CXCR2 agonists useful in conjunction with the compositions and methods described in this application include Gro -β T variants, for example, compared to Gro-β T, have one or more amino acid substitutions, insertions, and / or peptides having deletions, etc. In some embodiments, the CXCR2 agonist The sample has at least 85% sequence identity with respect to the amino acid sequence of sequence number (SEQ ID NO): 2. The peptide contains (for example, at least 85% of the amino acid sequence of SEQ ID NO: 2) (Peptides having 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity) This may occur. In some embodiments, the amino acid sequence of the CXCR2 agonist is one or more The amino acid sequence differs from that of sequence number (SEQ ID NO): 2 only by the above conservative amino acid substitution. In some embodiments, the amino acid sequence of the CXCR2 agonist is 20 or fewer, 15 or fewer. Below, the sequence number (SEQ I) is determined by non-conservative amino acid substitutions of 10 or fewer, 5 or fewer, or 1 or fewer. D NO): Different from the amino acid sequence of 2.

[0111] Additional examples of CXCR2 agonists useful in conjunction with the compositions and methods described in this application include Gro -β N69D variants, for example, Gro-β N69D, have one or more amino acid substitutions or insertions. These include peptides having, and / or deletions. In some embodiments, the CXCR2 The agonist is sequence-identical to at least 85% of the amino acid sequence of sequence number (SEQ ID NO): 3. Peptides with unisexuality (for example, less than the amino acid sequence of SEQ ID NO: 3) Peptides having sequence identity of 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% It may be (d). In some embodiments, the amino acid sequence of the CXCR2 agonist is The amino acid sequence of sequence number (SEQ ID NO): 3 can only be altered by one or more conservative amino acid substitutions. This differs from the above. In some embodiments, the amino acid sequence of the CXCR2 agonist is 20 or less. , by non-conservative amino acid substitutions of 15 or fewer, 10 or fewer, 5 or fewer, or 1 or fewer, the sequence number SEQ ID NO: The amino acid sequence is different from that of 3.

[0112] Additional examples of CXCR2 agonists useful in conjunction with the compositions and methods described in this application include Gro -βT N65D variants, for example, one or more amino acid substitutions compared to Gro-βT N65D, These include peptides having insertions and / or deletions. In some embodiments, the CX CR2 agonists contain at least 85% of the amino acid sequence of sequence number (SEQ ID NO): 4. Peptides with identical amino acid sequences (e.g., SEQ ID NO): 4 Peptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99%, or 100% sequence identity. It may be (Cyd). In some embodiments, the amino acid sequence of the CXCR2 agonist This is only possible by one or more conservative amino acid substitutions, as shown in SEQ ID NO: 4. This differs from the original. In some embodiments, the amino acid sequence of the CXCR2 agonist is 20 or less. Below, the sequence is modified by 15 or fewer, 10 or fewer, 5 or fewer, or 1 or fewer non-conservative amino acid substitutions. The amino acid sequence is different from that of sequence ID number 4.

[0113] CXCR4 Antagonist Exemplary CXCR4 antagonist for use in conjunction with the compositions and methods described in this application St is a compound represented by formula (I). Z-Linker-Z' (I) or a pharmaceutically acceptable salt thereof, where Z is: (i) A cyclic polyamine containing 9 to 32 ring members, where 2 to 8 ring members is a nitrogen atom separated from each other by two or more carbon atoms; or, (ii) Amine represented by formula (IA) [ka] Here, A is a monocyclic or bicyclic fused ring system containing at least one nitrogen atom. Including, and B is a substituent consisting of H or 1 to 20 atoms; Furthermore, here Z' is as follows: (i) A cyclic polyamine containing 9 to 32 ring members, where 2 to 8 ring members A nitrogen atom is a nitrogen atom separated from each other by two or more carbon atoms; (ii) Amine represented by formula (IB) [ka] Here, A' is a monocyclic or bicyclic fused ring containing at least one nitrogen atom. The system includes, and B' is a substituent consisting of H or 1 to 20 atoms; or, (iii) substituents represented by formula (IC) -N(R)-(CR2) n -X (IC) Here, each R is independently H or C1-C6 alkyl, and n is 1 or k is 2, and X is an aryl or heteroaryl group or a mercaptan; Here, the linker is bonded to an optionally substituted alkylene (for example, optionally Selectively substituted C1-C6 alkylenes, arbitrarily substituted heteroalkylenes (for example) (where C1-C6 heteroalkylenes are optionally substituted), (where alkeh is optionally substituted) Nylene (e.g., C2-C6 alkenylenes that are optionally substituted), optionally substituted heteroalkenylenes (e.g., C2-C6 heteroalkenylenes that have been selectively substituted), Arbitrarily substituted alkynylenes (e.g., arbitrarily substituted C2-C6 alkynylenes) Len), optionally substituted heteroalkynylene (e.g., optionally substituted C2-C6 heteroalkylenes), optionally substituted cycloalkylenes, optionally Substituted heterocycloalkylene, optionally substituted arylene, or optionally It is a heteroarylene that has been substituted.

[0114] In some embodiments, Z and Z' are each independently rings comprising 9 to 32 ring members. It is a polyamine, and of its 9 to 32 ring members, 2 to 8 are connected by two or more carbon atoms. They may be nitrogen atoms separated from each other. In some embodiments, Z and Z' are the same. It is a substituent of . For example, Z may be a cyclic polyamine containing 10 to 24 ring members. In some embodiments, Z may be a cyclic polyamine containing 14 ring members. In some embodiments, Z contains four nitrogen atoms. In some embodiments, Z contains 1,4 It is ,8,11-tetraazocyclotetradecane.

[0115] In some embodiments, the linker is represented by formula (ID), [ka] Here, ring D is an optionally substituted aryl group, an optionally substituted hetyl group. loaryl group, optionally substituted cycloalkyl group, or optionally substituted It is a heterocycloalkyl group; and, X and Y are each independently and optionally substituted alkylenes (for example, optionally Selectively substituted C1-C6 alkylenes, arbitrarily substituted heteroalkylenes (for example) (where C1-C6 heteroalkylenes are optionally substituted), (where alkeh is optionally substituted) Nylene (e.g., C2-C6 alkenylenes that are optionally substituted), optionally substituted heteroalkenylenes (e.g., C2-C6 heteroalkenylenes that have been selectively substituted), Arbitrarily substituted alkynylenes (e.g., arbitrarily substituted C2-C6 alkynylenes) Lenyl(, or optionally substituted heteroalkynylene (e.g., optionally substituted) It is a C2-C6 heteroalkylene.

[0116] For example, the linker may be represented by formula (IE). [ka] Here, ring D is an optionally substituted aryl group, or an optionally substituted hetero Aryl group, optionally substituted cycloalkyl group, or optionally substituted he It is a telocycloalkyl group; and, X and Y are each independently and optionally substituted alkylenes (for example, optionally Selectively substituted C1-C6 alkylenes, arbitrarily substituted heteroalkylenes (for example) (where C1-C6 heteroalkylene is optionally substituted, and C2-C6 heteroalkylene is optionally substituted) Lukenylene (e.g., C2-C6 alkenylenes that have been selectively substituted), selectively substituted A heteroalkenylene (e.g., a C2-C6 heteroalkenylene that has been selectively substituted) ), optionally substituted alkynylenes (e.g., optionally substituted C2-C6 alkynylenes) Quynylenes), or optionally substituted heteroalkyl quinylenes (e.g., optionally substituted It is a modified C2-C6 heteroalkylene. In some embodiments, X and Y are Each is independently and optionally substituted with a C1-C6 alkylene. In some embodiments, X and Y are identical substituents. In some embodiments, X and Y are, respectively, methyl The group is n-ethylene, n-propylene, n-butylene, n-pentylene, or n-hexylene. In some embodiments, X and Y are methylene groups, respectively.

[0117] The linker may be, for example, 1,3-phenylene, 2,6-pyridine, 3,5-pyridine, or 2,5-thioff. It may also be phenanthroline, 4,4'-(2,2'-bipyrimidine), 2,9-(1,10-phenanthroline), etc. In some embodiments, the linker is 1,4-phenylene-bis-(methylene).

[0118] A CXCR4 antagonist useful in combination with the compositions and methods described in this application. In formula (II), 1,1'-[1,4-phenylenebis(methylene)]-bis-1,4,8,11-tetra-azacic Prelixafor (represented herein as "AMD3100" and "Modivil"), which is represented by lotetradecane. This includes (also known as ) or its pharmaceutically acceptable salts. [ka]

[0119] Additional CXCR4 antagonists that may be used in conjunction with the compositions and methods described in this application Nist contains a variance of prelixafor, such as the compound described in U.S. Patent No. 5,583,131. Ant is included, and its contents relate to the CXCR4 antagonist, as referenced in this application. It is taken in more. In some embodiments, the CXCR4 antagonist is derived from the following: It may be a compound selected from the group: 1,1′-[1,3-phenylenebis(methylene)]- Bis-1,4,8,11-tetra-azacyclotetradecane; 1,1′-[1,4-phenylene-bis-(methyl (n)-bis-1,4,8,11-tetraazacyclotetradecane; 1,1′-[1,4-phenylene-bis-(me Bis-zinc complex or bis-copper complex of tetraene-bis-1,4,8,11-tetraazacyclotetradecane Body; 1,1′-[3,3′-biphenylene-bis-(methylene)]-bis-1,4,8,11-tetraazacrothene Tradecane; 11,11′-[1,4-phenylene-bis-(methylene)]-bis-1,4,7,11-tetraazasi Clotetradecane; 1,11′-[1,4-phenylene-bis-(methylene)]-1,4,8,11-tetraazasi Clotetradecane-1,4,7,11-tetraazacyclotetradecane; 1,1′-[2,6-pyridine-bi Su-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; 1,1-[3,5-pyridine-bis Su-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; 1,1′-[2,5-thiophene] -bis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; 1,1′-[4,4′-(2,2 '-bipyridine)-bis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; 1,1 '-[2,9-(1,10-phenanthroline)-bis-(methylene)]-bis-1,4,8,11-tetraazacyclo Tetradecane; 1,1′-[1,3-phenylene-bis-(methylene)]-bis-1,4,7,10-tetraazasi Clotetradecane; 1,1′-[1,4-phenylene-bis-(methylene)]-bis-1,4,7,10-tetraa Zacyclotetradecane; 1′-[5-nitro-1,3-phenylenebis(methylene)]bis-1,4,8,11- Tetraazacyclotetradecane; 1′,1′-[2,4,5,6-tetrachloro-1,3-phenylenebis( Methylene)bis-1,4,8,11-tetraazacyclotetradecane; 1,1′-[2,3,5,6-tetra-full Oro-1,4-phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane; 1, 1′-[1,4-naphthylene-bis-(methylene)]bis-1,4,8,11-tetraazacyclotetradecane; 1,1′-[1,3-phenylenebis-(methylene)]bis-1,5,9-triazacyclododecane; 1,1′-[1 ,4-phenylene-bis-(methylene)]-1,5,9-triazacyclododecane; 1,1′-[2,5-dimethyl -1,4-phenylenebis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane; 1,1 '-[2,5-dichloro-1,4-phenylenebis-(methylene)]-bis-1,4,8,11-tetraazacyclothe Tradecane; 1,1′-[2-bromo-1,4-phenylenebis-(methylene)]-bis-1,4,8,11-tetra Azacyclotetradecane; and 1,1′-[6-phenyl-2,4-pyridinebis-(methylene)]-bis -1,4,8,11-Tetraazacyclotetradecane.

[0120] In some embodiments, the CXCR4 antagonist is as described in US 2006 / 0035829. The compound in question is a compound that is related to the CXCR4 antagonist, and the disclosure is as described above. It is incorporated by reference in the application. In some embodiments, the CXCR4 antagonist is It may be a compound selected from the following group: 3,7,11,17-tetraazabicyclo (13.3.1) Heptadeca-1(17),13,15-triene; 4,7,10,17-tetraazabicyclo(13.3.1) Ptadeca-1(17),13,15-triene;1,4,7,10-tetraazacyclotetradecane;1,4,7-tri Azacyclotetradecane; and 4,7,10-triazabicyclo(13.3.1)heptadeca-1(17),13,1 5-Trien.

[0121] The aforementioned CXCR4 antagonist is a compound described in WO 2001 / 044229. The disclosed information relating to the CXCR4 antagonist is provided in this application by reference. It is incorporated. In some embodiments, the CXCR4 antagonist is a group consisting of the following: A more preferred compound may be: N-[4-(11-fluoro-1,4,7-triazacycotyl Radecanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine;N-[4-(11,11- Difluoro-1,4,7-triazacyclotetradecanyl)-1,4-phenylenebis(methylene)]-2-( (aminomethyl)pyridine; N-[4-(1,4,7-triazacyclotetradecane-2-onyl)-1,4-fe Nilenbis(methylene)]-2-(aminomethyl)pyridine; N-[12-(5-oxa-1,9-diazacyclo Tetradecanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[4- (11-Oxa-1,4,7-Triazacyclotetradecanyl)-1,4-Phenylenebis(methylene)]-2-( (aminomethyl)pyridine; N-[4-(11-thia-1,4,7-triazacyclotetradecanyl)-1,4-fu [Enilenbis(methylene)]-2-(aminomethyl)pyridine; N-[4-(11-sulfoxo-1,4,7-tri Azacyclotetradecanil)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine ; N-[4-(11-sulfono-1,4,7-triazacyclotetradecanyl)-1,4-phenylenebis(meth N-[4-(3-carboxo-1,4,7-triazacylated](Len)-2-(aminomethyl)pyridine; and [Tradecanil)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine.

[0122] Further CXCR4 antagonists useful in combination with the compositions and methods described in this application Nist contains compounds described in WO 2000 / 002870, the disclosure of which is CXCR4 As relating to antagonists, several facts are incorporated by reference in this application. In the application method, the CXCR4 antagonist is a compound selected from the group consisting of the following: Sometimes: N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis-(meth (Len)-2-(aminomethyl)pyridine; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4 -Phenylenebis(methylene)]-N-methyl-2-(aminomethyl)pyridine; N-[1,4,8,11-tetra Azacyclotetradecanyl-1,4-phenylenebis(methylene)]-4-(aminomethyl)pyridine ; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-3-( (Aminomethyl)pyridine; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenyl [methylene]-(2-aminomethyl-5-methyl)pyrazine; N-[1,4,8,11-tetraazacyx Rotetra-decanyl-1,4-phenylenebis(methylene)]-2-(aminoethyl)pyridine; N-[1, 4,8,11-Tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-2-(aminomethyl (Chill)thiophene; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylenebis( Methylene)-2-(aminomethyl)mercaptan; N-[1,4,8,11-tetraazacyclotetra-deca] Nyl-1,4-phenylenebis(methylene)]-2-aminobenzylamine; N-[1,4,8,11-tetraa Zacyclotetra-decanyl-1,4-phenylenebis(methylene)]-4-aminobenzylamine; N -[1,4,8,11-Tetraazacyclotetra-decanyl-1,4-phenylenebis(methylene)]-4-(Ami Noethyl)imidazole; N-[1,4,8,11-tetraazacyclotetra-decanyl-1,4-phenylene N-bis(methylene)]-benzylamine; N-[4-(1,4,7-triazacyclotetradecanyl)-1,4 -Phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[7-(4,7,10,17-tetraaza] Bicyclo[13.3.1]heptadeca-1(17),13,15-trienyl)-1,4-phenylenebis(methylene)] -2-(aminomethyl)pyridine; N-[7-(4,7,10-triazabicyclo[13.3.1]heptadeca-1(17) ,13,15-trienyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[1- (1,4,7-Triazacyclotetra-decanyl)-1,4-phenylenebis(methylene)]-2-(aminometh (Tyl)pyridine; N-[4-[4,7,10,17-tetraazabicyclo[13.3.1]heptadeca-1(17),13,15- [trienyl]-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[4-[4,7,10 -Triazabicyclo[13.3.1]heptadeca-1(17),13,15-trienyl]-1,4-phenylenbis( Methylene)-2-(aminomethyl)pyridine; N-[1,4,8,11-tetraazacyclotetradecanyl- 1,4-Phenylenebis(methylene)]-purine; 1-[1,4,8,11-tetraazacyclotetradecanyl] -1,4-phenylenebis(methylene)]-4-phenylpiperazine; N-[4-(1,7-diazacyclotet Radecanyl)-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; and N-[7-(4 ,10-diazabicyclo[13.3.1]heptadeca-1(17),13,15-trienyl)-1,4-phenylenbis( Methylene)-2-(aminomethyl)pyridine.

[0123] In some embodiments, the CXCR4 antagonist is selected from the group consisting of the following: The compound is: 1-[2,6-dimethoxypyrido-4-yl(methylene)]-1,4,8,11-tetraazacy Clotetradecane; 1-[2-chloropyrido-4-yl(methylene)]-1,4,8,11-tetraazacyclo Tetradecane; 1-[2,6-dimethylpyrido-4-yl(methylene)]-1,4,8,11-tetraazacyclo Tetradecane; 1-[2-methylpyrido-4-yl(methylene)]-1,4,8,11-tetraazacyclotet Radecane; 1-[2,6-dichloropyrido-4-yl(methylene)]-1,4,8,11-tetraazacyclotet Radecane; 1-[2-chloropyrido-5-yl(methylene)]-1,4,8,11-tetraazacyclotetrade Can; and 7-[4-methylphenyl (methylene)]-4,7,10,17-tetraazabicyclo[13.3.1] Heptadeca-1(17),13,15-triene.

[0124] In some embodiments, the CXCR4 antagonist is described in U.S. Patent No. 5,698,546. The compound in question is a CXCR4 antagonist, and the disclosure concerns this compound. In some embodiments, the CXCR4 antagonist This may be a compound selected from the group consisting of: 7,7′-[1,4-phenylene-bi [S(methylene)]bis-3,7,11,17-tetraazabicyclo[13.3.1]heptadeca-1(17),13,15-to Lien; 7,7′-[1,4-phenylene-bis(methylene)]bis[15-chloro-3,7,11,17-tetraaza Bicyclo[13.3.1]heptadeca-1 (17),13,15-triene]; 7,7′-[1,4-phenylene-bis( Methylene)bis[15-Methoxy-3,7,11,17-tetraazabicyclo[13.3.1]heptadeca-1(17), 13,15-triene]; 7,7′-[1,4-phenylene-bis(methylene)]bis-3,7,11,17-tetraaza Bicyclo[13.3.1]-heptadeca-13,16-triene-15-one; 7,7′-[1,4-phenylene-bis( Methylene)bis-4,7,10,17-tetraazabicyclo[13.3.1]-heptadeca-1(17),13,15-tri En; 8,8′-[1,4-phenylene-bis(methylene)]bis-4,8,12,19-tetraazabicyclo[15. 3.1] Nonadeca-1(19),15,17-triene; 6,6′-[1,4-phenylene-bis(methylene)]bis-3,6 ,9,15-tetraazabicyclo[11.3.1]pentadeca-1 (15),11,13-triene; 6,6′-[1,3-f [Enilen-bis(methylene)]bis-3,6,9,15-tetraazabicyclo[11.3.1]pentadeca-1 (15 ), 11,13-triene; and 17,17′-[1,4-phenylene-bis(methylene)]bis-3,6, 14,17,23 ,24-Hexaazatricyclo[17.3.1.1 8,12]Tetracosa-1(23),8,10,12(24),19,21-hexa En.

[0125] In some embodiments, the CXCR4 antagonist is described in U.S. Patent No. 5,021,409. The compound in question is a CXCR4 antagonist, and the disclosure concerns this compound. In some embodiments, the CXCR4 antagonist This may be a compound selected from the group consisting of: 2,2′-bicyclum, 6,6 ′-Bicyclum; 3,3′-(bis-1,5,9,13-tetraazacyclohexadecane); 3,3′-(Bi S-1,5,8,11,14-pentazacyclohexadecane; methylene (or polymethylene) Di-1-N-1,4,8,11-tetraazacine cyclotetradecane; 3,3′-bis-1,5,9,13-tetraazacine Chlohexadecane; 3,3′-Bis-1,5,8,11,14-Pentazacyclohexadecane; 5,5′-Bi Su-1,4,8,11-tetraazacyclotetradecane; 2,5′-bis-1,4,8,11-tetraazacyclotetradecane Tradecane; 2,6′-bis-1,4,8,11-tetraazacyclotetradecane; 11,11′-(1,2-eth (Propanediyl)bis-1,4,8,11-tetraazacyclotetradecane; 11,11′-(1,2-propanediyl) )bis-1,4,8,11-tetraazacyclotetradecane; 11,11′-(1,2-butanediyl)bis-1,4, 8,11-Tetraazacyclotetradecane; 11,11′-(1,2-pentanediyl)bis-1,4,8,11-Te Traazacyclotetradecane; and 11,11′-(1,2-hexanediyl)bis-1,4,8,11-teto Laazacyclotetradecane.

[0126] In some embodiments, the CXCR4 antagonist is as described in WO 2000 / 056729. The compound in question is a CXCR4 antagonist, and its disclosure is as follows: More incorporated herein. In some embodiments, the CXCR4 antagonist is It may be a compound selected from the following group: N-(2-pyridinylmethyl)-N′-( 6,7,8,9-Tetrahydro-5H-diclohepta[b]pyridine-9-yl)-1,4-benzenedimethaneamine N-(2-pyridinylmethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzene Dimethanamine; N-(2-pyridinylmethyl)-N′-(6,7-dihydro-5H-cyclopenta[b]pyridinylmethyl) Zin-7-yl)-1,4-benzenedimethaneamine; N-(2-pyridinylmethyl)-N′-(1,2,3,4-tetramethyl) Trahydro-1-naphthalenyl)-1,4-benzenedimethaneamine; N-(2-pyridinylmethyl)-N ′-(1-naphthalenyl)-1,4-benzenedimethaneamine; N-(2-pyridinylmethyl)-N′-(8-k Norinyl)-1,4-benzenedimethaneamine; N-(2-pyridinylmethyl)-N′-[2-[(2-pyridinyl [methyl)aminoethyl]-N′-(1-methyl-1,2,3,4-tetrahydro-8-quinolinyl)-1,4-be N-(2-pyridinylmethyl)-N′-[2-[(1H-imidazole-2-ylmeth [Aminoethyl]-N′-(1-methyl-1,2,3,4-tetrahydro-8-quinolinyl)-1,4-benzene Dimethanamine; N-(2-pyridinylmethyl)-N′-(1,2,3,4-tetrahydro-8-quinolinyl)-1 ,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-[(1H-imidazole-2-i [methyl)aminoethyl]-N′-(1,2,3,4-tetrahydro-1-naphthalenyl)-1,4-benzenedi Methaneamine; N-(2-pyridinylmethyl)-N′-(2-phenyl-5,6,7,8-tetrahydro-8-quino Linyl)-1,4-benzenedimethaneamine; N,N′-bis(2-pyridinylmethyl)-N′-(2-phenyl Lu-5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine;

[0127] N-(2-pyridinylmethyl)-N′-(5,6,7,8-tetrahydro-5-quinolinyl)-1,4-benzenedi Methaneamine; N-(2-pyridinylmethyl)-N′-(1H-imidazole-2-ylmethyl)-N′-(5,6 ,7,8-tetrahydro-5-quinolinyl)-1,4-benzenedimethaneamine; N-(2-pyridinylmeth (Lu)-N′-(1H-imidazole-2-ylmethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1 ,4-benzenedimethaneamine; N-(2-pyridinylmethyl)-N′-[(2-amino-3-phenyl)pro Pyr]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-(2- Pyridinylmethyl)-N′-(1H-imidazole-4-ylmethyl)-N′-(5,6,7,8-tetrahydro-8 -Quinolinyl)-1,4-Benzenedimethanamine; N-(2-Pyridinylmethyl)-N′-(2-Quinoline) Ilmethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-(2-pyridinylmethyl)-N′-(2-(2-naphthoyl)aminoethyl)-N′-(5,6,7,8-tetramethyl) Dro-8-quinolinyl)-1,4-benzenedimethaneamine; N-(2-pyridinylmethyl)-N′-[(S)-( 2-Acetylamino-3-phenyl)propyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1, 4-Benzene dimethanamine; N-(2-pyridinylmethyl)-N′-[(S)-(2-acetylamino-3-fu [Phenyl)propyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethane Min; N-(2-pyridinylmethyl)-N′-[3-((2-naphthalenylmethyl)amino)propyl]-N′- (5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinyl) Methyl)-N′-[2-(S)-pyrrolidineylmethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl) -1,4-Benzene dimethanamine; N-(2-pyridinylmethyl)-N′-[2-(R)-pyrrolidine methyl [Cyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-(2- Pyridinylmethyl)-N′-[3-pyrazoleylmethyl]-N′-(5,6,7,8-tetrahydro-8-quino Linyl)-1,4-Benzenedimethanamine; N-(2-Pyridinylmethyl)-N′-[2-Pyrroleylmethyl [Cyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-(2- Pyridinylmethyl)-N′-[2-thiophenylmethyl]-N′-(5,6,7,8-tetrahydro-8-quino Linyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-[2-thiazoleyl Methyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-(2 (-pyridinylmethyl)-N′-[2-furanylmethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl) )-1,4-benzenedimethaneamine; N-(2-pyridinylmethyl)-N′-[2-[(phenylmethyl)amine Minoethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-(2-pyridinylmethyl)-N′-(2-aminoethyl)-N′-(5,6,7,8-tetrahydro-8-quinoli Nyl)-1,4-benzenedimethaneamine; N-(2-pyridinylmethyl)-N′-3-pyrrolidinyl-N′- (5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinyl) Methyl)-N′-4-piperidinyl-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzene Dimethanamine; N-(2-pyridinylmethyl)-N′-[2-[(phenyl)amino]ethyl]-N′-(5,6 ,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-(2-pyridinylmeth (Lu)-N′-(7-methoxy-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benzenedimethane Min; N-(2-pyridinylmethyl)-N′-(6-methoxy-1,2,3,4-tetrahydro-2-naphthalenyl) )-1,4-benzenedimethaneamine; N-(2-pyridinylmethyl)-N′-(1-methyl-1,2,3,4-tetramethyl Rahydro-2-naphthalenyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′- (7-Methoxy-3,4-dihydronaphthalenyl)-1-(aminomethyl)-4-benzamide; N-(2-pyri) Dinylmethyl)-N′-(6-methoxy-3,4-dihydronaphthalenyl)-1-(aminomethyl)-4-ben Zuamide; N-(2-pyridinylmethyl)-N′-(1H-imidazole-2-ylmethyl)-N′-(7-meth Xy-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benzenedimethaneamine; N-(2-pyri Dinylmethyl)-N′-(8-hydroxy-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benze Dimethanamine; N-(2-pyridinylmethyl)-N′-(1H-imidazole-2-ylmethyl)-N′- (8-hydroxy-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benzenedimethaneamine; N -(2-pyridinylmethyl)-N′-(8-fluoro-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4- Benzene dimethanamine; N-(2-pyridinylmethyl)-N′-(1H-imidazole-2-ylmethyl) )-N′-(8-fluoro-1,2,3,4-tetrahydro-2-naphthalenyl)-1,4-benzenedimethane N-(2-pyridinylmethyl)-N′-(5,6,7,8-tetrahydro-7-quinolinyl)-1,4-benzene Dimethanamine; N-(2-pyridinylmethyl)-N′-(1H-imidazole-2-ylmethyl)-N′-(5 ,6,7,8-tetrahydro-7-quinolinyl)-1,4-benzenedimethaneamine; N-(2-pyridinyl (Tyl)-N′-[2-[(2-naphthalenylmethyl)amino]ethyl]-N′-(5,6,7,8-tetrahydro-8- Quinolinyl)-1,4-benzenedimethaneamine; N-(2-pyridinylmethyl)-N′-[2-(isobutyl [Luaminoethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethane Min; N-(2-pyridinylmethyl)-N′-[2-[(2-pyridinylmethyl)amino]ethyl]-N′-(5,6 ,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-(2-pyridinylmeth (Lu)-N′-[2-[(2-furanylmethyl)amino]ethyl]-N′-(5,6,7,8-tetrahydro-8-quinoli) Nyl)-1,4-benzenedimethaneamine; N-(2-pyridinylmethyl)-N′-(2-guanidinoethyl) )-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-(2-pyri Dinylmethyl)-N′-[2-[bis-[(2-methoxy)phenylmethyl]amino]ethyl]-N′-(5,6,7 ,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-(2-pyridinylmethyl) -N′-[2-[(1H-imidazole-4-ylmethyl)amino]ethyl]-N′-(5,6,7,8-tetrahydro- 8-Quinolinyl)-1,4-Benzenedimethanamine; N-(2-Pyridinylmethyl)-N′-[2-[(1H-I Midazole-2-ylmethyl)aminoethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1 ,4-benzenedimethaneamine; N-(2-pyridinylmethyl)-N′-[2-(phenylureido)eth [Lu]-N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-(2-Pi) Ridinylmethyl)-N′-[[N″-(n-butyl)carboxamide]methyl]-N′-(5,6,7,8-tetramethyl) (Dro-8-Quinolinyl)-1,4-Benzene Dimethanamine; N-(2-Pyridinylmethyl)-N'-(Cal Boxamidemethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethane Min; N-(2-pyridinylmethyl)-N′-[(N″-phenyl)carboxamidemethyl]-N′-(5,6,7 ,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-(2-pyridinylmethyl) -N′-(carboxymethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedime Tanamine; N-(2-pyridinylmethyl)-N′-(phenylmethyl)-N′-(5,6,7,8-tetrahydro Ro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(1H-ben Zuimidazole-2-ylmethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benze Dimethanamine; N-(2-pyridinylmethyl)-N′-(5,6-dimethyl-1H-benzimidazole) (-2-ylmethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethane (Hydrobromide salt); N-(2-pyridinylmethyl)-N′-(5-nitro-1H-benzimidazol) (Il-2-ylmethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethane Amine; N-(2-pyridinylmethyl)-N′-[(1H)-5-azabenzimidazole-2-ylmethyl]- N'-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N'-(2-pyridyl Nylmethyl)-N-(4-phenyl-1H-imidazole-2-ylmethyl)-N′-(5,6,7,8-tetrahydro) Ro-8-quinolinyl)-1,4-benzenedimethaneamine; N-(2-pyridinylmethyl)-N'-[2-(2-pyridinylmethyl) [Ridinyl)ethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethane Min; N-(2-pyridinylmethyl)-N′-(2-benzoxazolyl)-N′-(5,6,7,8-tetrahydro Ro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(tran (S-2-aminocyclohexyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzene Dimethanamine; N-(2-pyridinylmethyl)-N′-(2-phenylethyl)-N′-(5,6,7,8-tetramethyl) Rahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N′-(3 -phenylpropyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethane Amine; N-(2-pyridinylmethyl)-N′-(trans-2-aminocyclopentyl)-N′-(5,6,7, 8-Tetrahydro-8-Quinolinyl)-1,4-Benzenedimethanamine; N-[[4-[[(2-Pyridinyl [Tyl]aminomethyl]phenylmethyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-glycy N-amide; N-[[4-[[(2-pyridinylmethyl)aminomethyl]phenyl]methyl]-N-(5,6,7,8- Tetrahydro-8-quinolinyl)-(L)-alaninamide; N-[[4-[[(2-pyridinylmethyl)amide [N]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-(L)-aspart Amide; N-[[4-[[(2-pyridinylmethyl)aminomethyl]phenyl]methyl]-N-(5,6,7,8-the Trahydro-8-quinolinyl)-pyrazineamide; N-[[4-[[(2-pyridinylmethyl)amino]meth [L]phenylmethyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-(L)-prolineamide; N- [[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]-N-(5,6,7,8-tetrahydro -8-Quinolinyl)-(L)-Lysinamide; N-[[4-[[(2-Pyridinylmethyl)aminomethyl]methyl]phen [nyl]methyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-benzamide; N-[[4-[[(2-pyri [Dinylmethyl)aminomethyl]phenylmethyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl) )-picolinamide; N′-benzyl-N-[[4-[[(2-pyridinylmethyl)aminomethyl]pheny [Lu]methyl]-N-(5,6,7,8-tetrahydro-8-quinolinyl)-urea; N'-phenyl-N-[[4-[[(2 -pyridinylmethyl)aminomethyl]phenylmethyl]-N-(5,6,7,8-tetrahydro-8-quinol) Linyl)-urea; N-(6,7,8,9-tetrahydro-5H-cyclohepta[bacteriapyridin-9-i) (Lu)-4-[[(2-pyridinylmethyl)amino]methyl]benzamide; N-(5,6,7,8-tetrahydro- 8-Quinolinyl)-4-[[(2-Pyridinylmethyl)amino]methyl]benzamide; N,N′-Bis(2- Pyridinylmethyl)-N′-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethane Min; N,N′-bis(2-pyridinylmethyl)-N′-(6,7,8,9-tetrahydro-5H-cyclohepta[ Bacteriapyridin-9-yl)-1,4-benzenedimethaneamine; N,N'-bis(2-pyridinylmethyl (Tyl)-N′-(6,7-dihydro-5H-cyclopenta[bacterial pyridine-7-yl)-1,4-benzene Dimethanamine; N,N′-bis(2-pyridinylmethyl)-N′-(1,2,3,4-tetrahydro-1-naph Talenyl)-1,4-benzenedimethaneamine; N,N′-bis(2-pyridinylmethyl)-N′-[(5,6,7 ,8-tetrahydro-8-quinolinyl)methyl]-1,4-benzenedimethaneamine; N,N′-bis(2-p Ridinylmethyl)-N′[(6,7-dihydro-5H-cyclopenta[bacteriapyridin-7-yl)methyl [L]-1,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N-(2-methoxyethyl)-N′- (5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-(2-pyridinyl) Methyl)-N-[2-(4-methoxyphenyl)ethyl]-N′-(5,6,7,8-tetrahydro-8-quinolinyl) -1,4-Benzene dimethanamine; N,N′-Bis(2-pyridinylmethyl)-1,4-(5,6,7,8-tetra Hydro-8-quinolinyl)benzenedimethaneamine; N-[(2,3-dimethoxyphenyl)methyl]-N '-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimeth N-amine; N,N′-bis(2-pyridinylmethyl)-N-[1-(N″-phenyl-N″-methylureido)- 4-Piperidinyl]-1,3-Benzenedimethanamine; N,N′-Bis(2-Pyridinylmethyl)-N-[N [-p-toluenesulfonylphenylalanyl)-4-piperidinyl]-1,3-benzenedimethane Min; N,N′-bis(2-pyridinylmethyl)-N-[1-[3-(2-chlorophenyl)-5-methyl-iso Xazol-4-oil]-4-piperidinyl]-1,3-benzenedimethaneamine; N-[(2-hydroxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahyd Ro-5H-cyclohepta[bacteriapyridin-9-yl)-1,4-benzenedimethaneamine; N-[(4- [Cyanophenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-methyl Rohepta[bacteriapyridin-9-yl)-1,4-benzenedimethaneamine; N-[(4-cyanophen [(Nyl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4- Benzene dimethaneamine; N-[(4-acetamidophenyl)methyl]-N′-(2-pyridinylmethyl (Lu)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-[(4-Fe) [Noxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-methyl) Rohepta[bacteriapyridin-9-yl)-1,4-benzenedimethanamine; N-[(1-methyl-2- [Carboxamide)ethyl]-N,N′-bis(2-pyridinylmethyl)-1,3-benzenedimethaneamine ; N-[(4-benzyloxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-teto Lahydro-5H-cyclohepta[bacteriapyridine-9-yl)-1,4-benzenedimethanamine; N-[(thiophen-2-yl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5 H-cyclohepta[bacteriapyridin-9-yl)-1,4-benzenedimethanamine; N-[1-(ben [zyl)-3-pyrrolidinyl]-N,N′-bis(2-pyridinylmethyl)-1,3-benzenedimethaneamine; N-[[1-methyl-3-(pyrazole-3-yl)]propyl]-N,N′-bis(2-pyridinylmethyl)-1,3- Benzene dimethanamine; N-[1-(phenyl)ethyl]-N,N′-bis(2-pyridinylmethyl)-1, 3-Benzene dimethanamine; N-[(3,4-methylenedioxyphenyl)methyl]-N′-(2-pyrid Nylmethyl)-N-(6,7,8,9-tetrahydro-5H-diclohepta[b]pyridine-9-yl)-1,4-ben Zendimethanamine; N-[1-benzyl-3-carboxymethyl-4-piperidinyl]-N,N′-bis( 2-Pyridinylmethyl)-1,3-Benzenedimethanamine; N-[(3,4-Methylenedioxyphenyl )methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-ben Zendimethanamine; N-(3-pyridinylmethyl)-N′-(2-pyridinylmethyl)-N-(6,7,8,9- Tetrahydro-5H-diclohepta[b]pyridine-9-yl)-1,4-benzenedimethaneamine; N-[[ 1-Methyl-2-(2-tolyl)carboxamide]ethyl]-N,N′-bis(2-pyridinylmethyl)-1,3- Benzene dimethanamine; N-[(1,5-dimethyl-2-phenyl-3-pyrazolinone-4-yl)methyl ]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedi Methaneamine; N-[(4-propoxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8 ,9-tetrahydro-5H-diclohepta[b]pyridine-9-yl)-1,4-benzenedimethaneamine; N -(1-phenyl-3,5-dimethylpyrazoline-4-ylmethyl)-N′-(2-pyridinylmethyl)-N-(5, 6,7,8-Tetrahydro-8-Quinolinyl)-1,4-Benzene Dimethanamine; N-[H-Imidazole-4 -ylmethyl]-N,N′-bis(2-pyridinylmethyl)-1,3-benzenedimethaneamine; N-[(3-methyl Toxy-4,5-methylenedioxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9- Tetrahydro-5H-diclohepta[b]pyridine-9-yl)-1,4-benzenedimethaneamine; N-[( 3-Cyanophenyl)methyl]-N′-(2-Pyridinylmethyl)-N-(6,7,8,9-Tetrahydro-5H-di Chlohepta[b]pyridine-9-yl)-1,4-benzenedimethaneamine; N-[(3-cyanophenyl) Methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-ben Zendimethanamine; N-(5-ethylthiophen-2-ylmethyl)-N′-(2-pyridinylmethyl) -N-(6,7,8,9-tetrahydro-5H-diclohepta[b]pyridine-9-yl)-1,4-benzenedimethane Amine; N-(5-ethylthiophen-2-ylmethyl)-N′-(2-pyridinylmethyl)-N-(5,6,7,8- Tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-[(2,6-difluorophenyl [b](2-pyridinylmethyl)-N'-(6,7,8,9-tetrahydro-5H-diclohepta[b] Lysine-9-yl)-1,4-benzenedimethanamine; N-[(2,6-difluorophenyl)methyl]-N '-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimeth N-amine; N-[(2-difluoromethoxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(6 ,7,8,9-tetrahydro-5H-diclohepta[b]pyridine-9-yl)-1,4-benzenedimethaneamine N-(2-difluoromethoxyphenylmethyl)-N′-(2-pyridinylmethyl)-N-(5,6,7,8- Tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-(1,4-benzodioxane-6 -ylmethyl)-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-diclohepta[b] Pyridine-9-yl)-1,4-benzenedimethanamine; N,N′-bis(2-pyridinylmethyl)-N-[1 -(N″-phenyl-N″-methylureido)-4-piperidinyl]-1,4-benzenedimethaneamine; N ,N′-bis(2-pyridinylmethyl)-N-[N″-p-toluenesulfonylphenylalanyl)-4-py [Peridinyl]-1,4-benzenedimethaneamine; N-[1-(3-pyridinecarboxamide)-4-piperyl] [Dinyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethaneamine; N-[1-(cyclo [Propylcarboxamide)-4-piperidinyl]-N,N′-bis(2-pyridinylmethyl)-1,4-ben Zendimethanamine; N-[1-(1-phenylcyclopropylcarboxamide)-4-piperidinyl ]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethaneamine; N-(1,4-benzodioxy (san-6-ylmethyl)-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl) )-1,4-benzenedimethanamine; N-[1-[3-(2-chlorophenyl)-5-methyl-isoxazo [Lu-4-carboxamide]-4-piperidinyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzene Dimethanamine; N-[1-(2-thiomethylpyridine-3-carboxamide)-4-piperidinyl]-N,N '-Bis(2-pyridinylmethyl)-1,4-benzenedimethaneamine; N-[(2,4-difluorophenyl [Lu)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-B N-(1-methylpyrrole-2-ylmethyl)-N′-(2-pyridinylmethyl) -N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-[(2-hydro [Xyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinoline) (Lu)-1,4-benzenedimethaneamine; N-[(3-methoxy-4,5-methylenedioxyphenyl)meth [L]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzene Dimethanamine; N-(3-pyridinylmethyl)-N′-(2-pyridinylmethyl)-N-(5,6,7,8-teto Lahydro-8-quinolinyl)-1,4-benzenedimethanamine; N-[2-(N″-morpholinomethyl) -1-Cyclopentyl]-N,N′-Bis(2-pyridinylmethyl)-1,4-Benzenedimethanamine; N- [(1-methyl-3-piperidinyl)propyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzene Dimethanamine; N-(1-methylbenzimidazole-2-ylmethyl)-N′-(2-pyridinylmethyl N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-[1-(be [N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethane N-[[(1-phenyl-3-(N″-morpholino)]propyl]-N,N′-bis(2-pyridinylmethyl) )-1,4-benzenedimethanamine; N-[1-(isopropyl)-4-piperidinyl]-N,N′-bis(2- Pyridinylmethyl)-1,4-benzenedimethaneamine; N-[1-(ethoxycarbonyl)-4-piperyl [Dinyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-ben Zendimethanamine; N-[(1-methyl-3-pyrazolyl)propyl]-N′-(2-pyridinylmethyl) -N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-[1-methyl-2 -(N″,N″-diethylcarboxamide)ethyl]-N,N′-bis(2-pyridinylmethyl)-1,4-ben Zendimethaneamine; N-[(1-methyl-2-phenylsulfonyl)ethyl]-N′-(2-pyridinyl) Methyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-[(2- Chloro-4,5-methylenedioxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8- Tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-[1-methyl-2-[N″-(4- [Lolophenyl)carboxamide]ethyl]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetramethyl) (Dro-8-Quinolinyl)-1,4-Benzene Dimethanamine; N-(1-Acetoxyindole-3-yl) Methyl)-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-diclohepta[b]pyridinylmethyl) Zin-9-yl)-1,4-benzenedimethaneamine; N-[(3-benzyloxy-4-methoxyphenyl )methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-diclohepta[b]pyridinylmethyl) Zin-9-yl)-1,4-benzenedimethanamine; N-(3-quinolylmethyl)-N′-(2-pyridyl Nylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N -[(8-hydroxy)-2-quinolylmethyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-teto Rahydro-5H-diclohepta[b]pyridine-9-yl)-1,4-benzenedimethaneamine; N-(2-k (Nolylmethyl)-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-diclohept Ta[b]pyridine-9-yl)-1,4-benzenedimethaneamine; N-[(4-acetamidophenyl) [Tyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-diclohepta[b]pyridinylmethyl N-9-yl)-1,4-benzenedimethaneamine; N-[1H-imidazole-2-ylmethyl]-N,N′- Su(2-pyridinylmethyl)-1,4-benzenedimethanamine; N-(3-quinolylmethyl)-N′- (2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-diclohepta[b]pyridin-9-yl)- 1,4-Benzene dimethanamine; N-(2-thiazoleylmethyl)-N′-(2-pyridinylmethyl)- N-(6,7,8,9-tetrahydro-5H-diclohepta[b]pyridine-9-yl)-1,4-benzenedimethane Amine; N-(4-pyridinylmethyl)-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro- 5H-Diclohepta[b]pyridine-9-yl)-1,4-benzenedimethaneamine; N-[(5-benzyl o [Xy)benzo[b]pyrrole-3-ylmethyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzene Dimethanamine; N-(1-methylpyrazole-2-ylmethyl)-N′-(2-pyridinylmethyl)-N-( 6,7,8,9-Tetrahydro-5H-diclohepta[b]pyridine-9-yl)-1,4-benzenedimethaneamine N-[(4-methyl)-1H-imidazole-5-ylmethyl]-N,N′-bis(2-pyridinylmethyl)-1 ,4-benzenedimethaneamine; N-[[(4-dimethylamino)-1-naphthalenyl]methyl]-N,N′- Bis(2-pyridinylmethyl)-1,4-benzenedimethaneamine; N-[1,5-dimethyl-2-phenyl- [3-Pyrazolinone-4-ylmethyl]-N,N′-bis(2-pyridinylmethyl)-1,4-benzenedimethane Amine; N-[1-[(1-acetyl-2-(R)-prolinyl]-4-piperidinyl]-N-[2-(2-pyridinyl) Ethyl]-N′-(2-pyridinylmethyl)-1,3-benzenedimethaneamine; N-[1-[2-acetamine [dobenzoyl-4-piperidinyl]-4-piperidinyl]-N-[2-(2-pyridinyl)ethyl]-N′-(2- Pyridinylmethyl)-1,3-benzenedimethaneamine; N-[(2-cyano-2-phenyl)ethyl]-N ′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-diclohepta[b]pyridin-9-i (Lu)-1,4-benzenedimethanamine; N-[(N″-acetyltryptophanyl)-4-piperidinyl ]-N-[2-(2-pyridinyl)ethyl]-N′-(2-pyridinylmethyl)-1,3-benzenedimethaneamine ; N-[(N″-benzoylvalinyl)-4-piperidinyl]-N-[2-(2-pyridinyl)ethyl]-N′-(2- Pyridinylmethyl)-1,3-benzenedimethaneamine; N-[(4-dimethylaminophenyl)methyl [Lu]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-diclohepta[b]pyridinyl- 9-yl)-1,4-benzenedimethaneamine; N-(4-pyridinylmethyl)-N′-(2-pyridinylmethyl N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,4-benzenedimethaneamine; N-(1-methyl Rubenzimidazole-2-ylmethyl)-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetramethyl) Dro-5H-diclohepta[b]pyridine-9-yl)-1,4-benzenedimethaneamine; N-[1-butyl- 4-Piperidinyl]-N-[2-(2-pyridinyl)ethyl]-N′-(2-pyridinylmethyl)-1,3-benzene Dimethanamine; N-[1-benzoyl-4-piperidinyl]-N-[2-(2-pyridinyl)ethyl]-N′-( 2-Pyridinylmethyl)-1,3-Benzenedimethaneamine ; N-[1-(benzyl)-3-pyrrolidinyl]-N-[2-(2-pyridinyl)ethyl]-N′-(2-pyridinyl)ethyl (Tyl)-1,3-benzenedimethanamine; N-[(1-methyl)benzo[b]pyrrole-3-ylmethyl]-N -[2-(2-pyridinyl)ethyl]-N′-(2-pyridinylmethyl)-1,3-benzenedimethaneamine; N -[1H-imidazole-4-ylmethyl]-N-[2-(2-pyridinyl)ethyl]-N′-(2-pyridinylmethyl) N-[1-(benzyl)-4-piperidinyl]-N-[2-(2-pyridinyl] [(L)ethyl]-N′-(2-pyridinylmethyl)-1,4-benzenedimethaneamine; N-[1-methylbenn] [zuimidazole-2-ylmethyl]-N-[2-(2-pyridinyl)ethyl]-N′-(2-pyridinylmethyl) -1,4-Benzene dimethanamine; N-[(2-phenyl)benzo[b]pyrrole-3-ylmethyl]-N-[2 -(2-pyridinyl)ethyl]-N′-(2-pyridinylmethyl)-1,4-benzenedimethaneamine; N-[( 6-methylpyridin-2-yl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro -8-Quinolinyl)-1,4-Benzenedimethanamine; N-(3-methyl-1H-pyrazole-5-ylmeth (Lu)-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,3-benzene Dimethanamine; N-[(2-methoxyphenyl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7 ,8-tetrahydro-8-quinolinyl)-1,3-benzenedimethaneamine; N-[(2-ethoxyphenyl )methyl]-N′-(2-pyridinylmethyl)-N-(6,7,8,9-tetrahydro-5H-diclohepta[b]pyridinylmethyl) Zin-9-yl)-1,3-benzenedimethaneamine; N-(benzyloxyethyl)-N'-(2-pyridyl Nylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,3-benzenedimethaneamine; N- [(2-ethoxy-1-naphthalenyl)methyl]-N′-(2-pyridinylmethyl)-N-(5,6,7,8-tetramethyl) (Dro-8-Quinolinyl)-1,3-Benzene Dimethanamine; N-[(6-Methylpyridine-2-yl)methyl [Lu]-N'-(2-pyridinylmethyl)-N-(5,6,7,8-tetrahydro-8-quinolinyl)-1,3-benzene Dimethanamine; 1-[[4-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl]guani Zin; N-(2-pyridinylmethyl)-N-(8-methyl-8-azabicyclo[3.2.1]octan-3-yl)-1 ,4-benzenedimethaneamine; 1-[[4-[[(2-pyridinylmethyl)aminomethyl]phenyl]methyl [Tyl]homopiperazine; 1-[[3-[[(2-pyridinylmethyl)amino]methyl]phenyl]methyl] Homopiperazine; trans and cis-1-[[4-[[(2-pyridinylmethyl)amino]methyl]phen [nyl]methyl]-3,5-piperidinediamine; N,N′-[1,4-phenylenebis(methylene)]bis-4- (2-pyrimidyl)piperazine; 1-[[4-[[(2-pyridinylmethyl)aminomethyl]phenyl]methyl [Tyl]-1-(2-pyridinyl)methylamine; 2-(2-pyridinyl)-5-[[(2-pyridinylmethyl)amine Minomethyl]-1,2,3,4-tetrahydroisoquinoline; 1-[[4-[[(2-pyridinylmethyl)amine [no]methyl]phenyl]methyl]-3,4-diaminopyrrolidine; 1-[[4-[[(2-pyridinylmethyl) [amino]methyl]phenyl]methyl]-3,4-diacetylaminopyrrolidine; 8-[[4-[[(2-pyrid [4.3.0] ]nonane; and 8-[[4-[[(2-pyridinylmethyl)aminomethyl]phenyl]methyl]-2,5,8 -Triazabicyclo[4.3.0]nonan.

[0128] Further CXCR4 antagonists that may be used in conjunction with the compositions and methods described in this application As a nist, I am WO 2001 / 085196, WO 1999 / 050461, WO 2001 / 094420, and WO 2003 / 0905 The CXCR4 antagonists listed in 12 are mentioned, and the disclosures for each of these are CXC Compounds that inhibit R4 activity or expression are incorporated by reference in this application. ru.

[0129] Further CXCR4 agents may be used in conjunction with the compounds and methods described in this application. As for the antagonist, there is the CXCR4 antagonist described in WO 2015 / 063768, for example, BL-8040 (Bio Analog 4F-benzoyl TN14003 (4F-benzoyl), also known as LineRx Modi'in (Israel). Zoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH2; where, Nal Examples include naphthylalanine (=), citrulline (Cit), and D-lysine (DLys).

[0130] Further CXCR4 agents may be used in conjunction with the compounds and methods described in this application. As a gonist, anti-CXCR4 antibodies (including modified antibody fragments as described above) The following can be listed. Anti-CXCR4 may be used in conjunction with the compositions and methods described in this application. As for antibodies, urocuplumab (International Publication No. 2008 / 060367, F7; BMS-936564 or MDX-1338) Also known as Bristol-Myers Squibb, as well as the variants and fragments provided in Table 3. Examples include antibodies such as ant. [Table 3]

[0131] Methods for recombinant expression of peptides and proteins The peptides and proteins described in this application, for example, the corresponding peptide or protein By delivering nucleic acids encoding a substance to a host cell, the host cell can express the substance. The following sections describe the peptides and ta described in this application for the purpose of recombinant expression. Various methods that can be used for the purpose of introducing nucleic acids encoding proteins into host cells explain.

[0132] Transfection technology for recombinant expression Polynucleotides such as nucleic acids that encode polypeptides are used in cells (for example, mammalian cells). For example, techniques that can be used to introduce into human cells are known to those skilled in the art. In some embodiments, electroporation is used to apply electrostatic charge to the target cells. By applying a certain position, mammalian cells (e.g., human cells) can be made permeable. Mammalian cells, such as human cells, that have been exposed to an external electric field in this manner are then subjected to pretreatment. This process allows for the uptake of exogenous nucleic acids. Electroporation in mammalian cells is, for example, This is described in detail in Chu et al. (1987) Nucleic Acids Research 15:1311 (the opening The details shown are incorporated by reference in this application. Similar technologies include nucleoeffects (Nucleofection) TM ) the incorporation of exogenous polynucleotides into the nucleus of eukaryotic cells To stimulate, an applied electric field is used. Nucleofe is useful for carrying out this technique. ction TM The protocol is, for example, seen in Distler et al. (2005) Experimental Dermatology. Details are provided in 14:315 and US 2010 / 0317114, and each of these disclosures is further described. This is incorporated by reference in this application.

[0133] Transfection of host cells for the purpose of expressing recombinant peptides and proteins Further techniques useful for this include the squeeze-poration method. This method involves rapidly mechanically deforming cells to respond to the applied stress. This stimulates the uptake of exogenous DNA through the pores of the membrane that is formed. The technology does not require a vector to deliver nucleic acids to cells such as human cells. In this respect, it is advantageous. Squeeze-poration is, for example, Sharei et al. (201 3) Details are described in Journal of Visualized Experiments 81:e50980, and within its disclosures The contents are incorporated by reference in this application.

[0134] Lipofection is another technique useful for transfecting cells. Liposomes (these liposomes have cationic functional groups, for example, quaternary or protonated Nucleic acids are often placed inside the liposome (which usually presents the amine toward the outside of the liposome). This includes including the placement of the liposomes. As a result, due to the anionic nature of the cell membrane, the liposomes The electrostatic interaction between the liposome and the cell is promoted, and ultimately, for example, the liposome and By direct fusion with the cell membrane, or by the complex undergoing endocytosis This leads to the incorporation of exogenous nucleic acids. Regarding lipofection... For example, this is detailed in U.S. Patent No. 7,442,386, the disclosure of which is referenced in this application. It is taken up more easily. In order to induce the uptake of foreign nucleic acids, the ions of the cell membrane A similar technique that utilizes interactions involves connecting cells with a cationic polymer-nucleic acid complex. One example is bringing it into contact with the cell membrane. A poly(poly Exemplary cationic molecules that associate with nucleotides include activated dendrimers (e.g., Denn This information is described in ig(2003)Topics in Current Chemistry 228:227, and its disclosures are included in this application. (Incorporated by reference), and diethylaminoethyl (DEAE)-dextran (this Its use as a lancetion reagent is, for example, as described in Gulick et al. (1997) Current The details are described in Protocols in Molecular Biology 40:I:9.2:9.2.1, and the disclosures therein are the same as those in this application. (It is incorporated by reference). Magnetic beads are used in this way to incorporate nucleic acids. Because it utilizes an applied magnetic field, it is used to gently and efficiently transfect cells. It is a different tool. This technology is described in detail, for example, US 2010 / 0227406. The disclosed information is incorporated by reference in this application.

[0135] Another useful tool for inducing the uptake of exogenous nucleic acids by cells is laser technology. This is laserfection. This method gently makes the cells permeable, and To enable polynucleotides to penetrate the cell membrane, the cells are exposed to specific wavelengths. This involves exposure to electromagnetic waves. For more information on this method, see, for example, Rhodes et al. (2007). Methods in Cell Biology 82:309 is described in detail, and its disclosures are referenced in this application. It will be absorbed more.

[0136] Microvesicles recombinantly generate nucleic acids encoding the peptide or protein described in this application. To express this, it is a representative example of another carrier that can be used to introduce it into host cells. In some embodiments, the glycoprotein VSV-G is used, and genome modifiers such as nucleases are used. Using microvesicles that co-overexpress ornamental proteins, proteins can be efficiently delivered to cells. It is delivered to a target, and then the endogenous polynucleotide sequence is site-specifically cleaved as a catalyst. Then, the genome of the cell is shared by the polynucleotide of the purpose, such as a gene or regulatory sequence. Sometimes, they are incorporated in a conjugate manner. In order to genetically modify eukaryotic cells, Using such vesicles, also called gesicles, is possible, for example, as demonstrated by Quinn et al. ., Genetic Modification of Target Cells by Direct Delivery of Active Protein [ab [abstract]. In: Methylation changes in early embryonic genes in cancer [abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and C This is described in detail in ell Therapy; 2015 May 13, Abstract No. 122.

[0137] Viral vectors for nucleic acid delivery for recombinant expression The viral genome is exogenous, encoding the peptides and proteins described in this application. This can be used to efficiently deliver nucleic acids into host cells for recombinant expression. It is a rich source of vectors. Viral genomes are particularly useful for delivering genes. It is a suitable vector. This is because the polynucleotides contained within such genomes are, for example, For example, by performing generalized or specialized transduction, the genome of a cell can be incorporated. This is because it can be incorporated. These processes are the natural replication of viral vectors. It can occur as part of the cycle, and proteins induce gene integration. It is not necessary to add any substances or reagents. The peptide or protein described in this application It is used to introduce nucleic acid molecules that encode quality into host cells for recombinant expression. Examples of viral vectors include parvovirus, for example, adeno-associated virus AAV (Adenovirus), retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad4) 8) Coronaviruses, -chain RNA viruses, e.g., orthomyxoviruses (e.g., influenza) (Enza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), para Myxoviruses (e.g., measles and nephrosis), +-strand RNA viruses, e.g., picornaviruses and alphaviruses, as well as double-stranded DNA viruses (adenoviruses, herpesviruses) [For example, herpes simplex virus types 1 and 2, Epstein-Barr virus, site Megalovirus, and poxvirus [e.g., Vaccinia, modified Vaccinia an] Includes [modified vaccinia ankara (MVA)], fowlpox virus and canarypox virus, etc. (m), is included. Polynucleotides encoding peptides and proteins as described herein. Other viruses useful for delivering tide to host cells for recombinant expression include, for example, For example, Norwalk virus, Togavirus, Flavivirus, Reovirus, Papova This includes viruses, hepadnaviruses, and hepatitis viruses. Examples of retroviruses include... This includes avian leukemia-sarcoma, mammalian C-type, B-type, and D-type viruses, HTLV-BLV group, and rhynchophylaxis. Coffin virus, Supmavirus (Coffin, JM, Retroviridae: The viruses and their re plication, In Fundamental Virology, Third Edition, BN Fields, et al., Eds., L One example is ippincott-Raven Publishers, Philadelphia, 1996. Other examples include: Mouse leukemia virus, mouse sarcoma virus, mouse mammary cancer virus, bovine leukemia virus Feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus Virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey • Viruses, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiform virus Viruses are one example. Other examples of vectors include, for instance, U.S. Patent No. 5,801,030. This is described in the document, which concerns gene delivery and the development of recombinant proteins and peptides. This is incorporated by reference in this application as relating to the present.

[0138] Methods for in vivo genetic modification of hematopoietic stem cells and hematopoietic progenitor cells Using one or more methods described in this application, hematopoietic stem cells and hematopoietic progenitor cells are mobilized. Afterward, the recruited cells are subjected to, for example, editing (e.g., modification, destruction, etc.) of endogenous genes. This can sometimes lead to genetic modification.

[0139] nucleic acid In a particular embodiment, nucleic acids for in vivo transduction are used against viral infection. Originally an evolved system as an adaptive defense mechanism in cytoplasm and archaea. A cluster of regularly spaced short palindrome repeats Includes a (CRISPR) / Cas system. The CRISPR / Cas system is a pallination within plasmid DNA. This collection of DNA and proteins contains loam repeat sequences and associated Cas9 nucleases. Combination occurs when foreign DNA is first incorporated into the CRISPR gene locus, thereby merging the target sequence. It instructs a specific DNA cleavage. These foreign sequences and repeats-spacers at the CRISPR locus. The polynucleotides containing the elements are then transcribed in the host cell to generate guide RNA. This then anneals to the target sequence, localizing the Cas9 nuclease to this site. This allows for highly site-specific, Cas9-mediated DNA cleavage. This can occur in conventional polynucleotides, but this is in the very vicinity of the target DNA molecule. The interaction that brings Cas9 to the side is governed by RNA:DNA hybridization. This allows us to theoretically design a CRISPR / Cas system to achieve any desired outcome. Get DNA molecules can be cut. This technology is being developed for editing eukaryotic genomes. This was published (Hwang et al. (2013) Nature Biotechnology 31:227, and its disclosure is, (as incorporated by reference in this application), for example, genes encoding a target protein To cleave the DNA before the offspring is incorporated, the hematopoietic stem cell genome is edited in a site-specific manner. It is sometimes used as an efficient method to regulate gene expression. The use of is described, for example, in U.S. Patent No. 8,697,359, and its disclosure is in this application. It is incorporated by reference.

[0140] Before incorporating the target gene into hematopoietic stem cells, the genomic DNA is cut in a site-specific manner. As an alternative method, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases. - The use of effector nucleases (TALENs) is one example. This differs from the CRISPR / Cas system. These enzymes then act as guide polynucleotides for localization to specific target sequences. It does not contain ocides. Target specificity is instead determined by the DNA binding in these enzymes. Controlled by the main mechanism. In genome editing applications, the use of ZFNs and TALENs is... For example, Urnov et al. (2010) Nature Reviews Genetics 11:636; and Joung et al. (2013 Both of these disclosures are described in Nature Reviews Molecular Cell Biology 14:49. This is incorporated by reference in this application.

[0141] Further genetics may be used to incorporate nucleic acids into the genome of hematopoietic stem cells. As a sub-editing technique, it is possible to rationally design the genomic DNA to cut in a site-specific manner. Yes, ARCUS TM Meganucleases are one example. Using these enzymes is This is advantageous from the perspective of a given structure-activity relationship, which has been established for enzymes such as this one. To create a nuclease that selectively cuts DNA at a desired location, single-strand meganuclease -ase can be modified at a specific amino acid position, and in the nuclear DNA of hematopoietic stem cells, This makes it possible to integrate therapeutic genes in a site-specific manner. These single-chain nucleases For example, this is described in detail in U.S. Patent Nos. 8,021,867 and 8,445,251. Each of those disclosures is incorporated by reference in this application.

[0142] Other gene editing systems include Sleeping Beauty Transposase (S One example is the leeping Beauty Transposase) 100x (SB100x) system (Mates et al. (20 09) See Nat Genet 41(6):753-761. SB100x is a transposon and transposase. It is a synthetic transposon system consisting of (Tc1 / mariner type) SB Lansposase is found in the TA dinucleotide base pairs within the recipient DNA sequence. A transposon is inserted. According to the method described in this application, a therapeutic gene is trans It can be placed on a transposon, and after in vivo transduction, the transposon can be used to hematopoiesis. The TA dinucleotide is inserted into the genome of stem cells or hematopoietic progenitor cells at the appropriate position.

[0143] Other gene editing systems suitable for use with the method described in this application include: Examples include site-specific recombinases. When used in this application, the term "recombiner" is used. "Recombinase" or "site-specific recombinase" refers to one or more recombination sites (for example, 2, 3) Recombination reactions involving sites 4, 5, 7, 10, 12, 15, 20, 30, 50, etc., and excision Alternatively, examples include embedded proteins, enzymes, cofactors, or related proteins. These are wild-type proteins (Landy (1993) Current Opinion in Biotechnology) 3:699-707), or variants, derivatives (e.g., recombinant protein sequences or their fragments) This may include fusion proteins (containing ), fragments, and variants thereof. A list of recombinases suitable for use in specific embodiments of the present invention can be enumerated. Examples, though not limited to them, include Cre, Int, IHF, Xis, FLP, Fis, Hin, Gin, and OC. 31. Cin, Tn3 resolverase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCEl, and ParA are listed. It can be done.

[0144] Selective markers and selective drugs In certain embodiments, the method disclosed in this application involves (1) in vivo hematopoietic stem cells or A step of administering nucleic acids containing a selection marker for transducing hematopoietic progenitor cells, and (2) Hematopoietic stem cells or hematopoietic progenitor cells that have been transduced with nucleic acids containing the selection marker. A step of administering a selective agent for selection, thereby the nucleus containing the selection marker This includes the statement that hematopoietic stem cells or hematopoietic progenitor cells that were not transduced by acid do not survive.

[0145] In a particular embodiment, the selection marker is human O(6)-methylguanine-DNA-methyl It is a transferase (MGMT) variant.

[0146] In certain embodiments, the selected agent includes a methylating agent. The methylating agent is O6-benzylguanine (O6BG), bis-chloroethylnitrosourea. (BCNU), temozolomide, and combinations thereof are selected from the above nucleic acids. The hematopoietic stem cells or hematopoietic progenitor cells were then treated with a selection marker (e.g., human O(6)-methylguanine). It possesses a DNA-methyltransferase (MGMT) variant and is resistant to methylating agents. Cells that were not transduced survived, while those that were not transduced did not.

[0147] Therapeutic genes and targets for gene editing In a particular embodiment, the nucleic acid is (1) missing or defective in the subject A therapeutic gene that can be supplied to provide a gene, or (2) a defect in the subject This includes gene editing systems that modify a specific gene (gene target). Below, treatments A list of genes or gene targets (the cells in which they are expressed, and the cells in which they are destroyed) (Including diseases caused by this) Provide: HSC Fanconi Anemia ia)(FANC AF). Platelets :Hemophilia A (Factor VIII (F8)); Hemophilia B (Factor IX (F9)) Factor X deficiency (Factor X (F10)); Wiskott-Aldrich syndrome (Wiscott-Aldrich syndrome protein (WASP)) neutrophil :X-linked chronic meat Blastomatosis (cytochrome B-245 beta chain (CYBB)); Kostman syndrome (elastase neutrophil development) Currently (Elastase Neutrophil Expressed (ELANE))). red blood cells : Alpha-thalassemia (hemoglobin) Robin subunit alpha (HBA); beta-thalassemia and sickle cell disease (hemoglobin Robin subunit beta (HBB); pyruvate kinase deficiency (pyruvate kinase , liver and RBC(PKLR); Diamond-Blackfan Anemia ( Ribosomal protein S19 (RPS19). Single ball X-linked adrenoleukodystrophy (X-linked A drenoleukodystrophy) (ATP-binding cassette subfamily D member 1 (ABCD1)); metachromotic white Dystrophy (arylsulfatase A (ARSA)); Gaucher disease (glucosylceramide) Ze-beta (GBA); Hunter syndrome (iduronate 2-sulfatase (IDS)); Mucopolysaccharidosis I Type (idulonidase, alpha-L (IDUA)); osteopetrosis (T-cell immunomodulatory factor 1 (TCIRG1)). B cell Adenosine deaminase (ADA) deficiency severe combined immunodeficiency (Adenosine deaminase deficiency) ADA (Advanced Cardiac Disease): X-linked severe combined immunodeficiency (interleukin-2 receptor subunit γ-linked (IL2RG); Wiskott-Aldrich Syndrome (Wiskott-Aldrich Syndrome) Cott-Aldrich syndrome protein (WASP); X-linked agammaglobulinemia (Bult). Bruton's Tyrosine Kinase (BTK). T cells : Adenosine de Aminase (ADA) deficiency severe combined immunodeficiency (ADA); X-linked severe combined immunodeficiency (IL2RG); U Wiskott-Aldrich syndrome protein (WASP); X-linked High IgM syndrome (CD40 ligand (CD40LG)); IPEX syndrome (Forkhea Box P3 (Forkhea d Box P3 (FOXP3)); Early-onset inflammatory disease (Interleukin 4, 10, 13 (IL-4, 10, 13)) ); Hemophagocytic lymphohistiocytosis (perforin 1 (PRF1)); Cancer (artificial T cell receptor (TCR)) , cancer; Chimeric Antigen Receptor (CAR); Human Immunodeficiency Virus Rus (CC motif, chemokine, receptor 5 (CCR5)).

[0148] Viral delivery of nucleic acids Typically, a viral vector is a double-stranded circular DNA molecule derived from a virus. Using a viral vector, one or more therapeutic nucleic acids are delivered to target cells, and Sometimes, they are expressed in target cells. Certain viral vectors themselves They stably integrate themselves into the chromosome. Typically, viral vectors are located within the host cell. , enabling replication of one or more vectors encoding nucleic acids (e.g., therapeutic nucleic acids), It contains at least one promoter sequence. Viral vectors contain selection markers, etc. This application may optionally include one or more non-therapeutic components described herein.

[0149] The method described in this application is for the introduction of exogenous genes in vivo, particularly into humans. Retroviral vectors and adenovirus-derived recombinant gene delivery systems. This includes the use of genetically modified vectors and / or adeno-associated virus vectors. Recombinant retrovirus For producing viruses, and for infecting cells with such viruses in vitro or in vivo. The protocol for this is described in Current Protocols in Molecular Biology, Ausubel, FM. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and others. It is described in the standard laboratory manual.

[0150] DNA vectors, as well as adenoviruses, retroviruses, and lentiviruses. When carrying out the present invention, the virus used as a transduction agent for viral vectors is used. It may be used for this purpose. Examples of retroviruses are not limited to these. However, the following can be cited: Moloney mouse leukemia virus (M-MuLV), Moloney mouse meat Mouse sarcoma virus (MoMSV), Harvey mouse sarcoma virus (HaMuSV), mouse mammary cancer virus (Mu MTV, Gibbon's ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus , Friend mouse leukemia virus, mouse stem cell virus (MSCV), and Rouss sarcoma virus RSV and lentivirus. As used in this application, the term "lentivirus" is a multiple term. This refers to a group (or genus) of complex retroviruses. An example is a lentivirus. While not limited to these, examples include: HIV (Human Immunodeficiency Virus); Including HIV types 1 and HIV 2); Visna-Maedi virus (VMV); Ya Caprine arthritis-encephalitis virus (CAEV); equine infection Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV) Bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0151] In certain embodiments, adenovirus is used in accordance with the method described in this application. This can happen. The adenovirus genome, which encodes and expresses therapeutic genes... However, it is inactive in relation to its ability to replicate in a normal soluble virus life cycle. It may be manipulated to be sexualized. Adenovirus strain Ad type 5 dl324 or other strains. Suitable adenoviruses derived from adenoviruses (e.g., Ad2, Ad3, Ad7, etc.) The Kutar is known to those skilled in the art. Recombinant adenoviruses can infect non-dividing cells. It cannot be used to infect a wide variety of cell types, including epithelial cells. In that respect, it is advantageous in certain situations. Furthermore, virus particles are relatively It is relatively stable, suitable for purification and concentration, and, as described above, has no infectious properties. It can be modified to affect the culprit. In addition, the introduced adenovirus DNA (and the foreign DNA contained within it) is not incorporated into the host cell's genome, and is instead episodic. It remains a genome, and as a result, the introduced DNA is integrated into the host genome. Potential problems that may arise as a result of insertion mutations in retroviruses (e.g., retroviruses) It avoids (DNA). Furthermore, the ability of adenovirus genomes to load onto foreign DNA is also a factor in other genetic issues. It is larger compared to child delivery vectors (up to 8 kilobases).

[0152] Adeno-associated viruses are helpers for efficient replication and a productive life cycle. • As a virus, it requires another virus such as adenovirus or herpesvirus. It is a defective, naturally occurring virus. It also uses its DNA in non-dividing cells. It is also one of the few viruses that can be incorporated into it, and is incorporated stably at a high frequency. To be absorbed.

[0153] In certain embodiments, an embedded, helper-dependent adenovirus (e.g., HD) is used. -Ad5 / 35 ++ ) Uses a vector system. HD-Ad5 / 35 ++ The vectors are uniformly generated on the HSPC. The target is CD46, which is the receptor that manifests as [unclear]. For example, Wang et al (2019) Blood Ad See vances 3(19):2883-2894.

[0154] Treatment method In the case described in this application, in vivo transduction of hematopoietic stem cells and hematopoietic progenitor cells is performed by gene In the treatment of those who require it (for example, patients suffering from stem cell disorders) Hematopoietic stem cells and hematopoietic progenitor cells exhibit multipotency, and therefore, are limited. It is not something that can be done, but granulocytes (for example, promyelocytes, neutrophils, eosinophils, basophils), red blood cells ( For example, reticulocytes, red blood cells, platelets (thrombocytes) (for example, megakaryoblasts, platelets) ) Megakaryocytes, platelets, monocytes (e.g., monocytes, macrophages), dendritic cells, Microglia, osteoclasts, and lymphocytes (e.g., NK cells, B- cells, and T- cells) Hematopoietic stem cells can differentiate into multiple different blood lineages. Furthermore, hematopoietic stem cells are capable of self-renewal. Therefore, it is possible to produce daughter cells that have the same potential as the parent cell, and also, After undergoing in vivo transduction with a therapeutic gene, the hematopoietic stem cells rehom into the niche. Furthermore, it also features properties that re-establish productive and sustainable hematopoiesis. Thus, the traits are introduced. Hematopoietic stem cells and hematopoietic progenitor cells that have been treated are those that have a deficiency or defect in a certain cell type of the hematopoietic lineage. It will be a useful therapeutic modality for treating a wide range of diseases in the person. The defect is, for example, autoreactivity, such as T-lymphocytes or B-lymphocytes that cross-react with autoantigens. This is caused by a decrease in the population of endogenous hematopoietic cells due to the activation of immune cells. (For example, patients suffering from autoimmune disorders such as the autoimmune disorders described in this application) (In some cases). Furthermore, or instead, a deficiency or defect in cell activity leads to abnormal expression of the enzyme. This can therefore be caused by various metabolic disorders, such as those described in this application. (In the case of patients suffering from metabolic disorders.)

[0155] Therefore, using in vivo transduction of hematopoietic stem cells, one or more cell types of the hematopoietic lineage are missing. It may correct genes that are missing or deficient, thereby improving endogenous blood cell function. Treating pathological conditions associated with defects or reductions in hematopoietic stem cell populations. In vivo hematopoietic stem cell plasmapheresis. For example, use this to describe non-malignant hemoglobin disorders (e.g., sickle cell anemia, thalassemia). A. Fanconi anemia, aplastic anemia, and Wiscott-Aldrich syndrome. It can treat hemoglobin disorders selected from the group. In these cases, for example, , administered to the target of CXCR4 antagonists and / or CXCR2 agonists, for such treatment Accordingly, hematopoietic stem cells and hematopoietic progenitor cells accumulate in the circulating peripheral blood from stem cell niches such as bone marrow. This can trigger the release of hematopoietic stem cells and hematopoietic progenitor cells. Next, in nucleic acids (which may include, for example, therapeutic genes and selection markers), in v Transduction may be performed using ivo. When the selected drug is administered to the subject, the therapeutic gene and selection Hematopoietic stem cells or hematopoietic progenitor cells that were not transduced with nucleic acids containing markers survived. No. Transduced cells home to the hematopoietic stem cell niche and possess therapeutic genes. It is possible to reconstruct a population of cells.

[0156] Furthermore, or alternatively, hematopoietic stem cells and hematopoietic progenitor cells can be used to treat congenital immunodeficiency, etc. It can treat immunodeficiency. Furthermore, or alternatively, the compositions described in this application and Using the method, acquired immunodeficiency (e.g., selected from the group consisting of HIV and AIDS) It can treat congenital immunodeficiency. In these cases, for example, CXCR4 antagonists and / or administer a CXCR2 agonist to target stem cells in the bone marrow and other stem cell niches, and circulating peripheral blood. This can sometimes trigger the release of populations of hematopoietic stem cells and hematopoietic progenitor cells. The mobilized hematopoietic stem cells and hematopoietic progenitor cells are then transduced in vivo with nucleic acids. There is a method. After selection for nucleic acids, the selected cells are homing in the hematopoietic stem cell niche. This can occur, and immune cells that possess therapeutic genes (e.g., T lymphocytes, B lymphocytes) It may reassemble a population of globules, NK cells, or other immune cells.

[0157] Using hematopoietic stem cells and hematopoietic progenitor cells, metabolic disorders (e.g., glycogen storage disease) Mucopolysaccharidosis, Gaucher disease, Haller's disease, sphingolipidosis, metachromatic leukodystrophy, Globoid cell leukodystrophy and cerebral adrenal leukodystrophy. Metabolic disorders selected from the group consisting of cerebral adrenoleukodystrophy, etc. It can also treat the disorder. In these cases, for example, a CXCR4 antagonist and / or When a CXCR2 agonist is administered to the target, hematopoiesis is produced in the circulating peripheral blood from stem cell niches such as bone marrow. This can trigger the release of populations of stem cells and hematopoietic progenitor cells. Hematopoietic stem cells and hematopoietic progenitor cells may then be transduced in vivo with nucleic acids. After selection for nucleic acids, the selected cells home to the hematopoietic stem cell niche. This may involve reconstituting a population of hematopoietic cells that possess therapeutic genes.

[0158] Furthermore, or alternatively, hematopoietic stem cells or hematopoietic progenitor cells can be used to treat malignant tumors or proliferative diseases It can treat diseases (for example, blood cancer or myeloproliferative disorders). In the case of cancer treatment, For example, administering a CXCR4 antagonist and / or a CXCR2 agonist to the bone marrow, etc. This triggers the release of populations of hematopoietic stem cells and hematopoietic progenitor cells from the stem cell niche into the circulating peripheral blood. This can cause the mobilized hematopoietic stem cells and hematopoietic progenitor cells to then undergo nucleic acid treatment. Transduction may occur in vivo. After selection for nucleic acids, the selected cells are Hematopoietic cells that may home to the hematopoietic stem cell niche and that possess therapeutic genes. The population may be restructured. Treatment according to the compositions and methods described in this application. Exemplary blood cancers that can cause this include acute myeloid leukemia, acute lymphoblastic leukemia, and chronic myeloid leukemia. Diseases, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, and non-H1N1 In Dikin's lymphoma (non-Hodgkin's lymphoma) and other cancerous conditions including neuroblastoma be.

[0159] Further diseases that may be treated using the methods and compositions described in this application include: , but not limited to, adenosine deaminase deficiency and severe combined immunodeficiency Hyperimmune globulin M syndrome, Chediak-Dong disease, hereditary lymphohistiocytosis, bone marble Diseases, osteogenesis imperfecta, storage disorders, thalassemia major, systemic sclerosis, systemic lupus erythematosus Systemic lupus erythematosus (DIS), multiple sclerosis, and juvenile rheumatoid arthritis are among the conditions that can be treated. It can be listed.

[0160] Furthermore, using in vivo transduction of hematopoietic stem cells and hematopoietic progenitor cells, we can treat autoimmune diseases. It can be treated. In some embodiments, transduced hematopoietic stem cells and prehematopoietic cells are used. Progenitor cells can home in stem cell niches such as bone marrow and establish productive hematopoiesis. This then leads to the formation of autoreactive lymphocytes (e.g., autoreactive T-lymphocytes and / or This can occur due to the activation of autoreactive B lymphocytes, and is reduced during the process of eliminating autoimmune cells. It can be replaced by a small group of cells. It is a limited number of treatable autoimmune diseases. It is not something that can be treated, but it can be used for psoriasis, psoriatic arthritis, type 1 diabetes (type 1 diabetes), rheumatoid arthritis (RA), Human systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), lymphocytic Colitis, acute disseminated encephalomyelitis (ADEM), Addison's disease, alopecia universalis, ankylosing spondylitis, antiphosphate Lipid antibody syndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune Immunoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune oophoritis, Barlow Diseases such as Behçet's disease, bullous pemphigoid, cardiomyopathy, Chagas disease, and chronic fatigue immunodeficiency syndrome. (CFIDS), chronic inflammatory demyelinating polyneuropathy, Crohn's disease, scarring pemphigoid, ceria Rick's sprue herpetiform dermatitis, cold agglutinin disease, CREST syndrome, Degos disease, discoid lupus erythema Todes, autonomic neuropathy, endometriosis, essential mixed cryoglobulinemia, fibromyalgia - Fibromyitis, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto Thyroiditis, hidradenitis suppurativa, idiopathic and / or acute thrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA neuropathy, interstitial cystitis, juvenile arthritis, Kawasaki disease, lichen planus, Lyme disease, meningitis Yale disease, mixed connective tissue disease (MCTD), myasthenia gravis, neuromyotonia, opsoclaw Nus myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis, Pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, Polyarteritis nodosa, polyendocrine syndrome, polymyalgia rheumatica, primary agammaglobulinemia Primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, Lucoidosis, scleroderma, Sjögren's syndrome, Stiff Person syndrome, Takayasu's artery Inflammation, temporal arteritis (also known as giant cell arteritis), ulcerative colitis, collagenous colitis, Uveitis, vasculitis, vitiligo, vulvar pain (vulvar vestibular inflammation), and Wegener's granulomatosis, It can be listed.

[0161] Pharmacokinetics of CXCR2 agonists and CXCR4 antagonists, nucleic acid administration, and nucleic acid Selection For patients receiving both a CXCR4 antagonist and a CXCR2 agonist, The two drugs may be administered to the subject substantially simultaneously (for example, simultaneously or one at a time). (Place one immediately after the other). In some embodiments, the CXCR4 antagonist and the CXCR2 Agonists may be formulated together and administered as the same pharmaceutical composition. This can happen. Instead, the CXCR4 antagonist and the CXCR2 agonist can be treated as separate pharmaceuticals. The formulation may be prepared as a composition and administered to the subject separately but substantially simultaneously. ru.

[0162] In some embodiments, the CXCR2 agonist is administered after the administration of the CXCR4 antagonist. The subject is then administered the CXCR2 agonist to the CXCR4 Within approximately 12 hours after administration of the antagonist (for example, within approximately 10, 8, 6, 4, 2, or 1 hour). (to) Administer to the subject. In some embodiments, the CXCR2 agonist is administered to the CXC After administration of an R4 antagonist, approximately 30 minutes to 180 minutes, for example, approximately 40 minutes to 160 minutes, or approximately 50 minutes. Approximately 150 minutes, approximately 60 to 140 minutes, approximately 70 to 130 minutes, approximately 60 to 120 minutes, approximately 70 minutes to 110 minutes, or approximately 80 to 100 minutes (for example, approximately 30 minutes after administration of the CXCR4 antagonist) Approximately 35 minutes, approximately 40 minutes, approximately 45 minutes, approximately 50 minutes, approximately 55 minutes, approximately 60 minutes, approximately 65 minutes, approximately 70 minutes, approximately 75 minutes, approximately 80 minutes, Approximately 85 minutes, approximately 90 minutes, approximately 95 minutes, approximately 100 minutes, approximately 105 minutes, approximately 110 minutes, approximately 115 minutes, approximately 120 minutes, approximately 125 minutes, approximately 130 minutes, approximately 135 minutes, approximately 140 minutes, approximately 145 minutes, approximately 150 minutes, approximately 155 minutes, approximately 160 minutes, approximately 165 minutes, approximately 170 minutes, The drug is administered to the subject at approximately 175 minutes or approximately 180 minutes. In some embodiments, the CXCR2 is administered to the subject at approximately 175 minutes or approximately 180 minutes. The gonist is administered approximately 2 hours after the administration of the CXCR4 antagonist.

[0163] In a particular embodiment, nucleic acid administration for in vivo transduction is performed using CXCR4 antagonist After the completion of administration of the CXCR2 agonist, approximately 10 minutes to approximately 2 hours (for example, CXCR4 antagonist) After administration of the steroid and CXCR2 agonist, approximately 10 minutes to 1.9 hours, approximately 20 minutes to 1.8 hours, and approximately 25 minutes. (From 1 minute to approximately 1.7 hours, from approximately 30 minutes to approximately 1.6 hours, from approximately 40 minutes to approximately 1.5 hours, from approximately 1 hour to approximately 2 hours) In a particular embodiment, nucleic acid administration for in vivo transduction is performed using the CXCR4 antagonist. Approximately 10 minutes, 15 minutes, 20 minutes, 25 minutes, and 30 minutes after the completion of administration of the nist and CXCR2 agonist. It is performed for approximately 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, or 120 minutes. In this embodiment, nucleic acid administration for in vivo transduction is performed using a CXCR4 antagonist and a CXCR2 antagonist. After the completion of gonist administration, wait approximately 10 to 20 minutes (for example, CXCR4 antagonist and CXCR2 antagonist). After the completion of gonist administration, approximately 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, Perform this at approximately 17 minutes, 18 minutes, 19 minutes, or 20 minutes.

[0164] In certain embodiments, nucleic acid administration for in vivo transduction is performed using a CXCR2 agonist and After administration of the CXCR4 antagonist, approximately 2 to 10 hours, for example, approximately 2 to 3 hours. Duration: Approximately 2 to 4 hours, Approximately 2 to 5 hours, Approximately 2 to 6 hours, Approximately 2 to 7 hours Duration: Approximately 2 to 8 hours, Approximately 2 to 9 hours, Approximately 3 to 4 hours, Approximately 3 to 5 hours Duration: Approximately 3 to 6 hours, approximately 3 to 7 hours, approximately 3 to 8 hours, approximately 3 to 9 hours Duration: Approximately 3 to 10 hours, approximately 4 to 5 hours, approximately 4 to 6 hours, approximately 4 hours to 7 hours, approximately 4 to 8 hours, approximately 4 to 9 hours, approximately 4 to 10 hours, approximately 5 hours Approximately 6 hours, approximately 5 to 7 hours, approximately 5 to 8 hours, approximately 5 to 9 hours, approximately 5 hours Approximately 10 hours, approximately 6 to 7 hours, approximately 6 to 8 hours, approximately 6 to 9 hours, approximately 6 hours Approximately 10 hours, approximately 7 to 8 hours, approximately 7 to 9 hours, approximately 7 to 10 hours, approximately 8 hours It is performed for approximately 9 hours, approximately 8 to 10 hours, or approximately 9 to 10 hours.

[0165] In a particular embodiment, the selected drug is administered approximately 4 to 24 weeks after the administration of the nucleic acid. (For example, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks) Between, approximately 12 weeks, approximately 13 weeks, approximately 14 weeks, approximately 15 weeks, approximately 16 weeks, approximately 17 weeks, approximately 18 weeks, approximately 19 weeks It is administered between (approximately 20 weeks, 21 weeks, 22 weeks, 23 weeks, and 24 weeks). In this form, the selected drug is administered once. In a particular embodiment, the selected drug is administered as follows: After administration of the nucleic acid, the treatment begins approximately 4 to 10 weeks later, in 2, 3, or 4 cycles. Administer over 5, 6, 7, or 8 cycles. In this embodiment, the cycle is separated by 1 day, 2 days, 3 days , 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart They are separated, three weeks apart, or four weeks apart.

[0166] Routes of administration for CXCR2 agonists and CXCR4 antagonists The CXCR4 antagonist and CXCR2 agonist described in this application may be transmitted through various routes (for example, It may be administered to the patient intravenously, subcutaneously, intramuscularly, or parenterally. In the given case, the most suitable route of administration depends on the specific drug being administered, the patient, and the pharmaceutical formulation method. Method of administration (e.g., time of administration and route of administration), patient's age, weight, sex, and the disease being treated. It depends on the severity of the condition, the patient's diet, and the patient's rate of excretion.

[0167] Pharmaceutical composition The CXCR2 agonist and CXCR4 antagonist intended in this application are, respectively, for use in mammals. To administer to subjects such as elephants (for example, humans), the formulation is incorporated into the same pharmaceutical composition. There are such cases. For example, in this application, a CXCR2 agonist and / or a CXCR4 antagonist are used. A pharmaceutical composition is intended to be a mixture containing one or more suitable diluents, carriers, and / or excipients. The pharmaceutical composition may include a sterile aqueous suspension. For selecting a suitable formulation and Conventional procedures and ingredients for preparation are, for example, in Remington: The Science and Practice. of Pharmacy (2012, 22 nd ed.) and The United States Pharmacopeia: The National Form The information is described in Uulary (2015, USP 38 NF 33) (the entirety of which is included in this application). (Captured by light).

[0168] The pharmaceutical composition, either alone or in combination with a pharmaceutically acceptable carrier, can be used on a target (e.g., human). It may be administered to the target group, and the ratio of the active pharmaceutical ingredients (i.e., CXCR2 agonist and / Depending on the dosage (or CXCR4 antagonist), the selected route of administration, and standard pharmaceutical practices... Sometimes decisions are made based on this.

[0169] Dosage and administration of CXCR2 agonists and / or CXCR4 antagonists The intended CXCR2 agonist and CXCR4 antagonist are administered via one or more routes of administration. It may be administered to mammalian subjects (e.g., humans). CXCR2 agonists and CXCR4 antagonists are administered intravenously, intraperitoneally, intramuscularly, and arterially. It may be administered to the target patient via intravenous or subcutaneous injection.

[0170] The intended CXCR2 agonist is administered at a dose of approximately 0.001 mg / kg to approximately 1 mg / kg per subject's body weight, e.g. For example, from approximately 0.001 mg / kg to approximately 0.1 mg / kg, from approximately 0.05 mg / kg to approximately 0.1 mg / kg, and from approximately 0.05 mg / kg to approximately 0. It may be administered in doses of 0.7 mg / kg, and approximately 0.07 mg / kg to approximately 0.1 mg / kg.

[0171] The intended CXCR2 agonist is administered at a dose of approximately 0.001 mg / kg to less than approximately 0.05 mg / kg, for example, approximately 0.00 From 15 mg / kg to less than approximately 0.05 mg / kg, from approximately 0.002 mg / kg to less than approximately 0.05 mg / kg, or approximately 0.025 mg / kg From approximately less than 0.05 mg / kg, from approximately 0.003 mg / kg to approximately less than 0.05 mg / kg, from approximately 0.0035 mg / kg to approximately 0.05 Less than mg / kg, approximately 0.004 mg / kg to less than approximately 0.05 mg / kg, approximately 0.0045 mg / kg to less than approximately 0.05 mg / kg , approximately 0.005 mg / kg to less than approximately 0.05 mg / kg, approximately 0.0055 mg / kg to less than approximately 0.05 mg / kg, approximately 0.006 From mg / kg to less than approximately 0.05 mg / kg, from approximately 0.0065 mg / kg to less than approximately 0.05 mg / kg, or approximately 0.007 mg / kg From approximately 0.05 mg / kg to less than approximately 0.05 mg / kg, from approximately 0.075 mg / kg to less than approximately 0.05 mg / kg, and from approximately 0.008 mg / kg to approximately 0.05 mg / kg. Less than g / kg, approximately 0.0085 mg / kg to less than approximately 0.05 mg / kg, approximately 0.009 mg / kg to less than approximately 0.05 mg / kg , approximately 0.0095 mg / kg to less than approximately 0.05 mg / kg, approximately 0.01 mg / kg to less than approximately 0.05 mg / kg, approximately 0.015 mg / kg to less than approximately 0.05 mg / kg, approximately 0.02 to less than approximately 0.05 mg / kg, approximately 0.025 mg / kg to approximately 0.05 mg Less than g / kg; approximately 0.03 mg / kg to less than approximately 0.05 mg / kg, approximately 0.035 mg / kg to less than approximately 0.05 mg / kg, In amounts ranging from approximately 0.04 mg / kg to less than approximately 0.05 mg / kg, and from approximately 0.045 mg / kg to less than approximately 0.05 mg / kg It may be administered.

[0172] In a particular embodiment, the CXCR2 agonist is administered at a dose ranging from approximately 0.001 mg / kg to approximately 0.049 mg / kg For example, from approximately 0.001 mg / kg to approximately 0.045 mg / kg, from approximately 0.001 mg / kg to approximately 0.04 mg / kg, and approximately 0.001 From mg / kg to approximately 0.035 mg / kg, from approximately 0.001 mg / kg to approximately 0.03 mg / kg, from approximately 0.001 mg / kg to approximately 0.025 mg / kg, approximately 0.001 mg / kg to approximately 0.02 mg / kg, approximately 0.001 mg / kg to approximately 0.015 mg / kg, approximately 0.001 m It may be administered in amounts ranging from g / kg to approximately 0.01 mg / kg.

[0173] In a particular embodiment, the CXCR2 agonist is administered at a dose ranging from approximately 0.01 mg / kg to approximately 0.05 mg / kg. Full, approximately 0.01 mg / kg to approximately 0.049 mg / kg, approximately 0.01 mg / kg to approximately 0.045 mg / kg, approximately 0.01 mg / kg From approximately 0.04 mg / kg, from approximately 0.01 mg / kg to approximately 0.035 mg / kg, from approximately 0.01 mg / kg to approximately 0.03 mg / kg, approximately 0 From 0.01 mg / kg to approximately 0.025 mg / kg, from approximately 0.01 mg / kg to approximately 0.02 mg / kg, and from approximately 0.01 mg / kg to approximately It may be administered at a dose of 0.015 mg / kg.

[0174] In a particular embodiment, the CXCR2 agonist is administered at a dose ranging from approximately 0.02 mg / kg to approximately 0.05 mg / kg. Full, approximately 0.02 mg / kg to approximately 0.049 mg / kg, approximately 0.02 mg / kg to approximately 0.045 mg / kg, approximately 0.02 mg / kg From approximately 0.04 mg / kg, from approximately 0.02 mg / kg to approximately 0.035 mg / kg, from approximately 0.02 mg / kg to approximately 0.03 mg / kg, and It may be administered in doses ranging from approximately 0.02 mg / kg to approximately 0.025 mg / kg.

[0175] In a particular embodiment, the CXCR2 agonist is administered at a dose of approximately 0.03 mg / kg.

[0176] In a particular embodiment, the CXCR2 agonist is administered in a fixed dose of approximately 1 mg to approximately 8 mg. For example, the CXCR2 agonist is administered in doses of approximately 1 mg to 1.5 mg, approximately 1 mg to 2 mg, and approximately 1 mg to approximately 2.5 mg, approximately 1 mg to approximately 3 mg, approximately 1 mg to approximately 3.5 mg, approximately 1 mg to approximately 4 mg, approximately 1 mg From approximately 4.5 mg, from approximately 1 mg to approximately 5 mg, from approximately 1 mg to approximately 5.5 mg, from approximately 1 mg to approximately 6 mg, from approximately 1 mg Approximately 6.5 mg, approximately 1 mg to approximately 7 mg, approximately 1 mg to approximately 7.5 mg, approximately 1.5 mg to approximately 2 mg, approximately 1.5 mg to Approximately 2.5 mg, approximately 1.5 mg to approximately 3 mg, approximately 1.5 mg to approximately 3.5 mg, approximately 1.5 mg to approximately 4 mg, approximately 1.5 mg From approximately 4.5 mg, from approximately 1.5 mg to approximately 5 mg, from approximately 1.5 mg to approximately 5.5 mg, from approximately 1.5 mg to approximately 6 mg, and approximately 1. 5 mg to approximately 6.5 mg, approximately 1.5 mg to approximately 7 mg, approximately 1.5 mg to approximately 7.5 mg, approximately 1.5 mg to approximately 8 mg, Approximately 2 mg to approximately 2.5 mg, approximately 2 mg to approximately 3 mg, approximately 2 mg to approximately 3.5 mg, approximately 2 mg to approximately 4 mg, approximately 2 From mg to approximately 4.5 mg, from approximately 2 mg to approximately 5 mg, from approximately 2 mg to approximately 5.5 mg, from approximately 2 mg to approximately 6 mg, and from approximately 2 mg. From approximately 6.5 mg, approximately 2 mg to approximately 7 mg, approximately 2 mg to approximately 7.5 mg, approximately 2 mg to approximately 8 mg, approximately 2.5 mg Approximately 3 mg, approximately 2.5 mg to approximately 3.5 mg, approximately 2.5 mg to approximately 4 mg, approximately 2.5 mg to approximately 4.5 mg, approximately 2.5 mg From approximately 5 mg, from approximately 2.5 mg to approximately 5.5 mg, from approximately 2.5 mg to approximately 6 mg, from approximately 2.5 mg to approximately 6.5 mg, and approximately 2. 5 mg to approximately 7 mg, approximately 2.5 mg to approximately 7.5 mg, approximately 2.5 mg to approximately 8 mg, approximately 3 mg to approximately 3.5 mg, approximately 3 mg to approximately 4 mg, approximately 3 mg to approximately 4.5 mg, approximately 3 mg to approximately 5 mg, approximately 3 mg to approximately 5.5 mg, approximately 3 mg From approximately 6 mg, from approximately 3 mg to approximately 6.5 mg, from approximately 3 mg to approximately 7 mg, from approximately 3 mg to approximately 7.5 mg, from approximately 3 mg Approximately 8 mg, approximately 3.5 mg to approximately 4 mg, approximately 3.5 mg to approximately 4.5 mg, approximately 3.5 mg to approximately 5 mg, approximately 3.5 mg Approximately 5.5 mg, approximately 3.5 mg to approximately 6 mg, approximately 3.5 mg to approximately 6.5 mg, approximately 3.5 mg to approximately 7 mg, approximately 3.5 mg to approximately 7.5 mg, approximately 3.5 mg to approximately 8 mg, approximately 4 mg to approximately 4.5 mg, approximately 4 mg to approximately 5 mg, approximately 4 mg From approximately 5.5 mg, from approximately 4 mg to approximately 6 mg, from approximately 4 mg to approximately 6.5 mg, from approximately 4 mg to approximately 7 mg, from approximately 4 mg Approximately 7.5 mg, approximately 4 mg to approximately 8 mg, approximately 4.5 mg to approximately 5 mg, approximately 4.5 mg to approximately 5.5 mg, approximately 4.5 mg Approximately 6 mg, approximately 4.5 mg to approximately 6.5 mg, approximately 4.5 mg to approximately 7 mg, approximately 4.5 mg to approximately 7.5 mg, approximately 4.5 From mg to approximately 8 mg, from approximately 5 mg to approximately 5.5 mg, from approximately 5 mg to approximately 6 mg, from approximately 5 mg to approximately 6.5 mg, and from approximately 5 mg. From approximately 7 mg, approximately 5 mg to approximately 7.5 mg, approximately 5 mg to approximately 8 mg, approximately 5.5 mg to approximately 6 mg, approximately 5.5 mg Approximately 6.5 mg, approximately 5.5 mg to approximately 7 mg, approximately 5.5 mg to approximately 7.5 mg, approximately 5.5 mg to approximately 8 mg, approximately 6 mg From approximately 6.5 mg, approximately 6 mg to approximately 7 mg, approximately 6 mg to approximately 7.5 mg, approximately 6 mg to approximately 8 mg, approximately 6.5 mg Approximately 7 mg, approximately 6.5 mg to approximately 7.5 mg, approximately 6.5 mg to approximately 8 mg, approximately 7 mg to approximately 7.5 mg, approximately 7 mg to It is administered in a fixed dose of approximately 8 mg, or approximately 7.5 mg to 8 mg. In a particular embodiment, the CX Administer a CR2 agonist at a fixed dose of approximately 1.3 mg, 2.5 mg, or 5.5 mg.

[0177] In a particular embodiment, the CXCR2 agonist is administered at approximately 0.001 mg / kg / day, approximately 0.0015 mg / kg / day. g / day, about 0.002 mg / kg / day, about 0.0025 mg / kg / day, about 0.003 mg / kg / day, about 0.0035 mg / kg / day, Approximately 0.004 mg / kg / day, approximately 0.0045 mg / kg / day, approximately 0.005 mg / kg / day, approximately 0.0055 mg / kg / day, approximately 0.00 6 mg / kg / day, about 0.0065 mg / kg / day, about 0.007 mg / kg / day, about 0.0075 mg / kg / day, about 0.008 mg / k g / day, about 0.0085 mg / kg / day, about 0.009 mg / kg / day, about 0.0095 mg / kg / day, about 0.01 mg / kg / day, Approximately 0.015 mg / kg / day, approximately 0.02 mg / kg / day, approximately 0.025 mg / kg / day, approximately 0.03 mg / kg / day, approximately 0.035 mg / kg / day, approximately 0.04 mg / kg / day, approximately 0.045 mg / kg / day, approximately 0.049 mg / kg / day, or approximately 0.05 mg / kg / day Administer in an amount less than 1.3 ml. In a particular embodiment, the CXCR2 agonist is administered in an amount of approximately 1.3 ml. Administer in a fixed dose of g / day, 2.5 mg / day, or 5.5 mg / day.

[0178] In a particular embodiment, the CXCR2 agonist is administered at a fixed dose of approximately 1 mg / day to approximately 8 mg / day. Administer according to the prescribed dosage. For example, the CXCR2 agonist may be administered at approximately 1 mg / day, 1.5 mg / day, or 2 mg / day. , about 2.5 mg / day, about 3.5 mg / day, about 4 mg / day, about 5 mg / day, about 5.5 mg / day, about 6 mg / day, about 6.5 It may be administered in fixed doses of mg / day, approximately 7 mg / day, approximately 7.5 mg / day, or approximately 8 mg / day. .

[0179] In some embodiments, the CXCR4 antagonist is plerixafor or its pharmaceutical It is a generally acceptable salt. In some embodiments, the CXCR4 antagonist (e.g.) Subcutaneously administer plerixafor or a pharmaceutically acceptable salt thereof to the subject. In that embodiment, the CXCR4 antagonist (for example, prelixafor or its pharmaceutical agent) is used. A salt that is acceptable to the subject is administered at a dose of approximately 50 μg / kg to approximately 500 μg / kg per unit of body weight. For example, about 50 μg / kg, 55 μg / kg, 60 μg / kg, 65 μg / kg, 70 μg / kg, 75 μg / kg, 80 μg / kg, 85 μg / kg, 90 μg / kg, 95 μg / kg, 100 μg / kg, 105 μg / kg, 110 μg / kg, 115 μg / kg, 120 μg / kg, 125 μg / kg, 130 μg / kg, 135 μg / kg, 140 μg / kg, 145 μg / kg, 150 μg / kg, 155 μg / kg, 160 μg / kg, 165 μg / kg, 170 μg / kg, 175 μg / kg, 180 μg / kg, 185 μg / kg, 190 μg / kg, 195 μg / kg, 200 μg / kg, 205 μg / kg, 210 μg / kg, 215 μg / kg, 220 μg / kg, 225 μg / kg, 230 μg / kg, 235 μg / kg, 240 μg / kg, 245 μg / kg, 250 μg / kg, 255 μg / kg, 260 μg / kg, 265 μg / kg, 270 μg / kg, 275 μg / kg, 280 μg / kg, 285 μg / kg, 290 μg / kg, 295 μg / kg, 300 μg / kg, 305 μg / kg, 310 μg / kg, 315 μg / kg, 320 μg / kg, 325 μg / kg, 330 μg / kg, 335 μg / kg, 340 μg / kg, 345 μg / kg, 350 μg / kg, 355 μg / kg, 360 μg / kg, 365 μg / kg, 370 μg / kg, 375 μg / kg, 380 μg / kg, 385 μg / kg, 390 μg / kg, 395 μg / kg, 400 μg / kg, 405 μg / kg, 410 μg / kg, 415 μg / kg, 420 μg / kg, 425 μg / kg, 430 μg / kg, 435 μg / kg, 440 μg / kg, 445 μg / kg, 450 μg / kg, 455 μg / kg, 460 μg / kg, 465 μg / kg, 470 μg / kg, 475 μg / kg, 480 μg The above-mentioned subjects are administered the drug at doses of 485 μg / kg, 490 μg / kg, 495 μg / kg, or 500 μg / kg. In some embodiments, the CXCR4 antagonist (e.g., prelixafor) is used. (or a pharmaceutically acceptable salt thereof) in doses of approximately 200 μg / kg to approximately 300 μg / kg, for example, The drug is administered to the subjects at a dose of 240 μg / kg.

[0180] For example, in some embodiments, the CXCR4 antagonist (e.g., prelixa) (Lu or a pharmaceutically acceptable salt thereof) at doses ranging from approximately 50 μg / kg / day to approximately 500 μg / kg / day. For example, approximately 50 μg / kg / day, 55 μg / kg / day, 60 μg / kg / day, 65 μg / kg / day, 70 μg / kg / day, 75 μg / kg / day, 80 μg / kg / day, 85 μg / kg / day, 90 μg / kg / day, 95 μg / kg / day, 100 μg / kg / day, 105 μg / kg / day, 110 μg / kg / day, 115 μg / kg / day, 120 μg / kg / day, 125 μg / kg / day 130 μg / kg / day, 135 μg / kg / day, 140 μg / kg / day, 145 μg / kg / day, 150 μg / kg / day, 155 μg / kg / day μg / kg / day, 160 μg / kg / day, 165 μg / kg / day, 170 μg / kg / day, 175 μg / kg / day, 180 μg / k g / day, 185 μg / kg / day, 190 μg / kg / day, 195 μg / kg / day, 200 μg / kg / day, 205 μg / kg / day 210 μg / kg / day, 215 μg / kg / day, 220 μg / kg / day, 225 μg / kg / day, 230 μg / kg / day, 235 μg / kg / day μg / kg / day, 240 μg / kg / day, 245 μg / kg / day, 250 μg / kg / day, 255 μg / kg / day, 260 μg / k g / day, 265 μg / kg / day, 270 μg / kg / day, 275 μg / kg / day, 280 μg / kg / day, 285 μg / kg / day 290 μg / kg / day, 295 μg / kg / day, 300 μg / kg / day, 305 μg / kg / day, 310 μg / kg / day, 315 μg / kg / day μg / kg / day, 320 μg / kg / day, 325 μg / kg / day, 330 μg / kg / day, 335 μg / kg / day, 340 μg / k g / day, 345 μg / kg / day, 350 μg / kg / day, 355 μg / kg / day, 360 μg / kg / day, 365 μg / kg / day 370 μg / kg / day, 375 μg / kg / day, 380 μg / kg / day, 385 μg / kg / day, 390 μg / kg / day, 395 μg / kg / day, 400 μg / kg / day, 405 μg / kg / day, 410 μg / kg / day, 415 μg / kg / day, 420 μg / k g / day, 425 μg / kg / day, 430 μg / kg / day, 435 μg / kg / day, 440 μg / kg / day, 445 μg / kg / day, 450 μg / kg / day, 455 μg / kg / day, 460 μg / kg / day, 465 μg / kg / day, 470 μg / kg / day, 475 μg / kg / day, 480 μg / kg / day, 485 μg / kg / day, 490 μg / kg / day, 495 μg / kg / day, or 500 The subject is administered a dose of μg / kg / day. In some embodiments, the CXCR4 agent A gonist (e.g., prelixafor or a pharmaceutically acceptable salt thereof) is administered at approximately 200 μg / kg / The dose administered to the subject is approximately 300 μg / kg / day, for example, approximately 240 μg / kg / day. In some embodiments, the CXCR4 antagonist is administered as a single dose. In other embodiments, the CXCR4 antagonist is administered in two or more doses. There are things that need to be done.

[0181] The intended CXCR2 agonist and CXCR4 antagonist are administered to the target population at one or more doses. It may contribute to a single dose. For example, a CXCR2 agonist and / or a CXCR4 antagonist may be used in a single dose. It may be administered as a single dose, or in doses of 2, 3, 4, 5, or more. Multiple times When administering at this dose, the second and subsequent doses should be given on the same day as the first dose, or Administer the drug at least one day, at least one week, at least one month, or at least one year after the initial administration. For example, the intended CXCR2 agonist and CXCR4 antagonist described in this application The stimulant should be administered to subjects such as humans at least once a day, at least once a week, at least once a month, or at least once a year. The above factors include, for example, the subject's age, weight, sex, diet, and excretion rate. It may be administered depending on the child's needs.

[0182] In certain embodiments, the intended CXCR2 agonist and CXCR4 antagonist are used as follows: Each is administered once daily in a single dose. In certain embodiments, the intended CXCR2 jaw The nist and CXCR4 antagonist are administered for two consecutive days, respectively. In terms of dosage, the intended CXCR2 agonist and CXCR4 antagonist are administered once daily, respectively. A single dose is administered for two consecutive days. In certain embodiments, the intended CXCR2 agonist When stront and a CXCR4 antagonist are administered for two consecutive days, CD34 + Cell yield improved In a particular embodiment, the intended CXCR2 agonist and CXCR4 antagonist are 2 When administered for several consecutive days, a sufficient number of CD34 cells are produced for in vivo transduction. + Cells are mobilized. This becomes possible, where a one-day dose is insufficient. In a particular embodiment, The individuals described may have symptoms of insufficient mobilization of stem cells from the bone marrow.

[0183] Leukocytosis In certain embodiments, a CXCR2 agonist and optionally a CXCR4 antagonist are administered. This minimizes the change in leukocytosis (i.e., the change in the number of white blood cells in the blood is minimized). (It becomes a small number). In a particular embodiment, the leukocytes are neutrophils, eosinophils, basophils These are lymphocytes, monocytes, or a combination thereof. In contrast, G-CSF (which mobilizes neutrophils) Conventional treatment options for this purpose enhance leukocytosis, which, for example, sickle cell anemia. In patients with this condition, white blood cells such as neutrophils adhere to the endothelium, thereby affecting blood vessels. This is problematic because it increases the risk of serious and life-threatening complications such as obstructive crises. In a particular embodiment, a CXCR2 agonist and optionally a CXCR4 antagonist are used. When administered, a CXCR2 agonist and, optionally, a CXCR4 antagonist are administered, for example. Then, approximately 1 hour, 3 hours, 6 hours, 9 hours, 24 hours, or 48 hours later, per 1 ml of blood Approximately 30 x 1000 white blood cells per unit area, approximately 20 x 1000 white blood cells per 1 ml of blood, blood The amount of white blood cells present will be less than approximately 10 × 1000 per milliliter.

[0184] Cytokine levels In certain embodiments, a CXCR2 agonist and optionally a CXCR4 antagonist are administered. When administered, changes in IL-6 levels in the blood are minimized. In contrast, G-CSF reduces blood sugar levels. It increases the level of itokine, which is, for example, in patients with sickle cell disease. The title is as follows. Therefore, in a particular embodiment, a CXCR2 agonist and optionally a CXCR4 agonist are used. When an antagonist is administered, a CXCR2 agonist and, optionally, a CXCR4 antagonist are administered. For example, after about 1 hour, 3 hours, 6 hours, 9 hours, 24 hours, or 48 hours, IL-6 less than approximately 150 pg per 1 ml of blood, IL-6 less than approximately 100 pg per 1 ml of blood, or per 1 ml of blood This results in IL-6 levels of approximately 75 pg or less. In certain embodiments, a CXCR2 agonist and an optional Selective administration of CXCR4 antagonists can lead to the administration of CXCR2 agonists and, optionally, CXCR4 antagonists. Compared to the patient's serum IL-6 level before administering the tagonist, the elevated serum IL-6 level of the patient Substantially no increase is achieved. In some embodiments, a CXCR2 agonist and optional When a CXCR4 antagonist is administered selectively, CXCR2 agonists and CXCR4 antagonists are also administered selectively. Compared to the patient's serum IL-6 level before gonist administration, the patient's serum IL-6 level increased by 5%. An increase of less than 10%, an increase of less than 15%, an increase of less than 20%, an increase of less than 30%, or 50% This results in an increase of less than [a certain amount].

[0185] The pharmaceutical compositions described in this application may be administered to subjects in one or more doses. When administering in single doses, the second and subsequent doses should be administered at least one day after the first dose. It may be provided after a week or more, after a month or more, or after a year or more. For example, this application The pharmaceutical composition described may be used, for example, based on the age, weight, sex, and severity of the disease being treated. Depending on factors such as the diet and the rate of excretion, the frequency may be at least once a day, at least once a week, or once a month. Having suffered from one or more of the diseases, symptoms, or disorders described in this application, either once or at least once a year. It may be administered to individuals or other subjects who are present in the body. [Examples]

[0186] The following examples illustrate how the compositions and methods described in this application are used and prepared. To explain whether it can be evaluated, it is presented to those skilled in the art, and is purely illustrative. This is intended to be, and not intended to limit the scope of the invention.

[0187] Example 1: After in vivo transduction and selection, recruitment with Gro-β + prelixafor was performed using G-CS. F+ leads to in vivo transduction equivalent to that of prelixafor. In this example, hematopoietic stem cells and hematopoietic progenitor cells were treated with MGTA-145(Gro-βT) + plerixafor. It can be used to mobilize, and can be transformed in vivo. This demonstrates the following: As shown in Figure 1A, CD46-transgenic mice were given GCSF+ Mobilize with plerixafor (5 days) or Gro-β + plerixafor (administered subcutaneously at the same time). Next, after one hour, the HDAd5 / 35 will be installed. ++ Inject mgmt / GFP vector + HDAd-SB vector Administered (e.g., Li et al. (2018) Mol Ther Methods Clin Dev. 9:148-152). Both Dexamethasone was also used in the mobilization regimen.

[0188] LSK (system - cKit + Sca1 + ) The number of cells was measured at various time points after MGTA-145 injection administration. Measurement was performed by tubometry (Figure 1B). A certain amount of blood was placed in colony assay medium. By rating and scoring the number of colonies formed over time, (Figure 1C) was performed. As shown, when mobilization was carried out with MGTA-145 + prelixafor Mobilization peaked approximately 15 minutes after MGTA-145 injection. As shown in Figure 2, MGTA When mobilization is performed using -145 (labeled "Gro-β" in the figure) + prelixafor (labeled "AMD3100" in the figure), GC Fewer cells were recruited compared to when mobilizing with SF + prelixafor (labeled "AMD3100" in the figure). It's gone. However, surprisingly, as detailed below, MGTA-145 + Pre In vivo transduction of cells mobilized with lixafor was performed using GCSF + prelixafor. It was as effective as in vivo transduction of cells that had been mobilized.

[0189] Blood samples were collected at various time points after prelixafor administration and subjected to hemavet analysis. The leukemia (lyses) was subjected to the test. As shown in Figure 3A, the leukemia in the MGTA-145 + prelixafor group The leukocytosis was much lower in the G-CSF + prelixafor group. Similarly, Figure 3B shows the difference between using MGTA-145 + prelixafor and using G-CSF + prelixafor. Rather, fewer mononuclear cells (MNCs) are recruited one hour after the last injection of the drug. This graph shows the following. Based on these observations, mobilization using MGTA-145 + prelixafor Compared to mobilization using G-CSF + prelixafor, this method is more effective in treating blood disorders (e.g., sickle cell anemia). It is suggested that this is beneficial for patients with blood (blood). Figure 3C shows Brilliant. This shows the percentage of reticulocytes detected by Brilliant cresyl blue. .

[0190] As shown in the scheme in Figure 4, mobilization will be carried out, HDAd5 / 35 ++ mgmt / GFP vector + HDad-SB After administering Kutar by injection, at 4 weeks, 6 weeks, 8 weeks, and 10 weeks, O6-benzyl Guanine (O 6 BG) + bischloroethylnitrosourea (BCNU) IP for 4 rounds of selection The procedure was performed. Twelve weeks after in vivo transduction, the mice were sacrificed, and secondary For transplantation into recipients (lethally irradiated C57Bl / 6 mice), bone marrow lin - Cells were collected. These secondary transplanted mice were followed for 16 weeks for final analysis.

[0191] As shown in Figures 5A to C, the secondary recipient after transplantation has the same level of G FP+ cells were present. Figure 5A shows the percentage of cells in PBMCs at 10 and 12 weeks post-transplantation. This shows an increase in the CD3 levels in the MNCs of blood, spleen, and bone marrow at week 16. Figure 5B shows the CD3 levels in the MNCs of blood, spleen, and bone marrow. - Indicates the percentage of GFP expression in CD19- and Gr-1-positive cells. LSK cells in bone marrow samples. The analysis also revealed that the levels of GFP+ cells in the blood, spleen, and bone marrow were similar. This demonstrates that lineage-negative cells were obtained from the bone marrow 16 weeks after transduction. Figure 5C shows that lineage-negative cells were obtained from the bone marrow 16 weeks after transduction. The cells were isolated, and 2500 were seeded for a methylcellulose assay. Test results: This shows the percentage of GFP expression in pooled colony cells. In summary, this These results were obtained using either G-CSF + prelixafor or MGTA-145 + prelixafor. This indicates that in vivo transduction occurred at a similar level, regardless of whether cells were recruited. vinegar.

[0192] Next, engraftment is performed on human CD46 in PBMCs. + Flow cytometry for detecting cells The measurements were taken as shown in Figure 6A: G-CSF + prelixafor or MGTA-145 + p Similar levels of engraftment were observed regardless of which form of relixafor was used to recruit cells. Furthermore, as shown in Figure 6B, GFP in PBMCS was observed at various time points up to 16 weeks after transplantation. When expression was monitored, it was found that the introduced gene was stably maintained after transduction. This was shown.

[0193] Cellular composition of MNCs in the spleen and bone marrow of blood samples 16 weeks after secondary transplantation was measured, as shown in Figure 7. As shown in A. Each dot represents one animal. Naive animals that have not undergone trait introduction are shown. It was used as a control. Furthermore, it was a system-negative (Lin - ) cells, at 16 weeks, from the bone marrow They were isolated. 2500 cells were seeded for a methylcellulose assay. The number of negative cells was counted after 10 days and is shown in Figure 7B. According to these data, G-CSF + preliquid Regardless of whether safor or MGTA-145 + plerixafor was used to recruit cells, multiple It is shown that engraftment is observed in several cell lineages.

[0194] Cytokine levels in response to mobilization and transduction were analyzed and evaluated. Serum samples were collected for IL-6 ELISA at 1 hour and 6 hours later. (See Figure 8) Thus, mobilization with MGTA-145 + Prelixafor ("MGTA-145") is G-CSF + Prelixa Compared to mobilization by hormone ("G-CSF"), the induction of cytokine elevation was minimal. Each dot represents one animal. Samples derived from mice that have not been mobilized are used as control. Used as a rule. *, p<0.05.

[0195] Similar experiments were repeated in rhesus monkeys, showing high levels of gamma globin expression (this This is Sleeping Beauty Transposase Therefore, it was demonstrated that it was transformed into HSPC.

[0196] Furthermore, we will evaluate animal models of thalassemia and sickle cell disease. th3 / CD46tg mouse (Sa A disease model of raceemia was recruited using MGTA-145 + prelixafor. Blood samples were collected 15 minutes after administration. LSK (system) - cKit + Sca1 + ) The number of cells, Flow-S The measurements were taken by itchometry and are shown in Figure 9A. Colony-forming cells present in peripheral blood. The number was measured by a methylcellulose assay, as shown in Figure 9B. This represents one animal. These data are from the MGTA- in the mouse model of thalassemia. This suggests that HSCs can be efficiently mobilized with 145 + prelixafor.

[0197] Figure 10 shows Hb before treatment. th3 / CD46tg (thalassemia) and Hbb tm2 / CD46tg (Towns or sickle-shaped) This shows the phenotype of a disease model of erythrocyte disease (Townes or sickle cell disease model). Giemsa / May-Grunwald staining of the tissue sample revealed R The morphology of BCs was measured. The percentage of reticulocytes was determined by brilliant cresil blue staining. The measurements were taken using samples from CD46 mice as a "healthy" control.

[0198] Example 2: In vivo hematopoietic stem cells and hematopoietic progenitor cells for gene therapy of sickle cell anemia o Transfusion In this example, hematopoietic stem cells and hematopoietic progenitor cells are converted into Gro-β or MGTA-145 + preliquid. It is possible to mobilize using Sahor and add gamma genes, and via Cas-CRISPR editing HDAd5 / 35 is capable of reactivating endogenous gamma globin. ++ With mgmt vector, in It has been demonstrated that transformation can be achieved in vivo (e.g., Li et al. (2018) Blo See od 131(26):2915-2928 and Richter et al. (2016) Blood 128:2206-2217. sea ​​bream).

[0199] Townes / CD46tg Transgenic Mouse (This mouse is a mouse g The robin gene is replaced with the human globin gene (Ryan et al. (1997) Science 2) For 78(5339):873-876), GCSF + prelixafor (5 days), or Gro-β + prelixafor Xafor (2.5 mg / kg Gro-β or MGTA-145 and 5 mg / kg prelixafor administered subcutaneously simultaneously) (Given) Mobilization will be carried out, and after 1 hour, HDAd5 / 35 will be incorporated. ++ mgmt vector administered by injection (iv) In one cohort of animals (half), transduced HSCs / precursors were selected in vivo. To select, O 6 BG / BCNU treatment was performed. Specifically, O 6 BG / BCNU treatment, vector injection The procedure is started four weeks after administration, followed by a two-week interval, for a total of three cycles. Transduction was performed in vivo. The animals will be tracked for 18 weeks. During this time, blood samples will be taken for γ- and β- spectroscopy. δ - Globin expression (HPLC, qRT-PCR), target site cleavage (T7E1A assay), and phenotypic modification (hematology, reticulocyte We will analyze the globus and RBC morphology. At week 18, we will also analyze the bone marrow, spleen, and liver. We will analyze the T-cell response to genome editing enzymes (iCas, SB100x, Flpe) using spleen cells. .

[0200] Mice were sacrificed, and their blood / tissue was analyzed for phenotypic modification. . Bone marrow lin - The cells were transplanted into a secondary recipient that had been lethally irradiated, and Follow-up will continue for another 16 weeks. At the end of this period, the total volume will be compared to the pre-treatment sample. Based on NM sequencing and RNA / miRNA-Seq, (evaluate transcriptome changes) (To do so), and to evaluate the long-term genotoxic effects.

[0201] GCSF can cause complications in patients with sickle cell anemia, therefore Gro- Mobilization using β or MGTA-145 + prelixafor is considered safer. Mobilization using Gro-β or MGTA-145 + prelixafor can mobilize HSCs in their earlier stages. It is thought that they will be included.

[0202] Other embodiments All publications, patents, and patent applications referenced in this application are treated as if they were independent of each other. The publication or patent application is specifically and individually cited and incorporated by reference, This is incorporated by reference to this application to the extent that.

[0203] Although the present invention has been described in relation to its specific embodiments, further modifications are possible. This application is intended to be capable of, and generally, any modification of the present invention in accordance with the principles of the present invention. The use or adaptation of this invention will not be incorporated into known or customary practices within the art to which this invention relates. Departures from the present invention and the essential features described in this application and in accordance with the claims apply. Any modification, use, or adaptation of the present invention, including possible departures from the present invention, is included. It will be understood that this is the intended meaning.

[0204] Other embodiments are within the scope of the claims of this application.

Claims

1. This refers to a population of hematopoietic stem cells or hematopoietic progenitor cells mobilized from the bone marrow into the peripheral blood of mammals. Methods for introducing quality Here, the dose is approximately 0.001 mg / kg to approximately 0.1 mg / kg, or fixed at approximately 1 mg to approximately 8 mg. The CXCR2 is selected from the group consisting of Gro-β, Gro-β T, and their variants, based on the dose. Using an agonist, the target hematopoietic stem cells or hematopoietic progenitor cells are recruited into the peripheral blood. ru, The method described above includes: (a) Selective markers for transducing hematopoietic stem cells or hematopoietic progenitor cells in vivo. The steps include administering the nucleic acid to the subject, (b) Hematopoietic stem cells or hematopoietic progenitor cells transduced with nucleic acids containing a selection marker A step of administering a selection drug for selection, thereby shaping nucleic acids containing a selection marker. Hematopoietic stem cells or hematopoietic progenitor cells that have not undergone quality induction will not survive.

2. A method according to any of the prior claims, wherein the nucleic acid is a gene editing system or Includes components of a genetic engineering system.

3. The method according to claim 2, wherein the system is a CRISPR-Cas9 system, three pins Sleeping Beauty Transposase 100x (SB100x) The system and the FLP-FRT system are selected from among the options.

4. A method according to any of the preceding claims, wherein the nucleic acid further comprises a therapeutic gene.

5. The method according to claim 4, wherein the therapeutic gene is at least of the γ-globin gene Partially, or FANC AF; Factor VIII (F8); Factor IX (F9); Factor X (F10); Wiscott A Ludrich syndrome protein (WASP); cytochrome B-245 beta chain (CYBB); elastase Neutrophil expression (Elastase Neutrophil Expressed (ELANE)); hemoglobin subunit Alpha (HBA); hemoglobin subunit; beta (HBB); pyruvate kinase, liver Organs and RBCs (PKLR); ribosomal protein S19 (RPS19); ATP-binding cassette subfamily - D member 1 (ABCD1); aryl sulfatase A (ARSA); glucosylceramidase b T (GBA); Iduronate 2-sulfatase (IDS); Iduronidase, alpha-L (IDUA); T- Cellular immunomodulatory factor 1 (TCIRG1); adenosine deaminase (ADA); interleukin 2 receptor Pter subunit gamma (IL2RG); Bruton's tyrosine kinase sine kinase (BTK); adenosine deaminase (ADA); IL2RG; CD40 ligand (CD40LG); Forkhead Box P3 (FOXP3); Interleukin 4, 10, 13 (I L-4, 10, 13); Perforin 1 (PRF1); Artificial T cell receptor (TCR); Chimeric antigen receptor - (CAR); or at least a part of CC Motif Chemokine Receptor 5 (CCR5) Includes the genes that code for it.

6. A method according to any of the preceding claims, wherein the selected marker is human O(6)-methyl Contains ruguanine-DNA-methyltransferase (MGMT) variants.

7. A method according to any of the preceding claims, wherein the selected agent comprises a methylating agent.

8. The method according to claim 7, wherein the methylating agent is O6-benzylguanine (O6BG), S-chloroethylnitrosourea (BCNU), temozolomide, and combinations thereof, Selected.

9. A method according to any of the prior claims, wherein the nucleic acid is present in the vector.

10. The method according to claim 9, wherein the vector is a lentiviral vector, rAAV vector Select from the following: vector, and HDAd5 / 35++ vector.

11. A method according to any of the prior claims, wherein the nucleic acid is the CXCR2 agonist and The drug is administered approximately 10 minutes to 10 hours after administering the CXCR4 antagonist.

12. A method according to any of the preceding claims, wherein the selected agent is administered by administering the nucleic acid. The medication is administered approximately 4 weeks to 24 weeks after the initial treatment.

13. A method according to any of the preceding claims, wherein the dose is greater than about 0.015 mg / kg to about 0 It is less than 0.05 mg / kg.

14. A method according to any of the prior claims, wherein the CXCR2 agonist is Gro-β T include.

15. A method according to any of the prior claims, wherein the CXCR2 agonist is given in a dose of about 0.03 mg / kJ Administer in a dose of g.

16. A method according to any of the prior claims, wherein the method comprises the CXCR2 agonist The procedure further includes the step of administration.

17. A method according to any of the prior claims, wherein the CXCR2 agonist and the CXCR4 agonist A gonist is used to recruit the target hematopoietic stem cells or hematopoietic progenitor cells into the peripheral blood. 。

18. The method according to claim 17, wherein the CXCR4 antagonist is prelixafor ru.

19. The method according to claim 18, wherein the prelixafor is administered at a dose of approximately 240 μg / kg. Administer to the specified subjects.

20. The method according to any one of claims 17 to 19, wherein the CXCR2 agonist is This should be administered simultaneously with the CXCR4 antagonist.

21. The method according to any one of claims 17 to 19, wherein the CXCR2 agonist is This should be administered after the CXCR4 antagonist.

22. The method according to claim 21, wherein the CXCR2 agonist is the CXCR4 antagonist Administer within approximately 4 hours of administering the first drug.

23. The method according to claim 21 or 22, wherein the CXCR2 agonist is the CXCR4 agonist It is administered approximately two hours after the gonist.

24. The method according to any one of claims 17 to 23, wherein the CXCR2 agonist and Each CXCR4 antagonist is administered for two consecutive days.

25. The method according to claim 24, wherein the CXCR2 agonist and the CXCR4 antagonist Each of these medications is administered once a day for two consecutive days.

26. A method according to any of the preceding claims, wherein the fixed dose is approximately 2.5 mg to approximately 5.5 mg It is mg.

27. The method according to any of the preceding claims, wherein the fixed dose is approximately 1.3 mg.

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