Compositions and methods for non-genotoxic preconditioning

Abstract

Provided herein are methods and compositions relating to the use of anti-CD110 and anti-CD117 preconditioning agents to deplete endogenous hematopoietic stem cells in a subject prior to hematopoietic stem cell transplantation. Also provided are cell-based therapeutic methods and compositions.

Description

[0001] Cross-references to related applications This application claims the benefit and priority of International Application No. PCT / US2023 / 031423, filed on August 29, 2023, entitled Compositions and Methods for Non-Genogenic Pretreatment, which is hereby incorporated herein by reference in its entirety. Technical Field

[0002] This document provides methods and compositions relating to the use of antibody compositions to deplete the hematopoietic stem cells of a subject. The methods and compositions disclosed herein can be used, for example, for non-myeloablative conditioning prior to hematopoietic stem cell transplantation (HSCT) (e.g., allogeneic or autologous HSCT). Background Technology

[0003] Throughout an individual's life, the production of hematopoietic cells depends on a population of rare hematopoietic stem cells capable of self-renewal. Due to this unique nature, hematopoietic stem cell transplantation (HSCT) is an effective treatment with the potential to correct a variety of conditions, such as, but not limited to, hemoglobinopathies, autoimmune diseases, and hematologic malignancies. Prior to HSCT, recipients must undergo pretreatment aimed at: (1) resetting the immune system (in the case of non-autologous transplantation), (2) clearing the microenvironment, and (3) preparing the bone marrow microenvironment (niche) for donor cell transplantation so that the donor hematopoietic stem cells can rebuild the hematopoietic system. Traditional pretreatment protocols may involve the administration of chemotherapeutic agents, radiation, and / or immunosuppression. These methods are highly toxic in both the short and long term and can induce numerous life-threatening side effects, including hematologic malignancies, organ damage, organ failure, and infection (Gyurkocza et al., Blood (2014), 124:344-353), therefore there is a need for pretreatment regimens with less or no genotoxicity so that a wider range of patients can receive safer and more effective HSCT therapy.

[0004] Recent research has focused on developing pretreatment regimens that lack genotoxicity, including those using monoclonal antibodies that block hematopoietic stem cell survival factors, CAR T-mediated pretreatment, and antibody-drug conjugates (ADCs) (see, for example, Czechowicz et al., 318 (5854)). Science 1296-9 (2007); Arai et al., 26(5) Molecular Therapy 1181-1197 (2018); and Palchaudari et al., 34(7) Nature Biotechnology738-745 (2016). One such antibody-based approach targets CD117 to deplete hematopoietic stem cells. Although CD117 is highly expressed in hematopoietic stem cells and progenitor cells, strategies targeting CD117 alone are insufficient to prepare immune-active subjects for successful hematopoietic stem cell transplantation (see, for example, Xue et al., Blood 116, 5419-5422 (2010). Instead, anti-CD117 needs to be combined with CD47 blockade (see, for example, Chhabra et al., 10:8 (351)). Science Translational Medicine 351ra105 (2016)), or the CD117 antibody must be bound to the toxin to promote the depletion of endogenous hematopoietic stem cells and make it possible to transplant donor cells (see, for example, Czechowicz et al.). Nat Commun 10, 617 (2019). Therefore, there is a need for alternative antibody-based pretreatment regimens that can promote robust hematopoietic stem cell depletion and transplantation while significantly reducing the morbidity and mortality of HSCT. Summary of the Invention

[0005] This article provides methods and compositions relating to the depletion of endogenous hematopoietic stem cells in a subject by using anti-CD110 and anti-CD117 pretreatment agents (e.g., antibodies or antigen-binding fragments thereof) prior to HSCT. Cell-based therapeutic methods and compositions are also provided. While not intended to be bound by any particular operational theory, the examples provided below demonstrate that the concomitant targeting of CD110 and CD117 co-expressed on hematopoietic stem cells with antibodies utilizing Fc effector cell-mediated clearance leads to robust and synergistic depletion of endogenous hematopoietic stem cells and transplantation of donor hematopoietic stem cells, followed by multilineage reconstitution in immune-active mice. Because this non-genotoxic pretreatment does not require the use of non-selective myeloablative pretreatment agents (such as radiation or chemotherapy (e.g., 5-fluorouracil (5-FU))), the concomitant targeting of CD110 and CD117 has the potential to expand the use of hematopoietic stem cell transplantation therapy to a broader spectrum of patients across a variety of diseases and conditions.

[0006] Therefore, in one aspect, this document provides a method for depleting endogenous hematopoietic stem cells and / or hematopoietic pluripotent progenitor cells (HSPCs) in a subject, the method comprising administering a pharmaceutical composition to the subject comprising: (i) a first targeting portion specifically binding to CD117; and (ii) a second targeting portion specifically binding to CD110; wherein the first and second targeting portions comprise Fc regions capable of functionally binding to host FcRn and mediating effector functions in the subject. In some embodiments, the first and second targeting portions synergistically induce depletion of endogenous hematopoietic stem cells and / or hematopoietic pluripotent progenitor cells via Fc effector functions. In some embodiments, the first and second targeting portions bind to hematopoietic stem cells and / or hematopoietic pluripotent progenitor cells expressing both CD117 and CD110. In some embodiments, the hematopoietic stem cells expressing both CD117 and CD110 are long-term hematopoietic stem cells (LT-HSCs). In some embodiments, the first and second targeting portions do not contain toxins. In some embodiments, the subject is an immune-active, indicating a fully functional immune system. In other implementations, the subject is immune-impaired. In some implementations, the method does not include administering radiation or chemotherapy to the subject.

[0007] In some embodiments, the first targeting portion comprises a separable antibody or antigen-binding fragment thereof that specifically binds to CD117. In some embodiments, the separable antibody or antigen-binding fragment thereof that specifically binds to CD117 functionally disrupts signaling between stem cell factor (SCF) and CD117. In some embodiments, the second targeting portion comprises a separable antibody or antigen-binding fragment thereof that specifically binds to CD110. In some embodiments, the separable antibody or antigen-binding fragment thereof that specifically binds to CD110 functionally disrupts signaling between thrombopoietin (TPO) and CD110.

[0008] In some embodiments, the isolated antibodies for the first and / or second targeting moieties are monoclonal antibodies. In some embodiments, the antigen-binding fragments for the first and / or second targeting moieties are selected from: Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, scFv-Fc fragments, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (dsFv), fragments containing VL or VH domains, heavy chain antibodies (hcAb), single-domain antibodies (sdAb), microantibodies, and variable domains (VHH or nanobodies) derived from camel heavy chain antibodies.

[0009] In some embodiments, both the first and second targeting portions are contained on the same antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment is selected from: bifunctional antibodies (diabody), bifunctional antibody-Fc, single-chain bifunctional antibodies, tandem bifunctional antibodies (Tandab's), tandem scFv, tandem scFv-scFc, tandem di-scFv, tandem tri-scFv, multivalent antibodies, bivalent or bispecific single-chain variable fragments, bispecific IgG, and bispecific Fab-IgG.

[0010] In some embodiments, the isolated antibody or antigen-binding fragments of the first and / or second targeting portions are chimeric, humanized, or human. In some embodiments, the isolated antibody or antigen-binding fragments of the first and / or second targeting portions contain a human Fc region. In some embodiments, the target is a human being.

[0011] In another aspect, this document provides a method for hematopoietic stem cell transplantation in a desired subject, the method comprising: (a) depleting endogenous hematopoietic stem cells and / or hematopoietic pluripotent progenitor cells in the subject according to any of the HSPC depletion methods described herein; and (b) administering exogenous hematopoietic stem cells to the subject. In some embodiments, the administration of effective amounts of first and second targeting portions synergistically mediates the transplantation of exogenous hematopoietic stem cells in the subject. In some embodiments, the administration of exogenous hematopoietic stem cells to the subject results in at least 10% donor cell chimerism. In some embodiments, the donor cell chimerism is at least 55%. In some embodiments, the transplantation of exogenous hematopoietic stem cells results in multilineage reconstitution in the subject.

[0012] In some embodiments, the HSC transplantation method provided herein further includes monitoring the depletion of the subject's endogenous hematopoietic stem cells and / or hematopoietic pluripotent progenitor cells prior to administration of exogenous hematopoietic stem cells. In some embodiments, exogenous hematopoietic stem cells are administered to the subject after the first and second target portions have been substantially cleared from the subject's blood. In some embodiments, the administration of exogenous hematopoietic stem cells to the subject occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 days or more following co-administration of the first and second target portions to the subject.

[0013] In some embodiments, the exogenous hematopoietic stem cells are allogeneic hematopoietic stem cells. In some embodiments, the exogenous hematopoietic stem cells are autologous hematopoietic stem cells. In some embodiments, the exogenous hematopoietic stem cells include CD34+ hematopoietic stem cells and progenitor cells (HSPCs). In some embodiments, CD34+ HSPCs include CD34+ / CD38- / CD90+ HSPCs. In some embodiments, CD34+ HSPCs include CD34+ / CD38- / CD90+ / CD45RA- HSPCs.

[0014] In some embodiments, the HSC transplantation method provided herein further includes one or more of the following steps: (a) collecting hematopoietic stem cell populations from the subject before depletion; (b) culturing the collected hematopoietic stem cell populations; and (c) cryopreserving the collected hematopoietic stem cell populations. In some embodiments, collecting hematopoietic stem cell populations from the subject includes one or more of the following steps: (a) mobilizing the hematopoietic stem cell populations; and (b) collecting the hematopoietic stem cell populations via apheresis.

[0015] In some embodiments of the HSC transplantation methods provided herein, the exogenous hematopoietic stem cells are genetically modified. In some embodiments, the exogenous hematopoietic stem cells are genetically modified using one or more components of a gene editing system. In some embodiments, one or more components of the gene editing system are selected from: (i) CRISPR / Cas guide RNA, (ii) a DNA molecule encoding CRISPR / Cas guide RNA, (iii) a nucleic acid molecule encoding a CRISPR / Cas RNA guide polypeptide, (iv) a CRISPR / Cas RNA guide polypeptide, (v) a CRISPR / Cas guide RNA complexed with a CRISPR / Cas RNA guide polypeptide, (vi) a nucleic acid molecule encoding a zinc finger protein (ZFP), (vii) ZFP, (viii) a nucleic acid molecule encoding a transcription activator-like effector (TALE) protein, (ix) a TALE protein, and (x) a DNA donor polynucleotide.

[0016] In some embodiments, the CRISPR / Cas RNA-guided polypeptide is a base editor or a prime editor. In some embodiments, one or more components of the gene editing system contain a nuclease capable of generating double-strand breaks within a cellular locus. In some embodiments, one or more components of the gene editing system also contain a DNA donor polynucleotide. In some embodiments, the DNA donor polynucleotide contains non-overlapping 5' and 3' homologous arms, each homologous to a portion of the locus, so that once a double-strand break is generated within the locus by the nuclease, the donor polynucleotide sequence can be integrated into the locus via homology-directed repair (HDR). In some embodiments, the gene editing system contains a CRISPR nuclease and a single guide RNA (sgRNA) capable of hybridizing to a target sequence within the locus, wherein the sgRNA guides the CRISPR nuclease to the target sequence. In some embodiments, the CRISPR nuclease is a Cas protein. In some embodiments, the sgRNA and CRISPR nuclease are formed in a ribonucleoprotein (RNP) complex. In some implementations, gene modification corrects gene mutations, replaces mutant alleles carrying wild-type alleles, or inserts nucleic acid sequences encoding therapeutic proteins.

[0017] In some embodiments of the HSC depletion and transplantation methods provided herein, the subject suffers from a disease. In some embodiments, the disease is hemoglobinopathies. In some embodiments, the hemoglobinopathies are selected from sickle cell disease, alpha-thalassemia, beta-thalassemia, and delta-thalassemia.

[0018] On the other hand, this document provides compositions and kits comprising an antibody specifically binding to CD117 or an antigen-binding fragment thereof; an antibody specifically binding to CD110 or an antigen-binding fragment thereof; hematopoietic stem cells; and / or instructions for preparation or use thereof according to the methods described herein. The compositions, kits, and methods described herein can be used, for example, to treat cancer, autoimmune diseases, viral diseases, and blood disorders, as well as to induce tolerance. Attached Figure Description

[0019] Figure 1 Sensor diagrams depicting antibody binding to mouse CD110 or mouse CD117, as measured by ForteBio Octet. (A) Anti-mCD110 antibody binding to the extracellular domain (ECD) of recombinant mouse CD110 and (B) Anti-mCD117 antibody binding to the ECD of recombinant mouse CD117.

[0020] Figure 2A schematic diagram of a research design scheme for pretreatment of recipients with anti-mCD117 and anti-mCD110 antibodies is shown.

[0021] Figure 3 The study depicted the total chimerism of donor-derived hematopoietic cells in peripheral blood at (A) 4, 8, 12, and 16 post-transplantation after antibody-based pretreatment. (B) Gr-1 + Mac-1 + Myeloid cells, (C)CD19 + B cells, (D)CD3 + T cells and (E)NK1.1+ NK cells are donor-derived blood chimerism.

[0022] Figure 4 The total chimerism of donor-derived hematopoietic cells in the bone marrow 16 weeks post-transplantation is described. (A) Lin-CD117+Sca1+ (“LSK”) cells, (B) Lin - CD117 + Sca1 + SLAM + Flt3 - (LT-HSC) cells, (C) common myeloid progenitor cells ("CMP"): Lin - CD117 + Sca1 - CD16 / 32 - CD34 + (D) Granulocyte-monocyte progenitor cells (“GMP”: Lin - CD117 + Sca1 - CD16 / 32 + CD34 + (E) Megakaryocyte-erythrocyte progenitor cells ("MEP": Lin) - CD117 + Sca1 - CD16 / 32 - CD34 - (F) Common lymphoid progenitor cells (“CLP”: Lin) - CD117 + Sca1 + CD127 + () donor source bone marrow chimerism.

[0023] Figure 5 The study depicted the effects of antibody-based pretreatment with antibodies of different Fc forms 16 weeks post-transplantation on Lin. - CD117 + Sca1 +(LSK) cells and Lin - CD117 + Sca1 + SLAM + Flt3 - (“LT-HSC”) cell donor-derived hematopoietic chimerism. (A) In a regimen with G2a isotype mouse Fc carrying anti-mCD117 and anti-mCD110 antibodies, chimerism of donor-derived LSK (HSPC) and LT-HSC populations after antibody-based pretreatment. (B) In a regimen combining ACK2 (anti-mCD117 rat IgG2b) and AMM2 (anti-mCD110 rat IgG1) or anti-mCD110 and anti-mCD117 with mouse IgG2a Fc carrying a mutation (N297A) that reduces binding to the Fc γ receptor, chimerism of donor-derived LSK and LT-HSC populations after antibody-based pretreatment.

[0024] Figure 6 CD117 and CD110 receptor counts in the bone marrow of C57BL / 6J (“B6”) mice were depicted. (A) In Lin - and Lin - CD117 + Sca1 + (B) Median CD117 expression is shown in Lin-, LSK, LT-HSC, and common myeloid progenitor cells ("CMP"). - CD117 + Sca1 - CD16 / 32 - CD34 + ), granulocyte-monocyte progenitor cells (“GMP”: Lin - CD117 + Sca1 - CD16 / 32 + CD34 + Megakaryocyte-erythrocyte progenitor cells ("MEP") - CD117 + Sca1 - CD16 / 32 - CD34 - ) and common lymphoid progenitor cells (“CLP”: Lin - CD117 + Sca1 + CD127 + (C) The median expression of CD110 is shown in Lin. -LSK, LT-HSC, CMP, GMP, MEP and CLP groups.

[0025] Figure 7 illustrates (A) a schematic diagram of a study design evaluating HSC / HSPC depletion with anti-mCD117 and anti-mCD110 antibodies. (B) Total colony-forming units (CFU) formed by animal-derived HSPCs on days 7, 9, and 12 post-treatment, treated with a combination of anti-mCD117 and anti-mCD110 antibodies, anti-mCD117 antibody alone, or an allotype control. The graph represents the quantification of CFUs derived from triplicate plates, each of which was drawn from 5 animals in each treatment group, while the photograph depicts representative plates. (C) Lin-CD117+Sca1+ (“LSK”) cells and Lin... - CD117 + Sca1 + SLAM + Flt3 - (D) Quantification of the frequency and absolute count of (LT-HSC) cells. Total chimerism and lineage-specific chimerism of donor cells in peripheral blood at 8 weeks post-transplantation. Donor cells derived from bone marrow of animals treated with a combination of anti-mCD117 and anti-mCD110 antibodies, anti-CD117 antibody alone, or allotype control obtained at 7, 9, and 12 days post-transplantation.

[0026] Figure 8 Analysis of CD117 and CD110 expression in human bone marrow mononuclear cells isolated from bone marrow aspirates is provided. Sample gating was used to evaluate LT-HSCs (Lin-CD34+CD38-CD45RA-CD90+CD49f+) that showed CD117 and CD110 expression. Detailed Implementation

[0027] definition Unless otherwise defined herein, scientific and technical terms relating to this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definition. Furthermore, unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. In this application, unless otherwise stated, the use of “or” means “and / or”. Moreover, the use of the term “including” and other forms such as “includes” and “included” is not restrictive.

[0028] In general, the nomenclature and techniques described herein related to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques disclosed herein are generally performed according to conventional methods well-known in the art and as described in the various general and more specific references cited and discussed throughout this specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as is commonly done in the art or as described herein. The nomenclature, laboratory procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical and pharmaceutical chemistry described herein are well-known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and patient treatment.

[0029] As used herein, unless the context clearly indicates otherwise, the singular forms “a”, “an”, and “the” include plural referents.

[0030] The terms "about" and "approximately" indicate and encompass the indicated value as well as the range above and below that value. In some embodiments, the term "about" indicates a range within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of a given value or range. In some embodiments, the term "about" indicates a specified value ± one standard deviation of that value.

[0031] The term "the combination thereof" includes every possible combination of the elements that the term refers to.

[0032] The terms “CD110,” “c-MPL,” and “MPL” are used interchangeably in this document. CD110 is also referred to as a synonym, including the thrombopoietin receptor and myeloproliferative leukemia protein, among others. Unless otherwise stated, these terms include those naturally expressed by cells or derived from [specific sources]. c-MPL Any variant, isoform, and species homolog of human CD110 expressed in transfected cells. CD110 proteins include, for example, human CD110 (NCBI reference sequence: NP_005364.1). c-MPL Genes include, for example, the Homo sapiens MPL proto-oncogene, the thrombopoietin receptor (MPL), and the RefSeqGene (LRG_510) on chromosome 1 (NCBI reference sequence: NG_007525.1).

[0033] The terms “CD117” and “c-KIT” are used interchangeably in this document. CD117 is also referred to by synonyms including tyrosine protein kinase KIT and mast cell / stem cell growth factor receptor (SCFR), among others. Unless otherwise stated, these terms include those naturally expressed by cells or those produced by [unclear text - likely a tyrosine protein kinase]. c-KIT Any variant, isoform, and species homolog of human CD117 expressed in transfected cells. CD117 proteins include, for example, human CD117 (NCBI reference sequences: NP_000213.1; and NP_001087241.1). c-KIT Genes include, for example, the Homo sapiens KIT proto-oncogene, receptor tyrosine kinase (KIT), and RefSeqGene (LRG_307) on chromosome 4 (NCBI reference sequence: NG_007456.1).

[0034] The term "immunoglobulin" refers to a class of structurally related proteins that generally consist of two pairs of polypeptide chains: a pair of light (L) chains and a pair of heavy (H) chains. In a "complete immunoglobulin," all four chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Paul, Fundamental Immunology 7th Edition, Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. In short, each heavy chain typically contains a heavy chain variable region (V... H ) and heavy chain constant region (C H The heavy-chain constant region typically contains three structural domains, abbreviated as C. H1 C H2 and C H3 Each light chain typically contains a light chain variable region (V). L The light chain constant region typically contains a structural domain, abbreviated as C. L .

[0035] The term "antibody" describes a class of immunoglobulin molecules, and is used herein in its broadest sense. Antibodies specifically include complete antibodies (e.g., complete immunoglobulins) and antibody fragments. An antibody contains at least one antigen-binding domain. An example of an antigen-binding domain is formed by V... H -V L Antigen-binding domains formed by dimers. Antibodies described herein can be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific to different epitopes of a single target polypeptide, or may contain antigen-binding domains specific to more than one target polypeptide. See, for example, Tutt et al., (1991). J. Immunol. 147:60-69; Kufer et al., (2004). Trends Biotechnol.22:238-244; and Brinkmann and Kontermann, (2017). MABS, 9(2):182-212. The anti-CD110 and / or anti-CD117 antibodies described herein can be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical conjugation, gene fusion, non-covalent association, or other means) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody having a second binding specificity. In some embodiments, the bispecific or multispecific antibodies described herein contain binding specificity for both CD110 and CD117. In some embodiments, the multispecific antibodies described herein contain binding specificity for both CD110 and CD117.

[0036] "Antibody fragments" include portions of a complete antibody, such as the antigen-binding region or variable region of the complete antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, F(ab') fragments, scFv (sFv) fragments, scFv-Fc fragments, and nanobody fragments.

[0037] The “Fv” fragment is a non-covalently linked dimer of a heavy chain variable domain and a light chain variable domain.

[0038] In addition to the variable structural domains of the heavy and light chains, the “Fab” segment also contains the constant structural domain of the light chain and the first constant structural domain (C) of the heavy chain. H1 Fab fragments can be generated, for example, through recombinant methods or by digesting full-length antibodies with papain.

[0039] The “F(ab’)2” fragment contains two Fab’ fragments that are joined together near the hinge region by disulfide bonds. The F(ab’)2 fragment can be generated, for example, by recombinant methods or by digesting an intact antibody with pepsin. The F(ab’) fragment can be dissociated, for example, by treatment with β-mercaptoethanol.

[0040] A "single-chain Fv" or "sFv" or "scFv" antibody fragment contains V within a single polypeptide chain. H Domain and V L Domain. V H and V L They are generally linked via peptide linkers. See also A. (1994).

[0041] The “scFv-Fc” segment contains an scFv that is attached to the Fc structure field. For example, the Fc structure field can be attached to the C end of the scFv. The Fc structure field can be located at the V end. Hor V L Then, depending on the orientation of the variable structural domain in scFv (i.e., V) H V L or V L V H Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain contains the IgG1Fc domain.

[0042] Nanobody fragments contain only the variable domain of the heavy chain and lack the constant domains of the light and heavy chains. In some cases, nanobodies can be conjugated with other nanobodies and / or proteins to form multispecific proteins.

[0043] The antibodies described herein may also include other antibody variants, such as bifunctional antibodies, bifunctional antibody-Fc, single-chain bifunctional antibodies, tandem bifunctional antibodies (Tandab's), tandem scFv, tandem scFv-scFc, tandem di-scFv, tandem tri-scFv, "multivalent antibodies" (e.g., trivalent or quadrivalent antibodies), and bivalent or bispecific single-chain variable fragments, including bispecific IgG and bispecific Fab-IgG. Bi-scFv or di-scFv variants can be engineered by linking two scFv molecules with a linker. Bispecific antibodies may comprise two scFv molecules ((scFv)2) with different binding specificities. Linkage can be performed by creating a single peptide chain with two VH regions and two VL regions, resulting in a tandem scFv (see, for example, Kufer P. et al. (2004) Trends in Biotechnology 22(5):238-244). Bifunctional antibodies can be generated from scFv molecules with a linker peptide that is too short for the two variable regions to fold together (e.g., about 5 amino acids), thus forcing scFv dimerization. See, for example, Hollinger, Philipp et al. (July 1993) Proceedings of the National Academy of Sciences of the United States of America 90(14): 6444-8). Successfully purified multi-target affinities can be screened using a variety of in vitro and in vivo methods. Multi-target affinities that bind favorably to cells expressing both CD110 and CD117 can be screened using binding assays with engineered cell lines overexpressing only CD110 or CD117, or with variable combinations. Cells can be incubated with the multi-target affinity and then subjected to a fluorescently labeled secondary antibody. Flow cytometry can be used to detect antibody binding levels to engineered cells. Multi-target affinities are expected to bind favorably to cells simultaneously co-expressing both CD117 and CD110, thus confirming their bispecificity. If engineered cell lines are labeled using multiple methods, such as co-expressing fluorescent proteins (GFP, YFP, EBFP, etc.) as well as CD110 and CD117, flow cytometry can be used to track these lines. Alternatively, CellTrace proliferation dyes can be used to individually stain cells overexpressing the target receptor to label and monitor the binding of multi-target affinities. In addition to engineered cell lines, multi-target affinities can be tested on primary cells with known levels of the target receptor to confirm binding against the relevant cell type.

[0044] The term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies. A substantially homogeneous group of antibodies comprises antibodies that are substantially similar and bind to the same epitopes, in addition to variants that can be normally produced during monoclonal antibody production. Such variants generally exist only in small quantities. Monoclonal antibodies are typically obtained through a process involving the selection of a single antibody from a plurality of antibodies. For example, the selection process may involve choosing a unique clone from a pool of multiple clones, such as hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody may be further modified, for example, to increase its affinity for the target ("affinity maturation"), to humanize the antibody, to increase its yield in cell culture, and / or to reduce its immunogenicity in the target.

[0045] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a specific source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0046] A “humanized” form of a nonhuman antibody is a chimeric antibody containing a minimal sequence derived from a nonhuman antibody. A humanized antibody is generally a human immunoglobulin (receptor antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs from a nonhuman antibody (donor antibody). The donor antibody can be any suitable nonhuman antibody, such as mouse, rat, rabbit, chicken, or nonhuman primate antibodies with the desired specificity, affinity, or biological effect. In some cases, selected frame region residues of the recipient antibody are replaced by corresponding frame region residues from the donor antibody. Humanized antibodies may also contain residues not found in either the recipient or donor antibody. Such modifications can be performed to further improve antibody function. For more details, see Jones et al. Nature , 1986, 321:522-525; Riechmann et al., Nature ,1988,332:323-329; and Presta, Curr. Op. Struct. Biol. , 1992, 2:593-596, each of which is incorporated in its entirety by reference.

[0047] "Human antibody" refers to an antibody whose amino acid sequence corresponds to that of an antibody produced by a human or human cell, or is derived from a non-human antibody library or a human antibody coding sequence (e.g., obtained from a human source or designed de novo). Human antibodies explicitly exclude humanized antibodies.

[0048] "Isolated antibody" is an antibody that has been isolated and / or recovered from its native environmental components. Native environmental components may include enzymes, hormones, and other protein- or non-protein substances. In some embodiments, the isolated antibody is purified to a degree sufficient to obtain at least 15 N-terminal or internal amino acid sequence residues, for example, by using a rotary cup sequencer. In some embodiments, the isolated antibody is purified to homogeneity by gel electrophoresis (e.g., SDS-PAGE) under reducing or non-reducing conditions and detected by Coomassie brilliant blue or silver staining. Isolated antibodies include in situ antibodies from recombinant cells, since at least one component of the antibody's native environment is absent. In some aspects, the isolated antibody is prepared by at least one purification step.

[0049] In some embodiments, the isolated antibody is purified to at least 80%, 85%, 90%, 95%, or 99% by weight. In some embodiments, the isolated antibody is purified to at least 80%, 85%, 90%, 95%, or 99% by volume. In some embodiments, the isolated antibody is provided as a solution containing at least 85%, 90%, 95%, 98%, 99%, or 100% by weight. In some embodiments, the isolated antibody is provided as a solution containing at least 85%, 90%, 95%, 98%, 99%, or 100% by volume.

[0050] "Affinity" refers to the total strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, "binding affinity" as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can be expressed using the dissociation constant (K0). D Affinity can be measured using methods commonly known in the art, including those described herein. For example, affinity can be measured using surface plasmon resonance (SPR) techniques such as Biacore. The affinity is determined using instruments. In some implementations, affinity is determined at 25°C.

[0051] Regarding antibody binding to target molecules, the terms "specific binding," "specifically binds to," "specific for," "selectively binds," and "selective for" refer to a specific antigen (e.g., CD110 or CD117) or an epitope on a specific antigen, meaning a binding with a measurable difference from nonspecific or nonselective interactions. Specific binding can be measured, for example, by comparing the binding of a determining molecule to the binding of a control molecule. Specific binding can also be determined by competition with a control molecule at an antibody binding site on a simulated target. In this case, if the binding of the antibody to the target is competitively inhibited by the control molecule, specific binding is indicated. In some embodiments, "selective binding" refers to the ability of a selectively binding compound (e.g., an antibody or its antigen-binding fragment) to bind to a target protein (e.g., CD110 or CD117) with a higher affinity than its binding to non-target proteins. In some implementations, specific binding means that the binding to a target has an affinity that is at least 10, 50, 100, 250, 500, 1000 or more times higher than the affinity for a non-target.

[0052] As used herein, “functionally disrupted” or “functional disruption” refers to signal transduction between stem cell surface receptors (e.g., CD110 or CD117) and their homologous ligands (e.g., thrombopoietin or stem cell factor, respectively), meaning a weakening of the interaction between the receptor and ligand that would otherwise result in reduced normal biological activity (e.g., hematopoietic stem cell proliferation). In some embodiments, normal biological activity is eliminated. In some embodiments, functional disruption is achieved by an antibody or its antigen-binding fragment binding to the receptor or ligand and blocking or inhibiting the binding of the ligand to the receptor, and / or antagonizing the function of the ligand or receptor, preventing normal signal transduction between the ligand and receptor. In other embodiments, functional disruption is achieved through mechanisms other than direct binding or direct inhibition of the receptor or ligand. For example, functional disruption can be achieved by binding to and / or inhibiting cofactors, upstream signaling molecules, or downstream signaling molecules of the receptor or ligand, which may be, for example, necessary for effective signal transduction between the ligand and receptor. In some embodiments, functional impairment means a reduction in the binding or signaling between the receptor and its homologous ligand by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% relative to receptor-ligand signaling under physiological conditions. Any method known in the art that can be used to assess the biological activity resulting from signaling between the receptor and its homologous ligand can be used to assess functional impairment, including but not limited to cell proliferation assays and receptor competition assays. In other embodiments of the methods provided herein, the binding of an antibody or its antigen-binding fragment to a target protein does not functionally impair signaling but rather promotes immune-mediated depletion of such antibody-binding cells, for example, through ADCC, ADCP, or CDC.

[0053] As used herein, the term "synergy," referring to, for example, the depletion of endogenous hematopoietic stem cells in a subject and / or the transplantation of exogenous hematopoietic stem cells, means that the combination of pretreatment agents described herein (e.g., using anti-CD110 and anti-CD117 antibodies) is more effective than the additive effect of a single pretreatment agent. For example, the synergistic effect of antibody combinations allows for the use of one or more antibodies at lower doses and / or at lower frequencies of administration to the subject. The ability to utilize lower doses of antibodies and / or administer them at lower frequencies reduces the toxicities associated with administering the pretreatment agent to the subject without diminishing the efficacy of the pretreatment agent in depleting endogenous hematopoietic stem cells and transplanting exogenous hematopoietic stem cells. Furthermore, the synergistic effect can improve the efficacy of subsequent HSCT therapy in preventing, controlling, treating, or improving a given disease, such as hemoglobinopathies. Additionally, the synergistic effect of pretreatment agent combinations can avoid or reduce adverse or unwanted side effects associated with the use of any single pretreatment agent.

[0054] As used herein, the terms “subject,” “individual,” or “patient” are used interchangeably and refer to a warm-blooded animal, such as a mammal. In a particular embodiment, the term refers to a human being. A subject may have, be suspected of having, or be susceptible to a disease or condition to which HSCT may be beneficial (e.g., hemoglobinopathies). The term also includes livestock, pet animals, or animals used for research, including horses, cattle, sheep, poultry, pigs, cats, dogs, zoo animals, goats, primates (e.g., cynomolgus monkeys or rhesus monkeys), and rodents (e.g., mice and rats). A “subject in need” is a subject who has one or more symptoms of a disease or condition, has been diagnosed with, or is suspected of having, a disease or condition that can be treated with and / or may potentially benefit from HSCT as described herein.

[0055] As used herein, the term "application" refers to a method of giving a subject a dose of a composition (e.g., an antibody and / or cell therapy composition). Methods of application can vary depending on various factors, such as the pharmaceutical composition being applied and the severity of the condition, disease, or symptom being treated.

[0056] The term “treating” or “treatment” means any of the following: improving one or more symptoms of a disease or condition; preventing the appearance of such symptoms before their onset; slowing or completely halting the progression of a disease or condition (e.g., by means of longer relapse intervals, slowing or halting the worsening of symptoms); facilitating the onset of remission; slowing irreversible damage caused by a disease or condition in its progressive-chronic phase (primary and secondary phases); delaying the onset of said progressive phase; or any combination thereof.

[0057] "Effective amount" means the amount of a compound or composition as disclosed herein that effectively achieves a particular biological, therapeutic, or preventative outcome. Such outcomes include, but are not limited to, depletion of hematopoietic stem cells, transplantation of exogenous hematopoietic stem cells, and treatment of the diseases or conditions disclosed herein as determined by any suitable method in the art.

[0058] Methods to deplete endogenous hematopoietic stem cells This article provides methods and compositions for depleting endogenous hematopoietic stem cells from the bone marrow microenvironment prior to HSCT using selective non-genotoxic pretreatment agents. As described herein, the ablation of endogenous hematopoietic stem cells can be achieved by concurrently targeting CD110 and CD117 with selective antibodies or antibody fragments (“anti-CD110 and anti-CD117 pretreatment agents”).

[0059] In the field of bone marrow transplantation, pretreatment has a priori induced depletion of HSCs / HSPCs from the bone marrow microenvironment, thereby creating space for transplanted cells. As shown in the examples provided herein, the combined use of antibodies targeting CD117 and CD110 leads to phenotypic loss of both HSCs and HSPCs from the bone marrow, functional depletion of HSPCs indicated by a reduced capacity to form colony-forming units, and functional depletion of HSCs indicated by a reduced ability to support hematopoietic reconstitution in secondary bone marrow transplantation. Simultaneous targeting of CD117 and CD110 with antibodies utilizing Fc effector function results in synergistic depletion of HSCs and HSPCs compared to depletion from targeting CD117 or CD110 alone. Following the ablation of endogenous HSCs and HSPCs and after the pretreatment agent has been substantially removed from the recipient's blood circulation, exogenous donor hematopoietic stem cells can be introduced to occupy the same microenvironment previously occupied by the ablated cells.

[0060] The following describes in detail anti-CD110 and anti-CD117 pretreatment agents that can be used in the methods provided herein. In some embodiments, the pretreatment regimen does not involve the use of high-dose non-selective myeloablative agents, such as radiotherapy or chemotherapy (e.g., 5-FU), and optimally avoids their associated toxicities, including myelosuppression, mucositis, and organ and tissue toxicities (e.g., to cells of the gastrointestinal system, hair growth), as well as the risk of secondary malignancies. In particular, the compositions and methods of this disclosure combine the non-genotoxic selective ablation of endogenous hematopoietic stem cells with the administration of exogenous donor hematopoietic stem cells (e.g., genetically modified hematopoietic stem cells) to the recipient, resulting in highly efficient, long-term transplantation, multilineage hematopoietic reconstitution, and immune activity.

[0061] Anti-CD110 and anti-CD117 pretreatment agents CD110 CD110 (c-MPL), also known as the thrombopoietin receptor, is a mediator of thrombopoietin signaling and plays a crucial role in maintaining a quiescent, long-term hematopoietic stem cell population within the bone marrow microenvironment. Thrombopoietin-CD110 signaling stimulates megakaryocyte and platelet production and directly regulates hematopoietic stem cell proliferation, as both thrombopoietin and CD110 gene knockout mice exhibit severe hematopoietic stem cell loss. See, for example, Solar et al. Blood , 92 (1998), pp. 4-10; Yoshihara et al., Cell Stem Cell , 1 (2007), pp. 685-697; Qian et al., Cell Stem Cell , 1 (2007), pp. 671-684; and Nakamura-Ishizu and Suda, Ann. NY Acad. Sci. 1466 (2020), pp. 51-58.

[0062] Anti-CD110 pretreatment agents that can be used to practice the methods provided herein include antibodies that specifically bind to CD110 and their antigen-binding fragments. In some embodiments, the available anti-CD110 antibodies and their antigen-binding fragments are capable of functionally disrupting thrombopoietin-CD110 signaling. In other embodiments, the available anti-CD110 antibodies and their antigen-binding fragments do not functionally disrupt thrombopoietin-CD110 signaling. In some embodiments, the anti-CD110 pretreatment agent is an isolated monoclonal antibody that specifically binds to CD110. In some embodiments, the anti-CD110 pretreatment agent is an isolated bispecific antibody that specifically binds to CD110 and also specifically binds to a second antigen. In some embodiments, the second antigen is CD117. In some embodiments, the anti-CD110 pretreatment agent is an isolated antigen-binding fragment that specifically binds to CD110. In some embodiments, the isolated antigen-binding fragment that specifically binds to CD110 is selected from Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments. In some embodiments, the antibody that specifically binds to CD110 or its antigen-binding fragment is selected from bifunctional antibodies, bifunctional antibody-Fc, single-chain bifunctional antibodies, tandem bifunctional antibodies (Tandab's), tandem scFv, tandem scFv-scFc, tandem di-scFv, tandem tri-scFv, multivalent antibodies, bivalent or bispecific single-chain variable fragments, bispecific IgG, bispecific Fab-IgG, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (dsFv), fragments containing VL or VH domains, heavy chain antibodies (hcAb), single-domain antibodies (sdAb), microantibodies, and variable domains (VHH or nanobodies) derived from camel heavy chain antibodies. Other available antibody or antigen-binding fragment forms include Wilkinson & Hale (2022). mAbs The form described in , 14:1, DOI: 10.1080 / 19420862.2022.2123299. Suitable anti-CD110 pretreatment agents include fully human, humanized, or chimeric antibodies that specifically bind to CD110. Humanized antibodies are particularly suitable for in vivo application in humans due to their low antigenicity. Similarly, canine, feline, and other antibodies are particularly suitable for application in dogs, cats, and other species, respectively.

[0063] In certain embodiments, the anti-CD110 pretreatment agent is an anti-CD110 antibody or its antigen-binding fragment, which contains an Fc domain capable of binding to the host species' neonatal Fc receptor (FcRn). The FcRn functions as a circulating receptor or transcytosis receptor, responsible for maintaining IgG and albumin in the bloodstream and bidirectionally transporting these two ligands across polarized cell barriers. Therefore, the binding of the Fc domain of the anti-CD110 antibody to the recipient's FcRn can confer pharmacokinetics and half-life similar to those of the recipient's innate immunoglobulin (IgG). In some embodiments, similar effector functions, such as ADCC and ADCP functions and complement binding, are also conferred. In some such embodiments, the binding affinity of the Fc domain of the anti-CD110 pretreatment agent to the recipient's FcRn is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the binding affinity of the recipient's innate immunoglobulin (IgG) to its FcRn. In some embodiments, the anti-CD110 antibody is a human antibody, a humanized antibody, or a human chimeric antibody that contains an Fc domain (e.g., a human Fc domain) capable of binding to a human recipient's FcRn. In some such embodiments, the binding affinity of the Fc domain of the human antibody, humanized antibody, or human chimeric antibody to the human FcRn is at least within about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the binding affinity of the human recipient's innate immunoglobulin (IgG) to its FcRn. In other embodiments, the anti-CD110 antibody is a mouse antibody, a murine-derived antibody, or a murine chimeric antibody that contains an Fc domain (e.g., a mouse Fc domain) capable of binding to the recipient's mouse FcRn. In some embodiments, the anti-CD110 antibody induces Fc effector-mediated clearance of the recipient's HSCs and / or HSPCs. In some embodiments, the HSCs are LT-HSCs. Fc-mediated antibody effector functions may include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0064] In other embodiments, the anti-CD110 pretreatment agent is an anti-CD110 antibody or an antigen-binding fragment thereof comprising an Fc domain that has reduced binding affinity to the recipient's FcRn. In some such embodiments, the binding affinity of the Fc domain of the anti-CD110 pretreatment agent to the recipient's FcRn is less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the binding affinity of the recipient's innate immunoglobulin (IgG) to its FcRn. In some embodiments, the Fc domain of the anti-CD110 pretreatment agent is engineered to reduce effector function, such as reducing or ablating ADCC and ADCP function and complement binding. In some embodiments, the anti-CD110 pretreatment agent is an anti-CD110 antibody or an antigen-binding fragment thereof comprising an Fc domain with reduced binding affinity to one or more recipients' Fcγ receptors.

[0065] Non-limiting examples of suitable anti-CD110 antibodies include clones mAb-1.75, mAb-1.6, and mAb-1.111 (e.g., described in International Patent Publication No. WO 2011 / 060076, which is incorporated herein by reference in its entirety); MAb1.6.1 (C. Abbott et al., Hybridoma (Larchmt) , 29 (2010), pp. 103-113; and AMM2 (Yoshihara et al., Cell Stem Cell , 1 (2007), pp. 685-697; IBL-America (Immuno-Biological Laboratories). In some embodiments, the method includes using an anti-CD110 antibody comprising the heavy chain and light chain complementarity-determining regions (CDRs) of any of these antibodies. In some embodiments, the method includes using an anti-CD110 antibody comprising the three heavy chain CDRs and three light chain CDRs of any of these antibodies. In some embodiments, the method includes using an anti-CD110 antibody comprising the three heavy chain CDRs, three light chain CDRs, and a framework region of any of these antibodies. In some embodiments, the method includes using an anti-CD110 antibody comprising the variable heavy chain (V) of any of these antibodies. H ) and variable light chains (V LIn some embodiments, the anti-CD110 antibody is a chimeric human antibody. In some embodiments, the anti-CD110 antibody is humanized. In some embodiments, the anti-CD110 antibody is a human antibody. In some embodiments, the method includes using an anti-CD110 antibody comprising: (1) a heavy chain and light chain complementarity-determining region (CDR) of any of the above-described anti-CD110 antibodies; and (2) a human constant domain. In some embodiments, the method includes using an anti-CD110 antibody comprising: (1) three heavy chain CDRs and three light chain CDRs of any of the above-described anti-CD110 antibodies; and (2) a human constant domain. In some embodiments, the method includes using an anti-CD110 antibody comprising: (1) three heavy chain CDRs, three light chain CDRs, and a frame region of any of the above-described anti-CD110 antibodies; and (2) a human constant domain. In some embodiments, the method includes using an anti-CD110 antibody comprising: (1) a variable heavy chain (V110) of any of the above-described anti-CD110 antibodies. H ) and variable light chains (V L (1) and (2) human constant domain. Anti-CD110 antibodies can be any of the forms described herein.

[0066] In some embodiments, the anti-CD110 pretreatment agent is conjugated with a toxin. The anti-CD110 antibody-drug conjugate (ADC) is internalized upon binding to CD110 and its toxic payload is applied to ablate hematopoietic stem cells. In some embodiments, the toxin is selected from: saporin, saporin derivative, ricin, abrin, gelonin, momordin, apitoxin, shiga toxin, shiga-like toxin, T-2 mycotoxin, diphtheria toxin, busulfan, pseudomonas exotoxin A, ricin A chain derivative, trichosanthin, and luffin toxin. toxin, maytansine, amatoxin, mechlorethamine, cyclophosphamide, ethylenimine, methylmelamine, methotrexate, fluorouracil, fluxuridine, cytarabine, mercaptopurine, azathioprine, thioguanine, fludarabine phosphate, cladribine, dolastatin, auristatin, auristatin E, auristatin F F), MMAF, MMAE, MMAD, DMAF or DMAE, maytansine, DM1 or DM4, duocarmycin, calicheamicin, pyrrolobenzodiazepine, exatecan, and any combination thereof. In other embodiments, the anti-CD110 pretreatment agent is not conjugated with a toxin. In some embodiments, the anti-CD110 pretreatment agent is not an antibody-drug conjugate.

[0067] CD117 CD117 (c-Kit) is highly expressed in hematopoietic stem cells, pluripotent progenitor cells (MPPs), and lineage-restricted progenitor cells (such as common myeloid progenitor cells (CMPs), granulocyte-macrophage progenitor cells (GMPs), megakaryocyte-erythroid progenitor cells (MEPs), and common lymphoid progenitor cells (CLPs)), and is essential for hematopoiesis along with its ligand, stem cell factor (SCF). When CD117 binds to SCF, it forms a dimer, thereby activating its intrinsic tyrosine kinase activity, which in turn phosphorylates and activates signal transduction molecules that propagate signals within the cell. The signaling transmitted via CD117 after interaction with SCF is crucial for the survival, proliferation, and differentiation of hematopoietic stem cells. (See, for example, Edling and Hallberg, Int J Biochem Cell Biol. (2007), 39(11): 1995-1998; and Domen and Weissman, J Exp Med. (2000), 192(12): 1707-1718.

[0068] Anti-CD117 pretreatment agents that can be used to practice the methods provided herein include antibodies that specifically bind to CD117 and their antigen-binding fragments. In some embodiments, the available anti-CD117 antibodies and their antigen-binding fragments are capable of functionally disrupting SCF-CD117 signaling. In other embodiments, the available anti-CD110 antibodies and their antigen-binding fragments do not functionally disrupt SCF-CD117 signaling. In some embodiments, the anti-CD117 pretreatment agent is an isolated monoclonal antibody that specifically binds to CD117. In some embodiments, the anti-CD117 pretreatment agent is an isolated bispecific antibody that specifically binds to CD117 and also specifically binds to a second antigen. In some embodiments, the second antigen is CD110. In some embodiments, the anti-CD117 pretreatment agent is an isolated antigen-binding fragment that specifically binds to CD117. In some embodiments, the isolated antigen-binding fragment that specifically binds to CD117 is selected from Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments. In some implementations, the antibody or antigen-binding fragment that specifically binds to CD117 is selected from bifunctional antibodies, bifunctional antibody-Fc, single-chain bifunctional antibodies, tandem bifunctional antibodies (Tandabs), tandem scFv, tandem scFv-scFc, tandem di-scFv, tandem tri-scFv, multivalent antibodies, bivalent or bispecific single-chain variable fragments, bispecific IgG, bispecific Fab-IgG, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (dsFv), fragments containing VL or VH domains, heavy chain antibodies (hcAb), single-domain antibodies (sdAb), microantibodies, and variable domains (VHH or nanobodies) derived from camel heavy chain antibodies. Other available forms of antibody or antigen-binding fragments include Wilkinson & Hale (2022). mAbs The form described in , 14:1, DOI: 10.1080 / 19420862.2022.2123299. Suitable anti-CD117 pretreatment agents include fully human, humanized, or chimeric antibodies that specifically bind to CD117. Humanized antibodies are particularly suitable for in vivo human applications due to their low antigenicity. Similarly, canine, feline, mouse, and other antibodies are particularly suitable for applications in dogs, cats, and other species, respectively.

[0069] In certain embodiments, the anti-CD117 pretreatment agent is an anti-CD117 antibody or its antigen-binding fragment, which contains an Fc domain capable of binding to the host species neonatal Fc receptor (FcRn). The FcRn functions as a circulating receptor or transcytosis receptor, responsible for maintaining IgG and albumin in the bloodstream and bidirectionally transporting these two ligands across polarized cell barriers. Therefore, the binding of the Fc domain of the anti-CD117 antibody to the recipient's FcRn can confer pharmacokinetics and half-life similar to those of the recipient's innate immunoglobulin (IgG). In some embodiments, similar effector functions, such as ADCC and ADCP functions and complement binding, are also conferred. In some such embodiments, the binding affinity of the Fc domain of the anti-CD117 pretreatment agent to the recipient's FcRn is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the binding affinity of the recipient's innate immunoglobulin (IgG) to its FcRn. In some embodiments, the anti-CD117 antibody is a human antibody, a humanized antibody, or a human chimeric antibody that contains an Fc domain (e.g., a human Fc domain) capable of binding to a human recipient's FcRn. In some such embodiments, the binding affinity of the Fc domain of the human antibody, humanized antibody, or human chimeric antibody to the human FcRn is at least within 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the binding affinity of the human recipient's innate immunoglobulin (IgG) to its FcRn. In other embodiments, the anti-CD117 antibody is a mouse antibody, a murine-derived antibody, or a murine chimeric antibody that contains an Fc domain (e.g., a mouse Fc domain) capable of binding to the recipient's mouse FcRn. In some embodiments, the anti-CD117 antibody induces Fc effector-mediated clearance of the recipient's HSCs and / or HSPCs. In some embodiments, the HSCs are LT-HSCs. Fc-mediated antibody effector functions may include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0070] In other embodiments, the anti-CD117 pretreatment agent is an anti-CD117 antibody or an antigen-binding fragment thereof comprising an Fc domain that has reduced binding affinity to the recipient's FcRn. In some such embodiments, the binding affinity of the Fc domain of the anti-CD117 pretreatment agent to the recipient's FcRn is less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the binding affinity of the recipient's innate immunoglobulin (IgG) to its FcRn. In some embodiments, the Fc domain of the anti-CD117 pretreatment agent is engineered to reduce effector function, such as reducing or ablating ADCC and ADCP function and complement binding. In some embodiments, the anti-CD117 pretreatment agent is an anti-CD117 antibody or an antigen-binding fragment thereof comprising an Fc domain with reduced binding affinity to one or more recipients' Fcγ receptors.

[0071] Non-restrictive examples of suitable anti-CD117 antibodies include ACK-2 (see Czechowicz et al., Science (2007), 318:1296-9; eBioscience); SR-1 (Chandrasekaran et al., Hum Gene Ther. (2014) 25:1013-22); and AMG 191 (Pang et al., Biol Blood Marrow Transplant. (2018), 24:S230-S1 (Abstract 313)). In some embodiments, the method includes using an anti-CD117 antibody comprising the heavy chain and light chain CDRs of any of these antibodies. In some embodiments, the method includes using an anti-CD117 antibody comprising the three heavy chain CDRs and three light chain CDRs of any of these antibodies. In some embodiments, the method includes using an anti-CD117 antibody comprising the three heavy chain CDRs, three light chain CDRs, and a frame region of any of these antibodies. In some embodiments, the method includes using an anti-CD117 antibody comprising the V of any of these antibodies. H and V LIn some embodiments, the anti-CD117 antibody is a chimeric human antibody. In some embodiments, the anti-CD117 antibody is humanized. In some embodiments, the anti-CD117 antibody is a human antibody. In some embodiments, the method includes using an anti-CD117 antibody comprising: (1) a heavy chain and light chain complementarity-determining region (CDR) of any of the above-described anti-CD117 antibodies; and (2) a human constant domain. In some embodiments, the method includes using an anti-CD117 antibody comprising: (1) three heavy chain CDRs and three light chain CDRs of any of the above-described anti-CD117 antibodies; and (2) a human constant domain. In some embodiments, the method includes using an anti-CD117 antibody comprising: (1) three heavy chain CDRs, three light chain CDRs, and a frame region of any of the above-described anti-CD117 antibodies; and (2) a human constant domain. In some embodiments, the method includes using an anti-CD117 antibody comprising: (1) a variable heavy chain (VH) and a variable light chain (VL) of any of the above-described anti-CD117 antibodies; and (2) a human constant domain. The anti-CD117 antibody may be any of the forms described herein.

[0072] In some implementations, the anti-CD117 pretreatment agent is conjugated with a toxin. The anti-CD117 antibody-drug conjugate (ADC) is internalized upon binding to CD117 and its toxic payload is applied to ablate hematopoietic stem cells. In some implementations, the toxin is selected from: saponins, saponin derivatives, ricin, abrin, baicalin, momordicin, bee venom, shiga toxin, shiga-like toxin, T-2 mycotoxin, diphtheria toxin, busulfan, Pseudomonas exotoxin A, ricin A chain derivative, trichosanthin, loofah toxin, maytansin, amatoxin, nitrogen mustard, cyclophosphamide, ethyleneimine, methylmelamine, methotrexate, fluorouracil, fluorouracil, cytarabine, mercaptopurine, azathioprine, thioguanine, fludarabine phosphate, cladribine, sea hare toxin, auristatin, auristatin E, auristatin F, MMAF, MMAE, MMAD, DMAF or DMAE, maytansin, DM1 or DM4, pyroximin, calichimycin, pyrrolobenzodiazepine, eczetidine, and any combination thereof. In other embodiments, the anti-CD117 pretreatment agent is not conjugated to a toxin. In some embodiments, the anti-CD117 pretreatment agent is not an antibody-drug conjugate.

[0073] Drug pretreatment compositions and dosage forms In some embodiments, the effective dose of each of the anti-CD110 and anti-CD117 pretreatment agents of this disclosure is such that, when administered together, it consumes at least 10-fold, at least 100-fold, at least 1000-fold, at least 100,000-fold, or more of endogenous hematopoietic stem cells relative to the level of hematopoietic stem cells present in the recipient's bone marrow microenvironment prior to administration. In some embodiments, the endogenous hematopoietic stem cell consumption is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% of the endogenous hematopoietic stem cell level present in the recipient's bone marrow microenvironment prior to administration. In some embodiments, the endogenous hematopoietic stem cell consumption is about 10% to 80%, about 20% to 80%, about 30% to 80%, about 40% to 80%, about 50% to 80%, about 60% to 80%, or about 70% to 80% of the endogenous hematopoietic stem cell level present in the recipient's bone marrow microenvironment prior to administration. In some implementations, endogenous hematopoietic stem cells are consumed at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80% relative to the level of hematopoietic stem cells present in the recipient's bone marrow microenvironment prior to administration. In some embodiments, endogenous hematopoietic stem cells are depleted for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days or more after administration of anti-CD110 and anti-CD117 pretreatment agents. In certain embodiments, endogenous hematopoietic stem cells are depleted for at least 12 days after administration of anti-CD110 and anti-CD117 pretreatment agents. In some embodiments, depletion of the recipient's endogenous hematopoietic stem cells can be monitored after administration of anti-CD110 and anti-CD117 pretreatment agents. In some embodiments, depletion is determined by assessing endogenous lineage Sca-1+ c-Kit+ (LSK) cell levels. In some embodiments, depletion is determined by assessing endogenous long-term hematopoietic stem cell (LT-HSC) levels.In some embodiments, depletion is determined by assessing endogenous myeloid cell levels. In some embodiments, depletion is determined by assessing endogenous lineage-specific cell levels. In some embodiments, depletion is determined by assessing endogenous naive T cells. The production of T cells is used to determine this. Any known methods in the field for assessing endogenous hematopoietic stem cell depletion can be used in conjunction with publicly available methods.

[0074] Effective doses will vary depending on the individual and the specific pretreatment agent, but generally include at least about 50 μg / kg body weight, at least about 100 μg / kg, at least about 150 μg / kg, at least about 200 μg / kg, at least about 250 μg / kg, at least about 300 μg / kg, at least about 350 μg / kg, at least about 400 μg / kg, at least about 450 μg / kg, at least about 500 μg / kg, at least about 550 μg / kg, at least about 600 μg / kg, at least about 650 μg / kg, at least about 700 μg / kg, at least about 750 μg / kg, at least about 800 μg / kg, at least about 850 μg / kg, at least about 900 μg / kg, at least about 950 μg / kg, at least about 1 mg / kg, and up to about 2.5 mg / kg, up to about 5 mg / kg, up to about 7.5 mg / kg, up to about 10 mg / kg. The dosage is selected from 25 mg to 1000 mg, 25 mg to 750 mg, 25 mg to 650 mg, and 25 mg to 500 mg. In some embodiments, the dosage is selected from 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 325 mg, 500 mg, and 650 mg.

[0075] One or two pretreatment agents can be administered at doses over a period of time according to a schedule deemed appropriate by those skilled in the art to achieve the desired endogenous hematopoietic stem cell ablation. In some embodiments, the dose is administered daily. In some embodiments, the dose is administered twice daily. In some embodiments, the dose is administered three times daily. In some embodiments, the dose is administered four times daily. In some embodiments, the dose is administered in divided doses daily. In some embodiments, the dose is administered for approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, or approximately 7 days, 1 to 2 days, 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, 1 to 7 days, 1 to 10 days, or longer.

[0076] Anti-CD110 and anti-CD117 pretreatment agents can be formulated together or separately, but administered concurrently. As used herein, “concurrently” and “concurrently” mean administering at least two agents to a patient simultaneously or for a period of time while the effects of the first agent are still in effect. For example, the concurrent administration of the second agent may be performed within one to two days after the administration of the first agent, preferably within one to seven days after the administration of the first agent.

[0077] The anti-CD110 and anti-CD117 pretreatment agents of this disclosure may be formulated for administration in any form of composition that a person skilled in the art would find useful. In some embodiments, the anti-CD110 and anti-CD117 pretreatment agents are formulated as pills, capsules, tablets, syrups, ampoules, lozenges, or powders for individual oral administration. In some embodiments, the pretreatment agents are formulated for intravenous infusion or injection. In some embodiments, the pretreatment agents are pharmaceutical compositions or single-unit dosage forms. The pharmaceutical compositions and single-unit dosage forms provided herein comprise one or both of the anti-CD110 and anti-CD117 pretreatment agents in a preventive or therapeutically effective amount.

[0078] HSCT and transplantation methods Monitoring the removal of pretreatment agents before HSCT In some implementations, following co-administration of anti-CD110 and anti-CD117 preconditioning agents, the recipient's bone marrow microenvironment is cleared of endogenous hematopoietic stem cells, allowing exogenous donor hematopoietic stem cells to newly occupy the microenvironment. However, to avoid any residual preconditioning agents inadvertently clearing donor cells from the recipient, the pharmacokinetic levels of one or both preconditioning agents can be monitored in the recipient's blood prior to HSCT for clearance purposes. In some implementations, the recipient undergoes HSCT only after one or both of the anti-CD110 and anti-CD117 preconditioning agents have been substantially cleared from the recipient's blood circulation.

[0079] In some embodiments, the pretreatment agent is substantially cleared from circulation when the concentration of the anti-CD110 or anti-CD117 pretreatment agent (e.g., as assessed from a recipient's blood sample) is no longer detectable using any method known in the art for measuring the presence and / or activity of biological agents in blood or serum. In some embodiments, the pretreatment agent is substantially cleared from circulation when the concentration of the pretreatment agent is no longer detectable above a background threshold for an assay used to detect the pretreatment agent. Any method known in the art for detecting antibodies or antibody fragments, such as ELISA-based assays, immunoprecipitation techniques, and immunoblotting assays, can be used to assess the clearance of the pretreatment agent. In some embodiments, serum collected from the recipient at a specific time point after administration of the pretreatment agent can be contacted with stem cells (e.g., a sample of donor hematopoietic stem cells), and the binding of any pretreatment agent in the serum to the stem cells can be assessed using conventional methods. In other embodiments, growth inhibition of the contacted stem cells can be assessed in the presence of the recipient's serum.

[0080] In some embodiments, exogenous hematopoietic stem cells can be administered to the recipient once it has been confirmed that one or both of the anti-CD110 and anti-CD117 pretreatment agents have been adequately cleared from the recipient's blood circulation. In some embodiments, adequate clearance is achieved when the serum level of the pretreatment agent decreases by a factor of 5 below the peak level of the pretreatment agent after administration. In some embodiments, the pretreatment agent is at least 1 / 10, 1 / 100, 1 / 1000, 1 / 10,000, 1 / 100,000, 1 / 1,000,000, or less than 1 / 1,000,000 of the peak level before administration of exogenous donor hematopoietic stem cells. In other embodiments, exogenous donor hematopoietic stem cells can be administered based on the known or expected pharmacokinetics of the pretreatment agent. In some implementations, exogenous donor hematopoietic stem cells are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days after co-administration of anti-CD110 and anti-CD117 pretreatment agents.

[0081] hematopoietic stem cell transplantation In some embodiments, after administering pretreated antibodies and depleting endogenous hematopoietic stem cells, the method further includes administering a therapeutically effective amount of exogenous hematopoietic stem cells to the patient. In some embodiments of the methods provided herein, a therapeutically effective amount of hematopoietic stem cells and progenitor cells is administered to the patient. In some embodiments, the exogenous cells administered may include donor bone marrow cells, umbilical cord blood cells, hematopoietic stem cells and progenitor cells (HSPCs), peripheral blood CD34 cells, etc. + Cells, peripheral blood CD34 + and CD90 + Cells and any combination thereof.

[0082] Hematopoietic stem cells can be any hematopoietic stem cell that a skilled practitioner deems useful. In some implementations, exogenous hematopoietic stem cells can re-establish hematopoietic function in a patient upon transplantation. Hematopoietic function is defined by a hierarchy based on the expression of cell surface markers, initiated by hematopoietic stem cells that are both self-renewing and capable of differentiating into pluripotent progenitor cells, which then generate lineage-restricted progenitor cells and ultimately differentiate into terminally differentiated blood cells (Baum et al.). PNAS 89, 2804-2808 (1992); Majeti et al., Cell Stem Cell 1, 635-645 (2007); Doulatov et al., Cell Stem Cell 10, 120-136 (2012)). CD34 + Expression defines a heterogeneous population of HSPCs, which can be further divided into pluripotent progenitor cells (CD34). + / CD38 - / CD45RA - Long-term repopulating cells (CD34) in xenotransplanted mice + / CD38 - / CD90 + ) and highly enriched hematopoietic stem cell populations (CD34) + / CD38 - / CD90 + / CD45RA - ).

[0083] In some embodiments, hematopoietic stem cells belong to any subtype or colony-forming unit. In some embodiments, hematopoietic stem cells are colony-forming units-granulocyte-erythrocyte-monocyte-megakaryocyte. In some embodiments, hematopoietic stem cells are colony-forming units-erythrocyte. In some embodiments, hematopoietic stem cells are colony-forming units-granulocyte-macrophage. In some embodiments, hematopoietic stem cells are colony-forming units-megakaryocyte. In some embodiments, hematopoietic stem cells are colony-forming units-basophil. In some embodiments, hematopoietic stem cells are colony-forming units-eosinophil.

[0084] Hematopoietic stem cells can be derived from any source that a technician deems useful. In some embodiments, the hematopoietic stem cells are derived from a donor. In some embodiments, the donor is a patient. In some embodiments, the donor is another object of the same species, such as another person. In some embodiments, the hematopoietic stem cells are autologous. In some embodiments, the hematopoietic stem cells are allogeneic. In some embodiments, the hematopoietic stem cells are syngeneic.

[0085] Hematopoietic stem cells can be harvested using any technique that the technician deems useful. In some implementations, a hematopoietic stem cell mobilizing agent (such as Mozobil) is administered to the donor prior to harvesting. (G-CSF, GM-CSF). In some embodiments, hematopoietic stem cells are harvested from peripheral blood. In some embodiments, hematopoietic stem cells are harvested from umbilical cord blood. In some embodiments, hematopoietic stem cells are harvested from bone marrow. In some embodiments, donor cell populations can be obtained from products collected from a subject (such as a patient or subject requiring autologous HSCT). The product can be apheresis product containing a heterogeneous mixture of cells collected from the subject. The heterogeneous mixture of cells can contain primary cells as well as primary CD34+ cells and / or human stem cells and / or progenitor cells (HSPCs). CD34+ cells and / or HSPCs can be isolated or separated from other cells to obtain a stem cell population. After isolating CD34+ HSPCs, the resulting stem cell population is substantially free of non-CD34+ cells and is ready for subsequent genetic manipulation.

[0086] In some embodiments, flow cytometry is used to separate harvested hematopoietic stem cells from the primary cell population. In some cases, flow cytometry includes fluorescence-activated cell sorting (FACS). In some other embodiments, magnetic bead separation is used to separate harvested hematopoietic stem cells from the primary cell population. In some cases, magnetic bead separation includes magnetically activated cell sorting (MACS). In some other embodiments, harvested hematopoietic stem cells are separated using a device configured for hematopoietic stem cell enrichment, such as the Miltenyi Biotec CliniMACS cell manufacturing platform.

[0087] Methods for culturing or expanding primary hematopoietic stem cells are known in the art, including those described in International Patent Application No. PCT / US2022 / 72014, which is incorporated herein by reference in its entirety. Methods for culturing primary cells and their progeny are known, and suitable culture media, supplements, growth factors, etc., are also known and commercially available. Typically, human primary cells are maintained and expanded under serum-free conditions. Alternative culture media, supplements, and growth factors and / or their concentrations can be readily determined by those skilled in the art, and these are extensively described in the literature. In some embodiments, isolated or purified genetically modified cells can be expanded in vitro according to standard methods known to those skilled in the art.

[0088] In certain embodiments, HSCT can be performed using a freshly isolated cell population containing hematopoietic stem cells. In other specific embodiments, the HSCT of the methods described herein is performed using a cryopreserved cell population containing hematopoietic stem cells. Cells can be cryopreserved after hematopoietic stem cell harvesting or isolation, after culture initiation and activation, after modification (e.g., gene modification), or after expansion or any process step. Freeze-thaw cycles can provide a more homogeneous hematopoietic stem cell composition by removing non-hematopoietic stem cell populations. Hematopoietic stem cells can be stored using any technique that a person skilled in the art would find useful. In some embodiments, harvested cells are formulated in cryopreservation media and placed in cryogenic storage units, such as liquid nitrogen freezers (-195°C) or ultra-low temperature freezers (-65°C, -80°C, or -120°C), for long-term storage of at least one month, two months, three months, four months, six months, one year, two years, three years, or at least five years. In some embodiments, thawed cells are pretreated using the methods described herein.

[0089] Genetically modified hematopoietic stem cells The HSCT method described herein involves transplanting genetically modified hematopoietic stem cells, for example, to contain a therapeutic allogeneic donor polynucleotide sequence. The donor polynucleotide sequence described herein can be integrated into a variety of gene therapy constructs, for example, for delivering nucleic acids encoding proteins to a recipient. A vector construct refers to a polynucleotide molecule comprising all or part of a viral genome and an exogenous polynucleotide sequence. In some cases, gene transfer can be mediated by DNA viral vectors, such as adenovirus (Ad) or adeno-associated virus (AAV). Other vectors that can be used for gene therapy are known in the art. For example, constructs disclosed herein may include alphaviruses, herpesviruses, retroviruses, lentiviruses, or vaccinia viruses. The exogenous sequence generally encodes a recombinant molecule that will be expressed in cells, for example, for cell therapy. The processing steps of the method may also, or alternatively, include all or part of cell washing, dilution, selection, isolation, separation, culture, stimulation, packaging, and / or formulation. The method generally allows for large-scale cell processing (e.g., in compositions with a volume greater than or about 50 mL), for example, selection or isolation and / or transduction.

[0090] In some implementations, hematopoietic stem cells are genetically modified using gene-editing applications that utilize site-specific nucleases to knock out target genomic sequences or knock in exogenous sequences, and transfer the exogenous sequences into cells via viral transduction using recombinant viral vectors. In some such implementations, hematopoietic stem cells are collected via apheresis, enriched from the apheresis products, and then cryopreserved before any gene-editing method (e.g., gene knockout, gene knock-in, gene modification). Cryopreservation can be introduced after mobilization and collection (e.g., via apheresis) of stem cells and selection of hematopoietic stem cells. After cryopreservation, an assessment can be performed to determine whether a threshold number of hematopoietic stem cells has been collected from the donor for subsequent gene-editing steps. If a single round of mobilization, collection, selection, and cryopreservation does not reach the cell threshold number, subsequent rounds can be performed until the cell threshold number is reached. The threshold number of hematopoietic stem cells to be collected can vary depending on a variety of factors, including but not limited to the gene editing procedure performed (e.g., gene knockout, gene knock-in, gene correction), the target gene to be edited, the mechanism of modification of the target gene (e.g., homology-dependent repair (HDR)), the efficiency of the editing procedure (e.g., HDR efficiency), and the therapeutic threshold for treating a specific disease. In some embodiments, the threshold number of hematopoietic stem cells collected from the donor prior to gene editing is approximately 1 x 10⁻⁶. 4 Up to 1 x 10 5 1 x 10 5 Up to 1x 10 6 1 x 10 6 Up to 1 x 10 7 Cells / kg or more. In some implementations, at least approximately 1 x 10⁻⁶ cells are collected prior to gene editing. 5 Up to 1 x 10 7 Cells / kg. In some implementations, at least approximately 1 x 10⁻⁶ cells are collected before gene editing of the collected cells. 4 2 x 10 4 3 x 10 4 4 x 10 4 5 x 10 4 6 x 10 4 7 x 10 4 8 x 10 4 9 x 10 4 1 x 10 5 2 x 10 5 3 x 10 5 4 x 10 5 5 x 10 5 6 x 10 5 7 x 105 8 x 10 5 9 x 10 5 1 x 10 6 2 x 10 6 3 x 10 6 4 x 10 6 5 x 10 6 6 x 10 6 7 x 10 6 8 x 10 6 9 x 10 6 1 x 10 7 2 x 10 7 3 x 10 7 4 x 10 7 5 x 10 7 6 x 10 7 7 x 10 7 8 x 10 7 9 x 10 7 Or approximately 1 x 10 8 One hematopoietic stem cell per kg. Once the threshold number of hematopoietic stem cells has been mobilized, collected, selected, and cryopreserved, the cells can be thawed, cultured, and gene-edited.

[0091] In some embodiments, gene editing utilizes a nuclease introduced into the cell that can cause double-strand breaks near or within a genomic target site. This can be used to increase the frequency of homologous recombination and HDR at or near the cleavage site. In a preferred embodiment, the nuclease's recognition sequence is present only at the target site in the host cell genome, thereby minimizing any off-target genome binding and cleavage caused by the nuclease. Gene-editing nucleases that can be used in the methods provided herein include, but are not limited to, TAL effector DNA-binding domain-nuclease fusion proteins (TALENs), site-specific recombinases (e.g., serine or tyrosine recombinases), integrases (FLP, Cre, λ integrase), or dissociative enzymes; transposases, zinc finger nucleases (ZFNs), and clustered regularly spaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs, or modified versions thereof.

[0092] In some embodiments, genetically modified CD34+ stem cells are generated by introducing a CRISPR-associated Cas nuclease (e.g., Cas9), a guide RNA polynucleotide, and a donor polynucleotide sequence into primary CD34+ stem cells. By introducing these components into the cells, double-strand breaks can be introduced at specific sites, as directed by the guide polynucleotide sequence and the CRISPR-associated Cas9 nuclease. A donor polynucleotide containing the sequence of interest can be further introduced into the cells, and the sequence of interest can be inserted into the cells via homology-directed recombination. The transfer of the donor polynucleotide sequence can be performed via transduction. Methods for viral transfer, such as transduction, generally involve at least initiating transduction by incubating an input composition containing cells to be transduced and viral vector particles containing a vector in a centrifuge chamber, under conditions where transduction or initiation of transduction is performed in at least some cells of the input composition, wherein the method produces an output composition containing transduced cells.

[0093] Methods for introducing peptides, nucleic acids, and viral vectors (e.g., viral particles) into primary cells, target cells, or host cells are known in the art. Any known method can be used to introduce peptides or nucleic acids (e.g., nucleotide sequences encoding DNA nucleases or modified sgRNAs) into primary cells, such as human primary cells. Non-limiting examples of suitable methods include: electroporation (e.g., nuclear transfection), viral or bacteriophage infection, transfection, conjugation, protoplast fusion, liposome transfection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, etc.

[0094] In some embodiments, the Cas nuclease may be in the form of a protein. In some embodiments, the Cas nuclease may be in the form of a plasmid, thereby allowing cells carrying the expression construct to subsequently express the Cas nuclease. In other embodiments, the Cas nuclease is pre-complexed with guide RNA and introduced into the cell as a ribonucleoprotein (RNP). In some embodiments, the Cas nuclease and guide polynucleotide sequence are introduced into CD34+ cells via electroporation.

[0095] The introduction of donor polynucleotides can be performed via viral transduction using delivery vectors such as adeno-associated virus (AAV). Any serotype or pseudotype of AAV can be used. Some AAV vectors are derived from single-stranded (ss) DNA parvoviruses that are nonpathogenic to mammals. Briefly, in the generation of certain AAV vectors, the rep and cap viral genes, which can constitute up to 96% of the typical wild-type AAV genome, can be removed, leaving flanking inverted terminal repeat (ITR) sequences that can be used to initiate viral DNA replication, packaging, and integration. Wild-type AAV integrates into the human host cell genome at a preferential site specificity at chromosome 19q13.3. Alternatively, AAV can be maintained in vitro. To date, at least 12 human AAV serotypes (AAV serotypes 1 (AAV-1) to AAV-12) and more than 100 serotypes from non-human primates have been identified. Any of these serotypes and any combination thereof may be used within the scope of this disclosure. The serotype of the viral vector can be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the serotype is AAV6.

[0096] In some embodiments, viral transduction occurs within 30 minutes of electroporation. In some embodiments, viral transduction occurs simultaneously with electroporation. In some embodiments, viral transduction occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 minutes of electroporation.

[0097] In other implementations, hematopoietic stem cells are genetically modified using gene-editing applications that utilize base editors. Base editing is a CRISPR-Cas9-based genome editing technology that allows the introduction of point mutations into DNA without generating DSBs. Two main classes of base editors have been developed: cytosine base editors or CBEs that allow C>T conversions and adenine base editors or ABEs that allow A>G conversions (see, for example, Rees et al. (2018)). Nat Rev Genet 19:770-788).

[0098] In other embodiments, hematopoietic stem cells are genetically modified using a gene-editing application utilizing a leader editor. The leader editor (PE) consists of nCas9 fused with reverse transcriptase and used in conjunction with a leader-editing RNA (pegRNA, a guide RNA that includes a reverse-transcribed template region). Leader editing allows for the introduction of insertions, deletions, and conversions between 12 bases. Leader editing relies on the ability of a reverse transcriptase (RT) fused with a Cas nickase variant to convert the RNA sequence brought in by the leader-editing guide RNA (pegRNA) into DNA at the nick site generated by the Cas protein. The resulting DNA flap is then included or excluded from the target DNA sequence. See, for example, Anzalone et al. (2019). Nature 576:149-157. Non-restrictive examples of pilot editing systems include PE1, PEI-M1, PE1-M2, PE1-M3, PE1-M6, PE1-M15, PE1-M3inv, PE2, PE3, and PE3b.

[0099] In other embodiments, hematopoietic stem cells are genetically modified using a gene-editing application that utilizes DNA-guided peptides, such as *Halophilus gastroenteritidis* (Halophilus gastroenteritidis). Natronobacterium gregoryiArgonaute (NgAgo), RNA-guided peptides (e.g., Cas9, CasX, CasY, Cpf1, etc.); site-specific recombinases (e.g., Cre recombinase, Dre recombinase, Flp recombinase, KD recombinase, B2 recombinase, B3 recombinase, R recombinase, Hin recombinase, Tre recombinase, PhiC31 integrase, Bxb1 integrase, R4 integrase, λ integrase, HK022 integrase, HP1 integrase, etc.). Dissociative enzymes and / or invertases (e.g., Gln, Hin, γδ3, Tn3, Sin, β, etc.); transposons and / or DNA derived from transposons (e.g., bacterial transposons such as Tn3, Tn5, Tn7, Tn9, Tn10, Tn903, Tn1681, etc.; eukaryotic transposons such as Tc1 / mariner superfamily transposons, PiggyBac superfamily transposons, hAT superfamily transposons, PiggyBac, Sleeping Beauty, Frog Prince, Minos, Himar1, etc.), and CRISPR transposons that guide RNA guides through the DNA integration capability of naturally binding transposases and the targeting programmability of CRISPR-Cas (see, for example, Peters et al., Proc Natl Acad Sci USA 114:E7358-E7366 (2017); Klompe et al. Nature 571:219-225 (2019); and Halpin Healy et al., Nature 577:271-274 (2020).

[0100] Drug-based hematopoietic stem cell composition This document also provides methods, compositions, and kits for use with hematopoietic stem cells (e.g., genetically modified hematopoietic stem cells), including pharmaceutical compositions, treatment methods, and administration methods. Although the descriptions of the pharmaceutical compositions provided herein are primarily directed toward pharmaceutical compositions suitable for human administration, those skilled in the art will understand that such compositions are generally suitable for administration to any animal.

[0101] In some embodiments, the pharmaceutical composition comprises a modified host cell that is genetically engineered to contain an integrated donor sequence at a target gene locus. In some embodiments, the modified host cell is genetically engineered to contain an integrated functional donor sequence, such as an SNP donor, that corrects one or more mutations in a target gene (e.g., HBB) or inserts or replaces some or all of the mutated alleles with wild-type alleles. In a particular embodiment, the functional donor sequence is integrated into the translation start site at an endogenous locus of the target gene. In a particular embodiment, the functional donor sequence integrated into the host cell genome is expressed under the control of the natural promoter sequence of the target gene.

[0102] In some embodiments, the pharmaceutical composition comprises a plurality of modified host cells, and further comprises unmodified host cells and / or host cells that have undergone nuclease cleavage resulting in INDELS at the target gene locus, but without integration of the donor sequence. In some embodiments, the pharmaceutical composition consists of at least 5% modified host cells containing integrated donor sequences. In some embodiments, the pharmaceutical composition consists of approximately 9% to 50% modified host cells containing integrated donor sequences. In some embodiments, the pharmaceutical composition comprises at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50% or more of modified host cells containing an integrated donor sequence. The pharmaceutical compositions described herein may be formulated with one or more excipients to, for example: (1) increase stability; (2) alter biodistribution (e.g., target cells to specific tissues or cell types, such as hematopoietic stem cells); and / or (3) enhance transplantation in recipients.

[0103] The formulations disclosed herein may include, but are not limited to, saline, liposomes, lipid nanoparticles, polymers, peptides, proteins, and combinations thereof. Formulations of the pharmaceutical compositions described herein may be prepared by any method known in or developed thereafter in the field of pharmacology. As used herein, the term "pharmaceutical composition" refers to a composition comprising at least one active ingredient (e.g., exogenous hematopoietic stem cells) and optionally one or more pharmaceutically acceptable excipients. The pharmaceutical compositions of this disclosure may be sterile.

[0104] The relative amounts of the active ingredient (e.g., modified host cells), pharmaceutically acceptable excipients, and / or any other components in the pharmaceutical compositions according to this disclosure can vary depending on the identity, body size, and / or condition of the treated subject, and further depend on the route of administration of the composition. For example, the composition may include 0.1% to 99% (w / w) of the active ingredient. For instance, the composition may include 0.1% to 100% (w / w), such as 0.5% to 50%, 1% to 30%, 5% to 80%, or at least 80% (w / w) of the active ingredient.

[0105] As used herein, excipients include, but are not limited to, any and all solvents, dispersion media, diluents or other liquid media, dispersants or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, etc., to suit a desired specific dosage form. Various excipients used to formulate pharmaceutical compositions and techniques for preparing compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st edition, ARGennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). Within the scope of this disclosure, conventional excipient media may be considered for use unless any conventional excipient media is incompatible with the substance or its derivatives, such as producing any undesirable biological effects or otherwise interacting with any other component of the pharmaceutical composition in a harmful manner.

[0106] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, etc., and / or combinations thereof. Injectable formulations may be sterilized, for example, by filtration through a bacterial retention filter, and / or by incorporating a sterilizing agent into a sterile solid composition, which may be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

[0107] Dosage and administration In some embodiments, the method includes administering to an individual in need of treatment a composition containing an effective amount of hematopoietic stem cells (e.g., genetically modified hematopoietic stem cells). The therapeutically effective dose of hematopoietic stem cells can be in the range of approximately one million to approximately 200 billion cells, for example, one million to approximately 50 billion cells (e.g., approximately 5 million cells, approximately 25 million cells, approximately 500 million cells, approximately 1 billion cells, approximately 5 billion cells, approximately 20 billion cells, approximately 30 billion cells, approximately 40 billion cells, or a range defined by any two of the above values), such as approximately 10 million to approximately 100 billion cells (e.g., approximately 20 million cells, approximately 30 million cells, approximately 40 million cells, approximately 60 million cells, approximately 70 million cells, approximately 80 million cells). The number of cells may be approximately 10,000, 90 million, 10 billion, 25 billion, 50 billion, 75 billion, or 90 billion, or a range defined by any two of the above values, and in some cases, approximately 100 million to approximately 50 billion cells (e.g., approximately 120 million, 250 million, 350 million, 450 million, 650 million, 800 million, 900 million, 3 billion, 30 billion, or 45 billion) or any value between these ranges. In some embodiments, the method includes administering 2 x 10⁻⁶ cells per kg of body weight. 6 Up to 2 x 10 8 One surviving hematopoietic stem cell.

[0108] In some embodiments, a pharmaceutical composition comprising exogenous hematopoietic stem cells according to this disclosure can be delivered to a subject in a manner sufficient to deliver, for example, about 1 x 10⁻⁶ cells / day. 4 Up to 1 x 10 5 1 x 10 5 Up to 1 x 10 6 1 x 10 6 Up to 1 x 10 7The drug may be administered at dose levels of one or more cells, or in any amount sufficient to achieve the desired therapeutic or preventative effect. The desired dose of the modified host cell pharmaceutical composition of this disclosure may be administered once or multiple times. In some embodiments, delivery of the modified host cells to a subject provides a therapeutic effect for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more than 10 years. In some embodiments, a single dose is sufficient to achieve treatment or prevention of the disease or condition described herein. In other embodiments, a subject in need may receive more than one dose (e.g., 2, 3, or more than 3 doses) of the pharmaceutical hematopoietic stem cell composition described herein to achieve treatment or prevention of the disease or condition. Hematopoietic stem cells may be used sequentially or simultaneously with one or more other therapeutic, preventative, research, or diagnostic agents or medical procedures. Generally, each agent will be administered according to the dosage and / or schedule determined for that agent.

[0109] Infusion groups and their compositions can be administered to individuals in need using standard administration techniques, formulations, and / or devices. Formulations and administration devices, such as syringes and vials, are provided for storing and administering the compositions. Formulations or pharmaceutical compositions containing exogenous hematopoietic stem cells include those for intravenous, intraperitoneal, subcutaneous, intramuscular, or pulmonary administration. Compositions containing exogenous hematopoietic stem cells can be provided as sterile liquid products, such as isotonic solutions, suspensions, emulsions, dispersions, or viscous compositions, which in some respects may be buffered to a selected pH. Viscous compositions can be formulated within appropriate viscosity ranges to provide a longer contact time with a specific tissue. Liquid or viscous compositions may contain a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffer solutions, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof. Sterile injectable solutions can be prepared by incorporating hematopoietic stem cells into a solvent, such as by mixing with a suitable carrier, diluent, or excipient (such as sterile water, physiological saline, glucose, dextrose, etc.).

[0110] The exogenous hematopoietic stem cells included in the above-described pharmaceutical compositions can be administered via any route of delivery, systemic or local, to achieve therapeutic efficacy. These routes include, but are not limited to: enteric, gastrointestinal, epidural, oral, transdermal, intracerebral, intraventricular, epidermal, intradermal, subcutaneous, nasal, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intrasheath, intracerebral parenchyma, intraperitoneal, intrabladder, intravitreal, intracavitary, interstitial, intraperitoneal, intralymphatic, intramedullary, intrapulmonary, intraspinal, intrasynovial, intrasheath, intratubular, extra-gastric, percutaneous, periarticular, epidural, perineurial, periodontal, rectal, soft tissue, and surface. In a particular embodiment, the cells are administered intravenously. The pharmaceutical compositions can be administered to subjects in any amount and via any route of administration to effectively prevent, treat, or control the diseases described herein. The exact amount required will vary depending on the subject's species, age and general condition, disease severity, the specific composition, its method of administration, its mode of activity, etc.

[0111] In some embodiments, hematopoietic recovery, reconstitution, and / or donor chimerism in the recipient can be monitored after administration of exogenous donor hematopoietic stem cells as indicators of successful transplantation. In some embodiments, transplantation is determined by assessing donor lineage Sca-1+ c-Kit+ (LSK) cell chimerism. In some embodiments, transplantation is determined by assessing donor long-term hematopoietic stem cell (LT-HSC) chimerism. In some embodiments, transplantation is determined by assessing donor myeloid chimerism. In some embodiments, transplantation is determined by assessing lineage-specific chimerism. In some embodiments, transplantation is determined by assessing the generation of naïve T cells. Any method known in the art for assessing donor cell chimerism can be used in conjunction with the disclosed methods (see, for example, Pinkel et al.). Proc Natl Acad Sci USA (1996), 83: 2934-2938). In some implementations, after transplantation of donor stem cells, the recipient is a chimera or mixed chimera of donor cells. Mixed chimerism (MC) is defined as the presence of more than 5% host-derived cells in whole blood more than once. MC is further classified into high-level MC (95%–50% donor chimerism), low-level MC (49%–10% donor chimerism), or very low-level MC (<10% donor chimerism).

[0112] In some embodiments of the methods provided herein, administration of exogenous donor hematopoietic stem cells following co-administration of anti-CD110 and anti-CD117 pretreatment agents to the subject results in donor chimerism of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%. In some embodiments, donor chimerism is about 10% to 80%, about 20% to 80%, about 30% to 80%, about 40% to 80%, about 50% to 80%, about 60% to 80%, or about 70% to 80%. In some implementations, donor chimerism is at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%. In some implementations, donor chimerism is donor LSK cell chimerism. In some embodiments, donor chimerism is donor HSC chimerism. In some embodiments, donor HSC chimerism is donor LT-HSC chimerism. In some embodiments, donor chimerism is donor CMP cell chimerism. In some embodiments, donor chimerism is donor GMP cell chimerism. In some embodiments, donor chimerism is donor MEP cell chimerism. In some embodiments, donor chimerism is donor MEP cell chimerism. In some embodiments, donor chimerism is donor CLP cell chimerism. In some embodiments, donor chimerism is total donor cell chimerism.

[0113] Treatment The composition and method for hematopoietic stem cell depletion and transplantation provided in this article can be used as part of a treatment regimen for any disease or condition for which HSCT is applicable. HSCT can be used to treat a variety of conditions, including congenital and acquired conditions. In some implementation schemes, acquired conditions for which HSCT can be treated include, but are not limited to: (1) malignancies, including hematologic malignancies such as leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML)), lymphoma (e.g., Hodgkin's disease, non-Hodgkin's lymphoma)), myeloma (e.g., multiple myeloma (Kahler's disease)); solid tumors (e.g., neuroblastoma, desmoplastic small round cell tumor, Ewing's sarcoma). sarcoma, choriocarcinoma;(2) Hematologic disorders, including phagocyte disorders (e.g., chronic granulomatous disease), bone marrow failure disorders (e.g., myelodysplastic syndrome, Fanconi's anemia, dyskeratosis congenita), anemias (e.g., paroxysmal nocturnal hemoglobinuria, aplastic anemia, acquired pure red cell aplasia), and myeloproliferative disorders (e.g., polycythemia vera, essential thrombocythemia). (3) Metabolic disorders, including amyloidosis (e.g., amyloid light chain amyloidosis); (4) Environmentally induced diseases such as radiation poisoning; (5) Viral diseases (e.g., HTLV, HIV); and (6) Autoimmune diseases such as multiple sclerosis.

[0114] In some implementations, congenital conditions treatable with HSCT include, but are not limited to: (1) lysosomal storage disorders, including lipid deposition (disorders of lipid storage such as neuronal ceroid lipofuscinoses (e.g., infantile neuronal ceroid lipofuscinose (INCL, Santavuori disease) and Jansky-Bielschowsky disease (late infantile neuronal ceroid lipofuscinosis))); sphingolipidoses (e.g., Niemann-Pick disease and Gaucher disease); leukodystrophy (e.g., adrenoleukodystrophy, metachromatic leukodystrophy, Krabbe disease). Diseases (globoid cell leukodystrophy); mucopolysaccharidoses (e.g., Hurler syndrome (MPS IH, α-L-iduronidase deficiency), Schie syndrome (MPS IS), Hurler-Scheie syndrome (MPS I HS), Hunter syndrome (MPS II, idoronidase sulfate deficiency), Sanfilippo syndrome (MPS III), Morquio syndrome (MPS IV), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII)); glycoproteinoses (e.g., mucolipidosis II). II) (I-cell disease), fucosidosis, aspartylglucosaminuria, alpha-mannosidosis; and Wolman's disease (acid lipase deficiency).(2) Immunodeficiencies, including T-cell deficiencies (e.g., ataxia-telangiectasia and DiGeorge syndrome), combined T-cell and B-cell deficiencies (e.g., severe combined immunodeficiency (SCID), all types), well-defined syndromes (e.g., Wiskott-Aldrich syndrome), phagocytic disorders (e.g., Kostmann syndrome, Shwachman-Diamond syndrome), immune dysregulations (e.g., Griscelli syndrome, type II), and congenital immunodeficiency (e.g., NF-κ-B essential regulator (NEMO) deficiency (inhibitor deficiency of the κ light polypeptide gene enhancer in B-cell γ-kinase)); (3) Hematologic disorders, including hemoglobinopathy (e.g., sickle cell disease, thalassemia (e.g., β-thalassemia)), and anemia (e.g., aplastic anemias such as Diamond-Blackfan anemia and Fanconi anemia). Anemia, cytopenia (e.g., amegakaryocytic thrombocytopenia), and hemophagocytic syndrome (e.g., hemophagocytic lymphohistiocytosis, HLH).

[0115] In some implementations, the disease or condition is selected from hemoglobinopathies, viral infections, X-linked severe combined immune deficiency, Fanconi anemia, hemophilia, neoplasm, cancer, amyotrophic lateral sclerosis, alpha antitrypsin deficiency, Alzheimer's disease, Parkinson's disease, cystic fibrosis, blood disorders and conditions, inflammation, immune system disorders or conditions, metabolic diseases, liver diseases and conditions, kidney diseases and conditions, muscle diseases and conditions, bone or cartilage diseases and conditions, nervous system and neuronal diseases and conditions, cardiovascular diseases and conditions, lung diseases and conditions, and lysosomal storage diseases. In some implementations, hemoglobinopathies are selected from sickle cell disease, alpha-thalassemia, beta-thalassemia, and delta-thalassemia.

[0116] While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, modifications, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed to practice the invention. The following claims are intended to define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

[0117] Example Example 1: Co-administration of anti-CD110 and anti-CD117 pretreatment agents led to robust transplantation of HSPC. The results of this embodiment demonstrate that in immune-active recipient mice, the combination therapy with effector-competent anti-CD117 and anti-CD110 monoclonal antibodies has a synergistic effect, enabling robust transplantation of donor HSPCs (hematopoietic stem cells and progenitor cells) and multi-lineage reconstitution of hematopoietic cells.

[0118] Materials and methods Antibody Anti-mCD117 antibody ACK2 and anti-mCD110 antibody AMM2 are commercially available as rat immunoglobulins. The variable domain sequences of the antibodies were obtained by in-solution intracellular protease digestion, followed by liquid chromatography-tandem mass spectrometry and data analysis. Mouse IgG2a version antibodies were generated by fusing the variable domains with the constant regions of the mouse heavy and light chains. The mouse IgG2a_N297A version antibody was generated by inducing Asn in mouse Fc cells to Ala, which is homologous to Asn at position 297 in human Fc cells. The chimeric antibodies were transiently produced from CHO cells, purified, and their binding to their respective mouse antigens was confirmed in binding assays using ForteBio Octet assays. Figure 1 As shown. Recombinant mCD110-ECD-H6 or mCD117-ECD-H6 was captured on the sensor tip and transferred to anti-mCD110 mIgG2a or anti-mCD117 mIgG2a solutions at the following concentrations: 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 0 nM. After antibody association, the sensor tip was individually transferred to a buffer to assess antibody dissociation over time. Association and dissociation curves were calculated for each concentration level.

[0119] Preprocessing and transplantation B6.SJL- 8-10 weeks old Ptprc a Pepc b / BoyJ (“B6 CD45.1”) mice, according to Figure 2 The study design involved treatment with appropriate antibody regimens on day -7 after bone marrow transplantation. B6 CD45.1 mice were intravenously injected with 25 mg / kg mIgG2a isotype control antibody, 25 mg / kg anti-mCD117 mIgG2a antibody, 25 mg / kg anti-mCD110 mIgG2a antibody, or both 25 mg / kg anti-mCD117 antibody and 25 mg / kg anti-mCD110 antibody. Animals were also intravenously injected with 800,000 lineage-negative ("Lin") cells isolated from C57BL / 6J donor bone marrow cells. - "Donor cells. The chimerism result of this procedure is as follows..." Figure 3 and Figure 4 As shown.

[0120] Eight- to ten-week-old B6 mice were intravenously injected on day -7 with either A) 50 mg / kg mIgG2a allotype control antibody or a combination of 25 mg / kg anti-mCD117 mIgG2a antibody and 2.5 mg / kg anti-mCD110 mIgG2a antibody, or B) 25 mg / kg ACK2 (anti-mCD117 rIgG2b antibody) and 5 mg / kg AMM2 (anti-mCD110 rIgG1 antibody) or 25 mg / kg anti-mCD117 mIgG2a Fc-deletion antibody and 5 mg / kg anti-mCD110 mIgG2a Fc-deletion antibody, and bone marrow transplantation was performed on day 0. The chimerism results of this procedure are as follows: Figure 5 As shown.

[0121] Secondary transplant functional exhaustion like Figure 7A The data shows that B6.SJL- 8-10 weeks of age Ptprc a Pepc b / BoyJ mice (“B6 CD45.1”) were treated with anti-mCD117 and anti-mCD110 antibodies, and whole bone marrow was harvested on days 7, 9, and 12 post-pretreatment. Secondary recipients (CD45.2) who underwent lethal irradiation (10 Gy) received equal amounts of whole bone marrow cells from pretreated donor mice and GFP+ supported mice (CD45.2).

[0122] donor cell separation For separation Figure 3 and Figure 4 Donor cells were used from 8-10 week old B6 mice that were euthanized, and femurs, tibias, humeri, hip bones, and vertebrae were collected. The bones were crushed to separate the bone marrow, and RBCs were then lysed on ice using Gibco ACK lysis buffer for 7 minutes. Lineage-negative cells were collected using the Direct Lineage Cell Depletion Kit (Miltenyi Biotec) according to the manufacturer's instructions.

[0123] For separation Figure 5 Using the donor cells shown in Figure 7, 8–10 week old B6 CD45.1 mice were euthanized, and femurs, tibias, humeri, hip bones, and vertebrae were collected. The bones were fragmented to isolate the bone marrow, and RBCs were subsequently lysed on ice using Gibco ACK lysis buffer for 7 minutes. Whole bone marrow was used for functional assays and flow cytometry as shown in Figure 7. Figure 5Lineage-negative cells were collected using the Direct Lineage Cell Depletion Kit (Miltenyi Biotec) according to the manufacturer's instructions.

[0124] Co-expression analysis and receptor quantification For untreated ( B6 mice were euthanized, and femurs and tibias were collected. Bone marrow was extracted from the bones by centrifugation, and RBCs were lysed on ice for 7 minutes using Gibco ACK lysis buffer. Cells were stained with fluorescent antibodies specific to the following targets: Flt3, CD117, CD34, CD127, lineage (“Lin”: CD3e, Gr-1, CD11b, B220, TER119), Sca1, CD16 / 32, SLAM, and CD110. Samples were analyzed on a BD Fortessa X-20 cytometer and a BangsLabs MESF AF647 Ladder, enabling quantification of CD117 and CD110 receptors in the HSPC population. Figure 6 ).

[0125] Fresh human bone marrow extracts were processed using a Ficoll density gradient within 24 hours of collection. The resulting bone marrow mononuclear cells (BMMCs) were stained with antibodies and analyzed on a BD Fortessa X-20 cytometer. The human antibody group consists of the following antibody / clone / fluorophore combination: CD34(561)-APC, lineage dump CD3(UCHT1) / CD14(HCD14) / CD16(3G8) / CD19(HIB19) / CD20(2H7) / CD56(HCD56)-FITC, CD90(5E10)-BV421, CD45RA(HI100)-BV605, CD38(HIT2)-PE-Cy7, CD49f(GoH3)-BV510, CD110(1.6.1)-PE or CD117(104D2)-PE and viability dye FVS780-APC-Cy7 (Figure 7).

[0126] Chimerism analysis Monocytes were collected from peripheral blood HetaSep (Stemcell Technologies) and RBCs were lysed using GibcoACK lysis buffer. To detect myeloid and lymphoid cell chimerism in peripheral blood, cells were stained with fluorescent antibodies specific to the following targets: CD19, CD11b, Ter119, CD45.2, NK1.1, Gr-1, CD45.1, and CD3. Dead cells were labeled with fluorescent viability dyes. Furthermore, in a secondary transplant functional depletion study, CD45.1, CD45.2, and GFP (supporting bone marrow) expressing cells were evaluated in recipient peripheral blood using antibodies unique to CD45.1 and CD45.2. Samples were analyzed on a BD Fortessa X-20 cytometer.

[0127] To assess bone marrow cell chimerism, animals were euthanized, and femurs and tibias were subsequently harvested. Bone marrow was isolated from the bones by centrifugation, followed by lysis of RBCs using Gibco ACK lysis buffer. Bone marrow cells were isolated from untreated C57BL / 6J mice and stained with fluorescent antibodies specific to the following targets: Flt3, CD117, CD34, CD127, lineages (“Lin”: CD3e, Gr-1, CD11b, B220, TER119), Sca1, CD16 / 32, SLAM, and CD110. Dead cells were labeled with fluorescent viability dyes. Samples were analyzed on a BD Fortessa X-20 cytometer.

[0128] CFU analysis Figure 7B Total colony forming units (CFU) were assessed in whole bone marrow from the Methodcult GF3434 (StemCell Technologies) plating and quantified using StemVision (StemCell Technologies) after 7 days.

[0129] result Wild-type B6 CD45.1 mice were treated with 25 mg / kg of anti-mouse CD117 (mCD117) mIgG2a and 25 mg / kg of anti-mouse CD110 (mCD110) mIgG2a, both of which exhibited effector activity. Seven days after treatment, 800,000 lineage-depleted bone marrow cells from C57Bl6 mice were transplanted into the animals. These cells differed from syngeneic transplanted cells only in the CD45 allele, thus allowing for differentiation. Figure 2 Donor cell chimerism in peripheral blood was assessed at 4, 8, 12, and 16 weeks post-transplantation. Figure 3As shown, although mCD117 mIgG2a alone resulted in 10% of transplants and mCD110 mIgG2a alone did not result in any transplants, co-administration of the two antibodies resulted in synergistic transplantation, such as robust peripheral blood myeloid chimerism (Mac-1+Gr-1+ cells). Figure 3 B), B-cell chimerism (CD19+ cells); Figure 3 C) T cell chimerism (CD3+ cells); Figure 3 D) and NK cell chimerism (NK1.1+ cells; Figure 3 As indicated by E), these chimerisms increase over time. These results suggest that antibody-based pretreatment protocols that simultaneously target CD110 and CD117 produce a synergistic response, enabling stable transplantation of donor HSPCs and thereby achieving multilineage hematopoietic reconstitution.

[0130] Previous reports have described the use of monoclonal antibodies to effectively precondition the bone marrow microenvironment, in combination with anti-mCD117 or anti-mCD110 antibodies, but only in combination with chemotherapy and regimens requiring administration for several days prior to donor stem cell transplantation. For example, the rat anti-mouse CD117 antibody ACK2 has been combined with the hypomethylated chemotherapeutic agent 5-azacytidine (AZA) to enable transplantation in a mouse bone marrow transplantation model. This preconditioning regimen requires six consecutive days of administration of 5-azacytidine (see, for example, Bankova et al.). Blood Adv 5, 19 (2021)). Rat anti-mouse CD110 antibody AMM2 has been combined with the chemotherapeutic agent 5-fluorouracil (5-FU) to enable transplantation (see, for example, Arai et al., Ann. NY Acad Sci , 1176 (2009) and Yoshihara et al., Cell Stem Cell , 1 (2007). This is in contrast to the approach presented in this paper, which combines two monoclonal antibodies targeting CD117 and CD110, respectively, in a single dose without the use of chemotherapeutic agents, thus avoiding the harm to the recipient associated with genotoxic pretreatment.

[0131] In the prior art embodiments cited above, the degree of chimerism observed in antibody-chemotherapy combinations varied considerably. The ACK2-AZA combination resulted in 30%-60% bone marrow chimerism, while the AMM2-5-FU combination resulted in 6% chimerism. Notably, the results presented herein show that, in the absence of chemotherapy, chimerism can reach as high as 60%-80% when anti-CD117 and anti-CD110 antibodies are co-administered in a single dose, which is surprising and unexpected given the low activity of each individual antibody. Figure 4 As shown, no transplantation was observed after administration of anti-CD110 alone (compared to Arai et al., Ann. NY Acad SciConsistent with the results reported by 1176 (2009), and only 1-10% of transplants were observed with anti-CD117 alone (compared to Bankova et al., 1176, 2009). Blood Adv Consistent with the results reported on 5, 19 (2021). Arai et al. ( Ann. NY Acad Sci 1176 (2009) proposed that, based on the different effects of ACK2 and AMM2 on different subpopulations (cycled cells and quiescent cells, respectively) in the HSC population, the combination of ACK2 and AMM2 can achieve a certain degree of transplantation. In such cases, the effect of the combination of ACK2 and AMM2 on transplantation is expected to be at most additive, thereby expanding the cell types (cycled and quiescent) targeted by these antibodies. Therefore, we demonstrate synergistic transplantation when antibodies simultaneously target CD117 and CD110, which is unpredictable given the current technology.

[0132] Figure 4 The unexpectedly excellent fit shown was obtained Figure 6 Further results provided in the study support this, demonstrating that CD110 and CD117 do not label distinct true long-term HSC cell populations. (See also...) Figure 6 As shown in B and 6C, in LT-HSC (Lin-CD117+Sca-1+Slam+Flt3- cells, Figure 6 The highest expression of both CD110 and CD117 was observed in A). Similarly, CD110 and CD117 were also observed in human bone marrow LT-HSC (Lin-CD34+CD38-CD45RA-CD90+CD49f+). Figure 8 (This is an expression from the CCP.)

[0133] Furthermore, it was observed that the combination of ACK2 and AMM2 did not lead to synergistic transplantation, as evidenced by a lower percentage of donor-derived cells in the recipient's bone marrow at 16 weeks post-transplantation. Figure 5 As demonstrated in B), this differs from that shown in (Arai, 2009). ACK2 is a rat antibody against mouse CD117 with the IgG2b isotype; while AMM2 is a rat antibody against mouse CD110 with the IgG1 isotype. Due to interspecies differences in interactions with host proteins (including, but not limited to, receptors regulating antibody cycling half-life or effector function), rat isotype antibodies in mice do not fully recapitulate all their functional effects or potency. When the Fc regions of ACK2 and AMM2 are modified from rat to mouse isotype IgG2a, synergistic effects of anti-CD117 and CD110 antibodies (e.g., ...) are observed. Figure 3 , Figure 4 , Figure 5(As shown in A). To assess the role of effector function in these versions, and since mouse IgG2a has full effector activity, we reengineered ACK2 and AMM2 into mouse IgG2a antibodies by removing their effector activity through a mutation in the previously described Fc region (N297A), which removes a potential glycosylation site and reduces Fc receptor binding (see, for example, Shields RL et al., (2001) J Biol Chem 276: 6591-604). The combination of effector-deficient antiCD117 and antiCD110 antibodies did not result in co-donor cell transplantation, but rather produced less than 1% transplantation, similar to what was observed with the combination of ACK2 and AMM2 ( Figure 5 B).

[0134] High donor chimerism was observed with combined anti-mCD117 and anti-mCD110 antibodies. Figure 3 Previously, there was a long-term, persistent depletion of HSCs / HSPCs from the bone marrow (Figure 7). Wild-type B6 CD45.1 mice were treated with 25 mg / kg of each of the effector activities of anti-mouse CD117 (mCD117) mIgG2a and anti-mouse CD110 (mCD110) mIgG2a. Animals were euthanized at 7, 9, and 12 days post-treatment, and bone marrow was evaluated for HSC / HSPC function, depletion, and the ability to support the continued production of hematopoietic lineages after transplantation into secondary recipients. Figure 7A The combination of mCD110 mIgG2a and mCD117 mIgG2a promoted deep and sustained depletion of HSPCs, as indicated by a reduction in colony-forming units (CFU) at all tested time points. Previous data have shown that treatment with mCD110 rIgG1 (AMM2) did not result in a reduction in CFU (Yoshihara et al., 2007). Conversely, by day 12, animals treated with anti-mCD117 mIgG2a alone recovered their multilineage HSPC differentiation and proliferative potential, as observed by the regeneration of CFU. Figure 7B Flow-based / phenotypic analyses of bone marrow-derived cells from these animals at 7 and 9 days post-treatment showed loss of HSPC (LSK) and LT-HSC following treatment with anti-mCD117 mIgG2a or a combination of anti-mCD117 IgG2a and anti-mCD110 IgG2a. By day 12 post-treatment, variable recovery of LSK was observed in some animals treated with anti-mCD117 IgG2a alone, while the loss of LSK and LT-HSC persisted in all animals treated with the anti-mCD117 / mCD110 combination. Figure 7CFurthermore, transplantation of bone marrow cells recovered from antibody-treated animals into irradiated secondary recipients revealed that bone marrow recovered from animals treated with a combination of mCD117 and mCD110 antibodies could not support or severely impair the support for hematopoietic cell (granulocyte) production in recipient mice, revealing the true depth and long-term functional depletion of HSCs from donor animals. Figure 7D Regardless of whether bone marrow recovery occurred on day 7, day 9, or day 12 after administration, this was consistent with the facts. Conversely, bone marrow from animals treated with anti-mCD117 mIgG2a alone was able to de novo support for HSC / HSPC-derived cells (granulocytes), indicating that although HSC / HSPC cells were phenotypically reduced ( Figure 7C However, sufficient residual HSC in secondary acceptors maintains donor chimerism. This persistent depletion and purging of the microenvironment enables the realization of Figure 3 , Figure 4 and Figure 5 Robust chimerism observed in A. Figure 7D The low level of lymphoid chimerism observed in the study originated from residual lymphoid cells in donor bone marrow that were not targeted by anti-CD110 and anti-CD117 antibodies.

[0135] In the above-cited prior art embodiments illustrating transplantation using the ACK2-AZA protocol, transplantation requires administration of ACK2 prior to AZA treatment and is highly dependent on ACK2's ability to block SCF binding to CD117, as co-administration of SCF or use of another anti-mouse CD117 antibody (2B8) with only partial SCF-blocking ability negatively impacts LT-HSC depletion and / or subsequent transplantation (Bankova et al.). Blood Adv 5, 19 (2021)). We observed that only the effector-active versions of the anti-CD110 and anti-CD117 antibody combination achieved synergistic pretreatment, while the effector-deficient versions failed to lead to durable donor cell transplantation, suggesting a different mechanism of action compared to previously described antibody-chemotherapy regimens. There is also the hypothesis (Arai et al., 5, 19 (2021)). Ann. NY Acad Sci The lack of observed single-agent effect with AMM2 alone could be due to the negative impact of residual circulating antibodies on donor cell amplification after BMT. We observed a synergistic, durable long-term chimerism achieved by combining anti-CD117 and anti-CD110 antibodies, contrary to this hypothesis, and thus both novel and unexpected.

[0136] In summary, we describe a novel antibody-based pretreatment protocol that simultaneously targets CD110 and CD117 co-expressed on HSPCs and LT-HSCs, generating a synergistic response that enables stable transplantation of donor HSPCs and facilitates multi-lineage hematopoietic reconstitution.

[0137] All publications and patent applications referenced in this specification are incorporated herein by reference as if each individual publication or patent application were expressly and individually indicated to be incorporated by reference. Although the claimed subject matter has been described with reference to various embodiments, those skilled in the art will understand that various modifications, substitutions, omissions, and alterations may be made without departing from its spirit. Therefore, the scope of this subject matter is intended to be defined solely by the scope of the following claims (including their equivalents).

Claims

1. A method for depleting endogenous hematopoietic stem cells and / or hematopoietic pluripotent progenitor cells (HSPCs) in a subject, the method comprising administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising: i. A first targeting portion, wherein the first targeting portion specifically binds to CD117; and ii. A second targeting portion, which specifically binds to CD110; The first and second targeting portions include Fc regions in the object that are capable of functionally engaging the host FcRn and mediating effector functions.

2. The method of claim 1, wherein the first targeting portion and the second targeting portion synergistically induce the depletion of the endogenous hematopoietic stem cells and / or hematopoietic pluripotent progenitor cells via Fc effector functions.

3. The method of claim 1 or 2, wherein the first targeting portion and the second targeting portion are combined with hematopoietic stem cells and / or hematopoietic pluripotent progenitor cells expressing both CD117 and CD110.

4. The method of claim 3, wherein the hematopoietic stem cells expressing both CD117 and CD110 are long-term hematopoietic stem cells (LT-HSC).

5. The method of any one of claims 1 to 4, wherein the first targeting portion and the second targeting portion do not contain toxins.

6. The method of any one of claims 1 to 5, wherein the object is immune-active.

7. The method of any one of claims 1 to 6, wherein the method does not include administering radiotherapy or chemotherapy to the subject.

8. The method of any one of claims 1 to 7, wherein the first targeting portion comprises a separated antibody or an antigen-binding fragment thereof that specifically binds to CD117.

9. The method of claim 8, wherein the isolated antibody or its antigen-binding fragment that specifically binds to CD117 functionally disrupts signal transduction between stem cell factor (SCF) and CD117.

10. The method of any one of claims 1 to 9, wherein the second targeting portion comprises a separated antibody or an antigen-binding fragment thereof that specifically binds to CD110.

11. The method of claim 10, wherein the isolated antibody or its antigen-binding fragment that specifically binds to CD110 functionally disrupts the signal transduction between thrombopoietin (TPO) and CD110.

12. The method of any one of claims 8 to 11, wherein the isolated antibody of the first and / or second targeting portion is a monoclonal antibody.

13. The method of any one of claims 8 to 11, wherein the antigen-binding fragment of the first and / or second targeting portion is selected from: Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, scFv-Fc fragments, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (dsFv), fragments containing VL or VH domains, heavy chain antibodies (hcAb), single-domain antibodies (sdAb), microantibodies, and variable domains (VHH or nanobodies) derived from camel heavy chain antibodies.

14. The method of any one of claims 1 to 13, wherein the first targeting portion and the second targeting portion are both contained on the same antibody or its antigen-binding fragment.

15. The method of claim 14, wherein the antibody or its antigen-binding fragment is selected from: bifunctional antibodies, bifunctional antibody-Fc, single-chain bifunctional antibodies, tandem bifunctional antibodies (Tandab's), tandem scFv, tandem scFv-scFc, tandem di-scFv, tandem tri-scFv, multivalent antibodies, bivalent or bispecific single-chain variable fragments, bispecific IgG, and bispecific Fab-IgG.

16. The method of any one of claims 8 to 15, wherein the isolated antibody or antigen-binding fragment of the first and / or second targeting portion is chimeric, humanized, or human.

17. The method of any one of claims 1 to 16, wherein the isolated antibody or antigen-binding fragment of the first and / or second targeting portion comprises a human Fc region.

18. The method of any one of claims 1 to 17, wherein the object is a person.

19. A method for hematopoietic stem cell transplantation in a recipient, the method comprising: a. Depleting the endogenous hematopoietic stem cells and / or hematopoietic pluripotent progenitor cells in the subject according to any one of claims 1 to 19; and b. Administer exogenous hematopoietic stem cells to the subject.

20. The method of claim 19, wherein the administration of effective amounts of the first and second targeting portions synergistically mediates the transplantation of the exogenous hematopoietic stem cells in the subject.

21. The method of claim 20, wherein administration of exogenous hematopoietic stem cells to the subject results in at least 10% donor cell chimerism.

22. The method of claim 21, wherein the donor cell chimerism is at least 55%.

23. The method of any one of claims 19 to 22, wherein the transplantation of the exogenous hematopoietic stem cells results in multilineage reconstitution in the subject.

24. The method of any one of claims 19 to 23, further comprising monitoring the depletion of the subject's endogenous hematopoietic stem cells prior to administration of exogenous hematopoietic stem cells.

25. The method of any one of claims 19 to 24, wherein the exogenous hematopoietic stem cells are administered to the subject after the first and second target portions have been substantially removed from the subject's blood.

26. The method of any one of claims 19 to 25, wherein the administration of exogenous hematopoietic stem cells to the subject occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more days after the administration of the first and second targeting portions to the subject.

27. The method of any one of claims 19 to 26, wherein the exogenous hematopoietic stem cells are allogeneic hematopoietic stem cells.

28. The method of any one of claims 19 to 26, wherein the exogenous hematopoietic stem cells are autologous hematopoietic stem cells.

29. The method of any one of claims 19 to 28, wherein the exogenous hematopoietic stem cells comprise CD34+ hematopoietic stem cells and progenitor cells (HSPCs).

30. The method of claim 29, wherein the CD34+ HSPC comprises CD34+ / CD38- / CD90+ HSPC.

31. The method of claim 29, wherein the CD34+ HSPC comprises CD34+ / CD38- / CD90+ / CD45RA-HSPC.

32. The method of any one of claims 19 to 31, further comprising one or more of the following steps: a. Collect hematopoietic stem cell populations from the subject before depletion; b. Culture the collected hematopoietic stem cell population; and c. Cryopreservation of the collected hematopoietic stem cell population.

33. The method of claim 32, wherein collecting the hematopoietic stem cell population from the object comprises one or more of the following steps: a. Mobilizing the aforementioned hematopoietic stem cell population; and b. Collect the hematopoietic stem cell population using apheresis.

34. The method of any one of claims 19 to 33, wherein the exogenous hematopoietic stem cells are genetically modified.

35. The method of claim 34, wherein the exogenous hematopoietic stem cells are genetically modified using one or more components of a gene editing system.

36. The method of claim 35, wherein the one or more components of the gene editing system are selected from: (i) CRISPR / Cas guide RNA, (ii) a DNA molecule encoding CRISPR / Cas guide RNA, (iii) a nucleic acid molecule encoding a CRISPR / Cas RNA guide polypeptide, (iv) a CRISPR / Cas RNA guide polypeptide, (v) a CRISPR / Cas guide RNA complexed with a CRISPR / Cas RNA guide polypeptide, (vi) a nucleic acid molecule encoding a zinc finger protein (ZFP), (vii) ZFP, (viii) a nucleic acid molecule encoding a transcription activator-like effector (TALE) protein, (ix) a TALE protein, and (x) a DNA donor polynucleotide.

37. The method of claim 36, wherein the CRISPR / Cas RNA guide peptide is a base editor or a leader editor.

38. The method of claim 36, wherein one or more components of the gene editing system comprise a nuclease capable of generating double-strand breaks within a gene locus in a cell.

39. The method of claim 36, wherein one or more components of the gene editing system further comprise a DNA donor polynucleotide.

40. The method of claim 39, wherein the DNA donor polynucleotide comprises non-overlapping 5' and 3' homologous arms, wherein each homologous arm is homologous to a portion of the locus, so that once the double-strand break is generated within the locus by the nuclease, the donor polynucleotide sequence can be integrated into the locus via homology-directed repair (HDR).

41. The method of claim 36, wherein the gene editing system comprises a CRISPR nuclease and a single guide RNA (sgRNA) capable of hybridizing with a target sequence within the locus, wherein the sgRNA guides the CRISPR nuclease to the target sequence.

42. The method of claim 41, wherein the CRISPR nuclease is a Cas protein.

43. The method of claim 41, wherein the sgRNA and the CRISPR nuclease are formed in a ribonucleoprotein (RNP) complex.

44. The method of any one of claims 35 to 43, wherein the gene modification corrects a gene mutation, replaces a mutant allele carrying a wild-type allele, or inserts a nucleic acid sequence encoding a therapeutic protein.

45. The method of any one of claims 1 to 44, wherein the subject suffers from a disease.

46. ​​The method of claim 45, wherein the disease is hemoglobinopathies.

47. The method of claim 46, wherein the hemoglobinopathies are selected from sickle cell disease, α-thalassemia, β-thalassemia, and δ-thalassemia.

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