Compositions and methods for non-genotoxic conditioning

Abstract

Provided herein are methods and compositions for the use of anti-CD110 and anti-CD117 conditioning agents for depleting endogenous hematopoietic stem cells in a subject, for example, prior to hematopoietic stem cell transplantation. Also provided are cell-based therapeutic methods and compositions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS [1] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 401,910, filed August 29, 2022, which is incorporated herein by reference in its entirety.

[0002] [2] Provided herein are methods and compositions relating to the use of antibody compositions to deplete hematopoietic stem cells in subjects. The disclosed methods and compositions are useful, for example, for non-myeloablative conditioning prior to allogeneic and autologous hematopoietic stem cell transplantation (HSCT). [Background technology]

[0003] [3] Lifelong production of hematopoietic cells within an individual relies on a rare population of hematopoietic stem cells with the capacity for self-renewal. This unique property makes hematopoietic stem cell transplantation (HSCT) a powerful treatment with the potential to correct a variety of diseases, including, but not limited to, hemoglobinopathies, autoimmune diseases, and hematologic malignancies. Before undergoing HSCT, recipients must undergo conditioning, which has the following objectives: (1) resetting the immune system (in the case of non-autologous transplants), (2) removing the microenvironment, and (3) preparing the bone marrow niche for donor cell engraftment, thereby enabling hematopoietic reconstitution with donor hematopoietic stem cells. Traditional conditioning regimens include the administration of chemotherapy, radiation, and / or immunosuppression. These methods are highly toxic in the short and long term and can cause many life-threatening side effects, including hematologic malignancies, organ damage, organ failure, and infections (Gyurkocza et al. Blood (2014), 124:344-353). Therefore, there is a need for less genotoxic or non-genotoxic conditioning regimens that would enable a broader patient population to receive effective yet safer HSCT therapy.

[0004] [4] Recent efforts have focused on developing non-genotoxic conditioning regimens, including those utilizing monoclonal antibodies that block hematopoietic stem cell survival factors, CAR T cell-mediated conditioning, 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 Biotechnology 738-745(2016)). One such antibody-based approach targets CD117 for hematopoietic stem cell depletion. Although CD117 is highly expressed on hematopoietic stem and progenitor cells, strategies targeting CD117 alone are insufficient to prepare immunocompetent subjects for successful hematopoietic stem cell transplantation (see, e.g., Xue et al., Blood 116, 5419–5422 (2010)). Instead, combined anti-CD117 and CD47 blockade is required (see, e.g., Chhabra et al., Science Translational Medicine 351ra105 (2016)). Alternatively, CD117 antibodies must be combined with toxins to promote depletion of endogenous hematopoietic stem cells and allow donor cell engraftment (see, e.g., Czechowicz et al., Nat Commun 10, 617 (2019)). Therefore, additional antibody-based conditioning regimens are needed that can promote robust hematopoietic stem cell depletion and engraftment while significantly reducing HSCT morbidity and mortality. Summary of the Invention [Problem to be solved by the invention]

[0005] [5] Provided herein are methods and compositions related to the use of anti-CD110 and anti-CD117 conditioning agents, e.g., antibodies or antigen-binding fragments thereof, for depletion of endogenous hematopoietic stem cells in a subject, e.g., prior to HSCT. Also provided are cell-based therapeutic methods and compositions. While not intending to be bound by a particular theory of action, the examples provided below demonstrate that simultaneous targeting of CD110 and CD117, which are co-expressed on hematopoietic stem cells, with antibodies that utilize Fc effector cell-mediated removal results in potent and synergistic depletion of endogenous hematopoietic stem cells and engraftment of donor hematopoietic stem cells, followed by multilineage hematopoietic reconstitution in immunocompetent mice. Because this non-genotoxic conditioning occurs without the use of non-selective myeloablative conditioning agents, such as radiation or chemotherapy, simultaneous targeting of CD110 and CD117 has the potential to broaden the use of hematopoietic stem cell transplantation therapy across a broader range of patients with diverse diseases and conditions. [Means for solving the problem]

[0006] [6] Thus, in one aspect, a method for hematopoietic stem cell engraftment in a subject in need thereof is provided, comprising: (a) administering to the subject a pharmaceutical composition comprising: (i) a first targeting moiety that specifically binds to CD117; and (ii) a second targeting moiety that specifically binds to CD110, thereby depleting endogenous hematopoietic stem cells in the subject; and (b) administering exogenous hematopoietic stem cells to the subject; wherein administration of the pharmaceutical composition mediates engraftment of the exogenous hematopoietic stem cells, resulting in multilineage hematopoietic reconstitution in the subject. In some embodiments, the first and second moieties bind to hematopoietic stem cells (HSCs) that co-express CD117 and CD110. In some embodiments, the HSCs that co-express CD117 and CD110 are long-term hematopoietic stem cells (LT-HSCs).

[0007] [7] In some embodiments, the first targeting moiety comprises an isolated antibody or antigen-binding fragment thereof that specifically binds to CD117. In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to CD117 functionally interferes with signaling between stem cell factor (SCF) and CD117 and / or mediates elimination of CD117-expressing cells via Fc effector function. In some embodiments, the second targeting moiety comprises an isolated antibody or antigen-binding fragment thereof that specifically binds to CD110. In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to CD110 functionally interferes with signaling between thrombopoietin (TPO) and CD110 and / or mediates elimination of CD110-expressing cells via Fc effector function. In some embodiments, the isolated antibodies of the first and / or second targeting moieties are monoclonal antibodies. In some embodiments, the antigen-binding fragment of the first and / or second targeting moiety is selected from the group consisting of an Fv fragment, a Fab fragment, a F(ab')2 fragment, a Fab' fragment, an scFv (sFv) fragment, an scFv-Fc fragment, a single-chain Fv (scFv), a single-chain antibody, a disulfide-linked Fv (dsFv), a fragment comprising either a VL or VH domain, a heavy-chain antibody (hcAb), a single-domain antibody (sdAb), a minibody, and a variable domain derived from a camelid heavy-chain antibody (VHH or nanobody). In some embodiments, both the first targeting moiety and the second targeting moiety are comprised in the same antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a diabody, a diabody-Fc, a single-chain diabody, a tandem diabody (Tandab), a tandem scFv, a tandem scFv-scFc, a tandem di-scFv, a tandem tri-scFv, a multivalent antibody, a bivalent or bispecific single-chain variable fragment, a bispecific IgG, and a Fab-IgG bispecific. In some embodiments, the isolated antibody or antigen-binding fragment of the first and / or second targeting moiety comprises an Fc region capable of binding to the neonatal Fc receptor (FcRn) of the subject.In some embodiments, the isolated antibody or antigen-binding fragment of the first and / or second targeting moiety is chimeric, humanized, or human. In some embodiments, the isolated antibody or antigen-binding fragment of the first and / or second targeting moiety comprises a human Fc region.

[0008] [8] In another aspect, provided herein is a method for hematopoietic stem cell engraftment in a subject in need thereof, comprising: (a) co-administering to the subject: (i) an effective amount of a first isolated antibody or antigen-binding fragment thereof that specifically binds to CD117; and (ii) an effective amount of a second isolated antibody or antigen-binding fragment thereof that specifically binds to CD110, thereby depleting endogenous hematopoietic stem cells in the subject; and (b) administering exogenous hematopoietic stem cells to the subject, wherein co-administration of effective amounts of the first and second antibodies or fragments thereof synergistically mediates engraftment of the exogenous hematopoietic stem cells, resulting in multilineage hematopoietic reconstitution in the subject. In some embodiments, the first and second isolated antibodies or antigen-binding fragments thereof bind to hematopoietic stem cells that co-express CD117 and CD110. In some embodiments, the HSCs that co-express CD117 and CD110 are LT-HSCs.

[0009] [9] In some embodiments, the first isolated antibody or antigen-binding fragment thereof functionally interferes with signaling between stem cell factor (SCF) and CD117 and / or mediates elimination of CD117-expressing cells via Fc effector function. In some embodiments, the second isolated antibody or antigen-binding fragment thereof functionally interferes with signaling between thrombopoietin (TPO) and CD110 and / or mediates elimination of CD110-expressing cells via Fc effector function. In some embodiments, the first isolated antibody and / or the second isolated antibody are monoclonal antibodies. In some embodiments, the first isolated antibody and / or the second isolated antibody are bispecific antibodies. In some embodiments, the antigen-binding fragment that specifically binds to CD117 is selected from the group consisting of an Fv fragment, a Fab fragment, a F(ab')2 fragment, a Fab' fragment, an scFv (sFv) fragment, an scFv-Fc fragment, and a nanobody fragment. In some embodiments, the antigen-binding fragment that specifically binds to CD110 is selected from the group consisting of an Fv fragment, a Fab fragment, a F(ab')2 fragment, a Fab' fragment, an scFv (sFv) fragment, an scFv-Fc fragment, and a nanobody fragment. In some embodiments, the Fc region of the first isolated antibody and / or the second isolated antibody is capable of binding to the neonatal Fc receptor (FcRn) of the subject. In some embodiments, the first isolated antibody or antigen-binding fragment thereof and / or the second isolated antibody or antigen-binding fragment thereof is chimeric, humanized, or human. In some embodiments, the first isolated antibody or antigen-binding fragment thereof and / or the second isolated antibody or antigen-binding fragment thereof comprises a human Fc region. In some embodiments, the subject is a human.

[0010]

[10] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD117 and / or the antibody or antigen-binding fragment thereof that specifically binds to CD110 is conjugated to a toxin. In some embodiments, the toxin is selected from the group consisting of saporin, saporin derivatives, ricin, abrin, gelonin, momordin, apitoxin, shiga toxin, shiga-like toxin, T-2 mycotoxin, diphtheria toxin, busulfan, Pseudomonas exotoxin A, ricin A chain derivatives, trichosanthin, raffin toxin, maytansine, amatoxin, mechlorethamine, cyclophosphamide, ethyleneimine, methylmelamine, methotrexate, fluorouracil, floxuridine, cytarabine, mercaptopurine, azathioprine, thioguanine, fludarabine phosphate, cladribine, drostatin, auristatin, auristatin E, auristatin F, MMAF, MMAE, MMAD, DMAF, or DMAE, maytansine, DM1 or DM4, duocarmycin, calicheamicin, pyrrolobenzodiazepines, exetecan, and any combination thereof.

[0011]

[11] In some embodiments, the methods provided herein further include monitoring the subject for depletion of endogenous hematopoietic stem cells prior to administering the exogenous hematopoietic stem cells. In some embodiments, the exogenous hematopoietic stem cells are administered to the subject after the first and second targeting moieties, or the first and second isolated antibodies or antigen-binding fragment(s) thereof, have been substantially removed from the subject's blood. In some embodiments, the administration of the exogenous hematopoietic stem cells to the subject occurs within 3, 5, 7, or 10 days of co-administering the first and second targeting moieties, or the first and second isolated antibodies or antigen-binding fragment(s) thereof to the subject.

[0012]

[12] 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 comprise CD34+ hematopoietic stem progenitor cells (HSPCs). In some embodiments, the CD34+ HSPCs comprise CD34+ / CD38- / CD90+ HSPCs. In some embodiments, the CD34+ HSPCs comprise CD34+ / CD38- / CD90+ / CD45RA- HSPCs.

[0013]

[13] In some embodiments, the methods provided herein further comprise one or more of the following steps: (a) collecting a hematopoietic stem cell population from the subject prior to depletion; (b) culturing the collected hematopoietic stem cell population; and (c) cryopreserving the collected hematopoietic stem cell population. In some embodiments, collecting a hematopoietic stem cell population from the subject comprises one or more of the following steps: (i) mobilizing the hematopoietic stem cell population, and (ii) collecting the hematopoietic stem cell population by apheresis.

[0014]

[14] In some embodiments, 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, the one or more components of the gene editing system are selected from the group consisting of: (i) a CRISPR / Cas guide RNA, (ii) a DNA molecule encoding a CRISPR / Cas guide RNA, (iii) a nucleic acid molecule encoding a CRISPR / CasRNA guide polypeptide, (iv) a CRISPR / CasRNA guide polypeptide, (v) a CRISPR / Cas guide RNA complexed with a CRISPR / CasRNA guide polypeptide, (vi) a nucleic acid molecule encoding a zinc finger protein (ZFP), (vii) a ZFP, (viii) a nucleic acid molecule encoding a transcription activator-like effector (TALE) protein, (ix) a TALE protein, and (x) a DNA donor polynucleotide. In some embodiments, the CRISPR / CasRNA guide polypeptide is a base editor or a prime editor. In some embodiments, the one or more components of the gene editing system comprise a nuclease capable of generating a double-strand break within a genetic locus of the cell. In some embodiments, one or more components of the gene editing system further comprise a DNA donor polynucleotide. In some embodiments, the DNA donor polynucleotide comprises non-overlapping 5' and 3' homology arms, each homology arm being homologous to a portion of the locus, and upon creation of a double-stranded break within the locus by a nuclease, the donor polynucleotide sequence is integrated into the locus by homology-directed repair (HDR).

[0015]

[15] In some embodiments, the gene editing system includes a CRISPR nuclease and a single guide RNA (sgRNA) capable of hybridizing to a target sequence within the locus, where the sgRNA guides the CRISPR nuclease to the target sequence. In some embodiments, the CRISPR nuclease is a Cas protein. In some embodiments, the Cas protein is Cas9 or a high-fidelity variant thereof. In some embodiments, the sgRNA and the CRISPR nuclease form a ribonucleoprotein (RNP) complex. In some embodiments, the sgRNA includes one or more chemically modified nucleotides. In some embodiments, the modified nucleotides are selected from the group consisting of 2'-O-methyl nucleotides, 2'-O-methyl 3'-phosphorothioate nucleotides, and 2'-O-methyl 3'-thioPACE nucleotides. In some embodiments, the 5' end, 3' end, or a combination thereof of the modified sgRNA includes the modified nucleotide. In some embodiments, the method further includes contacting the stem cell population with an AAV vector comprising a donor polynucleotide sequence. In some embodiments, the genetic modification corrects a genetic mutation, replaces a mutant allele with a wild-type allele, or inserts a nucleic acid sequence encoding a therapeutic protein.

[0016]

[16] In some embodiments, the subject is suffering from a disease. In some embodiments, the disease is a hemoglobinopathy. In some embodiments, the hemoglobinopathy is selected from the group consisting of sickle cell disease, α-thalassemia, β-thalassemia, and δ-thalassemia.

[0017]

[17] In another aspect, provided herein is a method for depleting endogenous hematopoietic stem cells in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising: (a) a first targeting moiety that specifically binds to CD117; and (b) a second targeting moiety that specifically binds to CD110. In some embodiments, administration of the pharmaceutical composition mediates depletion of exogenous hematopoietic stem cells in the subject. In some embodiments, the first and second moieties bind to hematopoietic stem cells that co-express CD117 and CD110. In some embodiments, the hematopoietic stem cells that co-express CD117 and CD110 are LT-HSCs.

[0018]

[18] In another aspect, provided herein is a method for depleting endogenous hematopoietic stem cells in a subject in need thereof, the method comprising co-administering to the subject: (a) an effective amount of a first isolated antibody, or antigen-binding fragment thereof, that specifically binds to CD117; and (b) an effective amount of a second isolated antibody, or antigen-binding fragment thereof, that specifically binds to CD110. In some embodiments, co-administration of effective amounts of the first and second antibodies, or fragments thereof, synergistically mediates the depletion of exogenous hematopoietic stem cells in the subject.

[0019]

[19] In another aspect, provided herein are compositions and kits comprising an antibody or antigen-binding fragment thereof that specifically binds to CD117; an antibody or antigen-binding fragment thereof that specifically binds to CD110; hematopoietic stem cells, and / or instructions for their preparation or use according to the methods described herein. The compositions, kits, and methods described herein can be used, for example, to treat and induce tolerance to cancer, autoimmune disorders, viral diseases, and blood disorders. [Brief explanation of the drawings]

[0020] [Figure 1]

[20] Figure 1 shows sensorgrams demonstrating the binding of antibodies to mouse CD110 or mouse CD117 as measured by biolayer interferometry (ForteBio Octet). (A) Anti-mCD110 antibody binds to recombinant mouse CD110 extracellular domain (ECD), and (B) anti-mCD117 antibody binds to recombinant mouse CD117 (ECD). [Figure 2]

[21] Figure 2 is a schematic outlining the study design protocol for conditioning recipients with anti-mCD117 and anti-mCD110 antibodies. [Figure 3]

[22] Figure 3 shows (A) total chimerism of donor-derived hematopoietic cells in peripheral blood at 4, 8, 12, and 16 days after transplantation after antibody-based conditioning. (B) Donor-derived blood chimerism of Gr-1+Mac-1+ myeloid cells, (C) CD19+ B cells, (D) CD3+ T cells, and (E) NK1.1+ NK cells. [Figure 4]

[23] Figure 4 shows the total chimerism of donor-derived hematopoietic cells in bone marrow 16 weeks after transplantation. Donor-derived bone marrow chimerism of (A) Lin-CD117+Sca1+ ("LSK") cells, (B) Lin-CD117-Sca1+SLAM+Flt3- ("LT-HSC") cells, (C) common myeloid progenitor ("CMP": Lin-CD117+Sca1-CD16 / 32-CD34+), (D) granulocyte-monocyte progenitor ("GMP": Lin-CD117+Sca1-CD16 / 32+CD34+), (E) megakaryocyte-erythroid progenitor ("MEP": Lin-CD117+Sca1-CD16 / 32-CD34-), and (F) common lymphoid progenitor ("CLP": Lin-CD117+Sca1+CD127+) populations. [Figure 5]

[24] Figure 5 shows donor-derived hematopoietic chimerism of Lin-CD117+Sca1+ ("LSK") and Lin-CD117+Sca1+SLAM+Flt3- ("LT-HSC") cells at 16 weeks post-transplant after antibody-based conditioning with antibodies of different Fc formats. (A) Chimerism of donor-derived LSK and LT-HSC populations after antibody-based conditioning with a regimen in which anti-mCD117 and anti-mCD110 antibodies have mouse Fc isotypes of the G2a isotype. (B) Chimerism of donor-derived LSK and LT-HSC populations after antibody-based conditioning with a regimen combining either ACK2 (anti-mCD117 rat IgG2b) and AMM2 (anti-mCD110 rat IgG1) or anti-mCD110 and anti-mCD117 with mouse IgG2a Fc with a mutation (N297A) that reduces binding to the Fc gamma receptor. [Figure 6]

[25] Figure 6 depicts the number of CD117 and CD110 receptors in the bone marrow of C57BL / 6J ("B6") mice. A) Representative gating of mouse HSPCs is shown for Lin- and Lin-CD117+Sca1+ ("LSK") cells. B) Median CD117 expression is shown for Lin-, LSK, LT-HSC, common myeloid progenitor ("CMP": Lin-CD117+Sca1-CD16 / 32-CD34+), granulocyte-monocyte progenitor ("GMP": Lin-CD117+Sca1-CD16 / 32+CD34+), megakaryocyte-erythroid progenitor ("MEP": Lin-CD117+Sca1-CD16 / 32-CD34-), and common lymphoid progenitor ("CLP": Lin-CD117+Sca1+CD127+) populations. C) Median CD110 expression is shown for Lin-, LSK, LT-HSC, CMP, GMP, MEP, and CLP populations. [Figure 7]

[26] Figure 7 shows the analysis of CD117 and CD110 expression in human bone marrow mononuclear cells isolated from bone marrow aspirates. Sample gating for evaluation of LT-HSCs (Lin-CD34+CD38-CD45RA-CD90+CD49f+) shows the expression of CD117 and CD110. DETAILED DESCRIPTION OF THE INVENTION

[0021] definition

[27] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. However, the meaning and scope of terms shall be clear, and in the event of any potential ambiguity, definitions provided herein shall take precedence over dictionary or extensional definitions. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. In this application, the use of "or" means "and / or" unless otherwise stated. Furthermore, the use of the term "including," as well as other forms such as "includes," "included," etc., is not limiting.

[0022]

[28] Generally, the nomenclature used in connection with and techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods known in the art and as described in the various general and more specific references cited and discussed throughout the specification, unless otherwise noted. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly practiced in the art or as described herein. The nomenclature used in connection with, and laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0023]

[29] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[30] The terms "about" and "approximately" refer to and encompass a stated value and ranges above and below that value. In certain embodiments, the term "about" refers to a range within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of a given value or range. In certain embodiments, the term "about" refers to the specified value plus or minus one standard deviation of that value.

[0024]

[31] The term "combination thereof" includes all possible combinations of the elements referred to by that term.

[32] The terms "CD110," "c-MPL," and "MPL" are used interchangeably herein. CD110 is also known by synonyms, including, among others, thrombopoietin receptor and myeloproliferative leukemia protein. Unless otherwise specified, these terms include any variants, isoforms, and species homologs of human CD110 naturally expressed by cells or expressed by cells transfected with the c-MPL gene. CD110 proteins include, for example, human CD110 (NCBI Reference Sequence: NP_005364.1). c-MPL genes include, for example, the human (Homo Sapiens) MPL proto-oncogene, thrombopoietin receptor (MPL), located on chromosome 1 in RefSeqGene (LRG_510) (NCBI Reference Sequence: NG_007525.1).

[0025]

[33] The terms "CD117" and "c-KIT" are used interchangeably herein. CD117 is also known by synonyms including, among others, the tyrosine protein kinase KIT and the adipose / stem cell growth factor receptor (SCFR). Unless otherwise specified, these terms include any variants, isoforms, and species homologs of human CD117 naturally expressed by cells or expressed by cells transfected with the c-KIT gene. CD117 proteins include, for example, human CD117 (NCBI Reference Sequences: NP_000213.1; and NP_001087241.1). c-KIT genes include, for example, the human KIT proto-oncogene, receptor tyrosine kinase (KIT), located on chromosome 4 in RefSeqGene (LRG_307) (NCBI Reference Sequence: NG_007456.1).

[0026]

[34] The term "immunoglobulin" refers to a class of structurally related proteins that generally contain two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In an "intact immunoglobulin," all four chains are interconnected by disulfide bonds. The structure of immunoglobulins is well characterized. See, for example, Paul, Fundamental Immunology, 7th ed., Chapter 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain typically contains a heavy chain variable region (V H ) and the heavy chain constant region (C H The heavy chain constant region typically comprises C H1 , C H2 , and C H3 Each light chain typically contains three domains, abbreviated as V L ) and a light chain constant region. The light chain constant region typically contains one domain, which is C L It is abbreviated as:

[0027]

[35] The term "antibody" describes a type of immunoglobulin molecule and is used herein in the broadest sense. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins) and antibody fragments. An antibody contains at least one antigen-binding domain. An example of an antigen-binding domain is V H -V L The antigen-binding domain is formed by a dimer. The antibodies described herein may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains specific for 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 may be conjugated to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof can be operatively linked (e.g., chemically linked, genetically fused, non-covalently linked, etc.) to one or more other molecular entities, such as another antibody or antibody fragment, thereby generating a bispecific or multispecific antibody having a second binding specificity. In some embodiments, the bispecific or multispecific antibodies described herein comprise binding specificities for both CD110 and CD117. In some embodiments, the multispecific antibodies described herein comprise binding specificities for CD110 and CD117.

[0028]

[36] An "antibody fragment" comprises a portion of an intact antibody, e.g., the antigen-binding or variable region of the intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, F(ab')2 fragments, scFv (sFv) fragments, scFv-Fc fragments, and nanobody fragments.

[0029]

[37] An "Fv" fragment comprises a non-covalently associated dimer of one heavy- and one light-chain variable domain.

[38] A "Fab" fragment contains the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain of the heavy chain (C H1 Fab fragments can be produced, for example, by recombinant methods or by papain digestion of a full-length antibody.

[0030]

[39] An "F(ab')2" fragment contains two Fab' fragments joined by a disulfide bond near the hinge region. F(ab')2 fragments can be produced, for example, by recombinant methods or by pepsin digestion of intact antibodies. F(ab')2 fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0031]

[40] "Single-chain Fv" or "sFv" or "scFv" antibody fragments contain V in a single polypeptide chain. H Domains and V L Includes domain. V H and V L are generally linked by a peptide linker. See Pluckthun A. (1994).

[0032]

[41] An "scFv-Fc" fragment comprises an scFv linked to an Fc domain. For example, the Fc domain can be linked to the C-terminus of the scFv. The Fc domain is located in the direction of the variable domains in the scFv (i.e., V H V L or V L V H ) depending on V H or V L Any suitable Fc domain known in the art or described herein can be used. In some cases, the Fc domain comprises an IgG1 Fc domain.

[0033]

[42] A "nanobody" fragment contains only the variable domain of the heavy chain and lacks the light and heavy chain constant domains. In some cases, nanobodies can be conjugated to other nanobodies and / or proteins to create multispecific proteins.

[0034]

[43] The antibodies described herein may also include additional antibody variants, including bispecific IgG and Fab-IgG bispecific fragments, such as diabodies, diabody-Fc, single-chain diabodies, tandem diabodies (Tandab), tandem scFv, tandem scFv-scFc, tandem di-scFv, tandem tri-scFv, "multivalent antibodies" (e.g., trivalent or tetravalent antibodies), bivalent or bispecific single-chain variable fragments, etc. Bis-scFv or di-scFv variants can be engineered by joining two scFv molecules with a linker. Bispecific antibodies may comprise two scFv molecules ((scFv)2) with different binding specificities. Ligation can be performed by creating a single peptide chain using two VH and two VL regions, thereby generating a tandem scFv (see, e.g., Kufer P. et al. (2004) Trends in Biotechnology 22(5):238-244). Diabodies can be generated using scFv molecules with linker peptides that are too short (e.g., about 5 amino acids) for the two variable regions to fold together, forcing the scFv to dimerize. See, e.g., Hollinger, Philipp et al. (July 1993) Proceedings of the National Academy of Sciences of the United States of America 90(14):644-8. Successfully purified multitarget affinity agents can be screened using a variety of in vitro and in vivo methods. Binding assays using engineered cell lines overexpressing CD110 or CD117, alone or in various combinations, can be used to screen for multitarget affinity agents that bind well to cells expressing CD110 and CD117. Cells can be incubated with the multitarget affinity agent and then treated with a fluorescently labeled secondary antibody. Flow cytometry can be used to detect the level of antibody binding to the engineered cells. Multitarget affinity agents are expected to bind well to cells coexpressing CD117 and CD110, thereby confirming their bispecific nature.Engineered cell lines can be labeled using various methods, such as co-expression of fluorescent proteins (e.g., GFP, YFP, EBFP) with CD110 and CD117, and tracked by flow cytometry. Alternatively, cells overexpressing target receptors can be individually stained with CellTrace proliferation dyes to label and monitor binding of multitarget affinity agents. In addition to engineered cell lines, multitarget affinity agents can be tested against primary cells with known levels of target receptors to confirm binding to relevant cell types.

[0035]

[44] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population. A substantially homogeneous antibody population contains antibodies that are substantially similar and bind to the same epitope(s), except for variants that may normally arise during the production of monoclonal antibodies. Such variants are generally present in small amounts. Monoclonal antibodies are typically obtained by a process that includes the selection of a single antibody from multiple clones. For example, the selection process can be the selection of a unique clone from multiple clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further modified, for example, to improve affinity for the target ("affinity maturation"), humanize the antibody, improve productivity in cell culture, and / or reduce immunogenicity in a subject.

[0036]

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

[0037]

[46] "Humanized" forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Humanized antibodies are generally human immunoglobulins (recipient antibodies) in which residues from one or more CDRs have been replaced with residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody, that has the desired specificity, affinity, or biological effect. In some cases, selected framework region residues of the recipient antibody are substituted for residues from the corresponding framework region of the donor antibody. Humanized antibodies may also contain residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to further improve antibody function. For further 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 by reference in its entirety.

[0038]

[47] A "human antibody" is one having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source that utilizes the human antibody repertoire or human antibody-encoding sequences (e.g., obtained from a human source or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0039]

[48] ​​An "isolated antibody" is an antibody that has been separated and / or recovered from a component of its natural environment. Components of natural environment include enzymes, hormones, and other proteinaceous or non-proteinaceous substances. In some embodiments, the isolated antibody is purified sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example, using a spinning cup sequenator. In some embodiments, the isolated antibody is purified to homogeneity by gel electrophoresis (e.g., SDS-PAGE) under reducing or non-reducing conditions and detection by Coomassie blue or silver staining. An isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. In some aspects, the isolated antibody is prepared by at least one purification step.

[0040]

[49] 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 in a solution comprising at least 85%, 90%, 95%, 98%, 99%, or 100% by weight. In some embodiments, the isolated antibody is provided in a solution comprising at least 85%, 90%, 95%, 98%, 99%, or 100% by volume.

[0041]

[50] "Affinity" refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y is determined by the dissociation constant (K D ) Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined using surface plasmon resonance (SPR) technology, such as, for example, a Biacore® instrument. In some embodiments, affinity is determined at 25°C.

[0042]

[51] With respect to antibody binding to a target molecule, the terms "specific binding," "binds specifically," "specific for," "selectively binds," and "selective for" refer to binding that is measurably different from nonspecific or nonselective interactions with respect to a particular antigen (e.g., CD110 or CD117) or epitope on a particular antigen. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. Specific binding can also be determined by competition with a control molecule that mimics the antibody binding site on the target. Specific binding is then indicated if binding of the antibody to the target is competitively inhibited by the control molecule. In some embodiments, "selectively binds" refers to the ability of a selective binding compound, such as an antibody or antigen-binding fragment thereof, to bind to a target protein, such as CD110 or CD117, with higher affinity than it binds to a non-target protein. In certain embodiments, specific binding refers to binding to a target with an affinity that is at least 10-fold, 50-fold, 100-fold, 250-fold, 500-fold, 1000-fold, or more, greater than the affinity for a non-target.

[0043]

[52] As used herein, "functionally disrupting" or "functionally disrupting" signaling between a stem cell surface receptor (e.g., CD110 or CD117) and its cognate ligand (e.g., thrombopoietin or stem cell factor, respectively) means that the interaction between the receptor and ligand is reduced, and the normal biological activity that would otherwise result from their interaction (e.g., hematopoietic stem cell proliferation) is attenuated. In some embodiments, the normal biological activity is eliminated. In some embodiments, functional disruption is caused by an antibody or antigen-binding fragment thereof that binds to the receptor or ligand and blocks or attenuates binding of the ligand to the receptor and / or antagonizes the function of the ligand or receptor, preventing normal signaling between the ligand and receptor. In other embodiments, functional disruption is achieved by mechanisms other than direct binding or inhibition of the receptor or ligand. For example, functional disruption may be achieved by binding and / or inhibition of cofactors, upstream signaling molecules, or downstream signaling molecules to the receptor or ligand that may be necessary for effective signaling between the ligand and receptor. In some embodiments, reduced function means that binding or signaling between the receptor and its cognate ligand is reduced 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 signaling between the receptor and its ligand under physiological conditions. Any method known in the art useful for assessing biological activity resulting from signaling between a receptor and its cognate ligand can be used to assess functional interference, including, but not limited to, cell proliferation assays and receptor competition assays.In other embodiments of the methods provided herein, binding of the target protein by the antibody or antigen-binding fragment thereof does not functionally interfere with signal transduction, but instead promotes immune-mediated depletion of such antibody-bound cells, e.g., via ADCC, ADCP, or CDC.

[0044]

[53] As used herein, the term "synergistic," e.g., with respect to depletion of endogenous hematopoietic stem cells and / or engraftment of exogenous hematopoietic stem cells in a subject, refers to a combination of conditioning agents described herein (e.g., the use of an anti-CD110 antibody and an anti-CD117 antibody) that is more effective than the additive effects of the conditioning agents alone. For example, the synergistic effect of a combination of antibodies may allow for the use of lower doses and / or less frequent administration of one or more of the antibodies to a subject. The use of lower doses and / or less frequent administration of the antibodies may reduce the toxicity associated with administering the conditioning agent to a subject without reducing the effectiveness of the conditioning agent in depleting endogenous hematopoietic stem cells and engrafting exogenous hematopoietic stem cells. Furthermore, a synergistic effect may result in improved efficacy of subsequent HSCT therapy in preventing, managing, treating, or ameliorating certain diseases, such as hemoglobinopathies. Additionally, the synergistic effect of a combination of conditioning agents may avoid or reduce adverse or unwanted side effects associated with the use of any single conditioning agent.

[0045]

[54] As used herein, the terms "subject," "individual," or "patient" refer interchangeably to warm-blooded animals such as mammals. In certain embodiments, the terms refer to humans. A subject may have, be suspected of having, or be predisposed to a disease or disorder (e.g., a hemoglobinopathy) that may benefit from undergoing HSCT. The term also includes livestock, pet animals, or animals kept for research, such as horses, cattle, sheep, poultry, pigs, cats, dogs, zoo animals, goats, primates (e.g., cynomolgus or rhesus monkeys), and rodents (e.g., mice and rats). A "subject in need thereof" refers to a subject who has one or more symptoms of, has been diagnosed with, or is suspected of having or being predisposed to a disease or condition that may be treatable and / or potentially benefit from HSCT as described herein.

[0046]

[55] As used herein, the term "administration" refers to a method of providing a dose of a composition (e.g., an antibody and / or cell therapy composition) to a subject. The method of administration can vary depending on various factors (e.g., the pharmaceutical composition being administered and the severity of the condition, disease, or disorder being treated).

[0047]

[56] The term "treating" or "treatment" refers to any one of the following: ameliorating one or more symptoms of a disease or condition; preventing the onset of symptoms before they appear; slowing or completely preventing the progression of a disease or condition (which may be manifested by an increase in the period between relapse episodes, a delay or prevention of worsening of symptoms, etc.), promoting the onset of a period of remission; slowing irreversible damage that occurs in the progressive-chronic phase (both primary and secondary) of a disease or condition; delaying the onset of said progressive phase; or a combination thereof.

[0048]

[57] An "effective amount" refers to an amount of a compound or composition effective to achieve a particular biological, therapeutic, or prophylactic result as disclosed herein, including, but not limited to, hematopoietic stem cell depletion, exogenous hematopoietic stem cell engraftment, and treatment of a disease or condition as disclosed herein, as determined by any means suitable in the art.

[0049] Method for depleting endogenous hematopoietic stem cells

[58] Provided herein are methods and compositions that utilize selective, non-genotoxic conditioning agents to deplete endogenous hematopoietic stem cells from the bone marrow niche prior to HSCT. As described herein, ablation of endogenous hematopoietic stem cells can be achieved by simultaneously targeting CD110 and CD117 with selective antibodies or antibody fragments ("anti-CD110 and anti-CD117 conditioning agents"). After ablation and after the conditioning agent has been substantially removed from the recipient's circulation, exogenous donor hematopoietic stem cells can be introduced to occupy the same niche as the ablated endogenous hematopoietic stem cells. Anti-CD110 and anti-CD117 conditioning agents useful in the methods provided herein are described in detail below. In certain embodiments, the conditioning regimen does not include the use of high-dose non-selective myeloablative agents, such as radiation or chemotherapy, to optimally avoid concomitant toxicities such as myelosuppression, mucositis, and organ and tissue toxicity (e.g., gastrointestinal cells, hair growth), as well as the risk of secondary malignancies. In particular, the disclosed compositions and methods combine 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, which may promote efficient and long-term engraftment, multilineage hematopoietic reconstitution, and immune competence.

[0050] Anti-CD110 and anti-CD117 conditioning agents

[59] CD110

[60] CD110 (c-MPL), also known as the thrombopoietin receptor, is a mediator of thrombopoietin signaling and plays a key role in maintaining a quiescent, long-term hematopoietic stem cell population in the bone marrow niche. Thrombopoietin-CD110 signaling stimulates megakaryopoiesis and platelet production and directly regulates hematopoietic stem cell proliferation, as evidenced by the marked reduction in hematopoietic stem cells in mice knocked out of both thrombopoietin and CD110. 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.

[0051]

[61] Anti-CD110 conditioning agents useful in practicing the methods provided herein include targeting moieties, antibodies, and antigen-binding fragments thereof that specifically bind to CD110. In some embodiments, useful anti-CD110 targeting moieties, antibodies, and antigen-binding fragments thereof can functionally interfere with thrombopoietin-CD110 signaling. In other embodiments, useful anti-CD110 targeting moieties, antibodies, and antigen-binding fragments thereof do not functionally interfere with thrombopoietin-CD110 signaling. In some embodiments, an anti-CD110 conditioning agent is an isolated monoclonal antibody that specifically binds to CD110. In some embodiments, an anti-CD110 conditioning 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, an anti-CD110 conditioning 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 the group consisting of an Fv fragment, a Fab fragment, a F(ab')2 fragment, a Fab' fragment, an scFv (sFv) fragment, and an scFv-Fc fragment. In some embodiments, the antibody or antigen-binding fragment that specifically binds to CD110 is selected from the group consisting of a diabody, a diabody-Fc, a single-chain diabody, a tandem diabody (Tandab), a tandem scFv, a tandem scFv-scFc, a tandem di-scFv, a tandem tri-scFv, a multivalent antibody, a bivalent or bispecific single-chain variable fragment, a bispecific IgG, a Fab-IgG bispecific, a single-chain Fv (scFv), a single-chain antibody, a disulfide-linked Fv (dsFv), a fragment comprising either a VL or VH domain, a heavy-chain antibody (hcAb), a single-domain antibody (sdAb), a minibody, and a variable domain derived from a camelid heavy chain antibody (VHH or nanobody).Further useful antibody or antigen-binding fragment formats include those described in Wilkinson & Hale (2022), mAbs, 14:1, DOI:10.1080 / 19420862.2022.2123299. Suitable anti-CD110 conditioning agents include fully human, humanized, or chimeric antibodies that specifically bind to CD110. Humanized antibodies are particularly useful for in vivo applications in humans due to their low antigenicity. Similarly, caninized, feline, and murine antibodies are particularly useful for applications in dogs, cats, and other species, respectively.

[0052]

[62] In certain embodiments, the anti-CD110 conditioning agent is an anti-CD110 antibody or antigen-binding fragment thereof, comprising an Fc domain capable of binding to the host species' neonatal Fc receptor (FcRn). FcRn functions as a recycling or transcytosis receptor responsible for maintaining IgG and albumin in the circulation and transporting these two ligands bidirectionally across polarized cell barriers. Thus, binding of the Fc domain of an anti-CD110 antibody to the recipient's FcRn can confer to the anti-CD110 antibody pharmacodynamics and half-life similar to that of the recipient's native immunoglobulin (IgG). In some embodiments, similar effector functions, such as ADCC and ADCP functions and complement fixation, are also conferred. In some such embodiments, the binding affinity of the Fc domain of the anti-CD110 conditioning agent to the FcRn of a recipient is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the binding affinity of the recipient's native immunoglobulin (IgG) to the FcRn. In some embodiments, the anti-CD110 antibody is a human antibody, humanized antibody, or human chimeric antibody comprising an Fc domain (e.g., a human Fc domain) capable of binding to the FcRn of a human recipient. In some such embodiments, the binding affinity of the Fc domain of a human, humanized, or human chimeric antibody to human FcRn is within at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the binding affinity of the human recipient's native immunoglobulin (IgG) to the FcRn. In other embodiments, the anti-CD110 antibody is a murine, murine, or murine chimeric antibody that comprises an Fc domain (eg, a murine Fc domain) capable of binding to FcRn of the recipient mouse.

[0053]

[63] In other embodiments, the anti-CD110 conditioning agent is an anti-CD110 antibody or antigen-binding fragment thereof comprising an Fc domain with reduced binding to the recipient's FcRn. In some such embodiments, the binding affinity of the Fc domain of the anti-CD110 conditioning 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 native immunoglobulin (Ig) to the FcRn. In some embodiments, the Fc domain of the anti-CD110 conditioning agent is engineered to reduce effector function, such as ADCC and ADCP function and reduced or lost complement fixation. In some embodiments, the anti-CD110 conditioning agent is an anti-CD110 antibody or antigen-binding fragment thereof comprising an Fc domain with reduced binding to one or more of the recipient's Fc gamma receptors.

[0054]

[64] Non-limiting examples of suitable anti-CD110 antibodies include clones mAb-1.75, mAb-1.6, and mAb-1.111 (e.g., those described in International Patent Publication No. 2011 / 060076, incorporated 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 certain embodiments, the methods involve the use of an anti-CD110 antibody comprising the heavy and light chain complementarity determining regions (CDRs) of any of these antibodies. In certain embodiments, the methods involve the use of an anti-CD110 antibody comprising three heavy chain CDRs and three light chain CDRs of any of these antibodies. In certain embodiments, the methods involve the use of an anti-CD110 antibody comprising three heavy chain CDRs, three light chain CDRs, and framework regions of any of these antibodies. In certain embodiments, the methods involve the use of an anti-CD110 antibody comprising the variable heavy chain (V CDRs) of any of these antibodies. H ) and variable light chain (VL ) in a specific embodiment. In a specific embodiment, the anti-CD110 antibody is a chimeric human antibody. In a specific embodiment, the anti-CD110 antibody is a humanized antibody. In a specific embodiment, the anti-CD110 antibody is a human antibody. In some embodiments, the method comprises the use of an anti-CD110 antibody comprising: (1) the heavy and light chain complementarity determining regions (CDRs) of any of the above-mentioned anti-CD110 antibodies; and (2) a human Fc domain. In a specific embodiment, the method comprises the use of an anti-CD110 antibody comprising: (1) three heavy chain CDRs and three light chain CDRs of any of the above-mentioned anti-CD110 antibodies; and (2) a human Fc domain. In a specific embodiment, the method comprises the use of an anti-CD110 antibody comprising: (1) three heavy chain CDRs, three light chain CDRs, and framework regions of any of the above-mentioned anti-CD110 antibodies; and (2) a human Fc domain. In a specific embodiment, the method comprises the use of an anti-CD110 antibody comprising: (1) the three heavy chain CDRs, three light chain CDRs, and framework regions of any of the above-mentioned anti-CD110 antibodies; and (2) a human Fc domain. In a specific embodiment, the method comprises: (1) the variable heavy chain (V) of any of the above-mentioned anti-CD110 antibodies. H ) and variable light chain (V L ), and (2) the use of an anti-CD110 antibody comprising a human Fc domain. The anti-CD110 antibody may be in any format described herein.

[0055]

[65] In some embodiments, the anti-CD110 conditioning agent is conjugated to a toxin. Upon binding to CD110, the anti-CD110 antibody-drug conjugate (ADC) is internalized and delivers its toxic payload to ablate hematopoietic stem cells. In some embodiments, the toxin is selected from the group consisting of saporin, saporin derivatives, ricin, abrin, gelonin, mormoline, apitoxin, shiga toxin, shiga-like toxin, T-2 mycotoxin, diphtheria toxin, busulfan, Pseudomonas exotoxin A, ricin A chain derivatives, trichosanthin, raffintoxin, maytansine, amatoxin, mechlorethamine, cyclophosphamide, ethyleneimine, methylmelamine, methotrexate, fluorouracil, floxuridine, cytarabine, mercaptopurine, azathioprine, thioguanine, fludarabine phosphate, cladribine, drostatin, auristatin, auristatin E, auristatin F, MMAF, MMAE, MMAD, DMAF, or DMAE, maytansine, DM1 or DM4, duocarmycin, calicheamicin, pyrrolobenzodiazepines, exatecan, and any combination thereof.

[0056]

[66] CD117

[67] CD117 (c-Kit) is highly expressed on hematopoietic stem cells, multipotent progenitors (MPPs), and lineage-restricted progenitor cells, such as common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), megakaryocytic erythroid progenitors (MEPs), and common lymphoid progenitors (CLPs). Together with its ligand, stem cell factor (SCF), CD117 is essential for hematopoiesis. Upon binding to SCF, CD117 forms a dimer, activating its intrinsic tyrosine kinase activity, which then phosphorylates and activates signaling molecules that propagate intracellular signals. Signals transmitted through CD117 after interaction with SCF are important for the survival, proliferation, and differentiation of hematopoietic stem cells. (See, e.g., 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.

[0057]

[68] Anti-CD117 conditioning agents useful in practicing the methods provided herein include targeting moieties, antibodies, and antigen-binding fragments thereof that specifically bind to CD117. In some embodiments, useful anti-CD117 targeting moieties, antibodies, and antigen-binding fragments thereof can functionally disrupt SCF-CD117 signaling. In other embodiments, useful anti-CD110 targeting moieties, antibodies, and antigen-binding fragments thereof do not functionally disrupt SCF-CD117 signaling. In some embodiments, the anti-CD117 conditioning agent is an isolated monoclonal antibody that specifically binds to CD117. In some embodiments, the anti-CD117 conditioning 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 conditioning 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 the group consisting of an Fv fragment, a Fab fragment, a F(ab')2 fragment, a Fab' fragment, an scFv (sFv) fragment, and an scFv-Fc fragment. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD117 is selected from the group consisting of a diabody, a diabody-Fc, a single-chain diabody, a tandem diabody (Tandab), a tandem scFv, a tandem scFv-scFc, a tandem di-scFv, a tandem tri-scFv, a multivalent antibody, a bivalent or bispecific single-chain variable fragment, a bivalent or bispecific single-chain variable fragment, a bispecific IgG, a Fab-IgG bispecific, a single-chain Fv (scFv), a single-chain antibody, a disulfide-linked Fv (dsFv), a fragment comprising either a VL or a VH domain, a heavy-chain antibody (hcAb), a single-domain antibody (sdAb), a minibody, and a variable domain derived from a camelid heavy-chain antibody (VHH or nanobody).Further useful antibody or antigen-binding fragment formats include those described in Wilkinson & Hale (2022), mAbs, 14:1, DOI:10.1080 / 19420862.2022.2123299. Suitable anti-CD117 conditioning agents include fully human, humanized, or chimeric antibodies that specifically bind to CD117. Humanized antibodies are particularly useful for in vivo applications in humans due to their low antigenicity. Similarly, caninized, feline, and murine antibodies are particularly useful for applications in dogs, cats, and other species, respectively.

[0058]

[69] In certain embodiments, the anti-CD117 conditioning agent is an anti-CD117 antibody or antigen-binding fragment thereof containing an Fc domain capable of binding to the host species' neonatal Fc receptor (FcRn). FcRn functions as a recycling or transcytosis receptor responsible for maintaining IgG and albumin in the circulation and transporting these two ligands bidirectionally across the polarized cell barrier. Thus, binding of the Fc domain of the anti-CD117 antibody to the recipient's FcRn can confer to the anti-CD117 antibody pharmacodynamics and half-life similar to those of the recipient's native immunoglobulin (IgG). In some embodiments, similar effector functions, such as ADCC and ADCP functions and complement fixation, are also conferred. In some such embodiments, the binding affinity of the Fc domain of the anti-CD117 conditioning agent to the FcRn of the recipient is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the binding affinity of the recipient's native immunoglobulin (IgG) to the FcRn. In some embodiments, the anti-CD117 antibody is a human, humanized, or human chimeric antibody that comprises an Fc domain (e.g., a human Fc domain) capable of binding to the FcRn of a human recipient. In some such embodiments, the binding affinity of the Fc domain of a human, humanized, or human-chimeric antibody to human FcRn is at least 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 native immunoglobulin (IgG) to FcRn. In other embodiments, the anti-CD117 antibody is a murine, murinized, or murine-chimeric antibody that comprises an Fc domain (e.g., a murine Fc domain) capable of binding to the FcRn of a recipient mouse.

[0059]

[70] In other embodiments, the anti-CD117 conditioning agent is an anti-CD117 antibody or antigen-binding fragment thereof comprising an Fc domain with reduced binding to the recipient's FcRn. In some such embodiments, the binding affinity of the Fc domain of the anti-CD117 conditioning 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 native immunoglobulin (IgG) to the FcRn. In some embodiments, the Fc domain of the anti-CD117 conditioning agent is engineered to reduce effector functions, such as ADCC and ADCP function and reduced or absent complement fixation. In some embodiments, the anti-CD117 conditioning agent is an anti-CD117 antibody or antigen-binding fragment thereof comprising an Fc domain with reduced binding to one or more of the recipient's Fc gamma receptors.

[0060]

[71] Non-limiting examples of suitable anti-CD117 antibodies include ACK-2 (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 certain embodiments, the method comprises the use of an anti-CD117 antibody comprising the heavy and light chain CDRs of any of these antibodies. In certain embodiments, the method comprises the use of an anti-CD117 antibody comprising three heavy chain CDRs and three light chain CDRs of any of these antibodies. In certain embodiments, the method comprises the use of an anti-CD117 antibody comprising the three heavy chain CDRs, three light chain CDRs, and framework regions of any of these antibodies. In certain embodiments, the method comprises administering to a subject a subject the V H and V LIn certain embodiments, the method comprises the use of an anti-CD117 antibody comprising: (1) the heavy and light chain complementarity-determining regions (CDRs) of any of the above-mentioned anti-CD117 antibodies; and (2) a human Fc domain. In certain embodiments, the method comprises the use of an anti-CD117 antibody comprising: (1) three heavy chain CDRs and three light chain CDRs of any of the above-mentioned anti-CD117 antibodies; and (2) a human Fc domain. In certain embodiments, the method comprises the use of an anti-CD117 antibody comprising: (1) three heavy chain CDRs and three light chain CDRs of any of the above-mentioned anti-CD117 antibodies; and (2) a human Fc domain. In certain embodiments, the method comprises the use of an anti-CD117 antibody comprising: (1) three heavy chain CDRs, three light chain CDRs, and framework regions of any of the above-mentioned anti-CD117 antibodies; and (2) a human Fc domain. In certain embodiments, the method comprises the use of an anti-CD117 antibody comprising: (1) the variable heavy chain (VH) and variable light chain (VL) of any of the anti-CD117 antibodies described above, and (2) a human Fc domain. The anti-CD117 antibody may be in any format described herein.

[0061]

[72] In some embodiments, the anti-CD117 conditioning agent is conjugated to a toxin. Upon binding to CD117, the anti-CD117 antibody-drug conjugate (ADC) is internalized and delivers its toxic payload to ablate hematopoietic stem cells. In some embodiments, the toxin is selected from the group consisting of saporin, saporin derivatives, ricin, abrin, gelonin, mormoldin, apitoxin, shiga toxin, shiga-like toxin, T-2 mycotoxin, diphtheria toxin, busulfan, Pseudomonas exotoxin A, ricin A chain derivatives, trichosanthin, raffintoxin, maytansine, amatoxin, mechlorethamine, cyclophosphamide, ethyleneimine, methylmelamine, methotrexate, fluorouracil, floxuridine, cytarabine, mercaptopurine, azathioprine, thioguanine, fludarabine phosphate, cladribine, dolastatin, auristatin, auristatin E, auristatin F, MMAF, MMAE, MMAD, DMAF, or DMAE, maytansine, DM1 or DM4, duocarmycin, calicheamicin, pyrrolobenzodiazepines, exatecan, and any combination thereof.

[0062] Pharmaceutical Conditioning Compositions and Dosage Forms

[73] In some embodiments, an effective dose of each of the anti-CD110 and anti-CD117 conditioning agents of the present disclosure, when administered in combination, depletes endogenous hematopoietic stem cells by at least 10-fold, at least 100-fold, at least 1000-fold, at least 100,000-fold or more relative to the level of hematopoietic stem cells present in the recipient's bone marrow niche prior to administration. Effective doses will vary depending on the individual and the particular conditioning agent, but will generally be 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 g, 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, up to about 15 mg / kg, up to about 25 mg / kg, up to about 50 mg / kg, up to about 100 mg / kg. In certain embodiments, the dose 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 certain embodiments, the dose 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.

[0063]

[74] Doses of one or both conditioning agents can be administered over a period of time, on a schedule deemed appropriate by one of skill in the art to result in the desired ablation of endogenous hematopoietic stem cells. In certain embodiments, doses are administered daily. In certain embodiments, doses are administered twice daily. In certain embodiments, doses are administered three times daily. In certain embodiments, doses are administered four times daily. In certain embodiments, doses are administered daily in divided doses. In some embodiments, doses are administered for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days, 1-2 days, 1-3 days, 1-4 days, 1-5 days, 1-6 days, 1-7 days, 1-10 days, or more.

[0064]

[75] The anti-CD110 and anti-CD117 conditioning agents may be formulated together or separately, but are administered simultaneously. As used herein, "concurrently" and "simultaneously" refer to the administration of at least two agents to a patient either at the same time or within the time period during which the effects of the first agent are still acting on the patient. For example, the second agent can be administered 1-2 days, preferably 1-7 days, after the administration of the first agent.

[0065]

[76] The anti-CD110 and anti-CD117 conditioning agents of the present disclosure can be formulated for administration in any composition and by any technique deemed useful by one of ordinary skill in the art. In some embodiments, the anti-CD110 and anti-CD117 conditioning agents are formulated as tablets, capsules, pills, syrups, ampoules, lozenges, or powders for oral administration to an individual. In some embodiments, the conditioning agents are formulated for intravenous infusion or injection. In some embodiments, the conditioning agents are pharmaceutical compositions or single-unit dosage forms. The pharmaceutical compositions and single-unit dosage forms provided herein comprise a prophylactically or therapeutically effective amount of one or both of the anti-CD110 and anti-CD117 conditioning agents.

[0066] HSCT and engraftment methods

[77] Monitoring the removal of conditioning agents before HSCT

[78] In some embodiments, after co-administration of the anti-CD110 and anti-CD117 conditioning agents, endogenous hematopoietic stem cells are removed from the recipient's bone marrow niche, allowing exogenous donor hematopoietic stem cells to newly occupy the niche. However, to avoid inadvertent removal of donor cells by any conditioning agent remaining in the recipient, the pharmacokinetic levels of one or both conditioning agents can be monitored for removal from the recipient's blood prior to HSCT. In some embodiments, the recipient undergoes HSCT only after one or both of the anti-CD110 and anti-CD117 conditioning agents have been substantially removed from the recipient's circulation.

[0067] 79 In some embodiments, an anti-CD110 or anti-CD117 conditioning agent is substantially removed from the circulation when it is no longer detectable using any method known in the art for measuring the presence and / or activity of a biological substance in blood or serum, e.g., as assessed from a recipient blood sample. In some embodiments, a conditioning agent is substantially removed from the circulation when it is no longer detectable above the background threshold of the assay used to detect the conditioning agent. Any method useful in the art for detecting antibodies or antibody fragments, e.g., ELISA-based detection assays, immunoprecipitation techniques, and immunoblot assays, can be used to assess removal of the conditioning agent. In certain embodiments, serum collected from the recipient at a specific time point after administration of the conditioning agent can be contacted with a sample of stem cells, e.g., donor hematopoietic stem cells, and binding of any conditioning agent in the serum to the stem cells can be assessed using conventional methods. In other embodiments, the contacted stem cells can be assessed for growth inhibition in the presence of the recipient's serum.

[0068] In some embodiments, once it has been determined that one or both of the anti-CD110 and anti-CD117 conditioning agents have been sufficiently removed from the recipient's circulation, the recipient may be administered exogenous hematopoietic stem cells. In some embodiments, sufficient removal is achieved when the serum level of the conditioning agent has decreased by a certain factor from the peak level of the conditioning agent after administration. In some embodiments, the conditioning agent is at least 10-fold, 100-fold, 1000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, or greater than 1,000,000-fold below peak levels prior to administration of the exogenous hematopoietic stem cells. In other embodiments, the exogenous donor hematopoietic stem cells can be administered based on known or predicted pharmacokinetics of the conditioning agent. In some embodiments, the exogenous donor hematopoietic stem cells are administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, or more than 10 days after co-administration of the anti-CD110 and anti-CD117 conditioning agents.

[0069] hematopoietic stem cell transplantation

[81] In certain embodiments, after administering the conditioning antibody and depleting endogenous hematopoietic stem cells, the method further comprises 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 and progenitor cells is administered to the patient. In some embodiments, the administered exogenous cells are donor bone marrow cells, umbilical cord blood cells, hematopoietic stem and progenitor cells (HSPCs), peripheral blood CD34 cells, or the like. + Cells, peripheral blood CD34 + and CD90 + The present invention may include a method for treating a cancer, ...

[0070]

[82] Hematopoietic stem cells can be any hematopoietic stem cells deemed useful by those skilled in the art. In certain embodiments, once engrafted, the exogenous hematopoietic stem cells can reconstitute the patient's hematopoiesis. Human hematopoiesis is defined by a hierarchy based on cell surface marker expression initiated by hematopoietic stem cells that undergo both self-renewal and differentiation into multipotent progenitor cells, which then give rise to lineage-restricted progenitor cells and, ultimately, 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 HSPC population, which includes multipotent progenitor cells (CD34 + / CD38 - / CD45RA - ), long-term repopulating cells in xenografted mice (CD34 + / CD38 - / CD90 + ), and a population highly enriched for hematopoietic stem cells (CD34 + / CD38 - / CD90+ / CD45RA - ) can be further classified as

[0071]

[83] In certain embodiments, the hematopoietic stem cells are of any subtype or colony-forming unit. In certain embodiments, the hematopoietic stem cells are colony-forming unit-granulocyte-erythroid-monocyte-megakaryocyte cells. In certain embodiments, the hematopoietic stem cells are colony-forming unit-erythroid cells. In certain embodiments, the hematopoietic stem cells are colony-forming unit-granulocyte-macrophage cells. In certain embodiments, the hematopoietic stem cells are colony-forming unit-megakaryocyte cells. In certain embodiments, the hematopoietic stem cells are colony-forming unit-basophil cells. In certain embodiments, the hematopoietic stem cells are colony-forming unit-eosinophil cells.

[0072]

[84] Hematopoietic stem cells can be obtained from any source deemed useful to one of skill in the art. In certain embodiments, the hematopoietic stem cells are from a donor. In certain embodiments, the donor is a patient. In certain embodiments, the donor is another subject of the same species, e.g., another human. In certain embodiments, the hematopoietic stem cells are autologous. In certain embodiments, the hematopoietic stem cells are allogeneic. In certain embodiments, the hematopoietic stem cells are syngeneic.

[0073]

[85] Hematopoietic stem cells can be collected by any technique deemed useful to one of skill in the art. In some embodiments, the donor subject is administered a hematopoietic stem cell mobilizing agent (e.g., plerixafor (Mozobil®), G-CSF, GM-CSF) prior to collection. In certain embodiments, hematopoietic stem cells are collected from peripheral blood. In certain embodiments, hematopoietic stem cells are collected from umbilical cord blood. In certain embodiments, hematopoietic stem cells are collected from bone marrow. In certain embodiments, the donor cell population can be obtained from a product collected from a subject, such as a patient or a subject requiring autologous HSCT. The product can be an 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 and / or progenitor cells (HSPCs). To obtain the stem cell population, CD34+ cells and / or HSPCs can be isolated or separated from other cells. Following isolation of CD34+ HSPCs, the resulting stem cell population is substantially free of non-CD34+ cells and is suitable for subsequent genetic manipulation.

[0074]

[86] In some embodiments, the collected hematopoietic stem cells are separated from the primary cell population using flow cytometry. Optionally, the flow cytometry comprises fluorescence-activated cell sorting (FACS). In other specific embodiments, the collected hematopoietic stem cells are separated from the primary cell population using magnetic bead separation. Optionally, the magnetic bead separation comprises magnetic-activated cell sorting (MACS). In other specific embodiments, the collected hematopoietic stem cells are separated using a device configured for hematopoietic stem cell enrichment, such as the Miltenyi Biotec CliniMACS cell manufacturing platform.

[0075]

[87] Methods for culturing or expanding primary hematopoietic stem cells are known in the art, for example, as 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 appropriate culture media, supplements, growth factors, etc. are both known and commercially available. Typically, human primary cells are maintained and expanded under serum-free conditions. Alternative media, supplements, growth factors, and / or alternative concentrations can be readily determined by one of skill in the art and are widely 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.

[0076]

[88] 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 discussed herein is performed using a cryopreserved cell population containing hematopoietic stem cells. Cells can be cryopreserved after hematopoietic stem cell collection or isolation, after culture initiation and activation, after modification (e.g., genetic modification), or after expansion or any process step. Freeze-thaw cycles may result in a more homogeneous hematopoietic stem cell composition by removing non-hematopoietic stem cell populations. Hematopoietic stem cells can be preserved by any technique deemed useful by those skilled in the art. In certain embodiments, harvested cells are formulated in cryopreservation medium and placed in a cryopreservation unit, such as a liquid nitrogen freezer (-195°C) or an ultra-low temperature freezer (-65°C, -80°C, -120°C), for long-term storage of at least 1 month, 2 months, 3 months, 4 months, 6 months, 1 year, 2 years, 3 years, or at least 5 years. In some embodiments, the thawed cells are conditioned by the methods described herein.

[0077]

[89] Genetically modified hematopoietic stem cells

[90] The HSCT methods described herein include transplantation of hematopoietic stem cells genetically modified to contain, for example, a therapeutic xenogeneic donor polynucleotide sequence. The donor polynucleotide sequences described herein can be incorporated into a wide variety of gene therapy constructs, for example, to deliver nucleic acids encoding proteins to a subject in need thereof. 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 a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV). Other vectors useful in gene therapy methods are known in the art. For example, constructs of the present disclosure may comprise an alphavirus, herpesvirus, retrovirus, lentivirus, or vaccinia virus. The exogenous sequence typically encodes a recombinant molecule to be expressed in cells, for example, for use in cell therapy. The process steps of the methods can also include all or part of washing, diluting, selecting, isolating, separating, culturing, stimulating, packaging, and / or formulating the cells. The methods generally allow for large scale processing of cells (such as compositions in volumes of greater than or about 50 mL), for example selection or separation and / or transduction.

[0078]

[91] In some embodiments, hematopoietic stem cells are gene-edited using site-specific nuclease-based gene editing applications to knock out targeted genomic sequences or knock in exogenous sequences, and to introduce exogenous sequences into cells by viral transduction using recombinant viral vectors. In some such embodiments, before performing a gene editing method (e.g., gene knockout, gene knock-in, gene correction), hematopoietic stem cells are collected by apheresis, concentrated from the apheresis product, and then cryopreserved. Cryopreservation can be performed after stem cell mobilization and collection (e.g., by apheresis) and hematopoietic stem cell selection. After cryopreservation, it can be assessed whether a threshold number of hematopoietic stem cells has been collected from the donor to proceed to subsequent gene editing steps. If the threshold number of cells is not obtained after a single round of mobilization, collection, selection, and cryopreservation, subsequent procedures can be performed until the threshold number of cells is obtained. The threshold number of hematopoietic stem cells to be collected can vary depending on many factors, including, but not limited to, the gene editing procedure performed (e.g., gene knockout, gene knockin, gene correction), the target gene being edited, the mechanism by which the target gene is modified (e.g., homology-dependent repair (HDR)), the efficiency of the editing procedure (e.g., HDR efficiency), and the treatment threshold for treating a particular disease. In some embodiments, the threshold number of hematopoietic stem cells to be collected from a donor prior to gene editing is about 1 x 10 4 ~1×10 5 , 1×10 5 ~1×10 6 , 1×10 6 ~1×10 7 In some embodiments, prior to gene editing, the number of cells is at least about 1 x 10 5 ~1×10 7 In some embodiments, at least about 1 x 10 cells / kg are collected. 4 , 2 × 10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4, 9×10 4 , 1×10 5 , 2 × 10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , or approximately 1 × 10 8 of hematopoietic stem cells / kg are collected before proceeding to gene editing of the collected cells. Once a threshold number of hematopoietic stem cells have been mobilized, collected, selected, and cryopreserved, the cells are then proceeded to thaw, culture, and gene editing.

[0079]

[92] In some embodiments, gene editing utilizes a nuclease introduced into a cell that can create a double-stranded break near or within a genomic target site, which can be useful for increasing the frequency of homologous recombination and HDR at or near the break site. In preferred embodiments, the nuclease's recognition sequence is present in the host cell genome only at the target site, thereby minimizing off-target binding and cleavage of the genome by the nuclease. Gene editing nucleases useful 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, lambda integrase) or resolvases; transposases; zinc finger nucleases (ZFNs); and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. Non-limiting examples of Cas proteins include Cas 1, 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, homologs thereof, or modified versions thereof.

[0080]

[93] 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 cells, a double-strand break can be introduced at a specific site as directed by the guide polynucleotide sequence and the CRISPR-associated Cas9 nuclease. A donor polynucleotide containing a sequence of interest can be further introduced into the cells, allowing the sequence of interest to be inserted into the cells by homologous-directed recombination. The donor polynucleotide sequence can be introduced by transduction. Viral transfer methods, such as transduction, generally involve incubating an input composition containing cells to be transduced and viral vector particles containing a vector in a centrifuge chamber under conditions that transduce the cells or initiate transduction in at least some cells of the input composition, thereby at least initiating transduction, and producing an output composition containing transduced cells.

[0081]

[94] Methods for introducing polypeptides, 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 a polypeptide or nucleic acid (e.g., a nucleotide sequence encoding a DNA nuclease or a modified sgRNA) into primary cells, such as human primary cells. Non-limiting examples of suitable methods include electroporation (e.g., nucleofection), virus or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, 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.

[0082]

[95] In some embodiments, the Cas nuclease can be in the form of a protein. In some embodiments, the Cas nuclease can be in the form of a plasmid, which allows cells harboring this expression construct to then express the Cas nuclease. In other embodiments, the Cas nuclease is pre-complexed with a guide RNA and introduced into cells as a ribonucleoprotein (RNP). In some embodiments, the Cas nuclease and guide polynucleotide sequence are introduced into CD34+ cells by electroporation.

[0083]

[96] Introduction of the donor polynucleotide can occur by viral transduction using a delivery vector such as an adeno-associated virus (AAV). Any serotype or pseudotype of AAV can be used. Certain AAV vectors are derived from single-stranded (ss) DNA parvoviruses that are nonpathogenic to mammals. Briefly, certain AAV vectors can be generated by removing the rep and cap viral genes, which comprise 96% of the original wild-type AAV genome, leaving flanking inverted terminal repeats (ITRs) that can be used to initiate viral DNA replication, packaging, and integration. Wild-type AAV integrates preferentially into the human host cell genome in a site-specific manner, preferentially at chromosome 19q13.3. Alternatively, AAV may be maintained episomally. To date, at least 12 human serotypes of AAV (AAV serotype 1 (AAV-1) through AAV-12) and over 100 nonhuman primate serotypes have been discovered. Any of these serotypes, as well as any combination thereof, can be used within the scope of the present disclosure. The serotype of the viral vector can be selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the serotype is AAV6.

[0084]

[97] In some embodiments, viral transduction occurs within 30 minutes after 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.

[0085]

[98] In another embodiment, hematopoietic stem cells are genetically modified using a gene editing application that utilizes a base editor. Base editing is a CRISPR-Cas9-based genome editing technique that allows for the introduction of point mutations into DNA without generating DSBs. Two major classes of base editors have been developed: cytidine base editors, or CBEs, which allow for C>T conversions, and adenine base editors, or ABEs, which allow for A>G conversions (see, e.g., Rees et al. (2018) Nat Rev Genet 19:770-788).

[0086]

[99] In another embodiment, hematopoietic stem cells are genetically modified using a gene editing application that utilizes a prime editor. The prime editor (PE) consists of nCas9 fused to a reverse transcriptase used in combination with a prime editing RNA (pegRNA, a guide RNA containing a template region for reverse transcription). Prime editing can introduce insertions, deletions (indels), and 12-base pair changes. Prime editing relies on the ability of a reverse transcriptase (RT) fused to a Cas nickase variant to convert the RNA sequence provided by the prime editing guide RNA (pegRNA) into DNA at the nick site generated by the Cas protein. The DNA flap generated in this process may or may not be included in the target DNA sequence. See, for example, Anzalone et al. (2019) Nature 576:149-157. Non-limiting examples of prime editing systems include PE1, PEI-M1, PE1-M2, PE1-M3, PE1-M6, PE1-M15, PE1-M3inv, PE2, PE3, PE3b.

[0087]

[0100] In other embodiments, the hematopoietic stem cells are transduced with a DNA guide polypeptide such as NgAgo (Natronobacterium gregoryi Argonaute), an RNA guide polypeptide (e.g., Cas9, CasX, CasY, Cpf1, etc.); a site-specific recombinase (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, lambda integrase, HK022 integrase), or a combination thereof. , HP1 integrase, etc.); resolvases and / or invertases (e.g., Gin, Hin, γδ3, Tn3, Sin, Beta, etc.); transposons and / or DNA derived from transposons (e.g., bacterial transposons such as Tn3, Tn5, Tn7, Tn9, Tn10, Tn903, Tn1681; Tc1 / mariner superfamily transposons, PiggyBac superfamily transposons, hAT superfamily transposons, PiggyBac, Sleeping Eukaryotic transposons such as Beauty, Frog Prince, Minos, and Himar1) are genetically modified using gene editing applications that utilize CRISPR-transposons, which direct RNA-guided transposition by natively combining the DNA integration capabilities of transposases with the target programming capabilities of CRISPR-Cas (e.g., 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)).

[0088]

[0101] Pharmaceutical hematopoietic stem cell composition

[0102] Also provided herein are methods, compositions, and kits, e.g., pharmaceutical compositions, methods of treatment, and methods of administration, for the use of hematopoietic stem cells, e.g., genetically modified hematopoietic stem cells. While the description of pharmaceutical compositions provided herein primarily relates to pharmaceutical compositions suitable for administration to humans, one of skill in the art will understand that such compositions are generally suitable for administration to any animal.

[0089]

[0103] In some embodiments, the pharmaceutical composition comprises a modified host cell that has been genetically engineered to contain a donor sequence integrated into a targeted locus of the host cell. In some embodiments, the modified host cell is genetically engineered to contain an integrated functional donor sequence, such as, for example, a SNP donor that corrects one or more mutations in a target gene (e.g., HBB) or inserts or replaces some or all of the mutant alleles with a wild-type allele. In certain embodiments, the functional donor sequence is integrated into the translation start site of the endogenous locus of the target gene. In certain embodiments, the functional donor sequence integrated into the host cell genome is expressed under the control of the native promoter sequence of the target gene.

[0090]

[0104] In some embodiments, the pharmaceutical composition comprises a plurality of modified host cells, further comprising unmodified host cells and / or host cells that have undergone nuclease cleavage that results in an INDEL at the target locus but does not result in integration of the donor sequence. In some embodiments, the pharmaceutical composition comprises at least 5% modified host cells that contain an integrated donor sequence. In some embodiments, the pharmaceutical composition comprises about 9%-50% host cells that contain an integrated donor sequence. In some embodiments, the pharmaceutical composition comprises modified host cells that contain 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 integrated donor sequences. The pharmaceutical compositions described herein may be formulated with one or more excipients to, for example: (1) enhance stability; (2) alter biodistribution (e.g., targeting to particular tissues or cell types, e.g., hematopoietic stem cells); and / or (3) promote engraftment in the recipient.

[0091]

[0105] Formulations of the present disclosure may include, but are not limited to, saline, liposomes, lipid nanoparticles, polymers, peptides, proteins, and combinations thereof. The pharmaceutical composition formulations described herein can be prepared by any method known or hereafter developed in the field of pharmacy. 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 composition of the present disclosure may be sterile.

[0092]

[0106] The relative amounts of active ingredient (e.g., modified host cells), pharmaceutically acceptable excipient, and / or any additional components in a pharmaceutical composition according to the present disclosure can vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered. For example, the composition may contain 0.1% to 99% (w / w) active ingredient. By way of example, the composition may contain 0.1% to 100%, e.g., 0.5 to 50%, 1 to 30%, 5 to 80%, or at least 80% (w / w) active ingredient.

[0093]

[0107] As used herein, excipients include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, tonicity adjusting agents, thickening or emulsifying agents, preservatives, etc. Various excipients for formulating pharmaceutical compositions and techniques for preparing compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). The use of conventional excipients may be contemplated within the scope of the present disclosure, except where the conventional excipient may be incompatible with the substance or its derivatives, for example, by producing undesirable biological effects or otherwise interacting adversely with other component(s) of the pharmaceutical composition.

[0094]

[0108] 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, and the like, and / or combinations thereof. Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0095]

[0109] Dosing and Administration

[0110] In certain embodiments, the method includes administering to an individual in need of treatment a composition comprising an effective amount of hematopoietic stem cells (e.g., genetically modified hematopoietic stem cells). A therapeutically effective dose of hematopoietic stem cells can range from about 1 million to about 200 billion, e.g., 1 million to about 50 billion (e.g., about 5 million, about 25 million, about 500 million, about 1 billion, about 5 billion, about 20 billion, about 30 billion, about 40 billion, or a range defined by any two of the foregoing values), e.g., about 10 million to about 1 trillion (e.g., about 20 million, about 30 million, about 40 million, about 60 million, about 70 million, about 80 million, or about 100 million). In some embodiments, the concentration of IgG10 per kg of body weight can be 2×10, 3×10, 4×10, 5×10, 6×10, 7×10, 8×10, 9×10, 10 ... 6 ~2×10 8 The method includes administering viable hematopoietic stem cells.

[0096]

[0111] In certain embodiments, a pharmaceutical composition comprising exogenous hematopoietic stem cells according to the present disclosure may be administered in a concentration of, for example, about 1×10 4 ~1×10 5 , 1×10 5 ~1×10 6 , 1×10 6 ~1×10 7 The modified host cell pharmaceutical composition of the present disclosure can be administered in a dose sufficient to deliver one or more cells to a subject, or in any amount sufficient to achieve the desired therapeutic or prophylactic effect. The desired dose of the modified host cell pharmaceutical composition of the present disclosure can be administered once or multiple times. In some embodiments, administering 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, 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, only a single administration is sufficient to provide treatment or prevention of a disease or disorder described herein. In other embodiments, a subject in need of treatment or prevention of a disease or disorder may receive more than one administration, e.g., two, three, or more than three administrations of a pharmaceutical hematopoietic stem cell composition described herein, to provide such treatment or prevention. Hematopoietic stem cells can be used in combination, sequentially or concurrently, with one or more other therapeutic, prophylactic, research or diagnostic agents, or medical procedures, generally each agent being administered at a dose and / or time schedule determined for that agent.

[0097]

[0112] The infusion population and its composition can be administered to an individual in need thereof using standard administration techniques, formulations, and / or devices. Formulations and administrations using devices such as syringes and vials for storing and administering the compositions are provided. Formulations or pharmaceutical compositions containing exogenous hematopoietic stem cells include those for intravenous, intraperitoneal, subcutaneous, intramuscular, or pulmonary administration. The exogenous hematopoietic stem cell composition can be provided as a sterile liquid formulation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which in some embodiments can be buffered to a selected pH. Viscous compositions can be formulated within an appropriate viscosity range to extend the contact period with specific tissues. Liquid or viscous compositions can contain a carrier, which may be a solvent or dispersion medium, including, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof. Sterile injectable solutions can be prepared by incorporating the hematopoietic stem cells into a vehicle, for example, by mixing with a suitable carrier, diluent, or additive, such as sterile water, physiological saline, glucose, dextrose, or the like.

[0098]

[0113] The exogenous hematopoietic stem cells contained in the pharmaceutical composition can be administered by any delivery route, systemic or local, that results in a therapeutic effect. This includes, but is not limited to, enteral, gastrointestinal, epidural, oral, transdermal, intracerebral, intraventricular, subcuticular, intradermal, subcutaneous, intranasal, intravenous, intraarterial, intramuscular, intracardiac, intramedullary, intrathecal, intraparenchymal, intraperitoneal, intravesical, intravitreal, intracavity, intraintestinal, intraperitoneal, intralymphatic, intramedullary, intrapulmonary, intraspinal, intrasynovial, intradural, intrafallopian tube, parenteral, transdermal, periarticular, epidural, perineural, periodontal, rectal, soft tissue, and topical. In certain embodiments, the cells are administered intravenously. The pharmaceutical composition can be administered to a subject using any amount and any route of administration effective for the prevention, treatment, or management of the diseases described herein. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its route of administration, its mode of action, and the like.

[0099]

[0114] In some embodiments, after administration of exogenous donor hematopoietic stem cells, the recipient can be monitored for hematopoietic recovery, reconstitution, and / or donor chimerism as indicators of successful engraftment. In some embodiments, engraftment is determined by assessing donor bone marrow chimerism. In some embodiments, engraftment is determined by assessing lineage-specific chimerism. In some embodiments, engraftment is determined by assessing naive T cell production. Any method known in the art for assessing donor cell chimerism may be used in conjunction with the disclosed methods (see, e.g., Pinkel et al., Proc Natl Acad Sci USA (1996), 83:2934-2938). In certain embodiments, after donor stem cell transplantation, the recipient is chimeric or mixed chimeric with respect to donor cells. Mixed chimerism (MC) is defined as the presence of 5% or more host-derived cells in whole blood at one or more times. This is further classified as high-level MC (donor chimerism 95%-50%), low-level MC (donor chimerism 49%-10%), or ultra-low-level MC (donor chimerism less than 10%).

[0100] Treatment method

[0115] The hematopoietic stem cell depletion and engraftment compositions and methods provided herein can be used as part of a treatment regimen for any disease or condition for which HSCT is useful. HSCT can be used to treat a number of conditions, including congenital and acquired conditions. In some embodiments, acquired conditions treatable with HSCT include: (1) hematologic malignancies such as leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)), lymphoma (e.g., Hodgkin's disease, non-Hodgkin's lymphoma), myeloma (e.g., multiple myeloma (Carls syndrome)), and solid tumor cancers (e.g., neuroblastoma, desmoplastic small round cell tumor, Ewing's sarcoma, choriocarcinoma, and the like); (2) phagocytic disorders (e.g., chronic granulomatous disease), bone marrow failure disorders (e.g., bone marrow necrosis factor (BNF)), and the like. (3) blood diseases such as dysplastic syndromes, Fanconi anemia, dyskeratosis congenita), anemia (e.g., paroxysmal nocturnal hemoglobinuria, aplastic anemia, acquired pure erythropenia), and myeloproliferative disorders (e.g., polycythemia vera, essential thrombocythemia, myelofibrosis); (4) metabolic diseases such as amyloidosis (e.g., amyloid light chain (AL) amyloidosis); (5) environmentally induced diseases such as radiation poisoning; (6) viral diseases (e.g., HTLV, HIV), and (7) autoimmune diseases such as multiple sclerosis.

[0101]

[0116] In some embodiments, congenital diseases treatable with HSCT include: (1) lipoidosis (disorders of lipid storage such as neuronal ceroid lipofuscinosis (e.g., infantile neuronal ceroid lipofuscinosis (INCL, Santavoli disease) and Jansky-Bierschkowski disease (late-onset infantile neuronal ceroid lipofuscinosis)); sphingolipidoses (e.g., Niemann-Pick disease and Gaucher disease); leukodystrophies (e.g., adrenoleukodystrophy, metachromatic leukodystrophy, Krabbe disease (globoid cell leukodystrophy)); mucopolysaccharidoses (e.g., Hurler syndrome (MPS IH, alpha-L-iduronate hydrolase deficiency), Scheie syndrome (MPS IS), Hurler-Scheie syndrome (MPS IH-S), Hunter syndrome (MPS II, iduronidase sulfate deficiency), Sanfilippo syndrome (MPS III), Morquio syndrome (MPS IV), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII); glycoproteinopathies (e.g., mucolipidosis type II (I-cell disease), fucosemia, aspartylglucosaminuria, alpha-mannosidosis); and lysosomal storage disorders such as Wolman disease (acid lipase deficiency); (2) T-cell deficiencies (e.g., ataxia-telangiectasia and DiGeorge syndrome), combined T-cell and B-cell deficiencies (e.g., any type of severe combined immunodeficiency (SCID)), well-defined syndromes (e.g., Wiskott-Aldrich syndrome), phagocytic disorders (e.g., Kostmann syndrome, Shwachman-Diamond syndrome), immunoregulatory disorders (e.g., Grisselli syndrome, type II), congenital immunodeficiencies (e.g., NF-Kappa-B essential (3) hematological disorders such as hemoglobinopathies (e.g., sickle cell disease, thalassemia (e.g., beta thalassemia), anemia (e.g., aplastic anemia such as Diamond-Blackfan anemia and Fanconi anemia), cytopenias (e.g., megakaryocytic thrombocytopenia), and hemophagocytic syndromes (e.g., hemophagocytic lymphohistiocytosis (HLH)).

[0102]

[0117] In some embodiments, the disease or condition is selected from the group consisting of hemoglobinopathies, viral infections, X-linked severe combined immunodeficiency, Fanconi anemia, hemophilia, neoplasms, cancer, amyotrophic lateral sclerosis, alpha-antitrypsin deficiency, Alzheimer's disease, Parkinson's disease, cystic fibrosis, blood diseases and disorders, inflammation, immune system diseases or disorders, metabolic diseases, liver diseases and disorders, kidney diseases and disorders, muscle diseases and disorders, bone or cartilage diseases and disorders, neurological and nervous diseases and disorders, cardiovascular diseases and disorders, pulmonary diseases and disorders, and lysosomal storage disorders. In some embodiments, the hemoglobinopathies are selected from the group consisting of sickle cell disease, α-thalassemia, β-thalassemia, and δ-thalassemia.

[0103]

[0118] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will 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 may be employed in practicing the invention. The following claims are intended to define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby. [Example]

[0104] Example 1: Co-administration of anti-CD110 and anti-CD117 conditioning agents results in robust engraftment of transplanted HSPCs

[0119] In this example, we demonstrate that combined treatment with anti-CD117 and anti-CD110 monoclonal antibodies with effector function synergizes to enable robust engraftment of donor HSPCs (hematopoietic stem progenitor cells) and multilineage reconstitution of hematopoietic cells in immunocompetent recipient mice.

[0105] material and method

[0120] antibody

[0121] The anti-mCD117 antibody ACK2 and the anti-mCD110 antibody AMM2 are commercially available as rat immunoglobulins. The antibody variable domain sequences were obtained by in-solution endoproteinase digestion followed by liquid chromatography-tandem mass spectrometry and data analysis. A mouse IgG2a version of the antibody was generated by fusing the variable domains with mouse heavy and light chain constant regions. The mouse IgG2a_N297A version of the antibody was generated by mutating Asn at position 297 of the human Fc to Ala in the cognate mouse Fc. The chimeric antibodies were transiently produced in CHO cells, purified, and confirmed to bind to their respective mouse antigens in a binding assay using biolayer interferometry (ForteBio Octet), as shown in Figure 1. Recombinant mCD110-ECD-H6 or mCD117-ECD-H6 was captured onto a sensor chip, which was then transferred to a solution of anti-mCD110 mIgG2a or anti-mCD117 mIgG2a 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 binding, the sensor chip was transferred to buffer alone to assess antibody dissociation over time. Binding and dissociation curves were calculated for each concentration level.

[0106]

[0122] Conditioning and transplantation

[0123] 8-10 week old B6.SJL-Ptprc a Pepc b B6 / BoyJ ("B6 CD45.1") mice were treated with each antibody regimen according to the study design in Figure 2 on day -7 relative to bone marrow transplantation. B6 CD45.1 mice were intravenously injected with 25 mg / kg of isotype control antibody mIgG2a, 25 mg / kg of anti-mCD117 mIgG2a antibody, 25 mg / kg of anti-mCD110 mIgG2a antibody, or 25 mg / kg of anti-mCD117 antibody and 25 mg / kg of anti-mCD110 antibody. Animals were also treated with lineage-negative ("Lin") antibodies isolated from bone marrow cells of C57BL / 6J donors. -800,000 donor cells were injected intravenously. The chimerism results from this procedure are shown in Figures 3 and 4.

[0107]

[0124] Eight- to ten-week-old B6 mice were intravenously injected with either A) 50 mg / kg of mIgG2a isotype control antibody, or a combination of 25 mg / kg of anti-mCD117 mIgG2a antibody and 2.5 mg / kg of anti-mCD110 mIgG2a antibody; or B) 25 mg / kg of ACK2 (anti-mCD117 rIgG2b antibody) and 5 mg / kg of AMM2 (anti-mCD110 rIgG1 antibody) or 25 mg / kg of anti-mCD117 mIgG2a Fc-null antibody and 5 mg / kg of anti-mCD110 mIgG2a Fc-null antibody on day -7, and bone marrow transplantation was performed on day 0. The results of chimerism formation in this procedure are shown in Figure 5.

[0108]

[0125] Donor cell isolation

[0126] To isolate the donor cells used in Figures 3 and 4, 8-10 week old B6 mice were euthanized and the femur, tibia, humerus, hip bones, and vertebrae were collected. The bone marrow was isolated by crushing the bones, followed by lysis of RBCs using Gibco's ACK lysis buffer for 7 minutes on ice. Lineage-negative cells were collected using the Direct Lineage Cell Depletion Kit (Miltenyi Biotec) according to the manufacturer's instructions.

[0109]

[0127] To isolate the donor cells used in Figure 5, 8-10 week-old B6 CD45.1 mice were euthanized and the femur, tibia, humerus, hip, and vertebrae were collected. Bone marrow was isolated by crushing the bone marrow, followed by RBC lysis using Gibco's ACK lysis buffer for 7 minutes on ice. Lineage-negative cells were collected using the Direct Lineage Cell Depletion Kit (Miltenyi Biotec) according to the manufacturer's instructions.

[0110]

[0128] Coexpression analysis and receptor quantification

[0129] Naive B6 mice were euthanized and femurs and tibias were collected. Bone marrow was extracted from the bones by centrifugation, followed by RBC lysis using Gibco's ACK lysis buffer for 7 minutes on ice. Cells were stained with fluorescent antibodies against 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 with the BangsLabs MESF AF647 Ladder, allowing for quantification of CD117 and CD110 receptors in the HSPC population (Figure 6).

[0111]

[0130] Fresh human bone marrow aspirates were processed on a Ficoll density gradient within 24 hours of collection. The resulting bone marrow mononuclear cells (BMMCs) were stained with a panel of antibodies and analyzed on a BD Fortessa X-20 cytometer.

[0112]

[0131] The human antibody panel consisted of the following antibody / clone / fluorochrome combinations: CD34(561)-APC combined with the live cell stain FVS780-APC-Cy7, 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.

[0113]

[0132] Chimerism analysis

[0133] Mononuclear cells were collected from peripheral blood using HetaSep (Stemcell Technologies), followed by RBC lysis using Gibco ACK lysis buffer. To observe myeloid and lymphoid chimerism in peripheral blood, cells were stained with fluorescent antibodies specific for the following targets: CD19, CD11b, Ter119, CD45.2, NK1.1, Gr-1, CD45.1, and CD3. Dead cells were labeled with a fluorescent viability stain. Samples were analyzed on a BD Fortessa X-20 cytometer.

[0114]

[0134] To assess the chimeric status of bone marrow cells, animals were euthanized, followed by collection of femurs and tibiae. Bone marrow was isolated from the bones by centrifugation, followed by RBC lysis using Gibco ACK lysis buffer. Bone marrow cells were isolated from naive C57BL / 6J mice and stained with fluorescent antibodies specific for the following targets: Flt3, CD117, CD34, CD127, Lineage ("Lin": CD3e, Gr-1, CD11b, B220, TER119), Sca1, CD16 / 32, SLAM, and CD110. Dead cells were labeled with a fluorescent live cell stain. Samples were analyzed on a BD Fortessa X-20 cytometer.

[0115] result

[0135] Wild-type B6 CD45.1 mice were treated with 25 mg / kg of effector-competent anti-mouse CD117 (mCD117) mIgG2a and anti-mouse CD110 (mCD110) mIgG2a. Seven days after treatment, the animals were transplanted with 800,000 lineage-depleted bone marrow cells from C57B16 mice, which differ only in CD45 alleles, thereby distinguishing syngeneic transplants (Figure 2). Donor cell chimerism in peripheral blood was examined at 4, 8, 12, and 16 weeks after transplantation. As shown in Figure 3, mCD117 mIgG2a alone resulted in a 10% engraftment rate, whereas mCD110 mIgG2a alone did not. However, coadministration of both antibodies resulted in synergistic engraftment, as indicated by robust peripheral blood myeloid chimerism (Mac-1+Gr-1+ cells; Figure 3B), B cell chimerism (CD19+ cells; Figure 3C), T cell chimerism (CD3+ cells; Figure 3D), and NK cell chimerism (NK1.1+ cells; Figure 3E), which increased over time. These results demonstrate that antibody-based conditioning regimens simultaneously targeting CD110 and CD117 produce synergistic responses, enabling stable engraftment of donor HSPCs and subsequent multilineage hematopoietic reconstitution.

[0116]

[0136] The use of monoclonal antibodies for effective conditioning of the bone marrow niche has previously been reported with anti-mCD117 or anti-mCD110 antibodies, but only in combination with chemotherapy and in regimens requiring multiple days of administration prior to donor stem cell transplantation. For example, the rat anti-mouse CD117 antibody ACK2, when combined with the hypomethylating chemotherapy agent 5-azacytidine (AZA), enabled engraftment in a mouse bone marrow transplant model. This conditioning regimen required six consecutive days of 5-azacytidine administration (see, e.g., Bankova et al., Blood Adv 5, 19 (2021)). The rat anti-mouse CD110 antibody AMM2, when combined with the chemotherapy agent 5-fluorouracil (5-FU), enabled engraftment (see, e.g., Arai et al., Ann. NY Acad Sci, 1176 (2009) and Yoshihara et al., Cell Stem Cell, 1 (2007)). This is in contrast to the method provided herein, which combines two monoclonal antibodies targeting CD117 and CD110, respectively, in a single administration without the use of chemotherapeutic agents, thereby avoiding the risks to the recipient associated with genotoxic conditioning.

[0117]

[0137] In the prior art examples mentioned above, the degree of chimerism observed with antibody-chemotherapy combinations varied widely. 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, in which a single co-administration of anti-CD117 and anti-CD110 antibodies without chemotherapy achieved up to 60-80% chimerism, are surprising and unexpected given the low activity of each antibody alone. As shown in Figure 4, no engraftment was observed with anti-CD110 administration alone (consistent with the results reported by Arai et al., Ann. NYAcad Sci. 1176 (2009)), and only 1-10% engraftment was observed with anti-CD117 administration alone (consistent with the results reported by Bankova et al., Blood Adv. 5, 19 (2021)). Arai et al. (Ann. NY Acad Sci. 1176 (2009)) proposed that a certain degree of engraftment could be achieved by combining ACK2 and AMM2, based on the differential effects of ACK2 and AMM2 on different subpopulations within the HSC population (circulating versus quiescent cells, respectively). In such a scenario, the effects of combining ACK2 and AMM2 on engraftment would be expected to be, at most, additive, expanding the cell types (circulating and quiescent cells) targeted by these antibodies. Therefore, the demonstration of synergistic engraftment when CD117 and CD110 are simultaneously targeted by antibodies alone is unexpected in light of the prior art.

[0118]

[0138] The unexpectedly excellent chimerism shown in Figure 4 is supported by additional results shown in Figure 6, which demonstrate that CD110 and CD117 do not label distinct, true long-term HSC cell populations. As shown in Figures 6B and 6C, the highest expression of both CD110 and CD117 was observed in LT-HSCs (Lin-CD117+Sca-1+Slam+Flt3 cells, Figure 6A). Similarly, CD110 and CD117 are coexpressed in human bone marrow LT-HSCs (Lin-CD34+CD38-CD45RA-CD90+CD49f+, Figure 7).

[0119]

[0139] Furthermore, we observed that the combination of ACK2 and AMM2 (Arai, 2009) did not actually result in meaningful engraftment, as evidenced by the low percentage of donor-derived cells in the recipient's bone marrow 16 weeks after transplantation (Figure 5B). ACK2 is a rat antibody against mouse CD117 with an IgG2b isotype, while AMM2 is a rat antibody against mouse CD110 with an IgG1 isotype. As shown in Figures 3, 4, and 5A, synergistic effects using anti-CD117 and anti-CD110 antibodies were observed when the Fc regions of ACK2 and AMM2 were modified from rat to mouse isotypes of IgG2a. To assess the role of effector function in these versions, and because mouse IgG2a has full effector function, we engineered ACK2 and AMM2 as effector-deficient mouse IgG2a antibodies by a previously reported mutation (N297A) in the Fc region that removes a potential glycosylation site and may reduce Fc receptor binding (e.g., Shields RL et al. (2001) J Biol Chem 276:6591-604). The combination of effector-null anti-CD117 and anti-CD110 antibodies did not result in synergistic donor cell engraftment; the engraftment rate was less than 1%, similar to that observed with the combination of ACK2 and AMM2 (Figure 5B).

[0120]

[0140] Previous results demonstrating engraftment with the ACK2-AZA regimen required administration of ACK2 prior to AZA treatment and likely depended on ACK2's ability to block SCF binding to CD117, as coadministration of SCF or the use of another anti-mouse CD117 antibody (2B8), which only partially blocks SCF, negatively affected LT-HSC depletion and / or subsequent engraftment (Bankova et al., Blood Adv. 5, 19 (2021)). Our observation that synergistic preconditioning was achieved only with the combination of effector-functional versions of anti-CD110 and anti-CD117 antibodies, whereas effector-null versions did not result in sustained donor cell engraftment, suggests a different mechanism of action compared with previously reported antibody-chemotherapy regimens. Furthermore, it has been suggested that the lack of a single-agent effect of AMM2 alone may be due to the persistence of antibodies in the circulation that adversely affect the proliferation of donor cells after BMT (Arai et al., Ann. NY Acad Sci. 1176 (2009)). Our observation that the combination of anti-CD117 and anti-CD110 antibodies achieves long-term chimerism through a synergistic effect contradicts this hypothesis and is therefore novel and unexpected.

[0121]

[0141] In summary, we describe a novel antibody-based conditioning regimen targeting CD110 and CD117, co-expressed on HSPCs and LT-HSCs, that results in a synergistic response, enabling stable engraftment of donor HSPCs and thereby multilineage hematopoietic reconstitution.

[0122]

[0142] All publications and patent applications cited herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. While the claimed subject matter has been described in terms of various embodiments, those skilled in the art will recognize that various modifications, substitutions, omissions, and alterations can be made therein without departing from the spirit thereof. Accordingly, it is intended that the scope of the subject matter be limited only by the following claims, including equivalents thereof.

Claims

1. 1. A method for hematopoietic stem cell engraftment in a subject in need thereof, comprising: a. Subject to: i. a first targeting moiety that specifically binds to CD117; and ii. A second targeting moiety that specifically binds to CD110 depleting endogenous hematopoietic stem cells in a subject by administering a pharmaceutical composition comprising: b. Administering exogenous hematopoietic stem cells to a subject Including; The method, wherein administration of the pharmaceutical composition mediates engraftment of exogenous hematopoietic stem cells, resulting in multilineage hematopoietic reconstitution in the subject.

2. The method of claim 1 , wherein the first and second moieties bind to hematopoietic stem cells that co-express CD117 and CD110.

3. The method of claim 1 or 2, wherein the first targeting moiety comprises an isolated antibody or antigen-binding fragment thereof that specifically binds to CD117.

4. The method of claim 3, wherein the isolated antibody or antigen-binding fragment thereof that specifically binds to CD117 functionally interferes with signal transduction between stem cell factor (SCF) and CD117 and / or mediates elimination of CD117-expressing cells via Fc effector function.

5. The method of any one of claims 1 to 4, wherein the second targeting moiety comprises an isolated antibody, or antigen-binding fragment thereof, that specifically binds to CD110.

6. The method of claim 5, wherein the isolated antibody or antigen-binding fragment thereof that specifically binds to CD110 functionally interferes with signal transduction between thrombopoietin (TPO) and CD110 and / or mediates the elimination of CD110-expressing cells via Fc effector function.

7. The method of any one of claims 3 to 6, wherein the isolated antibody of the first and / or second targeting moiety is a monoclonal antibody.

8. 7. The method of any one of claims 3 to 6, wherein the antigen-binding fragment of the first and / or second targeting moiety is selected from the group consisting of an Fv fragment, a Fab fragment, an F(ab')2 fragment, a Fab' fragment, a scFv (sFv) fragment, a scFv-Fc fragment, a single-chain Fv (scFv), a single-chain antibody, a disulfide-linked Fv (dsFv), a fragment comprising either a VL or VH domain, a heavy-chain antibody (hcAb), a single-domain antibody (sdAb), a minibody, and a variable domain derived from a camelid heavy-chain antibody (VHH or nanobody).

9. The method of any one of claims 1 to 8, wherein both the first targeting moiety and the second targeting moiety are comprised in the same antibody or antigen-binding fragment thereof.

10. 10. The method of claim 9, wherein the antibody or antigen-binding fragment is selected from the group consisting of a diabody, a diabody-Fc, a single chain diabody, a tandem diabody (Tandab), a tandem scFv, a tandem scFv-scFc, a tandem di-scFv, a tandem tri-scFv, a multivalent antibody, a bivalent or bispecific single chain variable fragment, a bispecific IgG, and a Fab-IgG bispecific.

11. 11. The method of any one of claims 3 to 10, wherein the isolated antibody or antigen-binding fragment of the first and / or second targeting moiety comprises an Fc region capable of binding to the neonatal Fc receptor (FcRn) of the subject.

12. The method of any one of claims 3 to 11, wherein the isolated antibody or antigen-binding fragment of the first and / or second targeting moiety is chimeric, humanized, or human.

13. The method of any one of claims 3 to 12, wherein the isolated antibody or antigen-binding fragment of the first and / or second targeting moiety comprises a human Fc region.

14. 1. A method for hematopoietic stem cell engraftment in a subject in need thereof, comprising: a. Subject to: i. an effective amount of a first isolated antibody or antigen-binding fragment thereof that specifically binds to CD117; and ii. an effective amount of a second isolated antibody or antigen-binding fragment thereof that specifically binds to CD110. depleting endogenous hematopoietic stem cells in a subject by co-administering b. Administering exogenous hematopoietic stem cells to a subject Including; The method, wherein co-administration of effective amounts of a first and second antibody or fragment thereof synergistically mediates engraftment of exogenous hematopoietic stem cells, resulting in multilineage hematopoietic reconstitution in the subject.

15. The method of claim 14, wherein the first and second isolated antibodies, or antigen-binding fragments thereof, bind to hematopoietic stem cells that co-express CD117 and CD110.

16. The method of claim 14 or 15, wherein the first isolated antibody, or antigen-binding fragment thereof, functionally interferes with signal transduction between stem cell factor (SCF) and CD117 and / or mediates elimination of CD117-expressing cells via Fc effector function.

17. The method of any one of claims 14 to 16, wherein the second isolated antibody, or antigen-binding fragment thereof, functionally interferes with signaling between thrombopoietin (TPO) and CD110 and / or mediates elimination of CD110-expressing cells via Fc effector function.

18. The method of any one of claims 14 to 17, wherein the first isolated antibody and / or the second isolated antibody is a monoclonal antibody.

19. The method of any one of claims 14 to 17, wherein the first isolated antibody and / or the second isolated antibody is a bispecific antibody.

20. The method of any one of claims 14 to 19, wherein the antigen-binding fragment that specifically binds to CD117 is selected from the group consisting of an Fv fragment, a Fab fragment, an F(ab')2 fragment, a Fab' fragment, an scFv (sFv) fragment, an scFv-Fc fragment, and a nanobody fragment.

21. The method of any one of claims 14 to 20, wherein the antigen-binding fragment that specifically binds to CD110 is selected from the group consisting of an Fv fragment, a Fab fragment, an F(ab')2 fragment, a Fab' fragment, an scFv (sFv) fragment, an scFv-Fc fragment, and a nanobody fragment.

22. 22. The method of any one of claims 14 to 21, wherein the Fc region of the first isolated antibody and / or the second isolated antibody is capable of binding to the neonatal Fc receptor (FcRn) of the subject.

23. The method of any one of claims 14 to 22, wherein the first isolated antibody or antigen-binding fragment thereof and / or the second isolated antibody or antigen-binding fragment thereof is chimeric, humanized, or human.

24. The method of any one of claims 14 to 23, wherein the first isolated antibody or antigen-binding fragment thereof and / or the second isolated antibody or antigen-binding fragment thereof comprises a human Fc region.

25. The method of any one of claims 1 to 24, wherein the subject is a human.

26. The method of any one of claims 3 to 25, wherein the antibody or antigen-binding fragment thereof that specifically binds to CD117 and / or the antibody or antigen-binding fragment thereof that specifically binds to CD110 is conjugated to a toxin.

27. 27. The method of claim 26, wherein the toxin is selected from the group consisting of saporin, saporin derivatives, ricin, abrin, gelonin, momordin, apitoxin, shiga toxin, shiga-like toxin, T-2 mycotoxin, diphtheria toxin, busulfan, Pseudomonas exotoxin A, ricin A chain derivatives, trichosanthin, raffin toxin, maytansine, amatoxin, mechlorethamine, cyclophosphamide, ethyleneimine, methylmelamine, methotrexate, fluorouracil, floxuridine, cytarabine, mercaptopurine, azathioprine, thioguanine, fludarabine phosphate, cladribine, drostatin, auristatin, auristatin E, auristatin F, MMAF, MMAE, MMAD, DMAF, or DMAE, maytansine, DM1 or DM4, duocarmycin, calicheamicin, pyrrolobenzodiazepines, exatecan, and any combination thereof.

28. 28. The method of any one of claims 1 to 27, further comprising monitoring the subject for depletion of endogenous hematopoietic stem cells prior to administering the exogenous hematopoietic stem cells.

29. 29. The method of any one of claims 1-28, wherein the exogenous hematopoietic stem cells are administered to the subject after the first and second isolated antibodies, or antigen-binding fragment(s) thereof, have been substantially removed from the subject's blood.

30. 29. The method of any one of claims 1-28, wherein administering the exogenous hematopoietic stem cells to the subject occurs within 3, 5, 7, or 10 days of co-administering the first and second isolated antibodies, or antigen-binding fragment(s) thereof, to the subject.

31. The method of any one of claims 1 to 30, wherein the exogenous hematopoietic stem cells are allogeneic hematopoietic stem cells.

32. The method according to any one of claims 1 to 30, wherein the exogenous hematopoietic stem cells are autologous hematopoietic stem cells.

33. 33. The method of any one of claims 1 to 32, wherein the exogenous hematopoietic stem cells comprise CD34+ hematopoietic stem progenitor cells (HSPCs).

34. The method of claim 33, wherein the CD34+ HSPCs comprise CD34+ / CD38- / CD90+ HSPCs.

35. The method of claim 134, wherein the CD34+ HSPCs comprise CD34+ / CD38- / CD90+ / CD45RA- HSPCs.

36. Steps below: a. harvesting a hematopoietic stem cell population from a subject prior to depletion; b. Culturing the collected hematopoietic stem cell population; and c. Cryopreserving the collected hematopoietic stem cell population The method of any one of claims 1 to 35, further comprising one or more of:

37. Harvesting a hematopoietic stem cell population from a subject comprises the steps of: a. Mobilizing a hematopoietic stem cell population, and b. Harvesting a population of hematopoietic stem cells by apheresis 37. The method of claim 36, comprising one or more of:

38. The method of any one of claims 1 to 37, wherein the exogenous hematopoietic stem cells are genetically modified.

39. 39. The method of Claim 38, wherein the exogenous hematopoietic stem cells are genetically modified using one or more components of a gene editing system.

40. 40. The method of Claim 39, wherein one or more components of the gene editing system are selected from the group consisting of: (i) a CRISPR / Cas guide RNA, (ii) a DNA molecule encoding a 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) a ZFP, (viii) a nucleic acid molecule encoding a transcription activator-like effector (TALE) protein, (ix) a TALE protein, and (x) a DNA donor polynucleotide.

41. 41. The method of claim 40, wherein the CRISPR / Cas RNA guide polypeptide is a base editor or a prime editor.

42. 40. The method of Claim 39, wherein one or more components of the gene editing system comprise a nuclease capable of generating a double-stranded break within a locus of the cell.

43. 43. The method of Claim 42, wherein one or more components of the gene editing system further comprise a DNA donor polynucleotide.

44. 44. The method of claim 43, wherein the DNA donor polynucleotide comprises non-overlapping 5' and 3' homology arms, each homology arm being homologous to a portion of the locus, and wherein creation of a double-stranded break within the locus by a nuclease results in the donor polynucleotide sequence being integrated into the locus by homology-directed repair (HDR).

45. 40. The method of Claim 39, wherein the gene editing system comprises 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.

46. 46. ​​The method of claim 45, wherein the CRISPR nuclease is a Cas protein.

47. 47. The method of claim 46, wherein the Cas protein is Cas9 or a high-fidelity variant thereof.

48. 48. The method of any one of claims 45 to 47, wherein the sgRNA and CRISPR nuclease are formed into a ribonucleoprotein (RNP) complex.

49. 49. The method of any one of claims 45 to 48, wherein the sgRNA comprises one or more chemically modified nucleotides.

50. 50. The method of claim 49, wherein the modified nucleotides are selected from the group consisting of: 2'-O-methyl nucleotides, 2'-O-methyl 3'-phosphorothioate nucleotides, and 2'-O-methyl 3'-thioPACE nucleotides.

51. 51. The method of Claim 49 or 50, wherein the 5' end, 3' end, or a combination thereof of the modified sgRNA comprises modified nucleotides.

52. 52. The method of claim 51, further comprising contacting the stem cell population with an AAV vector comprising the donor polynucleotide sequence.

53. 53. The method of any one of claims 38 to 52, wherein the genetic modification corrects a genetic mutation, replaces a mutant allele with a wild-type allele, or inserts a nucleic acid sequence encoding a therapeutic protein.

54. 54. The method of any one of claims 1 to 53, wherein the subject is suffering from a disease.

55. 55. The method of claim 54, wherein the disease is a hemoglobinopathy.

56. 56. The method of claim 55, wherein the hemoglobinopathy is selected from the group consisting of sickle cell disease, alpha-thalassemia, beta-thalassemia, and delta-thalassemia.

57. 57. The method of any one of claims 1 to 56, wherein engraftment occurs without myeloablative conditioning.

58. 1. A method for depleting endogenous hematopoietic stem cells in a subject in need thereof, comprising administering to the subject: a. a first targeting moiety that specifically binds to CD117; and b. A second targeting moiety that specifically binds to CD110 Administering a pharmaceutical composition comprising:

59. 59. The method of claim 58, wherein administration of the pharmaceutical composition mediates depletion of exogenous hematopoietic stem cells in the subject.

60. 60. The method of claim 58 or 59, wherein the first and second moieties bind to hematopoietic stem cells that co-express CD117 and CD110.

61. 61. The method of any one of claims 58-60, wherein the first targeting moiety comprises an isolated antibody or antigen-binding fragment thereof that specifically binds to CD117.

62. 62. The method of any one of claims 58-61, wherein the targeting moiety that specifically binds to CD117 functionally interferes with signaling between stem cell factor (SCF) and CD117 and / or mediates elimination of CD117-expressing cells via Fc effector function.

63. 63. The method of any one of claims 58-62, wherein the second targeting moiety comprises an isolated antibody, or antigen-binding fragment thereof, that specifically binds to CD110.

64. The method of claim 63, wherein the isolated antibody or antigen-binding fragment thereof that specifically binds to CD110 functionally interferes with signal transduction between thrombopoietin (TPO) and CD110 and / or mediates elimination of CD110-expressing cells via Fc effector function.

65. 65. The method of any one of claims 61 to 64, wherein the isolated antibody of the first and / or second targeting moiety is a monoclonal antibody.

66. 65. The method of any one of claims 61 to 64, wherein the antigen-binding fragment of the first and / or second targeting moiety is selected from the group consisting of an Fv fragment, a Fab fragment, an F(ab')2 fragment, a Fab' fragment, a scFv (sFv) fragment, a scFv-Fc fragment, a single-chain Fv (scFv), a single-chain antibody, a disulfide-linked Fv (dsFv), a fragment comprising either a VL or VH domain, a heavy-chain antibody (hcAb), a single-domain antibody (sdAb), a minibody, and a variable domain derived from a camelid heavy-chain antibody (VHH or nanobody).

67. 67. The method of any one of claims 58 to 66, wherein both the first targeting moiety and the second targeting moiety are comprised in the same antibody or antigen-binding fragment thereof.

68. 68. The method of claim 67, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a diabody, a diabody-Fc, a single chain diabody, a tandem diabody (Tandab), a tandem scFv, a tandem scFv-scFc, a tandem di-scFv, a tandem tri-scFv, a multivalent antibody, a bivalent or bispecific single chain variable fragment, a bispecific IgG, and a Fab-IgG bispecific.

69. 69. The method of any one of claims 61-68, wherein the isolated antibody or antigen-binding fragment of the first and / or second targeting moiety comprises an Fc region capable of binding to the neonatal Fc receptor (FcRn) of the subject.

70. 70. The method of any one of claims 61 to 69, wherein the isolated antibody or antigen-binding fragment of the first and / or second targeting moiety is chimeric, humanized, or human.

71. 71. The method of any one of claims 61 to 70, wherein the isolated antibody or antigen-binding fragment of the first and / or second targeting moiety comprises a human Fc region.

72. 1. A method for depleting endogenous hematopoietic stem cells in a subject in need thereof, comprising administering to the subject: a. an effective amount of a first isolated antibody or antigen-binding fragment thereof that specifically binds to CD117; and b. an effective amount of a second isolated antibody or antigen-binding fragment thereof that specifically binds to CD110 The method of claim 1, further comprising co-administering

73. 73. The method of claim 72, wherein co-administration of effective amounts of the first and second antibodies or fragments thereof synergistically mediates depletion of exogenous hematopoietic stem cells in the subject.

74. 74. The method of claim 72 or 73, wherein the first and second isolated antibodies, or antigen-binding fragments thereof, bind to hematopoietic stem cells that co-express CD117 and CD110.

75. 75. The method of any one of claims 2, 15, 60 or 74, wherein the hematopoietic stem cells co-expressing CD117 and CD110 are LT-HSCs.

76. 76. The method of any one of claims 72-75, wherein the first isolated antibody, or antigen-binding fragment thereof, functionally interferes with signaling between stem cell factor (SCF) and CD117 and / or mediates elimination of CD117-expressing cells via Fc effector function.

77. The method of any one of claims 72 to 76, wherein the second isolated antibody, or antigen-binding fragment thereof, functionally interferes with signaling between thrombopoietin (TPO) and CD110 and / or mediates elimination of CD110-expressing cells via Fc effector function.

78. 78. The method of any one of claims 72 to 77, wherein the first isolated antibody and / or the second isolated antibody is a monoclonal antibody.

79. 79. The method of any one of claims 72 to 78, wherein the first isolated antibody and / or the second isolated antibody is a bispecific antibody.

80. 80. The method of any one of claims 72 to 79, wherein the antigen-binding fragment that specifically binds to CD117 is selected from the group consisting of an Fv fragment, a Fab fragment, an F(ab')2 fragment, a Fab' fragment, an scFv (sFv) fragment, an scFv-Fc fragment, and a nanobody fragment.

81. 81. The method of any one of claims 72 to 80, wherein the antigen-binding fragment that specifically binds to CD110 is selected from the group consisting of an Fv fragment, a Fab fragment, an F(ab')2 fragment, a Fab' fragment, an scFv (sFv) fragment, an scFv-Fc fragment, and a nanobody fragment.

82. 82. The method of any one of claims 72-81, wherein the Fc region of the first isolated antibody and / or the second isolated antibody is capable of binding to the neonatal Fc receptor (FcRn) of the subject.

83. 83. The method of any one of claims 72-82, wherein the first isolated antibody or antigen-binding fragment thereof and / or the second isolated antibody or antigen-binding fragment thereof is chimeric, humanized, or human.

84. 84. The method of any one of claims 72-83, wherein the first isolated antibody or antigen-binding fragment thereof and / or the second isolated antibody or antigen-binding fragment thereof comprises a human Fc region.

85. The method of any one of claims 72 to 84, wherein the subject is a human.

86. The method of any one of claims 72 to 85, wherein the antibody or antigen-binding fragment thereof that specifically binds to CD117 and / or the antibody or antigen-binding fragment thereof that specifically binds to CD110 is conjugated to a toxin.

87. 87. The method of claim 86, wherein the toxin is selected from the group consisting of saporin, saporin derivatives, ricin, abrin, gelonin, momordin, apitoxin, shiga toxin, shiga-like toxin, T-2 mycotoxin, diphtheria toxin, busulfan, Pseudomonas exotoxin A, ricin A chain derivatives, trichosanthin, raffin toxin, maytansine, amatoxin, mechlorethamine, cyclophosphamide, ethyleneimine, methylmelamine, methotrexate, fluorouracil, floxuridine, cytarabine, mercaptopurine, azathioprine, thioguanine, fludarabine phosphate, cladribine, dolastatin, auristatin, auristatin E, auristatin F, MMAF, MMAE, MMAD, DMAF, or DMAE, maytansine, DM1 or DM4, duocarmycin, calicheamicin, pyrrolobenzodiazepines, exatecan, and any combination thereof.

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