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WO2024173594A1 - Modified stem cell compositions and methods for use - Google Patents

Modified stem cell compositions and methods for use Download PDF

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Publication number
WO2024173594A1
WO2024173594A1 PCT/US2024/015853 US2024015853W WO2024173594A1 WO 2024173594 A1 WO2024173594 A1 WO 2024173594A1 US 2024015853 W US2024015853 W US 2024015853W WO 2024173594 A1 WO2024173594 A1 WO 2024173594A1
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WIPO (PCT)
Prior art keywords
modified
polypeptide
cell
antibody
sequence
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PCT/US2024/015853
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French (fr)
Inventor
Wendy PANG
Hye-Sook Kwon
Judith SHIZURU
Robert Sikorski
Leopold Daniel D'ESPAUX
Song Eun Lee
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Jasper Therapeutics Inc
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Jasper Therapeutics Inc
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Publication of WO2024173594A1 publication Critical patent/WO2024173594A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • HCT Hematopoietic cell transplantation
  • HSC autologous or allogeneic donor hematopoietic stem cells
  • HSPC hematopoietic stem and progenitor cells
  • HCT may be performed as part of therapy to treat a number of disorders, including cancers, such as leukemias, and immunodeficiency disorders.
  • HCT can result in the cure of a vast number of otherwise incurable and chronic diseases by replacing the defective or diseased blood-forming stem cells of the recipient with those from a healthy donor or with gene-corrected cells. While transplants can potentially cure disease, stem cells must reach and engraft in the bone marrow to have a disease-modifying effect.
  • unmodified stem cell grafts do not provide any inherent advantage relative to endogenous stem cells to enable homing to the bone marrow niche.
  • stem cell transplant In order to increase the likelihood of stem cell engraftment, stem cell transplant currently requires toxic conditioning to deplete the patient’s existing stem cells in the marrow and donor lymphocytes to overcome the immune barrier. Even with these additions, a significant number of patients still face graft failure. Furthermore, there are significant complications associated with intensive conditioning as well as graft versus host disease. As a result, despite the curative capacity of HCT, access to transplant is limited to only a fraction of patients who could benefit due to toxicities and unwanted complications associated with the procedure.
  • a significant barrier to the safety and efficacy of stem-cell based therapies is the failure of healthy donor or gene-corrected stem cells to engraft in a patient’s bone marrow.
  • HCT hematopoietic stem cells
  • HSPC hematopoietic stem and progenitor cells
  • the disclosure provides a modified or engineered cell comprising a nucleic acid encoding a CD117 polypeptide, including variants thereof.
  • the modified CD117 polypeptides are capable of signaling in HSCs and/or HSPCs in the absence of stem cell factor (SCF) binding.
  • the modified CD117 polypeptides provide for constitutive signaling and/or CD117-mediated kinase activity when expressed or present in cells, e.g., HSCs and/or HSPCs. Accordingly, in particular embodiments, when expressed or present in HSCs and/or HSPCs, the modified CD117 polypeptides allow CD117 signaling when bound by antibodies that block SCF binding to CD117.
  • an HSC or HSPC is transduced with a vector disclosed herein or a modified messenger (mRNA) encoding the CD117 polypeptide, e.g., the modified cell comprises an exogenous or introduced polynucleotide sequence encoding the CD117 polypeptide.
  • mRNA modified messenger
  • a modified hematopoietic stem cell (HSC) or hematopoietic stem and progenitor cell (HSPC) comprises a nucleic acid encoding a modified CD117 polypeptide, optionally wherein the modified CD117 polypeptide has constitutive CD117 signaling and/or kinase activity, optionally wherein the modified cell is capable of proliferation and/or survival when contacted with an CD117 monoclonal antibody capable of inhibiting proliferation and/or survival of an HSPC expressing only a wild-type CD117, and optionally wherein the nucleic acid comprises the following elements, optionally from 5’ to 3’: a 5’ HBA1 UTR; a CleanCap Reagent AG 3’ OMe 5’ terminal cap sequence; a sequence encoding the modified CD117 polypeptide; a TAATAA stop codon; and a 3’ HBB1 UTR.
  • a nucleic acid sequence of the disclosure comprises a sequence of Table 1 or Table 2, e.g., SEQ ID NOS: 64-71 or SEQ ID NOS: 73-76.
  • a nucleic acid sequence of the disclosure e.g., a vector sequence or mRNA comprises a sequence of Table 1 or Table 2, or a variant or fragment thereof, e.g., having at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.
  • an mRNA sequence of the disclosure comprises one or more of the following modifications: pseudouridine substitution of one or more uridine; N1-methyl-pseudouridine substitution of one or more uridine; 5 methoxyuridine substitution of one or more uridine; 5-methylcytidine substitution of one or more cytidine; a m7G(5')ppp(5')(2'OMeA)pG cap sequence; or a m7(3'OMeG)(5')ppp(5')(2'OMeA)pG cap sequence.
  • the polypeptide or the polynucleotide is humanized.
  • the disclosure provides a modified CD117 polypeptide comprising one or more amino acid modifications as compared to a wild type CD117 polypeptide, e.g., one or more amino acid substitutions, insertions, or deletions.
  • the modified CD117 polypeptide comprises one or more amino acid substitutions at one or more of the following amino acids present in wild type human CD117: N505 or D816, e.g., a D816V substitution and/or a N505I substitution.
  • the one or more amino acid modifications is located within surface exposed amino acid residues or regions of the extracellular domain, the membrane spanning region, or the intracellular domain of the wild type CD117 polypeptide, e.g., the juxtamembrane region or a kinase domain.
  • the modified CD117 polypeptide has at least 90%, at least 95%, at least 98%, or at least 99% sequence homology to the wild type CD117 polypeptide, or a functional fragment thereof.
  • the wild type CD117 polypeptide is a wild type human CD117 polypeptide, optionally having one of the following amino acid sequences: MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRY
  • the modified CD117 polypeptide substantially retains kinase activity as compared to the wild type CD117 polypeptide, and in some embodiments, the modified CD117 polypeptide has substantially increased kinase activity as compared to the wild type CD117 polypeptide. In particular embodiments, the modified CD117 polypeptide substantially retains or has increased kinase activity, optionally constitutive kinase activity, in the absence of SCF binding, as compared to the kinase activity of wild type CD117 polypeptide in the presence of SCF binding. In particular embodiments, the modified CD117 polypeptide substantially retains kinase activity, optionally in response to SCF binding, as compared to the wild type CD117 polypeptide.
  • the modified CD117 polypeptide has increased kinase activity, optionally in response to SCF binding, as compared to the wild type CD117 polypeptide.
  • the modified CD117 has constitutive kinase activity, optionally in the absence of SCF binding, of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of kinase activity in response to SCF binding in cells, e.g., HSCs and/or HSPCs, expressing the wild type CD117 polypeptide.
  • the one or more amino acid modifications do not substantially inhibit or reduce CD117 signaling or cell proliferation, optionally in response to SCF binding, by the modified CD117 polypeptide expressed in cells, e.g., HSCs and/or HSPCs, as compared to the wild type CD117 polypeptide.
  • the one or more amino acid modifications do not substantially inhibit or reduce binding of an anti-c-Kit antibody (also referred to as a CD117 antibody) to the modified CD117 polypeptide expressed in cells, e.g., HSCs and/or HSPCs, as compared to the wild type CD117 polypeptide.
  • the CD117 antibody disrupts or blocks binding of SCF to wild type CD117 and/or the modified CD117 polypeptide.
  • the CD117 antibody comprises the six CDRs present in any one of JSP191, AB85, CDX-0159, or FSI-174.
  • the CD117 antibody in any one of JSP191, AB85, CDX-0159, or FSI-174.
  • the one or more amino acid modifications do not substantially inhibit or reduce binding of stem cell factor (SCF) to the modified CD117 polypeptide expressed in cells as compared to the wild type CD117 polypeptide.
  • SCF stem cell factor
  • the disclosure provides a nucleic acid encoding the modified CD117 polypeptide.
  • the nucleic acid comprises RNA, DNA, or a combination thereof. In one embodiment, the nucleic acid comprises a modified mRNA. In particular embodiments, the nucleic acid is associated with one or more lipids, optionally wherein the nucleic acid is present within a lipid nucleic acid particle, a lipid nanoparticle, or a liposome. [0018] In a further related embodiment, the disclosure provides a vector comprising the nucleic acid encoding the modified CD117 polypeptide. In certain embodiments, the vector is an expression vector, e.g., an AAV vector or a lentiviral vector. In particular embodiments, the vector is capable of transducing hematopoietic stem cells.
  • the disclosure provides a host cell comprising a vector comprising the nucleic acid encoding the modified CD117 polypeptide.
  • the host cell is a bacterial or mammalian cell.
  • the disclosure provides a modified cell comprising the modified CD117 polypeptide and/or the nucleic acid encoding the modified CD117 polypeptide.
  • the cell expresses both the modified CD117 polypeptide and wild type CD117 polypeptide.
  • the cell was transduced with the vector.
  • the cell is a stem cell.
  • the cell is a hematopoietic stem and progenitor cell (HSPC) or a hematopoietic stem cell (HSC).
  • the cell is CD34+, and in some embodiments, the cell is CD34+/CD90+, CD34+/CD38-, CD34+/CD38-/CD90+, or CD34+/CD133+.
  • the cell is a human cell. In some embodiments, the cell was obtained from a mammalian donor, e.g, a human donor.
  • the mammalian donor is a subject in need of a hematopoietic stem cell transplant (autologous donor), wherein in other embodiments, the mammalian donor is not the subject in need of the hematopoietic stem cell transplant (allogeneic donor).
  • the cell expresses the modified CD117 polypeptide, optionally wherein the modified cell expresses the modified CD117 polypeptide transiently.
  • the modified CD117 polypeptide is expressed on the cell surface or in the cell membrane, and in certain embodiments, the cell is capable of proliferating in the presence of an anti-CD117 antibody and/or in the absence of SCF.
  • the anti-CD117 antibody is capable of inhibiting proliferation and/or survival of a cell expressing only the wild-type CD117 but does not substantially inhibit proliferation and/or survival of a cell expressing the modified CD117 polypeptide. In some embodiments, the anti-CD117 antibody induces apoptosis or death of a cell expressing only the wild-type CD117 but does not substantially induce apoptosis or death of a cell expressing the modified CD117 polypeptide. In certain embodiments, contact with or the presence of the anti-CD117 antibody results in less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% as much cell death in cells expressing the modified CD117 polypeptide as compared to in cells expressing only the wild-type CD117 polypeptide.
  • the anti-CD117 antibody is selected from the group consisting of: JSP191, CDX-0159, AB85, and FSI-174.
  • the disclosure provides a pharmaceutical composition comprising the modified cells, e.g., HSCs and/or HSPCs, comprising the nucleic acid encoding the modified CD117 polypeptide, and a pharmaceutically acceptable excipient, carrier, or diluent.
  • the pharmaceutical composition further comprises an anti- CD117 antibody.
  • the pharmaceutical composition further comprises one or more anti-CD47, anti-CD40L, anti-CD122, anti-CD4, and/or anti-CD8 antibody.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable excipient, carrier, or diluent and a modified hematopoietic stem cell (HSC) or a hematopoietic stem and progenitor cell (HSPC), wherein the modified HSC or HSPC comprises a modified CD117 polypeptide, optionally wherein the modified CD117 polypeptide has constitutive CD117 signaling and/or kinase activity, and optionally wherein the modified cell is capable of proliferation and/or survival when contacted with an CD117 monoclonal antibody capable of inhibiting proliferation and/or survival of an HSPC expressing only a wild-type CD117.
  • HSC hematopoietic stem cell
  • HSPC hematopoietic stem and progenitor cell
  • the CD117 signaling and/or kinase activity of the modified CD117 is not substantially inhibited by an CD117 monoclonal antibody.
  • the CD117 monoclonal antibody inhibits binding of SCF to CD117.
  • the CD117 monoclonal antibody comprises one or more of the six CDRs present in any one of JSP191, AB85, CDX-0159, or FSI-174.
  • the CD117 monoclonal antibody is any one of JSP191, AB85, CDX- 0159, or FSI-174.
  • the CD117 antibody is JSP191.
  • the CD117 antibody is FSI-174.
  • the modified CD117 comprises one or more amino acid modifications as compared to the wild-type CD117 polypeptide.
  • the one or more amino acid modifications comprise one or more amino acid substitutions, insertions, or deletions.
  • one or more of the amino acid modifications are present within surface exposed amino acid residues of the extracellular domain, within the membrane spanning domain, or within an intracellular domain of the modified CD117 polypeptide.
  • the modified CD117 polypeptide comprises substitution or deletion of one or more of the following amino acids present in wild type human CD117: N505 or D816.
  • the modified CD117 polypeptide comprises a D816V substitution and/or a N505I substitution as compared to wild type human CD117.
  • the modified CD117 polypeptide has at least 90%, at least 95%, at least 98%, or at least 99% sequence homology to wild type CD117 polypeptide.
  • the modified cell expresses both the modified CD117 polypeptide and a wild type CD117 polypeptide.
  • the modified cell expresses the modified CD117 polypeptide transiently.
  • the HSC or HSPC is CD34+, optionally wherein the HSPC is CD34+/CD90+, CD34+/CD38-, or CD34+/CD38-/CD90+, or CD34+CD133+.
  • the cell is a human cell. In certain embodiments, the cell was obtained from a mammalian donor.
  • the mammalian donor is a subject in need of a hematopoietic cell transplant (HCT).
  • the mammalian donor is a healthy donor.
  • the cell obtained from the mammalian donor was modified ex vivo.
  • the pharmaceutical composition further comprises an anti-CD117 antibody, an anti-CD47, anti-CD40L, anti- CD122, anti-CD4, and/or an anti-CD8 antibody.
  • the disclosure includes a method of modifying a cell, e.g., an HSC and/or HSPC, comprising introducing a nucleic acid or vector encoding a modified CD117 polypeptide into the cell, optionally wherein the cell is transiently modified, and optionally wherein the method is for preparing modified cells for hematopoietic cell transplantation (HCT) into a mammalian subject.
  • the nucleic acid or vector is introduced into the cell by transfection, transduction, infection, electroporation, or nanopore technology.
  • the disclosure includes a method of treating a mammalian subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising modified cells, e.g., HSCs and/or HSPCs, comprising the nucleic acid encoding the modified CD117 polypeptide and/or the modified CD117 polypeptide.
  • the method further comprises administering to the subject a conditioning regimen to facilitate or increase engraftment of the modified cells, or deplete endogenous, wild-type HSCs, wherein the conditioning regimen is administered prior to or concurrent with the administering of the pharmaceutical composition.
  • the conditioning regimen comprises or consists of an anti-CD117 antibody, optionally JSP191.
  • the conditioning regimen comprises one or more of: chemotherapy (optionally a nucleoside analog and/or an alkylating agent), monoclonal antibody therapy, and radiation, optionally radiation to the entire body.
  • chemotherapy optionally a nucleoside analog and/or an alkylating agent
  • monoclonal antibody therapy optionally radiation to the entire body.
  • the conditioning regimen is milder than would be used if the subject was being administered hematopoietic stem cells that did not comprise the modified CD117 polypeptide.
  • the subject is not administered a conditioning regimen to facilitate or increase engraftment of the modified cells, prior to or concurrent with the administering of the pharmaceutical composition, or the conditioning regimen only comprises the anti-CD117 antibody.
  • the method results in reduced toxicity, reduced morbidity, or reduced graft-versus-host disease, as compared to a method wherein a subject is administered hematopoietic stem cells that do not comprise the modified CD117 polypeptide in combination with a conditioning regimen, e.g., a reduction of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% in toxicity, morbidity, and/or graft-versus-host disease.
  • a conditioning regimen e.g., a reduction of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% in toxicity, morbidity, and/or graft-versus-host disease.
  • methods of cell transplant disclosed here are used to treat a hematologic diseases that could benefit from hematopoietic stem cell transplantation.
  • the method is used to treat a disease or disorder selected from the group consisting of: a cancer, a cardiac disorder, a neural disorder, an autoimmune disease, an immunodeficiency, a metabolic disorder, and a genetic disorder.
  • the cancer is a solid tissue cancer or a blood cancer, e.g., a leukemia, a lymphoma, or a myelodysplastic syndrome, such as acute myeloid leukemia (AML).
  • the immunodeficiency is severe combined immunodeficiency (SCID).
  • the genetic disorder is sickle cell disease or Fanconi anemia.
  • the methods further comprises administering to the subject another therapeutic agent for treatment of the disease or disorder.
  • FIG. 1 is a graph showing the OD595 of wild type BaF3 cells (BaF3), or BaF3 cells expressing wild-type CD117 (CD117) or the CD117-D816V mutant in the presence of the indicated concentrations of stem cell factor (SCF), and in the presence or absence of the anti- CD117 antibody, JSP191.
  • SCF stem cell factor
  • FIG. 2 is a table showing various myeloablative, reduced intensity myeloablative, and non-myeloablative conditioning regimens, reproduced from Atilla, Erden et al. “A Review of Myeloablative vs Reduced Intensity/Non-Myeloablative Regimens in Allogeneic Hematopoietic Stem Cell Transplantations.” Balkan Medical Journal, Vol.
  • FIG.3 is a graph showing cKit expression kinetics following electroporation of mRNAs encoding the indicated cKit into human CD34+ cells. At about 20 hours, the lines from top to bottom correspond to: wild type cKit, unidentified cKit mutant, unidentified cKit mutant, cKit N505I, control, and mock electroporation (EP). The cKit construct is ct180 with N1m- pseudouridine instead of uridine, [0028] FIG. 4 shows viability and live cell numbers of CD34+ cells one day following electroporation with the indicated mRNA or control.
  • FIG.5 shows the design of the segmented polyA tail A140S, in which the 140 adenine bases are segmented into 2x 70 adenine sequences.
  • HCT Hematopoietic stem cell transplantation
  • HSCs healthy hematopoietic stem cells
  • HSPCs hematopoietic stem and progenitor cells
  • HCT graft-vs-host disease
  • Certain HCT procedures include conditioning a patient prior to HCT by treatment with an CD117 antibody that inhibits stem cell factor (SCF) from binding to CD117 on the surface of a patient’s endogenous HSCs, which depletes endogenous HSCs prior to transplant of HSCs and/or HSPCs into the patient.
  • SCF stem cell factor
  • the present disclosure provides compositions and methods that augment the ability of donor or autologous gene-corrected HSCs and/or HSPCs to engraft and/or persist in recipients, thereby increasing the likelihood of success of an HCT procedure, and reducing the toxicities associated with HCT.
  • the disclosure provides modified HSCs and/or HSPCs for transplant that comprise a modified CD117 polypeptide having constitutive activity or retaining activity (e.g., CD117 kinase activity), even in the presence of CD117 antibodies that inhibit SCF binding to the modified CD117 polypeptide.
  • the modified CD117 polypeptide has constitutive activity even in the presence of the CD117 antibody JSP191.
  • the modified HSCs and/or HSPCs have activity (e.g., constitutive CD117 kinase activity) even when not bound by SCF.
  • the modified HSCs and/or HPSCs comprising a modified CD117 polypeptide are not substantially depleted by CD117 antibodies used in conditioning therapies. Accordingly, the modified cells can be transplanted into the subject in the presence of CD117 antibodies without being subject to depletion, thus providing an improved method of conditioning a patient for HCT and potentially allowing a reduced washout period and/or other advantages.
  • the HCT methods provided herein may reduce the need for intensive chemotherapy, radiation, and/or donor lymphocytes or other cells used to facilitate HSC and/or HSPC engraftment, thereby reducing the toxicity of HCT.
  • the compositions and methods disclosed herein may be used to treat all disorders for which blood stem cell transplantation is indicated.
  • Binding of SCF and/or CD117 antibodies to the modified CD117 polypeptides may be determined by a variety of methods known in the art. For example, binding may be determined using transiently transfected HEK293T cells that express the modified CD117. Following transfection, cells are incubated with SCF and/or CD117 antibodies.
  • Bound monoclonal antibodies may be detected using an Alexa Fluor 488-conjugated secondary antibody and cellular fluorescence determined by flow cytometry.
  • Bound SCF may be determined using a fluorescently-labeled antibody that binds SCF and flow cytometry.
  • the disclosure provides for compositions and methods for the ex vivo introduction of the CD117 variants and mutants (modified CD117), by RNA-based and/or DNA-based methods, into HSCs and/or HSPCs, including but not limited to CD34+ cells or subsets of CD34+ cells, such that the HSCs and/or HSPCs are able to be successfully transplanted into recipients.
  • the modified CD117 may be expressed transiently in the modified HSCs and/or HSPCs.
  • a nucleic acid encoding a modified CD117 may be transiently introduced into HSCs/and/or HSPCs prior to transplant, where it expresses the modified CD117.
  • the modified CD117 may be expressed in addition to the endogenous wild type CD117. Transplantation of these modified HSCs and/or HSPCs may be done after or in combination with conditioning therapies, including treatment with antibodies (such as anti- CD117 antibodies).
  • These HSCs and/or HSPCs may be transplanted alone or in combination with other cells.
  • antibody includes reference to an immunoglobulin molecule immunologically reactive with a particular antigen, and includes both polyclonal and monoclonal antibodies.
  • the term also includes genetically engineered forms such as humanized antibodies, chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies.
  • antibody also includes antigen binding forms of antibodies, including fragments with antigen-binding capability (e.g., Fab', F(ab')2, Fab, Fv and rIgG. The term also refers to recombinant single chain Fv fragments (scFv). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies.
  • a "humanized antibody” is an immunoglobulin molecule which contains minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody non-human species
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Kabat provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. Unless otherwise specified numbering of positions within the variable regions of antibodies is Kabat numbering.
  • Kabat a certain definition of CDRs (e.g., Kabat) that definition specifies the minimum number of CDR residues present in the antibody (i.e., the Kabat CDRs). It does not exclude that other residues falling within another conventional CDR definition but outside the specified definition are also present.
  • an antibody comprising CDRs defined by Kabat includes among other possibilities, an antibody in which the CDRs contain Kabat CDR residues and no other CDR residues, and an antibody in which CDR H1 is a composite Chothia-Kabat CDR H1 and other CDRs contain Kabat CDR residues and no additional CDR residues based on other definitions.
  • polynucleotide refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs or mixtures thereof.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide or nucleoside analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the term polynucleotide, as used herein, includes, but is not limited to, double- and single-stranded molecules, and mixtures thereof.
  • any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form, whether as RNA or DNA, or a mixture thereof.
  • polypeptide refers to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, to include disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.
  • a polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences.
  • sequence identity refers to the percentage identity obtained when sequences are aligned for maximum correspondence over a comparison window (e.g., a specified region of each of the sequences), which may be calculated by any of the algorithms described herein using default parameters, which are expected to generate the same alignment, in most cases, when applied to similar sequences. Identity is calculated, unless specified otherwise, across the full length of the reference sequence.
  • a sequence-of-interest “shares at least x% identity to” a reference sequence if, when the sequence-of-interest is aligned to the reference sequence, at least x% (rounded down) of the residues in the sequence-of-interest are aligned as an exact match to a corresponding residue in the reference sequence. Gaps may be introduced into the sequence- of-interest and/or the reference sequence to maximize correspondence over the comparison window.
  • Sequence similarity i.e., identity
  • sequences can be aligned using the methods and computer programs, including BLAST, available over the worldwide web at ncbi.nlm.nih.gov/BLAST/.
  • sequence identity is determined using the BLAST algorithm (e.g., bl2seq) with default parameters.
  • FASTA Genetics Computing Group
  • GCG Genetics Computing Group
  • Other techniques for alignment are described in Methods in Enzymology, vol.266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA.
  • alignment programs that permit gaps in the sequence.
  • the Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol.
  • the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol.48: 443-453 (1970). [0049] Of interest is the BestFit program using the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482-489 (1981) to determine sequence identity.
  • the gap generation penalty will generally range from 1 to 5, usually 2 to 4 and in many embodiments will be 3.
  • the gap extension penalty will generally range from about 0.01 to 0.20 and in many instances will be 0.10.
  • the program has default parameters determined by the sequences inputted to be compared.
  • the sequence identity is determined using the default parameters determined by the program.
  • FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based upon the following parameters: Mismatch Penalty: 1.00; Gap Penalty: 1.00; Gap Size Penalty: 0.33; and Joining Penalty: 30.0.
  • a "vector” as used herein refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which can be used to mediate delivery of the polynucleotide to a cell.
  • Illustrative vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery vehicles.
  • An "expression vector” as used herein encompasses a vector, e.g., plasmid, minicircle, viral vector, liposome, and the like, as discussed herein or as known in the art, comprising a polynucleotide which encodes a gene product of interest, and is used for effecting the expression of a gene product in an intended target cell.
  • An expression vector also comprises control elements operatively linked to the encoding region to facilitate expression of the gene product in the target.
  • control elements e.g., promoters, enhancers, UTRs, miRNA targeting sequences, etc.
  • a "promoter” as used herein encompasses a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. Promoters and corresponding protein or polypeptide expression may be ubiquitous, meaning strongly active in a wide range of cells, tissues, and species, or it may be cell-type specific, tissue-specific, or species specific.
  • Promoters may be “constitutive,” meaning continually active, or “inducible,” meaning the promoter can be activated or deactivated by the presence or absence of biotic or abiotic factors.
  • "Operatively linked” or “operably linked” refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.
  • mutant refers to a mutant of a reference polynucleotide or polypeptide sequence (e.g., a native or wild-type) polynucleotide or polypeptide sequence, i.e., having less than 100% sequence identity with the reference polynucleotide or polypeptide sequence.
  • a variant comprises at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to a reference polynucleotide sequence, e.g., a native polynucleotide or polypeptide sequence.
  • a variant may be a polynucleotide having a sequence identity of 50% or more, 60% or more, or 70% or more with a full-length native polynucleotide sequence, e.g., an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the full-length native polynucleotide sequence.
  • a variant may be a polypeptide having a sequence identity of 70% or more with a full-length native polypeptide sequence, e.g., an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the full- length native polypeptide sequence.
  • Variants may also include variant fragments of a reference sequence, e.g., a native sequence sharing a sequence identity of 70% or more with a fragment of the reference, e.g., native, sequence, e.g., an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the native sequence.
  • modified CD117 polypeptides comprises a modification as disclosed herein and a deletion, such as an N-terminal and/or C-terminal deletion, yet substantially retain CD117 activity, e.g., kinase activity.
  • the modified CD117 has constitutive kinase activity.
  • stem cell refers to a mammalian cell that has the ability both to self-renew, and to generate differentiated progeny (see Morrison et al. (1997) Cell 88:287- 298). Endogenous stem cells may be characterized by the presence of markers associated with specific epitopes.
  • Hematopoietic stem cells are multipotent cells that reside in the bone marrow (BM) and are responsible for the life-long production of mature blood cells.
  • Hematopoietic stem and progenitor cells include HSCs as well as hematopoietic progenitor cells that reside in bone marrow and are capable of differentiating into mature blood cells.
  • HSC and/or HSPC engraftment cells may be fresh, frozen, or subject to prior culture.
  • HSC and/or HSPC may be obtained from fetal liver, bone marrow, cord blood, or peripheral blood, by a donor (allogeneic), the patient themselves (autologous), or any other conventional source.
  • administering or “introducing” or “providing”, as used herein, refer to delivery of a composition to a cell, to cells, tissues and/or organs of a subject, or to a subject. Such administering or introducing may take place in vivo, in vitro, or ex vivo.
  • treatment or “treating” and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, e.g., reducing the likelihood that the disease or symptom thereof occurs in the subject, and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, and includes: (a) inhibiting the disease, i.e., arresting its development; or (b) relieving the disease, i.e., causing regression of the disease.
  • the therapeutic agent may be administered before, during or after the onset of disease or injury.
  • the treatment of ongoing disease where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest.
  • the terms "individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).
  • the term “substantially” means by a significant or large amount or degree.
  • to “substantially” increase may mean to increase by at least two-fold, at least three-fold, at least four-fold, at least five-fold, or at least ten-fold
  • to “substantially” decrease may mean to decrease by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.
  • CD117 Variant Polypeptides and Polynucleotides
  • CD117 also known as c-Kit or stem cell factor receptor (SCFR)
  • SCFR stem cell factor receptor
  • RTK type III receptor tyrosine kinase
  • PDGF platelet-derived growth factor
  • CSF-1 macrophage colony-stimulating factor 1
  • CD117 is essential for the development of normal hematopoietic cells and plays an important role in the survival, proliferation, and differentiation of mast cells, melanocytes, and germ cells.
  • CD117 includes: an approximately 519 amino acid extracellular domain comprised of five immunoglobulin-like domains; a transmembrane segment; a juxtamembrane domain; and a protein kinase domain that contains an insert of about 80 amino acid residues. Approximately 184 amino acids of the extracellular domain are surface exposed, which were identified based on x-ray crystallographic studies.
  • CD117 The crystallographic structure of CD117 is provided in, e.g., Mol, et al., Accelerated Publications, Volume 278, ISSUE 34, P31461-31464, August 22, 2003; Ogg et al., RCSB Protein Data Bank, 6XV9, Crystal structure of the kinase domain of human c-KIT in complex with a type-II inhibitor, DOI: 10.2210/pdb6XV9/pdb; McAuley et al., RCSB Protein Data Bank Alkynyl Benzoxazines and Dihydroquinazolines as Cysteine Targeting Covalent Warheads and Their Application in Identification of Selective Irreversible Kinase Inhibitors, DOI: 10.1021/jacs.9b13391; Schimpl et al., RCSB Protein Data Bank 6GQM, Crystal structure of human c-KIT kinase domain in complex with a small molecule inhibitor, AZD3229, DOI
  • the disclosure provides a modified CD117 polypeptide comprising one or more amino acid modifications as compared to a wild type CD117 polypeptide.
  • the one or more amino acid modifications comprise one or more amino acid substitutions, insertions, or deletions.
  • the one or more amino acid modifications are located in the extracellular domain of the CD117 polypeptide.
  • the one or more amino acid modifications are located in one or more surface exposed amino acids or regions of the CD117 polypeptide’s extracellular domain.
  • the modified CD117 comprises one or more modification within the juxtamembrane region or the kinase domain.
  • the modified CD117 polypeptides comprise one or more deletions, e.g., an N-terminal or C-terminal deletion, optionally wherein the deletion does not substantially impair biological activity, e.g., signaling, of the modified CD117 polypeptide.
  • the modified CD117 polypeptides retain or have at least 90%, at least 95%, at least 98%, or at least 99% sequence homology to the wild type CD117 polypeptide.
  • the one or more amino acid modifications comprise one or more amino acid substitutions or deletions of an amino acid residue selected from the following in human CD117: N505 or D816.
  • the one or more amino acid modifications comprise one or more amino acid substitutions, e.g., of any of these residues.
  • the amino acid residue is substituted by any other amino acid, by alanine.
  • the amino acid substitution is a conservative amino acid substitution.
  • conservative substitution denotes that one or more amino acids are replaced by another, biologically similar residue. [0066] In the scheme below, conservative substitutions of amino acids are grouped by the indicated physicochemical properties. I: neutral, hydrophilic, II: acids and amides, III: basic, IV: hydrophobic, V: aromatic, bulky amino acids.
  • the one of more amino acid substitutions comprises a D816V substitution and/or a N505I substitution.
  • the wild type CD117 polypeptide upon which the variant is based is a human CD117 polypeptide, while in other embodiments, it is another mammalian CD117 polypeptide. Sequences of human and mammalian CD117 polypeptides are known in the art.
  • the human CD117 polypeptide is expressed as various isoforms, and any of these may be used according to the disclosure.
  • These isoforms include two GNNK+ and GNNK ⁇ isoforms (also denoted CD117 and CD117A, respectively), which differ by the presence or absence of four amino acids, 510-GNNK-513 (SEQ ID NO: 25) in the extra-cellular domain adjacent to the trans-membrane domain, and which are co-expressed in most tissues, although the GNNK ⁇ isoform usually predominates.
  • Isoforms may also differ in the presence or absence of a Ser residue at position 715 in the inter- kinase domain, and the disclosure also includes isoforms of CD117, including those shown below, in which Ser175 is either present or absent.
  • These isoforms may comprise any of the modifications disclosed herein, including, e.g., an N505I or D816V modification, and variants thereof, e.g., comprising at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.
  • the wild type CD117 polypeptide is the GNNK+ or GNNK- isoform and comprises or consists of one of the following amino acid sequences (the GNNK tetrapeptide (SEQ ID NO: 25), and the N505 and D816 residues are in bold; numbering is based on GNNK- isoform): MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNY
  • the modified CD117 polypeptide comprises or consists of either of the following sequences: MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTY
  • the variant retains the N505I or D816V amino acid substitution present in the modified CD117.
  • the CD117 variant comprises a different amino acid modification that confers constitutive activity to the modified CD117.
  • a fragment substantially retains CD117 kinase activity, e.g., retains at least 50% CD117 kinase activity as wild type CD117.
  • the modified CD117 polypeptide substantially retains kinase activity as compared to the wild type CD117 polypeptide.
  • the modified CD117 polypeptide has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the kinase activity of the wild type CD117 polypeptide when bound by SCF, and in certain embodiments, the modified CD117 has this activity even in the absence of SCF binding. In some embodiments, the modified CD117 polypeptide has increased kinase activity as compared to the wild type CD117 polypeptide. In particular embodiments, the modified CD117 polypeptide has at least 150%, at least 200%, at least 300%, at least 500%, at least 750%, or at least 1000% of the kinase activity of the wild type CD117 polypeptide.
  • the modified CD117 has constitutive kinase activity, even in the absence of SCF binding or in the presence of the CD117 antibody.
  • Kinase activity may be determined using assays known in the art, including the ADP ⁇ GloTM Kinase Assay, which is a luminescent kinase assay that measures ADP formed from a kinase reaction; ADP is converted into ATP, which is converted into light by Ultra ⁇ GloTM Luciferase (available from Promega Corporation, Madison, WI).
  • the modified CD117 polypeptide constitutively phosphorylates Gab2, Shc, SHP-2 and/or Cbl.
  • the one or more amino acid modifications do not substantially inhibit or reduce binding of stem cell factor (SCF) to the modified CD117 polypeptide when expressed in cells, as compared to the binding of SCF to the wild type CD117 polypeptide. In particular embodiments, the one or more amino acid modifications do not substantially inhibit or reduce binding of stem cell factor (SCF) to the modified CD117 polypeptide expressed in cells as compared to the wild type CD117 polypeptide.
  • the modified CD117 is a modified CD117 having constitutive signaling or kinase activity, e.g., without bound SCF ligand. In certain embodiments, the modified CD117 has constitutive autophosphorylation activity, e.g., without bound SCF.
  • modified CD117 have been identified, e.g., in cancer cells, and any of these may be used according to the compositions and methods disclosed herein.
  • activating or gain-of-function CD117 modifications include, but are not limited to, N505I, V559D, D816V, D816H, V568F, V570F, or Y703F, modifications or mutation of amino acid residues corresponding to 505, 522, 816, 557, 558, 559, 568, 569, 570, 703, 816, or deletion of codon 579 (Asp).
  • the amino acid modification is in the region between the transmembrane and tyrosine kinase domains.
  • Mutations causing constitutive activation of CD117 have been shown to be causative in some forms of mastocytosis, and several types of mutations have been associated with myeloproliferative disorders (MPDs), acute myelogenous leukemia (AML), sinonasal lymphomas, and gastrointestinal stromal tumors (GIST). These may be considered activating mutation of two types — ‘regulatory type’ mutations, which affect regulation of the kinase molecule, and ‘enzymatic pocket type’ mutations, which alter the amino acid sequence directly forming the enzymatic site. Either type of mutation may be used according to various embodiments of the disclosure, including any of those disclosed in Longley et al., Leukemia Research, Vol.
  • the one or more amino acid modifications do not result in cells expressing only the modified CD117 having substantially inhibited or reduce CD117 signaling or proliferation, optionally in response to SCF binding, as compared to the signaling in cells only expressing the wild type CD117 polypeptide.
  • the modified CD117 retains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% CD117 signaling and/or proliferation, optionally in response to SCF binding, as compared to the corresponding wild type CD117.
  • the modified CD117 retains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% CD117 signaling and/or proliferation, in the absence of SCF binding, as compared to the corresponding wild type CD117.
  • the one or more amino acid modifications do not substantially inhibit or reduce binding of an CD117 antibody to the modified CD117 polypeptide expressed in cells as compared to the wild type CD117 polypeptide.
  • the CD117 antibody comprises the six CDRs present in any one of JSP191, AB85, CDX-0159, or FSI-174.
  • the CD117 antibody in any one of JSP191, AB85, CDX-0159, or FSI-174.
  • the CD117 antibody is JSP191 or comprises the six CDRs present in JSP191.
  • the CD117 antibody is AB85 or comprises the six CDRs present in AB85.
  • the CD117 antibody is CDX-0159 or comprises the six CDRs present in CDX-0159.
  • the CD117 antibody is FSI-174 or comprises the six CDRs present in FSI-174.
  • the disclosure also provides nucleic acid or polynucleotides encoding a modified CD117 polypeptide disclosed herein.
  • the nucleic acid comprises RNA, DNA, or a combination thereof, and in particular embodiments, the nucleic acid comprises single-stranded and/or double-stranded regions, or a mixture thereof.
  • the nucleic acid is a double-stranded DNA, and in certain embodiments, the nucleic acid is a single stranded RNA, e.g., a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the nucleic acid comprises a modified mRNA.
  • polynucleotides described herein are codon-optimized, e.g., to enhance expression of the encoded polypeptide in a host cell.
  • polynucleotide variants comprise one or more modified nucleotide or nucleoside.
  • Modified mRNAs comprising one or more modified nucleoside have been described as having advantages over unmodified mRNAs, including increase stability, higher expression levels and reduced immunogenicity.
  • modifications to mRNAs that may be present in the nucleic acids encoding the modified CD117 polypeptides are described, e.g., in PCT Patent Application Publication Nos.
  • the sequence encoding a modified CD117 protein comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the modified CD117 encoding sequences disclosed herein.
  • the mRNA construct encoding a CD47 protein comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the CD47 encoding sequences disclosed herein or comprises an mRNA construct sequence disclosed herein, optionally without the promoter sequence and optionally with a polyA tail.
  • the modified mRNA comprises one or more nucleoside modification.
  • the modified mRNA sequence comprises at least one modification as compared to an unmodified A, G, U or C ribonucleoside.
  • uridine can a similar nucleoside such as pseudouridine ( ⁇ ) or N1-methyl-pseudouridine (m1 ⁇ ), and cytosine can be replaced by 5-methylcytosine.
  • the at least one modified nucleosides include N1-methyl-pseudouridine and/or 5-methylcytidine.
  • one or more uridines are replaced by 5- methoxyuridine (5moU).
  • the modified mRNA comprises a 5’ terminal cap sequence followed by a sequence encoding the modified CD117 polypeptide, followed by a 3’ tailing sequence, such as a polyA or a polyA-G sequence.
  • the nucleic acid sequence encoding the modified CD117 comprises 5’ and/or 3’ cellular or viral untranslated regions (UTRs) relative to the sequence encoding the CD117 polypeptide.
  • the UTR improves mRNA stability, localization and/or expression.
  • the UTR is tissue specific.
  • the 5’ UTR comprises a UTR sequence from alpha-globin.
  • the nucleic acid comprises a Kozak sequence.
  • the 3’UTR comprises a UTR from an alpha-globin and/or a beta-globin gene, i.e., a 5’ UTR from hemoglobin alpha 1 (HBA1) and/or a 3’ UTR from one or more of HBA1 or hemoglobin beta 1 (HBB1) gene.
  • the nucleic acid sequence encoding the modified CD117 comprises a 5’ UTR with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% identity to a 5’UTR sequence of HBA1: ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCACC (SEQ ID NO: 47).
  • the nucleic acid sequence encoding the modified CD117 comprises a Kozak sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% identity to the following: GCCGCCACC.
  • the nucleic acid sequence encoding the modified CD117 comprises a 3’UTR nucleic acid sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% identity to a 3’UTR of HBB1: GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAA CTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATA AAAAACATTTATTTTCATTGC (SEQ ID NO: 48).
  • the nucleic acid sequence encoding the modified CD117 comprises a 3’UTR nucleic acid sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% to a 3’UTR of HBA1: GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCT CCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCA (SEQ ID NO: 49).
  • the nucleic acid sequence encoding the modified CD117 comprises an extra stop codon downstream of TAA to avoid run-off translation of an mRNA.
  • the extra stop codon is TGA.
  • the nucleic acid sequence encoding the modified CD117 comprises a TCTAGA sequence to linearize a plasmid as a template for transcription.
  • the nucleic acid sequence encoding the modified CD117 encodes a poly-adenine or poly guanine tail.
  • a polyA or polyA-G tail improves mRNA stability and manufacturability.
  • the polyA tail may be from 20 to 180 adenine bases in length.
  • the polyA tail may be from 35 to 140 bases in length.
  • the polyA tail may be from 70 to 150 bases in length.
  • the polyA tail is segmented with a linker to reduce recombination during plasmid production in prokaryotic cells.
  • the polyA tail is 70 adenine bases in length.
  • the linker is a series of bases other than adenine.
  • the linker is a series of bases including adenine.
  • the linker is about 3 to about 10 bases in length.
  • the linker is about 5 to about 20 bases in length.
  • the linker comprises the sequence TATGCA.
  • the sequence encoding the modified CD117 protein comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the modified CD117 encoding sequences disclosed herein.
  • a modified mRNA comprises a 5’ terminal cap sequence followed by a sequence encoding the modified CD117 polypeptide, including one or more 5’ or 3’ UTRs, followed by a 3’ tailing sequence, such as a polyA or a polyA-G tail sequence.
  • the mRNA encoding CD117 (including modified forms or variants thereof) comprises a wild type 5’ terminal cap sequence, and in certain embodiments, the mRNA encoding CD117 (including modified forms or variants thereof) comprises a modified 5’ terminal cap, not limited to but including, e.g., m7G(5')ppp(5')(2'OMeA)pG (CleanCap® Reagent AG for co-transcriptional capping of mRNA; TriLink Biotechnologies, USA) or m7(3'OMeG)(5')ppp(5')(2'OMeA)pG (CleanCap Reagent AG (3' OMe) for co- transcriptional capping of mRNA; TriLink Biotechnologies, USA).
  • m7G(5')ppp(5')(2'OMeA)pG CleanCap® Reagent AG for co-transcriptional capping of mRNA; TriLink Biotechnologies, USA
  • the mRNA encoding the modified CD117 comprises the modified 5’ terminal cap, 3 ⁇ -O-Me- m7G(5')ppp(5')G (Anti Reverse Cap Analog (ARCA); APExBIO, USA).
  • a vaccinia virus mRNA cap methyltransferase adds 7-methylguanylate cap structures (Cap-0) to the 5’ end of RNA.
  • a vaccinia Cap 2’-O- Methyltransferase adds a methyl group at the 2 ⁇ -O position of the first nucleotide adjacent to the cap structure at the 5’ end of the RNA.
  • a modified CD117 mRNA construct comprises the following elements, optionally from 5’ to 3’: a 5’ HBA1 UTR; a CleanCap Reagent AG 3’ OMe 5’ terminal cap sequence; a sequence encoding the modified CD117 polypeptide; a TAATAA stop codon; and a 3’ HBB1 UTR.
  • the modified CD117 mRNA construct comprising the aforementioned elements from 5’ to 3’ has constitutive CD117 signaling and/or kinase activity.
  • a modified cell comprising the modified mRNA construct comprising the aforementioned elements from 5’ to 3’ is capable of proliferation and/or survival when contacted with an CD117 monoclonal antibody capable of inhibiting proliferation and/or survival of an HSPC expressing only a wild-type CD117.
  • the sequence encoding the modified CD117 is codon- optimized and/or humanized.
  • the construct further comprises a polyA sequence, e.g., after the 3’ HBB1 UTR, optionally an A140S polyA.
  • the mRNA construct comprises any of the sequences disclosed herein. FIG.
  • the mRNA construct comprises any of the polynucleotide sequences disclosed herein, e.g, a sequence comprises a sequence of Table 1 or Table 2, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity to any of the sequences disclosed herein.
  • an mRNA comprises a sequence of Table 1 or Table 2, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity thereto, comprises one or more of the following modifications: pseudouridine substitution of one or more uridine; N1-methyl-pseudouridine substitution of one or more uridine; 5 methoxyuridine substitution of one or more uridine; 5-methylcytidine substitution of one or more cytidine; a m7G(5')ppp(5')(2'OMeA)pG cap sequence; or a m7(3'OMeG)(5')ppp(5')(2'OMeA)pG cap sequence.
  • saRNA self-amplifying RNA
  • saRNA is similar to mRNA, and saRNA is a linear, single-stranded RNA molecule that is synthesized with a 5 ⁇ cap, 3 ⁇ polyA tail and 5 ⁇ and 3 ⁇ UTRs.
  • saRNA also encodes four extra proteins in addition to the transgene, e.g., CD117, being expressed.
  • the nucleic acid e.g., a modified mRNA
  • the nucleic acid is associated with one or more lipids, e.g., to facilitate delivery across the cell membrane, shield its negative charge, and/or to protect against degradation by nucleases.
  • the nucleic acid is associated with or present within a lipid nucleic acid particle, a lipid nanoparticle, or a liposome.
  • the lipid nucleic acid particle, a lipid nanoparticle, or a liposome facilitates delivery or uptake of the nucleic acid by a cell.
  • mRNA optionally modified mRNA, is co-formulation into lipid nanoparticles (LNPs).
  • mRNA-LNP formulations comprise: (1) an ionizable or cationic lipid or polymeric material bearing tertiary or quaternary amines to encapsulate the polyanionic mRNA; (2) a zwitterionic lipid (e.g., 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine [DOPE]) that resembles the lipids in the cell membrane; (3) cholesterol to stabilize the lipid bilayer of the LNP; and (4) a polyethylene glycol (PEG)-lipid to lend the nanoparticle a hydrating layer, improve colloidal stability, and reduce protein absorption.
  • DOPE 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine
  • the nucleic acid encoding the CD117 polypeptide which encompasses functional fragments or variants thereof, is present in a vector.
  • the vector is capable of delivering the nucleic acid into mammalian HSCs and/or HSPCs or other stem cells, e.g., into the nucleus of the HSCs, HSPCs or stem cells.
  • the vector is an episomal vector, e.g., a plasmid.
  • the vector is an expression vector comprising a promoter sequence operatively linked to a nucleic acid sequence encoding the CD117 polypeptide.
  • the expression vector comprises a promoter sequence that facilitates expression of the encoded modified CD117 polypeptide in HSCs, HSPCs and/or other stem cells.
  • the expression vector comprises 5’ and/or 3’ cellular or viral UTRs or the derivatives thereof upstream and downstream, respectively, of the sequence encoding the modified CD117 polypeptide.
  • the vector is a viral vector, optionally an AAV vector, a cytomegalovirus vector, an adenovirus vector, or a lentiviral vector.
  • a viral vector infects an HSC and/or HSPCs when viral vector and the cells are incubated together for at least about 24 hours in a culture medium.
  • modified cells e.g., HSCs and/or HSPCs
  • the modified CD117 polypeptide comprises one or more amino acid substitutions, e.g., at one or more of the following amino acids present in wild type human CD117: N505 or D816.
  • the modified CD117 polypeptide comprises a D816V substitution and/or a N505I substitution.
  • the nucleic acid encoding the modified CD117 polypeptide is transiently present in the modified cell, and it is not present within the genome of the cell.
  • the modified cell expresses and/or comprises the modified CD117 polypeptide, and in particular embodiments, the modified CD117 polypeptide is present on the cell surface, e.g., with the extracellular domain present outside the modified cell.
  • the modified cell is transduced with or infected with an expression vector, optionally a viral vector.
  • the modified cell expresses and/or comprises both the modified CD117 polypeptide and a wild type, endogenous CD117 polypeptide, and in particular embodiments, both the modified CD117 polypeptide and the wild type, endogenous CD117 polypeptide are present on the cell surface, e.g., with their extracellular domains present outside the modified cell.
  • the modified CD117 polypeptide is present on the cell surface, e.g., with its extracellular domain present outside the modified cell.
  • the CD117s are human CD117 or modified forms thereof.
  • any of the modifications disclosed herein may be present in cells that are to be transplanted into a subject, e.g., to treat a disease or disorder in the subject.
  • the modifications are made to cells that would benefit from avoiding immune detection by natural killer (NK) cells or T cells, or from avoiding phagocytosis, when administered to a subject.
  • NK natural killer
  • Illustrative cell types include, but are not limited to, stem cells, induced pluripotent stem cells (iPSCs), T cells, cardiac cells, pancreatic islet cells, NK cells, B cells.
  • the mammalian cells are HSCs and/or HSPCs.
  • the cell is obtained from a mammalian, e.g., a human donor.
  • the mammalian donor is a subject in need of a hematopoietic cell transplant (HCT) or is a healthy donor.
  • the cell obtained from the mammalian donor was modified ex vivo.
  • the modified cell is a stem cell or pluripotent cell, and in certain embodiments, the stem cell is a hematopoietic stem cell (HSC) or an HSPC.
  • the stem cell is a mammalian cell that has the ability both to self-renew, and to generate differentiated progeny.
  • the stem cell is a human cell.
  • the stem cell may have one or more of the following properties: an ability to undergo asynchronous, or symmetric replication, that is where the two daughter cells after division can have different phenotypes; extensive self-renewal capacity; capacity for existence in a mitotically quiescent form; and clonal regeneration of all the tissue in which they exist, for example the ability of hematopoietic stem cells to reconstitute all hematopoietic lineages.
  • HSCs Hematopoietic stem cells
  • HSCs are maintained throughout life (self-renewing). They produce hematopoietic progenitor cells that differentiate into every type of mature blood cell within a well-defined hierarchy.
  • the HSCs and/or HSPCs are obtained from bone marrow, peripheral blood, or umbilical cord blood and subsequently modified by introduction of the nucleic acid encoding the CD117 polypeptide into the cell.
  • HSCs and/or HSPCs can also be generated in vitro, for example from pluripotent embryonic stem cells, induced pluripotent cells, and the like. For example, see Sugimura et al. (2017) Nature 545:432- 438, herein specifically incorporated by reference, which details a protocol for generation of HSCs and/or HSPCs.
  • the cells may be fresh, frozen, or have been subject to prior culture. They may be fetal, neonate, adult, etc.
  • Hematopoietic stem cells and HSPCs may be obtained from fetal liver, bone marrow, blood, particularly G-CSF or GM-CSF mobilized peripheral blood, or any other conventional source. Cells for engraftment are optionally isolated from other cells, where the manner in which the stem cells are separated from other cells of the hematopoietic or other lineage is not critical to this invention. If desired, a substantially homogeneous population of stem or progenitor cells may be obtained by selective isolation of cells free of markers associated with differentiated cells, while displaying epitopic characteristics associated with the stem cells. [00111] Modified HSCs and/or HSPCs may be produced using HSCs and/or HSPCs obtained from a mammalian donor.
  • the donor is a subject in need of a hematopoietic stem cell transplant, e.g., a subject diagnosed with a disease or disorder that can be treated with HCT.
  • the modified HSCs and/or HSPCs may be produced using HSCs and/or HSPCs obtained from a healthy donor, e.g., wherein the modified HSCs and/or HSPCs are to be used to treat a different subject with HCT.
  • the modified HSCs and/or HSPCs may be autologous or allogeneic to a subject in need for HCT.
  • the bone marrow Prior to harvesting stem cells from a donor, the bone marrow can be primed with granulocyte colony-stimulating factor (G-CSF; filgrastim [Neupogen]) to increase the stem cell count.
  • G-CSF granulocyte colony-stimulating factor
  • Mobilization of stem cells from the bone marrow into peripheral blood by cytokines such as G-CSF or GM-CSF has led to the widespread adoption of peripheral blood progenitor cell collection by apheresis for hematopoietic stem cell transplantation.
  • the dose of G-CSF used for mobilization may be about 10 ug/kg/day. In autologous donors who are heavily pretreated, however, doses of up to about 40 ug/kg/day can be given.
  • Mozobil may be used in conjunction with G-CSF to mobilize hematopoietic stem cells to peripheral blood for collection.
  • HSC hematopoietic stem cell
  • CD34 is well known for its unique expression on HSCs and HSPCs.
  • the modified cell is a CD34+ cell.
  • the modified cell is a subset of HSCs or HSPCs that has one of the following patterns or combinations of cell surface marker expression: CD34+/CD90+, CD34+/CD38-, or CD34+/CD38-/CD90+.
  • the CD34+ and/or CD90+ cells may be selected by affinity methods, including without limitation magnetic bead selection, flow cytometry, and the like from the donor hematopoietic cell sample.
  • the HSC and/or HSPC composition may be at least about 50% pure, as defined by the percentage of cells that are CD34+ in the population, may be at least about 75% pure, at least about 85% pure, at least about 95% pure, or more.
  • a composition comprising hematopoietic stem cells (HSCs) and/or hematopoietic stem and progenitor cells (HSPCs), may be administered to a patient.
  • the HSCs and/or HSPCs are optionally, although not necessarily, purified.
  • the subject is administered a cell population enriched for CD34+ hematopoietic stem cells, comprising HSCs and/or HSPCs.
  • the cell populations are enriched for expression of CD34, e.g., by art recognized methods such as the cliniMACS.RTM. system, by flow cytometry, etc.
  • Cell populations single enriched for CD34 may be from about 50% up to about 90% CD34+ cells, e.g., at least about 85% CD34+ cells, at least about 90% CD34+ cells, at least about 95% CD34+ cells and may be up to about 99% CD34+ cells or more.
  • unmanipulated bone marrow or mobilized peripheral blood populations are used.
  • the disclosure provides a method of modifying cells, including stem cells such as HSCs and/or HSPCs, comprising introducing the nucleic acid encoding a modified CD117 polypeptide into the cell.
  • the introduced nucleic acid is present within a viral vector.
  • the nucleic acid is associated with or present in a lipid nanoparticle, liposome, or the like.
  • the nucleic acid remains present in the modified cell only transiently, or the nucleic acid only transiently expresses the modified CD117 polypeptide in the cell.
  • the method is used to prepare modified cells for HCT treatment of a mammalian subject.
  • the nucleic acid or vector may be introduced into the cell by a variety of methods known in the art, such as transfection, transduction, infection, electroporation, or nanopore technology.
  • mRNA e.g., modified mRNA is introduced into the cells using lipid nucleic acid particles (LNPs) or nanoparticles.
  • LNPs lipid nucleic acid particles
  • cells e.g., HSCs and/or HSPCS may be modified by introducing a nucleic acid encoding a modified CD117 polypeptide into the HSCs and/or HSPCs according to a variety of methods available in the art, e.g., electroporation.
  • the modified cell comprising a modified CD117 polypeptide and/or the encoding nucleic acid is a host cell, such as, e.g., an HEK293 cell that may be used to produce modified CD117 polypeptides or mRNA encoding a CD117 polypeptide.
  • a host cell such as, e.g., an HEK293 cell that may be used to produce modified CD117 polypeptides or mRNA encoding a CD117 polypeptide.
  • any host cells may be employed, including but not limited to, for example, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast.
  • the disclosure provides a plasmid that may be used to produce an mRNA encoding a CD117 polypeptide.
  • the plasmid includes a T7 promoter sequence.
  • the plasmid comprises any of the sequences disclosed herein.
  • the modified cells e.g., stem cells
  • the cells are further modified to provide a replacement nucleic acid or protein to the cells, e.g., where the cells are obtained from a subject having a genetic disorder resulting in reduced or lack of expression of a gene or protein, or the expression of a mutant form of a gene or protein.
  • the cells e.g., HSCs and/HSPCs
  • the cells may be contacted with a gene therapy vector that results in the insertion into the cellular genome of an expression cassette that expresses the correct form of a mutated gene or protein.
  • the gene therapy may replace the mutated gene or a mutated region thereof, e.g., via homologous recombination.
  • the HSCs and/or HSPCs are modified to correct a mutated gene using gene editing or base editing methods, such as, e.g., a CRISPR-Cas9 system that targets the mutated gene.
  • the HSCs and/or HSPCs are modified to correct a mutated gene using Zinc-finger nucleases (ZFNs), meganucleases, or transcription activator-like effector nucleases (TALENs) that target the mutated gene. Correction of a genetic mutation may be done prior to, at the same time as, or following introduction of the nucleic acid encoding CD117, or a fragment or variant thereof.
  • ZFNs Zinc-finger nucleases
  • TALENs transcription activator-like effector nucleases
  • the disclosure provides a method of modifying cells, and the resulting modified cells, including stem cells such as HSCs and/or HSPCs, comprising modifying one or more endogenous CD117 genes or alleles within the cells, e.g., by homologous recombination or gene editing according to a variety of methods available in the art.
  • a CD117 gene in HSCs and/or HSPCs is edited by any of a variety of methods known and available in the art, including but not limited to: transcription activator- like effector nucleases (TALENs), megaTALs, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) systems, zinc finger nucleases, homing endonucleases, or meganucleases.
  • the CD117 gene is edited by a base editing method.
  • a gene-editing system is a system comprising one or more proteins or polynucleotides capable of editing an endogenous target gene or locus in a sequence specific manner.
  • the gene-editing system is a protein-based gene regulating system comprising a protein comprising one or more zinc-finger binding domains and an enzymatic domain.
  • the protein-based gene regulating system comprises a protein comprising a Transcription activator-like effector nuclease (TALEN) domain and an enzymatic domain.
  • TALENs Transcription activator-like effector nuclease
  • the gene editing system comprises a nucleic acid sequence corresponding to a region of the CD117 gene and comprising a modification thereof.
  • Zinc finger-based systems comprise a fusion protein comprising two protein domains: a zinc finger DNA binding domain and an enzymatic domain.
  • a “zinc finger DNA binding domain”, “zinc finger protein”, or “ZFP” is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion.
  • the zinc finger domain by binding to a target DNA sequence, directs the activity of the enzymatic domain to the vicinity of the sequence and, hence, induces modification of the endogenous target gene in the vicinity of the target sequence.
  • a zinc finger domain can be engineered to bind to virtually any desired sequence.
  • one or more zinc finger binding domains can be engineered to bind to one or more target DNA sequences in the target genetic locus.
  • Expression of a fusion protein comprising a zinc finger binding domain and an enzymatic domain in a cell effects modification in the target genetic locus.
  • a zinc finger binding domain comprises one or more zinc fingers. Miller et al. (1985) EMBO J. 4:16010-1714; Rhodes (1993) Scientific American Febuary:56-65; U.S. Pat. No. 6,453,242.
  • a single zinc finger domain is about 30 amino acids in length.
  • An individual zinc finger binds to a three-nucleotide (i.e., triplet) sequence (or a four-nucleotide sequence which can overlap, by one nucleotide, with the four- nucleotide binding site of an adjacent zinc finger). Therefore the length of a sequence to which a zinc finger binding domain is engineered to bind (e.g., a target sequence) will determine the number of zinc fingers in an engineered zinc finger binding domain.
  • the DNA-binding domains of individual ZFNs comprise between three and six individual zinc finger repeats and can each recognize between 9 and 18 base pairs.
  • Zinc finger binding domains can be engineered to bind to a sequence of choice. See, for example, Beerli et al. (2002) Nature Biotechnol.20:135-141; Pabo et al. (2001) Ann. Rev. Biochem.70:313-340; Isalan et al. (2001) Nature Biotechnol.19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416.
  • An engineered zinc finger binding domain can have a novel binding specificity, compared to a naturally-occurring zinc finger protein.
  • a target DNA sequence for binding by a zinc finger domain can be accomplished, for example, according to the methods disclosed in U.S. Pat. No. 6,453,242. It will be clear to those skilled in the art that simple visual inspection of a nucleotide sequence can also be used for selection of a target DNA sequence. Accordingly, any means for target DNA sequence selection can be used in the methods described herein.
  • a target site generally has a length of at least 9 nucleotides and, accordingly, is bound by a zinc finger binding domain comprising at least three zinc fingers.
  • the zinc finger binding domains bind to a target DNA sequence that is at least 90% identical to a target DNA sequence (e.g., epitope-encoding) within a target locus of a target CD117 gene.
  • the zinc finger binding domains bind to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence within a target locus of a target gene. In some embodiments, the zinc finger binding domains bind to a target DNA sequence that is 100% identical to a target DNA sequence within a target locus of a target gene.
  • the enzymatic domain portion of the zinc finger fusion proteins can be obtained from any endo- or exonuclease. Exemplary endonucleases from which an enzymatic domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases.
  • restriction endonucleases suitable for use as an enzymatic domain of the ZFPs described herein are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci.
  • fusion proteins comprise the enzymatic domain from at least one Type IIS restriction enzyme and one or more zinc finger binding domains.
  • An exemplary Type IIS restriction enzyme whose cleavage domain is separable from the binding domain, is Fok I. This particular enzyme is active as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575.
  • two fusion proteins each comprising a FokI enzymatic domain, can be used to reconstitute a catalytically active cleavage domain.
  • a single polypeptide molecule containing a zinc finger binding domain and two FokI enzymatic domains can also be used.
  • Exemplary ZFPs comprising FokI enzymatic domains are described in US Patent No.9,782,437.
  • TALEN-based systems comprise a protein comprising a TAL effector DNA binding domain and an enzymatic domain.
  • TAL effectors are proteins that are secreted by Xanthomonas bacteria via their type III secretion system when they infect plants.
  • the DNA binding domain contains a repeated, highly conserved, 33–34 amino acid sequence with divergent 12th and 13th amino acids. These two positions, referred to as the Repeat Variable Diresidue (RVD), are highly variable and strongly correlated with specific nucleotide recognition.
  • RVD Repeat Variable Diresidue
  • the TAL effector domains can be engineered to bind specific target DNA sequences by selecting a combination of repeat segments containing the appropriate RVDs.
  • the nucleic acid specificity for RVD combinations is as follows: HD targets cytosine, NI targets adenenine, NG targets thymine, and NN targets guanine (though, in some embodiments, NN can also bind adenenine with lower specificity).
  • the TAL effector domains bind to a target DNA sequence that is at least 90% identical to a target DNA sequence (e.g., epitope-enoding) within a target locus of a CD117 gene.
  • the TAL effector domains bind to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence within a target locus of a target gene. In some embodiments, the TAL effector domains bind to a target DNA sequence that is 100% identical to a target DNA sequence within a target locus of a target gene.
  • Methods and compositions for assembling the TAL-effector repeats are known in the art. See e.g., Cermak et al, Nucleic Acids Research, 39:12, 2011, e82. Plasmids for constructions of the TAL-effector repeats are commercially available from Addgene.
  • the gene-editing system is a combination gene-regulating system comprising a site-directed modifying polypeptide and a nucleic acid guide molecule.
  • a site-directed modifying polypeptide refers to a polypeptide that binds to a nucleic acid guide molecule, is targeted to a target nucleic acid sequence, such as, for example, a DNA sequence, by the nucleic acid guide molecule to which it is bound, and modifies the target DNA sequence (e.g., cleavage, mutation, or methylation of target DNA).
  • a site-directed modifying polypeptide comprises two portions, a portion that binds the nucleic acid guide and an activity portion.
  • a site-directed modifying polypeptide comprises an activity portion that exhibits site-directed enzymatic activity (e.g., DNA methylation, DNA cleavage, histone acetylation, histone methylation, etc.), wherein the site of enzymatic activity is determined by the guide nucleic acid.
  • the nucleic acid guide comprises two portions: a first portion that is complementary to, and capable of binding with, an endogenous target DNA sequence (referred to herein as a “DNA-binding segment”), and a second portion that is capable of interacting with the site-directed modifying polypeptide (referred to herein as a “protein- binding segment”).
  • the DNA-binding segment and protein-binding segment of a nucleic acid guide are comprised within a single polynucleotide molecule. In some embodiments, the DNA-binding segment and protein-binding segment of a nucleic acid guide are each comprised within separate polynucleotide molecules, such that the nucleic acid guide comprises two polynucleotide molecules that associate with each other to form the functional guide. [00131] The nucleic acid guide mediates the target specificity of the combined protein/nucleic gene regulating systems by specifically hybridizing with a target DNA sequence comprised within the DNA sequence of a target gene.
  • target gene encompasses the full-length DNA sequence for that particular gene and a full-length DNA sequence for a particular target gene will comprise a plurality of target genetic loci, which refer to portions of a particular target gene sequence (e.g., an exon or an intron). Within each target genetic loci are shorter stretches of DNA sequences referred to herein as “target DNA sequences” or “target sequences” that can be modified by the gene-regulating systems described herein. Further, each target genetic loci comprises a “target modification site,” which refers to the precise location of the modification induced by the gene-regulating system (e.g., the location of an insertion, a deletion, or mutation, the location of a DNA break, or the location of an epigenetic modification).
  • the gene-regulating systems described herein may comprise a single nucleic acid guide, or may comprise a plurality of nucleic acid guides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleic acid guides).
  • the CRISPR/Cas systems described below are exemplary embodiments of a combination protein/nucleic acid system.
  • the gene editing systems described herein are CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR Associated) nuclease systems.
  • the site-directed modifying polypeptide is a CRISPR- associated endonuclease (a “Cas” endonuclease) and the nucleic acid guide molecule is a guide RNA (gRNA).
  • a Cas polypeptide refers to a polypeptide that can interact with a gRNA molecule and, in concert with the gRNA molecule, homes or localizes to a target DNA sequence and includes naturally occurring Cas proteins and engineered, altered, or otherwise modified Cas proteins that differ by one or more amino acid residues from a naturally-occurring Cas sequence.
  • the Cas protein is a Cas9 protein.
  • Cas9 is a multi-domain enzyme that uses an HNH nuclease domain to cleave the target strand of DNA and a RuvC- like domain to cleave the non-target strand.
  • mutants of Cas9 can be generated by selective domain inactivation enabling the conversion of WT Cas9 into an enzymatically inactive mutant (e.g., dCas9), which is unable to cleave DNA, or a nickase mutant, which is able to produce single-stranded DNA breaks by cleaving one or the other of the target or non-target strand.
  • a guide RNA typically comprises two segments, a DNA-binding segment and a protein-binding segment.
  • the protein-binding segment of a gRNA is comprised in one RNA molecule and the DNA-binding segment is comprised in another separate RNA molecule.
  • Such embodiments are referred to herein as “double-molecule gRNAs” or “two-molecule gRNA” or “dual gRNAs.”
  • the gRNA is a single RNA molecule and is referred to herein as a "single-guide RNA” or an "sgRNA.”
  • the term "guide RNA” or "gRNA” is inclusive, referring both to two-molecule guide RNAs and sgRNAs.
  • the protein-binding segment of a gRNA typically comprises, in part, two complementary stretches of nucleotides that hybridize to one another to form a double stranded RNA duplex (dsRNA duplex), which facilitates binding to the Cas protein.
  • the DNA-binding segment (or "DNA-binding sequence") of a gRNA comprises a nucleotide sequence that is complementary to and capable of binding to a specific sequence target DNA sequence or RNA sequence.
  • the protein-binding segment of the gRNA interacts with a Cas polypeptide and the interaction of the gRNA molecule and site-directed modifying polypeptide results in Cas binding to the endogenous DNA or RNA and produces one or more modifications within or around the target DNA sequence.
  • the precise location of the target modification site is determined by both (i) base-pairing complementarity between the gRNA and the target DNA or RNA sequence; and (ii) the location of a short motif, referred to as the protospacer adjacent motif (PAM), in the target DNA sequence.
  • the PAM sequence is required for Cas binding to the target DNA sequence.
  • a variety of PAM sequences are known in the art and are suitable for use with a particular Cas endonuclease (e.g., a Cas9 endonuclease) are known in the art (See e.g., Nat Methods.2013 Nov; 10(11): 1116–1121 and Sci Rep.2014; 4: 5405).
  • the PAM sequence is located within 50 base pairs of the target modification site. In some embodiments, the PAM sequence is located within 10 base pairs of the target modification site.
  • the DNA or RNA sequences that can be targeted by this method are limited only by the relative distance of the PAM sequence to the target modification site and the presence of a unique 20 base pair sequence to mediate sequence-specific, gRNA- mediated Cas binding.
  • the target modification site is located at the 5’ terminus of the target locus. In some embodiments, the target modification site is located at the 3’ end of the target locus. In some embodiments, the target modification site is located within an intron or an exon of the target locus.
  • the guide RNA binds to a CD117 polynucleotide sequence and includes a region complementary to a target CD117 sequence.
  • the guide RNA targets or binds a region of CD117 polynucleotide sequence that encodes one of the following amino acid residues: N505 or D816.
  • the present disclosure provides a polynucleotide encoding a gRNA.
  • a gRNA-encoding nucleic acid is comprised in an expression vector, e.g., a recombinant expression vector.
  • the present disclosure provides a polynucleotide encoding a site-directed modifying polypeptide.
  • the polynucleotide encoding a site-directed modifying polypeptide is comprised in an expression vector, e.g., a recombinant expression vector.
  • the site-directed modifying polypeptide is a Cas protein, e.g., a Cas9 protein.
  • Cas molecules of a variety of species can be used in the methods and compositions described herein, including Cas molecules derived from S. pyogenes, S. aureus, N. meningitidis, S. thermophiles, etc.
  • the Cas protein is a Cas9 protein or a Cas9 ortholog and is selected from the group consisting of SpCas9, SpCas9-HF1, SpCas9- HF2, SpCas9-HF3, SpCas9-HF4, SaCas9, FnCpf, FnCas9, eSpCas9, and NmeCas9.
  • the Cas9 protein is a naturally-occurring Cas9 protein. Exemplary naturally occurring Cas9 molecules are described in Chylinski et al., RNA Biology 201310:5, 727-737.
  • a Cas9 protein comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a Cas9 amino acid sequence described in Chylinski et al., RNA Biology 201310:5, 727-737; Hou et al., PNAS Early Edition 2013, 1-6).
  • a Cas polypeptide comprises one or more of the following activities: a) a nickase activity, i.e., the ability to cleave a single strand, e.g., the non- complementary strand or the complementary strand, of a nucleic acid molecule; b) a double stranded nuclease activity, i.e., the ability to cleave both strands of a double stranded nucleic acid and create a double stranded break, which in an embodiment is the presence of two nickase activities; c) an endonuclease activity; d) an exonuclease activity; and/or e) a helicase activity, i.e., the ability to unwind the helical structure of a double stranded nucleic acid.
  • a nickase activity i.e., the ability to cleave a single strand, e.g., the non-
  • the Cas9 is a wildtype (WT) Cas9 protein or ortholog.
  • WT Cas9 comprises two catalytically active domains (HNH and RuvC). Binding of WT Cas9 to DNA based on gRNA specificity results in double-stranded DNA breaks that can be repaired by non- homologous end joining (NHEJ) or homology-directed repair (HDR).
  • NHEJ non- homologous end joining
  • HDR homology-directed repair
  • Cas9 is fused to heterologous proteins that recruit DNA-damage signaling proteins, exonucleases, or phosphatases to further increase the likelihood or the rate of repair of the target sequence by one repair mechanism or another.
  • a WT Cas9 is co- expressed with a nucleic acid repair template to facilitate the incorporation of an exogenous nucleic acid sequence by homology-directed repair.
  • different Cas9 proteins i.e., Cas9 proteins from various species
  • Cas9 proteins from various species may be advantageous to use in the various provided methods in order to capitalize on various enzymatic characteristics of the different Cas9 proteins (e.g., for different PAM sequence preferences; for increased or decreased enzymatic activity; for an increased or decreased level of cellular toxicity; to change the balance between NHEJ, homology-directed repair, single strand breaks, double strand breaks, etc.).
  • the Cas polypeptides are engineered to alter one or more properties of the Cas polypeptide.
  • the Cas polypeptide comprises altered enzymatic properties, e.g., altered nuclease activity, (as compared with a naturally occurring or other reference Cas molecule) or altered helicase activity.
  • the present disclosure provides guide RNAs (gRNAs) that direct a site-directed modifying polypeptide to a specific target DNA sequence.
  • a gRNA comprises a DNA- targeting segment and protein-binding segment.
  • the DNA-targeting segment of a gRNA comprises a nucleotide sequence that is complementary to a sequence in the target DNA sequence.
  • the DNA-targeting segment of a gRNA interacts with a target DNA in a sequence-specific manner via hybridization (i.e., base pairing), and the nucleotide sequence of the DNA-targeting segment determines the location within the target DNA that the gRNA will bind.
  • the DNA-targeting segment of a gRNA can be modified (e.g., by genetic engineering) to hybridize to any desired sequence within a target DNA sequence.
  • the protein-binding segment of a guide RNA interacts with a site-directed modifying polypeptide (e.g., a Cas9 protein) to form a complex.
  • a site-directed modifying polypeptide e.g., a Cas9 protein
  • the guide RNA guides the bound polypeptide to a specific nucleotide sequence within target DNA via the above-described DNA-targeting segment.
  • the protein-binding segment of a guide RNA comprises two stretches of nucleotides that are complementary to one another and which form a double stranded RNA duplex.
  • a gRNA comprises two separate RNA molecules.
  • each of the two RNA molecules comprises a stretch of nucleotides that are complementary to one another such that the complementary nucleotides of the two RNA molecules hybridize to form the double-stranded RNA duplex of the protein-binding segment.
  • a gRNA comprises a single RNA molecule (sgRNA).
  • the specificity of a gRNA for a target loci is mediated by the sequence of the DNA- binding segment, which comprises about 20 nucleotides that are complementary to a target DNA sequence within the target locus. In some embodiments, the corresponding target DNA sequence is approximately 20 nucleotides in length. In some embodiments, the DNA-binding segments of the gRNA sequences of the present invention are at least 90% complementary to a target DNA sequence within a target locus. In some embodiments, the DNA-binding segments of the gRNA sequences of the present disclosure are at least 95%, 96%, 97%, 98%, or 99% complementary to a target DNA sequence within a target locus, e.g., CD117.
  • the DNA-binding segments of the gRNA sequences of the present invention are 100% complementary to a target DNA sequence within a target locus.
  • the DNA-binding segments of the gRNA sequences bind to a target DNA sequence that is at least 90% identical to a target DNA sequence within a target locus of a CD117 gene.
  • the DNA-binding segments of the gRNA sequences bind to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence within a target locus of a target gene.
  • the DNA-binding segments of the gRNA sequences bind to a target DNA sequence that is 100% identical to a target DNA sequence within a target locus of a target gene.
  • the DNA-binding segments of the gRNA sequences described herein are designed to minimize off-target binding using algorithms known in the art (e.g., Cas- OFF finder) to identify target sequences that are unique to a particular target locus or target gene.
  • the gRNAs described herein can comprise one or more modified nucleosides or nucleotides which introduce stability toward nucleases.
  • these modified gRNAs may elicit a reduced innate immune as compared to a non-modified gRNA.
  • innate immune response includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.
  • the gRNAs described herein are modified at or near the 5' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of their 5' end).
  • the 5' end of a gRNA is modified by the inclusion of a eukaryotic mRNA cap structure or cap analog (e.g., a G(5 ')ppp(5 ')G cap analog, a m7G(5 ')ppp(5 ')G cap analog, or a 3 '-0-Me-m7G(5 ')ppp(5 ')G anti reverse cap analog (ARCA)).
  • a eukaryotic mRNA cap structure or cap analog e.g., a G(5 ')ppp(5 ')G cap analog, a m7G(5 ')ppp(5 ')G cap analog, or a 3 '-0-Me-m7G(5 ')ppp(5 ')G anti reverse cap analog (ARCA)
  • an in vitro transcribed gRNA is modified by treatment with a phosphatase (e.g., calf intestinal alkaline phosphatas
  • a gRNA comprises a modification at or near its 3' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of its 3' end).
  • the 3' end of a gRNA is modified by the addition of one or more (e.g., 25-200) adenine (A) residues.
  • modified nucleosides and modified nucleotides can be present in a gRNA, but also may be present in other gene-regulating systems, e.g., mRNA, RNAi, or siRNA- based systems.
  • modified nucleosides and nucleotides can include one or more of.
  • alteration e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage;
  • alteration e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar;
  • wholesale replacement of the phosphate moiety with "dephospho" linkers d) modification or replacement of a naturally occurring nucleobase; e) replacement or modification of the ribose-phosphate backbone; f) modification of the 3' end or 5' end of the oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety; and g) modification of the sugar.
  • a modified nucleoside or nucleotide can have a modified sugar and a modified nucleobase.
  • every base of a gRNA is modified.
  • each of the phosphate groups of a gRNA molecule are replaced with phosphorothioate groups.
  • a software tool can be used to optimize the choice of gRNA within a user's target sequence, e.g., to minimize total off-target activity across the genome. Off target activity may be other than cleavage.
  • cleavage efficiency at each off-target sequence can be predicted, e.g., using an experimentally-derived weighting scheme.
  • Each possible gRNA can then be ranked according to its total predicted off-target cleavage; the top-ranked gRNAs represent those that are likely to have the greatest on-target and the least off-target cleavage.
  • CRISPR-Cas systems and components thereof are described in, US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233 and US8999641 and applications related thereto; and WO2014/018423, WO2014/093595, WO2014/093622, WO2014/093635, WO2014/093655, WO2014/093661, WO2014/093694, WO2014/093701, WO2014/093709, WO2014/093712, WO2014/093718, WO2014/145599, WO2014/204723, WO2014/204724, WO2014/204725, WO2014/204726, WO2014/204727, WO2014/204728, WO2014/204729, WO2015/065964, WO2015/089351,
  • the gene editing methods comprise or consist of base editing methods.
  • Base editing activity involves chemically altering a base within a polynucleotide, e.g., converting a first base to a second base.
  • the base editing activity is cytidine deaminase activity, e.g., converting target C•G to T•A.
  • the base editing activity is adenosine or adenine deaminase activity, e.g., converting A•T to G•C.
  • the base editing activity is cytidine deaminase activity, e.g., converting target C•G to T•A and adenosine or adenine deaminase activity, e.g., converting A•T to G•C.
  • the base editing methods comprise single nucleotide base editing, such as nucleotide deamination, i.e., A ⁇ G or C ⁇ T.
  • Base editing systems may edit genomic DNA or transcribed RNA.
  • Adenosine and cytidine base editors that may be used include, but are not limited to, base editors described in Antoniou P. et al., Base and Prime Editing Technologies for Blood Disorders, Front. Genome Ed., 28 January 2021.
  • base editing methods comprise C ⁇ G conversion as described in Kurt, I. C. et al. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat. Biotechnol. (2020).
  • dual editors facilitate simultaneous C ⁇ T and A ⁇ G conversion as described in Zhao, D. et al. New base editors change C to A in bacteria and C to G in mammalian cells. Nat. Biotechnol. (2020).
  • a base editor system generally refers to a system for editing a nucleobase of a target nucleotide sequence.
  • a base editor (BE) system comprises: (1) a polynucleotide programmable nucleotide binding domain, a deaminase domain (e.g., cytidine deaminase or adenosine deaminase) for deaminating nucleobases in the target nucleotide sequence; and (2) one or more guide polynucleotides (e.g., guide RNA) in conjunction with the polynucleotide programmable nucleotide binding domain.
  • a deaminase domain e.g., cytidine deaminase or adenosine deaminase
  • guide polynucleotides e.g., guide RNA
  • the base editor (BE) system comprises a nucleobase editor domain selected from an adenosine deaminase or a cytidine deaminase, and a domain having nucleic acid sequence specific binding activity.
  • the base editor system comprises: (1) a base editor (BE) comprising a polynucleotide programmable DNA binding domain and a deaminase domain for deaminating one or more nucleobases in a target nucleotide sequence; and (2) one or more guide RNAs in conjunction with the polynucleotide programmable DNA binding domain.
  • the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain.
  • the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE) or a cytidine base editor (CBE).
  • Cas9 or Cas9 domain refers to an RNA guided nuclease comprising a Cas9 protein, or a fragment or variant thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9).
  • a Cas9 protein or a fragment or variant thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9).
  • a variety of different Cas9 proteins, and fragments and variants thereof, are known and available in the art.
  • a guide polynucleotide is a polynucleotide that is specific for a target sequence (e.g., specifically hybridizes to a target polynucleotide sequence, such as a CD117 gene or mRNA) and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cpfl).
  • the guide polynucleotide is a guide RNA (gRNA).
  • gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule.
  • gRNA is used to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules gRNAs, and gRNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs).
  • gRNAs that exist as single RNA species may comprise two domains: (1) a domain that shares homology to a target nucleic acid, and thus directs binding of a Cas9 complex to the target nucleic acid; and (2) a domain that binds a Cas9 protein.
  • domain (2) is a sequence known as a tracrRNA, which comprises a stem-loop structure.
  • domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al., Science 337:816-821(2012).
  • gRNAs e.g., those including domain 2 are described, e.g., in US20160208288, entitled “Switchable Cas9 Nucleases and Uses Thereof," and US 9,737,604, entitled “Delivery System For Functional Nucleases.”
  • a gRNA comprises two or more of domains (1) and (2), which may be referred to as an extended gRNA.
  • An extended gRNA will bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions.
  • the gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to the target site, providing the sequence specificity of the nuclease: RNA complex.
  • the base editing method e.g., a single nucleotide base editing method, targets a polynucleotide encoding a CD117 polypeptide.
  • CD117 polynucleotide sequence follows: >NM_000222.2 Homo sapiens KIT proto-oncogene, receptor tyrosine kinase (KIT), transcript variant 1 mRNA: TCTGGGGGCTCGGCTTTGCCGCGCTCGCTGCACTTGGGCGAGAGCTGGAACG TGGACCAGAGCTCGGATCCCATCGCAGCTACCGCGATGAGAGGCGCTCGCG GCGCCTGGGATTTTCTCTGCGTTCTGCTCCTACTGCTTCGCGTCCAGACAGGC TCTTCTCAACCATCTGTGAGTCCAGGGGAACCGTCTCCACCATCCATCCATCC AGGAAAATCAGACTTAATAGTCCGCGTGGGCGACGAGATTAGGCTGTTATGC ACTGATCCGGGCTTTGTCAAATGGACTTTTGAGATCCTGGATGAAACGAATG AGAATAAGCAGAATGAATGGATCACGGAAAAGGCAGAAGCCACCAACACCG GCAAATACACGTGCACCAACAAACAAACAA
  • the guide RNA binds to a CD117 polynucleotide sequence and includes a region complementary to a target CD117 sequence.
  • the guide RNA targets or binds a region of CD117 polynucleotide sequence that encodes one of the following amino acid residues: N505 or D816.
  • base editing methods, systems, and components thereof that may be used according to the methods and compositions disclosed herein is described in PCT Application Publication No. WO2021041945.
  • the base editing method comprises use of a modified CRISPR protein, bound to a guide RNA, and a base editing enzyme, such as a deaminase, wherein the modified CRISPR protein does not cause a double- stranded break.
  • the modified CRISPR protein is a nucleobase editor polypeptide or nucleic acid programmable-DNA binding protein (napDNAbp), as disclosed in PCT Application Publication Nos.
  • the method of base editing a polynucleotide encoding a CD117 polypeptide comprises expressing in a cell a nucleobase editor polypeptide, wherein the nucleobase editor polypeptide comprises a napDNAbp and a deaminase, and contacting the cell with a guide RNA capable of targeting the polynucleotide encoding a CD117 polypeptide.
  • base editing may refer to RNA base editing methods, e.g., as described in Porto E. et al. Base editing: advances and therapeutic opportunities, Nature Reviews Drug Discovery volume 19, pages 839–859 (2020).
  • any of the gene editing including base editing methods disclosed herein or known in the art may be used to modify one or more amino acids within an epitope of wild type human CD117 bound by an anti-CD117 antibody, optionally wherein the epitope comprises one or more of the following amino acids present in the wild type human CD117: N505 or D816, including but not limited to any of these recited amino acid residues.
  • the method introduces a A ⁇ G or C ⁇ T mutation into one or both alleles of the CD117 gene, which results in the gene encoding a different amino acid by the codon that was mutated.
  • the disclosure provides a modified cell, e.g., HSPC or HSC, that comprises one or more components of a gene editing, e.g., base editing, system disclosed herein.
  • the one or more component comprises a nucleic acid that binds to a CD117 gene or encoded mRNA, e.g., at a site to be modified to result in the encoding and/or expression of a modified CD117 disclosed herein, such as, e.g., a guide RNA.
  • the guide RNA binds to a CD117 polynucleotide sequence and includes a region complementary to a target CD117 sequence.
  • the guide RNA targets or binds a region of CD117 polynucleotide sequence that encodes one of the following amino acid residues: N505 or D816.
  • the one or more component comprises a base editing enzyme, e.g., any of those disclosed herein or in references cited herein.
  • a modified cell expressing a modified CD117 polypeptide is not substantially inhibited, eliminated, or killed by monoclonal antibodies (mAbs) that bind endogenous or wild-type cell-surface CD117 and inhibit proliferation of or kill a cell expressing only the wild-type CD117 and not a modified CD117 polypeptide disclosed herein.
  • proliferation of the modified cell expressing the modified CD117 polypeptide is inhibited by less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%, as compared to proliferation of the same cell type that is not modified, e.g., only expresses wild-type CD117.
  • the modified cell expressing the modified CD117 polypeptide is not substantially inhibited, eliminated, depleted, or killed by monoclonal antibodies (mAbs) that bind endogenous or wild-type cell-surface CD117 and inhibit proliferation of or kill a cell expressing only the wild-type CD117 and not a modified CD117 polypeptide disclosed herein.
  • proliferation of the modified cell expressing the modified CD117 polypeptide is inhibited, eliminated, depleted, or killed by less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%, as compared to proliferation of the same cell type that is not modified, e.g., only expresses wild-type CD117.
  • Anti-CD117 Antibodies Compositions and methods disclosed herein may be applicable to any CD117 antibody, particularly monoclonal anti-human CD117 antibodies.
  • methods disclosed herein include a conditioning regimen that comprises administration of an anti- CD117 antibody, wherein the anti-CD117 antibody depletes or reduces endogenous HSPCs.
  • Illustrative CD117 antibodies include, but are not limited to, SR-1, JSP191, 8D7, K45, 104D2, CK6, YB5.B8, AF-2-1, AF11, AF12, AF112, AF-3, AF-1-1, NF, NF-2-1, NF11, NF12, NF112, NF-3, HF11, HF12, and HF112.
  • a number of antibodies contemplated by the disclosure that specifically bind human CD117 are known in the art and commercially available, including without limitation SR1, 2B8, ACK2, YB5-B8, 57A5, 104D2, etc.
  • the anti-CD117 antibody is selected from the group consisting of: JSP191 (Jasper Therapeutics; Redwood City, CA); CDX-0159 (Celldex Therapeutics, Hampton, NJ); MGTA-117 (AB85) (Magenta Therapeutics, Cambridge, MA); CK6 (Magenta Therapeutics, Cambridge, MA); AB249 (Magenta Therapeutics, Cambridge, MA); and FSI-174 (Gilead, Foster City, CA).
  • Antibodies from Magenta Therapeutics contemplated by the disclosure include but are not limited to those that are disclosed in US Patent Application Publication No.20190153114, PCT Application Publication Nos. WO2019084064, WO2020/219748, and WO2020/219770.
  • the FSI-174 antibody is disclosed in PCT application Publication No. WO2020/112687 and U.S. Patent Application Publication No.20200165337.
  • the disclosure includes but is not limited to any CD117 antibodies and/or CDR sets disclosed in any of the patent application disclosed herein, which are all incorporated by reference in their entireties.
  • the CD117 antibody binds to the extracellular region of CD117, i.e., amino acids 26-524.
  • the antibody is the humanized form of SR1, which is a murine CD117 antibody disclosed in U.S. Patent Nos. 5,919,911 and 5,489,516.
  • the humanized antibody referred to as JSP191 (formerly referred to as AMG191), is described in U.S. Pat. Nos. 8,436,150, 8,791,249, and 7,915,391, and U.S. Patent Application Publication No. 20110223165.
  • JSP191 is an aglycosylated IgG1 humanized antibody.
  • JSP191 is a humanized monoclonal antibody in clinical development as a conditioning agent to clear hematopoietic stem cells from bone marrow.
  • JSP191 specifically binds to human CD117, a receptor for stem cell factor (SCF), which is expressed on the surface of hematopoietic stem and progenitor cells (HSPCs). JSP191 blocks SCF from binding to CD117 and disrupts critical survival signals, leading to the depletion of hematopoietic stem cells.
  • SCF stem cell factor
  • HSPCs hematopoietic stem and progenitor cells
  • the sequences of the heavy and light chains of JSP191 are: Heavy Chain: MDWTWRVFCLLAVAPGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMH WVRQAPGQGLEWMGVIYSGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDT AVYYCARERDTRFGNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
  • variable light chain domain of JSP191 comprises the following sequence: DIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMHWYQQKPGQPPKLLIYLASNL ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDP YTFGGGTKVEIK (SEQ ID NO:11).
  • CDX-0159 is a humanized monoclonal antibody that specifically binds the receptor tyrosine kinase KIT with high specificity and potently inhibits its activity. CDX-0159 is designed to block KIT activation by disrupting both SCF binding and KIT dimerization.
  • CDX- 0159 and other CD117 antibodies are described in U.S. Patent No.10,781,267, and in particular embodiments, an CD117 disclosed herein comprises the CDRs of any of the antibodies disclosed therein.
  • the CD117 antibody comprises: (i) a light chain variable region ("VL") comprising the amino acid sequence: DIVMTQSPSX K1 LSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKX K2 LIYSASYR YSGVPDRFX K3 GSGSGTDFTLTISSLQX K4 EDFAX K5 YX K6 CQQYNSYPRTFGGGTKVEIK (SEQ ID NO:12), wherein X K1 is an amino acid with an aromatic or aliphatic hydroxyl side chain, X K2 is an amino acid with an aliphatic or aliphatic hydroxyl side chain, X K3 is an amino acid with an aliphatic hydroxyl side chain, X K4 is an amino acid with an aliphatic hydroxyl side chain or is P, XK5 is an amino acid with a charged or acidic side chain, and XK6 is an amino acid with an aromatic side chain; and (ii)
  • antibodies e.g., human or humanized antibodies
  • VH CDRs of a VH domain comprising the amino acid sequence: QVQLKQSGAELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEWIARIYPG SGNTYYNEKFKGKATLTAEKSSSTAYMQLSSLTSEDSAVYFCARGVYYFDYWGQ GTTLTVSS (SEQ ID NO:14) or QVQLKQSGAELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEWIARIYPG SGNTYYNEKFKGKATLTAEKSSSTAYMQLSSLTSEDSAVYFCARGVYYFDYWGQ GTTLTVSA (SEQ ID NO:15), and (ii) VL CDRs of a VL domain comprising the amino acid sequence DIVMTQSQKFMSTSVGDRVSVTCKCK
  • MGTA-117 (AB85) is a CD117-targeted antibody engineered for the transplant setting and conjugated to amanitin, which is being developed for patients undergoing immune reset through either autologous or allogeneic stem cell transplant. MGTA-117 depletes hematopoietic stem and progenitor cells, and this antibody and others contemplated by the disclosure are described in U.S> Application No. 20200407440 and/or PCT Application No. WO2019084064. Epitope analysis of AB85 binding to CD177 is described in PCT Application Publication No.
  • WO2020219770 which identified the following two epitopes within CD117: EKAEATNTGKYTCTNKHGLSNSIYVFVRDPA (amino acids 60-90; (SEQ ID NO:17)), and RCPLTDPEVTNYSLKGCQGKP (amino acids 100-130; (SEQ ID NO:18)).
  • EKAEATNTGKYTCTNKHGLSNSIYVFVRDPA amino acids 60-90; (SEQ ID NO:17)
  • RCPLTDPEVTNYSLKGCQGKP amino acids 100-130; (SEQ ID NO:18)
  • the heavy chain variable region (VH) amino acid sequence of AB85 is: EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMAIINPRDS DTRYRPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYEGYEGAFDI WGQGTLVTVSS (SEQ ID NO:19).
  • the VH CDR amino acid sequences of AB85 are as follows: NYWIG (VH CDR1; SEQ ID NO: 32); IINPRDSDTRYRPSFQG (VH CDR2; SEQ ID NO: 33); and HGRGYEGYEGAFDI (VH CDR3; SEQ ID NO: 34).
  • the light chain variable region (VL) amino acid sequence of AB85 is: DIQMTQSPSSLSASVGDRVTITCRSSQGIRSDLGWYQQKPGKAPKLLIYDASNLETGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANGFPLTFGGGTKVEIK (SEQ ID NO:20).
  • the VL CDR amino acid sequences of AB85 are as follows: RSSQGIRSDLG (VL CDR1; SEQ ID NO: 35); DASNLET (VL CDR2; SEQ ID NO: 36); and QQANGFPLT (VL CDR3; SEQ ID NO: 37).
  • FSI-174 is an anti-cKIT antibody being developed in combination with 5F9 as a non- toxic transplant conditioning regimen, as well as a treatment for targeted hematologic malignancies.
  • the sequences of FSI-174 are disclosed in PCT Application Publication No. 2020/112687, U.S. Patent Application Publication No. 20200165337, and U.S. Patent No. 11,041,022.
  • an CD117 antibody comprises the three CDRs or variable heavy chain regions present in any of AH1, AH2, AH3, AH4, or AH5 disclosed therein, and/or the three CDRs or variable heavy chain regions present in any of AL1 or AL2 disclosed therein.
  • the antibody comprises any of the heavy chain variable region sequences (AH2, AH3, AH4) and/or light chain variable chain region sequences provided below (AL2), or the CDRs therein shown underlined: AH2: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYMNHWVRQAPGQGLEWMGVIYSG NGDTSYAQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQG TLVTVSS (SEQ ID NO:21) AH3: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYMNHWVRQAPGQGLEWMGVIYSG NGDTSYNQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQG TLVTVSS (SEQ ID NO:22) AH4: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYMNHWVRQAPGQGLEW
  • CK6 is described in US Patent Application No.2012/0288506 (and U.S. Pat. No.8,552,157).
  • CK6 has the following heavy chain CDR amino acid sequences: CDR-H1 with SYWIG (SEQ ID NO: 50); CDR-H2 with IIYPGDSDTRYSPSFQG (SEQ ID NO: 51); CDR-H3 with HGRGYNGYEGAFDI (SEQ ID NO: 52).
  • CK6 has the following light chain CDR amino acid sequences: CDR-L1 with RASQGISSALA (SEQ ID NO: 53); CDR-L2 with DASSLES (SEQ ID NO: 54); and CDR-L3 with CQQFNSYPLT (SEQ ID NO: 55).
  • any of the CDRS disclosed herein may be exchanged for a sequence within an example heavy chain variable domain, e.g., using the methods and variable heavy chain and variable light chain sequences identified respectively in US Patent No. 6,054,297: [00188]
  • Example variable heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISENGS DTYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGGAVSYFDV WGQGTLVTVSS (SEQ ID NO: 56).
  • Example variable light chain DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT (SEQ ID NO: 57).
  • Ab249 was derived from antibody CK6, as an antagonist anti-CD117 antibody, as disclosed in PCT Application No. WO2020092655A1. Ab249 has improved binding characteristics over the parent CK6.
  • Ab249 has the following heavy chain CDRS: TSWIG (VH CDR1; SEQ ID NO: 58) IIYPGDSDTRYSPSFQG (VH CDR2; SEQ ID NO: 51); and HGLGYNGYEGAFDI (VH CDR3; SEQ ID NO: 59).
  • Ab249 has the following light chain CDRS: RASQGIGSALA (VL CDR1; SEQ ID NO: 60); DASNLET (VL CDR2; SEQ ID NO: 36); and QQLNGYPLT (VL CDR3; SEQ ID NO: 61).
  • Ab249 has the following variable heavy chain sequence (CDRS are underlined): EVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGWVRQMPGKGLEWMGIIYPGDS DTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYEGAFDI WGQGTLVTVSS(SEQ ID NO: 62).
  • Ab249 has the following variable light chain sequence (CDRS are underlined): DIQMTQSPSSLSASVGDRVTITCRASQGIGSALAWYQQKPGKAPKLLIYDASNLETGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPLTFGQGTRLEIK (SEQ ID NO: 63).
  • the antibody may include one or more CDR with at least 70%, 80%, 90%, 95%, or 99% amino acid or nucleotide sequence identity to a CDR present in a humanized monoclonal antibody that binds CD117, e.g., an antibody derived from any of the mouse antibodies SR1, ACK2, ACK4, 2B8, 3C11, MR-1, and CD122.
  • the antibody blocks the binding of stem cell factor (SCF) to stem cell factor receptor (CD117).
  • SCF stem cell factor
  • CD117 antibodies include JSP191, as well as those described in WO2007127317A2 and US20200165337A1, both incorporated herein in their entirety.
  • the anti-CD117 antibody comprises the full heavy chain and/or full light chain of any of the antibodies disclosed herein, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity to a heavy or light chain disclosed herein, e.g., a JSP191 heavy or light chain.
  • the anti-CD117 antibody comprises the variable region of a heavy chain and/or light chain of any of the antibodies disclosed herein, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity to the variable region of a heavy or light chain disclosed herein, e.g., a JSP191 heavy or light chain variable region.
  • the anti-CD117 antibody comprises a heavy chain and/or a light chain comprising one or more CDRs of an antibody disclosed herein, e.g., two, three, four, five or six CDRs of an antibody disclosed herein, e.g., a JSP191 antibody.
  • the anti-CD117 antibody comprises a heavy chain or variable region thereof comprising one, two, or three heavy chain CDRs disclosed herein, e.g., a JSP191 heavy chain.
  • the anti-CD117 antibody comprises a light chain or variable region thereof comprising one, two, or three light chain CDRs disclosed herein, e.g., a JSP191 light chain.
  • the antibody binds to a region of wild-type CD117 or an epitope of wild-type CD117 that is modified in a modified CD117 polypeptides disclosed herein. In particular embodiments, the antibody does not bind a modified CD117 polypeptide disclosed herein, or binds to a modified CD117 polypeptide disclosed herein with reduced affinity, e.g., less than 50%, less than 25%, or less than 10%.
  • Antibody affinity to a particular polypeptide such as wild-type CD117 or a modified CD117 may be determined, e.g., by measuring the equilibrium dissociation constant between the antibody and its antigen (K D ), which may be determined by routine methods in the art, e.g., by surface plasmon resonance, as described in Hearty, Stephen, Paul Leonard, and Richard O’Kennedy. "Measuring antibody– antigen binding kinetics using surface plasmon resonance.” Antibody Engineering: Methods and Protocols, Second Edition (2012): 411-442.
  • the modified cell expressing the modified CD117 polypeptide is capable of proliferating or surviving in the presence of an anti-CD117 antibody, e.g., an anti-CD117 antibody that blocks or inhibits binding of SCF to CD117 on the cell surface.
  • proliferation and/or survival of the modified cell expressing the modified CD117 polypeptide, in the presence of an anti-CD117 antibody is at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, or at least 40% the level of proliferation and/or survival in the absence of the anti-CD117 antibody.
  • the anti-CD117 antibody is capable of inhibiting proliferation of or inducing death or apoptosis of a cell expressing only the wild-type CD117 and not a modified CD117 polypeptide disclosed herein.
  • the anti-CD117 antibody is selected from the group consisting of: SR1, 2B8, ACK2, YB5-B8, 57A5, 104D2, JSP191, CDX-0159, MGTA-117 (AB85), and FSI-174.
  • the antibody is JSP191.
  • the modified CD117 polypeptides disclosed herein when expressed on a HSC and/or HSPC surface, are capable of substantially binding SCF in the presence of an anti-CD117 antibody that inhibit binding of SCF to endogenous, wild-type CD117 on the cell surface.
  • the modified CD117 polypeptides disclosed herein, when expressed on an HSC surface are capable of intracellular signaling when bound by SCF, in the absence of and in the presence of an anti-CD117 antibody that inhibit binding of SCF to endogenous, wild-type CD117 on the cell surface.
  • SCF binding and/or SCF-mediating signaling is in not substantially reduced in the presence of the anti-CD117 antibody, e.g., binding and/or signaling of the modified cell expressing the modified CD117 polypeptide is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the level of binding and/or signaling observed in the same cell type that is not modified, e.g., only expresses wild-type CD117.
  • CD117 signaling or proliferation or viability may be determined using methods standard in the art.
  • CD117 signaling or proliferation (e.g., in response to SCF), of cells comprising a modified CD117 polypeptide is determined using a cell line (e.g., Ba/F3 cells) engineered to express the modified CD117 polypeptide.
  • a cell line e.g., Ba/F3 cells
  • Cells are cultured in the presence of IL-3, with or without stem cell factor (SCF), and in the presence or absence of an anti-CD117 antibody, e.g., JSP191.
  • SCF stem cell factor
  • an anti-CD117 antibody e.g., JSP191.
  • Control parental Ba/F3 cells do not proliferate in the absence of IL-3.
  • parental Ba/F3 cells do not express CD117 and are not responsive to SCF signaling.
  • the disclosure also provides methods of preparing HSCs and/or HSPCs for HCT, comprising introducing a polynucleotide sequence encoding a modified CD117 described herein into the HSCs and/or HSPCs,
  • the polynucleotide sequence encoding the modified CD117 is present within an mRNA or an expression vector, and the modified CD117 is transiently or constitutively expressed after it is introduced into the HSCs and/or HSPCs.
  • the polynucleotide and/or vector may comprise nucleotide modifications, including any of those disclosed herein or known in the art, e.g., to increase expression or stability of the polynucleotide or vector.
  • a composition comprising HSCs and/or HSPCs, is administered to a patient. Such methods are well known in the art.
  • the stem cells are optionally, although not necessarily, purified. Abundant reports explore various methods for purification of stem cells and subsequent engraftment, including flow cytometry; an isolex system (Klein et al. (2001) Bone Marrow Transplant. 28(11):1023-9; Prince et al.
  • the present disclosure also includes pharmaceutical compositions comprising one or more modified CD117 polypeptides, one or more polynucleotides or vectors comprising a sequence encoding a modified CD117 polypeptide (e.g., a modified mRNA), or a modified cell comprising a polynucleotide or vector encoding a modified CD117 polypeptide and/or expressing a modified CD117, in combination with one or more pharmaceutically acceptable diluent, carrier, or excipient.
  • a modified CD117 polypeptide e.g., a modified mRNA
  • a modified cell comprising a polynucleotide or vector encoding a modified CD117 polypeptide and/or expressing a modified CD117
  • the present invention discloses a pharmaceutical composition
  • a pharmaceutical composition comprising a modified cell comprising a modified CD117 polypeptide (or nucleic acid sequence encoding the modified CD117 polypeptide) described herein and one or more pharmaceutically acceptable diluent, carrier, or excipient.
  • the cell is a heterologous cell or an autologous cell obtained from the subject to be treated.
  • the cell is a stem cell, e.g., a HSC and/or HSPC.
  • the pharmaceutical composition further comprises one or more additional active agents.
  • the one or more additional active agent comprises an anti-CD117 antibody.
  • the anti-CD117 antibody is selected from the group consisting of: SR1, 2B8, ACK2, YB5-B8, 57A5, 104D2, JSP191, CDX-0159, MGTA-117 (AB85), and FSI-174.
  • the antibody is JSP191.
  • the one or more additional active agent comprises one or more anti-CD47, anti-CD40L, anti-CD122, anti-CD4, and/or anti-CD8 antibody.
  • polynucleotides, polypeptides, and cells described herein can be combined with pharmaceutically-acceptable carriers, diluents and reagents useful in preparing a formulation that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for mammalian, e.g., human or primate, use.
  • the pharmaceutical composition is a solution or suspension comprising modified cells disclosed herein.
  • carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulations.
  • Solutions or suspensions used for the formulations can include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; detergents such as Tween 20 to prevent aggregation; and compounds for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the pharmaceutical compositions are sterile.
  • suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the carrier can be, e.g., a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the composition is sterile and may be fluid to the extent that easy syringability exists.
  • a pharmaceutical composition include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the internal compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • the disclosure provides methods of treating a mammalian subject in need thereof, comprising administering to the subject modified cells, e.g., HSCs or HSPCs, comprising a modified CD117 polypeptide described herein and/or a nucleic acid encoding the modified CD117 polypeptide.
  • the subject is in need of HCT or a hematopoietic stem cell transplant.
  • the transplant may be autologous, allogeneic, or xenogeneic, including without limitation allogeneic haploidentical stem cells, mismatched allogeneic stem cells, genetically engineered autologous or allogeneic cells, etc.
  • the modified HSCs or HSPCs are infused into the subject, e.g., by intravenous infusion, e.g., through a central vein over a period of several minutes to several hours.
  • the modified HSCs or HSPCs transiently express the modified CD117 polypeptide, which is constitutively active, e.g., has constitutive kinase activity.
  • the modified CD117 has constitutive autophosphorylation activity, e.g., without bound SCF.
  • the modified HSCs or HSPCs that transiently express the modified CD117 are resistant to ablation by an CD117 antibody, such as, e.g., JSP191.
  • a subject in need of HCT may be conditioned using an CD117 monoclonal antibody such as JSP191 prior to, with, or following HCT in order to ablate diseased HSPCs, whereas the transplanted modified HSCs or HSPCs are less susceptible to ablation by any monoclonal antibody in the subject following transplant, since they transiently express the modified CD117, which provides transient constitutive CD117 signaling, even in the presence of the antibody.
  • the donor is allogeneic to the recipient
  • the HLA type of the donor and recipient may be tested for a match, or haploidentical cells may be used.
  • haploidentical cells may be used.
  • cells obtained from HLA-haploidentical donors or HLA-identical donors are used.
  • HLA- haploidentical donors can be manipulated by CD34 or CD34/CD90 selection.
  • HLA matching traditionally, the loci critical for matching are HLA-A, HLA-B, and HLA-DR.
  • HLA- C and HLA-DQ are also now considered when determining the appropriateness of a donor.
  • a completely matched sibling donor is generally considered the ideal donor.
  • a complete match or a single mismatch is considered acceptable for most transplantation, although in certain circumstances, a greater mismatch is tolerated.
  • matching is both serologic and molecular.
  • the degree of tolerable HLA disparity is much greater, and a match of three or four out of the six HLA-A, HLA-B and HLA-DRB1 antigens is typically sufficient for transplantation.
  • Immunocompetent donor T cells may be removed using a variety of methods to reduce or eliminate the possibility that graft versus host disease (GVHD) will develop.
  • GVHD graft versus host disease
  • the HCT methods disclosed use modified HSCs comprising a modified CD117 polypeptide or nucleic acid encoding the modified CD117 polypeptide.
  • the methods are believed to result in reduced toxicity, reduced morbidity, or reduced graft-versus-host disease, as compared to HCT wherein a subject is administered HSCs that do not comprise the modified CD117 polypeptide or nucleic acid encoding the modified CD117 polypeptide.
  • the methods of the invention are also believed to provide for improved engraftment of stem cells after transplantation into a recipient.
  • the disclosure provides a method for producing a population of cell comprising a plurality of modified HSCs and/or HSPCs, comprising: i) obtaining HSCs and/or HSPCs from a donor subject, optionally a mammal, e.g., a human; ii) introducing a polynucleotide sequence, e.g., an mRNA or plasmid, encoding a modified CD117 polypeptide into the HSC and/or HSPCs, optionally wherein the modified CD117 polypeptide comprises a sequence disclosed herein, or a functional variant or fragment thereof; and iii) optionally, modifying the HSCs and/or HSPCs, e.g., by introducing a gene therapy vector, or by gene editing or base editing, e.g., to correct a gene mutation in the subject; and iv) providing to a recipient subject in need of HCT the modified HSCs and/or HSPCs, comprising: i
  • the introduced polynucleotide sequence is an mRNA that is expressed in the HSCs and/or HSPCs following introduction into the cells.
  • the gene therapy vector or reagents used to perform the gene editing are introduced into cells obtained from a subject to undergo HCT using the modified HSCs and/or HSPCs, i.e., autologous HCT.
  • the polynucleotide sequence encoding the modified CD117 polypeptide and the gene therapy vector or reagents used for gene editing are introduced into the cells at the same time, or either may be introduced before or after the other, optionally within 10 minutes, 20 minutes, 30 minutes, one hour, two hours, four hours, eight hours, 12 hours, 24 hours, or 48 hours of each other.
  • the polynucleotide sequence encoding the modified CD117 polypeptide is an mRNA, and it is introduced into the cells by electroporation.
  • the subject is administered a conditioning regimen to facilitate or increase engraftment of the modified cells.
  • the conditioning regimen depletes endogenous normal or disease HSCs of the subject.
  • Conditioning regimens may be given prior to transplant to reduce the number of blood stem cells in the bone marrow to make space for donor blood stem cells to engraft and cure the patient.
  • the conditioning regimen is administered prior to and/or concurrent with the administering of the pharmaceutical composition.
  • a variety of conditioning regimens are known and available in the art. These include myeloablative, reduced intensity, and non-myeloablative conditioning regimens. Illustrative conditioning regimens are described in Figure 1, and any of these may be used according to the methods disclosed herein, although the conditioning regimen is not limited to those disclosed in Figure 1.
  • the modified cells are administered to a subject in combination with a non-myeloablative conditioning regimen.
  • the conditioning regimen comprises one or more of: chemotherapy (optionally a nucleoside analog and/or an alkylating agent), monoclonal antibody therapy, and radiation, optionally radiation to the entire body.
  • chemotherapy optionally a nucleoside analog and/or an alkylating agent
  • monoclonal antibody therapy optionally radiation to the entire body.
  • radiation optionally radiation to the entire body.
  • two or more conditioning agents are used, they are administered at the same or different times, or two or more may be administered at the same time, and the other(s) at different times.
  • the various conditioning agents are administered to the subject or present within the subject during an overlapping time period prior to the subject being administered the modified HSPCs/HSCs.
  • the conditioning regimen comprises administration of an anti- CD117 antibody, wherein the anti-CD117 antibody depletes endogenous HSCs expressing wild-type CD117, but the anti-CD117 antibody does not deplete the administered modified HSCs.
  • the anti-CD117 antibody is selected from the group consisting of: SR1, 2B8, ACK2, YB5-B8, 57A5, 104D2, JSP191, CDX-0159, MGTA-117 (AB85), and FSI-174.
  • the antibody is JSP191.
  • the conditioning regimen comprises an anti-CD117 antibody alone.
  • an effective dose of CD117 antibody is the dose that depletes endogenous hematopoietic stem cells.
  • the effective dose will depend on the individual and the specific antibody, but it will generally be up to about 100 ⁇ g/kg body weight, up to about 250 ⁇ g/kg, up to about 500 ⁇ g/kg, up to about 750 ⁇ g/kg, up to about 1 mg/kg, up to about 1.2 mg/kg, up to about 1.5 mg/kg, up to about 3 mg/kg, up to about 5 mg/kg, up to about 10 mg/kg.
  • the subject is administered about 0.01 mg/kg to about 2 mg/kg of the CD117 antibody, e.g., JSP191, and optionally the subject is administered about 0.1 mg/kg to about 1 mg/kg of the CD117 antibody, e.g., JSP191.
  • CD117 antibody may be administered to a subject in a dose about 0.01 mg/kg to about 2 mg/kg of the subject’s body weight, or about 0.1 mg/kg to about 1 mg/kg of the subject’s body weight.
  • the CD117 signaling antibodies are administered in a dose of about 0.6 mg/kg.
  • HCT is performed after the CD117 antibody (e.g., JSP-191) serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml.
  • the conditioning regimen comprises administration of an anti- CD117 antibody in combination with one or more additional antibodies.
  • the one or more additional antibodies comprise one or more of: anti-CD47, anti- CD40L, anti-CD122, anti-CD4, and/or anti-CD8 antibody.
  • the conditioning regimen comprises administration of an anti- CD117 antibody, alone or in combination with a myeloablative (MA) conditioning, reduced intensity conditioning (RIC), or other non-MA (NMA) conditioning regimen.
  • MA myeloablative
  • RIC reduced intensity conditioning
  • NMA non-MA
  • the conditioning regimen is a genotoxic conditioning regimen and/or may comprise one or more of: chemotherapy (optionally a nucleoside analog and/or an alkylating agent), monoclonal antibody therapy, and radiation, optionally radiation to the entire body.
  • the conditioning regimen is milder than would be used if the subject was being administered cells, e.g., HSCs, that did not comprise the modified CD117 polypeptide.
  • the conditioning regimen comprises use of an anti-CD117 antibody in combination with chemotherapy (optionally a nucleoside analog and/or an alkylating agent), other monoclonal antibody therapy, and/or radiation
  • the amount of chemotherapy, other monoclonal antibody therapy, and/or radiation is reduced as compared to the amount used when not in combination with an anti-CD117 antibody, such as JSP191.
  • either or both the amount and/or duration of other conditioning therapy may be reduced by at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, or by about 100%.
  • the subject is not administered a myeloablative or genotoxic conditioning regimen prior to or concurrent with the administering of the pharmaceutical composition.
  • the recipient may be immunocompetent, and the transplantation may be performed in the absence of myeloablative conditioning, i.e., in the absence of radiation and/or chemotherapeutic drugs.
  • the recipient may be conditioned with the combined administration a set of agents selected according to the cells and HLA match.
  • Total Body Irradiation (TBI) [00215]
  • TBI Total Body Irradiation
  • the main purpose of TBI in HSC engraftment conditioning is to suppress the patient’s immune system prior to engraftment.
  • the entire patient may be treated with a single radiation beam, with a distance of about 3-6 meters from the radiation source to reduce the dose rate.
  • TBI in extant therapies is typically given in low doses, several times per day, over a period of three to five days. TBI causes significant apoptosis of rapidly dividing cells in radiosensitive organs such as the blood, bone marrow, and the GI tract immediately after radiation exposure.
  • TBI may be given as a single dose as part of a combination conditioning therapy in which an anti-CD117 antibody and a chemotherapy are also administered prior to HSC engraftment.
  • the subject is administered TBI of about 500 cGy to about 5Gy, optionally of about 1 to about 4 Gy or about 1 to about 3 Gy.
  • the total body irradiation may include a single or fractionated irradiation dose within the range of about 50 cGy – 15 Gy, about 50 cGy – 10 Gy, about 50 cGy – 5 Gy, about 50 cGy – 1 Gy, about 50 cGy – 500 cGy, 0.5-1 Gy (500 cGy -1000 cGy), about 0.5-1.5 Gy, about 0.5-2.5 Gy, about 0.5-5 Gy, about 0.5-7.5 Gy, about 0.5-10 Gy, about 0.5-15 Gy, about 1-1.5 Gy, about 1- 2 Gy, about 1-2.5 Gy, about 1-3 Gy, about 1-3.5 Gy, about 1-4 Gy, about 1-4.5 Gy, about 1- 5.5 Gy, about 1-7.5 Gy, about 1-10 Gy, about 2-3 Gy, about 2-4 Gy, about 2-5 Gy, about 2-6 Gy, or about 2-7 Gy.
  • the TBI is administered in a single dose of about 2 Gy, optionally within 24 hours prior to the transplant.
  • the subject is administered twice daily about 2-Gy fractions given over 3 days (total dose about 12 Gy); twice-daily about 1.5-Gy fractions over 4-4.5 days (total dose about 12-13.5 Gy); three-times- daily about 1.2-Gy fractions over 4 days (total dose about 12-13.2 Gy); and once-daily about 3-Gy fractions for 4 days (total dose about 12 Gy).
  • a subject is administered low dose TBI, i.e., less than or equal to 5 Gy, e.g., about 1-3 Gy or about 2-4 Gy given in one or two fractions.
  • the subject is administered at total of less than about 5 Gy, less than about 4 Gy, less than about 3 Gy, or less than about 2 Gy of TBI, which may be administered in one or more fraction or dose.
  • the subject is administered at total of less than about 5 Gy, less than or about 4 Gy, less than or about 3 Hy, less than or about 2 Gy, less than or about 1 Gy, less than about 500 cGy, less than about 250 cGy, less than about 100 cGy, or less than about 50 cGy of TBI, which may be administered in one or more fraction or dose.
  • it is administered as a single dose on the day of HCT.
  • the TBI is administered 5, 4, 3, 2, or 1 days prior to the HCT. In other embodiments the TBI is administered the day of the HCT prior to engraftment. In particular embodiments, the TBI is administered once, e.g., on any of the indicated days. In some embodiments, the subject is administered TBI of about 1 to about 3 Gy, about 1-2 days prior to, or on the day of the transplant (day 0). Chemotherapy [00218] Chemotherapy may refer to any anti-cancer drug that targets rapidly dividing cells.
  • Chemotherapy i.e., anti-cancer or anti-neoplastic agents may include, but are not limited to, fludarabine, clorafabine, cytarabine, an anthracycline drug, such as daunorubicin (daunomycin) or idarubicin, cladribine (2-CdA), mitoxantrone, etoposide (VP-16), 6-thioguanine (6-TG), hydroxyurea, 6-mercaptopurine (6-MP), azacytidine, and/or decitabine.
  • the chemotherapy is fludarabine.
  • Chemotherapies may be administered to partially or completely ablate the patient’s bone marrow cells in preparation for donor HSC cell engraftment and/or as part of continuing treatment thereafter.
  • the subject is administered about 10-50 mg/m2/day of chemotherapy, optionally about 30 mg/m2/day, wherein optionally the chemotherapy is fludarabine and/or clofarabine.
  • the subject is administered about 10 to about 50 mg/m2/day of the chemotherapy, optionally 20 mg/m2/day, 25 mg/m2/day, or about 30 mg/m2/day for about one to about six days.
  • the subject is administered about 10-50 mg/m2/day of the chemotherapy, optionally about 30 mg/m2/day of the fludarabine and/or clofarabine about 10 to about 1 days prior to the HCT.
  • the chemotherapy is administered on days -10, -9, -8, -6, -7, -5 -4, -3, -2, and/or -1 days prior to the HCT. In certain embodiments, the chemotherapy is administered daily during any of these time periods.
  • the disclosure provides methods for conditioning a subject for HCT, the method comprising administering to the subject an CD117 antibody, total body irradiation (TBI), and a chemotherapeutic agent.
  • the method comprises administering to the subject a JSP191 antibody or variant thereof, TBI, and fludarabine.
  • the CD117 antibody, the total body irradiation (TBI), and the chemotherapeutic agent are administered at the same or different times, or two or more may be administered at the same time, and the other at a different time.
  • the CD117 antibody, the total body irradiation (TBI), and the chemotherapeutic agent are administered to the subject or present within the subject during an overlapping time period prior to the subject receiving HCT.
  • the CD117 antibody is administered about 5 to about 20 days before the HCT. In some embodiments, the CD117 antibody is administered on one or more of days 10 through 14 before the HCT. In some embodiments, the CD117 antibody is administered on one or more of days 5, 6, or 7 through about 10 to about 14 days prior to the HCT. In certain embodiments, the CD117 antibody is administered daily during any of these time periods.
  • the day of transplant may in some embodiments be determined by the CD117 antibody blood concentration of the patient: e.g., the day of transplant may be within about 4 to about10 days from the day the subject’s CD117 antibody blood concentration of about 2000 ng/ml or less.
  • the day of transplant is determined by the CD117 antibody (e.g., JSP- 191) serum or plasma concentration, wherein the serum or plasma concentration is less than 100 ng/ml in serum or plasma, less than 200 ng/ml in serum or plasma, less than 300 ng/ml in serum or plasma, less than 400 ng/ml in serum or plasma, less than 500 ng/ml in serum or plasma, less than 600 ng/ml in serum or plasma, less than 700 ng/ml in serum or plasma, or less than 800 ng/ml in serum or plasma on or before the day of transplant.
  • the CD117 antibody (e.g., JSP-191) serum or plasma concentration is less than 500 ng/ml on the day of transplant.
  • the transplant (e.g., HCT) or graft is performed within 0-5 days or 0-3 days following the CD117 antibody (e.g., JSP-191) serum or concentration levels falling below 500 ng/ml.
  • the transplant (e.g., HCT) or graft is performed within three days of the CD117 antibody (e.g., JSP- 191) serum or concentration levels falling below 100 ng/ml, below 200 ng/ml, below 300 ng/ml, below 400 ng/ml, below 500 ng/ml, below 600 ng/ml, below 700 ng/ml, or below 800 ng/ml, e.g., on the same day that the CD117 antibody (e.g., JSP-191) serum or concentration levels fall below any of these concentrations, within one day following, within two days following, or within three days following the CD117 antibody serum or concentration level falling below any of these concentrations.
  • the CD117 antibody is JSP-191, and in particular embodiments, the CD117 antibody level falls below 500 ng/ml before transplant or graft is performed.
  • the TBI is administered 5, 4, 3, 2, or 1 days prior to the HCT. In other embodiments the TBI is administered the day of the HCT prior to engraftment. In particular embodiments, the TBI is administered once, e.g., on any of the indicated days.
  • the chemotherapy is administered on days -10, -9, -8, -6, -7, -5 -4, -3, -2, and/or -1 days prior to the HCT. In certain embodiments, the chemotherapy is administered daily during any of these time periods.
  • the CD117 antibody e.g., JSP191 or a humanized CD117 antibody as described in US20200165337A1
  • the chemotherapy e.g., fludarabine
  • the TBI is administered on the day of the transplant, prior to engraftment.
  • the antibody and/or chemotherapy is administered daily during any of these time periods.
  • the TBI is administered only on a single day.
  • the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml.
  • the subject is administered about 0.01 mg/kg to about 2 mg/kg of the CD117 antibody, e.g., JSP191, optionally the subject is administered about 0.1 mg/kg to about 1 mg/kg of the CD117 antibody, e.g., JSP191.
  • CD117 antibody may be administered to a subject in a dose about 0.01 mg/kg to about 2 mg/kg of the subject’s body weight, or about 0.1 mg/kg to about 1 mg/kg of the subject’s body weight.
  • the CD117 signaling antibodies are administered in a dose of about 0.6 mg/kg, optionally on one or more of days 14 through 10 prior to HCT.
  • the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml.
  • the subject is administered TBI of about 500 cGy to about 5Gy, optionally of about 1 to about 4 Gy or about 1 to about 3 Gy.
  • the total body irradiation (TBI) may include a single or fractionated irradiation dose within the range of about 50 cGy – 15 Gy, about 50 cGy – 10 Gy, about 50 cGy – 5 Gy, about 50 cGy – 1 Gy, about 50 cGy – 500 cGy, 0.5-1 Gy (500 cGy -1000 cGy), about 0.5-1.5 Gy, about 0.5-2.5 Gy, about 0.5-5 Gy, about 0.5-7.5 Gy, about 0.5-10 Gy, about 0.5-15 Gy, about 1-1.5 Gy, about 1- 2 Gy, about 1-2.5 Gy, about 1-3 Gy, about 1-3.5 Gy, about 1-4 Gy, about 1-4.5 Gy, about 1- 5.5 Gy, about 1-7.5 Gy, about 1-10 Gy, about 2-3 Gy, about 2-4 Gy
  • the TBI is administered in a single dose of about 2 Gy, optionally within 24 hours prior to the transplant.
  • the subject is administered twice daily about 2-Gy fractions given over 3 days (total dose about 12 Gy); twice-daily about 1.5-Gy fractions over 4-4.5 days (total dose about 12-13.5 Gy); three-times- daily about 1.2-Gy fractions over 4 days (total dose about 12-13.2 Gy); and once-daily about 3-Gy fractions for 4 days (total dose about 12 Gy).
  • a subject is administered low dose TBI, i.e., less than or equal to 5 Gy, e.g., about 1-3 Gy or about 2-4 Gy given in one or two fractions.
  • the subject is administered at total of less than about 5 Gy, less than about 4 Gy, less than about 3 Gy, or less than about 2 Gy of TBI, which may be administered in one or more fraction or dose.
  • the subject is administered at total of less than about 5 Gy, less than or about 4 Gy, less than or about 3 Hy, less than or about 2 Gy, less than or about 1 Gy, less than about 500 cGy, less than about 250 cGy, less than about 100 cGy, or less than about 50 cGy of TBI, which may be administered in one or more fraction or dose.
  • it is administered as a single dose on the day of HCT.
  • the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml.
  • the subject is administered about 10-50 mg/m2/day of chemotherapy, optionally about 30 mg/m2/day, wherein optionally the chemotherapy is fludarabine and/or clofarabine.
  • the subject is administered about 10 to about 50 mg/m2/day of the chemotherapy, optionally 20 mg/m2/day, 25 mg/m2/day, or about 30 mg/m2/day for about one to about six days.
  • the subject is administered about 0.1 to about 1.0 mg/kg of the CD117 antibody (e.g., JSP191 or a humanized CD117 antibody as described in US20200165337A1), about 0.5 to about 3 Gy of the TBI, and about 10-50 mg/m2/day of chemotherapy (e.g., fludarabine), before HCT.
  • the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml.
  • the CD117 antibody (e.g., JSP191 or a humanized CD117 antibody as described in US20200165337A1) is administered on one or more of days 14 through 10 prior to HCT in a dose of about 0.6 mg/kg
  • the chemotherapy e.g., fludarabine
  • the TBI is administered on the day of the transplant, prior to engraftment in a dose of about 2 Gy.
  • the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml.
  • the CD117 antibody is administered in a dose of about 0.1 mg/kg to about 1 mg/kg of the CD117 antibody about 5 to about 20 days before the HCT.
  • the subject is administered TBI of about 1 to about 3 Gy, about 1-2 days prior to, or on the day of the transplant (day 0).
  • the subject is administered about 10-50 mg/m2/day of the chemotherapy, optionally about 30 mg/m2/day of the fludarabine and/or clofarabine about 10 to about 1 days prior to the HCT.
  • the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml.
  • the CD117 antibody e.g., JSP191 or a humanized CD117 antibody as described in US20200165337A1
  • the chemotherapy e.g., fludarabine
  • the TBI is administered on the day of the transplant, prior to engraftment in a dose of about 2 Gy.
  • the chemotherapy is administered daily during any of these time periods.
  • the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml.
  • the CD117 antibody (e.g., JSP191 or a humanized CD117 antibody as described in US20200165337A1) is administered on one or more of days 14 through 10 prior to HSC transplant in a dose of about 0.6 mg/kg
  • the chemotherapy e.g., fludarabine
  • the TBI is administered on the day of the transplant, prior to engraftment in a dose of about 3 Gy.
  • the chemotherapy is administered daily during any of these time periods.
  • the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml.
  • the CD117 antibody e.g., JSP191 or a humanized CD117 antibody as described in US20200165337A1
  • the chemotherapy e.g., fludarabine
  • the TBI is administered on the day of the transplant, prior to engraftment in a dose of about 2 Gy.
  • the chemotherapy is administered daily during any of these time periods.
  • the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml.
  • the CD117 antibody (e.g., JSP191 or a humanized CD117 antibody as described in US20200165337A1) is administered one or more of days 14 through 10 prior to HSC transplant in a dose of about 0.6 mg/kg
  • the chemotherapy e.g., fludarabine
  • the TBI is administered on the day of the transplant, prior to engraftment in a dose of about 3 Gy.
  • the chemotherapy is administered daily during any of these time periods.
  • the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml.
  • the dose of stem cells, e.g., modified HSCs comprising a modified CD117 polypeptide and/or nucleic acid encoding a modified CD117 polypeptide, administered to a subject may depend on the purity of the infused cell composition, and the source of the cells. In particular embodiments, the dose administered is at least or about 1-2x10 6 CD34+ cells/kg body weight for autologous and allogeneic transplants.
  • Higher doses can include, for example, at least or about 3x10 6 , at least or about 4x10 6 , at least or about 5x10 6 , at least or about 6x10 6 , at least or about 7x10 6 , at least or about 8x10 6 , at least or about 9x10 6 , at least or about 10 7 or more CD34+ cells/kg body weight for autologous and allogeneic transplants.
  • the dose is limited by the number of available cells, and the methods disclosed encompass delivering less cells when necessary or limited.
  • the dose is calculated by the number of CD34+ cells present.
  • a maximum number of CD3+ cells delivered with the modified HSC composition is not more than about 10 7 CD3+ cells/kg of recipient body weight, not more than about 10 6 CD3+ cells/kg of recipient body weight, not more than about 10 5 CD3+ cells/kg of recipient body weight, or not more than about 10 4 CD3+ cells/kg of recipient body weight.
  • cell populations may be selected for expression of CD34 and CD90, which cell populations may be highly purified, e.g., at least about 85% CD34+ CD90+ cells, at least about 90% CD34+ CD90+ cells, at least about 95% CD34+ CD90+ cells and may be up to about 99% CD34+ CD90+ cells or more.
  • the disclosure includes a method of treating a mammalian subject in need thereof, comprising administering to the subject modified cells, e.g., HSCs and/or HSPCs, comprising a modified CD117 polypeptide disclosed herein, e.g., a modified CD117 with constitutive CD117 signaling or kinase activity.
  • the modified CD117 has constitutive autophosphorylation activity, e.g., without bound SCF.
  • the modified CD117 polypeptide is transiently expressed in the cells, e.g., for about one day, about two days, about three days, about four days, about five days, or about a week.
  • the subject is also administered a conditioning regimen to facilitate or increase engraftment of the modified cells following transplantation, wherein the conditioning regimen is administered prior to or concurrent with the administering of the pharmaceutical composition.
  • the conditioning regimen comprises administration of an CD117 antibody, e.g., any disclosed herein (such as, e.g., JSP191), to the subject.
  • the CD117 antibody is administered to the subject prior to administration of the pharmaceutical composition to the subject.
  • This period of time allows clearance of the CD117 antibody (or other agent used for conditioning).
  • the period of time required for clearance of the ablative agent may be empirically determined, or may be based on prior experience of the pharmacokinetics of the agent. Historically, the time for clearance was usually the time sufficient for the level of ablative agent, e.g., CD117 antibody, to decrease at least about 10-fold from peak levels, usually at least about 100-fold, 1000-fold, 10,000-fold, or more.
  • the modified cells being administered to the subject according to the methods disclosed herein comprise a modified CD117 polypeptide that is not bound by the ablative CD117 antibody used for conditioning
  • the disclosed methods do not require a wash- out period, or they require only a reduced wash-out period as compared to when unmodified cells are transplanted.
  • the wash-out period is less than five days, less than four days, less than 3 days, less than two days, or less than one day.
  • the method comprises administering the CD117 antibody and the pharmaceutical composition or modified cells, e.g., modified HSCs and/or HSPCs, during an overlapping period of time or at about the same time.
  • the method comprises administering the CD117 antibody to the subject after administration of the pharmaceutical composition or modified cells, e.g., modified HSCs and/or HSPCs, optionally for a period of time of at least one day, at least two days, at least three days, at least four days, at least five days, or at least one week.
  • modified HSCs and/or HSPCs e.g., modified HSCs and/or HSPCs
  • This may continue to ablate endogenous HSCs and/or HSPCs following administration of the modified HSCs and/or HSPCs, thus allowing greater engraftment.
  • the method comprises: (i) selectively ablating endogenous hematopoietic stem cells in the subject by administering to the subject an CD117 antibody, e.g., JSP-191; (ii) optionally, waiting for a period of time following administration of the CD117 antibody; and (iii) following (ii), administering to the subject the pharmaceutical composition comprising the modified cells, e.g., modified HSCs and/or modified HSPCs, in a dose effective to achieve multilineage peripheral blood chimerism.
  • the period of time of step (ii) is less than five days, less than four days, less than 3 days, less than two days, or less than one day, or there is no period of time.
  • the modified CD117 has constitutive CD117 signaling or kinase activity.
  • the modified CD117 polypeptide is transiently expressed in the cells, e.g., for about one day, about two days, about three days, about four days, about five days, or about a week.
  • the modified CD117 polypeptide has at least 90%, at least 95%, at least 98%, or at least 99% identity to a wild-type human CD117 polypeptide and comprises an amino acid substitution at a position referred to as N505 or D816, such as, e.g., N505I or D816V.
  • the method of treating a subject in need of HCT comprises: (i) administering a conditioning regimen to the subject, wherein the conditioning regimen comprises an anti-CD117 monoclonal antibody, e.g., JSP191; and (ii) administering modified HSCs to the subject, wherein the modified HSCs comprise a modified CD117 polypeptide, wherein the modified CD117 polypeptide is expressed on the cell surface, and wherein the modified HSCs are not depleted by the conditioning regimen to the same extent as endogenous HSCs that comprise only wild type CD117 polypeptide and/or wherein the modified HSCs have a proliferative advantage as compared to the endogenous HSCs.
  • the conditioning regimen comprises an anti-CD117 monoclonal antibody, e.g., JSP191
  • modified HSCs comprise a modified CD117 polypeptide, wherein the modified CD117 polypeptide is expressed on the cell surface
  • the modified HSCs are not depleted by the conditioning regimen to the same extent as endogenous H
  • the modified CD117 has constitutive CD117 signaling or kinase activity. In certain embodiments, the modified CD117 has constitutive autophosphorylation activity, e.g., without bound SCF. In particular embodiments, the modified CD117 polypeptide is transiently expressed in the cells, e.g., for about one day, about two days, about three days, about four days, about five days, or about a week. In particular embodiments, the conditioning regimen comprises a monoclonal CD117 antibody, e.g., JSP191.
  • the modified CD117 polypeptide has at least 90%, at least 95%, at least 98%, or at least 99% identity to a wild-type human CD117 polypeptide and comprises an amino acid substitution at a position referred to as N505 or D816, such as, e.g., N505I or D816V.
  • the actual location of the “N505” or “D816” modification may differ depending on the particular isotype of CD117 polypeptide.
  • the modified CD117 polypeptide has the sequence shown in any one of SEQ ID NOs: 3-6. [00241]
  • the transplantation is performed in the absence of myeloablative conditioning.
  • the recipient is immunocompetent.
  • the administration of the pre-transplantation conditioning regimen is repeated as necessary to achieve the desired level of ablation.
  • the recipient may be a chimera or mixed chimera for the donor cells.
  • the modified cells and methods disclosed herein may be used to treat a variety of indications amenable to treatment with stem cell transplantation, including hematological diseases.
  • the cells and methods disclosed herein may be used in the context of any hematopoietic cell transplant to treat any disease or disorder requiring such a transplant. Examples include, gene therapy, cord blood transplant, treatment of leukemias and cancers, and treatment of non-cancer diseases.
  • the modified cells and methods may be used to treat a leukemia or a severe combined immunodeficiency (SCID). They may also be used to treat various bone marrow failure states and diseases, as well as hemoglobinopathies.
  • the methods comprise: (i) conditioning a subject by administration of an anti-CD117 antibody, e.g., JSP191, alone or in combination with one or more additional conditioning agent; and administering to the subject modified HSPCs/HSCs comprising a modified CD117 disclosed herein, e.g., CD117 E73A.
  • the modified CD117 cells comprise one or more additional modifications.
  • they may comprise one or more introduced genes to replace a missing, mutated, or dysfunctional gene or protein product in a diseased cell.
  • they are used to treat any of the following disorders: multiple myeloma, non-Hodgkin lymphoma, Hodgkin disease, acute myeloid leukemia, neuroblastoma, germ cell tumors, and autoimmune disorders, e.g., systemic lupus erythematosus (SLE), systemic sclerosis, or amyloidosis, for example, by autologous HCT.
  • SLE systemic lupus erythematosus
  • amyloidosis for example, by autologous HCT.
  • they are used to treat any of the following disorders: acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia; chronic lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndromes, multiple myeloma, non- Hodgkin lymphoma, Hodgkin disease, aplastic anemia, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, thalassemias, thalassemia major, sickle cell anemia, combined immunodeficiency, severe combined immunodeficiency (SCID), Wiskott- Aldrich syndrome, hemophagocytic lymphohistiocytosis (HLH), inborn errors of metabolism (e.g., mucopolysaccharidosis, Gaucher disease, metachromatic leukodystrophies, and adrenoleukodystrophies), epidermolysis bullosa, severe
  • the methods disclosed are used to treat a solid tissue cancer or a blood cancer, such as a leukemia, a lymphoma, or a myelodysplastic syndrome.
  • the leukemia is acute myeloid leukemia (AML).
  • the lymphoma is diffuse large B-cell lymphoma.
  • the methods disclosed are used to treat an immunodeficiency.
  • the immunodeficiency is severe combined immunodeficiency (SCID).
  • the immunodeficiency is immunoglobulin G subclass deficiency, selective immunoglobulin A deficiency, DiGeorge syndrome, hyper-immunoglobulin M (HIGM) syndrome, selective IgM deficiency, Wiskott- Aldrich syndrome, or X-linked agammaglobulinemia (XLA).
  • the methods disclosed are used to treat a genetic disorder.
  • the genetic disorder is sickle cell disease or Fanconi anemia.
  • Sickle cell diseases that may be treat include, but are not limited to: HbS disease; drepanocytic anemia; meniscocytosis, and chronic hemolytic anemia.
  • the method further comprises administering to the subject a therapeutic agent for treatment of the disease or disorder being treated by the HCT method.
  • a therapeutic agent for treatment of the disease or disorder being treated by the HCT method.
  • wild-type CD117 Ba/F3 cells expressing wild-type human CD117 (CD117) and mutant human CD117-D816V were cultured in the absence of IL-3, in varying concentrations of stem cell factor (SCF), and in the presence or absence of anti-CD117 antibody JSP191.
  • SCF stem cell factor
  • Control parental Ba/F3 cells did not proliferate in the absence of IL-3 ( Figure 1 and Figure 3). Further, parental Ba/F3 cells did not express CD117 and were not responsive to SCF signaling. Therefore, control parental Ba/F3 cells did not proliferate in the presence of increasing concentrations of SCF, and there was no effect on viability or proliferation with the addition of JSP191.
  • the Ba/F3 cell line expressing wild-type human CD117 (CD117, sequence of ct180, Table 1) showed dose-responsive proliferation to SCF, which was inhibited in the presence of anti-CD117 antibody JSP191 ( Figure 1).
  • the Ba/F3 cell line expressing the CD117-D816V (Table 1) mutant was able to proliferate in the absence of SCF and proliferation was not inhibited by the presence of the anti-CD117 antibody JSP191 ( Figure 1).
  • Wild type CD117 was expressed at higher levels than CD117 N505I (sequence in Table 1), but both showed greater expression than control or mock electroporated cells. Cell viability is shown in FIG.4.
  • Illustrative CD117 mRNA constructs and vector sequences are provided in Table 1 below. For all constructs/sequences disclosed herein, in the context of mRNA, the Ts are Us, and in particular embodiments, the Ts are fully substituted with N1mPsU. Coding sequence is capitalized. Note that throughout the specification, construct sequences are generally specified T7 promoter ⁇ 3’UTR and do not include poly A tail sequences, except for A140S. mRNA constructs electroporated into cells may lack the T7 promoter sequence.
  • the T7 promoter sequence comprises the sequence TAATACGACTCACTATAG.
  • the T7 polymerase starts transcription at the underlined G in the promoter sequence.
  • the polymerase then transcribes using the opposite strand as a template from 5’->3’.
  • the first base in the transcript will be a G.
  • Examples of illustrative self-amplifying RNA constructs are shown in Table 2 below Illustrative plasmid and mRNA constructs/sequences are provided in the table below. For all constructs/sequences disclosed herein, in the context of RNA, the Ts are Us, and in particular embodiments, the Ts are fully substituted with N1mPsU. Coding sequence is capitalized.
  • construct sequences are generally specified T7 promoter ⁇ 3’UTR and do not include poly A tail sequences, except for A140S.
  • mRNA constructs electroporated into cells may lack the T7 promoter sequence.
  • the T7 promoter sequence comprises the sequence TAATACGACTCACTATAG (SEQ ID NO: 72).
  • the T7 polymerase starts transcription at the underlined G in the promoter sequence.
  • the polymerase then transcribes using the opposite strand as a template from 5’->3’.
  • the first base in the transcript will be a G.

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Abstract

Provided herein are modified CD117 polypeptides comprising one or more amino acid modifications that provide for constitutive signaling and/or CD117-mediated kinase activity when expressed or present in cells, e.g., hematopoietic stem cells (HSC) and/or hematopoietic stem and progenitor cells (HSPC), used in stem cell transplant (HCT). As provided herein, the constitutively active CD117 polypeptides may increase the survival of transplanted cells, e.g., cells transplanted after a conditioning regimen comprising a CD117 antibody.

Description

MODIFIED STEM CELL COMPOSITIONS AND METHODS FOR USE RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63/485,195, filed February 15, 2023, and U.S. Provisional Patent Application Serial No. 63/593,909, filed October 27, 2023, which are incorporated herein by reference in their entireties. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING [0002] The contents of the electronic sequence listing (JATH_015_02WO_SeqList_ST26.xml; Size: 153,050 bytes; and Date of Creation: February 10, 2024) are herein incorporated by reference in its entirety. FIELD OF THE INVENTION [0003] The present disclosure relates to modified hematopoietic stem and hematopoietic stem and progenitor cells, e.g., comprising a constitutively active modified CD117, and their use for hematopoietic cell transplantation. BACKGROUND [0004] Hematopoietic cell transplantation (HCT) generally involves the intravenous infusion of autologous or allogeneic donor hematopoietic stem cells (HSC), or hematopoietic stem and progenitor cells (HSPC), obtained from bone marrow, peripheral blood, or umbilical cord blood into a subject whose bone marrow or immune system is damaged or defective. HCT may be performed as part of therapy to treat a number of disorders, including cancers, such as leukemias, and immunodeficiency disorders. [0005] HCT can result in the cure of a vast number of otherwise incurable and chronic diseases by replacing the defective or diseased blood-forming stem cells of the recipient with those from a healthy donor or with gene-corrected cells. While transplants can potentially cure disease, stem cells must reach and engraft in the bone marrow to have a disease-modifying effect. Currently, unmodified stem cell grafts do not provide any inherent advantage relative to endogenous stem cells to enable homing to the bone marrow niche. In fact, patients today are infused with many more stem cells than are expected to engraft in the bone marrow, because so many are lost along the way. [0006] In order to increase the likelihood of stem cell engraftment, stem cell transplant currently requires toxic conditioning to deplete the patient’s existing stem cells in the marrow and donor lymphocytes to overcome the immune barrier. Even with these additions, a significant number of patients still face graft failure. Furthermore, there are significant complications associated with intensive conditioning as well as graft versus host disease. As a result, despite the curative capacity of HCT, access to transplant is limited to only a fraction of patients who could benefit due to toxicities and unwanted complications associated with the procedure. A significant barrier to the safety and efficacy of stem-cell based therapies is the failure of healthy donor or gene-corrected stem cells to engraft in a patient’s bone marrow. [0007] There is clearly a need in the art for improved compositions and methods for HCT, including methods with increased engraftment. The present disclosure addresses this need. BRIEF SUMMARY OF THE INVENTION [0008] The present disclosure provides inter alia novel modified CD117 polypeptides, hematopoietic stem cells (HSC) and hematopoietic stem and progenitor cells (HSPC), and related compositions and methods of use thereof in stem cell transplant. In one embodiment, the disclosure provides a modified or engineered cell comprising a nucleic acid encoding a CD117 polypeptide, including variants thereof. In particular embodiments, the modified CD117 polypeptides are capable of signaling in HSCs and/or HSPCs in the absence of stem cell factor (SCF) binding. In certain embodiments, the modified CD117 polypeptides provide for constitutive signaling and/or CD117-mediated kinase activity when expressed or present in cells, e.g., HSCs and/or HSPCs. Accordingly, in particular embodiments, when expressed or present in HSCs and/or HSPCs, the modified CD117 polypeptides allow CD117 signaling when bound by antibodies that block SCF binding to CD117. [0009] In some embodiments, an HSC or HSPC is transduced with a vector disclosed herein or a modified messenger (mRNA) encoding the CD117 polypeptide, e.g., the modified cell comprises an exogenous or introduced polynucleotide sequence encoding the CD117 polypeptide. In some embodiments of the disclosure provided herein, a modified hematopoietic stem cell (HSC) or hematopoietic stem and progenitor cell (HSPC) comprises a nucleic acid encoding a modified CD117 polypeptide, optionally wherein the modified CD117 polypeptide has constitutive CD117 signaling and/or kinase activity, optionally wherein the modified cell is capable of proliferation and/or survival when contacted with an CD117 monoclonal antibody capable of inhibiting proliferation and/or survival of an HSPC expressing only a wild-type CD117, and optionally wherein the nucleic acid comprises the following elements, optionally from 5’ to 3’: a 5’ HBA1 UTR; a CleanCap Reagent AG 3’ OMe 5’ terminal cap sequence; a sequence encoding the modified CD117 polypeptide; a TAATAA stop codon; and a 3’ HBB1 UTR. [0010] In some embodiments, a nucleic acid sequence of the disclosure comprises a sequence of Table 1 or Table 2, e.g., SEQ ID NOS: 64-71 or SEQ ID NOS: 73-76. In some embodiments, a nucleic acid sequence of the disclosure, e.g., a vector sequence or mRNA comprises a sequence of Table 1 or Table 2, or a variant or fragment thereof, e.g., having at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto. In some embodiments an mRNA sequence of the disclosure comprises one or more of the following modifications: pseudouridine substitution of one or more uridine; N1-methyl-pseudouridine substitution of one or more uridine; 5 methoxyuridine substitution of one or more uridine; 5-methylcytidine substitution of one or more cytidine; a m7G(5')ppp(5')(2'OMeA)pG cap sequence; or a m7(3'OMeG)(5')ppp(5')(2'OMeA)pG cap sequence. In some embodiments, the polypeptide or the polynucleotide is humanized. [0011] In one aspect, the disclosure provides a modified CD117 polypeptide comprising one or more amino acid modifications as compared to a wild type CD117 polypeptide, e.g., one or more amino acid substitutions, insertions, or deletions. In certain embodiments, the modified CD117 polypeptide comprises one or more amino acid substitutions at one or more of the following amino acids present in wild type human CD117: N505 or D816, e.g., a D816V substitution and/or a N505I substitution. In particular embodiments, the one or more amino acid modifications is located within surface exposed amino acid residues or regions of the extracellular domain, the membrane spanning region, or the intracellular domain of the wild type CD117 polypeptide, e.g., the juxtamembrane region or a kinase domain. In particular embodiments, the modified CD117 polypeptide has at least 90%, at least 95%, at least 98%, or at least 99% sequence homology to the wild type CD117 polypeptide, or a functional fragment thereof. In certain embodiments, the wild type CD117 polypeptide is a wild type human CD117 polypeptide, optionally having one of the following amino acid sequences: MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPV DVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKE QIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYID PTQLPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPS AHLTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRK RDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRS VRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNIL LTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYG IFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLK RPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLV HDDV (SEQ ID NO:1); or MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPV DVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKEQIHP HTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQ LPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAH LTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRD SFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRI GSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTH GRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFL WELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRP TFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHD DV (SEQ ID NO:2). [0012] In particular embodiments, the modified CD117 polypeptide substantially retains kinase activity as compared to the wild type CD117 polypeptide, and in some embodiments, the modified CD117 polypeptide has substantially increased kinase activity as compared to the wild type CD117 polypeptide. In particular embodiments, the modified CD117 polypeptide substantially retains or has increased kinase activity, optionally constitutive kinase activity, in the absence of SCF binding, as compared to the kinase activity of wild type CD117 polypeptide in the presence of SCF binding. In particular embodiments, the modified CD117 polypeptide substantially retains kinase activity, optionally in response to SCF binding, as compared to the wild type CD117 polypeptide. In particular embodiments, the modified CD117 polypeptide has increased kinase activity, optionally in response to SCF binding, as compared to the wild type CD117 polypeptide. [0013] In particular embodiments, the modified CD117 has constitutive kinase activity, optionally in the absence of SCF binding, of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of kinase activity in response to SCF binding in cells, e.g., HSCs and/or HSPCs, expressing the wild type CD117 polypeptide. [0014] In particular embodiments, the one or more amino acid modifications do not substantially inhibit or reduce CD117 signaling or cell proliferation, optionally in response to SCF binding, by the modified CD117 polypeptide expressed in cells, e.g., HSCs and/or HSPCs, as compared to the wild type CD117 polypeptide. [0015] In particular embodiments, the one or more amino acid modifications do not substantially inhibit or reduce binding of an anti-c-Kit antibody (also referred to as a CD117 antibody) to the modified CD117 polypeptide expressed in cells, e.g., HSCs and/or HSPCs, as compared to the wild type CD117 polypeptide. In certain embodiments, the CD117 antibody disrupts or blocks binding of SCF to wild type CD117 and/or the modified CD117 polypeptide. In particular embodiments, the CD117 antibody comprises the six CDRs present in any one of JSP191, AB85, CDX-0159, or FSI-174. In particular embodiments, the CD117 antibody in any one of JSP191, AB85, CDX-0159, or FSI-174. [0016] In particular embodiments, the one or more amino acid modifications do not substantially inhibit or reduce binding of stem cell factor (SCF) to the modified CD117 polypeptide expressed in cells as compared to the wild type CD117 polypeptide. [0017] In a related embodiment, the disclosure provides a nucleic acid encoding the modified CD117 polypeptide. In particular embodiments, the nucleic acid comprises RNA, DNA, or a combination thereof. In one embodiment, the nucleic acid comprises a modified mRNA. In particular embodiments, the nucleic acid is associated with one or more lipids, optionally wherein the nucleic acid is present within a lipid nucleic acid particle, a lipid nanoparticle, or a liposome. [0018] In a further related embodiment, the disclosure provides a vector comprising the nucleic acid encoding the modified CD117 polypeptide. In certain embodiments, the vector is an expression vector, e.g., an AAV vector or a lentiviral vector. In particular embodiments, the vector is capable of transducing hematopoietic stem cells. In related embodiments, the disclosure provides a host cell comprising a vector comprising the nucleic acid encoding the modified CD117 polypeptide. In particular embodiments, the host cell is a bacterial or mammalian cell. [0019] In another related embodiment, the disclosure provides a modified cell comprising the modified CD117 polypeptide and/or the nucleic acid encoding the modified CD117 polypeptide. In particular embodiments, the cell expresses both the modified CD117 polypeptide and wild type CD117 polypeptide. In certain embodiments, the cell was transduced with the vector. In certain embodiments, the cell is a stem cell. In certain embodiments, the cell is a hematopoietic stem and progenitor cell (HSPC) or a hematopoietic stem cell (HSC). In particular embodiments, the cell is CD34+, and in some embodiments, the cell is CD34+/CD90+, CD34+/CD38-, CD34+/CD38-/CD90+, or CD34+/CD133+. In some embodiments, the cell is a human cell. In some embodiments, the cell was obtained from a mammalian donor, e.g, a human donor. In certain embodiments, the mammalian donor is a subject in need of a hematopoietic stem cell transplant (autologous donor), wherein in other embodiments, the mammalian donor is not the subject in need of the hematopoietic stem cell transplant (allogeneic donor). In certain embodiments, the cell expresses the modified CD117 polypeptide, optionally wherein the modified cell expresses the modified CD117 polypeptide transiently. In particular embodiments, the modified CD117 polypeptide is expressed on the cell surface or in the cell membrane, and in certain embodiments, the cell is capable of proliferating in the presence of an anti-CD117 antibody and/or in the absence of SCF. In certain embodiments, the anti-CD117 antibody is capable of inhibiting proliferation and/or survival of a cell expressing only the wild-type CD117 but does not substantially inhibit proliferation and/or survival of a cell expressing the modified CD117 polypeptide. In some embodiments, the anti-CD117 antibody induces apoptosis or death of a cell expressing only the wild-type CD117 but does not substantially induce apoptosis or death of a cell expressing the modified CD117 polypeptide. In certain embodiments, contact with or the presence of the anti-CD117 antibody results in less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% as much cell death in cells expressing the modified CD117 polypeptide as compared to in cells expressing only the wild-type CD117 polypeptide. In some embodiments, the anti-CD117 antibody is selected from the group consisting of: JSP191, CDX-0159, AB85, and FSI-174. [0020] In a further related embodiment, the disclosure provides a pharmaceutical composition comprising the modified cells, e.g., HSCs and/or HSPCs, comprising the nucleic acid encoding the modified CD117 polypeptide, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition further comprises an anti- CD117 antibody. In certain embodiments, the pharmaceutical composition further comprises one or more anti-CD47, anti-CD40L, anti-CD122, anti-CD4, and/or anti-CD8 antibody. [0021] In certain embodiments, the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient, carrier, or diluent and a modified hematopoietic stem cell (HSC) or a hematopoietic stem and progenitor cell (HSPC), wherein the modified HSC or HSPC comprises a modified CD117 polypeptide, optionally wherein the modified CD117 polypeptide has constitutive CD117 signaling and/or kinase activity, and optionally wherein the modified cell is capable of proliferation and/or survival when contacted with an CD117 monoclonal antibody capable of inhibiting proliferation and/or survival of an HSPC expressing only a wild-type CD117. In certain embodiments, the CD117 signaling and/or kinase activity of the modified CD117 is not substantially inhibited by an CD117 monoclonal antibody. In certain embodiments, the CD117 monoclonal antibody inhibits binding of SCF to CD117. In certain embodiments, the CD117 monoclonal antibody comprises one or more of the six CDRs present in any one of JSP191, AB85, CDX-0159, or FSI-174. In certain embodiments, the CD117 monoclonal antibody is any one of JSP191, AB85, CDX- 0159, or FSI-174. In certain embodiments, the CD117 antibody is JSP191. In certain embodiments, the CD117 antibody is FSI-174. In certain embodiments, the modified CD117 comprises one or more amino acid modifications as compared to the wild-type CD117 polypeptide. In certain embodiments, the one or more amino acid modifications comprise one or more amino acid substitutions, insertions, or deletions. In certain embodiments, one or more of the amino acid modifications are present within surface exposed amino acid residues of the extracellular domain, within the membrane spanning domain, or within an intracellular domain of the modified CD117 polypeptide. In certain embodiments, the modified CD117 polypeptide comprises substitution or deletion of one or more of the following amino acids present in wild type human CD117: N505 or D816. In certain embodiments, the modified CD117 polypeptide comprises a D816V substitution and/or a N505I substitution as compared to wild type human CD117. In certain embodiments, the modified CD117 polypeptide has at least 90%, at least 95%, at least 98%, or at least 99% sequence homology to wild type CD117 polypeptide. In certain embodiments, the modified cell expresses both the modified CD117 polypeptide and a wild type CD117 polypeptide. In certain embodiments, the modified cell expresses the modified CD117 polypeptide transiently. In certain embodiments, the HSC or HSPC is CD34+, optionally wherein the HSPC is CD34+/CD90+, CD34+/CD38-, or CD34+/CD38-/CD90+, or CD34+CD133+. In certain embodiments, the cell is a human cell. In certain embodiments, the cell was obtained from a mammalian donor. In certain embodiments, the mammalian donor is a subject in need of a hematopoietic cell transplant (HCT). In certain embodiments, the mammalian donor is a healthy donor. In certain embodiments, the cell obtained from the mammalian donor was modified ex vivo. In certain embodiments, the pharmaceutical composition further comprises an anti-CD117 antibody, an anti-CD47, anti-CD40L, anti- CD122, anti-CD4, and/or an anti-CD8 antibody. [0022] In a related aspect, the disclosure includes a method of modifying a cell, e.g., an HSC and/or HSPC, comprising introducing a nucleic acid or vector encoding a modified CD117 polypeptide into the cell, optionally wherein the cell is transiently modified, and optionally wherein the method is for preparing modified cells for hematopoietic cell transplantation (HCT) into a mammalian subject. In certain embodiments, the nucleic acid or vector is introduced into the cell by transfection, transduction, infection, electroporation, or nanopore technology. [0023] In another aspect, the disclosure includes a method of treating a mammalian subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising modified cells, e.g., HSCs and/or HSPCs, comprising the nucleic acid encoding the modified CD117 polypeptide and/or the modified CD117 polypeptide. In some embodiments, the method further comprises administering to the subject a conditioning regimen to facilitate or increase engraftment of the modified cells, or deplete endogenous, wild-type HSCs, wherein the conditioning regimen is administered prior to or concurrent with the administering of the pharmaceutical composition. In some embodiments, the conditioning regimen comprises or consists of an anti-CD117 antibody, optionally JSP191. In some embodiments, the conditioning regimen comprises one or more of: chemotherapy (optionally a nucleoside analog and/or an alkylating agent), monoclonal antibody therapy, and radiation, optionally radiation to the entire body. In particular embodiments, the conditioning regimen is milder than would be used if the subject was being administered hematopoietic stem cells that did not comprise the modified CD117 polypeptide. In some embodiments, the subject is not administered a conditioning regimen to facilitate or increase engraftment of the modified cells, prior to or concurrent with the administering of the pharmaceutical composition, or the conditioning regimen only comprises the anti-CD117 antibody. In particular embodiments, the method results in reduced toxicity, reduced morbidity, or reduced graft-versus-host disease, as compared to a method wherein a subject is administered hematopoietic stem cells that do not comprise the modified CD117 polypeptide in combination with a conditioning regimen, e.g., a reduction of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% in toxicity, morbidity, and/or graft-versus-host disease. [0024] In particular embodiments, methods of cell transplant disclosed here are used to treat a hematologic diseases that could benefit from hematopoietic stem cell transplantation. In certain embodiments, the method is used to treat a disease or disorder selected from the group consisting of: a cancer, a cardiac disorder, a neural disorder, an autoimmune disease, an immunodeficiency, a metabolic disorder, and a genetic disorder. In certain embodiments, the cancer is a solid tissue cancer or a blood cancer, e.g., a leukemia, a lymphoma, or a myelodysplastic syndrome, such as acute myeloid leukemia (AML). In certain embodiments, the immunodeficiency is severe combined immunodeficiency (SCID). In certain embodiments, the genetic disorder is sickle cell disease or Fanconi anemia. In some embodiments, the methods further comprises administering to the subject another therapeutic agent for treatment of the disease or disorder. In particular embodiments, transiently modified CD34+ HSPCs are administered by a single intravenous infusion following a reduced intensity conditioning regimen. BRIEF DESCRIPTION OF THE DRAWINGS [0025] FIG. 1 is a graph showing the OD595 of wild type BaF3 cells (BaF3), or BaF3 cells expressing wild-type CD117 (CD117) or the CD117-D816V mutant in the presence of the indicated concentrations of stem cell factor (SCF), and in the presence or absence of the anti- CD117 antibody, JSP191. These results show that the CD117-D816V mutant confers a proliferative advantage to cells, even in the absence of SCF and the presence of the anti-CD117 antibody, JSP191. At SCF (5 ng/ml), the lines of the graph from top to bottom are: D816V; D816V + JSP191; CD117; BaF3; and CD117 + JSP191. The cKit construct is ct180 with N1m- pseudouridine instead of uridine. [0026] FIG. 2 is a table showing various myeloablative, reduced intensity myeloablative, and non-myeloablative conditioning regimens, reproduced from Atilla, Erden et al. “A Review of Myeloablative vs Reduced Intensity/Non-Myeloablative Regimens in Allogeneic Hematopoietic Stem Cell Transplantations.” Balkan Medical Journal, Vol. 34, 1 (2017):1-9. doi:10.4274/balkanmedj.2017.0055. [0027] FIG.3 is a graph showing cKit expression kinetics following electroporation of mRNAs encoding the indicated cKit into human CD34+ cells. At about 20 hours, the lines from top to bottom correspond to: wild type cKit, unidentified cKit mutant, unidentified cKit mutant, cKit N505I, control, and mock electroporation (EP). The cKit construct is ct180 with N1m- pseudouridine instead of uridine, [0028] FIG. 4 shows viability and live cell numbers of CD34+ cells one day following electroporation with the indicated mRNA or control. [0029] FIG.5 shows the design of the segmented polyA tail A140S, in which the 140 adenine bases are segmented into 2x 70 adenine sequences. DETAILED DESCRIPTION OF THE INVENTION [0030] Hematopoietic stem cell transplantation (HCT) can be a curative therapy for many diseases, based on the principle that healthy hematopoietic stem cells (HSCs) and/or hematopoietic stem and progenitor cells (HSPCs) replace abnormal HSCs. However, HCT is not widely used due to the toxicities associated with the current practices of this procedure. The deleterious effects of HCT include substantial tissue injury and even mortality from the use of chemotherapy and/or radiation prior to transplant (which are needed to prepare recipients to accept donor or autologous gene-corrected cells) and graft-vs-host disease (GVHD) caused by donor lymphocytes that are contained within allogeneic hematopoietic grafts. Despite the known complications caused by chemotherapy and/or radiation, and the infusion of donor lymphocytes in the allograft, these modalities are incorporated into HCTs, because they facilitate the engraftment of donor HSCs and/or HSPCs. Furthermore, HCTs can fail because donor cells fail to engraft and/or fail to persist following the HCT procedure. [0031] Certain HCT procedures include conditioning a patient prior to HCT by treatment with an CD117 antibody that inhibits stem cell factor (SCF) from binding to CD117 on the surface of a patient’s endogenous HSCs, which depletes endogenous HSCs prior to transplant of HSCs and/or HSPCs into the patient. However, this typically requires a significant washout period of about a week or more following administration of the CD117 antibody, to minimize CD117 antibody depletion of the newly transplanted HSCs and/or HSPCs. [0032] The present disclosure provides compositions and methods that augment the ability of donor or autologous gene-corrected HSCs and/or HSPCs to engraft and/or persist in recipients, thereby increasing the likelihood of success of an HCT procedure, and reducing the toxicities associated with HCT. In particular, the disclosure provides modified HSCs and/or HSPCs for transplant that comprise a modified CD117 polypeptide having constitutive activity or retaining activity (e.g., CD117 kinase activity), even in the presence of CD117 antibodies that inhibit SCF binding to the modified CD117 polypeptide. In some embodiments, the modified CD117 polypeptide has constitutive activity even in the presence of the CD117 antibody JSP191. [0033] In particular embodiments, the modified HSCs and/or HSPCs have activity (e.g., constitutive CD117 kinase activity) even when not bound by SCF. In particular embodiments, the modified HSCs and/or HPSCs comprising a modified CD117 polypeptide are not substantially depleted by CD117 antibodies used in conditioning therapies. Accordingly, the modified cells can be transplanted into the subject in the presence of CD117 antibodies without being subject to depletion, thus providing an improved method of conditioning a patient for HCT and potentially allowing a reduced washout period and/or other advantages. [0034] The HCT methods provided herein may reduce the need for intensive chemotherapy, radiation, and/or donor lymphocytes or other cells used to facilitate HSC and/or HSPC engraftment, thereby reducing the toxicity of HCT. The compositions and methods disclosed herein may be used to treat all disorders for which blood stem cell transplantation is indicated. [0035] Binding of SCF and/or CD117 antibodies to the modified CD117 polypeptides may be determined by a variety of methods known in the art. For example, binding may be determined using transiently transfected HEK293T cells that express the modified CD117. Following transfection, cells are incubated with SCF and/or CD117 antibodies. Bound monoclonal antibodies may be detected using an Alexa Fluor 488-conjugated secondary antibody and cellular fluorescence determined by flow cytometry. Bound SCF may be determined using a fluorescently-labeled antibody that binds SCF and flow cytometry. [0036] In certain embodiments, the disclosure provides for compositions and methods for the ex vivo introduction of the CD117 variants and mutants (modified CD117), by RNA-based and/or DNA-based methods, into HSCs and/or HSPCs, including but not limited to CD34+ cells or subsets of CD34+ cells, such that the HSCs and/or HSPCs are able to be successfully transplanted into recipients. The modified CD117 may be expressed transiently in the modified HSCs and/or HSPCs. For example, a nucleic acid encoding a modified CD117 may be transiently introduced into HSCs/and/or HSPCs prior to transplant, where it expresses the modified CD117. Thus, the modified CD117 may be expressed in addition to the endogenous wild type CD117. Transplantation of these modified HSCs and/or HSPCs may be done after or in combination with conditioning therapies, including treatment with antibodies (such as anti- CD117 antibodies). These HSCs and/or HSPCs may be transplanted alone or in combination with other cells. [0037] It is to be understood that this invention is not limited to the particular methodology, products, apparatus and factors described, as such methods, apparatus and formulations may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by appended claims. [0038] It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a drug candidate" refers to one or mixtures of such candidates, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art, and so forth. [0039] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art. [0040] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. [0041] As used herein, "antibody" includes reference to an immunoglobulin molecule immunologically reactive with a particular antigen, and includes both polyclonal and monoclonal antibodies. The term also includes genetically engineered forms such as humanized antibodies, chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies. The term "antibody" also includes antigen binding forms of antibodies, including fragments with antigen-binding capability (e.g., Fab', F(ab')2, Fab, Fv and rIgG. The term also refers to recombinant single chain Fv fragments (scFv). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. [0042] A "humanized antibody" is an immunoglobulin molecule which contains minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. [0043] The assignment of amino acids to each VL and VH domain (and the CDRs therein) is in accordance with any conventional definition of CDRs. Conventional definitions include: the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991); the Chothia definition (Chothia & Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); a composite of Chothia Kabat CDR in which CDR-H1 is a composite of Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modelling software; and, the contact definition of Martin et al. (world wide web bioinfo.org.uk/abs). Kabat provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. Unless otherwise specified numbering of positions within the variable regions of antibodies is Kabat numbering. When an antibody is said to comprise CDRs by a certain definition of CDRs (e.g., Kabat) that definition specifies the minimum number of CDR residues present in the antibody (i.e., the Kabat CDRs). It does not exclude that other residues falling within another conventional CDR definition but outside the specified definition are also present. For example, an antibody comprising CDRs defined by Kabat includes among other possibilities, an antibody in which the CDRs contain Kabat CDR residues and no other CDR residues, and an antibody in which CDR H1 is a composite Chothia-Kabat CDR H1 and other CDRs contain Kabat CDR residues and no additional CDR residues based on other definitions. [0044] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs or mixtures thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide or nucleoside analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, includes, but is not limited to, double- and single-stranded molecules, and mixtures thereof. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form, whether as RNA or DNA, or a mixture thereof. [0045] As used herein, the terms "polypeptide," "peptide," and "protein" refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, to include disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. [0046] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. As understood in the art, sequence identity refers to the percentage identity obtained when sequences are aligned for maximum correspondence over a comparison window (e.g., a specified region of each of the sequences), which may be calculated by any of the algorithms described herein using default parameters, which are expected to generate the same alignment, in most cases, when applied to similar sequences. Identity is calculated, unless specified otherwise, across the full length of the reference sequence. Thus, a sequence-of-interest “shares at least x% identity to” a reference sequence if, when the sequence-of-interest is aligned to the reference sequence, at least x% (rounded down) of the residues in the sequence-of-interest are aligned as an exact match to a corresponding residue in the reference sequence. Gaps may be introduced into the sequence- of-interest and/or the reference sequence to maximize correspondence over the comparison window. [0047] Sequence similarity (i.e., identity) can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the worldwide web at ncbi.nlm.nih.gov/BLAST/. Unless indicated to the contrary, sequence identity is determined using the BLAST algorithm (e.g., bl2seq) with default parameters. [0048] Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol.266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Of particular interest are alignment programs that permit gaps in the sequence. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol.70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol.48: 443-453 (1970). [0049] Of interest is the BestFit program using the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482-489 (1981) to determine sequence identity. The gap generation penalty will generally range from 1 to 5, usually 2 to 4 and in many embodiments will be 3. The gap extension penalty will generally range from about 0.01 to 0.20 and in many instances will be 0.10. The program has default parameters determined by the sequences inputted to be compared. Preferably, the sequence identity is determined using the default parameters determined by the program. This program is available also from Genetics Computing Group (GCG) package, from Madison, Wis., USA. [0050] Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based upon the following parameters: Mismatch Penalty: 1.00; Gap Penalty: 1.00; Gap Size Penalty: 0.33; and Joining Penalty: 30.0. [0051] A "vector" as used herein refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which can be used to mediate delivery of the polynucleotide to a cell. Illustrative vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery vehicles. [0052] An "expression vector" as used herein encompasses a vector, e.g., plasmid, minicircle, viral vector, liposome, and the like, as discussed herein or as known in the art, comprising a polynucleotide which encodes a gene product of interest, and is used for effecting the expression of a gene product in an intended target cell. An expression vector also comprises control elements operatively linked to the encoding region to facilitate expression of the gene product in the target. The combination of control elements, e.g., promoters, enhancers, UTRs, miRNA targeting sequences, etc., and a gene or genes to which they are operably linked for expression is sometimes referred to as an "expression cassette." Many such control elements are known and available in the art or can be readily constructed from components that are available in the art. [0053] A "promoter" as used herein encompasses a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. Promoters and corresponding protein or polypeptide expression may be ubiquitous, meaning strongly active in a wide range of cells, tissues, and species, or it may be cell-type specific, tissue-specific, or species specific. Promoters may be “constitutive,” meaning continually active, or “inducible,” meaning the promoter can be activated or deactivated by the presence or absence of biotic or abiotic factors. [0054] "Operatively linked" or "operably linked" refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained. [0055] The term "native" or “wild-type” as used herein refers to a nucleotide sequence, e.g., gene, or gene product, e.g., RNA or polypeptide, that is present in a wild-type cell, tissue, organ or organism, e.g., a wild-type human gene or protein sequence. The term “variant” as used herein refers to a mutant of a reference polynucleotide or polypeptide sequence (e.g., a native or wild-type) polynucleotide or polypeptide sequence, i.e., having less than 100% sequence identity with the reference polynucleotide or polypeptide sequence. Put another way, a variant comprises at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to a reference polynucleotide sequence, e.g., a native polynucleotide or polypeptide sequence. For example, a variant may be a polynucleotide having a sequence identity of 50% or more, 60% or more, or 70% or more with a full-length native polynucleotide sequence, e.g., an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the full-length native polynucleotide sequence. As another example, a variant may be a polypeptide having a sequence identity of 70% or more with a full-length native polypeptide sequence, e.g., an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the full- length native polypeptide sequence. Variants may also include variant fragments of a reference sequence, e.g., a native sequence sharing a sequence identity of 70% or more with a fragment of the reference, e.g., native, sequence, e.g., an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the native sequence. In particular embodiments, modified CD117 polypeptides comprises a modification as disclosed herein and a deletion, such as an N-terminal and/or C-terminal deletion, yet substantially retain CD117 activity, e.g., kinase activity. In particular embodiments of any modified CD117 polypeptide disclosed herein, the modified CD117 has constitutive kinase activity. [0056] The term “stem cell” as used herein refers to a mammalian cell that has the ability both to self-renew, and to generate differentiated progeny (see Morrison et al. (1997) Cell 88:287- 298). Endogenous stem cells may be characterized by the presence of markers associated with specific epitopes. Hematopoietic stem cells (HSC) are multipotent cells that reside in the bone marrow (BM) and are responsible for the life-long production of mature blood cells. Hematopoietic stem and progenitor cells (HSPCs) include HSCs as well as hematopoietic progenitor cells that reside in bone marrow and are capable of differentiating into mature blood cells. In some embodiments, HSC and/or HSPC engraftment cells may be fresh, frozen, or subject to prior culture. HSC and/or HSPC may be obtained from fetal liver, bone marrow, cord blood, or peripheral blood, by a donor (allogeneic), the patient themselves (autologous), or any other conventional source. [0057] The terms "administering" or "introducing" or “providing”, as used herein, refer to delivery of a composition to a cell, to cells, tissues and/or organs of a subject, or to a subject. Such administering or introducing may take place in vivo, in vitro, or ex vivo. [0058] The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, e.g., reducing the likelihood that the disease or symptom thereof occurs in the subject, and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, and includes: (a) inhibiting the disease, i.e., arresting its development; or (b) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. The subject therapy may be administered before or during the symptomatic stage of the disease. [0059] The terms "individual," "host," "subject," and "patient" are used interchangeably herein, and refer to a mammal, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.). [0060] As used herein, the term “substantially” means by a significant or large amount or degree. For example, to “substantially” increase may mean to increase by at least two-fold, at least three-fold, at least four-fold, at least five-fold, or at least ten-fold, and to “substantially” decrease may mean to decrease by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. [0061] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention. [0062] Generally, conventional methods of protein synthesis, recombinant cell culture and protein isolation, and recombinant DNA techniques within the skill of the art are employed in the present invention. Such techniques are explained fully in the literature, see, e.g., Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory Manual (1982); Sambrook, Russell and Sambrook, Molecular Cloning: A Laboratory Manual (2001); Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: Portable Protocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988). CD117 Variant Polypeptides and Polynucleotides [0063] CD117, also known as c-Kit or stem cell factor receptor (SCFR), has a molecular weight of 145 kDa as a mature protein and is a member of the type III receptor tyrosine kinase (RTK) family that includes platelet-derived growth factor (PDGF) receptors and the macrophage colony-stimulating factor 1 (CSF-1) (c-fms) receptor. CD117 is essential for the development of normal hematopoietic cells and plays an important role in the survival, proliferation, and differentiation of mast cells, melanocytes, and germ cells. It is expressed by hematopoietic cells in the embryonic liver throughout development, and by more committed progenitors, such as myeloid, erythroid, megakaryocytic, natural killer, and dendritic progenitor cells. [0064] CD117 includes: an approximately 519 amino acid extracellular domain comprised of five immunoglobulin-like domains; a transmembrane segment; a juxtamembrane domain; and a protein kinase domain that contains an insert of about 80 amino acid residues. Approximately 184 amino acids of the extracellular domain are surface exposed, which were identified based on x-ray crystallographic studies. The crystallographic structure of CD117 is provided in, e.g., Mol, et al., Accelerated Publications, Volume 278, ISSUE 34, P31461-31464, August 22, 2003; Ogg et al., RCSB Protein Data Bank, 6XV9, Crystal structure of the kinase domain of human c-KIT in complex with a type-II inhibitor, DOI: 10.2210/pdb6XV9/pdb; McAuley et al., RCSB Protein Data Bank Alkynyl Benzoxazines and Dihydroquinazolines as Cysteine Targeting Covalent Warheads and Their Application in Identification of Selective Irreversible Kinase Inhibitors, DOI: 10.1021/jacs.9b13391; Schimpl et al., RCSB Protein Data Bank 6GQM, Crystal structure of human c-KIT kinase domain in complex with a small molecule inhibitor, AZD3229, DOI: 10.1021/acs.jmedchem.8b00938; and Lin et al., RCSB Protein Data Bank Identification of a Multitargeted Tyrosine Kinase Inhibitor for the Treatment of Gastrointestinal Stromal Tumors and Acute Myeloid Leukemia, DOI: 10.1021/acs.jmedchem.9b01229. Binding of CD117 to its ligand (stem cell factor; SCF) induces receptor dimerization, trans autophosphorylation of the kinase domain, recruitment of signaling proteins via phosphotyrosine binding or Src homology 2 (SH2) domains, and subsequent signal transduction. [0065] In one aspect, the disclosure provides a modified CD117 polypeptide comprising one or more amino acid modifications as compared to a wild type CD117 polypeptide. In particular embodiments, the one or more amino acid modifications comprise one or more amino acid substitutions, insertions, or deletions. In certain embodiments, the one or more amino acid modifications are located in the extracellular domain of the CD117 polypeptide. In certain embodiments, the one or more amino acid modifications are located in one or more surface exposed amino acids or regions of the CD117 polypeptide’s extracellular domain. In certain embodiments, the modified CD117 comprises one or more modification within the juxtamembrane region or the kinase domain. In particular embodiments, the modified CD117 polypeptides comprise one or more deletions, e.g., an N-terminal or C-terminal deletion, optionally wherein the deletion does not substantially impair biological activity, e.g., signaling, of the modified CD117 polypeptide. In certain embodiments, the modified CD117 polypeptides retain or have at least 90%, at least 95%, at least 98%, or at least 99% sequence homology to the wild type CD117 polypeptide. In particular embodiments, the one or more amino acid modifications comprise one or more amino acid substitutions or deletions of an amino acid residue selected from the following in human CD117: N505 or D816. In certain embodiments, the one or more amino acid modifications comprise one or more amino acid substitutions, e.g., of any of these residues. In certain embodiments, the amino acid residue is substituted by any other amino acid, by alanine. In certain embodiments, the amino acid substitution is a conservative amino acid substitution. The term "conservative substitution" as used herein denotes that one or more amino acids are replaced by another, biologically similar residue. [0066] In the scheme below, conservative substitutions of amino acids are grouped by the indicated physicochemical properties. I: neutral, hydrophilic, II: acids and amides, III: basic, IV: hydrophobic, V: aromatic, bulky amino acids.
Figure imgf000022_0001
[0067] In the scheme below, conservative substitutions of amino acids are grouped by the indicated physicochemical properties. VI: neutral or hydrophobic, VII: acidic, VIII: basic, IX: polar, X: aromatic.
Figure imgf000022_0002
[0068] In particular embodiments, the one of more amino acid substitutions comprises a D816V substitution and/or a N505I substitution. [0069] In certain embodiments, the wild type CD117 polypeptide upon which the variant is based is a human CD117 polypeptide, while in other embodiments, it is another mammalian CD117 polypeptide. Sequences of human and mammalian CD117 polypeptides are known in the art. Due to alternative splicing of the CD117 gene, the human CD117 polypeptide is expressed as various isoforms, and any of these may be used according to the disclosure. These isoforms include two GNNK+ and GNNKí isoforms (also denoted CD117 and CD117A, respectively), which differ by the presence or absence of four amino acids, 510-GNNK-513 (SEQ ID NO: 25) in the extra-cellular domain adjacent to the trans-membrane domain, and which are co-expressed in most tissues, although the GNNKí isoform usually predominates. Isoforms may also differ in the presence or absence of a Ser residue at position 715 in the inter- kinase domain, and the disclosure also includes isoforms of CD117, including those shown below, in which Ser175 is either present or absent. These isoforms may comprise any of the modifications disclosed herein, including, e.g., an N505I or D816V modification, and variants thereof, e.g., comprising at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. [0070] In particular embodiments, the wild type CD117 polypeptide is the GNNK+ or GNNK- isoform and comprises or consists of one of the following amino acid sequences (the GNNK tetrapeptide (SEQ ID NO: 25), and the N505 and D816 residues are in bold; numbering is based on GNNK- isoform): MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPV DVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNK EQIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYI DPTQLPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLK PSAHLTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRR KRDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRR SVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNI LLTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSY GIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPL KRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLL VHDDV (SEQ ID NO:1); or MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPV DVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKEQIHP HTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQ LPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAH LTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRD SFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRI GSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTH GRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFL WELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRP TFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHD DV (SEQ ID NO:2). [0071] In certain embodiments, the modified CD117 polypeptide comprises or consists of either of the following sequences: MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPV DVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNK EQIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYI DPTQLPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLK PSAHLTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRR KRDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRR SVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNI LLTHGRITKICDFGLARDIKNVSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSY GIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPL KRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLL VHDDV (SEQ ID NO:3); MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPV DVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKEQIHP HTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQ LPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAH LTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRD SFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRI GSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTH GRITKICDFGLARDIKNVSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFL WELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRP TFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHD DV (SEQ ID NO:4); MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPV DVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFIFAFKGNNKE QIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYID PTQLPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPS AHLTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRK RDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRS VRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNIL LTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYG IFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLK RPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLV HDDV (SEQ ID NO:5); or MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPV DVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFIFAFKEQIHPH TLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLP YDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLT EREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFI CSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRIGS YIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHG RITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLW ELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRPTF KQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV (SEQ ID NO:6), or a variant or fragment thereof, e.g., having at least 90%, at least 95%, at least 98%, or at least 99% identity thereto. In particular embodiments, the variant retains the N505I or D816V amino acid substitution present in the modified CD117. In certain embodiments, the CD117 variant comprises a different amino acid modification that confers constitutive activity to the modified CD117. In particular embodiments, a fragment substantially retains CD117 kinase activity, e.g., retains at least 50% CD117 kinase activity as wild type CD117. [0072] In certain embodiments, the modified CD117 polypeptide substantially retains kinase activity as compared to the wild type CD117 polypeptide. In particular embodiments, the modified CD117 polypeptide has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the kinase activity of the wild type CD117 polypeptide when bound by SCF, and in certain embodiments, the modified CD117 has this activity even in the absence of SCF binding. In some embodiments, the modified CD117 polypeptide has increased kinase activity as compared to the wild type CD117 polypeptide. In particular embodiments, the modified CD117 polypeptide has at least 150%, at least 200%, at least 300%, at least 500%, at least 750%, or at least 1000% of the kinase activity of the wild type CD117 polypeptide. In particular embodiments, the modified CD117 has constitutive kinase activity, even in the absence of SCF binding or in the presence of the CD117 antibody. Kinase activity may be determined using assays known in the art, including the ADP^Glo™ Kinase Assay, which is a luminescent kinase assay that measures ADP formed from a kinase reaction; ADP is converted into ATP, which is converted into light by Ultra^Glo™ Luciferase (available from Promega Corporation, Madison, WI). In certain embodiments, the modified CD117 polypeptide constitutively phosphorylates Gab2, Shc, SHP-2 and/or Cbl. [0073] In particular embodiments, the one or more amino acid modifications do not substantially inhibit or reduce binding of stem cell factor (SCF) to the modified CD117 polypeptide when expressed in cells, as compared to the binding of SCF to the wild type CD117 polypeptide. In particular embodiments, the one or more amino acid modifications do not substantially inhibit or reduce binding of stem cell factor (SCF) to the modified CD117 polypeptide expressed in cells as compared to the wild type CD117 polypeptide. [0074] In certain embodiments, the modified CD117 is a modified CD117 having constitutive signaling or kinase activity, e.g., without bound SCF ligand. In certain embodiments, the modified CD117 has constitutive autophosphorylation activity, e.g., without bound SCF. A variety of such modified CD117 have been identified, e.g., in cancer cells, and any of these may be used according to the compositions and methods disclosed herein. Illustrative examples of activating or gain-of-function CD117 modifications include, but are not limited to, N505I, V559D, D816V, D816H, V568F, V570F, or Y703F, modifications or mutation of amino acid residues corresponding to 505, 522, 816, 557, 558, 559, 568, 569, 570, 703, 816, or deletion of codon 579 (Asp). See, e.g., Akin and Metcalfe, Journal of Allergy and Clinical Immunology, Vol.114, Issue 1, p13-19, July 1, 2004; Hirotakoji et al., Science 23, Jan 1998, Vol.279, Issue 5350, pp.577-580; Sanlorenzo et al., J Proteomics 2016 July 20, 144: 140-147, and references cited in any of the aforementioned, all of which are hereby incorporated by reference in their entireties. In particular embodiments, the amino acid modification is in the region between the transmembrane and tyrosine kinase domains. Mutations causing constitutive activation of CD117 have been shown to be causative in some forms of mastocytosis, and several types of mutations have been associated with myeloproliferative disorders (MPDs), acute myelogenous leukemia (AML), sinonasal lymphomas, and gastrointestinal stromal tumors (GIST). These may be considered activating mutation of two types — ‘regulatory type’ mutations, which affect regulation of the kinase molecule, and ‘enzymatic pocket type’ mutations, which alter the amino acid sequence directly forming the enzymatic site. Either type of mutation may be used according to various embodiments of the disclosure, including any of those disclosed in Longley et al., Leukemia Research, Vol. 25, Issue 7, July 2001, pp. 571-576, and references cited therein, all of which are incorporated herein by reference in its entireties. [0075] In particular embodiments, the one or more amino acid modifications do not result in cells expressing only the modified CD117 having substantially inhibited or reduce CD117 signaling or proliferation, optionally in response to SCF binding, as compared to the signaling in cells only expressing the wild type CD117 polypeptide. In particular embodiments, the modified CD117 retains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% CD117 signaling and/or proliferation, optionally in response to SCF binding, as compared to the corresponding wild type CD117. In particular embodiments, the modified CD117 retains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% CD117 signaling and/or proliferation, in the absence of SCF binding, as compared to the corresponding wild type CD117. [0076] In particular embodiments, the one or more amino acid modifications do not substantially inhibit or reduce binding of an CD117 antibody to the modified CD117 polypeptide expressed in cells as compared to the wild type CD117 polypeptide. [0077] In particular embodiments, the CD117 antibody comprises the six CDRs present in any one of JSP191, AB85, CDX-0159, or FSI-174. In particular embodiments, the CD117 antibody in any one of JSP191, AB85, CDX-0159, or FSI-174. [0078] In some embodiments, the CD117 antibody is JSP191 or comprises the six CDRs present in JSP191. [0079] In some embodiments, the CD117 antibody is AB85 or comprises the six CDRs present in AB85. [0080] In some embodiments, the CD117 antibody is CDX-0159 or comprises the six CDRs present in CDX-0159. [0081] In some embodiments, the CD117 antibody is FSI-174 or comprises the six CDRs present in FSI-174. [0082] The disclosure also provides nucleic acid or polynucleotides encoding a modified CD117 polypeptide disclosed herein. In particular embodiments, the nucleic acid comprises RNA, DNA, or a combination thereof, and in particular embodiments, the nucleic acid comprises single-stranded and/or double-stranded regions, or a mixture thereof. In certain embodiments, the nucleic acid is a double-stranded DNA, and in certain embodiments, the nucleic acid is a single stranded RNA, e.g., a messenger RNA (mRNA). In certain embodiments, the nucleic acid comprises a modified mRNA. In particular embodiments, the polynucleotides described herein, e.g., modified mRNA, are codon-optimized, e.g., to enhance expression of the encoded polypeptide in a host cell. [0083] In particular embodiments, polynucleotide variants comprise one or more modified nucleotide or nucleoside. Modified mRNAs comprising one or more modified nucleoside have been described as having advantages over unmodified mRNAs, including increase stability, higher expression levels and reduced immunogenicity. Non-limiting examples of modifications to mRNAs that may be present in the nucleic acids encoding the modified CD117 polypeptides are described, e.g., in PCT Patent Application Publication Nos. WO2011/130624, WO2012/138453, WO2013052523, WO2013151666, WO2013/071047, WO2013/078199, WO2012045075, WO2014081507, WO2014093924, WO2014164253, US Patent Nos: US 8,278,036 (describing modified mRNAs comprising pseudouridine), US 8,691,966 (describing modified mRNAs comprising pseudouridine and/or N1-methylpseudouridine), US 8,835,108 (describing modified mRNAs comprising 5-methylcytidine, US 8,748,089 (describing modified mRNAs comprising pseudouridine or 1-methylpseudouridine). [0084] In certain embodiments, the sequence encoding a modified CD117 protein comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the modified CD117 encoding sequences disclosed herein. In certain embodiments, the mRNA construct encoding a CD47 protein comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the CD47 encoding sequences disclosed herein or comprises an mRNA construct sequence disclosed herein, optionally without the promoter sequence and optionally with a polyA tail. [0085] In particular embodiments, the modified mRNA comprises one or more nucleoside modification. In particular embodiments, the modified mRNA sequence comprises at least one modification as compared to an unmodified A, G, U or C ribonucleoside. For example, uridine can a similar nucleoside such as pseudouridine (ψ) or N1-methyl-pseudouridine (m1ψ), and cytosine can be replaced by 5-methylcytosine. In particular embodiments, the at least one modified nucleosides include N1-methyl-pseudouridine and/or 5-methylcytidine. In certain embodiments, one or more uridines are replaced by 5- methoxyuridine (5moU). In certain embodiments, all uridines in the modified mRNA are replaced with a similar nucleoside such as pseudouridine (ψ) or N1-methyl-pseudouridine (m1ψ), and/or all cytosines in the modified mRNA are substituted with a similar nucleoside such as 5-methylcytosine. [0086] In particular embodiments, the mRNA comprises a 5’ terminal cap sequence followed by a sequence encoding the modified CD117 polypeptide, followed by a 3’ tailing sequence, such as a polyA or a polyA-G sequence. [0087] In particular embodiments, the nucleic acid sequence encoding the modified CD117 comprises 5’ and/or 3’ cellular or viral untranslated regions (UTRs) relative to the sequence encoding the CD117 polypeptide. In some embodiments, the UTR improves mRNA stability, localization and/or expression. In some embodiments the UTR is tissue specific. In some embodiments, the 5’ UTR comprises a UTR sequence from alpha-globin. In some embodiments the nucleic acid comprises a Kozak sequence. In some embodiments the 3’UTR comprises a UTR from an alpha-globin and/or a beta-globin gene, i.e., a 5’ UTR from hemoglobin alpha 1 (HBA1) and/or a 3’ UTR from one or more of HBA1 or hemoglobin beta 1 (HBB1) gene. [0088] In some embodiments, the nucleic acid sequence encoding the modified CD117 comprises a 5’ UTR with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% identity to a 5’UTR sequence of HBA1: ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCACC (SEQ ID NO: 47). [0089] In some embodiments, the nucleic acid sequence encoding the modified CD117 comprises a Kozak sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% identity to the following: GCCGCCACC. [0090] In some embodiments, the nucleic acid sequence encoding the modified CD117 comprises a 3’UTR nucleic acid sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% identity to a 3’UTR of HBB1: GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAA CTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATA AAAAACATTTATTTTCATTGC (SEQ ID NO: 48). [0091] In some embodiments, the nucleic acid sequence encoding the modified CD117 comprises a 3’UTR nucleic acid sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% to a 3’UTR of HBA1: GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCT CCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCA (SEQ ID NO: 49). [0092] In some embodiments, the nucleic acid sequence encoding the modified CD117 comprises an extra stop codon downstream of TAA to avoid run-off translation of an mRNA. In some embodiments, the extra stop codon is TGA. In some embodiments the nucleic acid sequence encoding the modified CD117 comprises a TCTAGA sequence to linearize a plasmid as a template for transcription. [0093] In some embodiments, the nucleic acid sequence encoding the modified CD117 encodes a poly-adenine or poly guanine tail. A polyA or polyA-G tail improves mRNA stability and manufacturability. In some embodiments, the polyA tail may be from 20 to 180 adenine bases in length. In some embodiments, the polyA tail may be from 35 to 140 bases in length. In some embodiments, the polyA tail may be from 70 to 150 bases in length. In some embodiments, the polyA tail is segmented with a linker to reduce recombination during plasmid production in prokaryotic cells. In some embodiments the polyA tail is 70 adenine bases in length. In some embodiments, the linker is a series of bases other than adenine. In some embodiments, the linker is a series of bases including adenine. In some embodiments, the linker is about 3 to about 10 bases in length. In some embodiments, the linker is about 5 to about 20 bases in length. In some embodiments the linker comprises the sequence TATGCA. [0094] In certain embodiments, the sequence encoding the modified CD117 protein comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the modified CD117 encoding sequences disclosed herein. [0095] In particular embodiments, a modified mRNA comprises a 5’ terminal cap sequence followed by a sequence encoding the modified CD117 polypeptide, including one or more 5’ or 3’ UTRs, followed by a 3’ tailing sequence, such as a polyA or a polyA-G tail sequence. [0096] In particular embodiments, the mRNA encoding CD117 (including modified forms or variants thereof) comprises a wild type 5’ terminal cap sequence, and in certain embodiments, the mRNA encoding CD117 (including modified forms or variants thereof) comprises a modified 5’ terminal cap, not limited to but including, e.g., m7G(5')ppp(5')(2'OMeA)pG (CleanCap® Reagent AG for co-transcriptional capping of mRNA; TriLink Biotechnologies, USA) or m7(3'OMeG)(5')ppp(5')(2'OMeA)pG (CleanCap Reagent AG (3' OMe) for co- transcriptional capping of mRNA; TriLink Biotechnologies, USA). In certain embodiments, the mRNA encoding the modified CD117 comprises the modified 5’ terminal cap, 3´-O-Me- m7G(5')ppp(5')G (Anti Reverse Cap Analog (ARCA); APExBIO, USA). In some embodiments, a vaccinia virus mRNA cap methyltransferase adds 7-methylguanylate cap structures (Cap-0) to the 5’ end of RNA. In some embodiments, a vaccinia Cap 2’-O- Methyltransferase adds a methyl group at the 2´-O position of the first nucleotide adjacent to the cap structure at the 5’ end of the RNA. [0097] In some embodiments, a modified CD117 mRNA construct comprises the following elements, optionally from 5’ to 3’: a 5’ HBA1 UTR; a CleanCap Reagent AG 3’ OMe 5’ terminal cap sequence; a sequence encoding the modified CD117 polypeptide; a TAATAA stop codon; and a 3’ HBB1 UTR. [0098] In some embodiments, the modified CD117 mRNA construct comprising the aforementioned elements from 5’ to 3’ has constitutive CD117 signaling and/or kinase activity. In some embodiments, a modified cell comprising the modified mRNA construct comprising the aforementioned elements from 5’ to 3’ is capable of proliferation and/or survival when contacted with an CD117 monoclonal antibody capable of inhibiting proliferation and/or survival of an HSPC expressing only a wild-type CD117. [0099] In particular embodiments, the sequence encoding the modified CD117 is codon- optimized and/or humanized. In particular embodiments, the construct further comprises a polyA sequence, e.g., after the 3’ HBB1 UTR, optionally an A140S polyA. In particular embodiments, the mRNA construct comprises any of the sequences disclosed herein. FIG. 6 shows the design of the segmented polyA tail A140S, in which the 140 adenine bases are segmented into 2x 70 adenine sequences. [00100] In particular embodiments, the mRNA construct comprises any of the polynucleotide sequences disclosed herein, e.g, a sequence comprises a sequence of Table 1 or Table 2, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity to any of the sequences disclosed herein. In some embodiments an mRNA comprises a sequence of Table 1 or Table 2, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity thereto, comprises one or more of the following modifications: pseudouridine substitution of one or more uridine; N1-methyl-pseudouridine substitution of one or more uridine; 5 methoxyuridine substitution of one or more uridine; 5-methylcytidine substitution of one or more cytidine; a m7G(5')ppp(5')(2'OMeA)pG cap sequence; or a m7(3'OMeG)(5')ppp(5')(2'OMeA)pG cap sequence. [00101] Certain embodiments contemplate the use of a self-amplifying RNA (saRNA) that encodes a CD117 polypeptide, in order to modify cells, e.g., HSCs/HSPCs. saRNA is similar to mRNA, and saRNA is a linear, single-stranded RNA molecule that is synthesized with a 5ƍ cap, 3ƍ polyA tail and 5ƍ and 3ƍ UTRs. However, saRNA also encodes four extra proteins in addition to the transgene, e.g., CD117, being expressed. These four extra proteins encode a replicase, which enables amplification of the original strand of RNA upon delivery into the cell, thus yielding a higher amount of protein expression and thus a minimal dose of RNA required. [00102] In certain embodiments, the nucleic acid, e.g., a modified mRNA, is associated with one or more lipids, e.g., to facilitate delivery across the cell membrane, shield its negative charge, and/or to protect against degradation by nucleases. In certain embodiments, the nucleic acid is associated with or present within a lipid nucleic acid particle, a lipid nanoparticle, or a liposome. In certain embodiments, the lipid nucleic acid particle, a lipid nanoparticle, or a liposome facilitates delivery or uptake of the nucleic acid by a cell. In certain embodiments, mRNA, optionally modified mRNA, is co-formulation into lipid nanoparticles (LNPs). In particular embodiments, mRNA-LNP formulations comprise: (1) an ionizable or cationic lipid or polymeric material bearing tertiary or quaternary amines to encapsulate the polyanionic mRNA; (2) a zwitterionic lipid (e.g., 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine [DOPE]) that resembles the lipids in the cell membrane; (3) cholesterol to stabilize the lipid bilayer of the LNP; and (4) a polyethylene glycol (PEG)-lipid to lend the nanoparticle a hydrating layer, improve colloidal stability, and reduce protein absorption. In certain embodiments, the nucleic acid encoding the CD117 polypeptide, which encompasses functional fragments or variants thereof, is present in a vector. In particular embodiments, the vector is capable of delivering the nucleic acid into mammalian HSCs and/or HSPCs or other stem cells, e.g., into the nucleus of the HSCs, HSPCs or stem cells. In certain embodiments, the vector is an episomal vector, e.g., a plasmid. In particular embodiments, the vector is an expression vector comprising a promoter sequence operatively linked to a nucleic acid sequence encoding the CD117 polypeptide. In particular embodiments, the expression vector comprises a promoter sequence that facilitates expression of the encoded modified CD117 polypeptide in HSCs, HSPCs and/or other stem cells. In particular embodiments, the expression vector comprises 5’ and/or 3’ cellular or viral UTRs or the derivatives thereof upstream and downstream, respectively, of the sequence encoding the modified CD117 polypeptide. [00103] In certain embodiments, the vector is a viral vector, optionally an AAV vector, a cytomegalovirus vector, an adenovirus vector, or a lentiviral vector. In certain embodiments, a viral vector infects an HSC and/or HSPCs when viral vector and the cells are incubated together for at least about 24 hours in a culture medium. Modified Hematopoietic Stem Cells and Pharmaceutical Compositions [00104] In a related aspect, the disclosure provides modified cells, e.g., HSCs and/or HSPCs, comprising a nucleic acid encoding a modified CD117 polypeptide described herein. In certain embodiments, the modified CD117 polypeptide comprises one or more amino acid substitutions, e.g., at one or more of the following amino acids present in wild type human CD117: N505 or D816. In particular embodiments, the modified CD117 polypeptide comprises a D816V substitution and/or a N505I substitution. [00105] In certain embodiments, the nucleic acid encoding the modified CD117 polypeptide is transiently present in the modified cell, and it is not present within the genome of the cell. In particular embodiments, the modified cell expresses and/or comprises the modified CD117 polypeptide, and in particular embodiments, the modified CD117 polypeptide is present on the cell surface, e.g., with the extracellular domain present outside the modified cell. In certain embodiments, the modified cell is transduced with or infected with an expression vector, optionally a viral vector. In particular embodiments, the modified cell expresses and/or comprises both the modified CD117 polypeptide and a wild type, endogenous CD117 polypeptide, and in particular embodiments, both the modified CD117 polypeptide and the wild type, endogenous CD117 polypeptide are present on the cell surface, e.g., with their extracellular domains present outside the modified cell. In certain embodiments, the modified CD117 polypeptide is present on the cell surface, e.g., with its extracellular domain present outside the modified cell. In particular embodiments, the CD117s are human CD117 or modified forms thereof. [00106] The modifications disclosed herein may be made to any type of cell, e.g., any mammalian cell. In particular embodiments, any of the modifications disclosed herein may be present in cells that are to be transplanted into a subject, e.g., to treat a disease or disorder in the subject. In certain embodiments, the modifications are made to cells that would benefit from avoiding immune detection by natural killer (NK) cells or T cells, or from avoiding phagocytosis, when administered to a subject. Illustrative cell types include, but are not limited to, stem cells, induced pluripotent stem cells (iPSCs), T cells, cardiac cells, pancreatic islet cells, NK cells, B cells. In particular embodiments, the mammalian cells are HSCs and/or HSPCs. [00107] In particular embodiments the cell is obtained from a mammalian, e.g., a human donor. In come embodiments, the mammalian donor is a subject in need of a hematopoietic cell transplant (HCT) or is a healthy donor. In particular embodiments, the cell obtained from the mammalian donor was modified ex vivo. [00108] In particular embodiments, the modified cell is a stem cell or pluripotent cell, and in certain embodiments, the stem cell is a hematopoietic stem cell (HSC) or an HSPC. In some embodiments, the stem cell is a mammalian cell that has the ability both to self-renew, and to generate differentiated progeny. In certain embodiments, the stem cell is a human cell. The stem cell may have one or more of the following properties: an ability to undergo asynchronous, or symmetric replication, that is where the two daughter cells after division can have different phenotypes; extensive self-renewal capacity; capacity for existence in a mitotically quiescent form; and clonal regeneration of all the tissue in which they exist, for example the ability of hematopoietic stem cells to reconstitute all hematopoietic lineages. [00109] Hematopoietic stem cells (HSCs) are maintained throughout life (self-renewing). They produce hematopoietic progenitor cells that differentiate into every type of mature blood cell within a well-defined hierarchy. In certain embodiments, the HSCs and/or HSPCs are obtained from bone marrow, peripheral blood, or umbilical cord blood and subsequently modified by introduction of the nucleic acid encoding the CD117 polypeptide into the cell. HSCs and/or HSPCs can also be generated in vitro, for example from pluripotent embryonic stem cells, induced pluripotent cells, and the like. For example, see Sugimura et al. (2017) Nature 545:432- 438, herein specifically incorporated by reference, which details a protocol for generation of HSCs and/or HSPCs. [00110] The cells may be fresh, frozen, or have been subject to prior culture. They may be fetal, neonate, adult, etc. Hematopoietic stem cells and HSPCs may be obtained from fetal liver, bone marrow, blood, particularly G-CSF or GM-CSF mobilized peripheral blood, or any other conventional source. Cells for engraftment are optionally isolated from other cells, where the manner in which the stem cells are separated from other cells of the hematopoietic or other lineage is not critical to this invention. If desired, a substantially homogeneous population of stem or progenitor cells may be obtained by selective isolation of cells free of markers associated with differentiated cells, while displaying epitopic characteristics associated with the stem cells. [00111] Modified HSCs and/or HSPCs may be produced using HSCs and/or HSPCs obtained from a mammalian donor. In particular embodiments, the donor is a subject in need of a hematopoietic stem cell transplant, e.g., a subject diagnosed with a disease or disorder that can be treated with HCT. In other embodiments, the modified HSCs and/or HSPCs may be produced using HSCs and/or HSPCs obtained from a healthy donor, e.g., wherein the modified HSCs and/or HSPCs are to be used to treat a different subject with HCT. Thus, the modified HSCs and/or HSPCs may be autologous or allogeneic to a subject in need for HCT. [00112] Prior to harvesting stem cells from a donor, the bone marrow can be primed with granulocyte colony-stimulating factor (G-CSF; filgrastim [Neupogen]) to increase the stem cell count. Mobilization of stem cells from the bone marrow into peripheral blood by cytokines such as G-CSF or GM-CSF has led to the widespread adoption of peripheral blood progenitor cell collection by apheresis for hematopoietic stem cell transplantation. The dose of G-CSF used for mobilization may be about 10 ug/kg/day. In autologous donors who are heavily pretreated, however, doses of up to about 40 ug/kg/day can be given. Mozobil may be used in conjunction with G-CSF to mobilize hematopoietic stem cells to peripheral blood for collection. [00113] Among hematopoietic stem cell (HSC) markers, CD34 is well known for its unique expression on HSCs and HSPCs. In certain embodiments, the modified cell is a CD34+ cell. In particular embodiments, the modified cell is a subset of HSCs or HSPCs that has one of the following patterns or combinations of cell surface marker expression: CD34+/CD90+, CD34+/CD38-, or CD34+/CD38-/CD90+. The CD34+ and/or CD90+ cells may be selected by affinity methods, including without limitation magnetic bead selection, flow cytometry, and the like from the donor hematopoietic cell sample. The HSC and/or HSPC composition may be at least about 50% pure, as defined by the percentage of cells that are CD34+ in the population, may be at least about 75% pure, at least about 85% pure, at least about 95% pure, or more. [00114] For engraftment purposes, a composition comprising hematopoietic stem cells (HSCs) and/or hematopoietic stem and progenitor cells (HSPCs), may be administered to a patient. The HSCs and/or HSPCs are optionally, although not necessarily, purified. Methods are available for purification of stem cells and subsequent engraftment, including flow cytometry; an isolex system (Klein et al. (2001) Bone Marrow Transplant. 28(11):1023-9; Prince et al. (2002) Cytotherapy 4(2):137-45); immunomagnetic separation (Prince et al. (2002) Cytotherapy 4(2):147-55; Handgretinger et al. (2002) Bone Marrow Transplant. 29(9):731-6; Chou et al. (2005) Breast Cancer. 12(3):178-88); and the like. Each of these references is herein specifically incorporated by reference, particularly with respect to procedures, cell compositions and doses for hematopoietic stem cell transplantation. In particular embodiments, the subject is administered a cell population enriched for CD34+ hematopoietic stem cells, comprising HSCs and/or HSPCs. In some embodiments the cell populations are enriched for expression of CD34, e.g., by art recognized methods such as the cliniMACS.RTM. system, by flow cytometry, etc. Cell populations single enriched for CD34 may be from about 50% up to about 90% CD34+ cells, e.g., at least about 85% CD34+ cells, at least about 90% CD34+ cells, at least about 95% CD34+ cells and may be up to about 99% CD34+ cells or more. Alternatively, unmanipulated bone marrow or mobilized peripheral blood populations are used. [00115] In certain embodiments, the disclosure provides a method of modifying cells, including stem cells such as HSCs and/or HSPCs, comprising introducing the nucleic acid encoding a modified CD117 polypeptide into the cell. In particular embodiments, the introduced nucleic acid is present within a viral vector. In certain embodiments, the nucleic acid is associated with or present in a lipid nanoparticle, liposome, or the like. In certain embodiments, the nucleic acid remains present in the modified cell only transiently, or the nucleic acid only transiently expresses the modified CD117 polypeptide in the cell. In certain embodiments, the method is used to prepare modified cells for HCT treatment of a mammalian subject. In particular embodiments, the nucleic acid or vector may be introduced into the cell by a variety of methods known in the art, such as transfection, transduction, infection, electroporation, or nanopore technology. In particular embodiments, mRNA, e.g., modified mRNA is introduced into the cells using lipid nucleic acid particles (LNPs) or nanoparticles. Thus, cells, e.g., HSCs and/or HSPCS may be modified by introducing a nucleic acid encoding a modified CD117 polypeptide into the HSCs and/or HSPCs according to a variety of methods available in the art, e.g., electroporation. [00116] In certain embodiments, the modified cell comprising a modified CD117 polypeptide and/or the encoding nucleic acid is a host cell, such as, e.g., an HEK293 cell that may be used to produce modified CD117 polypeptides or mRNA encoding a CD117 polypeptide. In preparing the subject compositions, any host cells may be employed, including but not limited to, for example, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. In certain embodiments, the disclosure provides a plasmid that may be used to produce an mRNA encoding a CD117 polypeptide. In particular embodiments, the plasmid includes a T7 promoter sequence. In particular embodiments, the plasmid comprises any of the sequences disclosed herein. [00117] In certain embodiments, the modified cells, e.g., stem cells, are further modified to provide a replacement nucleic acid or protein to the cells, e.g., where the cells are obtained from a subject having a genetic disorder resulting in reduced or lack of expression of a gene or protein, or the expression of a mutant form of a gene or protein. Thus, the cells, e.g., HSCs and/HSPCs, may be further genetically altered to correct a genetic defect present in the cells, e.g., HSCs and/or HSPCs. In certain embodiments, the cells may be contacted with a gene therapy vector that results in the insertion into the cellular genome of an expression cassette that expresses the correct form of a mutated gene or protein. In certain embodiments, the gene therapy may replace the mutated gene or a mutated region thereof, e.g., via homologous recombination. In certain embodiments, the HSCs and/or HSPCs are modified to correct a mutated gene using gene editing or base editing methods, such as, e.g., a CRISPR-Cas9 system that targets the mutated gene. In certain embodiments, the HSCs and/or HSPCs are modified to correct a mutated gene using Zinc-finger nucleases (ZFNs), meganucleases, or transcription activator-like effector nucleases (TALENs) that target the mutated gene. Correction of a genetic mutation may be done prior to, at the same time as, or following introduction of the nucleic acid encoding CD117, or a fragment or variant thereof. [00118] In certain embodiments, the disclosure provides a method of modifying cells, and the resulting modified cells, including stem cells such as HSCs and/or HSPCs, comprising modifying one or more endogenous CD117 genes or alleles within the cells, e.g., by homologous recombination or gene editing according to a variety of methods available in the art. In certain embodiments, a CD117 gene in HSCs and/or HSPCs is edited by any of a variety of methods known and available in the art, including but not limited to: transcription activator- like effector nucleases (TALENs), megaTALs, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) systems, zinc finger nucleases, homing endonucleases, or meganucleases. In certain embodiments, the CD117 gene is edited by a base editing method. As used herein, a gene-editing system is a system comprising one or more proteins or polynucleotides capable of editing an endogenous target gene or locus in a sequence specific manner. In some embodiments, the gene-editing system is a protein-based gene regulating system comprising a protein comprising one or more zinc-finger binding domains and an enzymatic domain. In some embodiments, the protein-based gene regulating system comprises a protein comprising a Transcription activator-like effector nuclease (TALEN) domain and an enzymatic domain. Such embodiments are referred to herein as “TALENs.” In particular embodiments, the gene editing system comprises a nucleic acid sequence corresponding to a region of the CD117 gene and comprising a modification thereof. [00119] Zinc finger-based systems comprise a fusion protein comprising two protein domains: a zinc finger DNA binding domain and an enzymatic domain. A “zinc finger DNA binding domain”, “zinc finger protein”, or “ZFP” is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The zinc finger domain, by binding to a target DNA sequence, directs the activity of the enzymatic domain to the vicinity of the sequence and, hence, induces modification of the endogenous target gene in the vicinity of the target sequence. A zinc finger domain can be engineered to bind to virtually any desired sequence. Accordingly, after identifying a target genetic locus containing a target DNA sequence at which cleavage or recombination is desired (e.g., a target locus or epitope identified herein), one or more zinc finger binding domains can be engineered to bind to one or more target DNA sequences in the target genetic locus. Expression of a fusion protein comprising a zinc finger binding domain and an enzymatic domain in a cell, effects modification in the target genetic locus. [00120] In some embodiments, a zinc finger binding domain comprises one or more zinc fingers. Miller et al. (1985) EMBO J. 4:16010-1714; Rhodes (1993) Scientific American Febuary:56-65; U.S. Pat. No. 6,453,242. Typically, a single zinc finger domain is about 30 amino acids in length. An individual zinc finger binds to a three-nucleotide (i.e., triplet) sequence (or a four-nucleotide sequence which can overlap, by one nucleotide, with the four- nucleotide binding site of an adjacent zinc finger). Therefore the length of a sequence to which a zinc finger binding domain is engineered to bind (e.g., a target sequence) will determine the number of zinc fingers in an engineered zinc finger binding domain. In some embodiments, the DNA-binding domains of individual ZFNs comprise between three and six individual zinc finger repeats and can each recognize between 9 and 18 base pairs. [00121] Zinc finger binding domains can be engineered to bind to a sequence of choice. See, for example, Beerli et al. (2002) Nature Biotechnol.20:135-141; Pabo et al. (2001) Ann. Rev. Biochem.70:313-340; Isalan et al. (2001) Nature Biotechnol.19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416. An engineered zinc finger binding domain can have a novel binding specificity, compared to a naturally-occurring zinc finger protein. Engineering methods include, but are not limited to, rational design and various types of selection. [00122] Selection of a target DNA sequence for binding by a zinc finger domain can be accomplished, for example, according to the methods disclosed in U.S. Pat. No. 6,453,242. It will be clear to those skilled in the art that simple visual inspection of a nucleotide sequence can also be used for selection of a target DNA sequence. Accordingly, any means for target DNA sequence selection can be used in the methods described herein. A target site generally has a length of at least 9 nucleotides and, accordingly, is bound by a zinc finger binding domain comprising at least three zinc fingers. However, binding of, for example, a 4-finger binding domain to a 12-nucleotide target site, a 5-finger binding domain to a 15-nucleotide target site or a 6-finger binding domain to an 18-nucleotide target site, is also possible. As will be apparent, binding of larger binding domains (e.g., 7-, 8-, 9-finger and more) to longer target sites is also possible. [00123] In some embodiments, the zinc finger binding domains bind to a target DNA sequence that is at least 90% identical to a target DNA sequence (e.g., epitope-encoding) within a target locus of a target CD117 gene. In some embodiments, the zinc finger binding domains bind to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence within a target locus of a target gene. In some embodiments, the zinc finger binding domains bind to a target DNA sequence that is 100% identical to a target DNA sequence within a target locus of a target gene. [00124] The enzymatic domain portion of the zinc finger fusion proteins can be obtained from any endo- or exonuclease. Exemplary endonucleases from which an enzymatic domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes which cleave DNA are known (e.g., 51 Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains. Exemplary restriction endonucleases (restriction enzymes) suitable for use as an enzymatic domain of the ZFPs described herein are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise the enzymatic domain from at least one Type IIS restriction enzyme and one or more zinc finger binding domains. [00125] An exemplary Type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is Fok I. This particular enzyme is active as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575. Thus, for targeted double-stranded DNA cleavage using zinc finger-Fok I fusions, two fusion proteins, each comprising a FokI enzymatic domain, can be used to reconstitute a catalytically active cleavage domain. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two FokI enzymatic domains can also be used. Exemplary ZFPs comprising FokI enzymatic domains are described in US Patent No.9,782,437. [00126] TALEN-based systems comprise a protein comprising a TAL effector DNA binding domain and an enzymatic domain. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). The FokI restriction enzyme described above is an exemplary enzymatic domain suitable for use in TALEN-based gene regulating systems. [00127] TAL effectors are proteins that are secreted by Xanthomonas bacteria via their type III secretion system when they infect plants. The DNA binding domain contains a repeated, highly conserved, 33–34 amino acid sequence with divergent 12th and 13th amino acids. These two positions, referred to as the Repeat Variable Diresidue (RVD), are highly variable and strongly correlated with specific nucleotide recognition. Therefore, the TAL effector domains can be engineered to bind specific target DNA sequences by selecting a combination of repeat segments containing the appropriate RVDs. The nucleic acid specificity for RVD combinations is as follows: HD targets cytosine, NI targets adenenine, NG targets thymine, and NN targets guanine (though, in some embodiments, NN can also bind adenenine with lower specificity). [00128] In some embodiments, the TAL effector domains bind to a target DNA sequence that is at least 90% identical to a target DNA sequence (e.g., epitope-enoding) within a target locus of a CD117 gene. In some embodiments, the TAL effector domains bind to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence within a target locus of a target gene. In some embodiments, the TAL effector domains bind to a target DNA sequence that is 100% identical to a target DNA sequence within a target locus of a target gene. Methods and compositions for assembling the TAL-effector repeats are known in the art. See e.g., Cermak et al, Nucleic Acids Research, 39:12, 2011, e82. Plasmids for constructions of the TAL-effector repeats are commercially available from Addgene. [00129] In some embodiments, the gene-editing system is a combination gene-regulating system comprising a site-directed modifying polypeptide and a nucleic acid guide molecule. Herein, a "site-directed modifying polypeptide" refers to a polypeptide that binds to a nucleic acid guide molecule, is targeted to a target nucleic acid sequence, such as, for example, a DNA sequence, by the nucleic acid guide molecule to which it is bound, and modifies the target DNA sequence (e.g., cleavage, mutation, or methylation of target DNA). A site-directed modifying polypeptide comprises two portions, a portion that binds the nucleic acid guide and an activity portion. In some embodiments, a site-directed modifying polypeptide comprises an activity portion that exhibits site-directed enzymatic activity (e.g., DNA methylation, DNA cleavage, histone acetylation, histone methylation, etc.), wherein the site of enzymatic activity is determined by the guide nucleic acid. [00130] In particular embodiments, the nucleic acid guide comprises two portions: a first portion that is complementary to, and capable of binding with, an endogenous target DNA sequence (referred to herein as a “DNA-binding segment”), and a second portion that is capable of interacting with the site-directed modifying polypeptide (referred to herein as a “protein- binding segment”). In some embodiments, the DNA-binding segment and protein-binding segment of a nucleic acid guide are comprised within a single polynucleotide molecule. In some embodiments, the DNA-binding segment and protein-binding segment of a nucleic acid guide are each comprised within separate polynucleotide molecules, such that the nucleic acid guide comprises two polynucleotide molecules that associate with each other to form the functional guide. [00131] The nucleic acid guide mediates the target specificity of the combined protein/nucleic gene regulating systems by specifically hybridizing with a target DNA sequence comprised within the DNA sequence of a target gene. Reference herein to a target gene encompasses the full-length DNA sequence for that particular gene and a full-length DNA sequence for a particular target gene will comprise a plurality of target genetic loci, which refer to portions of a particular target gene sequence (e.g., an exon or an intron). Within each target genetic loci are shorter stretches of DNA sequences referred to herein as “target DNA sequences” or “target sequences” that can be modified by the gene-regulating systems described herein. Further, each target genetic loci comprises a “target modification site,” which refers to the precise location of the modification induced by the gene-regulating system (e.g., the location of an insertion, a deletion, or mutation, the location of a DNA break, or the location of an epigenetic modification). The gene-regulating systems described herein may comprise a single nucleic acid guide, or may comprise a plurality of nucleic acid guides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleic acid guides). [00132] The CRISPR/Cas systems described below are exemplary embodiments of a combination protein/nucleic acid system. [00133] In some embodiments, the gene editing systems described herein are CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR Associated) nuclease systems. In such embodiments, the site-directed modifying polypeptide is a CRISPR- associated endonuclease (a “Cas" endonuclease) and the nucleic acid guide molecule is a guide RNA (gRNA). [00134] A Cas polypeptide refers to a polypeptide that can interact with a gRNA molecule and, in concert with the gRNA molecule, homes or localizes to a target DNA sequence and includes naturally occurring Cas proteins and engineered, altered, or otherwise modified Cas proteins that differ by one or more amino acid residues from a naturally-occurring Cas sequence. [00135] In some embodiments, the Cas protein is a Cas9 protein. Cas9 is a multi-domain enzyme that uses an HNH nuclease domain to cleave the target strand of DNA and a RuvC- like domain to cleave the non-target strand. In some embodiments, mutants of Cas9 can be generated by selective domain inactivation enabling the conversion of WT Cas9 into an enzymatically inactive mutant (e.g., dCas9), which is unable to cleave DNA, or a nickase mutant, which is able to produce single-stranded DNA breaks by cleaving one or the other of the target or non-target strand. [00136] A guide RNA (gRNA) typically comprises two segments, a DNA-binding segment and a protein-binding segment. In some embodiments, the protein-binding segment of a gRNA is comprised in one RNA molecule and the DNA-binding segment is comprised in another separate RNA molecule. Such embodiments are referred to herein as "double-molecule gRNAs" or "two-molecule gRNA" or “dual gRNAs.” In some embodiments, the gRNA is a single RNA molecule and is referred to herein as a "single-guide RNA" or an "sgRNA." The term "guide RNA" or "gRNA" is inclusive, referring both to two-molecule guide RNAs and sgRNAs. [00137] The protein-binding segment of a gRNA typically comprises, in part, two complementary stretches of nucleotides that hybridize to one another to form a double stranded RNA duplex (dsRNA duplex), which facilitates binding to the Cas protein. [00138] The DNA-binding segment (or "DNA-binding sequence") of a gRNA comprises a nucleotide sequence that is complementary to and capable of binding to a specific sequence target DNA sequence or RNA sequence. The protein-binding segment of the gRNA interacts with a Cas polypeptide and the interaction of the gRNA molecule and site-directed modifying polypeptide results in Cas binding to the endogenous DNA or RNA and produces one or more modifications within or around the target DNA sequence. The precise location of the target modification site is determined by both (i) base-pairing complementarity between the gRNA and the target DNA or RNA sequence; and (ii) the location of a short motif, referred to as the protospacer adjacent motif (PAM), in the target DNA sequence. The PAM sequence is required for Cas binding to the target DNA sequence. A variety of PAM sequences are known in the art and are suitable for use with a particular Cas endonuclease (e.g., a Cas9 endonuclease) are known in the art (See e.g., Nat Methods.2013 Nov; 10(11): 1116–1121 and Sci Rep.2014; 4: 5405). In some embodiments, the PAM sequence is located within 50 base pairs of the target modification site. In some embodiments, the PAM sequence is located within 10 base pairs of the target modification site. The DNA or RNA sequences that can be targeted by this method are limited only by the relative distance of the PAM sequence to the target modification site and the presence of a unique 20 base pair sequence to mediate sequence-specific, gRNA- mediated Cas binding. In some embodiments, the target modification site is located at the 5’ terminus of the target locus. In some embodiments, the target modification site is located at the 3’ end of the target locus. In some embodiments, the target modification site is located within an intron or an exon of the target locus. [00139] In particular embodiments, the guide RNA binds to a CD117 polynucleotide sequence and includes a region complementary to a target CD117 sequence. In certain embodiments, the guide RNA targets or binds a region of CD117 polynucleotide sequence that encodes one of the following amino acid residues: N505 or D816. In some embodiments, the present disclosure provides a polynucleotide encoding a gRNA. In some embodiments, a gRNA-encoding nucleic acid is comprised in an expression vector, e.g., a recombinant expression vector. In some embodiments, the present disclosure provides a polynucleotide encoding a site-directed modifying polypeptide. In some embodiments, the polynucleotide encoding a site-directed modifying polypeptide is comprised in an expression vector, e.g., a recombinant expression vector. [00140] In some embodiments, the site-directed modifying polypeptide is a Cas protein, e.g., a Cas9 protein. Cas molecules of a variety of species can be used in the methods and compositions described herein, including Cas molecules derived from S. pyogenes, S. aureus, N. meningitidis, S. thermophiles, etc. In some embodiments, the Cas protein is a Cas9 protein or a Cas9 ortholog and is selected from the group consisting of SpCas9, SpCas9-HF1, SpCas9- HF2, SpCas9-HF3, SpCas9-HF4, SaCas9, FnCpf, FnCas9, eSpCas9, and NmeCas9. In some embodiments, the Cas9 protein is a naturally-occurring Cas9 protein. Exemplary naturally occurring Cas9 molecules are described in Chylinski et al., RNA Biology 201310:5, 727-737. [00141] In some embodiments, a Cas9 protein comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a Cas9 amino acid sequence described in Chylinski et al., RNA Biology 201310:5, 727-737; Hou et al., PNAS Early Edition 2013, 1-6). [00142] In some embodiments, a Cas polypeptide comprises one or more of the following activities: a) a nickase activity, i.e., the ability to cleave a single strand, e.g., the non- complementary strand or the complementary strand, of a nucleic acid molecule; b) a double stranded nuclease activity, i.e., the ability to cleave both strands of a double stranded nucleic acid and create a double stranded break, which in an embodiment is the presence of two nickase activities; c) an endonuclease activity; d) an exonuclease activity; and/or e) a helicase activity, i.e., the ability to unwind the helical structure of a double stranded nucleic acid. [00143] In some embodiments, the Cas9 is a wildtype (WT) Cas9 protein or ortholog. WT Cas9 comprises two catalytically active domains (HNH and RuvC). Binding of WT Cas9 to DNA based on gRNA specificity results in double-stranded DNA breaks that can be repaired by non- homologous end joining (NHEJ) or homology-directed repair (HDR). In some embodiments, Cas9 is fused to heterologous proteins that recruit DNA-damage signaling proteins, exonucleases, or phosphatases to further increase the likelihood or the rate of repair of the target sequence by one repair mechanism or another. In some embodiments, a WT Cas9 is co- expressed with a nucleic acid repair template to facilitate the incorporation of an exogenous nucleic acid sequence by homology-directed repair. [00144] In some embodiments, different Cas9 proteins (i.e., Cas9 proteins from various species) may be advantageous to use in the various provided methods in order to capitalize on various enzymatic characteristics of the different Cas9 proteins (e.g., for different PAM sequence preferences; for increased or decreased enzymatic activity; for an increased or decreased level of cellular toxicity; to change the balance between NHEJ, homology-directed repair, single strand breaks, double strand breaks, etc.). [00145] In some embodiments, the Cas polypeptides are engineered to alter one or more properties of the Cas polypeptide. For example, in some embodiments, the Cas polypeptide comprises altered enzymatic properties, e.g., altered nuclease activity, (as compared with a naturally occurring or other reference Cas molecule) or altered helicase activity. [00146] The present disclosure provides guide RNAs (gRNAs) that direct a site-directed modifying polypeptide to a specific target DNA sequence. A gRNA comprises a DNA- targeting segment and protein-binding segment. The DNA-targeting segment of a gRNA comprises a nucleotide sequence that is complementary to a sequence in the target DNA sequence. As such, the DNA-targeting segment of a gRNA interacts with a target DNA in a sequence-specific manner via hybridization (i.e., base pairing), and the nucleotide sequence of the DNA-targeting segment determines the location within the target DNA that the gRNA will bind. The DNA-targeting segment of a gRNA can be modified (e.g., by genetic engineering) to hybridize to any desired sequence within a target DNA sequence. [00147] The protein-binding segment of a guide RNA interacts with a site-directed modifying polypeptide (e.g., a Cas9 protein) to form a complex. The guide RNA guides the bound polypeptide to a specific nucleotide sequence within target DNA via the above-described DNA-targeting segment. The protein-binding segment of a guide RNA comprises two stretches of nucleotides that are complementary to one another and which form a double stranded RNA duplex. [00148] In some embodiments, a gRNA comprises two separate RNA molecules. In such embodiments, each of the two RNA molecules comprises a stretch of nucleotides that are complementary to one another such that the complementary nucleotides of the two RNA molecules hybridize to form the double-stranded RNA duplex of the protein-binding segment. In some embodiments, a gRNA comprises a single RNA molecule (sgRNA). [00149] The specificity of a gRNA for a target loci is mediated by the sequence of the DNA- binding segment, which comprises about 20 nucleotides that are complementary to a target DNA sequence within the target locus. In some embodiments, the corresponding target DNA sequence is approximately 20 nucleotides in length. In some embodiments, the DNA-binding segments of the gRNA sequences of the present invention are at least 90% complementary to a target DNA sequence within a target locus. In some embodiments, the DNA-binding segments of the gRNA sequences of the present disclosure are at least 95%, 96%, 97%, 98%, or 99% complementary to a target DNA sequence within a target locus, e.g., CD117. In some embodiments, the DNA-binding segments of the gRNA sequences of the present invention are 100% complementary to a target DNA sequence within a target locus. [00150] In some embodiments, the DNA-binding segments of the gRNA sequences bind to a target DNA sequence that is at least 90% identical to a target DNA sequence within a target locus of a CD117 gene. In some embodiments, the DNA-binding segments of the gRNA sequences bind to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence within a target locus of a target gene. In some embodiments, the DNA-binding segments of the gRNA sequences bind to a target DNA sequence that is 100% identical to a target DNA sequence within a target locus of a target gene. [00151] In some embodiments, the DNA-binding segments of the gRNA sequences described herein are designed to minimize off-target binding using algorithms known in the art (e.g., Cas- OFF finder) to identify target sequences that are unique to a particular target locus or target gene. In some embodiments, the gRNAs described herein can comprise one or more modified nucleosides or nucleotides which introduce stability toward nucleases. In such embodiments, these modified gRNAs may elicit a reduced innate immune as compared to a non-modified gRNA. The term "innate immune response" includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, particularly the interferons, and cell death. [00152] In some embodiments, the gRNAs described herein are modified at or near the 5' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of their 5' end). In some embodiments, the 5' end of a gRNA is modified by the inclusion of a eukaryotic mRNA cap structure or cap analog (e.g., a G(5 ')ppp(5 ')G cap analog, a m7G(5 ')ppp(5 ')G cap analog, or a 3 '-0-Me-m7G(5 ')ppp(5 ')G anti reverse cap analog (ARCA)). In some embodiments, an in vitro transcribed gRNA is modified by treatment with a phosphatase (e.g., calf intestinal alkaline phosphatase) to remove the 5' triphosphate group. In some embodiments, a gRNA comprises a modification at or near its 3' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of its 3' end). For example, in some embodiments, the 3' end of a gRNA is modified by the addition of one or more (e.g., 25-200) adenine (A) residues. [00153] In some embodiments, modified nucleosides and modified nucleotides can be present in a gRNA, but also may be present in other gene-regulating systems, e.g., mRNA, RNAi, or siRNA- based systems. In some embodiments, modified nucleosides and nucleotides can include one or more of. a) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage; b) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar; c) wholesale replacement of the phosphate moiety with "dephospho" linkers; d) modification or replacement of a naturally occurring nucleobase; e) replacement or modification of the ribose-phosphate backbone; f) modification of the 3' end or 5' end of the oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety; and g) modification of the sugar. [00154] In some embodiments, the modifications listed above can be combined to provide modified nucleosides and nucleotides that can have two, three, four, or more modifications. For example, in some embodiments, a modified nucleoside or nucleotide can have a modified sugar and a modified nucleobase. In some embodiments, every base of a gRNA is modified. In some embodiments, each of the phosphate groups of a gRNA molecule are replaced with phosphorothioate groups. [00155] In some embodiments, a software tool can be used to optimize the choice of gRNA within a user's target sequence, e.g., to minimize total off-target activity across the genome. Off target activity may be other than cleavage. For example, for each possible gRNA choice using S. pyogenes Cas9, software tools can identify all potential off-target sequences (preceding either NAG or NGG PAMs) across the genome that contain up to a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base-pairs. The cleavage efficiency at each off-target sequence can be predicted, e.g., using an experimentally-derived weighting scheme. Each possible gRNA can then be ranked according to its total predicted off-target cleavage; the top-ranked gRNAs represent those that are likely to have the greatest on-target and the least off-target cleavage. Other functions, e.g., automated reagent design for gRNA vector construction, primer design for the on-target Surveyor assay, and primer design for high- throughput detection and quantification of off-target cleavage via next-generation sequencing, can also be included in the tool. [00156] Additional information regarding CRISPR-Cas systems and components thereof are described in, US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233 and US8999641 and applications related thereto; and WO2014/018423, WO2014/093595, WO2014/093622, WO2014/093635, WO2014/093655, WO2014/093661, WO2014/093694, WO2014/093701, WO2014/093709, WO2014/093712, WO2014/093718, WO2014/145599, WO2014/204723, WO2014/204724, WO2014/204725, WO2014/204726, WO2014/204727, WO2014/204728, WO2014/204729, WO2015/065964, WO2015/089351, WO2015/089354, WO2015/089364, WO2015/089419, WO2015/089427, WO2015/089462, WO2015/089465, WO2015/089473 and WO2015/089486, WO2016205711, WO2017/106657, WO2017/127807 and applications related thereto. [00157] In certain embodiments, the gene editing methods comprise or consist of base editing methods. Various base editing methods are well known in the art and may be adapted to edit any target nucleic acid sequence within a cell. Base editing activity involves chemically altering a base within a polynucleotide, e.g., converting a first base to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, e.g., converting target C•G to T•A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, e.g., converting A•T to G•C. In another embodiment, the base editing activity is cytidine deaminase activity, e.g., converting target C•G to T•A and adenosine or adenine deaminase activity, e.g., converting A•T to G•C. In some embodiments, the base editing methods comprise single nucleotide base editing, such as nucleotide deamination, i.e., A→G or C→T. Base editing systems may edit genomic DNA or transcribed RNA. [00158] A variety of base editing methods are known and used in the art, including but not limited to those disclosed in the references cited herein. Adenosine and cytidine base editors that may be used include, but are not limited to, base editors described in Antoniou P. et al., Base and Prime Editing Technologies for Blood Disorders, Front. Genome Ed., 28 January 2021. In some embodiments, base editing methods comprise C→G conversion as described in Kurt, I. C. et al. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat. Biotechnol. (2020). In some embodiments, dual editors facilitate simultaneous C→T and A→G conversion as described in Zhao, D. et al. New base editors change C to A in bacteria and C to G in mammalian cells. Nat. Biotechnol. (2020). [00159] A base editor system generally refers to a system for editing a nucleobase of a target nucleotide sequence. In certain embodiments, a base editor (BE) system comprises: (1) a polynucleotide programmable nucleotide binding domain, a deaminase domain (e.g., cytidine deaminase or adenosine deaminase) for deaminating nucleobases in the target nucleotide sequence; and (2) one or more guide polynucleotides (e.g., guide RNA) in conjunction with the polynucleotide programmable nucleotide binding domain. In various embodiments, the base editor (BE) system comprises a nucleobase editor domain selected from an adenosine deaminase or a cytidine deaminase, and a domain having nucleic acid sequence specific binding activity. In some embodiments, the base editor system comprises: (1) a base editor (BE) comprising a polynucleotide programmable DNA binding domain and a deaminase domain for deaminating one or more nucleobases in a target nucleotide sequence; and (2) one or more guide RNAs in conjunction with the polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE) or a cytidine base editor (CBE). [00160] Cas9 or Cas9 domain refers to an RNA guided nuclease comprising a Cas9 protein, or a fragment or variant thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). A variety of different Cas9 proteins, and fragments and variants thereof, are known and available in the art. [00161] A guide polynucleotide is a polynucleotide that is specific for a target sequence (e.g., specifically hybridizes to a target polynucleotide sequence, such as a CD117 gene or mRNA) and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cpfl). In certain embodiments, the guide polynucleotide is a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. gRNA is used to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules gRNAs, and gRNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs). gRNAs that exist as single RNA species may comprise two domains: (1) a domain that shares homology to a target nucleic acid, and thus directs binding of a Cas9 complex to the target nucleic acid; and (2) a domain that binds a Cas9 protein. In some embodiments, domain (2) is a sequence known as a tracrRNA, which comprises a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al., Science 337:816-821(2012). Other examples of gRNAs (e.g., those including domain 2) are described, e.g., in US20160208288, entitled "Switchable Cas9 Nucleases and Uses Thereof," and US 9,737,604, entitled "Delivery System For Functional Nucleases.” In some embodiments, a gRNA comprises two or more of domains (1) and (2), which may be referred to as an extended gRNA. An extended gRNA will bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions. The gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to the target site, providing the sequence specificity of the nuclease: RNA complex. [00162] In some embodiments, the base editing method, e.g., a single nucleotide base editing method, targets a polynucleotide encoding a CD117 polypeptide. An illustrative CD117 polynucleotide sequence follows: >NM_000222.2 Homo sapiens KIT proto-oncogene, receptor tyrosine kinase (KIT), transcript variant 1 mRNA: TCTGGGGGCTCGGCTTTGCCGCGCTCGCTGCACTTGGGCGAGAGCTGGAACG TGGACCAGAGCTCGGATCCCATCGCAGCTACCGCGATGAGAGGCGCTCGCG GCGCCTGGGATTTTCTCTGCGTTCTGCTCCTACTGCTTCGCGTCCAGACAGGC TCTTCTCAACCATCTGTGAGTCCAGGGGAACCGTCTCCACCATCCATCCATCC AGGAAAATCAGACTTAATAGTCCGCGTGGGCGACGAGATTAGGCTGTTATGC ACTGATCCGGGCTTTGTCAAATGGACTTTTGAGATCCTGGATGAAACGAATG AGAATAAGCAGAATGAATGGATCACGGAAAAGGCAGAAGCCACCAACACCG GCAAATACACGTGCACCAACAAACACGGCTTAAGCAATTCCATTTATGTGTT TGTTAGAGATCCTGCCAAGCTTTTCCTTGTTGACCGCTCCTTGTATGGGAAAG AAGACAACGACACGCTGGTCCGCTGTCCTCTCACAGACCCAGAAGTGACCAA TTATTCCCTCAAGGGGTGCCAGGGGAAGCCTCTTCCCAAGGACTTGAGGTTT ATTCCTGACCCCAAGGCGGGCATCATGATCAAAAGTGTGAAACGCGCCTACC ATCGGCTCTGTCTGCATTGTTCTGTGGACCAGGAGGGCAAGTCAGTGCTGTC GGAAAAATTCATCCTGAAAGTGAGGCCAGCCTTCAAAGCTGTGCCTGTTGTG TCTGTGTCCAAAGCAAGCTATCTTCTTAGGGAAGGGGAAGAATTCACAGTGA CGTGCACAATAAAAGATGTGTCTAGTTCTGTGTACTCAACGTGGAAAAGAGA AAACAGTCAGACTAAACTACAGGAGAAATATAATAGCTGGCATCACGGTGA CTTCAATTATGAACGTCAGGCAACGTTGACTATCAGTTCAGCGAGAGTTAAT GATTCTGGAGTGTTCATGTGTTATGCCAATAATACTTTTGGATCAGCAAATGT CACAACAACCTTGGAAGTAGTAGATAAAGGATTCATTAATATCTTCCCCATG ATAAACACTACAGTATTTGTAAACGATGGAGAAAATGTAGATTTGATTGTTG AATATGAAGCATTCCCCAAACCTGAACACCAGCAGTGGATCTATATGAACAG AACCTTCACTGATAAATGGGAAGATTATCCCAAGTCTGAGAATGAAAGTAAT ATCAGATACGTAAGTGAACTTCATCTAACGAGATTAAAAGGCACCGAAGGA GGCACTTACACATTCCTAGTGTCCAATTCTGACGTCAATGCTGCCATAGCATT TAATGTTTATGTGAATACAAAACCAGAAATCCTGACTTACGACAGGCTCGTG AATGGCATGCTCCAATGTGTGGCAGCAGGATTCCCAGAGCCCACAAT AGATTGGTATTTTTGTCCAGGAACTGAGCAGAGATGCTCTGCTTCTGTACTGC CAGTGGATGTGCAGACACTAAACTCATCTGGGCCACCGTTTGGAAAGCTAGT GGTTCAGAGTTCTATAGATTCTAGTGCATTCAAGCACAATGGCACGGTTGAA TGTAAGGCTTACAACGATGTGGGCAAGACTTCTGCCTATTTTAACTTTGCATT TAAAGGTAACAACAAAGAGCAAATCCATCCCCACACCCTGTTCACTCCTTTG CTGATTGGTTTCGTAATCGTAGCTGGCATGATGTGCATTATTGTGATGATTCT GACCTACAAATATTTACAGAAACCCATGTATGAAGTACAGTGGAAGGTTGTT GAGGAGATAAATGGAAACAATTATGTTTACATAGACCCAACACAACTTCCTT ATGATCACAAATGGGAGTTTCCCAGAAACAGGCTGAGTTTTGGGAAAACCCT GGGTGCTGGAGCTTTCGGGAAGGTTGTTGAGGCAACTGCTTATGGCTTAATT AAGTCAGATGCGGCCATGACTGTCGCTGTAAAGATGCTCAAGCCGAGTGCCC ATTTGACAGAACGGGAAGCCCTCATGTCTGAACTCAAAGTCCTGAGTTACCT TGGTAATCACATGAATATTGTGAATCTACTTGGAGCCTGCACCATTGGAGGG CCCACCCTGGTCATTACAGAATATTGTTGCTATGGTGATCTTTTGAATTTTTT GAGAAGAAAACGTGATTCATTTATTTGTTCAAAGCAGGAAGATCATGCAGAA GCTGCACTTTATAAGAATCTTCTGCATTCAAAGGAGTCTTCCTGCAGCGATAG TACTAATGAGTACATGGACATGAAACCTGGAGTTTCTTATGTTGTCCCAACC AAGGCCGACAAAAGGAGATCTGTGAGAATAGGCTCATACATAGAAAGAGAT GTGACTCCCGCCATCATGGAGGATGACGAGTTGGCCCTAGACTTAGAAGACT TGCTGAGCTTTTCTTACCAGGTGGCAAAGGGCATGGCTTTCCTCGCCTCCAAG AATTGTATTCACAGAGACTTGGCAGCCAGAAATATCCTCCTTACTCATGGTC GGATCACAAAGATTTGTGATTTTGGTCTAGCCAGAGACATCAAGAATGATTC TAATTATGTGGTTAAAGGAAACGCTCGACTACCTGTGAAGTGGATGGCACCT GAAAGCATTTTCAACTGTGTATACACGTTTGAAAGTGACGTCTGGTCCTATG GGATTTTTCTTTGGGAGCTGTTCTCTTTAGGAAGCAGCCCCTATCCTGGAATG CCGGTCGATTCTAAGTTCTACAAGATGATCAAGGAAGGCTTCCGGATGCTCA GCCCTGAACACGCACCTGCTGAAATGTATGACATAATGAAGACTTGCTGGGA TGCAGATCCCCTAAAAAGACCAACATTCAAGCAAATTGTTCAGCTAATTGAG AAGCAGATTTCAGAGAGCACCAATCATATTTACTCCAACTTAGCAAACTGCA GCCCCAACCGACAGAAGCCCGTGGTAGACCATTCTGTGCGGATCAATTCTGT CGGCAGCACCGCTTCCTCCTCCCAGCCTCTGCTTGTGCACGACGATGTCTGAG CAGAATCAGTGTTTGGGTCACCCCTCCAGGAATGATCTCTTCTTTTGGCTTCC ATGATGGTTATTTTCTTTTCTTTCAACTTGCATCCAACTCCAGGATAGTGGGC ACCCCACTGCAATCCTGTCTTTCTGAGCACACTTTAGTGGCCGATGATTTTTG TCATCAGCCACCATCCTATTGCAAAGGTTCCAACTGTATATATTCCCAATAGC AACGTAGCTTCTACCATGAACAGAAAACATTCTGATTTGGAAAAAGAGAGG GAGGTATGGACTGGGGGCCAGAGTCCTTTCCAAGGCTTCTCCAATTCTGCCC AAAAATATGGTTGATAGTTTACCTGAATAAATGGTAGTAATCACAGTTGGCC TTCAGAACCATCCATAGTAGTATGATGATACAAGATTAGAAGCTGAAAACCT AAGTCCTTTATGTGGAAAACAGAACATCATTAGAACAAAGGACAGAGTATG AACACCTGGGCTTAAGAAATCTAGTATTTCATGCTGGGAATGAGACATAGGC CATGAAAAAAATGATCCCCAAGTGTGAACAAAAGATGCTCTTCTGTGGACCA CTGCATGAGCTTTTATACTACCGACCTGGTTTTTAAATAGAGTTTGCTATTAG AGCATTGAATTGGAGAGAAGGCCTCCCTAGCCAGCACTTGTATATACGCATC TATAAATTGTCCGTGTTCATACATTTGAGGGGAAAACACCATAAGGTTTCGTT TCTGTATACAACCCTGGCATTATGTCCACTGTGTATAGAAGTAGATTAAGAG CCATATAAGTTTGAAGGAAACAGTTAATACCATTTTTTAAGGAAACAATATA ACCACAAAGCACAGTTTGAACAAAATCTCCTCTTTTAGCTGATGAACTTATTC TGTAGATTCTGTGGAACAAGCCTATCAGCTTCAGAATGGCATTGTACTCAAT GGATTTGATGCTGTTTGACAAAGTTACTGATTCACTGCATGGCTCCCACAGG AGTGGGAAAACACTGCCATCTTAGTTTGGATTCTTATGTAGCAGGAAATAAA GTATAGGTTTAGCCTCCTTCGCAGGCATGTCCTGGACACCGGGCCAGTATCT ATATATGTGTATGTACGTTTGTATGTGTGTAGACAAATATTTGGAGGGGTATT TTTGCCCTGAGTCCAAGAGGGTCCTTTAGTACCTGAAAAGTAACTTGGCTTTC ATTATTAGTACTGCTCTTGTTTCTTTTCACATAGCTGTCTAGAGTAGCTTACCA GAAGCTTCCATAGTGGTGCAGAGGAAGTGGAAGGCATCAGTCCCTATGTATT TGCAGTTCACCTGCACTTAAGGCACTCTGTTATTTAGACTCATCTTACTGTAC CTGTTCCTTAGACCTTCCATAATGCTACTGTCTCACTGAAACATTTAAATTTT ACCCTTTAGACTGTAGCCTGGATATTATTCTTGTAGTTTACCTCTTTAAAAAC AAAACAAAACAAAACAAAAAACTCCCCTTCCTCACTGCCCAATATAAAAGG CAAATGTGTACATGGCAGAGTTTGTGTGTTGTCTTGAAAGATTCAGGTATGTT GCCTTTATGGTTTCCCCCTTCTACATTTCTTAGACTACATTTAGAGAACTGTG GCCGTTATCTGGAAGTAACCATTTGCACTGGAGTTCTATGCTCTCGCACCTTT CCAAAGTTAACAGATTTTGGGGTTGTGTTGTCACCCAAGAGATTGTTGTTTGC CATACTTTGTCTGAAAAATTCCTTTGTGTTTCTATTGACTTCAATGATAGTAA GAAAAGTGGTTGTTAGTTATAGATGTCTAGGTACTTCAGGGGCACTTCATTG AGAGTTTTGTCTTGGATATTCTTGAAAGTTTATATTTTTATAATTTTTTCTTAC ATCAGATGTTTCTTTGCAGTGGCTTAATGTTTGAAATTATTTTGTGGCTTTTTT TGTAAATATTGAAATGTAGCAATAATGTCTTTTGAATATTCCCAAGCCCATGA GTCCTTGAAAATATTTTTTATATATACAGTAACTTTATGTGTAAATACATAAG CGGCGTAAGTTTAAAGGATGTTGGTGTTCCACGTGTTTTATTCCTGTATGTTG TCCAATTGTTGACAGTTCTGAAGAATTCTAATAAAATGTACATATATAAATC AAAAAAAAAAAAAAAA (SEQ ID NO: 46). [00163] In particular embodiments, the guide RNA binds to a CD117 polynucleotide sequence and includes a region complementary to a target CD117 sequence. In certain embodiments, the guide RNA targets or binds a region of CD117 polynucleotide sequence that encodes one of the following amino acid residues: N505 or D816. [00164] One example of base editing methods, systems, and components thereof that may be used according to the methods and compositions disclosed herein is described in PCT Application Publication No. WO2021041945. In some embodiments, the base editing method comprises use of a modified CRISPR protein, bound to a guide RNA, and a base editing enzyme, such as a deaminase, wherein the modified CRISPR protein does not cause a double- stranded break. In some embodiments, the modified CRISPR protein is a nucleobase editor polypeptide or nucleic acid programmable-DNA binding protein (napDNAbp), as disclosed in PCT Application Publication Nos. WO2021041945 or WO2021163587In some embodiments, the method of base editing a polynucleotide encoding a CD117 polypeptide comprises expressing in a cell a nucleobase editor polypeptide, wherein the nucleobase editor polypeptide comprises a napDNAbp and a deaminase, and contacting the cell with a guide RNA capable of targeting the polynucleotide encoding a CD117 polypeptide. [00165] In other embodiments, base editing may refer to RNA base editing methods, e.g., as described in Porto E. et al. Base editing: advances and therapeutic opportunities, Nature Reviews Drug Discovery volume 19, pages 839–859 (2020). [00166] In particular embodiments, any of the gene editing, including base editing methods disclosed herein or known in the art may be used to modify one or more amino acids within an epitope of wild type human CD117 bound by an anti-CD117 antibody, optionally wherein the epitope comprises one or more of the following amino acids present in the wild type human CD117: N505 or D816, including but not limited to any of these recited amino acid residues. In certain embodiments, the method introduces a A→G or C→T mutation into one or both alleles of the CD117 gene, which results in the gene encoding a different amino acid by the codon that was mutated. [00167] In certain embodiments, the disclosure provides a modified cell, e.g., HSPC or HSC, that comprises one or more components of a gene editing, e.g., base editing, system disclosed herein. In particular embodiments, the one or more component comprises a nucleic acid that binds to a CD117 gene or encoded mRNA, e.g., at a site to be modified to result in the encoding and/or expression of a modified CD117 disclosed herein, such as, e.g., a guide RNA. In particular embodiments, the guide RNA binds to a CD117 polynucleotide sequence and includes a region complementary to a target CD117 sequence. In certain embodiments, the guide RNA targets or binds a region of CD117 polynucleotide sequence that encodes one of the following amino acid residues: N505 or D816. In particular embodiments, the one or more component comprises a base editing enzyme, e.g., any of those disclosed herein or in references cited herein. [00168] In particular embodiments, a modified cell expressing a modified CD117 polypeptide is not substantially inhibited, eliminated, or killed by monoclonal antibodies (mAbs) that bind endogenous or wild-type cell-surface CD117 and inhibit proliferation of or kill a cell expressing only the wild-type CD117 and not a modified CD117 polypeptide disclosed herein. In certain embodiments, proliferation of the modified cell expressing the modified CD117 polypeptide is inhibited by less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%, as compared to proliferation of the same cell type that is not modified, e.g., only expresses wild-type CD117. [00169] In particular embodiments, the modified cell expressing the modified CD117 polypeptide is not substantially inhibited, eliminated, depleted, or killed by monoclonal antibodies (mAbs) that bind endogenous or wild-type cell-surface CD117 and inhibit proliferation of or kill a cell expressing only the wild-type CD117 and not a modified CD117 polypeptide disclosed herein. In certain embodiments, proliferation of the modified cell expressing the modified CD117 polypeptide is inhibited, eliminated, depleted, or killed by less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%, as compared to proliferation of the same cell type that is not modified, e.g., only expresses wild-type CD117. Anti-CD117 Antibodies [00170] Compositions and methods disclosed herein may be applicable to any CD117 antibody, particularly monoclonal anti-human CD117 antibodies. In certain embodiments, methods disclosed herein include a conditioning regimen that comprises administration of an anti- CD117 antibody, wherein the anti-CD117 antibody depletes or reduces endogenous HSPCs. Illustrative CD117 antibodies include, but are not limited to, SR-1, JSP191, 8D7, K45, 104D2, CK6, YB5.B8, AF-2-1, AF11, AF12, AF112, AF-3, AF-1-1, NF, NF-2-1, NF11, NF12, NF112, NF-3, HF11, HF12, and HF112. A number of antibodies contemplated by the disclosure that specifically bind human CD117 are known in the art and commercially available, including without limitation SR1, 2B8, ACK2, YB5-B8, 57A5, 104D2, etc. In certain embodiments, the anti-CD117 antibody is selected from the group consisting of: JSP191 (Jasper Therapeutics; Redwood City, CA); CDX-0159 (Celldex Therapeutics, Hampton, NJ); MGTA-117 (AB85) (Magenta Therapeutics, Cambridge, MA); CK6 (Magenta Therapeutics, Cambridge, MA); AB249 (Magenta Therapeutics, Cambridge, MA); and FSI-174 (Gilead, Foster City, CA). Antibodies from Magenta Therapeutics contemplated by the disclosure include but are not limited to those that are disclosed in US Patent Application Publication No.20190153114, PCT Application Publication Nos. WO2019084064, WO2020/219748, and WO2020/219770. The FSI-174 antibody is disclosed in PCT application Publication No. WO2020/112687 and U.S. Patent Application Publication No.20200165337. The disclosure includes but is not limited to any CD117 antibodies and/or CDR sets disclosed in any of the patent application disclosed herein, which are all incorporated by reference in their entireties. [00171] In certain embodiments, the CD117 antibody binds to the extracellular region of CD117, i.e., amino acids 26-524. The sequence of this region is shown below: QPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWIT EKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDP EVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVLSE KFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQTKLQ EKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGF INIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENES NIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGML QCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFK HNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTP (SEQ ID NO:7). [00172] In particular embodiments, the antibody is the humanized form of SR1, which is a murine CD117 antibody disclosed in U.S. Patent Nos. 5,919,911 and 5,489,516. The humanized antibody, referred to as JSP191 (formerly referred to as AMG191), is described in U.S. Pat. Nos. 8,436,150, 8,791,249, and 7,915,391, and U.S. Patent Application Publication No. 20110223165. JSP191 is an aglycosylated IgG1 humanized antibody. JSP191 is a humanized monoclonal antibody in clinical development as a conditioning agent to clear hematopoietic stem cells from bone marrow. JSP191 specifically binds to human CD117, a receptor for stem cell factor (SCF), which is expressed on the surface of hematopoietic stem and progenitor cells (HSPCs). JSP191 blocks SCF from binding to CD117 and disrupts critical survival signals, leading to the depletion of hematopoietic stem cells. [00173] The sequences of the heavy chains and light chains of JSP191 are disclosed as SEQ ID NO: 4 in U.S. Patent No. 8,436,150 and SEQ ID NO: 2 in U.S. Patent No. 8,436,150, respectively. The sequences of the heavy and light chains of JSP191 are: Heavy Chain: MDWTWRVFCLLAVAPGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMH WVRQAPGQGLEWMGVIYSGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDT AVYYCARERDTRFGNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK (SEQ ID NO:8) Light Chain: MVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMH WYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQN NEDPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC (SEQ ID NO:9) [00174] In certain embodiments, the variable heavy domain of JSP191 comprises the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSG NGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQG TLVTVSS (SEQ ID NO:10). [00175] In certain embodiments, the variable light chain domain of JSP191 comprises the following sequence: DIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMHWYQQKPGQPPKLLIYLASNL ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDP YTFGGGTKVEIK (SEQ ID NO:11). [00176] The CDRs present in JSP191 are as follows: VH CDR1 = YNMH (SEQ ID NO: 26); VH CDR2 = IYSGNGDTSYNQKFKG (SEQ ID NO: 27); VH CDR3 = ERDTRFGN (SEQ ID NO: 28); VL CDR1 = RASESVDIYGNSFMH (SEQ ID NO: 29); VL CDR2 = LASNLES (SEQ ID NO: 30); and VL CDR3 = QQNNEDPYT (SEQ ID NO: 31). [00177] CDX-0159 is a humanized monoclonal antibody that specifically binds the receptor tyrosine kinase KIT with high specificity and potently inhibits its activity. CDX-0159 is designed to block KIT activation by disrupting both SCF binding and KIT dimerization. CDX- 0159 and other CD117 antibodies are described in U.S. Patent No.10,781,267, and in particular embodiments, an CD117 disclosed herein comprises the CDRs of any of the antibodies disclosed therein. In certain embodiments, the CD117 antibody comprises: (i) a light chain variable region ("VL") comprising the amino acid sequence: DIVMTQSPSXK1LSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKXK2LIYSASYR YSGVPDRFXK3GSGSGTDFTLTISSLQXK4EDFAXK5YXK6CQQYNSYPRTFGGGTKVEIK (SEQ ID NO:12), wherein XK1 is an amino acid with an aromatic or aliphatic hydroxyl side chain, XK2 is an amino acid with an aliphatic or aliphatic hydroxyl side chain, XK3 is an amino acid with an aliphatic hydroxyl side chain, XK4 is an amino acid with an aliphatic hydroxyl side chain or is P, XK5 is an amino acid with a charged or acidic side chain, and XK6 is an amino acid with an aromatic side chain; and (ii) a heavy chain variable region ("VH") comprising the amino acid sequence: QVQLVQSGAEXH1KKPGASVKXH2SCKASGYTFTDYYINAVVXH3QAPGKGLEWIARI YPGSGNTYYNEKFKGRXH4TXH5TAXH6KSTSTAYMXH7LSSLRSEDXH8AVYFCARGV YYFDYWGQGTTVTVSS (SEQ ID NO:13), wherein XH1 is an amino acid with an aliphatic side chain, XH2 is an amino acid with an aliphatic side chain, XH3 is an amino acid with a polar or basic side chain, XH4 is an amino acid with an aliphatic side chain, XH5 is an amino acid with an aliphatic side chain, XH6 is an amino acid with an acidic side chain, XH7 is an amino acid with an acidic or amide derivative side chain, and XH8 is an amino acid with an aliphatic hydroxyl side chain. In specific aspects, described herein are antibodies (e.g., human or humanized antibodies), including antigen-binding fragments thereof, comprising: (i) VH CDRs of a VH domain comprising the amino acid sequence: QVQLKQSGAELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEWIARIYPG SGNTYYNEKFKGKATLTAEKSSSTAYMQLSSLTSEDSAVYFCARGVYYFDYWGQ GTTLTVSS (SEQ ID NO:14) or QVQLKQSGAELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEWIARIYPG SGNTYYNEKFKGKATLTAEKSSSTAYMQLSSLTSEDSAVYFCARGVYYFDYWGQ GTTLTVSA (SEQ ID NO:15), and (ii) VL CDRs of a VL domain comprising the amino acid sequence DIVMTQSQKFMSTSVGDRVSVTCKASQNVRTNVAWYQQKPGQSPKALIYSASYRYS GVPDRFTGSGSGTDFTLTI SNVQSEDLADYFCQQYNSYPRTFGGGTKLEIKR (SEQ ID NO:16). [00178] MGTA-117 (AB85) is a CD117-targeted antibody engineered for the transplant setting and conjugated to amanitin, which is being developed for patients undergoing immune reset through either autologous or allogeneic stem cell transplant. MGTA-117 depletes hematopoietic stem and progenitor cells, and this antibody and others contemplated by the disclosure are described in U.S> Application No. 20200407440 and/or PCT Application No. WO2019084064. Epitope analysis of AB85 binding to CD177 is described in PCT Application Publication No. WO2020219770, which identified the following two epitopes within CD117: EKAEATNTGKYTCTNKHGLSNSIYVFVRDPA (amino acids 60-90; (SEQ ID NO:17)), and RCPLTDPEVTNYSLKGCQGKP (amino acids 100-130; (SEQ ID NO:18)). [00179] The sequences of the variable heavy chain and variable light chains of AB85 are disclosed as SEQ ID NO: 143 and SEQ ID NO: 144 from PCT Application No. WO2019084064, respectively. [00180] The heavy chain variable region (VH) amino acid sequence of AB85 is: EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMAIINPRDS DTRYRPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYEGYEGAFDI WGQGTLVTVSS (SEQ ID NO:19). [00181] The VH CDR amino acid sequences of AB85 are as follows: NYWIG (VH CDR1; SEQ ID NO: 32); IINPRDSDTRYRPSFQG (VH CDR2; SEQ ID NO: 33); and HGRGYEGYEGAFDI (VH CDR3; SEQ ID NO: 34). [00182] The light chain variable region (VL) amino acid sequence of AB85 is: DIQMTQSPSSLSASVGDRVTITCRSSQGIRSDLGWYQQKPGKAPKLLIYDASNLETGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANGFPLTFGGGTKVEIK (SEQ ID NO:20). [00183] The VL CDR amino acid sequences of AB85 are as follows: RSSQGIRSDLG (VL CDR1; SEQ ID NO: 35); DASNLET (VL CDR2; SEQ ID NO: 36); and QQANGFPLT (VL CDR3; SEQ ID NO: 37). [00184] FSI-174 is an anti-cKIT antibody being developed in combination with 5F9 as a non- toxic transplant conditioning regimen, as well as a treatment for targeted hematologic malignancies. The sequences of FSI-174 are disclosed in PCT Application Publication No. 2020/112687, U.S. Patent Application Publication No. 20200165337, and U.S. Patent No. 11,041,022. In particular embodiments, an CD117 antibody comprises the three CDRs or variable heavy chain regions present in any of AH1, AH2, AH3, AH4, or AH5 disclosed therein, and/or the three CDRs or variable heavy chain regions present in any of AL1 or AL2 disclosed therein. [00185] In certain embodiments, the CDRs present in FSI-174 and related antibodies are as follows: VH CDR1 = SYNMH (SEQ ID NO: 38); VH CDR2 = VIYSGNGDTSY(A/N)QKF(K/Q)G (SEQ ID NO: 39); VH CDR3 = ERDTRFGN (SEQ ID NO: 40); VL CDR1 = RAS(D/E)SVDIYG(N/Q)SFMH (SEQ ID NO: 41); VL CDR2 = LASNLES (SEQ ID NO: 42); and VL CDR3 = QQNNEDPYT (SEQ ID NO: 31). A/N and the like indicate that the amino acid position may be either of the two amino acids, in this example, A or N. In certain embodiments, CDRs present in the heavy variable region are CDRs H1, H2 and H3 as defined by Kabat: H1 = SYNMH (SEQ ID NO: 38); H2 = VIYSGNGDTSYAQKFKG (SEQ ID NO: 44); H3 = ERDTRFGN (SEQ ID NO: 28); and the CDRs present in the light variable region are CDRs L1, L2 and L3 as defined by Kabat: L1 = RASESVDIYGQSFMH (SEQ ID NO: 45); L2 = LASNLES (SEQ ID NO: 30); and L3 = QQNNEDPYT (SEQ ID NO: 31), respectively except that 1, 2, or 3 CDR residue substitutions is/are present selected from N to A at heavy chain position 60, K to Q at heavy chain position 64 and N to Q at light chain position 30, positions being numbered according to Kabat. In certain embodiments, the antibody comprises any of the heavy chain variable region sequences (AH2, AH3, AH4) and/or light chain variable chain region sequences provided below (AL2), or the CDRs therein shown underlined: AH2: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYMNHWVRQAPGQGLEWMGVIYSG NGDTSYAQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQG TLVTVSS (SEQ ID NO:21) AH3: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYMNHWVRQAPGQGLEWMGVIYSG NGDTSYNQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQG TLVTVSS (SEQ ID NO:22) AH4: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYMNHWVRQAPGQGLEWMGVIYSG NGDTSYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQG TLVTVSS (SEQ ID NO:23) AL2: DIVMTQSPLSLPVTPGEPASISCRASESVDIYGQSFMHWYQQKPGQPPKLLIYLASNLE SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQNNEDPYTFGGGTKVEIK (SEQ ID NO:24) [00186] CK6 is anti-CD117 antibody developed to selectively deplete endogenous hematopoietic stem cells prior to the stem cell transplants in the treatment of various hematopoietic diseases, metabolic disorders, cancers, and autoimmune diseases. CK6 is described in US Patent Application No.2012/0288506 (and U.S. Pat. No.8,552,157). CK6 has the following heavy chain CDR amino acid sequences: CDR-H1 with SYWIG (SEQ ID NO: 50); CDR-H2 with IIYPGDSDTRYSPSFQG (SEQ ID NO: 51); CDR-H3 with HGRGYNGYEGAFDI (SEQ ID NO: 52). CK6 has the following light chain CDR amino acid sequences: CDR-L1 with RASQGISSALA (SEQ ID NO: 53); CDR-L2 with DASSLES (SEQ ID NO: 54); and CDR-L3 with CQQFNSYPLT (SEQ ID NO: 55). [00187] In some embodiments, any of the CDRS disclosed herein may be exchanged for a sequence within an example heavy chain variable domain, e.g., using the methods and variable heavy chain and variable light chain sequences identified respectively in US Patent No. 6,054,297: [00188] Example variable heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISENGS DTYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGGAVSYFDV WGQGTLVTVSS (SEQ ID NO: 56). [00189] Example variable light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT (SEQ ID NO: 57). [00190] Ab249 was derived from antibody CK6, as an antagonist anti-CD117 antibody, as disclosed in PCT Application No. WO2020092655A1. Ab249 has improved binding characteristics over the parent CK6. Ab249 has the following heavy chain CDRS: TSWIG (VH CDR1; SEQ ID NO: 58) IIYPGDSDTRYSPSFQG (VH CDR2; SEQ ID NO: 51); and HGLGYNGYEGAFDI (VH CDR3; SEQ ID NO: 59). Ab249 has the following light chain CDRS: RASQGIGSALA (VL CDR1; SEQ ID NO: 60); DASNLET (VL CDR2; SEQ ID NO: 36); and QQLNGYPLT (VL CDR3; SEQ ID NO: 61). [00191] Ab249 has the following variable heavy chain sequence (CDRS are underlined): EVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGWVRQMPGKGLEWMGIIYPGDS DTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYEGAFDI WGQGTLVTVSS(SEQ ID NO: 62). [00192] Ab249 has the following variable light chain sequence (CDRS are underlined): DIQMTQSPSSLSASVGDRVTITCRASQGIGSALAWYQQKPGKAPKLLIYDASNLETGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPLTFGQGTRLEIK (SEQ ID NO: 63). [00193] In particular embodiments, the antibody may include one or more CDR with at least 70%, 80%, 90%, 95%, or 99% amino acid or nucleotide sequence identity to a CDR present in a humanized monoclonal antibody that binds CD117, e.g., an antibody derived from any of the mouse antibodies SR1, ACK2, ACK4, 2B8, 3C11, MR-1, and CD122. In some embodiments, the antibody blocks the binding of stem cell factor (SCF) to stem cell factor receptor (CD117). Illustrative embodiments of CD117 antibodies that may be used include JSP191, as well as those described in WO2007127317A2 and US20200165337A1, both incorporated herein in their entirety. [00194] In certain embodiments, the anti-CD117 antibody comprises the full heavy chain and/or full light chain of any of the antibodies disclosed herein, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity to a heavy or light chain disclosed herein, e.g., a JSP191 heavy or light chain. In certain embodiments, the anti-CD117 antibody comprises the variable region of a heavy chain and/or light chain of any of the antibodies disclosed herein, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity to the variable region of a heavy or light chain disclosed herein, e.g., a JSP191 heavy or light chain variable region. In certain embodiments, the anti-CD117 antibody comprises a heavy chain and/or a light chain comprising one or more CDRs of an antibody disclosed herein, e.g., two, three, four, five or six CDRs of an antibody disclosed herein, e.g., a JSP191 antibody. In particular embodiments, the anti-CD117 antibody comprises a heavy chain or variable region thereof comprising one, two, or three heavy chain CDRs disclosed herein, e.g., a JSP191 heavy chain. In particular embodiments, the anti-CD117 antibody comprises a light chain or variable region thereof comprising one, two, or three light chain CDRs disclosed herein, e.g., a JSP191 light chain. [00195] In particular embodiments, the antibody binds to a region of wild-type CD117 or an epitope of wild-type CD117 that is modified in a modified CD117 polypeptides disclosed herein. In particular embodiments, the antibody does not bind a modified CD117 polypeptide disclosed herein, or binds to a modified CD117 polypeptide disclosed herein with reduced affinity, e.g., less than 50%, less than 25%, or less than 10%. Antibody affinity to a particular polypeptide, such as wild-type CD117 or a modified CD117 may be determined, e.g., by measuring the equilibrium dissociation constant between the antibody and its antigen (KD), which may be determined by routine methods in the art, e.g., by surface plasmon resonance, as described in Hearty, Stephen, Paul Leonard, and Richard O’Kennedy. "Measuring antibody– antigen binding kinetics using surface plasmon resonance." Antibody Engineering: Methods and Protocols, Second Edition (2012): 411-442. [00196] In particular embodiments, the modified cell expressing the modified CD117 polypeptide is capable of proliferating or surviving in the presence of an anti-CD117 antibody, e.g., an anti-CD117 antibody that blocks or inhibits binding of SCF to CD117 on the cell surface. In particular embodiments, proliferation and/or survival of the modified cell expressing the modified CD117 polypeptide, in the presence of an anti-CD117 antibody (e.g., an anti-CD117 antibody that blocks or inhibits binding of SCF to CD117 on the cell surface), is at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, or at least 40% the level of proliferation and/or survival in the absence of the anti-CD117 antibody. In certain embodiments, the anti-CD117 antibody is capable of inhibiting proliferation of or inducing death or apoptosis of a cell expressing only the wild-type CD117 and not a modified CD117 polypeptide disclosed herein. In particular embodiments, the anti-CD117 antibody is selected from the group consisting of: SR1, 2B8, ACK2, YB5-B8, 57A5, 104D2, JSP191, CDX-0159, MGTA-117 (AB85), and FSI-174. In particular embodiments, the antibody is JSP191. Thus, in particular embodiments, the modified CD117 polypeptides disclosed herein, when expressed on a HSC and/or HSPC surface, are capable of substantially binding SCF in the presence of an anti-CD117 antibody that inhibit binding of SCF to endogenous, wild-type CD117 on the cell surface. Similarly, in particular embodiments, the modified CD117 polypeptides disclosed herein, when expressed on an HSC surface, are capable of intracellular signaling when bound by SCF, in the absence of and in the presence of an anti-CD117 antibody that inhibit binding of SCF to endogenous, wild-type CD117 on the cell surface. In particular embodiments, SCF binding and/or SCF-mediating signaling is in not substantially reduced in the presence of the anti-CD117 antibody, e.g., binding and/or signaling of the modified cell expressing the modified CD117 polypeptide is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the level of binding and/or signaling observed in the same cell type that is not modified, e.g., only expresses wild-type CD117. [00197] CD117 signaling or proliferation or viability may be determined using methods standard in the art. For example, in certain embodiments, CD117 signaling or proliferation (e.g., in response to SCF), of cells comprising a modified CD117 polypeptide is determined using a cell line (e.g., Ba/F3 cells) engineered to express the modified CD117 polypeptide. Cells are cultured in the presence of IL-3, with or without stem cell factor (SCF), and in the presence or absence of an anti-CD117 antibody, e.g., JSP191. Control parental Ba/F3 cells do not proliferate in the absence of IL-3. Further, parental Ba/F3 cells do not express CD117 and are not responsive to SCF signaling. Proliferation in response to SCF binding may this be determined for cells overexpressing the modified CD117, e.g., in the presence and absence of the anti-CD117 antibody. [00198] The disclosure also provides methods of preparing HSCs and/or HSPCs for HCT, comprising introducing a polynucleotide sequence encoding a modified CD117 described herein into the HSCs and/or HSPCs, In particular embodiments, the polynucleotide sequence encoding the modified CD117 is present within an mRNA or an expression vector, and the modified CD117 is transiently or constitutively expressed after it is introduced into the HSCs and/or HSPCs. The polynucleotide and/or vector may comprise nucleotide modifications, including any of those disclosed herein or known in the art, e.g., to increase expression or stability of the polynucleotide or vector. [00199] For engraftment purposes, a composition comprising HSCs and/or HSPCs, is administered to a patient. Such methods are well known in the art. The stem cells are optionally, although not necessarily, purified. Abundant reports explore various methods for purification of stem cells and subsequent engraftment, including flow cytometry; an isolex system (Klein et al. (2001) Bone Marrow Transplant. 28(11):1023-9; Prince et al. (2002) Cytotherapy 4(2):137-45); immunomagnetic separation (Prince et al. (2002) Cytotherapy 4(2):147-55; Handgretinger et al. (2002) Bone Marrow Transplant. 29(9):731-6; Chou et al. (2005) Breast Cancer.12(3):178-88); and the like. Each of these references is herein specifically incorporated by reference, particularly with respect to procedures, cell compositions and doses for hematopoietic stem cell transplantation. [00200] The present disclosure also includes pharmaceutical compositions comprising one or more modified CD117 polypeptides, one or more polynucleotides or vectors comprising a sequence encoding a modified CD117 polypeptide (e.g., a modified mRNA), or a modified cell comprising a polynucleotide or vector encoding a modified CD117 polypeptide and/or expressing a modified CD117, in combination with one or more pharmaceutically acceptable diluent, carrier, or excipient. [00201] The present invention discloses a pharmaceutical composition comprising a modified cell comprising a modified CD117 polypeptide (or nucleic acid sequence encoding the modified CD117 polypeptide) described herein and one or more pharmaceutically acceptable diluent, carrier, or excipient. In particular embodiments, the cell is a heterologous cell or an autologous cell obtained from the subject to be treated. In particular embodiments, the cell is a stem cell, e.g., a HSC and/or HSPC. In certain embodiments, the pharmaceutical composition further comprises one or more additional active agents. In certain embodiments, the one or more additional active agent comprises an anti-CD117 antibody. In particular embodiments, the anti-CD117 antibody is selected from the group consisting of: SR1, 2B8, ACK2, YB5-B8, 57A5, 104D2, JSP191, CDX-0159, MGTA-117 (AB85), and FSI-174. In particular embodiments, the antibody is JSP191. In certain embodiments, the one or more additional active agent comprises one or more anti-CD47, anti-CD40L, anti-CD122, anti-CD4, and/or anti-CD8 antibody. [00202] The polynucleotides, polypeptides, and cells described herein can be combined with pharmaceutically-acceptable carriers, diluents and reagents useful in preparing a formulation that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for mammalian, e.g., human or primate, use. In certain embodiments, the pharmaceutical composition is a solution or suspension comprising modified cells disclosed herein. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulations. Solutions or suspensions used for the formulations can include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; detergents such as Tween 20 to prevent aggregation; and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In particular embodiments, the pharmaceutical compositions are sterile. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In certain embodiments, it is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, e.g., a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. In some cases, the composition is sterile and may be fluid to the extent that easy syringability exists. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In certain embodiments, a pharmaceutical composition include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the internal compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Methods of Use [00203] In further aspects, the disclosure provides methods of treating a mammalian subject in need thereof, comprising administering to the subject modified cells, e.g., HSCs or HSPCs, comprising a modified CD117 polypeptide described herein and/or a nucleic acid encoding the modified CD117 polypeptide. In particular embodiments, the subject is in need of HCT or a hematopoietic stem cell transplant. The transplant may be autologous, allogeneic, or xenogeneic, including without limitation allogeneic haploidentical stem cells, mismatched allogeneic stem cells, genetically engineered autologous or allogeneic cells, etc. In particular embodiments, the modified HSCs or HSPCs are infused into the subject, e.g., by intravenous infusion, e.g., through a central vein over a period of several minutes to several hours. In particular embodiments, the modified HSCs or HSPCs transiently express the modified CD117 polypeptide, which is constitutively active, e.g., has constitutive kinase activity. In certain embodiments, the modified CD117 has constitutive autophosphorylation activity, e.g., without bound SCF. In particular embodiments, the modified HSCs or HSPCs that transiently express the modified CD117 are resistant to ablation by an CD117 antibody, such as, e.g., JSP191. Accordingly, a subject in need of HCT may be conditioned using an CD117 monoclonal antibody such as JSP191 prior to, with, or following HCT in order to ablate diseased HSPCs, whereas the transplanted modified HSCs or HSPCs are less susceptible to ablation by any monoclonal antibody in the subject following transplant, since they transiently express the modified CD117, which provides transient constitutive CD117 signaling, even in the presence of the antibody. [00204] Where the donor is allogeneic to the recipient, the HLA type of the donor and recipient may be tested for a match, or haploidentical cells may be used. In certain embodiments, cells obtained from HLA-haploidentical donors or HLA-identical donors are used. HLA- haploidentical donors can be manipulated by CD34 or CD34/CD90 selection. For HLA matching, traditionally, the loci critical for matching are HLA-A, HLA-B, and HLA-DR. HLA- C and HLA-DQ are also now considered when determining the appropriateness of a donor. A completely matched sibling donor is generally considered the ideal donor. For unrelated donors, a complete match or a single mismatch is considered acceptable for most transplantation, although in certain circumstances, a greater mismatch is tolerated. Preferably matching is both serologic and molecular. Where the donor cells are from umbilical cord blood, the degree of tolerable HLA disparity is much greater, and a match of three or four out of the six HLA-A, HLA-B and HLA-DRB1 antigens is typically sufficient for transplantation. Immunocompetent donor T cells may be removed using a variety of methods to reduce or eliminate the possibility that graft versus host disease (GVHD) will develop. [00205] The HCT methods disclosed use modified HSCs comprising a modified CD117 polypeptide or nucleic acid encoding the modified CD117 polypeptide. The methods are believed to result in reduced toxicity, reduced morbidity, or reduced graft-versus-host disease, as compared to HCT wherein a subject is administered HSCs that do not comprise the modified CD117 polypeptide or nucleic acid encoding the modified CD117 polypeptide. The methods of the invention are also believed to provide for improved engraftment of stem cells after transplantation into a recipient. [00206] In certain embodiments, the disclosure provides a method for producing a population of cell comprising a plurality of modified HSCs and/or HSPCs, comprising: i) obtaining HSCs and/or HSPCs from a donor subject, optionally a mammal, e.g., a human; ii) introducing a polynucleotide sequence, e.g., an mRNA or plasmid, encoding a modified CD117 polypeptide into the HSC and/or HSPCs, optionally wherein the modified CD117 polypeptide comprises a sequence disclosed herein, or a functional variant or fragment thereof; and iii) optionally, modifying the HSCs and/or HSPCs, e.g., by introducing a gene therapy vector, or by gene editing or base editing, e.g., to correct a gene mutation in the subject; and iv) providing to a recipient subject in need of HCT the modified HSCs and/or HSPCs resulting from step iii or step iv. [00207] In particular embodiments, the introduced polynucleotide sequence is an mRNA that is expressed in the HSCs and/or HSPCs following introduction into the cells. In particular embodiments, the gene therapy vector or reagents used to perform the gene editing are introduced into cells obtained from a subject to undergo HCT using the modified HSCs and/or HSPCs, i.e., autologous HCT. In particular embodiments, the polynucleotide sequence encoding the modified CD117 polypeptide and the gene therapy vector or reagents used for gene editing are introduced into the cells at the same time, or either may be introduced before or after the other, optionally within 10 minutes, 20 minutes, 30 minutes, one hour, two hours, four hours, eight hours, 12 hours, 24 hours, or 48 hours of each other. In particular embodiments, the polynucleotide sequence encoding the modified CD117 polypeptide is an mRNA, and it is introduced into the cells by electroporation. [00208] In particular embodiments of any of the methods of treatment disclosed herein, the subject is administered a conditioning regimen to facilitate or increase engraftment of the modified cells. In certain embodiments, the conditioning regimen depletes endogenous normal or disease HSCs of the subject. Conditioning regimens may be given prior to transplant to reduce the number of blood stem cells in the bone marrow to make space for donor blood stem cells to engraft and cure the patient. Typically, the conditioning regimen is administered prior to and/or concurrent with the administering of the pharmaceutical composition. A variety of conditioning regimens are known and available in the art. These include myeloablative, reduced intensity, and non-myeloablative conditioning regimens. Illustrative conditioning regimens are described in Figure 1, and any of these may be used according to the methods disclosed herein, although the conditioning regimen is not limited to those disclosed in Figure 1. In particular embodiments, the modified cells are administered to a subject in combination with a non-myeloablative conditioning regimen. [00209] In certain embodiments, the conditioning regimen comprises one or more of: chemotherapy (optionally a nucleoside analog and/or an alkylating agent), monoclonal antibody therapy, and radiation, optionally radiation to the entire body. In certain embodiments where two or more conditioning agents are used, they are administered at the same or different times, or two or more may be administered at the same time, and the other(s) at different times. In particular embodiments, the various conditioning agents are administered to the subject or present within the subject during an overlapping time period prior to the subject being administered the modified HSPCs/HSCs. [00210] In certain embodiments, the conditioning regimen comprises administration of an anti- CD117 antibody, wherein the anti-CD117 antibody depletes endogenous HSCs expressing wild-type CD117, but the anti-CD117 antibody does not deplete the administered modified HSCs. In particular embodiments, the anti-CD117 antibody is selected from the group consisting of: SR1, 2B8, ACK2, YB5-B8, 57A5, 104D2, JSP191, CDX-0159, MGTA-117 (AB85), and FSI-174. In particular embodiments, the antibody is JSP191. In particular embodiments, the conditioning regimen comprises an anti-CD117 antibody alone. In particular embodiments, the subject is administered the anti-CD117 antibody prior to administration of the modified HSCs, e.g., as a single dose. [00211] An effective dose of CD117 antibody is the dose that depletes endogenous hematopoietic stem cells. The effective dose will depend on the individual and the specific antibody, but it will generally be up to about 100 ^g/kg body weight, up to about 250 ^g/kg, up to about 500 ^g/kg, up to about 750 ^g/kg, up to about 1 mg/kg, up to about 1.2 mg/kg, up to about 1.5 mg/kg, up to about 3 mg/kg, up to about 5 mg/kg, up to about 10 mg/kg. In some embodiments, the subject is administered about 0.01 mg/kg to about 2 mg/kg of the CD117 antibody, e.g., JSP191, and optionally the subject is administered about 0.1 mg/kg to about 1 mg/kg of the CD117 antibody, e.g., JSP191. In some embodiments, CD117 antibody may be administered to a subject in a dose about 0.01 mg/kg to about 2 mg/kg of the subject’s body weight, or about 0.1 mg/kg to about 1 mg/kg of the subject’s body weight. In some embodiments, the CD117 signaling antibodies are administered in a dose of about 0.6 mg/kg. In particular embodiments, HCT is performed after the CD117 antibody (e.g., JSP-191) serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml. [00212] In certain embodiments, the conditioning regimen comprises administration of an anti- CD117 antibody in combination with one or more additional antibodies. In certain embodiments, the one or more additional antibodies comprise one or more of: anti-CD47, anti- CD40L, anti-CD122, anti-CD4, and/or anti-CD8 antibody. [00213] In certain embodiments, the conditioning regimen comprises administration of an anti- CD117 antibody, alone or in combination with a myeloablative (MA) conditioning, reduced intensity conditioning (RIC), or other non-MA (NMA) conditioning regimen. Examples of various conditioning regimens are provided in Fig.2. In certain embodiments, the conditioning regimen is a genotoxic conditioning regimen and/or may comprise one or more of: chemotherapy (optionally a nucleoside analog and/or an alkylating agent), monoclonal antibody therapy, and radiation, optionally radiation to the entire body. In particular embodiments, since the subject is being administered modified cells, e.g., HSCs, comprising a modified CD117 described herein and/or an anti-CD117 antibody, the conditioning regimen is milder than would be used if the subject was being administered cells, e.g., HSCs, that did not comprise the modified CD117 polypeptide. In particular embodiments, wherein the conditioning regimen comprises use of an anti-CD117 antibody in combination with chemotherapy (optionally a nucleoside analog and/or an alkylating agent), other monoclonal antibody therapy, and/or radiation, the amount of chemotherapy, other monoclonal antibody therapy, and/or radiation is reduced as compared to the amount used when not in combination with an anti-CD117 antibody, such as JSP191. For example, either or both the amount and/or duration of other conditioning therapy may be reduced by at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, or by about 100%. [00214] However, in other embodiments, the subject is not administered a myeloablative or genotoxic conditioning regimen prior to or concurrent with the administering of the pharmaceutical composition. For example, the recipient may be immunocompetent, and the transplantation may be performed in the absence of myeloablative conditioning, i.e., in the absence of radiation and/or chemotherapeutic drugs. The recipient may be conditioned with the combined administration a set of agents selected according to the cells and HLA match. Total Body Irradiation (TBI) [00215] The main purpose of TBI in HSC engraftment conditioning is to suppress the patient’s immune system prior to engraftment. In certain embodiments, the entire patient may be treated with a single radiation beam, with a distance of about 3-6 meters from the radiation source to reduce the dose rate. TBI in extant therapies is typically given in low doses, several times per day, over a period of three to five days. TBI causes significant apoptosis of rapidly dividing cells in radiosensitive organs such as the blood, bone marrow, and the GI tract immediately after radiation exposure. However, in some embodiments, TBI may be given as a single dose as part of a combination conditioning therapy in which an anti-CD117 antibody and a chemotherapy are also administered prior to HSC engraftment. [00216] In some embodiments, the subject is administered TBI of about 500 cGy to about 5Gy, optionally of about 1 to about 4 Gy or about 1 to about 3 Gy. In some embodiments, the total body irradiation (TBI) may include a single or fractionated irradiation dose within the range of about 50 cGy – 15 Gy, about 50 cGy – 10 Gy, about 50 cGy – 5 Gy, about 50 cGy – 1 Gy, about 50 cGy – 500 cGy, 0.5-1 Gy (500 cGy -1000 cGy), about 0.5-1.5 Gy, about 0.5-2.5 Gy, about 0.5-5 Gy, about 0.5-7.5 Gy, about 0.5-10 Gy, about 0.5-15 Gy, about 1-1.5 Gy, about 1- 2 Gy, about 1-2.5 Gy, about 1-3 Gy, about 1-3.5 Gy, about 1-4 Gy, about 1-4.5 Gy, about 1- 5.5 Gy, about 1-7.5 Gy, about 1-10 Gy, about 2-3 Gy, about 2-4 Gy, about 2-5 Gy, about 2-6 Gy, or about 2-7 Gy. In some embodiments, the TBI is administered in a single dose of about 2 Gy, optionally within 24 hours prior to the transplant. In some embodiments, the subject is administered twice daily about 2-Gy fractions given over 3 days (total dose about 12 Gy); twice-daily about 1.5-Gy fractions over 4-4.5 days (total dose about 12-13.5 Gy); three-times- daily about 1.2-Gy fractions over 4 days (total dose about 12-13.2 Gy); and once-daily about 3-Gy fractions for 4 days (total dose about 12 Gy). In certain embodiments, a subject is administered low dose TBI, i.e., less than or equal to 5 Gy, e.g., about 1-3 Gy or about 2-4 Gy given in one or two fractions. In particular embodiments, the subject is administered at total of less than about 5 Gy, less than about 4 Gy, less than about 3 Gy, or less than about 2 Gy of TBI, which may be administered in one or more fraction or dose. In particular embodiments, the subject is administered at total of less than about 5 Gy, less than or about 4 Gy, less than or about 3 Hy, less than or about 2 Gy, less than or about 1 Gy, less than about 500 cGy, less than about 250 cGy, less than about 100 cGy, or less than about 50 cGy of TBI, which may be administered in one or more fraction or dose. In particular embodiments, it is administered as a single dose on the day of HCT. [00217] In some embodiments, the TBI is administered 5, 4, 3, 2, or 1 days prior to the HCT. In other embodiments the TBI is administered the day of the HCT prior to engraftment. In particular embodiments, the TBI is administered once, e.g., on any of the indicated days. In some embodiments, the subject is administered TBI of about 1 to about 3 Gy, about 1-2 days prior to, or on the day of the transplant (day 0). Chemotherapy [00218] Chemotherapy may refer to any anti-cancer drug that targets rapidly dividing cells. Chemotherapy, i.e., anti-cancer or anti-neoplastic agents may include, but are not limited to, fludarabine, clorafabine, cytarabine, an anthracycline drug, such as daunorubicin (daunomycin) or idarubicin, cladribine (2-CdA), mitoxantrone, etoposide (VP-16), 6-thioguanine (6-TG), hydroxyurea, 6-mercaptopurine (6-MP), azacytidine, and/or decitabine. In certain embodiments, the chemotherapy is fludarabine. Chemotherapies may be administered to partially or completely ablate the patient’s bone marrow cells in preparation for donor HSC cell engraftment and/or as part of continuing treatment thereafter. [00219] In some embodiments, the subject is administered about 10-50 mg/m2/day of chemotherapy, optionally about 30 mg/m2/day, wherein optionally the chemotherapy is fludarabine and/or clofarabine. In some embodiments, the subject is administered about 10 to about 50 mg/m2/day of the chemotherapy, optionally 20 mg/m2/day, 25 mg/m2/day, or about 30 mg/m2/day for about one to about six days. In some embodiments, the subject is administered about 10-50 mg/m2/day of the chemotherapy, optionally about 30 mg/m2/day of the fludarabine and/or clofarabine about 10 to about 1 days prior to the HCT. [00220] In some embodiments, the chemotherapy is administered on days -10, -9, -8, -6, -7, -5 -4, -3, -2, and/or -1 days prior to the HCT. In certain embodiments, the chemotherapy is administered daily during any of these time periods. Combination therapies for hematopoietic stem cell (HSC) transplant conditioning [00221] In certain embodiments, the disclosure provides methods for conditioning a subject for HCT, the method comprising administering to the subject an CD117 antibody, total body irradiation (TBI), and a chemotherapeutic agent. In certain embodiments, the method comprises administering to the subject a JSP191 antibody or variant thereof, TBI, and fludarabine. In certain embodiments, the CD117 antibody, the total body irradiation (TBI), and the chemotherapeutic agent are administered at the same or different times, or two or more may be administered at the same time, and the other at a different time. In particular embodiments, the CD117 antibody, the total body irradiation (TBI), and the chemotherapeutic agent are administered to the subject or present within the subject during an overlapping time period prior to the subject receiving HCT. [00222] In some embodiments, the CD117 antibody is administered about 5 to about 20 days before the HCT. In some embodiments, the CD117 antibody is administered on one or more of days 10 through 14 before the HCT. In some embodiments, the CD117 antibody is administered on one or more of days 5, 6, or 7 through about 10 to about 14 days prior to the HCT. In certain embodiments, the CD117 antibody is administered daily during any of these time periods. The day of transplant may in some embodiments be determined by the CD117 antibody blood concentration of the patient: e.g., the day of transplant may be within about 4 to about10 days from the day the subject’s CD117 antibody blood concentration of about 2000 ng/ml or less. In some embodiments, the day of transplant is determined by the CD117 antibody (e.g., JSP- 191) serum or plasma concentration, wherein the serum or plasma concentration is less than 100 ng/ml in serum or plasma, less than 200 ng/ml in serum or plasma, less than 300 ng/ml in serum or plasma, less than 400 ng/ml in serum or plasma, less than 500 ng/ml in serum or plasma, less than 600 ng/ml in serum or plasma, less than 700 ng/ml in serum or plasma, or less than 800 ng/ml in serum or plasma on or before the day of transplant. In some embodiments, the CD117 antibody (e.g., JSP-191) serum or plasma concentration is less than 500 ng/ml on the day of transplant. In particular embodiments, the transplant (e.g., HCT) or graft is performed within 0-5 days or 0-3 days following the CD117 antibody (e.g., JSP-191) serum or concentration levels falling below 500 ng/ml. In particular embodiments, the transplant (e.g., HCT) or graft is performed within three days of the CD117 antibody (e.g., JSP- 191) serum or concentration levels falling below 100 ng/ml, below 200 ng/ml, below 300 ng/ml, below 400 ng/ml, below 500 ng/ml, below 600 ng/ml, below 700 ng/ml, or below 800 ng/ml, e.g., on the same day that the CD117 antibody (e.g., JSP-191) serum or concentration levels fall below any of these concentrations, within one day following, within two days following, or within three days following the CD117 antibody serum or concentration level falling below any of these concentrations. In particular embodiments, the CD117 antibody is JSP-191, and in particular embodiments, the CD117 antibody level falls below 500 ng/ml before transplant or graft is performed. [00223] In some embodiments, the TBI is administered 5, 4, 3, 2, or 1 days prior to the HCT. In other embodiments the TBI is administered the day of the HCT prior to engraftment. In particular embodiments, the TBI is administered once, e.g., on any of the indicated days. [00224] In some embodiments, the chemotherapy is administered on days -10, -9, -8, -6, -7, -5 -4, -3, -2, and/or -1 days prior to the HCT. In certain embodiments, the chemotherapy is administered daily during any of these time periods. [00225] In some embodiments, the CD117 antibody (e.g., JSP191 or a humanized CD117 antibody as described in US20200165337A1) is administered on one or more of days 14 through 10 prior to HCT, the chemotherapy (e.g., fludarabine) is administered on days 4 through 2 prior to HCT, and the TBI is administered on the day of the transplant, prior to engraftment. In certain embodiments, the antibody and/or chemotherapy is administered daily during any of these time periods. In certain embodiments, the TBI is administered only on a single day. In particular embodiments, the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml. [00226] In some embodiments, the subject is administered about 0.01 mg/kg to about 2 mg/kg of the CD117 antibody, e.g., JSP191, optionally the subject is administered about 0.1 mg/kg to about 1 mg/kg of the CD117 antibody, e.g., JSP191. In some embodiments, CD117 antibody may be administered to a subject in a dose about 0.01 mg/kg to about 2 mg/kg of the subject’s body weight, or about 0.1 mg/kg to about 1 mg/kg of the subject’s body weight. In some embodiments, the CD117 signaling antibodies are administered in a dose of about 0.6 mg/kg, optionally on one or more of days 14 through 10 prior to HCT. In particular embodiments, the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml. [00227] In some embodiments, the subject is administered TBI of about 500 cGy to about 5Gy, optionally of about 1 to about 4 Gy or about 1 to about 3 Gy. In some embodiments, the total body irradiation (TBI) may include a single or fractionated irradiation dose within the range of about 50 cGy – 15 Gy, about 50 cGy – 10 Gy, about 50 cGy – 5 Gy, about 50 cGy – 1 Gy, about 50 cGy – 500 cGy, 0.5-1 Gy (500 cGy -1000 cGy), about 0.5-1.5 Gy, about 0.5-2.5 Gy, about 0.5-5 Gy, about 0.5-7.5 Gy, about 0.5-10 Gy, about 0.5-15 Gy, about 1-1.5 Gy, about 1- 2 Gy, about 1-2.5 Gy, about 1-3 Gy, about 1-3.5 Gy, about 1-4 Gy, about 1-4.5 Gy, about 1- 5.5 Gy, about 1-7.5 Gy, about 1-10 Gy, about 2-3 Gy, about 2-4 Gy, about 2-5 Gy, about 2-6 Gy, or about 2-7 Gy. In some embodiments, the TBI is administered in a single dose of about 2 Gy, optionally within 24 hours prior to the transplant. In some embodiments, the subject is administered twice daily about 2-Gy fractions given over 3 days (total dose about 12 Gy); twice-daily about 1.5-Gy fractions over 4-4.5 days (total dose about 12-13.5 Gy); three-times- daily about 1.2-Gy fractions over 4 days (total dose about 12-13.2 Gy); and once-daily about 3-Gy fractions for 4 days (total dose about 12 Gy). In certain embodiments, a subject is administered low dose TBI, i.e., less than or equal to 5 Gy, e.g., about 1-3 Gy or about 2-4 Gy given in one or two fractions. In particular embodiments, the subject is administered at total of less than about 5 Gy, less than about 4 Gy, less than about 3 Gy, or less than about 2 Gy of TBI, which may be administered in one or more fraction or dose. In particular embodiments, the subject is administered at total of less than about 5 Gy, less than or about 4 Gy, less than or about 3 Hy, less than or about 2 Gy, less than or about 1 Gy, less than about 500 cGy, less than about 250 cGy, less than about 100 cGy, or less than about 50 cGy of TBI, which may be administered in one or more fraction or dose. In particular embodiments, it is administered as a single dose on the day of HCT. In particular embodiments, the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml. [00228] In some embodiments, the subject is administered about 10-50 mg/m2/day of chemotherapy, optionally about 30 mg/m2/day, wherein optionally the chemotherapy is fludarabine and/or clofarabine. In some embodiments, the subject is administered about 10 to about 50 mg/m2/day of the chemotherapy, optionally 20 mg/m2/day, 25 mg/m2/day, or about 30 mg/m2/day for about one to about six days. [00229] In some embodiments, the subject is administered about 0.1 to about 1.0 mg/kg of the CD117 antibody (e.g., JSP191 or a humanized CD117 antibody as described in US20200165337A1), about 0.5 to about 3 Gy of the TBI, and about 10-50 mg/m2/day of chemotherapy (e.g., fludarabine), before HCT. In particular embodiments, the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml. [00230] In some embodiments, the CD117 antibody (e.g., JSP191 or a humanized CD117 antibody as described in US20200165337A1) is administered on one or more of days 14 through 10 prior to HCT in a dose of about 0.6 mg/kg, the chemotherapy (e.g., fludarabine) is administered on days 4 through 2 prior to HCT in a dose of about 30 mg/m2/day and the TBI is administered on the day of the transplant, prior to engraftment in a dose of about 2 Gy. In particular embodiments, the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml. [00231] In some embodiments, the CD117 antibody is administered in a dose of about 0.1 mg/kg to about 1 mg/kg of the CD117 antibody about 5 to about 20 days before the HCT. In some embodiments, the subject is administered TBI of about 1 to about 3 Gy, about 1-2 days prior to, or on the day of the transplant (day 0). In some embodiments, the subject is administered about 10-50 mg/m2/day of the chemotherapy, optionally about 30 mg/m2/day of the fludarabine and/or clofarabine about 10 to about 1 days prior to the HCT. In particular embodiments, the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml. [00232] In some embodiments, the CD117 antibody (e.g., JSP191 or a humanized CD117 antibody as described in US20200165337A1) is administered on one or more of days 14 through 10 prior to HSC transplant in a dose of about 0.6 mg/kg, the chemotherapy (e.g., fludarabine) is administered on three (optionally consecutive) days, e.g., days 4 through 2, prior to HSC transplant in a dose of about 30 mg/m2/day, and the TBI is administered on the day of the transplant, prior to engraftment in a dose of about 2 Gy. In certain embodiments, the chemotherapy is administered daily during any of these time periods. In particular embodiments, the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml. [00233] In some embodiments, the CD117 antibody (e.g., JSP191 or a humanized CD117 antibody as described in US20200165337A1) is administered on one or more of days 14 through 10 prior to HSC transplant in a dose of about 0.6 mg/kg, the chemotherapy (e.g., fludarabine) is administered on three (optionally consecutive) days, e.g., on days 4 through 2, prior to HSC transplant in a dose of about 30 mg/m2/day, and the TBI is administered on the day of the transplant, prior to engraftment in a dose of about 3 Gy. In certain embodiments, the chemotherapy is administered daily during any of these time periods. In particular embodiments, the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml. [00234] In some embodiments, the CD117 antibody (e.g., JSP191 or a humanized CD117 antibody as described in US20200165337A1) is administered on one or more of days 14 through 10 prior to HSC transplant in a dose of about 0.6 mg/kg, the chemotherapy (e.g., fludarabine) is administered on five (optionally consecutive) days, e.g., days 6 through 2, prior to HSC transplant in a dose of about 30 mg/m2/day, and the TBI is administered on the day of the transplant, prior to engraftment in a dose of about 2 Gy. In certain embodiments, the chemotherapy is administered daily during any of these time periods. In particular embodiments, the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml. [00235] In some embodiments, the CD117 antibody (e.g., JSP191 or a humanized CD117 antibody as described in US20200165337A1) is administered one or more of days 14 through 10 prior to HSC transplant in a dose of about 0.6 mg/kg, the chemotherapy (e.g., fludarabine) is administered for five (optionally consecutive) days, e.g., on days 6 through 2, prior to HSC transplant in a dose of about 30 mg/m2/day, and the TBI is administered on the day of the transplant, prior to engraftment in a dose of about 3 Gy. In certain embodiments, the chemotherapy is administered daily during any of these time periods. In particular embodiments, the HCT is performed after the CD117 antibody serum or plasma level falls below 500 ng/ml, e.g., within 0-3 days following the CD117 antibody serum or plasma level falling below 500 ng/ml. [00236] The dose of stem cells, e.g., modified HSCs comprising a modified CD117 polypeptide and/or nucleic acid encoding a modified CD117 polypeptide, administered to a subject may depend on the purity of the infused cell composition, and the source of the cells. In particular embodiments, the dose administered is at least or about 1-2x106 CD34+ cells/kg body weight for autologous and allogeneic transplants. Higher doses can include, for example, at least or about 3x106, at least or about 4x106, at least or about 5x106, at least or about 6x106, at least or about 7x106, at least or about 8x106, at least or about 9x106, at least or about 107 or more CD34+ cells/kg body weight for autologous and allogeneic transplants. Frequently, the dose is limited by the number of available cells, and the methods disclosed encompass delivering less cells when necessary or limited. Typically, regardless of the source, the dose is calculated by the number of CD34+ cells present. The percent number of CD34+ cells can be low for unfractionated bone marrow or mobilized peripheral blood; in which case the total number of cells administered may be higher. [00237] In certain embodiments, a maximum number of CD3+ cells delivered with the modified HSC composition is not more than about 107 CD3+ cells/kg of recipient body weight, not more than about 106 CD3+ cells/kg of recipient body weight, not more than about 105 CD3+ cells/kg of recipient body weight, or not more than about 104 CD3+ cells/kg of recipient body weight. Alternatively, cell populations may be selected for expression of CD34 and CD90, which cell populations may be highly purified, e.g., at least about 85% CD34+ CD90+ cells, at least about 90% CD34+ CD90+ cells, at least about 95% CD34+ CD90+ cells and may be up to about 99% CD34+ CD90+ cells or more. [00238] In certain embodiments, the disclosure includes a method of treating a mammalian subject in need thereof, comprising administering to the subject modified cells, e.g., HSCs and/or HSPCs, comprising a modified CD117 polypeptide disclosed herein, e.g., a modified CD117 with constitutive CD117 signaling or kinase activity. In certain embodiments, the modified CD117 has constitutive autophosphorylation activity, e.g., without bound SCF. In particular embodiments, the modified CD117 polypeptide is transiently expressed in the cells, e.g., for about one day, about two days, about three days, about four days, about five days, or about a week. In particular embodiments, the subject is also administered a conditioning regimen to facilitate or increase engraftment of the modified cells following transplantation, wherein the conditioning regimen is administered prior to or concurrent with the administering of the pharmaceutical composition. In particular embodiments, the conditioning regimen comprises administration of an CD117 antibody, e.g., any disclosed herein (such as, e.g., JSP191), to the subject. In some embodiments, the CD117 antibody is administered to the subject prior to administration of the pharmaceutical composition to the subject. In particular embodiments, there is a “washout” period following administration of the CD117 antibody and before administration of the modified cells (i.e., the HCT). This period of time allows clearance of the CD117 antibody (or other agent used for conditioning). The period of time required for clearance of the ablative agent may be empirically determined, or may be based on prior experience of the pharmacokinetics of the agent. Historically, the time for clearance was usually the time sufficient for the level of ablative agent, e.g., CD117 antibody, to decrease at least about 10-fold from peak levels, usually at least about 100-fold, 1000-fold, 10,000-fold, or more. However, since the modified cells being administered to the subject according to the methods disclosed herein comprise a modified CD117 polypeptide that is not bound by the ablative CD117 antibody used for conditioning, the disclosed methods do not require a wash- out period, or they require only a reduced wash-out period as compared to when unmodified cells are transplanted. In certain embodiments, the wash-out period is less than five days, less than four days, less than 3 days, less than two days, or less than one day. In certain embodiments, the method comprises administering the CD117 antibody and the pharmaceutical composition or modified cells, e.g., modified HSCs and/or HSPCs, during an overlapping period of time or at about the same time. In particular embodiments, the method comprises administering the CD117 antibody to the subject after administration of the pharmaceutical composition or modified cells, e.g., modified HSCs and/or HSPCs, optionally for a period of time of at least one day, at least two days, at least three days, at least four days, at least five days, or at least one week. This may continue to ablate endogenous HSCs and/or HSPCs following administration of the modified HSCs and/or HSPCs, thus allowing greater engraftment. [00239] In one embodiment, the method comprises: (i) selectively ablating endogenous hematopoietic stem cells in the subject by administering to the subject an CD117 antibody, e.g., JSP-191; (ii) optionally, waiting for a period of time following administration of the CD117 antibody; and (iii) following (ii), administering to the subject the pharmaceutical composition comprising the modified cells, e.g., modified HSCs and/or modified HSPCs, in a dose effective to achieve multilineage peripheral blood chimerism. In particular embodiments, the period of time of step (ii) is less than five days, less than four days, less than 3 days, less than two days, or less than one day, or there is no period of time. In particular embodiments, the modified CD117 has constitutive CD117 signaling or kinase activity. In particular embodiments, the modified CD117 polypeptide is transiently expressed in the cells, e.g., for about one day, about two days, about three days, about four days, about five days, or about a week. In particular embodiments, the modified CD117 polypeptide has at least 90%, at least 95%, at least 98%, or at least 99% identity to a wild-type human CD117 polypeptide and comprises an amino acid substitution at a position referred to as N505 or D816, such as, e.g., N505I or D816V. [00240] In certain embodiments, the method of treating a subject in need of HCT comprises: (i) administering a conditioning regimen to the subject, wherein the conditioning regimen comprises an anti-CD117 monoclonal antibody, e.g., JSP191; and (ii) administering modified HSCs to the subject, wherein the modified HSCs comprise a modified CD117 polypeptide, wherein the modified CD117 polypeptide is expressed on the cell surface, and wherein the modified HSCs are not depleted by the conditioning regimen to the same extent as endogenous HSCs that comprise only wild type CD117 polypeptide and/or wherein the modified HSCs have a proliferative advantage as compared to the endogenous HSCs. In particular embodiments, the modified CD117 has constitutive CD117 signaling or kinase activity. In certain embodiments, the modified CD117 has constitutive autophosphorylation activity, e.g., without bound SCF. In particular embodiments, the modified CD117 polypeptide is transiently expressed in the cells, e.g., for about one day, about two days, about three days, about four days, about five days, or about a week. In particular embodiments, the conditioning regimen comprises a monoclonal CD117 antibody, e.g., JSP191. In particular embodiments, the modified CD117 polypeptide has at least 90%, at least 95%, at least 98%, or at least 99% identity to a wild-type human CD117 polypeptide and comprises an amino acid substitution at a position referred to as N505 or D816, such as, e.g., N505I or D816V. The actual location of the “N505” or “D816” modification may differ depending on the particular isotype of CD117 polypeptide. In particular embodiments, the modified CD117 polypeptide has the sequence shown in any one of SEQ ID NOs: 3-6. [00241] In some embodiments, the transplantation is performed in the absence of myeloablative conditioning. In some embodiments the recipient is immunocompetent. The administration of the pre-transplantation conditioning regimen is repeated as necessary to achieve the desired level of ablation. Following transplantation with donor stem cells, the recipient may be a chimera or mixed chimera for the donor cells. [00242] The modified cells and methods disclosed herein may be used to treat a variety of indications amenable to treatment with stem cell transplantation, including hematological diseases. In particular embodiments, the cells and methods disclosed herein may be used in the context of any hematopoietic cell transplant to treat any disease or disorder requiring such a transplant. Examples include, gene therapy, cord blood transplant, treatment of leukemias and cancers, and treatment of non-cancer diseases. In particular embodiments, the modified cells and methods may be used to treat a leukemia or a severe combined immunodeficiency (SCID). They may also be used to treat various bone marrow failure states and diseases, as well as hemoglobinopathies. For example, in certain embodiments, the methods comprise: (i) conditioning a subject by administration of an anti-CD117 antibody, e.g., JSP191, alone or in combination with one or more additional conditioning agent; and administering to the subject modified HSPCs/HSCs comprising a modified CD117 disclosed herein, e.g., CD117 E73A. In particular embodiments, the modified CD117 cells comprise one or more additional modifications. For example, they may comprise one or more introduced genes to replace a missing, mutated, or dysfunctional gene or protein product in a diseased cell. [00243] In particular embodiments, they are used to treat any of the following disorders: multiple myeloma, non-Hodgkin lymphoma, Hodgkin disease, acute myeloid leukemia, neuroblastoma, germ cell tumors, and autoimmune disorders, e.g., systemic lupus erythematosus (SLE), systemic sclerosis, or amyloidosis, for example, by autologous HCT. In particular embodiments, they are used to treat any of the following disorders: acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia; chronic lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndromes, multiple myeloma, non- Hodgkin lymphoma, Hodgkin disease, aplastic anemia, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, thalassemias, thalassemia major, sickle cell anemia, combined immunodeficiency, severe combined immunodeficiency (SCID), Wiskott- Aldrich syndrome, hemophagocytic lymphohistiocytosis (HLH), inborn errors of metabolism (e.g., mucopolysaccharidosis, Gaucher disease, metachromatic leukodystrophies, and adrenoleukodystrophies), epidermolysis bullosa, severe congenital neutropenia, Shwachman- Diamond syndrome, Diamond-Blackfan anemia, leukocyte adhesion deficiency, and the like, for example, by allogeneic HCT. [00244] In particular embodiments, the methods disclosed are used to treat a solid tissue cancer or a blood cancer, such as a leukemia, a lymphoma, or a myelodysplastic syndrome. In particular embodiments, the leukemia is acute myeloid leukemia (AML). [00245] In particular embodiments, the lymphoma is diffuse large B-cell lymphoma. [00246] In particular embodiments, the methods disclosed are used to treat an immunodeficiency. In particular embodiments, the immunodeficiency is severe combined immunodeficiency (SCID). In particular embodiments, the immunodeficiency is immunoglobulin G subclass deficiency, selective immunoglobulin A deficiency, DiGeorge syndrome, hyper-immunoglobulin M (HIGM) syndrome, selective IgM deficiency, Wiskott- Aldrich syndrome, or X-linked agammaglobulinemia (XLA). [00247] In particular embodiments, the methods disclosed are used to treat a genetic disorder. In particular embodiments, the genetic disorder is sickle cell disease or Fanconi anemia. Sickle cell diseases that may be treat include, but are not limited to: HbS disease; drepanocytic anemia; meniscocytosis, and chronic hemolytic anemia. [00248] In certain embodiments of any of the HCT methods disclosed, the method further comprises administering to the subject a therapeutic agent for treatment of the disease or disorder being treated by the HCT method. EXAMPLES Example 1 PROLIFERATION OF HEMATOPOIETIC CELLS EXPRESSING A CD117 VARIANT IS NOT INHIBITED BY AN ANTI-CD117 ANTIBODY [00249] To demonstrate that expression of a mutated CD117 confers a proliferative advantage for hematopoietic cells expressing mutant CD117 vs. wild-type CD117, Ba/F3 cells expressing wild-type human CD117 (CD117) and mutant human CD117-D816V were cultured in the absence of IL-3, in varying concentrations of stem cell factor (SCF), and in the presence or absence of anti-CD117 antibody JSP191. [00250] Control parental Ba/F3 cells did not proliferate in the absence of IL-3 (Figure 1 and Figure 3). Further, parental Ba/F3 cells did not express CD117 and were not responsive to SCF signaling. Therefore, control parental Ba/F3 cells did not proliferate in the presence of increasing concentrations of SCF, and there was no effect on viability or proliferation with the addition of JSP191. [00251] The Ba/F3 cell line expressing wild-type human CD117 (CD117, sequence of ct180, Table 1) showed dose-responsive proliferation to SCF, which was inhibited in the presence of anti-CD117 antibody JSP191 (Figure 1). [00252] The Ba/F3 cell line expressing the CD117-D816V (Table 1) mutant was able to proliferate in the absence of SCF and proliferation was not inhibited by the presence of the anti-CD117 antibody JSP191 (Figure 1). [00253] These studies demonstrate that cells comprising a constitutively active modified CD117 are capable of proliferating in the presence of anti-CD117 antibodies that inhibit SCF binding to CD117, and thus the modified CD117 confers a proliferative advantage to the modified cells as compared to wild type cells, particularly in the presence of the anti-CD117 antibody. Example 2 CD117 EXPRESSION [00254] Various CD117 mRNAs encoding wild type or various mutant CD117s were electroporated into human CD34+ cells, and CD117 expression was analyzed. As shown in FIG.3, the cells expressed CD117 highly within hours of electroporation and for about one day after electroporation. Wild type CD117 was expressed at higher levels than CD117 N505I (sequence in Table 1), but both showed greater expression than control or mock electroporated cells. Cell viability is shown in FIG.4. [00255] Illustrative CD117 mRNA constructs and vector sequences are provided in Table 1 below. For all constructs/sequences disclosed herein, in the context of mRNA, the Ts are Us, and in particular embodiments, the Ts are fully substituted with N1mPsU. Coding sequence is capitalized. Note that throughout the specification, construct sequences are generally specified T7 promoter → 3’UTR and do not include poly A tail sequences, except for A140S. mRNA constructs electroporated into cells may lack the T7 promoter sequence. The T7 promoter sequence comprises the sequence TAATACGACTCACTATAG. The T7 polymerase starts transcription at the underlined G in the promoter sequence. The polymerase then transcribes using the opposite strand as a template from 5’->3’. The first base in the transcript will be a G.
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
[00256] Examples of illustrative self-amplifying RNA constructs are shown in Table 2 below Illustrative plasmid and mRNA constructs/sequences are provided in the table below. For all constructs/sequences disclosed herein, in the context of RNA, the Ts are Us, and in particular embodiments, the Ts are fully substituted with N1mPsU. Coding sequence is capitalized. Note that throughout the specification, construct sequences are generally specified T7 promoter → 3’UTR and do not include poly A tail sequences, except for A140S. mRNA constructs electroporated into cells may lack the T7 promoter sequence. The T7 promoter sequence comprises the sequence TAATACGACTCACTATAG (SEQ ID NO: 72). The T7 polymerase starts transcription at the underlined G in the promoter sequence. The polymerase then transcribes using the opposite strand as a template from 5’->3’. The first base in the transcript will be a G.
Figure imgf000089_0002
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
[00257] The various embodiments described above can be combined to provide further embodiments. [00258] Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, patent applications, and publications to provide yet further embodiments. [00259] These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure [00260] All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety.

Claims

Claims 1. A modified hematopoietic stem cell (HSC) or hematopoietic stem and progenitor cell (HSPC), wherein the modified HSC or HSPC comprises a nucleic acid encoding a modified CD117 polypeptide, optionally wherein the modified CD117 polypeptide has constitutive CD117 signaling and/or kinase activity, optionally wherein the modified cell is capable of proliferation and/or survival when contacted with an CD117 monoclonal antibody capable of inhibiting proliferation and/or survival of an HSPC expressing only a wild-type CD117, and optionally wherein the nucleic acid comprises the following elements, optionally from 5’ to 3’: a 5’ HBA1 UTR; a CleanCap Reagent AG 3’ OMe 5’ terminal cap sequence; a sequence encoding the modified CD117 polypeptide; a TAATAA stop codon; and a 3’ HBB1 UTR.
2. The modified HSC or HSPC of claim 1, wherein the CD117 signaling and/or kinase activity of the modified CD117 is not substantially inhibited by an CD117 monoclonal antibody.
3. The modified HSC or HSPC of claim 2, wherein the CD117 monoclonal antibody inhibits binding of SCF to CD117.
4. The modified HSC or HSPC of claim 2, wherein the CD117 monoclonal antibody comprises one or more of the six CDRs present in any one of JSP191, AB85, CDX-0159, or FSI-174.
5. The modified HSC or HSPC of claim 2, wherein the CD117 monoclonal antibody is any one of JSP191, AB85, CDX-0159, or FSI-174.
6. The modified HSC or HSPC of claim 5, wherein the CD117 antibody is JSP191.
7. The modified HSC or HSPC of claim 5, wherein the CD117 antibody is FSI-174.
8. The modified HSC or HSPC of any one of claims 1-7, wherein the modified CD117 comprises one or more amino acid modifications as compared to the wild-type CD117 polypeptide.
9. The modified HSC or HSPC of claim 8, wherein the one or more amino acid modifications comprise one or more amino acid substitutions, insertions, or deletions.
10. The modified HSC or HSPC of claim 8, wherein one or more of the amino acid modifications are present within surface exposed amino acid residues of the extracellular domain, within the membrane spanning domain, or within an intracellular domain of the modified CD117 polypeptide.
11. The modified HSC or HSPC of claim 8, wherein the modified CD117 polypeptide comprises substitution or deletion of one or more of the following amino acids present in wild type human CD117: N505 or D816.
12. The modified HSC or HSPC of claim 11, wherein the modified CD117 polypeptide comprises a D816V substitution and/or a N505I substitution as compared to wild type human CD117.
13. The modified HSC or HSPC of any one of claims 1-12, wherein the modified CD117 polypeptide has at least 90%, at least 95%, at least 98%, or at least 99% sequence homology to a wild type CD117 polypeptide.
14. The modified HSC or HSPC of any one of claims 8-13, wherein the wild type CD117 polypeptide is a wild type human CD117 polypeptide, optionally having one of the following amino acid sequences: MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPV DVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKE QIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYID PTQLPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPS AHLTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRK RDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRS VRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNIL LTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYG IFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLK RPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLV HDDV (SEQ ID NO:1); or MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPV DVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKEQIHP HTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQ LPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAH LTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRD SFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRI GSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTH GRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFL WELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRP TFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHD DV (SEQ ID NO:2), or a variant or fragment thereof of either sequence, e.g., having at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.
15. The modified HSC or HSPC of any one of claims 1-14, wherein the modified cell expresses both the modified CD117 polypeptide and a wild type CD117 polypeptide.
16. The modified HSC or HSPC of any one of claims 1-15, wherein the modified CD117 polypeptide sequence is encoded by a polynucleotide sequence that comprises or consists of one of the following: taatacgactcactataAGgactcttctggtccccacagactcagagagaacccaccgccgccAccATGAGAGGCGCC CGGGGCGCATGGGACTTCTTATGTGTATTATTGCTCCTCCTGAGGGTTCAGACCG GGAGTAGCCAACCTTCCGTGTCCCCTGGTGAACCATCGCCACCATCGATCCACCC TGGCAAATCCGACCTGATCGTGAGGGTCGGCGATGAAATCCGGCTCCTATGCAC GGACCCTGGTTTTGTGAAATGGACCTTTGAGATCCTCGATGAAACCAATGAGAAT AAACAGAACGAGTGGATTACAGAAAAGGCTGAGGCCACAAATACAGGTAAATA CACATGCACCAATAAGCACGGCCTCTCGAACTCTATCTACGTTTTTGTGCGAGAT CCTGCAAAACTTTTCCTCGTCGATCGCTCCTTATACGGAAAGGAGGACAATGATA CACTGGTCAGATGCCCTTTAACAGACCCCGAGGTTACAAACTATTCACTCAAGGG TTGTCAGGGTAAACCTCTGCCAAAAGACCTGCGCTTTATCCCTGATCCTAAAGCC GGCATCATGATAAAATCTGTAAAGCGGGCCTACCACCGTCTCTGCTTGCACTGTA GCGTTGATCAAGAAGGCAAGTCAGTTTTAAGCGAGAAATTCATCCTGAAGGTGC GCCCAGCCTTCAAGGCTGTGCCTGTAGTGTCAGTGTCTAAGGCCTCATACCTGCT CCGGGAGGGGGAGGAATTTACTGTGACCTGCACGATAAAAGACGTTTCCTCTTCT GTGTACTCTACTTGGAAGAGGGAAAATAGCCAGACCAAGCTGCAGGAGAAGTAC AACAGCTGGCATCATGGTGACTTCAACTACGAGAGACAGGCCACTCTGACTATTT CATCTGCACGCGTGAATGACTCCGGTGTGTTTATGTGCTACGCAAATAACACCTT CGGCAGTGCCAATGTGACAACTACCCTGGAAGTCGTTGACAAGGGCTTCATTAAC ATCTTTCCAATGATCAATACAACCGTCTTTGTTAACGATGGCGAAAACGTGGACC TGATCGTTGAATATGAAGCATTCCCCAAACCAGAGCACCAGCAGTGGATCTACAT GAATCGCACTTTCACCGACAAGTGGGAAGACTACCCCAAATCCGAGAACGAGTC TAACATCCGCTATGTGTCGGAACTCCACCTGACTAGACTTAAGGGTACGGAAGG AGGCACCTACACCTTTTTGGTGAGCAATAGCGACGTGAACGCGGCAATTGCTTTT AACGTATACGTGAATACGAAACCCGAAATATTGACATATGACCGTCTGGTGAAC GGAATGCTTCAGTGTGTGGCCGCAGGCTTTCCTGAACCAACCATCGACTGGTACT TTTGCCCTGGTACCGAGCAGCGGTGCTCCGCGAGCGTGCTGCCTGTGGACGTCCA GACGCTAAATTCTAGTGGGCCACCTTTTGGAAAACTGGTGGTTCAGTCGTCAATT GATTCTTCTGCATTTAAGCATAATGGGACAGTGGAGTGTAAAGCTTACAACGATG TGGGGAAGACAAGCGCCTATTTCAtCTTTGCCTTTAAGGGGAACAATAAAGAGCA GATTCATCCACACACCTTGTTCACTCCTTTATTGATCGGGTTTGTGATCGTGGCGG GAATGATGTGTATTATCGTTATGATTTTGACTTATAAGTACCTGCAGAAGCCTAT GTATGAAGTGCAGTGGAAAGTGGTGGAAGAGATCAACGGGAACAATTACGTTTA TATCGACCCCACCCAGTTGCCATATGACCACAAATGGGAATTTCCCAGGAATCGC TTGAGCTTCGGGAAGACACTCGGTGCCGGAGCCTTCGGAAAGGTAGTAGAAGCA ACGGCTTACGGGCTAATCAAGTCAGATGCCGCTATGACTGTTGCGGTGAAAATGT TGAAGCCATCGGCTCATCTGACAGAGCGGGAGGCTCTGATGAGCGAACTCAAGG TTCTCAGTTACCTCGGCAATCACATGAACATTGTAAATCTCCTTGGGGCCTGTAC GATCGGCGGTCCCACCCTCGTCATAACAGAATACTGCTGCTATGGCGATCTGCTG AACTTCCTCCGGCGCAAGAGGGATTCCTTTATATGTAGCAAACAAGAAGACCAC GCGGAGGCCGCTCTATACAAAAACCTGTTACACAGTAAAGAGTCTTCATGCAGC GACAGTACGAATGAATACATGGACATGAAACCTGGGGTAAGTTATGTTGTGCCT ACTAAAGCCGACAAGCGGCGCAGCGTCAGGATCGGATCCTATATTGAGAGGGAC GTGACACCTGCTATTATGGAAGATGACGAGTTAGCATTGGACCTCGAGGACCTTC TATCCTTTTCATATCAGGTGGCCAAGGGCATGGCCTTCCTGGCGTCTAAAAACTG TATTCACCGCGATTTGGCCGCGAGAAACATCCTGCTCACACATGGAAGAATCACC AAAATTTGCGACTTTGGCCTGGCCAGAGATATCAAGAACGACTCAAACTATGTG GTGAAGGGAAATGCACGTCTGCCCGTGAAGTGGATGGCACCAGAGTCAATCTTT AATTGTGTGTATACGTTCGAAAGTGATGTCTGGTCATACGGAATCTTCCTGTGGG AGTTGTTCTCCCTGGGGTCTTCCCCCTACCCAGGGATGCCTGTGGACTCTAAATTC TACAAGATGATCAAGGAGGGCTTCCGCATGTTATCACCAGAGCACGCACCCGCT GAGATGTACGATATTATGAAAACTTGCTGGGACGCTGATCCCCTAAAGCGGCCA ACTTTCAAACAGATTGTTCAGCTTATTGAAAAGCAGATCAGTGAATCTACAAACC ATATCTATAGTAATCTGGCCAATTGCTCACCTAACCGACAGAAGCCCGTGGTTGA TCACTCCGTTAGGATCAACTCTGTGGGCAGCACTGCAAGCTCCAGTCAGCCCCTG CTTGTCCACGATGATGTCtaaTaAgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaact actaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgc; or taatacgactcactataAGgactcttctggtccccacagactcagagagaacccaccgccgccAccATGAGAGGCGCC CGGGGCGCATGGGACTTCTTATGTGTATTATTGCTCCTCCTGAGGGTTCAGACCG GGAGTAGCCAACCTTCCGTGTCCCCTGGTGAACCATCGCCACCATCGATCCACCC TGGCAAATCCGACCTGATCGTGAGGGTCGGCGATGAAATCCGGCTCCTATGCAC GGACCCTGGTTTTGTGAAATGGACCTTTGAGATCCTCGATGAAACCAATGAGAAT AAACAGAACGAGTGGATTACAGAAAAGGCTGAGGCCACAAATACAGGTAAATA CACATGCACCAATAAGCACGGCCTCTCGAACTCTATCTACGTTTTTGTGCGAGAT CCTGCAAAACTTTTCCTCGTCGATCGCTCCTTATACGGAAAGGAGGACAATGATA CACTGGTCAGATGCCCTTTAACAGACCCCGAGGTTACAAACTATTCACTCAAGGG TTGTCAGGGTAAACCTCTGCCAAAAGACCTGCGCTTTATCCCTGATCCTAAAGCC GGCATCATGATAAAATCTGTAAAGCGGGCCTACCACCGTCTCTGCTTGCACTGTA GCGTTGATCAAGAAGGCAAGTCAGTTTTAAGCGAGAAATTCATCCTGAAGGTGC GCCCAGCCTTCAAGGCTGTGCCTGTAGTGTCAGTGTCTAAGGCCTCATACCTGCT CCGGGAGGGGGAGGAATTTACTGTGACCTGCACGATAAAAGACGTTTCCTCTTCT GTGTACTCTACTTGGAAGAGGGAAAATAGCCAGACCAAGCTGCAGGAGAAGTAC AACAGCTGGCATCATGGTGACTTCAACTACGAGAGACAGGCCACTCTGACTATTT CATCTGCACGCGTGAATGACTCCGGTGTGTTTATGTGCTACGCAAATAACACCTT CGGCAGTGCCAATGTGACAACTACCCTGGAAGTCGTTGACAAGGGCTTCATTAAC ATCTTTCCAATGATCAATACAACCGTCTTTGTTAACGATGGCGAAAACGTGGACC TGATCGTTGAATATGAAGCATTCCCCAAACCAGAGCACCAGCAGTGGATCTACAT GAATCGCACTTTCACCGACAAGTGGGAAGACTACCCCAAATCCGAGAACGAGTC TAACATCCGCTATGTGTCGGAACTCCACCTGACTAGACTTAAGGGTACGGAAGG AGGCACCTACACCTTTTTGGTGAGCAATAGCGACGTGAACGCGGCAATTGCTTTT AACGTATACGTGAATACGAAACCCGAAATATTGACATATGACCGTCTGGTGAAC GGAATGCTTCAGTGTGTGGCCGCAGGCTTTCCTGAACCAACCATCGACTGGTACT TTTGCCCTGGTACCGAGCAGCGGTGCTCCGCGAGCGTGCTGCCTGTGGACGTCCA GACGCTAAATTCTAGTGGGCCACCTTTTGGAAAACTGGTGGTTCAGTCGTCAATT GATTCTTCTGCATTTAAGCATAATGGGACAGTGGAGTGTAAAGCTTACAACGATG TGGGGAAGACAAGCGCCTATTTCAACTTTGCCTTTAAGGGGAACAATAAAGAGC AGATTCATCCACACACCTTGTTCACTCCTTTATTGATCGGGTTTGTGATCGTGGCG GGAATGATGTGTATTATCGTTATGATTTTGACTTATAAGTACCTGCAGAAGCCTA TGTATGAAGTGCAGTGGAAAGTGGTGGAAGAGATCAACGGGAACAATTACGTTT ATATCGACCCCACCCAGTTGCCATATGACCACAAATGGGAATTTCCCAGGAATCG CTTGAGCTTCGGGAAGACACTCGGTGCCGGAGCCTTCGGAAAGGTAGTAGAAGC AACGGCTTACGGGCTAATCAAGTCAGATGCCGCTATGACTGTTGCGGTGAAAAT GTTGAAGCCATCGGCTCATCTGACAGAGCGGGAGGCTCTGATGAGCGAACTCAA GGTTCTCAGTTACCTCGGCAATCACATGAACATTGTAAATCTCCTTGGGGCCTGT ACGATCGGCGGTCCCACCCTCGTCATAACAGAATACTGCTGCTATGGCGATCTGC TGAACTTCCTCCGGCGCAAGAGGGATTCCTTTATATGTAGCAAACAAGAAGACC ACGCGGAGGCCGCTCTATACAAAAACCTGTTACACAGTAAAGAGTCTTCATGCA GCGACAGTACGAATGAATACATGGACATGAAACCTGGGGTAAGTTATGTTGTGC CTACTAAAGCCGACAAGCGGCGCAGCGTCAGGATCGGATCCTATATTGAGAGGG ACGTGACACCTGCTATTATGGAAGATGACGAGTTAGCATTGGACCTCGAGGACCT TCTATCCTTTTCATATCAGGTGGCCAAGGGCATGGCCTTCCTGGCGTCTAAAAAC TGTATTCACCGCGATTTGGCCGCGAGAAACATCCTGCTCACACATGGAAGAATCA CCAAAATTTGCGACTTTGGCCTGGCCAGAgtgATCAAGAACGACTCAAACTATGTG GTGAAGGGAAATGCACGTCTGCCCGTGAAGTGGATGGCACCAGAGTCAATCTTT AATTGTGTGTATACGTTCGAAAGTGATGTCTGGTCATACGGAATCTTCCTGTGGG AGTTGTTCTCCCTGGGGTCTTCCCCCTACCCAGGGATGCCTGTGGACTCTAAATTC TACAAGATGATCAAGGAGGGCTTCCGCATGTTATCACCAGAGCACGCACCCGCT GAGATGTACGATATTATGAAAACTTGCTGGGACGCTGATCCCCTAAAGCGGCCA ACTTTCAAACAGATTGTTCAGCTTATTGAAAAGCAGATCAGTGAATCTACAAACC ATATCTATAGTAATCTGGCCAATTGCTCACCTAACCGACAGAAGCCCGTGGTTGA TCACTCCGTTAGGATCAACTCTGTGGGCAGCACTGCAAGCTCCAGTCAGCCCCTG CTTGTCCACGATGATGTCtaaTaAgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaact actaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgc or a variant or fragment thereof of either sequence, e.g., having at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, or a corresponding mRNA sequence, optionally wherein the mRNA sequence comprises one or more of the following modifications: pseudouridine substitution of one or more uridine; N1-methyl-pseudouridine substitution of one or more uridine; 5 methoxyuridine substitution of one or more uridine; 5-methylcytidine substitution of one or more cytidine; a m7G(5')ppp(5')(2'OMeA)pG cap sequence; or a m7(3'OMeG)(5')ppp(5')(2'OMeA)pG cap sequence.
17. The modified HSC or HSPC of any one of claims 1-16, wherein the polypeptide or polynucleotide sequence is humanized.
18. The modified HSC or HSPC of any one of claims 1-17, wherein the cell is a human cell.
19. The modified HSC or HSPC of any one of claims 1-18, wherein the cell was obtained from a mammalian donor.
20. The pharmaceutical composition of claim 19, wherein the mammalian donor is a subject in need of a hematopoietic cell transplant (HCT) or a healthy donor.
21. The modified HSC or HSPC of any one of claims 19-20, wherein the cell obtained from the mammalian donor was modified ex vivo.
22. A pharmaceutical composition comprising the modified HSC or HSPC of any one of claims 1-21.
23. The pharmaceutical composition of claim 1-22, further comprising an anti-CD117 antibody.
24. The pharmaceutical composition of any one of claims 22-23, further comprising one or more anti-CD47, anti-CD40L, anti-CD122, anti-CD4, and/or anti-CD8 antibody.
25. A method of treating a mammalian subject in need thereof, comprising administering to the subject the modified HSC or HSPC of any one of claims 1-22 or the pharmaceutical composition of any one of claims 22-24.
26. The method of claim 25, wherein the subject is administered a conditioning regimen to facilitate or increase engraftment of the modified cells, wherein the conditioning regimen is administered prior to or concurrent with the administering of the pharmaceutical composition.
27. The method of claim 26, wherein the conditioning regimen is also administered subsequent to the administering of the pharmaceutical composition.
28. The method of claim 26 or claim 27, wherein the conditioning regimen is milder than would be used if the subject was being administered hematopoietic stem cells that did not comprise the modified CD117 polypeptide.
29. The method of claim 25, wherein the subject is not administered a conditioning regimen to facilitate or increase engraftment of the modified cells, prior to or concurrent with the administering of the pharmaceutical composition.
30. The method of any one of claims 25-29, wherein the method results in reduced toxicity, reduced morbidity, or reduced graft-versus-host disease, as compared to a method wherein a subject is administered hematopoietic stem cells that do not comprise the modified CD117 polypeptide in combination with a conditioning regimen.
31. The method of any one of claims 25-30, wherein the conditioning regimen comprises or consists of administration of an anti-CD117 monoclonal antibody, optionally JSP191.
32. The method of any one of claims 25-31, wherein the subject is treated for a disease or disorder selected from the group consisting of: a cancer, a cardiac disorder, a neural disorder, an autoimmune disease, an immunodeficiency, a metabolic disorder, and a genetic disorder.
33. The method of claim 32, wherein the cancer is a solid tissue cancer or a blood cancer.
34. The method of claim 33, wherein the blood cancer is a leukemia, a lymphoma, or a myelodysplastic syndrome.
35. The method of claim 34, wherein the leukemia is acute myeloid leukemia (AML).
36. The method of claim 32, wherein the immunodeficiency is severe combined immunodeficiency (SCID).
37. The method of claim 32, wherein the genetic disorder is sickle cell disease or Fanconi anemia.
38. The method of any one of claims 25-37, further comprising administering to the subject a therapeutic agent for treatment of the disease or disorder.
39. A modified CD117 polypeptide comprising one or more amino acid modifications as compared to a wild type CD117 polypeptide, wherein a CD117 monoclonal antibody does not substantially inhibit or reduce CD117 signaling by the modified CD117 polypeptide expressed in cells as compared to the wild type CD117 polypeptide.
40. The modified CD117 polypeptide of claim 39, wherein the modified CD117 polypeptide has constitutive CD117 signaling and/or kinase activity.
41. The modified CD117 polypeptide of claim 39, wherein the modified CD117 polypeptide substantially retains kinase activity as compared to the wild type CD117 polypeptide.
42. The modified CD117 polypeptide of any one of claims 39-41, wherein the modified CD117 polypeptide has substantially increased CD117 signaling and/or kinase activity, optionally in response to SCF binding, as compared to the wild type CD117 polypeptide.
43. The modified CD117 polypeptide of any one of claims 39-42, wherein the CD117 antibody comprises one or more of the six CDRs present in any one of JSP191, AB85, CDX-0159, or FSI-174.
44. The modified CD117 polypeptide of claim 43, wherein the CD117 antibody is any one of JSP191, AB85, CDX-0159, or FSI-174.
45. The modified CD117 polypeptide of claim 43, wherein the CD117 antibody comprises one or more, optionally six, CDRs present in JSP191 and/or FSI-174.
46. The modified CD117 polypeptide of claim 45, wherein the CD117 antibody is JSP191 or FSI-174.
47. The modified CD117 polypeptide of any one of claims 39-46, wherein the one or more amino acid modifications comprise one or more amino acid substitutions, insertions, or deletions.
48. The modified CD117 polypeptide of claim 47, wherein one or more of the amino acid modifications are present within surface exposed amino acid residues of the extracellular domain, within the membrane spanning domain, or within an intracellular domain of the wild type CD117 polypeptide.
49. The modified CD117 polypeptide of claim 47 or claim 48, wherein the one or more amino acid modifications comprise one or more amino acid substitutions or deletions.
50. The modified CD117 polypeptide of claim 49, wherein the one or more amino acid modifications comprise one or more amino acid substitutions.
51. The modified CD117 polypeptide of any one of claims 47-50, wherein the one of more amino acid substitutions or deletions comprises substitution or deletion of one or more of the following amino acids present in wild type human CD117: N505 or D816.
52. The modified CD117 polypeptide of claim 50 or claim 51, wherein the one of more amino acid substitutions comprises a D816V substitution and/or a N505I substitution.
53. The modified CD117 polypeptide of any one of claims 39-52, wherein the modified CD117 polypeptide has at least 90%, at least 95%, at least 98%, or at least 99% sequence homology to the wild type CD117 polypeptide.
54. The modified CD117 polypeptide of any one of claims 39-53, wherein the wild type CD117 polypeptide is a wild type human CD117 polypeptide, optionally having one of the following amino acid sequences: MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPV DVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKE QIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYID PTQLPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPS AHLTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRK RDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRS VRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNIL LTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYG IFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLK RPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLV HDDV (SEQ ID NO:1); or MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPV DVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKEQIHP HTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQ LPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAH LTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRD SFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRI GSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTH GRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFL WELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRP TFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHD DV (SEQ ID NO:2).
55. A nucleic acid encoding the modified CD117 polypeptide of any one of claims 39-54, or comprising a sequence of SEQ ID NOS: 64-71 or SEQ ID NOS: 73-76.
56. The nucleic acid of claim 55, wherein the nucleic acid comprises RNA, DNA, or a combination thereof.
57. The nucleic acid of claim 56, wherein the nucleic acid comprises a modified mRNA.
58. The nucleic acid of any one of claims 55-57, wherein the nucleic acid is associated with one or more lipids, optionally wherein the nucleic acid is present within a lipid nucleic acid particle, a lipid nanoparticle, or a liposome.
59. A vector comprising the nucleic acid of any one of claims 55-58.
60. The vector of claim 59, wherein the vector is an expression vector.
61. The vector of claim 59 or claim 60, wherein the vector is a viral vector, optionally an AAV vector or a lentiviral vector.
62. The vector of any one of claims 59-61, wherein the vector is capable of transducing hematopoietic stem cells.
63. A modified cell comprising the modified CD117 polypeptide of any one of claims 39-54 and/or the nucleic acid of any one of claims 55-58.
64. The modified cell of claim 63, wherein the cell expresses both the modified CD117 polypeptide and a wild type CD117 polypeptide.
65. The modified cell of claim 63 or claim 64 wherein the cell was transduced with the vector of any one of claims 59-62.
66. The modified cell of any one of claims 63-65, wherein the cell is a stem cell or a pluripotent cell.
67. The modified cell of claim 66, wherein the stem cell is a hematopoietic stem cell (HSC) or a hematopoietic stem and progenitor cell (HSPC).
68. The modified cell of any one of claims 63-67, wherein the cell is CD34+, optionally wherein the cell is CD34+/CD90+, CD34+/CD38-, or CD34+/CD38-/CD90+, or CD34+CD133+.
69. The modified cell of any one of claims 63-68, wherein the cell is a human cell.
70. The modified cell of any one of claims 63-69, wherein the cell was obtained from a mammalian donor.
71. The modified cell of claim 70, wherein the mammalian donor is a subject is in need of a hematopoietic cell transplant (HCT).
72. The modified cell of claim 70, wherein the mammalian donor is a healthy donor.
73. The modified cell of any one of claims 70-72, wherein the cell obtained from the mammalian donor was modified ex vivo.
74. The modified cell of any one of claims 63-73, wherein the cell expresses the modified CD117 polypeptide, optionally wherein the modified cell expresses the modified CD117 polypeptide transiently.
75. The modified cell of claim 74, wherein the modified CD117 polypeptide is expressed on the cell surface or in the cell membrane.
76. The modified cell of any one of claims 63-75, wherein the cell is capable of proliferating and/or surviving in the presence of an anti-CD117 antibody.
77. The modified cell of claim 76, wherein the anti-CD117 antibody is capable of inhibiting proliferation and/or survival of a cell expressing only the wild-type CD117.
78. The modified cell of claim 76 or claim 77, wherein the anti-CD117 antibody is selected from the group consisting of: JSP191, CDX-0159, AB85, and FSI-174.
79. A method of modifying a cell, comprising introducing the nucleic acid of any one of claims 55-58 or the vector of any one of claims 59-62 into the cell, optionally wherein the cell is transiently modified, and optionally wherein the method is for preparing modified cells for hematopoietic cell transplantation (HCT) into a mammalian subject.
80. The method of claim 79, wherein the nucleic acid or vector is introduced into the cell by transfection, transduction, infection, electroporation, or nanopore technology.
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WO2023069961A1 (en) * 2021-10-18 2023-04-27 Jasper Therapeutics, Inc. Modified stem cell compositions and methods for use

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022173861A1 (en) * 2021-02-09 2022-08-18 Jasper Therapeutics, Inc. Modified stem cell compositions and methods for use
WO2023069961A1 (en) * 2021-10-18 2023-04-27 Jasper Therapeutics, Inc. Modified stem cell compositions and methods for use

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