WO2023019270A1 - 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
WO2023019270A1
WO2023019270A1 PCT/US2022/074942 US2022074942W WO2023019270A1 WO 2023019270 A1 WO2023019270 A1 WO 2023019270A1 US 2022074942 W US2022074942 W US 2022074942W WO 2023019270 A1 WO2023019270 A1 WO 2023019270A1
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cell
cxcr4
modified
hspcs
subject
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PCT/US2022/074942
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French (fr)
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Wendy PANG
Rajiv TIWARI
Hye-Sook Kwon
Judith SHIZURU
Robert Sikorski
Leopold Daniel D’ESPAUX
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Jasper Therapeutics, Inc.
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Publication of WO2023019270A1 publication Critical patent/WO2023019270A1/en

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    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • 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
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/145—Thrombopoietin [TPO]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
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    • 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.
  • transplant today 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 sickle cell disease 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.
  • the present disclosure addresses this need.
  • BRIEF SUMMARY OF THE INVENTION The present disclosure provides inter alia novel modified HSCs and HSPCs and related compositions and methods of use thereof in hematopoietic stem cell transplant.
  • the disclosure provides a modified or engineered cell comprising a nucleic acid encoding a CXCR4 polypeptide (including variants thereof), optionally wherein the cell is transduced with a vector disclosed herein or a modified mRNA encoding the CXCR4 polypeptide, e.g., the modified cell comprises an exogenous or introduced polynucleotide sequence encoding the CXCR4 polypeptide.
  • the cell is a stem cell, e.g., a HSC or HSPC.
  • 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.
  • the cell was obtained from a mammalian 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 CXCR4 polypeptide, optionally wherein the modified cell expresses the CXCR4 polypeptide transiently.
  • the CXCR4 polypeptides is a human CXCR4 polypeptide, or a variant or fragment thereof.
  • the CXCR4 polypeptide is a modified CXCR4 polypeptide, e.g., with increased or constitutive activity as compared to the corresponding wild type CXCR4 polypeptide.
  • the CXCR4 polypeptide comprises an amino acid substitution at amino acid 119, e.g., a 119S substitution.
  • the CXCR4 polypeptide comprises a WHIM mutation or a C-terminal deletion, e.g., a deletion of about 5-25 amino acid residues, about 10-25 amino acid residues, or about 15-20 amino acid residues, e.g., the t19 deletion corresponding to deletion of the C-terminal 19 amino acid residues of CXCR4.
  • the exogenous or introduced polynucleotide is an mRNA
  • the mRNA is modified, e.g., to comprise 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 modified cell comprises one or more additional modification.
  • the modified cell may further comprise an introduced polynucleotide sequence that expresses a therapeutic protein, such as, for example, a wild type or functional form of a protein that is not expressed or has reduced activity in an HCT recipient, possibly due to a gene mutation in the HCT recipient.
  • a therapeutic protein such as, for example, a wild type or functional form of a protein that is not expressed or has reduced activity in an HCT recipient, possibly due to a gene mutation in the HCT recipient.
  • the modified cell comprises a polynucleotide sequence that has been gene edited, e.g., by Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) together with a CRISPR-associated protein (Cas), transcription activator-like effector nuclease (TALEN), or zinc finger nuclease gene editing technology.
  • CRISPR Clustered Regulatory Interspaced Short Palindromic Repeats
  • Cas CRISPR-associated protein
  • TALEN transcription activator-like effector nu
  • the disclosure provides a pharmaceutical composition comprising the modified cells, e.g., HSCs and/or HSPCs, comprising the nucleic acid encoding the CXCR4 polypeptide, and a pharmaceutically acceptable excipient, carrier, or diluent.
  • the pharmaceutical composition comprises a preparation of human allogeneic transiently modified hematopoietic stem and progenitor cells (HSPCs) comprising an introduced nucleic acid sequence, such as, e.g., chemically modified mRNA, encoding a modified version of CXCR4 into CD34+ HSPCs selected from mobilized peripheral blood.
  • HSPCs human allogeneic transiently modified hematopoietic stem and progenitor cells
  • the disclosure includes a method of modifying a cell, e.g., an HSC or HSPC, comprising introducing a nucleic acid or vector encoding a CXCR4 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 nucleic acid e.g., mRNA
  • LNPs lipid nanoparticles
  • the nucleic acid e.g., mRNA
  • the nucleic acid is an mRNA, such as, e.g., chemically modified mRNA, encoding a modified version of CXCR4.
  • 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 CXCR4 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 or HSPCs, 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 chemotherapy (optionally a nucleoside analog and/or an alkylating agent), monoclonal antibody therapy, or radiation, optionally radiation to the entire body (total body irradiation or TBI).
  • chemotherapy optionally a nucleoside analog and/or an alkylating agent
  • monoclonal antibody therapy optionally radiation to the entire body (total body irradiation or TBI).
  • radiation optionally radiation to the entire body (total body irradiation or TBI).
  • TBI total body irradiation or TBI.
  • CXCR4 transient expression in HSCs and/or HSPCs improves the ability of HSCs/HSPCs that are injected intravenously to home to the bone marrow.
  • CXCR4 transient expression in HSCs and/or HSPCs improves the ability of HSCs/HSPCs to engraft in the bone marrow, measured by higher donor myeloid chimerism in the bone marrow and in the peripheral blood.
  • CXCR4 transient expression in HSCs and/or HSPCs improves neutrophil and platelet recovery following transplantation.
  • an improvement correlates to an increase in measured value of the improved property or characteristic of at least 10%, at least 20%, at least 50%, at least 100%, at least two- fold, at least three-fold, or at least five-fold.
  • 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).
  • SCID severe combined immunodeficiency
  • the genetic disorder is sickle cell disease or Fanconi anemia.
  • the methods further comprise administering to the subject another therapeutic agent for treatment of the disease or disorder.
  • transiently modified CD34+ HSPCs are administered by a single intravenous infusion following a reduced intensity conditioning regimen.
  • C Mean fluorescence intensity (MFI) measured by flow cytometry of CXCR4 protein expression on the surface of CD34+ HSPCs that were not electroporated (Control), mock electroporated, and CXCR4 mRNA electroporated over time.
  • the mRNA used was CXCR4 wild type with 5moU modification.
  • D Viability as measured by flow cytometry staining of live cells of CD34+ HSPCs that were not electroporated (Control), mock electroporated, and CXCR4 mRNA electroporated over time post-electroporation.
  • the mRNA used was CXCR4 wild type with 5moU modification.
  • HCT Hematopoietic stem cell transplantation
  • HSCs healthy hematopoietic stem cells
  • HSPCs hematopoietic stem and progenitor cells
  • 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.
  • compositions and methods disclosed herein reduce the risk of graft failure for patients and increase the number of healthy donor or gene corrected HSPCs that stick and stay in the bone marrow. By promoting more cells migrating towards and engrafting in the marrow, they also reduce the need for intensive conditioning regimens.
  • compositions and methods disclosed herein may be used to treat all disorders for which blood stem cell (e.g., HSC and/or HSPC) transplantation is indicated.
  • the compositions and methods disclosed herein are used to introduce CXCR4 mRNA into allogeneic normal HSPCs to improve engraftment and replace a patient’s diseased stem cells with a healthy hematopoietic system.
  • the disclosure provides for compositions and methods for the ex vivo introduction of a polynucleotide encoding a CXCR4 polypeptide, e.g., a human CXCR4 polypeptide), 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.
  • Transplantation of these modified or engineered (e.g., genetically engineered) HSPCs may be done after or in combination with a conditioning regimen, including treatment with antibodies (such as anti-CD117 antibodies).
  • modified or engineered HSPCs may be transplanted alone or in combination with other cells.
  • this invention is not limited to the particular methodology, products, apparatus and factors described, as such methods, apparatus and formulations may, of course, vary.
  • the terminology used herein is for the purpose of describing particular embodiments only, and it is not intended to limit the scope of the present invention which will be limited only by appended claims. [0025] 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.
  • 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).
  • 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
  • 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.
  • identity refers to the percentage of exact matching residues in an alignment of a sequence provided herein to a reference sequence, such as an alignment generated by a BLAST algorithm or other alignment algorithms known in the art. Identity may be calculated based on an alignment of a full-length sequence provided herein and a full-length reference sequence. Identity may also be calculated based on a partial alignment of a sequence provided herein and a reference sequence, if the reference sequence is longer than a sequence provided herein. Identity may also be calculated based on a partial alignment of a sequence provided herein and a reference sequence, if the reference sequence is shorter than a sequence provided herein.
  • a query sequence “shares at least x % identity to” a subject sequence if in the alignment of the two sequences, at least x % (rounded down) of the residues in the subject sequence are aligned as an exact match to a corresponding residue in the query sequence, wherein the numerator is the number of exact matches and the denominator is the length of the query sequence.
  • the denominator may alternatively be the length of the query sequence minus any gaps of two or more non-matching residues.
  • BLAST software is available over the worldwide web at ncbi.nlm.nih.gov/BLAST/.
  • FASTA is available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc.
  • 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. Biol.
  • GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol.48: 443-453 (1970). 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. This program is available also from Genetics Computing Group (GCG) package, from Madison, Wis., USA.
  • GCG Genetics Computing Group
  • 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. The combination of control elements, e.g.
  • promoters 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, for example a native 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, e.g., 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.
  • 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.
  • Treatment covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) 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.).
  • CXCR4 Polypeptides and Polynucleotides [0044] C-X-C chemokine receptor type 4 (CXCR4), also known as fusin or CD184 (cluster of differentiation 184), is a protein that in humans is encoded by the CXCR4 gene.
  • the CXCR4 protein is a CXC chemokine receptor.
  • CXCR4 is expressed by most cells, including hematopoietic and endothelial cells (ECs), neurons and stem cells (embryonic and adult).
  • the CXCR4 polypeptide including functional fragments and variants, binds to the chemokine stromal cell derived factor-1 (SDF-1, also known as CXCL12), which is a constitutively expressed and inducible chemokine that regulates multiple physiological processes, including embryonic development and organ homeostasis.
  • SDF-1 is expressed in several organs including lung, liver, skin and bone marrow.
  • CXCR4 functional fragments and variants bind to SDF-1 with at least 50%, at least 75%, at least 90% specificity and affinity as a corresponding wild type CXCR4 protein.
  • Any CXCR4 protein, including functional fragments or variants thereof, may be used according to aspects of the disclosure.
  • the CXCR4 polypeptide is a human CXCR4 polypeptide, while in other embodiments, it is another mammalian CXCR4 polypeptide. Sequences of human and mammalian CXCR4 polypeptides are known in the art.
  • the CXCR4 polypeptide sequence comprises or consists of one of the following amino acid sequences: MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVI LVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVI YTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFA NVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQK RKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCC LNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS (SEQ ID NO: 1); or MSIPLPLLQIYTSDNYTEEM
  • the CXCR4 polypeptide sequence, or fragment or variant thereof is encoded by a polynucleotide sequence that comprises or consists of one of the following: ATGTCCATTC CTTTGCCTCT TTTGCAGATA TACACTTCAG ATAACTACAC CGAGGAAATG GGCTCAGGGG ACTATGACTC CATGAAGGAA CCCTGTTTCC GTGAAGAAAA TGCTAATTTC AATAAAATCT TCCTGCCCAC CATCTACTCC ATCATCTTCT TAACTGGCAT TGTGGGCAAT GGATTGGTCA TCCTGGTCAT GGGTTACCAG AAGAAACTGA GAAGCATGAC GGACAAGTAC AGGCTGCACC TGTCAGTGGC CGACCTCCTC TTTGTCATCA CGCTTCCCTT CTGGGCAGTT GATGCCGTGG CAAACTGGTA CTTTGGGAAC TTCCTATGCA AGGCAGTCCA TGTCATCTAC ACAGTCAACC TCTACAGCAG TG
  • the CXCR4 polypeptide comprises one or more modifications, e.g., modifications that can improve HSPC functional activity.
  • modifications e.g., modifications that can improve HSPC functional activity.
  • CXCR4 with a point mutation at amino acid position 119 e.g., 119S
  • Zhang, W.-b., et al., A point mutation that confers constitutive activity to CXCR4 reveals that T140 is an inverse agonist and that AMD3100 and ALX40-4C are weak partial agonists. Journal of Biological Chemistry, 2002. 277(27): p.24515-24521].
  • CXCR4 hyperactive mutants associated with WHIM a disorder causing Warts, Hypogammaglobulinemia, Infections, and Myelokathexis, which have been shown to improve short-term BM engraftment [ Gao, J.L., et al., Cxcr4-haploinsufficient bone marrow transplantation corrects leukopenia in an unconditioned WHIM syndrome model. J Clin Invest, 2018. 128(8): p. 3312-3318], and the disclosure includes the use of any CXCR4 mutations disclosed therein.
  • the WHIM CXCR4 mutant is WHIMt19, which corresponds to CXCR4 with a truncation of the C-terminal 19 amino acids.
  • the CXCR4 polypeptide of any of the embodiments disclosed herein comprises one or more modifications, e.g., amino acid insertions, substitutions, or deletions.
  • the CXCR4 has increased or constitutive activity as compared to wild-type CXCR4.
  • the CXCR4 polypeptide is a modified or variant CXCR4 polypeptide comprising a point mutation at the amino acid residue corresponding to position 119, e.g., a 119S point mutation, or a WHIM mutation, e.g., a C-terminal deletion, such as, e.g., a truncation or deletion of about the C-terminal 19 amino acids.
  • the remainder of the CXCR4 polypeptide is unmodified as compared to wild-type CXCR4 or retains at least 90%, at least 95%, at least 98%, or at least 99% identity to the wild-type CXCR4, e.g., human CXCR4.
  • the polypeptide or polynucleotide sequence is humanized.
  • the polynucleotide or nucleic acid sequence 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
  • mRNA chemical compositions including nucleoside modifications and/or 5’ cap modifications have been shown to improve mRNA stability, translation efficiency, and prevent cellular responses against certain RNA moieties.
  • the nucleic acid comprises a modified mRNA.
  • 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 CXCR4 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).
  • the modified mRNA comprises one or more nucleoside modification.
  • the mRNAs are fully modified at all occurrences of one or more nucleoside, e.g., A, G, U or C.
  • 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 (PsU or ⁇ ) or N1-methyl- pseudouridine (N1mPsU or m1 ⁇ ), and/or cytosine can be replaced by 5-methylcytosine.
  • the at least one modified nucleoside includes modification of U to 5- methoxyuridine (5moU).
  • the at least one modified nucleoside includes N1-methyl-pseudouridine and/or 5-methylcytidine and/or 5-methoxyuridine.
  • all uridines in the modified mRNA are replaced with a similar nucleoside such as pseudouridine ( ⁇ ) or N1-methyl-pseudouridine (m1 ⁇ ) or 5-methoxyuridine, and/or all cytosines in the modified mRNA are substituted with a similar nucleoside such as 5- methylcytosine.
  • the modified mRNA comprises a 5’ terminal cap sequence followed by a sequence encoding the CXCR4 polypeptide, followed by a 3’ tailing sequence, such as a polyA or a polyA-G sequence.
  • the mRNA encoding CXCR4 (including modified forms or variants thereof) comprises a wild type 5’ terminal cap sequence, and in certain embodiments, the mRNA encoding CXCR4 (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
  • the mRNA encoding CXCR4 comprises the modified 5’ terminal cap, 3 ⁇ -O-Me-m7G(5')ppp(5')G (Anti Reverse Cap Analog (ARCA); APExBIO, USA).
  • the disclosure provides: an unmodified mRNA encoding a wild-type CDCR4 polypeptide; a modified mRNA encoding a wild type CXCR4 polypeptide; a modified mRNA encoding a wild type CXCR4 with 5moU modification(s); a modified mRNA encoding a wild type CXCR4 with N1mPsU/5mC modification(s); a modified mRNA encoding a wild type CXCR4 with PsU modification(s); a modified mRNA encoding a t19 mutant with N1mPsU modification(s); and a modified mRNA encoding a wild type CXCR4 with N1mPsU modification(s).
  • the mRNA modification(s) occur at every such residue in the modified mRNA.
  • the modified mRNA comprises: CleanCapAG and 5moU modification(s); CleanCapAG-3’OMe and N1mPsU modification(s) (NEB HiScribe); CleanCapAG and N1mPsU modification(s) (NEB HiScribe); or CleanCapAG-3’OMe and N1mPsU (Ambion Mega).
  • 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 CXCR4 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 other 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 CXCR4 polypeptide.
  • the expression vector comprises a promoter sequence that facilitates expression of the encoded CXCR4 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 CXCR4 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 HSPC when viral vector and the HSC and/or HSPCs are incubated together for at least about 24 hours in a culture medium.
  • modified Hematopoietic Stem Cells and Pharmaceutical Compositions [0057]
  • the disclosure provides modified cells, e.g., HSCs and/or HSPCs, comprising a nucleic acid encoding a CXCR4 polypeptide as described herein.
  • the nucleic acid encoding the CXCR4 polypeptide is transiently present in the modified cell, and/or is not present within the genome of the cell.
  • the modified cell expresses and/or comprises the CXCR4 polypeptide, and in particular embodiments, the CXCR4 polypeptide is present on the cell surface.
  • the modified cell is transduced with or infected with an expression vector, optionally a viral vector.
  • the modified cell is transduced with a modified mRNA.
  • the modifications disclosed herein may be made to any type of cell, e.g., any mammalian cell.
  • 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.
  • 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 modified cell is a stem cell and/or progenitor cell, and in certain embodiments, the stem cell is an HSC or an HSPC.
  • the cell is a mammalian cell that has the ability both to self-renew, and to generate differentiated progeny, e.g., an HSC or an HSPC.
  • the stem cell and/or progenitor cell is a human cell.
  • the stem cell and/or progenitor 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
  • 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 CXCR4 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.
  • the cells may be fresh, frozen, or have been subject to prior culture. They may be fetal, neonate, adult, etc. HSCs and/or 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 HSCs and/or HSPCs are separated from other cells of the hematopoietic or other lineage is not critical to this invention.
  • a substantially homogeneous population of HSCs and/or HSPCs may be obtained by selective isolation of cells free of markers associated with differentiated cells, while displaying epitopic characteristics associated with the stem cells.
  • 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.
  • G-CSF granulocyte colony-stimulating factor
  • Nepogen filgrastim
  • G-CSF cytokines
  • GM-CSF GM-CSF
  • 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 and/or HSPC markers CD34 is well known for its unique expression on HSCs and/or HSPCs.
  • the modified cell is a CD34+ cell.
  • the modified cell is a subset of HSC and/or HSPC 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.
  • 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.
  • 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 HSCs and/or HSPCs, comprising introducing the nucleic acid encoding a CXCR4 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. In certain embodiments, the nucleic acid remains present in the modified cell only transiently, or the nucleic acid only transiently expresses the CXCR4 polypeptide in the cell. In certain embodiments, the method is used to prepare modified cells, e.g., HSCs and/or HSPCs, 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.
  • 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
  • LNPs lipid nucleic acid particles
  • cells may be modified by introducing a nucleic acid encoding a CXCR4 polypeptide into the HSCs and/or HSPCs according to a variety of methods available in the art.
  • a composition comprising HSCs and/or HSPCs, is administered to a patient. Such methods are well known in the art.
  • the HSCs and/or HSPCs are optionally, although not necessarily, purified.
  • compositions comprising one or more CXCR4 polypeptides, one or more polynucleotides or vectors comprising a sequence encoding a CXCR4 polypeptide (e.g., a modified mRNA), or a modified cell, e.g., HSC and/or HSPC, comprising a polynucleotide or vector encoding a CXCR4 polypeptide and/or expressing a CXCR4 polypeptide, in combination with one or more pharmaceutically acceptable diluent, carrier, or excipient.
  • a CXCR4 polypeptide e.g., a modified mRNA
  • a modified cell e.g., HSC and/or HSPC
  • the present invention discloses a pharmaceutical composition
  • a pharmaceutical composition comprising a modified cell comprising a CXCR4 polypeptide (or nucleic acid sequence encoding the CXCR4 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.
  • 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 and/or HSPCs, comprising a CXCR4 polypeptide described herein and/or a nucleic acid encoding the CXCR4 polypeptide.
  • the subject is in need of HCT.
  • 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 and/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 HLA type of the donor and recipient may be tested for a match, or 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.
  • 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.
  • Preferably matching is both serologic and molecular.
  • 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.
  • GVHD graft versus host disease
  • the HCT methods disclosed use modified HSCs and/or HSPCs comprising an exogenous or introduced CXCR4 polypeptide or nucleic acid encoding the CXCR4 polypeptide.
  • CXCR4 expression in HSCs and/or HSPCs improves HSCs/HSPCs ability to migrate towards SDF-1 in vitro and in vivo.
  • CXCR4 expression in HSCs and/or HSPCs improves HSCs/HSPCs that are injected intravenously to home to the bone marrow.
  • CXCR4 transient expression in HSCs and/or HSPCs improves HSCs/HSPCs engraftment in the bone marrow, measured by higher donor myeloid chimerism in the bone marrow and in the peripheral blood.
  • CXCR4 transient expression in HSCs and/or HSPCs improves neutrophil and platelet recovery following transplantation.
  • 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 encoding a CXCR4 polypeptide into the HSC and/or HSPCs, optionally wherein the CXCR4 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, e.g., to correct a gene mutation in the subject.
  • 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 CXCR4 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 CXCR4 polypeptide is an mRNA, and it is introduced into the cells by electroporation.
  • the disclosure provides a method for HCT comprising: providing to a subject in need thereof a population of cells comprising a plurality of modified or engineered HSCs and/or HSPCs, wherein the modified HSCs and/or HSPCs: i) were obtained from a donor, optionally a mammal, e.g., a human; ii) comprise an introduced polynucleotide sequence encoding a CXCR4 polypeptide into the HSC and/or HSPCs, optionally wherein the CXCR4 polypeptide comprises a sequence disclosed herein, or a functional variant or fragment thereof, and wherein the modified HSCs and/or HSPCs express the encoded CXCR4 polypeptide; and iii) optionally, were further modified by comprising a gene therapy vector, or by gene editing, e.g., to correct a gene mutation in the subject.
  • the introduced polynucleotide sequence is an mRNA that is expressed in the HSCs and/or HSPCs following introduction into the cells.
  • the HCT is autologous or allogeneic.
  • the subject being treated is the same or different from the donor.
  • the gene therapy vector or reagents used to perform the gene editing were introduced into HSCs and/or HSPCs obtained from the subject being treated by HCT.
  • the polynucleotide sequence encoding the CXCR4 polypeptide and the gene therapy vector or reagents used for gene editing were introduced into the cells at the same time, or either was 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 CXCR4 polypeptide is an mRNA, and it was introduced into the cells by electroporation.
  • the disclosure provides a method for HCT, comprising: i) obtaining HSCs and/or HSPCs from a donor, optionally a mammal, e.g., a human; ii) introducing a polynucleotide sequence encoding a CXCR4 polypeptide into the HSCs and/or HSPCs, optionally wherein the CXCR4 polypeptide comprises a sequence disclosed herein, or a functional variant or fragment thereof, to produce modified HSCs and/or HCPCs; iii) optionally, modifying the HSCs and/or HSPCs, e.g., by introducing a gene therapy vector, or by gene 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.
  • a donor optionally a mammal, e.g.,
  • the introduced polynucleotide sequence is an mRNA that is expressed in the HSCs and/or HSPCs following introduction into the cells.
  • the HCT is autologous or allogeneic.
  • the subject being treated is the same or different from the donor.
  • 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 CXCR4 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 CXCR4 polypeptide is an mRNA, and it is introduced into the cells by electroporation.
  • the method results in one or more of the following clinical outcomes: Engraftment: >95% (three consecutive days with absolute neutrophil count ⁇ 500/ ⁇ L by 28 days post-transplantation); Use of non-myeloablative conditioning; Oral mucositis: ⁇ 10% at 30 days; Veno-occlusive disease (VOD): ⁇ 2% at 100 days; Grade 3-4 acute graft-versus-host disease: ⁇ 5% at 100 days; or Chronic graft-versus-host disease: ⁇ 5% at 2 years.
  • VOD Veno-occlusive disease
  • the subject is administered a conditioning regimen to facilitate or increase engraftment of the modified cells, e.g., prior to and/or concurrent with the modified HSCs and/or HSPCs being provided or administered to the subject.
  • the conditioning regimen depletes endogenous normal or diseased HSCs and/or HSPCs 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 modified HSCs and/or HSPCs or pharmaceutical composition disclosed herein.
  • 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 6, 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 6.
  • 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.
  • 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 is milder than would be used if the subject was being administered cells, e.g., HSPCs or HSCs, that did not comprise the modified CXCR4 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.
  • 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 agents used according to the conditioning regimen are administered to the subject prior to and/or concurrently with the administration of the modified HSPCs/HSCs or pharmaceutical composition to the subject.
  • the period of time required for clearance of the conditioning agent may be empirically determined, or may be based on prior experience of the pharmacokinetics of the agent.
  • the time for clearance was usually the time sufficient for the level of conditioning agent, e.g., anti-c-Kit antibody, to decrease at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold from peak levels, e.g., at least about 100-fold, 1000-fold, 10,000-fold, or more.
  • the wash-out period is between 2 days and three weeks or between 5 days and two weeks, e.g., about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days about 17 days, about 18 days, about 19 days, or about 20 days.
  • the conditioning regimen comprises providing to the subject an anti-CD117 antibody, e.g., an anti-CD117 monoclonal antibody that inhibits stem cell factor from binding to CD117 on the cell surface, such as e.g., JSP191.
  • the conditioning regimen comprises providing to the subject total body irradiation (TBI).
  • the conditioning regimen comprises providing to the subject a chemotherapeutic agent, such as, e.g., fludarabine or azacytidine.
  • the conditioning regimen comprises a combination of TBI and a chemotherapeutic agent.
  • the conditioning regimen comprises a combination of an anti-CD117 monoclonal antibody, e.g., JSP191 and TBI and fludarabine, or a combination of an anti-CD117 monoclonal antibody, e.g., JSP191 and azacytidine.
  • Anti-c-Kit Antibodies [0087] In certain embodiments, the conditioning regimen comprises administration of an anti- CD117 antibody, wherein the anti-CD117 antibody depletes or reduces endogenous HSPCs.
  • 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-74.
  • the antibody is JSP191.
  • the antibody is the humanized form of SR1, JSP191, described in U.S. Pat. Nos.8,436,150 and 7,915,391.
  • Compositions and methods disclosed herein may be applicable to any anti-CD117 antibody, particularly monoclonal anti-human CD117 antibodies, e.g., those that block or inhibit binding of SCF to CD117.
  • An anti-CD117 antibody may refer to an antibody that binds to CD117, e.g., human CD117, or an antigen-binding fragment thereof.
  • CD117 e.g., human CD117
  • a number of antibodies contemplated by the disclosure that specifically bind human CD117 are known in the art and commercially available, including without limitation, JSP-191, 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 anti-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 anti-CD117 antibody binds to the extracellular region of CD117 i i id 26524 Th f thi i i h b l
  • Illustrative anti-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.
  • the anti-CD117 antibody is selected from the group consisting of: JSP191, CDX-0159 (from Celldex Therapeutics, Hampton, NJ), MGTA-117 (AB85) (from Magenta Therapeutics, Cambridge, MA), CK6 (from Magenta Therapeutics, Cambridge, MA), AB249 (from Magenta Therapeutics, Cambridge, MA), and FSI-174 (from Gilead, South San Francisco, CA).
  • JSP191, CDX-0159 from Celldex Therapeutics, Hampton, NJ
  • MGTA-117 AB85
  • Magenta Therapeutics Cambridge, MA
  • CK6 from Magenta Therapeutics, Cambridge, MA
  • AB249 from Magenta Therapeutics, Cambridge, MA
  • FSI-174 from Gilead, South San Francisco, CA.
  • the antibodies from Magenta Therapeutics are disclosed in US Patent Application Publication No.20190153114.
  • the antibody is one disclosed in any of US Pat. Nos. 7,915,391, US 8,436,150, or US 8,791,249. In certain embodiments, the antibody is one disclosed in US Pat. Application Publ. No 20200165337 or any of PCT Publication Nos. WO 2020/112687, WO2020/219748, WO 2020/219770, or WO 2019/084064. [0092] In particular embodiments, the antibody is a humanized form of SR1, a murine anti- CD117 antibody described in U.S. Pat. Nos. 5,919,911 and 5,489,516. The humanized form, JSP191, is disclosed in U.S. Patent Nos. 7,915,391, 8,436,150, and 8,791,249.
  • JSP191 is an aglycosylated IgG1 humanized antibody. 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. JSP191 blocks SCF from binding to CD117 and disrupts stem cell factor (SCF) signaling, leading to the depletion of hematopoietic stem cells.
  • SCF stem cell factor
  • JSP191 is a heterotetramer consisting of 2 heavy chains of the IgG1 subclass and 2 light chains of the kappa subclass, which are covalently linked through disulfide bonds.
  • variable heavy domain of JSP191 comprises the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSGNG DTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQGTLVT VSS (SEQ ID NO: 8) [0095]
  • the variable light chain domain of JSP191 comprises the following sequence: DIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMHWYQQKPGQPPKLLIYLASNLES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDP YTFGGGTKVEIK (SEQ ID NO: 8)
  • 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 anti-CD117 antibodies are described in U.S. Patent No. 10,781,267, and in particular embodiments, an anti-CD117 disclosed herein comprises the CDRs of any of the antibodies disclosed therein.
  • the anti-CD117 antibody comprises: (i) a light chain variable region ("VL") comprising the amino acid sequence: DIVMTQSPSX K1 LSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKX K2 LIYSASYRYS GVPDRFX K3 GSGSGTDFTLTISSLQX K4 EDFAX K5 YX K6 CQQYNSYPRTFGGGTKVEIK (SEQ ID NO: 16), 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, 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
  • antigen-binding fragments thereof comprising: (i) VH CDRs of a VH domain comprising the amino acid sequence GTTLTVSA (SEQ ID NO: 19), and (ii) VL CDRs of a VL domain comprising the amino acid sequence DIVMTQSQKFMSTSVGDRVSVTCKASQNVRTNVAWYQQKPGQSPKALIYSASYRYSGV PDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYNSYPRTFGGGTKLEIKR (SEQ ID NO: 20).
  • 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 Publication No.20200407440 and/or PCT Application Publication 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: 21), and RCPLTDPEVTNYSLKGCQGKP (amino acids 100-130; SEQ ID NO: 22).
  • EKAEATNTGKYTCTNKHGLSNSIYVFVRDPA amino acids 60-90; SEQ ID NO: 21
  • RCPLTDPEVTNYSLKGCQGKP amino acids 100-130; SEQ ID NO: 22.
  • the heavy chain variable region (VH) amino acid sequence of Ab85 is: EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMAIINPRDSDT RYRPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYEGYEGAFDIWGQG TLVTVSS (SEQ ID NO: 23).
  • the VH CDR amino acid sequences of AB85 are as follows: NYWIG (SEQ ID NO: 24; VH CDR1); IINPRDSDTRYRPSFQG (SEQ ID NO: 25; VH CDR2); and HGRGYEGYEGAFDI (SEQ ID NO: 26; VH CDR3).
  • the light chain variable region (VL) amino acid sequence of AB85 is: DIQMTQSPSSLSASVGDRVTITCRSSQGIRSDLGWYQQKPGKAPKLLIYDASNLETGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQANGFPLTFGGGTKVEIK (SEQ ID NO: 27).
  • the VL CDR amino acid sequences of AB85 are as follows: RSSQGIRSDLG (SEQ ID NO: 28; VL CDR1); DASNLET (SEQ ID NO: 29; VL CDR2); and QQANGFPLT (SEQ ID NO: 30; VL CDR3).
  • FSI-174 is an anti-CD117 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 anti-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: 39).
  • 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: 40); CDR-H2 with IIYPGDSDTRYSPSFQG (SEQ ID NO: 41); CDR-H3 with HGRGYNGYEGAFDI (SEQ ID NO: 42).
  • CK6 has the following light chain CDR amino acid sequences: CDR-L1 with RASQGISSALA (SEQ ID NO: 43); CDR-L2 with DASSLES (SEQ ID NO: 44); and CDR-L3 with CQQFNSYPLT (SEQ ID NO: 45).
  • 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:
  • Example variable heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISEN GSDTYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGGAVSYFDV WGQGTLVTVSS (SEQ ID NO: 46)
  • Example variable light chain DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT (SEQ ID NO: 47)
  • Ab249 was derived from antibody CK6, as an antagonist anti-CD117 antibody, as disclosed in PCT Application No.
  • Ab249 has improved binding characteristics over the parent CK6.
  • Ab249 has the following heavy chain CDRS: TSWIG (SEQ ID NO: 48; VH CDR1) IIYPGDSDTRYSPSFQG (SEQ ID NO: 41; VH CDR2); and HGLGYNGYEGAFDI (SEQ ID NO: 49; VH CDR3).
  • Ab249 has the following light chain CDRS: RASQGIGSALA (SEQ ID NO: 50; VL CDR1); DASNLET (SEQ ID NO: 29; VL CDR2); and QQLNGYPLT (SEQ ID NO: 51; VL CDR3).
  • Ab249 has the following variable heavy chain sequence (CDRS are underlined): EVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGWVRQMPGKGLEWMGIIYPGDSDT RYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYEGAFDIWGQG TLVTVSS (SEQ ID NO: 52).
  • Ab249 has the following variable light chain sequence (CDRS are underlined): DIQMTQSPSSLSASVGDRVTITCRASQGIGSALAWYQQKPGKAPKLLIYDASNLETGVPS RFSGSGTDFTLTISSLQPEDFATYYCQQLNGYPLTFGQGTRLEIK (SEQ ID NO: 53) [00108]
  • 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 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 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.
  • JSP191 is an aglycosylated IgG1 humanized antibody.
  • JSP191 (formerly AMG191) 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.
  • SCF stem cell factor
  • JSP191 blocks SCF from binding to CD117 and disrupts critical survival signals, leading to the depletion of hematopoietic stem cells.
  • the conditioning regimen comprises an anti-CD117 antibody alone.
  • the subject is administered the anti-CD117 antibody prior to administration of the modified HSCs and/or HSPCs, e.g., as a single dose.
  • the subject is administered about 0.01 mg/kg to about 10 mg/kg of the anti-c-kit antibody, e.g., JSP191, about 0. 1 mg/kg to about 10 mg/kg of the anti-c-kit antibody, e.g., JSP191, about 1.0 mg/kg to about 10 mg/kg of the anti-c-kit antibody, e.g., JSP191.
  • the subject is administered about 0.01 mg/kg to about 2 mg/kg of the anti- c-kit antibody, e.g., JSP191, optionally the subject is administered about 0.1 mg/kg to about 1 mg/kg of the anti-c-Kit antibody, e.g., JSP191.
  • anti-c-Kit 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 anti- c-Kit signaling antibodies are administered in a dose of about 0.6 mg/kg, optionally on days 14 through 10 prior to HCT.
  • the anti-c-Kit antibody is administered in a dose of about 0.1 mg/kg to about 1 mg/kg of the anti-c-Kit antibody about 5 to about 20 days before the HCT.
  • the anti-c-Kit antibody e.g., JSP191 is administered about 5 to about 20 days before the HCT (administration of the modified stem cells).
  • the anti-c-Kit antibody is administered on days 10 through 14 before the HCT.
  • the anti-c-Kit antibody is administered on days 5, 6, or 7 through about 10 to about 14 days prior to the HCT.
  • the anti-c-Kit antibody is administered daily during any of these time periods.
  • the day of transplant may in some embodiments be determined by the anti-c-Kit 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 anti-c-Kit antibody blood concentration of about 2000 ng/ml or less.
  • 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.
  • 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 [00118] 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/m 2 /day of chemotherapy, optionally about 30 mg/m 2 /day, wherein optionally the chemotherapy is fludarabine and/or clofarabine.
  • the subject is administered about 10 to about 50 mg/m 2 /day of the chemotherapy, optionally 20 mg/m2/day, 25 mg/m 2 /day, or about 30 mg/m 2 /day for about one to about six days.
  • the subject is administered about 10-50 mg/m 2 /day of the chemotherapy, optionally about 30 mg/m 2 /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 anti-c-Kit 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 anti-c-Kit 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 anti-c-Kit 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 anti-c-Kit antibody is administered about 5 to about 20 days before the HCT. In some embodiments, the anti-c-Kit antibody is administered on days 10 through 14 before the HCT. In some embodiments, the anti-c-Kit antibody is administered on days 5, 6, or 7 through about 10 to about 14 days prior to the HCT. In certain embodiments, the anti-c-Kit antibody is administered daily during any of these time periods.
  • the day of transplant may in some embodiments be determined by the anti-c-Kit 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 anti-c-Kit antibody blood concentration of about 2000 ng/ml or less.
  • the TBI is administered 5, 4, 3, 2, or 1 days prior to the HCT.
  • the TBI is administered the day of the HCT prior to engraftment.
  • 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 anti-c-Kit antibody e.g., JSP191 or a humanized c-kit 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 subject is administered about 0.01 mg/kg to about 2 mg/kg of the anti-c-kit antibody, e.g., JSP191, optionally the subject is administered about 0.1 mg/kg to about 1 mg/kg of the anti-c-Kit antibody, e.g., JSP191.
  • anti-c-Kit 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 anti-c-Kit signaling antibodies are administered in a dose of about 0.6 mg/kg, optionally on days 14 through 10 prior to HCT.
  • 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 subject is administered about 10-50 mg/m 2 /day of chemotherapy, optionally about 30 mg/m 2 /day, wherein optionally the chemotherapy is fludarabine and/or clofarabine. In some embodiments, the subject is administered about 10 to about 50 mg/m 2 /day of the chemotherapy, optionally 20 mg/m2/day, 25 mg/m 2 /day, or about 30 mg/m 2 /day for about one to about six days.
  • the subject is administered about 0.1 to about 1.0 mg/kg of the anti-c-Kit antibody (e.g., JSP191 or a humanized c-kit antibody as described in US20200165337A1), about 0.5 to about 3 Gy of the TBI, and about 10-50 mg/m 2 /day of chemotherapy (e.g., fludarabine), before HCT.
  • the anti-c-Kit antibody e.g., JSP191 or a humanized c-kit antibody as described in US20200165337A1
  • chemotherapy e.g., fludarabine
  • the anti-c-Kit antibody e.g., JSP191 or a humanized c-kit 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 anti-c-Kit antibody is administered in a dose of about 0.1 mg/kg to about 1 mg/kg of the anti-c-Kit 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/m 2 /day of the chemotherapy, optionally about 30 mg/m 2 /day of the fludarabine and/or clofarabine about 10 to about 1 days prior to the HCT.
  • the anti-c-Kit antibody e.g., JSP191 or a humanized c-kit 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 anti-c-Kit antibody e.g., JSP191 or a humanized c-kit 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 3 Gy.
  • the chemotherapy is administered daily during any of these time periods.
  • the anti-c-Kit antibody e.g., JSP191 or a humanized c-kit 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 anti-c-Kit antibody e.g., JSP191 or a humanized c-kit 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 3 Gy.
  • the chemotherapy is administered daily during any of these time periods.
  • the dose of stem cells e.g., modified HSCs and/or HSPCs comprising an exogenous CXCR4 polypeptide and/or nucleic acid encoding a CXCR4 polypeptide, administered to a subject may depend on the purity of the infused cell composition, and the source of the cells.
  • 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 HSPC 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 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 and/or HSPCs to the subject, wherein the modified HSCs and/or HSPCs comprise an exogenous or introduced CXCR4 polypeptide and/or an exogenous or introduced nucleic acid sequence encoding the CXCR4 polypeptide.
  • the nucleic acid sequence is operably linked to a promoter sequence.
  • the anti-c-Kit antibody and/or chemotherapy may be delivered orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.
  • the anti-c-Kit antibody e.g., JSP191
  • the chemotherapy e.g., fludarabine
  • the TBI is administered in a single dose of radiation.
  • the methods disclosed herein may be used to treat a variety of indications amenable to stem cell transplantation.
  • HCT methods disclosed herein are 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, hemoglobinopathies, and a genetic disorder.
  • 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.
  • 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, hemoglobinopathies, and a genetic disorder.
  • they are used to treat any of the following disorders: multiple myeloma,
  • 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 bull
  • 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.
  • a blood cancer such as a leukemia, a lymphoma, or a myelodysplastic syndrome.
  • the disease is a blood cancer, optionally a leukemia, a lymphoma, or a myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • the methods disclosed are used to treat acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), acute lymphoblastic leukemia (ALL), hodgkin lymphoma, non-hodgkin lymphoma, clonal hematopoiesis of indeterminate potential (CHIP), clonal cytopenia of undetermined significance (CCUS) myelodysplastic syndromes (MDS), idiopathic cytopenia of undetermined significance (ICUS), or myeloproliferative neoplasms (MPN).
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • CMML chronic myelomonocytic leukemia
  • ALL acute lymphoblastic leukemia
  • CHIP clonal hematopoiesis of indeterminate potential
  • CCUS clonal cytopenia of undetermined significance
  • MDS my
  • the disease or disorder is multiple myeloma, chronic myelogenous leukemia (CML) myelodysplastic syndromes (MDS), a myeloproliferative neoplasm, or a myeloid leukemia, e.g., acute myeloid leukemia (AML) or chronic myeloid leukemia (CML).
  • CML chronic myelogenous leukemia
  • MDS myelodysplastic syndromes
  • a myeloproliferative neoplasm or a myeloid leukemia, e.g., acute myeloid leukemia (AML) or chronic myeloid leukemia (CML).
  • the disease is MDS or AML.
  • the cancer is a lymphoid leukemia, e.g., acute lymphocytic leukemia (ALL) or chronic lymphocytic leukemia (CLL).
  • ALL acute lymphocytic leukemia
  • CLL chronic lymphocytic leuk
  • the cancer is a myelodysplastic/myeloproliferative neoplasm (MDS/MPN), such as, e.g., chronic myelomonocytic leukemia (CMML).
  • MDS/MPN myelodysplastic/myeloproliferative neoplasm
  • CMML myelodysplastic/myeloproliferative neoplasm
  • CMML is considered to be one of the myelodysplastic/myeloproliferative neoplasms (MDS/MPN), a type of chronic blood cancer in which a person's bone marrow does not make blood effectively.
  • MDS/MPN myelodysplastic/myeloproliferative neoplasms
  • the subject has a hematopoietic cell transplant comorbidity index (HCT-CI) greater than or equal to 3 (Sorror ML, et al. Hematopoietic cell transplantation (HCT)- specific comorbidity index: a new tool for risk assessment before allogeneic HCT. Blood. 2005;106(8):2912-2919.).
  • the subject has a hematopoietic cell transplant comorbidity index (HCT-CI) less than or equal to 3.
  • HCT-CI hematopoietic cell transplant comorbidity index
  • the disease or disorder is multiple myeloma, severe combined immune deficiency (SCID), chronic myelogenous leukemia (CML), myelodysplastic syndromes (MDS), a myeloproliferative neoplasm, or acute myeloid leukemia (AML).
  • SCID severe combined immune deficiency
  • CML chronic myelogenous leukemia
  • MDS myelodysplastic syndromes
  • AML acute myeloid leukemia
  • the disease treated according to the disclosure is referred to as MDS/AML, which includes both MDS and AML.
  • MDS Myelodysplastic syndromes
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndromes
  • AML acute myeloid leukemia
  • the disease is characterized by an overproduction of immature blood cells.
  • the resulting lack of mature, healthy blood cells causes anemia and an increased risk for infection and bleeding.
  • Around 5-10% of patients with solid tumors who are treated with chemotherapy, radiation or autologous stem cell transplantation develop treatment-related MDS or AML.
  • MDS Myelodysplastic syndromes
  • hematopoietic neoplasms characterized by abnormal differentiation and cytomorphology (i.e., dysplasia) of pluripotent hematopoietic progenitor cells (i.e., stem cells) residing in the myeloid compartment of the bone marrow (BM).
  • cytomorphology i.e., dysplasia
  • pluripotent hematopoietic progenitor cells i.e., stem cells
  • BM myeloid compartment of the bone marrow
  • Methods disclosed herein may be used to treat various forms of MDS, including but not limited to those shown in the table below, which is reproduced from Chung, US Pharm.2021;46(9):39-44. In certain embodiments, the methods result in decreased cytopenia.
  • the methods disclosed are used to treat an immunodeficiency.
  • the immunodeficiency is severe combined immunodeficiency (SCID).
  • the methods disclosed are used to treat a genetic disorder.
  • the genetic disorder is sickle ceH 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 an additional therapeutic agent for treatment of the disease or disorder being treated by the HCT method.
  • Improvement in the engraftment efficiency of HSPCs would also improve outcomes globally in HCT settings where the number of HSPCs are limiting, including ex vivo gene therapy of autologous HSPCs.
  • a platform for transient mRNA-based modification of human CD34+ HSPCs was developed by testing multiple electroporation systems, including ones from Lonza, ThermoFisher, Maxcyte, and Miltenyi.
  • DNA templates were synthesized by commercial vendors (Twist, IDT) to contain desired sequences (e.g., genes, mutants, non-coding elements such as untranslated regions) downstream of a T7 promoter and used to transcribe mRNA. Chemical modifications were included in IVT reactions, e.g., replacing uracil by 5-methoxyuridine or N1-methyl-pseudouridine, or adding 5’ cap analogs such as CleanCapAG (TriLink). DNA templates were removed by DNase digestion. PolyA tails were either encoded in the DNA templates or added enzymatically.
  • mRNAs were purified using silica membrane columns (Qiagen) and analyzed by capillary electrophoresis (Agilent Fragment Analyzer) for size composition, and by UV absorbance (Nanodrop) for purity. All mRNAs were of the expected size and highly purified (A260/280 and As260/230 > 1.8; band purity > 80%) (Figure 1). Yields varied by condition (chemical modification) but were generally >100 ug for ⁇ 40 ul reactions—sufficient for characterization and testing for functional activity (in vitro cell-based assays and in vivo mouse transplantation models).
  • Human CD34+ HSPCs were successfully isolated from mobilized peripheral blood obtained from healthy subjects. Purchased apheresis products of donors administered with G-CSF were used to mobilize CD34+ HSPCs to the bloodstream (Miltenyi and AllCells). Enrichment was carried out using magnetic anti-CD34 beads on a Miltenyi Prodigy system. Flow cytometry demonstrated 95% CD34+ cell purity (Figure 2), 97% viability, and 75% recovery after enrichment (data not shown). These cells were aliquoted and cryopreserved for subsequent studies.
  • CXCR4 mRNA were electroporated into human CD34+ HSPCs using a Miltenyi Elmo platform to characterize mRNAs and process conditions for functional activity.
  • frozen human CD34+ HSPCs were thawed and cultured in X-VIVO media (Lonza) supplemented with cytokines—stem cell factor (SCF), FLT3-ligand (FLT3-L) and thrombopoietin (TPO)—for 24 hours, then electroporated with candidate mRNAs.
  • cytokines stem cell factor
  • FLT3-L FLT3-ligand
  • TPO thrombopoietin
  • mice was sacrificed one day later and the human cell chimerism in the bone marrow (BM) was measured.
  • BM homing varied dramatically by CXCR4 mRNA chemical modification ( Figure 5A).
  • Hyperactive CXCR4 mutant sequences were examined, including the common WHIM mutant t19 (truncation of the C-terminal 19 amino acids) and the CXCR4 point mutant (119S), which in a transient mRNA context may lead to improved activity or allow the use of a lower dose of introduced CXCR4 mRNA.
  • Engraftment present at end of the 26-week period demonstrates the engraftment durability of CXCR4 mRNA-electroporated HSPCs in transplanted NSG mice.
  • CD34+ HSPCs are obtained from at least 3 healthy human donors, with approximately 3-6 recipient mice per human donor.
  • CXCR4 mRNA electroporated human CD34+ HSPCs is compared to mock electroporated CD34+ HSPCs (no mRNA) and non-electroporated CD34+ HSPCs.
  • Human chimerism is measured as percent human CD45 reconstitution in the bone marrow of engrafted NSG mice at 26 weeks following transplantation.
  • Multilineage reconstitution in bone marrow of engrafted NSG mice at 26 weeks following transplantation is measured as percent human CD3+ T cells, percent human CD19+ B cells, and percent human CD13+ and/or CD33+ granulocyte/monocytes). It is expected that CD34+ HSPCs electroporated with CXCR4 mRNA will demonstrate at least 20% improvement in their homing and engraftment efficiency in vivo in long-term engraftment studies.
  • CXCR4 mRNAs are assessed to confer desirable HSPC functional activity for their ability to ameliorate disease in the Townes SCD mouse model.
  • Townes mice were previously used as a preclinical model of SCD to demonstrate successful engraftment of an allogeneic HSPC graft (Bankova, A.K., Pang, W.W., Velasco,B.J., Pyser, J., Long-Boyle, J.R., Shizuru,J,A,.
  • Anti-CD117 Antibody Synergizes with 5-Azacytidine to Augment Engraftment of Hematopoietic Stem Cells in Mice with Sickle Cell Disease in Transplantation & Cellular Therapy Meetings.2021. Online).
  • CXCR4 mRNA By transiently introducing CXCR4 mRNA into the HSPCs, we aim to achieve faster and more complete engraftment. Because CXCR4 is highly conserved between mouse and human and human CXCR4 responds to mouse SDF-1, it is believed that lead human CXCR4 will have activity when electroporated into mouse HSPCs.
  • CD117+ HSPCs are evaluated for short-term engraftment in the bone marrow of Townes mice between 1-4 days after transplantation. Since mouse HSPCs do not express CD34, CD117 is used as the phenotypic marker to isolate HSPCs by fluorescence activated cell sorting (FACS). CXCR4 mRNA electroporated, mock electroporated, and non-electroporated CD117+ HSPCs are transplanted intravenously into Townes mice conditioned with sublethal methods, including anti-CD117 antibody (ACK2)-based combinations with azacitidine and low dose irradiation, which are sacrificed between 1-4 days later.
  • ACK2 anti-CD117 antibody
  • Bone marrow is analyzed for the presence of allogeneic HSPCs by flow cytometry.
  • CXCR4 mRNA electroporated allogeneic mouse CD117+ HSPCs are also evaluated for long-term engraftment and multilineage reconstitution needed to sustain long-term hematopoiesis, including erythropoiesis, in vivo.
  • Allogeneic HSPC engraftment are evaluated in Townes mice throughout a 16 week monitoring period following transplantation, which includes measurements of complete blood counts and donor chimerism using flow cytometric analyses of allelic biomarkers (CD45.1 vs CD45.2), blood hemoglobin, and RBC morphology and count.
  • CXCR4 expression in the CD117+ HSPCs will result in a 20% or greater improvement in donor chimerism, reticulocyte cell counts, and blood hemoglobin at ⁇ 1 month and ⁇ 4 months after transplantation.
  • These studies are expected to establish improvement in engraftment of human CD34+ HSPCs and amelioration of disease in the SCD mouse model, with statistically significant improvement engraftment and in disease outcomes, e.g., 20% or more sustained increased in donor chimerism after 1-4 months of transplantation from mRNA-engineered vs. unmanipulated and mock-electroporated HSPCs.
  • the various embodiments described herein can be combined to provide further embodiments.

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Abstract

Modified stem cells and methods of use for stem cell transplant are provided.

Description

MODIFIED STEM CELL COMPOSITIONS AND METHODS FOR USE RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63/232,898, filed August 13, 2021, and U.S. Provisional Patent Application Serial No.63/314,942, filed February 28, 2022, which are incorporated herein by reference in their entireties. SEQUENCE LISTING [0002] This application is being filed electronically via Patent Center and includes an electronically submitted sequence listing in .xml format. The .xml file contains a sequence listing entitled JATH_007_02WO_SeqList_ST26.xml created on August 10, 2022 and having a size of 60 kilobytes. The sequence listing contained in this .txt file is part of the specification and is incorporated herein by reference in its entirety. FIELD OF THE INVENTION [0003] The present disclosure relates to modified hematopoietic stem and progenitor cells, 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) and/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, transplant today 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 sickle cell disease 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 HSCs and HSPCs and related compositions and methods of use thereof in hematopoietic stem cell transplant. In one embodiment, the disclosure provides a modified or engineered cell comprising a nucleic acid encoding a CXCR4 polypeptide (including variants thereof), optionally wherein the cell is transduced with a vector disclosed herein or a modified mRNA encoding the CXCR4 polypeptide, e.g., the modified cell comprises an exogenous or introduced polynucleotide sequence encoding the CXCR4 polypeptide. In certain embodiments, the cell is a stem cell, e.g., a HSC or HSPC. 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. 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 CXCR4 polypeptide, optionally wherein the modified cell expresses the CXCR4 polypeptide transiently. In certain embodiments, the CXCR4 polypeptides is a human CXCR4 polypeptide, or a variant or fragment thereof. In particular embodiments, the CXCR4 polypeptide is a modified CXCR4 polypeptide, e.g., with increased or constitutive activity as compared to the corresponding wild type CXCR4 polypeptide. In particular embodiments, the CXCR4 polypeptide comprises an amino acid substitution at amino acid 119, e.g., a 119S substitution. In particular embodiments, the CXCR4 polypeptide comprises a WHIM mutation or a C-terminal deletion, e.g., a deletion of about 5-25 amino acid residues, about 10-25 amino acid residues, or about 15-20 amino acid residues, e.g., the t19 deletion corresponding to deletion of the C-terminal 19 amino acid residues of CXCR4. In certain embodiments, the exogenous or introduced polynucleotide is an mRNA, and in some embodiments, the mRNA is modified, e.g., to comprise 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 certain embodiments, the modified cell comprises one or more additional modification. For example, the modified cell may further comprise an introduced polynucleotide sequence that expresses a therapeutic protein, such as, for example, a wild type or functional form of a protein that is not expressed or has reduced activity in an HCT recipient, possibly due to a gene mutation in the HCT recipient. In certain embodiments, the modified cell comprises a polynucleotide sequence that has been gene edited, e.g., by Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) together with a CRISPR-associated protein (Cas), transcription activator-like effector nuclease (TALEN), or zinc finger nuclease gene editing technology. For example, gene editing may have been performed to correct a genetic mutation present in the cell, e.g., in the context of autologous HCT. [0009] 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 CXCR4 polypeptide, and a pharmaceutically acceptable excipient, carrier, or diluent. In particular embodiments, the pharmaceutical composition comprises a preparation of human allogeneic transiently modified hematopoietic stem and progenitor cells (HSPCs) comprising an introduced nucleic acid sequence, such as, e.g., chemically modified mRNA, encoding a modified version of CXCR4 into CD34+ HSPCs selected from mobilized peripheral blood. [0010] In a related aspect, the disclosure includes a method of modifying a cell, e.g., an HSC or HSPC, comprising introducing a nucleic acid or vector encoding a CXCR4 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. In particular embodiments, the nucleic acid, e.g., mRNA, is introduced into the cell using lipid nanoparticles (LNPs), liposomes, nanomechanical methods, or other modalities. The nucleic acid, e.g., mRNA, may be present within or bound to an LNP or liposome. In particular embodiments, the nucleic acid is an mRNA, such as, e.g., chemically modified mRNA, encoding a modified version of CXCR4. [0011] 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 CXCR4 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 or HSPCs, 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 chemotherapy (optionally a nucleoside analog and/or an alkylating agent), monoclonal antibody therapy, or radiation, optionally radiation to the entire body (total body irradiation or TBI). [0012] In particular embodiments, expression of CXCR4 (e.g., transiently) in HSCs and/or HSPCs improves HCT. In particular embodiments, CXCR4 transient expression in HSCs and/or HSPCs improves HSCs/HSPCs ability to migrate towards SDF-1 in vitro. In particular embodiments, CXCR4 transient expression in HSCs and/or HSPCs improves the ability of HSCs/HSPCs that are injected intravenously to home to the bone marrow. In particular embodiments, CXCR4 transient expression in HSCs and/or HSPCs improves the ability of HSCs/HSPCs to engraft in the bone marrow, measured by higher donor myeloid chimerism in the bone marrow and in the peripheral blood. In particular embodiments, CXCR4 transient expression in HSCs and/or HSPCs improves neutrophil and platelet recovery following transplantation. In certain embodiments, an improvement correlates to an increase in measured value of the improved property or characteristic of at least 10%, at least 20%, at least 50%, at least 100%, at least two- fold, at least three-fold, or at least five-fold. [0013] 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 comprise 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 [0014] Figure 1. Synthesis of 5 CXCR4 mRNA variants by IVT. Five CXCR4 mRNA variants, using different uracil analogs, were synthesized via IVT and fragment analyses of the products demonstrated constructs of the anticipated size and of high purity. The IVT mRNA shown in the lanes of the gel to the right are as follows: 1 = CXCR4 wild type with full N1mPsU modification; 2 = CXCR4 t19 mutant with full N1mPsU modification; 3= CXCR4 119S mutant with full N1mPsU modification; 4 = ShGFP control with full N1mPsU modification; and 5 = GSP control with full N1mPsU modification. [0015] Figure 2. Isolation of human CD34+ HSPCs from mobilized peripheral blood using Prodigy system. Representative flow cytometric analysis demonstrated significant enrichment of human CD34+ HSPCs from a single mobilized peripheral blood apheresis product using the Prodigy system, which yielded about 95% pure CD34+ HSPCs, about 75% recovery, and about 97% viability. [0016] Figure 3. Electroporation of human CD34+HSPCs with CXCR4 mRNA. (A) mRNAs encoding various CXCR4 sequences containing various chemical modifications were electroporated and characterized. (B) Surface expression of CXCR4 was measured by flow cytometry for CXCR4 mRNA variants (colored) vs mock electroporated control (black); The peaks from left to right correspond to the following mRNAs: unmodified CXCR4 wild type; CXCR4 wild type with 5moU modification; CXCR4 wild type with N1mPsU/5mC modification; CSCR4 wild type with PsU modification; CXCR4 t19 mutant with N1mPsU modification; and CXCR4 wild type with N1mPsU modification. (C) Mean fluorescence intensity (MFI) measured by flow cytometry of CXCR4 protein expression on the surface of CD34+ HSPCs that were not electroporated (Control), mock electroporated, and CXCR4 mRNA electroporated over time. The mRNA used was CXCR4 wild type with 5moU modification. (D) Viability as measured by flow cytometry staining of live cells of CD34+ HSPCs that were not electroporated (Control), mock electroporated, and CXCR4 mRNA electroporated over time post-electroporation. The mRNA used was CXCR4 wild type with 5moU modification. (E) Representative flow cytometry analyses of CD34+ HSPCs cultured for 4 or 24 hours after no electroporation (Control), mock electroporation, and CXCR4 mRNA electroporation. The mRNA used was CXCR4 wild type with 5moU modification. [0017] Figure 4. Effect of mRNA chemistry on CXCR4 expression and in vitro HSPC migration towards SDF-1. (Left graph) CXCR4 mRNAs containing various chemistries vs expression level. The mRNAs correspond to: X1 = CleanCapAG, 5moU (TriLink); A = CleanCapAG-3’OMe, N1mPsU (NEB HiScribe); B = CleanCapAG, N1mPsU (NEB HiScribe); and D = CleanCapAG- 3’OMe, N1mPsU (Ambion Mega). All mRNAs encode wild type CXCR4. (Right graph) Various mRNA chemistries led to differences in transwell migration. mRNA “A” contained N1m- pseudouridine U-substitution, and CleanCapAG-3’OMe (TriLink). [0018] Figure 5. Homing of CXCR4 mRNA electroporated human CD34+ HSPCs into the bone marrow of NSG mice one day after transplantation. (A) Frequency of HSPCs homing to the BM at ~16 hours post injection. mRNAs encoding WT CXCR4 were synthesized with the indicated nucleoside substitutions. All mRNAs encoded wild type CXCR4. (B) mRNAs with the same chemistry (N1mPsU) but the indicated CXCR4 mutant sequences. [0019] Figure 6. 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. DETAILED DESCRIPTION OF THE INVENTION [0020] Hematopoietic stem cell transplantation (HCT) can be 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 and/or HSPCs. However, there are significant complications that reduce the HCT success and usefulness for certain patients. [0021] 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. By promoting faster and more complete engraftment, compositions and methods disclosed herein reduce the risk of graft failure for patients and increase the number of healthy donor or gene corrected HSPCs that stick and stay in the bone marrow. By promoting more cells migrating towards and engrafting in the marrow, they also reduce the need for intensive conditioning regimens. Older, frailer patients as well as very young patients currently restricted from receiving transplant due to conditioning toxicity may gain greater access to this life-saving therapy. [0022] Compositions and methods disclosed herein may be used to treat all disorders for which blood stem cell (e.g., HSC and/or HSPC) transplantation is indicated. In particular embodiments, the compositions and methods disclosed herein are used to introduce CXCR4 mRNA into allogeneic normal HSPCs to improve engraftment and replace a patient’s diseased stem cells with a healthy hematopoietic system. [0023] In certain embodiments, the disclosure provides for compositions and methods for the ex vivo introduction of a polynucleotide encoding a CXCR4 polypeptide, e.g., a human CXCR4 polypeptide), 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. Transplantation of these modified or engineered (e.g., genetically engineered) HSPCs may be done after or in combination with a conditioning regimen, including treatment with antibodies (such as anti-CD117 antibodies). These modified or engineered HSPCs may be transplanted alone or in combination with other cells. [0024] 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 it is not intended to limit the scope of the present invention which will be limited only by appended claims. [0025] 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. [0026] 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. [0027] 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 both of those included limits are also included in the invention. [0028] 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. [0029] 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. [0030] 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. [0031] 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. [0032] As used herein, the terms “identity” and “identical,” when referring to a comparison of two sequences, refers to the percentage of exact matching residues in an alignment of a sequence provided herein to a reference sequence, such as an alignment generated by a BLAST algorithm or other alignment algorithms known in the art. Identity may be calculated based on an alignment of a full-length sequence provided herein and a full-length reference sequence. Identity may also be calculated based on a partial alignment of a sequence provided herein and a reference sequence, if the reference sequence is longer than a sequence provided herein. Identity may also be calculated based on a partial alignment of a sequence provided herein and a reference sequence, if the reference sequence is shorter than a sequence provided herein. Thus, when aligning two sequences, according to the aforementioned, a query sequence “shares at least x % identity to” a subject sequence if in the alignment of the two sequences, at least x % (rounded down) of the residues in the subject sequence are aligned as an exact match to a corresponding residue in the query sequence, wherein the numerator is the number of exact matches and the denominator is the length of the query sequence. In some embodiments, the denominator may alternatively be the length of the query sequence minus any gaps of two or more non-matching residues. Where the subject sequence has variable positions (e.g., residues denoted X), an alignment to any residue in the query sequence is counted as a match. [0033] BLAST software is available over the worldwide web at ncbi.nlm.nih.gov/BLAST/. 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). 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. This program is available also from Genetics Computing Group (GCG) package, from Madison, Wis., USA. 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. [0034] 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. [0035] 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. [0036] 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. [0037] "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. [0038] 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. The term “variant” as used herein refers to a mutant of a reference polynucleotide or polypeptide sequence, for example a native 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, e.g., 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. [0039] 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. [0040] 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) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) 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 will desirably be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease. [0041] 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.). [0042] 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. [0043] 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). CXCR4 Polypeptides and Polynucleotides [0044] C-X-C chemokine receptor type 4 (CXCR4), also known as fusin or CD184 (cluster of differentiation 184), is a protein that in humans is encoded by the CXCR4 gene. The CXCR4 protein is a CXC chemokine receptor. CXCR4 is expressed by most cells, including hematopoietic and endothelial cells (ECs), neurons and stem cells (embryonic and adult). In certain embodiments, the CXCR4 polypeptide, including functional fragments and variants, binds to the chemokine stromal cell derived factor-1 (SDF-1, also known as CXCL12), which is a constitutively expressed and inducible chemokine that regulates multiple physiological processes, including embryonic development and organ homeostasis. SDF-1 is expressed in several organs including lung, liver, skin and bone marrow. In certain embodiments, CXCR4 functional fragments and variants bind to SDF-1 with at least 50%, at least 75%, at least 90% specificity and affinity as a corresponding wild type CXCR4 protein. [0045] Any CXCR4 protein, including functional fragments or variants thereof, may be used according to aspects of the disclosure. In certain embodiments, the CXCR4 polypeptide is a human CXCR4 polypeptide, while in other embodiments, it is another mammalian CXCR4 polypeptide. Sequences of human and mammalian CXCR4 polypeptides are known in the art. In particular embodiments, the CXCR4 polypeptide sequence comprises or consists of one of the following amino acid sequences: MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVI LVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVI YTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFA NVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQK RKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCC LNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS (SEQ ID NO: 1); or MSIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGN GLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAV HVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPD FIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKG HQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFF HCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS (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. [0046] In particular embodiments, the CXCR4 polypeptide sequence, or fragment or variant thereof, is encoded by a polynucleotide sequence that comprises or consists of one of the following: ATGTCCATTC CTTTGCCTCT TTTGCAGATA TACACTTCAG ATAACTACAC CGAGGAAATG GGCTCAGGGG ACTATGACTC CATGAAGGAA CCCTGTTTCC GTGAAGAAAA TGCTAATTTC AATAAAATCT TCCTGCCCAC CATCTACTCC ATCATCTTCT TAACTGGCAT TGTGGGCAAT GGATTGGTCA TCCTGGTCAT GGGTTACCAG AAGAAACTGA GAAGCATGAC GGACAAGTAC AGGCTGCACC TGTCAGTGGC CGACCTCCTC TTTGTCATCA CGCTTCCCTT CTGGGCAGTT GATGCCGTGG CAAACTGGTA CTTTGGGAAC TTCCTATGCA AGGCAGTCCA TGTCATCTAC ACAGTCAACC TCTACAGCAG TGTCCTCATC CTGGCCTTCA TCAGTCTGGA CCGCTACCTG GCCATCGTCC ACGCCACCAA CAGTCAGAGG CCAAGGAAGC TGTTGGCTGA AAAGGTGGTC TATGTTGGCG TCTGGATCCC TGCCCTCCTG CTGACTATTC CCGACTTCAT CTTTGCCAAC GTCAGTGAGG CAGATGACAG ATATATCTGT GACCGCTTCT ACCCCAATGA CTTGTGGGTG GTTGTGTTCC AGTTTCAGCA CATCATGGTT GGCCTTATCC TGCCTGGTAT TGTCATCCTG TCCTGCTATT GCATTATCAT CTCCAAGCTG TCACACTCCA AGGGCCACCA GAAGCGCAAG GCCCTCAAGA CCACAGTCAT CCTCATCCTG GCTTTCTTCG CCTGTTGGCT GCCTTACTAC ATTGGGATCA GCATCGACTC CTTCATCCTC CTGGAAATCA TCAAGCAAGG GTGTGAGTTT GAGAACACTG TGCACAAGTG GATTTCCATC ACCGAGGCCC TAGCTTTCTT CCACTGTTGT CTGAACCCCA TCCTCTATGC TTTCCTTGGA GCCAAATTTA AAACCTCTGC CCAGCACGCA CTCACCTCTG TGAGCAGAGG GTCCAGCCTC AAGATCCTCT CCAAAGGAAA GCGAGGTGGA CATTCATCTG TTTCCACTGA GTCTGAGTCT TCAAGTTTTC ACTCCAGC (SEQ ID NO: 3); or ATGGAGGGGA TCAGTATATA CACTTCAGAT AACTACACCG AGGAAATGGG CTCAGGGGAC TATGACTCCA TGAAGGAACC CTGTTTCCGT GAAGAAAATG CTAATTTCAA TAAAATCTTC CTGCCCACCA TCTACTCCAT CATCTTCTTA ACTGGCATTG TGGGCAATGG ATTGGTCATC CTGGTCATGG GTTACCAGAA GAAACTGAGA AGCATGACGG ACAAGTACAG GCTGCACCTG TCAGTGGCCG ACCTCCTCTT TGTCATCACG CTTCCCTTCT GGGCAGTTGA TGCCGTGGCA AACTGGTACT TTGGGAACTT CCTATGCAAG GCAGTCCATG TCATCTACAC AGTCAACCTC TACAGCAGTG TCCTCATCCT GGCCTTCATC AGTCTGGACC GCTACCTGGC CATCGTCCAC GCCACCAACA GTCAGAGGCC AAGGAAGCTG TTGGCTGAAA AGGTGGTCTA TGTTGGCGTC TGGATCCCTG CCCTCCTGCT GACTATTCCC GACTTCATCT TTGCCAACGT CAGTGAGGCA GATGACAGAT ATATCTGTGA CCGCTTCTAC CCCAATGACT TGTGGGTGGT TGTGTTCCAG TTTCAGCACA TCATGGTTGG CCTTATCCTG CCTGGTATTG TCATCCTGTC CTGCTATTGC ATTATCATCT CCAAGCTGTC ACACTCCAAG GGCCACCAGA AGCGCAAGGC CCTCAAGACC ACAGTCATCC TCATCCTGGC TTTCTTCGCC TGTTGGCTGC CTTACTACAT TGGGATCAGC ATCGACTCCT TCATCCTCCT GGAAATCATC AAGCAAGGGT GTGAGTTTGA GAACACTGTG CACAAGTGGA TTTCCATCAC CGAGGCCCTA GCTTTCTTCC ACTGTTGTCT GAACCCCATC CTCTATGCTT TCCTTGGAGC CAAATTTAAA ACCTCTGCCC AGCACGCACT CACCTCTGTG AGCAGAGGGT CCAGCCTCAA GATCCTCTCC AAAGGAAAGC GAGGTGGACA TTCATCTGTT TCCACTGAGT CTGAGTCTTC AAGTTTTCAC TCCAGC (SEQ ID NO: 4) 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. [0047] In certain embodiments, the CXCR4 polypeptide comprises one or more modifications, e.g., modifications that can improve HSPC functional activity. Several hyperactive CXCR4 mutants have been reported, including CXCR4 with a point mutation at amino acid position 119 (e.g., 119S) that can increase or abrogate SDF-1 response [ Zhang, W.-b., et al., A point mutation that confers constitutive activity to CXCR4 reveals that T140 is an inverse agonist and that AMD3100 and ALX40-4C are weak partial agonists. Journal of Biological Chemistry, 2002. 277(27): p.24515-24521]. Others include CXCR4 hyperactive mutants associated with WHIM, a disorder causing Warts, Hypogammaglobulinemia, Infections, and Myelokathexis, which have been shown to improve short-term BM engraftment [ Gao, J.L., et al., Cxcr4-haploinsufficient bone marrow transplantation corrects leukopenia in an unconditioned WHIM syndrome model. J Clin Invest, 2018. 128(8): p. 3312-3318], and the disclosure includes the use of any CXCR4 mutations disclosed therein. In one embodiment, the WHIM CXCR4 mutant is WHIMt19, which corresponds to CXCR4 with a truncation of the C-terminal 19 amino acids. In particular embodiments, the CXCR4 polypeptide of any of the embodiments disclosed herein comprises one or more modifications, e.g., amino acid insertions, substitutions, or deletions. In particular embodiments, the CXCR4 has increased or constitutive activity as compared to wild-type CXCR4. In particular embodiments, the CXCR4 polypeptide is a modified or variant CXCR4 polypeptide comprising a point mutation at the amino acid residue corresponding to position 119, e.g., a 119S point mutation, or a WHIM mutation, e.g., a C-terminal deletion, such as, e.g., a truncation or deletion of about the C-terminal 19 amino acids. In particular embodiments, the remainder of the CXCR4 polypeptide is unmodified as compared to wild-type CXCR4 or retains at least 90%, at least 95%, at least 98%, or at least 99% identity to the wild-type CXCR4, e.g., human CXCR4. [0048] In particular embodiments, the polypeptide or polynucleotide sequence is humanized. In particular embodiments, the polynucleotide or nucleic acid sequence 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). [0049] A modified mRNA encoding a CXCR4 polypeptide, including variants thereof, wherein the modified mRNA comprises one or more nucleoside modification or substitutions and/or a cap sequence. mRNA chemical compositions including nucleoside modifications and/or 5’ cap modifications have been shown to improve mRNA stability, translation efficiency, and prevent cellular responses against certain RNA moieties. In certain embodiments, the nucleic acid comprises a modified mRNA. 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 CXCR4 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). [0050] In particular embodiments, the modified mRNA comprises one or more nucleoside modification. In particular embodiments, the mRNAs are fully modified at all occurrences of one or more nucleoside, e.g., A, G, U or C. 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 (PsU or Ψ) or N1-methyl- pseudouridine (N1mPsU or m1Ψ), and/or cytosine can be replaced by 5-methylcytosine. In certain embodiments, the at least one modified nucleoside includes modification of U to 5- methoxyuridine (5moU). In particular embodiments, the at least one modified nucleoside includes N1-methyl-pseudouridine and/or 5-methylcytidine and/or 5-methoxyuridine. In certain embodiments, all uridines in the modified mRNA are replaced with a similar nucleoside such as pseudouridine (Ψ) or N1-methyl-pseudouridine (m1Ψ) or 5-methoxyuridine, and/or all cytosines in the modified mRNA are substituted with a similar nucleoside such as 5- methylcytosine. [0051] In particular embodiments, the modified mRNA comprises a 5’ terminal cap sequence followed by a sequence encoding the CXCR4 polypeptide, followed by a 3’ tailing sequence, such as a polyA or a polyA-G sequence. [0052] In particular embodiments, the mRNA encoding CXCR4 (including modified forms or variants thereof) comprises a wild type 5’ terminal cap sequence, and in certain embodiments, the mRNA encoding CXCR4 (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 CXCR4 comprises the modified 5’ terminal cap, 3´-O-Me-m7G(5')ppp(5')G (Anti Reverse Cap Analog (ARCA); APExBIO, USA). [0053] In particular embodiments, the disclosure provides: an unmodified mRNA encoding a wild-type CDCR4 polypeptide; a modified mRNA encoding a wild type CXCR4 polypeptide; a modified mRNA encoding a wild type CXCR4 with 5moU modification(s); a modified mRNA encoding a wild type CXCR4 with N1mPsU/5mC modification(s); a modified mRNA encoding a wild type CXCR4 with PsU modification(s); a modified mRNA encoding a t19 mutant with N1mPsU modification(s); and a modified mRNA encoding a wild type CXCR4 with N1mPsU modification(s). In particular embodiments, the mRNA modification(s) occur at every such residue in the modified mRNA. In particular embodiments, the modified mRNA comprises: CleanCapAG and 5moU modification(s); CleanCapAG-3’OMe and N1mPsU modification(s) (NEB HiScribe); CleanCapAG and N1mPsU modification(s) (NEB HiScribe); or CleanCapAG-3’OMe and N1mPsU (Ambion Mega). In particular embodiments, the encode wild type CXCR4. (Right graph) Various mRNA chemistries led to differences in transwell migration. [0054] 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. [0055] In certain embodiments, the nucleic acid encoding the CXCR4 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 other 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 CXCR4 polypeptide. In particular embodiments, the expression vector comprises a promoter sequence that facilitates expression of the encoded CXCR4 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 CXCR4 polypeptide. [0056] 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 HSPC when viral vector and the HSC and/or HSPCs are incubated together for at least about 24 hours in a culture medium. Modified Hematopoietic Stem Cells and Pharmaceutical Compositions [0057] In a related aspect, the disclosure provides modified cells, e.g., HSCs and/or HSPCs, comprising a nucleic acid encoding a CXCR4 polypeptide as described herein. In certain embodiments, the nucleic acid encoding the CXCR4 polypeptide is transiently present in the modified cell, and/or is not present within the genome of the cell. In particular embodiments, the modified cell expresses and/or comprises the CXCR4 polypeptide, and in particular embodiments, the CXCR4 polypeptide is present on the cell surface. 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 is transduced with a modified mRNA. [0058] 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. [0059] In particular embodiments, the modified cell is a stem cell and/or progenitor cell, and in certain embodiments, the stem cell is an HSC or an HSPC. In some embodiments, the cell is a mammalian cell that has the ability both to self-renew, and to generate differentiated progeny, e.g., an HSC or an HSPC. In certain embodiments, the stem cell and/or progenitor cell is a human cell. The stem cell and/or progenitor 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. [0060] 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 CXCR4 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. [0061] The cells may be fresh, frozen, or have been subject to prior culture. They may be fetal, neonate, adult, etc. HSCs and/or 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 HSCs and/or HSPCs are separated from other cells of the hematopoietic or other lineage is not critical to this invention. If desired, a substantially homogeneous population of HSCs and/or HSPCs may be obtained by selective isolation of cells free of markers associated with differentiated cells, while displaying epitopic characteristics associated with the stem cells. [0062] 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. [0063] Prior to harvesting HSCs and/or HSPCs 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. [0064] Among HSC and/or HSPC markers, CD34 is well known for its unique expression on HSCs and/or HSPCs. In certain embodiments, the modified cell is a CD34+ cell. In particular embodiments, the modified cell is a subset of HSC and/or HSPC 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. [0065] 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. [0066] In certain embodiments, the disclosure provides a method of modifying cells, including HSCs and/or HSPCs, comprising introducing the nucleic acid encoding a CXCR4 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 CXCR4 polypeptide in the cell. In certain embodiments, the method is used to prepare modified cells, e.g., HSCs and/or HSPCs, 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 CXCR4 polypeptide into the HSCs and/or HSPCs according to a variety of methods available in the art. [0067] For engraftment purposes, a composition comprising HSCs and/or HSPCs, is administered to a patient. Such methods are well known in the art. The HSCs and/or HSPCs 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. [0068] The present disclosure also includes pharmaceutical compositions comprising one or more CXCR4 polypeptides, one or more polynucleotides or vectors comprising a sequence encoding a CXCR4 polypeptide (e.g., a modified mRNA), or a modified cell, e.g., HSC and/or HSPC, comprising a polynucleotide or vector encoding a CXCR4 polypeptide and/or expressing a CXCR4 polypeptide, in combination with one or more pharmaceutically acceptable diluent, carrier, or excipient. [0069] The present invention discloses a pharmaceutical composition comprising a modified cell comprising a CXCR4 polypeptide (or nucleic acid sequence encoding the CXCR4 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. [0070] 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 [0071] 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 and/or HSPCs, comprising a CXCR4 polypeptide described herein and/or a nucleic acid encoding the CXCR4 polypeptide. In particular embodiments, the subject is in need of HCT. 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 and/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. [0072] 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. [0073] The HCT methods disclosed use modified HSCs and/or HSPCs comprising an exogenous or introduced CXCR4 polypeptide or nucleic acid encoding the CXCR4 polypeptide. CXCR4 expression in HSCs and/or HSPCs improves HSCs/HSPCs ability to migrate towards SDF-1 in vitro and in vivo. CXCR4 expression in HSCs and/or HSPCs improves HSCs/HSPCs that are injected intravenously to home to the bone marrow. CXCR4 transient expression in HSCs and/or HSPCs improves HSCs/HSPCs engraftment in the bone marrow, measured by higher donor myeloid chimerism in the bone marrow and in the peripheral blood. CXCR4 transient expression in HSCs and/or HSPCs improves neutrophil and platelet recovery following transplantation. The methods of the invention are also believed to provide for improved engraftment of stem cells after transplantation into a recipient. [0074] 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 encoding a CXCR4 polypeptide into the HSC and/or HSPCs, optionally wherein the CXCR4 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, e.g., to correct a gene mutation in the subject. [0075] 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 CXCR4 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 CXCR4 polypeptide is an mRNA, and it is introduced into the cells by electroporation. [0076] In certain embodiments, the disclosure provides a method for HCT comprising: providing to a subject in need thereof a population of cells comprising a plurality of modified or engineered HSCs and/or HSPCs, wherein the modified HSCs and/or HSPCs: i) were obtained from a donor, optionally a mammal, e.g., a human; ii) comprise an introduced polynucleotide sequence encoding a CXCR4 polypeptide into the HSC and/or HSPCs, optionally wherein the CXCR4 polypeptide comprises a sequence disclosed herein, or a functional variant or fragment thereof, and wherein the modified HSCs and/or HSPCs express the encoded CXCR4 polypeptide; and iii) optionally, were further modified by comprising a gene therapy vector, or by gene editing, e.g., to correct a gene mutation in the subject. [0077] 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 certain embodiments, the HCT is autologous or allogeneic. Thus, in certain embodiments, the subject being treated is the same or different from the donor. In particular embodiments, the gene therapy vector or reagents used to perform the gene editing were introduced into HSCs and/or HSPCs obtained from the subject being treated by HCT. In particular embodiments, the polynucleotide sequence encoding the CXCR4 polypeptide and the gene therapy vector or reagents used for gene editing were introduced into the cells at the same time, or either was 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 CXCR4 polypeptide is an mRNA, and it was introduced into the cells by electroporation. [0078] In certain embodiments, the disclosure provides a method for HCT, comprising: i) obtaining HSCs and/or HSPCs from a donor, optionally a mammal, e.g., a human; ii) introducing a polynucleotide sequence encoding a CXCR4 polypeptide into the HSCs and/or HSPCs, optionally wherein the CXCR4 polypeptide comprises a sequence disclosed herein, or a functional variant or fragment thereof, to produce modified HSCs and/or HCPCs; iii) optionally, modifying the HSCs and/or HSPCs, e.g., by introducing a gene therapy vector, or by gene 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. [0079] 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 certain embodiments, the HCT is autologous or allogeneic. Thus, in certain embodiments, the subject being treated is the same or different from the donor. 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 CXCR4 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 CXCR4 polypeptide is an mRNA, and it is introduced into the cells by electroporation. [0080] In particular embodiments of methods of treatment disclosed herein, the method results in one or more of the following clinical outcomes: Engraftment: >95% (three consecutive days with absolute neutrophil count ≥500/µL by 28 days post-transplantation); Use of non-myeloablative conditioning; Oral mucositis: <10% at 30 days; Veno-occlusive disease (VOD): <2% at 100 days; Grade 3-4 acute graft-versus-host disease: <5% at 100 days; or Chronic graft-versus-host disease: <5% at 2 years. [0081] 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, e.g., prior to and/or concurrent with the modified HSCs and/or HSPCs being provided or administered to the subject. In certain embodiments, the conditioning regimen depletes endogenous normal or diseased HSCs and/or HSPCs 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 modified HSCs and/or HSPCs or pharmaceutical composition disclosed herein. [0082] 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 6, 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 6. In particular embodiments, the modified cells are administered to a subject in combination with a non-myeloablative conditioning regimen. [0083] 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. [0084] In particular embodiments, since the subject is being administered modified cells, e.g., HSCs, comprising a modified CXCR4 described herein, the conditioning regimen is milder than would be used if the subject was being administered cells, e.g., HSPCs or HSCs, that did not comprise the modified CXCR4 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%. [0085] In some embodiments, the agents used according to the conditioning regimen are administered to the subject prior to and/or concurrently with the administration of the modified HSPCs/HSCs or pharmaceutical composition to the subject. In particular embodiments, there is a “washout” period following administration of the anti-c-Kit antibody and before administration of the modified cells. This period of time allows clearance of the anti-c-Kit antibody (or any other agent used for conditioning). The period of time required for clearance of the conditioning 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 conditioning agent, e.g., anti-c-Kit antibody, to decrease at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold from peak levels, e.g., at least about 100-fold, 1000-fold, 10,000-fold, or more. In certain embodiments, the wash-out period is between 2 days and three weeks or between 5 days and two weeks, e.g., about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days about 17 days, about 18 days, about 19 days, or about 20 days. [0086] In certain embodiments, the conditioning regimen comprises providing to the subject an anti-CD117 antibody, e.g., an anti-CD117 monoclonal antibody that inhibits stem cell factor from binding to CD117 on the cell surface, such as e.g., JSP191. In certain embodiments, the conditioning regimen comprises providing to the subject total body irradiation (TBI). In certain embodiments, the conditioning regimen comprises providing to the subject a chemotherapeutic agent, such as, e.g., fludarabine or azacytidine. In some embodiment, the conditioning regimen comprises a combination of TBI and a chemotherapeutic agent. In some embodiments, the conditioning regimen comprises a combination of an anti-CD117 monoclonal antibody, e.g., JSP191 and TBI and fludarabine, or a combination of an anti-CD117 monoclonal antibody, e.g., JSP191 and azacytidine. Anti-c-Kit Antibodies [0087] In certain embodiments, the conditioning regimen comprises administration of an anti- CD117 antibody, wherein the anti-CD117 antibody depletes or reduces endogenous HSPCs. 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-74. In particular embodiments, the antibody is JSP191. In particular embodiments, the antibody is the humanized form of SR1, JSP191, described in U.S. Pat. Nos.8,436,150 and 7,915,391. [0088] Compositions and methods disclosed herein may be applicable to any anti-CD117 antibody, particularly monoclonal anti-human CD117 antibodies, e.g., those that block or inhibit binding of SCF to CD117. An anti-CD117 antibody may refer to an antibody that binds to CD117, e.g., human CD117, or an antigen-binding fragment thereof. [0089] A number of antibodies contemplated by the disclosure that specifically bind human CD117 are known in the art and commercially available, including without limitation, JSP-191, 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 anti-CD117 antibodies and/or CDR sets disclosed in any of the patent application disclosed herein, which are all incorporated by reference in their entireties. [0090] In certain embodiments, the anti-CD117 antibody binds to the extracellular region of CD117 i i id 26524 Th f thi i i h b l
Figure imgf000033_0001
[0091] Illustrative anti-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 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, CDX-0159 (from Celldex Therapeutics, Hampton, NJ), MGTA-117 (AB85) (from Magenta Therapeutics, Cambridge, MA), CK6 (from Magenta Therapeutics, Cambridge, MA), AB249 (from Magenta Therapeutics, Cambridge, MA), and FSI-174 (from Gilead, South San Francisco, CA). The antibodies from Magenta Therapeutics are disclosed in US Patent Application Publication No.20190153114. In certain embodiments, the antibody is one disclosed in any of US Pat. Nos. 7,915,391, US 8,436,150, or US 8,791,249. In certain embodiments, the antibody is one disclosed in US Pat. Application Publ. No 20200165337 or any of PCT Publication Nos. WO 2020/112687, WO2020/219748, WO 2020/219770, or WO 2019/084064. [0092] In particular embodiments, the antibody is a humanized form of SR1, a murine anti- CD117 antibody described in U.S. Pat. Nos. 5,919,911 and 5,489,516. The humanized form, JSP191, is disclosed in U.S. Patent Nos. 7,915,391, 8,436,150, and 8,791,249. JSP191 is an aglycosylated IgG1 humanized antibody. 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. JSP191 blocks SCF from binding to CD117 and disrupts stem cell factor (SCF) signaling, leading to the depletion of hematopoietic stem cells. JSP191 is a heterotetramer consisting of 2 heavy chains of the IgG1 subclass and 2 light chains of the kappa subclass, which are covalently linked through disulfide bonds. There are no N-linked glycans on JSP191 due to an intentional substitution from an asparagine to glutamine at heavy chain residue 297. The sequences of the heavy chains and light chains of JSP191 are disclosed as SEQ ID NO: 4 from US8436150 and SEQ ID NO: 2 from US8436150, respectively. [0093] The sequences of the heavy chains and light chains of JSP191 are disclosed as SEQ ID NO: 4 from U.S. Patent No. 8,436,150 and SEQ ID NO: 2 from U.S. Patent No. 8,436,150, respectively. The sequences of the heavy and light chains of JSP191 are: Heavy Chain:
Figure imgf000034_0001
and Light Chain: MVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMHW
Figure imgf000034_0002
[0094] In certain embodiments, the variable heavy domain of JSP191 comprises the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSGNG DTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQGTLVT VSS (SEQ ID NO: 8) [0095] In certain embodiments, the variable light chain domain of JSP191 comprises the following sequence: DIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMHWYQQKPGQPPKLLIYLASNLES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDP YTFGGGTKVEIK (SEQ ID NO: 9). [0096] The CDRs present in JSP191 are as follows: VH CDR1 = YNMH (SEQ ID NO: 10); VH CDR2 = IYSGNGDTSYNQKFKG (SEQ ID NO: 11); VH CDR3 = ERDTRFGN (SEQ ID NO: 12); VL CDR1 = RASESVDIYGNSFMH (SEQ ID NO: 13); VL CDR2 = LASNLES (SEQ ID NO: 14); and VL CDR3 = QQNNEDPYT (SEQ ID NO: 15). [0097] 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 anti-CD117 antibodies are described in U.S. Patent No. 10,781,267, and in particular embodiments, an anti-CD117 disclosed herein comprises the CDRs of any of the antibodies disclosed therein. In certain embodiments, the anti-CD117 antibody comprises: (i) a light chain variable region ("VL") comprising the amino acid sequence: DIVMTQSPSXK1LSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKXK2LIYSASYRYS GVPDRFXK3GSGSGTDFTLTISSLQXK4EDFAXK5YXK6CQQYNSYPRTFGGGTKVEIK (SEQ ID NO: 16), 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: QVQLVQSGAEXH1KKPGASVKXH2SCKASGYTFTDYYINAVVXH3QAPGKGLEWIARIYP GSGNTYYNEKFKGRXH4TXH5TAXH6KSTSTAYMXH7LSSLRSEDXH8AVYFCARGVYYFD YWGQGTTVTVSS (SEQ ID NO: 17), 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
Figure imgf000036_0001
GTTLTVSA (SEQ ID NO: 19), and (ii) VL CDRs of a VL domain comprising the amino acid sequence DIVMTQSQKFMSTSVGDRVSVTCKASQNVRTNVAWYQQKPGQSPKALIYSASYRYSGV PDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYNSYPRTFGGGTKLEIKR (SEQ ID NO: 20). [0098] 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 Publication No.20200407440 and/or PCT Application Publication 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: 21), and RCPLTDPEVTNYSLKGCQGKP (amino acids 100-130; SEQ ID NO: 22). [0099] 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 Publication No. WO2019084064, respectively. [00100] The heavy chain variable region (VH) amino acid sequence of Ab85 is: EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMAIINPRDSDT RYRPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYEGYEGAFDIWGQG TLVTVSS (SEQ ID NO: 23). [00101] The VH CDR amino acid sequences of AB85 are as follows: NYWIG (SEQ ID NO: 24; VH CDR1); IINPRDSDTRYRPSFQG (SEQ ID NO: 25; VH CDR2); and HGRGYEGYEGAFDI (SEQ ID NO: 26; VH CDR3). [00102] The light chain variable region (VL) amino acid sequence of AB85 is: DIQMTQSPSSLSASVGDRVTITCRSSQGIRSDLGWYQQKPGKAPKLLIYDASNLETGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQANGFPLTFGGGTKVEIK (SEQ ID NO: 27). [00103] The VL CDR amino acid sequences of AB85 are as follows: RSSQGIRSDLG (SEQ ID NO: 28; VL CDR1); DASNLET (SEQ ID NO: 29; VL CDR2); and QQANGFPLT (SEQ ID NO: 30; VL CDR3). [00104] FSI-174 is an anti-CD117 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 anti-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. [00105] In certain embodiments, the CDRs present in FSI-174 and related antibodies are as follows: VH CDR1 = SYNMH (SEQ ID NO: 31); VH CDR2 = VIYSGNGDTSY(A/N)QKF(K/Q)G (SEQ ID NO: 32); VH CDR3 = ERDTRFGN (SEQ ID NO: 12); VL CDR1 = RAS(D/E)SVDIYG(N/Q)SFMH (SEQ ID NO: 33); VL CDR2 = LASNLES (SEQ ID NO: 14); and VL CDR3 = QQNNEDPYT (SEQ ID NO: 15). 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: 31); H2 = VIYSGNGDTSYAQKFKG (SEQ ID NO: 34); H3 = ERDTRFGN (SEQ ID NO: 12); and the CDRs present in the light variable region are CDRs L1, L2 and L3 as defined by Kabat: L1 = RASESVDIYGQSFMH (SEQ ID NO: 35); L2 = LASNLES (SEQ ID NO: 14); and L3 = QQNNEDPYT (SEQ ID NO: 15), 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:
Figure imgf000038_0001
39). [00106] 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: 40); CDR-H2 with IIYPGDSDTRYSPSFQG (SEQ ID NO: 41); CDR-H3 with HGRGYNGYEGAFDI (SEQ ID NO: 42). CK6 has the following light chain CDR amino acid sequences: CDR-L1 with RASQGISSALA (SEQ ID NO: 43); CDR-L2 with DASSLES (SEQ ID NO: 44); and CDR-L3 with CQQFNSYPLT (SEQ ID NO: 45). [00107] 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: Example variable heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISEN GSDTYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGGAVSYFDV WGQGTLVTVSS (SEQ ID NO: 46) Example variable light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT (SEQ ID NO: 47) 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 (SEQ ID NO: 48; VH CDR1) IIYPGDSDTRYSPSFQG (SEQ ID NO: 41; VH CDR2); and HGLGYNGYEGAFDI (SEQ ID NO: 49; VH CDR3). Ab249 has the following light chain CDRS: RASQGIGSALA (SEQ ID NO: 50; VL CDR1); DASNLET (SEQ ID NO: 29; VL CDR2); and QQLNGYPLT (SEQ ID NO: 51; VL CDR3). Ab249 has the following variable heavy chain sequence (CDRS are underlined): EVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGWVRQMPGKGLEWMGIIYPGDSDT RYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYEGAFDIWGQG TLVTVSS (SEQ ID NO: 52). Ab249 has the following variable light chain sequence (CDRS are underlined): DIQMTQSPSSLSASVGDRVTITCRASQGIGSALAWYQQKPGKAPKLLIYDASNLETGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPLTFGQGTRLEIK (SEQ ID NO: 53) [00108] 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. [00109] 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. [00110] 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. [00111] JSP191 is an aglycosylated IgG1 humanized antibody. JSP191 (formerly AMG191) 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. JSP191 blocks SCF from binding to CD117 and disrupts critical survival signals, leading to the depletion of hematopoietic stem cells. 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 and/or HSPCs, e.g., as a single dose. [00112] In some embodiments, the subject is administered about 0.01 mg/kg to about 10 mg/kg of the anti-c-kit antibody, e.g., JSP191, about 0. 1 mg/kg to about 10 mg/kg of the anti-c-kit antibody, e.g., JSP191, about 1.0 mg/kg to about 10 mg/kg of the anti-c-kit antibody, e.g., JSP191. In some embodiments, the subject is administered about 0.01 mg/kg to about 2 mg/kg of the anti- c-kit antibody, e.g., JSP191, optionally the subject is administered about 0.1 mg/kg to about 1 mg/kg of the anti-c-Kit antibody, e.g., JSP191. In some embodiments, anti-c-Kit 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 anti- c-Kit signaling antibodies are administered in a dose of about 0.6 mg/kg, optionally on days 14 through 10 prior to HCT. In some embodiments, the anti-c-Kit antibody is administered in a dose of about 0.1 mg/kg to about 1 mg/kg of the anti-c-Kit antibody about 5 to about 20 days before the HCT. [00113] In some embodiments, the anti-c-Kit antibody, e.g., JSP191 is administered about 5 to about 20 days before the HCT (administration of the modified stem cells). In some embodiments, the anti-c-Kit antibody is administered on days 10 through 14 before the HCT. In some embodiments, the anti-c-Kit antibody is administered on days 5, 6, or 7 through about 10 to about 14 days prior to the HCT. In certain embodiments, the anti-c-Kit antibody is administered daily during any of these time periods. The day of transplant may in some embodiments be determined by the anti-c-Kit 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 anti-c-Kit antibody blood concentration of about 2000 ng/ml or less. [00114] 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. Total Body Irradiation (TBI) [00115] 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. [00116] 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. [00117] 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 [00118] 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. [00119] 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. [00120] 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 [00121] In certain embodiments, the disclosure provides methods for conditioning a subject for HCT, the method comprising administering to the subject an anti-c-Kit 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 anti-c-Kit 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 anti-c-Kit 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. [00122] In some embodiments, the anti-c-Kit antibody is administered about 5 to about 20 days before the HCT. In some embodiments, the anti-c-Kit antibody is administered on days 10 through 14 before the HCT. In some embodiments, the anti-c-Kit antibody is administered on days 5, 6, or 7 through about 10 to about 14 days prior to the HCT. In certain embodiments, the anti-c-Kit antibody is administered daily during any of these time periods. The day of transplant may in some embodiments be determined by the anti-c-Kit 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 anti-c-Kit antibody blood concentration of about 2000 ng/ml or less. [00123] 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. [00124] 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. [00125] In some embodiments, the anti-c-Kit antibody (e.g., JSP191 or a humanized c-kit antibody as described in US20200165337A1) is administered on 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. [00126] In some embodiments, the subject is administered about 0.01 mg/kg to about 2 mg/kg of the anti-c-kit antibody, e.g., JSP191, optionally the subject is administered about 0.1 mg/kg to about 1 mg/kg of the anti-c-Kit antibody, e.g., JSP191. In some embodiments, anti-c-Kit 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 anti-c-Kit signaling antibodies are administered in a dose of about 0.6 mg/kg, optionally on days 14 through 10 prior to HCT. [00127] 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. [00128] 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. [00129] In some embodiments, the subject is administered about 0.1 to about 1.0 mg/kg of the anti-c-Kit antibody (e.g., JSP191 or a humanized c-kit 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. [00130] In some embodiments, the anti-c-Kit antibody (e.g., JSP191 or a humanized c-kit antibody as described in US20200165337A1) is administered on 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. [00131] In some embodiments, the anti-c-Kit antibody is administered in a dose of about 0.1 mg/kg to about 1 mg/kg of the anti-c-Kit 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. [00132] In some embodiments, the anti-c-Kit antibody (e.g., JSP191 or a humanized c-kit antibody as described in US20200165337A1) is administered on 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. [00133] In some embodiments, the anti-c-Kit antibody (e.g., JSP191 or a humanized c-kit antibody as described in US20200165337A1) is administered on 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. [00134] In some embodiments, the anti-c-Kit antibody (e.g., JSP191 or a humanized c-kit antibody as described in US20200165337A1) is administered on 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. [00135] In some embodiments, the anti-c-Kit antibody (e.g., JSP191 or a humanized c-kit antibody as described in US20200165337A1) is administered on 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. [00136] The dose of stem cells, e.g., modified HSCs and/or HSPCs comprising an exogenous CXCR4 polypeptide and/or nucleic acid encoding a CXCR4 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. [00137] In certain embodiments, a maximum number of CD3+ cells delivered with the modified HSPC 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. [00138] 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 and/or HSPCs to the subject, wherein the modified HSCs and/or HSPCs comprise an exogenous or introduced CXCR4 polypeptide and/or an exogenous or introduced nucleic acid sequence encoding the CXCR4 polypeptide. In particular embodiments, the nucleic acid sequence is operably linked to a promoter sequence. [00139] The anti-c-Kit antibody and/or chemotherapy may be delivered orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. In some embodiments, the anti-c-Kit antibody, e.g., JSP191, is administered to the subject intravenously, the chemotherapy, e.g., fludarabine, is administered to the subject intravenously, and the TBI is administered in a single dose of radiation. [00140] The methods disclosed herein may be used to treat a variety of indications amenable to stem cell transplantation. In particular embodiments, HCT methods disclosed herein are 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, hemoglobinopathies, and a genetic disorder. 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. [00141] 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. [00142] 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. [00143] In some embodiments, the disease is a blood cancer, optionally a leukemia, a lymphoma, or a myelodysplastic syndrome (MDS). In particular embodiments, the methods disclosed are used to treat acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), acute lymphoblastic leukemia (ALL), hodgkin lymphoma, non-hodgkin lymphoma, clonal hematopoiesis of indeterminate potential (CHIP), clonal cytopenia of undetermined significance (CCUS) myelodysplastic syndromes (MDS), idiopathic cytopenia of undetermined significance (ICUS), or myeloproliferative neoplasms (MPN). In particular embodiments, the leukemia is acute myeloid leukemia (AML). [00144] In some embodiments, the disease or disorder is multiple myeloma, chronic myelogenous leukemia (CML) myelodysplastic syndromes (MDS), a myeloproliferative neoplasm, or a myeloid leukemia, e.g., acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). In some embodiments, the disease is MDS or AML. In some embodiments, the cancer is a lymphoid leukemia, e.g., acute lymphocytic leukemia (ALL) or chronic lymphocytic leukemia (CLL). [00145] In some embodiments, the cancer is a myelodysplastic/myeloproliferative neoplasm (MDS/MPN), such as, e.g., chronic myelomonocytic leukemia (CMML). MDS/MPN have both "dysplastic" and "proliferative" features that cannot be classified as either myelodysplastic syndromes (MDS) or myeloproliferative neoplasms (MPN), and for this reason have been categorized as an overlap syndrome with its own distinct characteristics (MDS/MPN). CMML is cancer of the blood. CMML is considered to be one of the myelodysplastic/myeloproliferative neoplasms (MDS/MPN), a type of chronic blood cancer in which a person's bone marrow does not make blood effectively. [00146] In some embodiments, the subject has a hematopoietic cell transplant comorbidity index (HCT-CI) greater than or equal to 3 (Sorror ML, et al. Hematopoietic cell transplantation (HCT)- specific comorbidity index: a new tool for risk assessment before allogeneic HCT. Blood. 2005;106(8):2912-2919.). In some embodiments, the subject has a hematopoietic cell transplant comorbidity index (HCT-CI) less than or equal to 3. [00147] In some embodiments, the disease or disorder is multiple myeloma, severe combined immune deficiency (SCID), chronic myelogenous leukemia (CML), myelodysplastic syndromes (MDS), a myeloproliferative neoplasm, or acute myeloid leukemia (AML). [00148] In certain embodiments, the disease treated according to the disclosure is referred to as MDS/AML, which includes both MDS and AML. Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) exist along a continuous disease spectrum starting with early-stage MDS, which may progress to advanced MDS, AML, cured AML or resistant AML. The disease is characterized by an overproduction of immature blood cells. The resulting lack of mature, healthy blood cells causes anemia and an increased risk for infection and bleeding. Around 5-10% of patients with solid tumors who are treated with chemotherapy, radiation or autologous stem cell transplantation develop treatment-related MDS or AML. [00149] Myelodysplastic syndromes (MDS) are a group of hematopoietic neoplasms characterized by abnormal differentiation and cytomorphology (i.e., dysplasia) of pluripotent hematopoietic progenitor cells (i.e., stem cells) residing in the myeloid compartment of the bone marrow (BM). These abnormalities lead to ineffective hematopoiesis and to cytopenia (i.e., lower- than-normal peripheral blood cell counts) of one or more lineages of the myeloid progenitor cells that manifests as anemia, neutropenia, and/or thrombocytopenia. Methods disclosed herein may be used to treat various forms of MDS, including but not limited to those shown in the table below, which is reproduced from Chung, US Pharm.2021;46(9):39-44. In certain embodiments, the methods result in decreased cytopenia.
Table 1
Figure imgf000051_0001
[00150] In particular embodiments, the methods disclosed are used to treat an immunodeficiency. In particular embodiments, the immunodeficiency is severe combined immunodeficiency (SCID).
[00151] In particular embodiments, the methods disclosed are used to treat a genetic disorder. In particular embodiments, the genetic disorder is sickle ceH 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.
[00152] In certain embodiments of any of the HCT methods disclosed, the method further comprises administering to the subject an additional therapeutic agent for treatment of the disease or disorder being treated by the HCT method.
EXAMPLES Example 1 TRANSIENT MRNA EXPRESSION OF CXCR4 IN HSPCS IMPROVES ENGRAFTMENT [00153] Experiments were conducted to demonstrate that increased CXCR4 protein expression by CD34+HSPCs stimulates efficient engraftment of normal HSPCs, by facilitating their homing as well as retention in the bone marrow and increasing their proliferation and repopulation potential. The transient nature of the expression modification de-risks against permanent chromosomal changes that may be associated with non-mRNA modalities, and the improved engraftment of healthy human CD34+ HSPCs eliminates the need for highly toxic HCT conditioning and non-HSPC cellular populations that cause GVHD. Improvement in the engraftment efficiency of HSPCs would also improve outcomes globally in HCT settings where the number of HSPCs are limiting, including ex vivo gene therapy of autologous HSPCs. Studies were conducted to develop and evaluate a CXCR4 mRNA transiently introduced into human CD34+ HSPCs via electroporation. A platform for transient mRNA-based modification of human CD34+ HSPCs was developed by testing multiple electroporation systems, including ones from Lonza, ThermoFisher, Maxcyte, and Miltenyi. The electroporation experiments described were conducted using the Miltenyi Elmo electroporator that can perform small- and large- GMP-scale electroporation (~100 ul to ~200 ml or ~ 0.5 M to ~ 1 B cells), which is scalable to a clinical platform. Synthesis of CXCR4 mRNA by in vitro transcription (IVT) [00154] IVT was performed to create mRNAs encoding CXCR4 containing various sequence and chemistry elements for characterization and functional testing. Briefly, DNA templates were synthesized by commercial vendors (Twist, IDT) to contain desired sequences (e.g., genes, mutants, non-coding elements such as untranslated regions) downstream of a T7 promoter and used to transcribe mRNA. Chemical modifications were included in IVT reactions, e.g., replacing uracil by 5-methoxyuridine or N1-methyl-pseudouridine, or adding 5’ cap analogs such as CleanCapAG (TriLink). DNA templates were removed by DNase digestion. PolyA tails were either encoded in the DNA templates or added enzymatically. mRNAs were purified using silica membrane columns (Qiagen) and analyzed by capillary electrophoresis (Agilent Fragment Analyzer) for size composition, and by UV absorbance (Nanodrop) for purity. All mRNAs were of the expected size and highly purified (A260/280 and As260/230 > 1.8; band purity > 80%) (Figure 1). Yields varied by condition (chemical modification) but were generally >100 ug for ~40 ul reactions—sufficient for characterization and testing for functional activity (in vitro cell-based assays and in vivo mouse transplantation models). Isolation of human CD34+ HSPCs from mobilized peripheral blood [00155] Human CD34+ HSPCs were successfully isolated from mobilized peripheral blood obtained from healthy subjects. Purchased apheresis products of donors administered with G-CSF were used to mobilize CD34+ HSPCs to the bloodstream (Miltenyi and AllCells). Enrichment was carried out using magnetic anti-CD34 beads on a Miltenyi Prodigy system. Flow cytometry demonstrated 95% CD34+ cell purity (Figure 2), 97% viability, and 75% recovery after enrichment (data not shown). These cells were aliquoted and cryopreserved for subsequent studies. Electroporation of human CD34+ HSPCs with CXCR4 mRNA [00156] CXCR4 mRNA were electroporated into human CD34+ HSPCs using a Miltenyi Elmo platform to characterize mRNAs and process conditions for functional activity. Generally, frozen human CD34+ HSPCs were thawed and cultured in X-VIVO media (Lonza) supplemented with cytokines—stem cell factor (SCF), FLT3-ligand (FLT3-L) and thrombopoietin (TPO)—for 24 hours, then electroporated with candidate mRNAs. Controls included human CD34+ HSPCs that were non-electroporated, or mock-electroporated with no mRNA. Expression of CXCR4 on the cell surface was evaluated by flow cytometry. mRNAs increased CXCR4 expression >100 fold, dependent on mRNA quantity and chemical composition (Figure 3B). CleanCapAG, N1-methyl- pseudouridine, and other modifications dramatically improved expression level per amount of mRNA. mRNA-induced expression of CXCR4 was transient and no longer detected ~72 hours after electroporation (Figure 3C). Viability of CD34+ HSPCS remained approximately 90% for ~7 days after electroporation, in both CXCR4 mRNA-electroporated and control CD34+ HSPCs (Figure 3D). HSPC phenotype (CD34+CD38-) was maintained in ~60-70% of cells up to ~48-72 hours, similarly between CXCR4 mRNA-electroporated and control CD34+ HSPCs (Figure 3E). [00157] These pilot data show that electroporation of CXCR4 mRNA in CD34+ HSPCs can successfully induce tunable and transient CXCR4 protein expression while maintaining viability and HSPC phenotype. >99% of cells expressing target mRNA were routinely observed with relatively low cell-to-cell variability and >90% viability. Improved in vitro homing of CXCR4 mRNA-electroporated CD34+ HSPCs towards SDF-1. [00158] The functional activity of various modified mRNAs as measured by the migration of CD34+ HSPCs in vitro towards SDF-1 was examined using a Transwell migration assay [ Kollet, O., et al., Rapid and efficient homing of human CD34(+)CD38(-/low)CXCR4(+) stem and progenitor cells to the bone marrow and spleen of NOD/SCID and NOD/SCID/B2m(null) mice. Blood, 2001. 97(10): p. 3283-91]. Electroporation of CXCR4 mRNAs containing different chemistry modifications dramatically affected expression level (Figure 4A), and significantly increased the number of migrated cells vs. cells (Figure 4B). Improving in vivo short-term engraftment of CXCR4 mRNA-electroporated human CD34+ HSPCs into the bone marrow of NSG mice [00159] The ability of CXCR4 electroporated human CD34+ HSPCs to home in vivo into the bone marrow of sub-lethally irradiated NOD-scid IL2Rg(null) (NSG) mice was evaluated. Briefly, after intravenous transplantation of CXCR4 mRNA-electroporated or control human CD34+ HSPCs, the mice was sacrificed one day later and the human cell chimerism in the bone marrow (BM) was measured. BM homing varied dramatically by CXCR4 mRNA chemical modification (Figure 5A). Hyperactive CXCR4 mutant sequences were examined, including the common WHIM mutant t19 (truncation of the C-terminal 19 amino acids) and the CXCR4 point mutant (119S), which in a transient mRNA context may lead to improved activity or allow the use of a lower dose of introduced CXCR4 mRNA. Evidence demonstrated that these hyperactive CXCR4 sequences may be associated with a BM homing advantage compared with WT CXCR4 (Figure 5B). Long-term engraftment of CXCR4 mRNA-electroporated human CD34+ HSPCs into the bone marrow of NSG mice [00160] CXCR4 mRNA electroporated human CD34+ HSPCs are evaluated for long-term engraftment and multilineage reconstitution needed to sustain long-term hematopoiesis in vivo. Human cell engraftment is evaluated in NSG mice throughout a 26-week monitoring period following transplantation. Engraftment present at end of the 26-week period demonstrates the engraftment durability of CXCR4 mRNA-electroporated HSPCs in transplanted NSG mice. CD34+ HSPCs are obtained from at least 3 healthy human donors, with approximately 3-6 recipient mice per human donor. CXCR4 mRNA electroporated human CD34+ HSPCs is compared to mock electroporated CD34+ HSPCs (no mRNA) and non-electroporated CD34+ HSPCs. Human chimerism is measured as percent human CD45 reconstitution in the bone marrow of engrafted NSG mice at 26 weeks following transplantation. Multilineage reconstitution in bone marrow of engrafted NSG mice at 26 weeks following transplantation is measured as percent human CD3+ T cells, percent human CD19+ B cells, and percent human CD13+ and/or CD33+ granulocyte/monocytes). It is expected that CD34+ HSPCs electroporated with CXCR4 mRNA will demonstrate at least 20% improvement in their homing and engraftment efficiency in vivo in long-term engraftment studies. Example 2 AMELIORATION OF DISEASE IN A MOUSE MODEL OF SICKLE CELL DISEASE (SCD) [00161] Curing SCD can be achieved by hematopoietic cell transplantation (HCT), but the morbidity and mortality of the procedure have precluded the more widespread use of this therapy. Graft failure remains one significant challenge unique to this population. In addition, SCD patients who undergo HCT generally have severe manifestations of the disease. Hence, they are more fragile and prone to complications. Graft-vs-host disease (GVHD), risk of infertility, neurologic toxicities, and chronic pain are factors that negatively impact survival and quality of life post- HCT. Thus, the major challenges of HCT for SCD are to develop transplantation procedures that minimize toxicity without compromising engraftment and to infuse grafts that do not elicit GVHD. [00162] CXCR4 mRNAs are assessed to confer desirable HSPC functional activity for their ability to ameliorate disease in the Townes SCD mouse model. Townes mice were previously used as a preclinical model of SCD to demonstrate successful engraftment of an allogeneic HSPC graft (Bankova, A.K., Pang, W.W., Velasco,B.J., Pyser, J., Long-Boyle, J.R., Shizuru,J,A,. Anti-CD117 Antibody Synergizes with 5-Azacytidine to Augment Engraftment of Hematopoietic Stem Cells in Mice with Sickle Cell Disease in Transplantation & Cellular Therapy Meetings.2021. Online). By transiently introducing CXCR4 mRNA into the HSPCs, we aim to achieve faster and more complete engraftment. Because CXCR4 is highly conserved between mouse and human and human CXCR4 responds to mouse SDF-1, it is believed that lead human CXCR4 will have activity when electroporated into mouse HSPCs. [00163] Initially, electroporated allogeneic mouse CD117+ HSPCs are evaluated for short-term engraftment in the bone marrow of Townes mice between 1-4 days after transplantation. Since mouse HSPCs do not express CD34, CD117 is used as the phenotypic marker to isolate HSPCs by fluorescence activated cell sorting (FACS). CXCR4 mRNA electroporated, mock electroporated, and non-electroporated CD117+ HSPCs are transplanted intravenously into Townes mice conditioned with sublethal methods, including anti-CD117 antibody (ACK2)-based combinations with azacitidine and low dose irradiation, which are sacrificed between 1-4 days later. Bone marrow is analyzed for the presence of allogeneic HSPCs by flow cytometry. CXCR4 mRNA electroporated allogeneic mouse CD117+ HSPCs are also evaluated for long-term engraftment and multilineage reconstitution needed to sustain long-term hematopoiesis, including erythropoiesis, in vivo. Allogeneic HSPC engraftment are evaluated in Townes mice throughout a 16 week monitoring period following transplantation, which includes measurements of complete blood counts and donor chimerism using flow cytometric analyses of allelic biomarkers (CD45.1 vs CD45.2), blood hemoglobin, and RBC morphology and count. It is expected that CXCR4 expression in the CD117+ HSPCs will result in a 20% or greater improvement in donor chimerism, reticulocyte cell counts, and blood hemoglobin at ~1 month and ~4 months after transplantation. [00164] These studies are expected to establish improvement in engraftment of human CD34+ HSPCs and amelioration of disease in the SCD mouse model, with statistically significant improvement engraftment and in disease outcomes, e.g., 20% or more sustained increased in donor chimerism after 1-4 months of transplantation from mRNA-engineered vs. unmanipulated and mock-electroporated HSPCs. [00165] The various embodiments described herein can be combined to provide further embodiments. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, application and publications to provide yet further embodiments. 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 [00168] All of the U.S. patents, U.S. patent application publications, U.S. patent application, foreign patents, foreign patent application 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 cell comprising an exogenous or introduced nucleic acid sequence encoding a CXCR4 polypeptide, including functional fragments or variants thereof, optionally wherein the CXCR4 polypeptide is transiently expressed in the cell.
2. The modified cell of claim 1, wherein the cell is a stem cell or a progenitor cell.
3. The modified cell of claim 2, wherein the cell is a hematopoietic stem cell (HSC) or a hematopoietic stem and progenitor cell (HSPC).
4. The modified cell of any one of claims 1-3, wherein the cell is CD34+, optionally wherein the cell is CD34+/CD90+, CD34+/CD38-, or CD34+/CD38-/CD90+, or CD34+CD133+.
5. The modified cell of any one of claims 1-4, wherein the cell is a human cell.
6. The modified cell of any one of claims 1-5, wherein the cell was obtained from a mammalian donor, optionally a human donor.
7. The modified cell of claim 6, wherein the mammalian donor is a healthy donor or a subject in need of a hematopoietic stem cell transplant.
8. The modified cell of any one of claims 1-7, wherein the cell expresses the CXCR4 polypeptide, optionally wherein the modified cell expresses the CXCR4 polypeptide transiently.
9. The modified cell of claim 8, wherein the CXCR4 polypeptide is expressed on the cell surface.
10. The modified cell of any one of claims 1-9, wherein the CXCR4 polypeptide comprises one of the following sequences:
Figure imgf000058_0001
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, optionally wherein the CXCR4 polypeptide or variant or fragment thereof comprises an amino acid substitution at the amino acid corresponding to position 119, such as a 119S substitution, or a C-terminal deletion, such as a truncation of the C-terminal about 19 amino acids.
11. The modified cell of any one of claims 1-9, wherein the exogenous or introduced sequence is encoded by a polynucleotide sequence that comprises or consists of one of the following: ATGTCCATTC CTTTGCCTCT TTTGCAGATA TACACTTCAG ATAACTACAC
Figure imgf000058_0002
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
( Q ) 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.
12. The modified cell of any one of claims 1-11, wherein the polypeptide or polynucleotide sequence is humanized.
13. A pharmaceutical composition comprising a population of modified cells of any one of claims 1-12, and a pharmaceutically acceptable excipient, carrier, or diluent.
14. A method of treating a mammalian subject in need thereof, comprising administering to the subject the modified cell of any one of claims 1-11 or the pharmaceutical composition of claim 13.
15. The method of claim 35, wherein the subject is also 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.
16. The method of any one of claims 14-15, wherein the conditioning regimen comprises or consists of one or more of: an anti-CD117 monoclonal antibody, optionally JSP191, total body irradiation, or a chemotherapeutic agent, optionally azacytidine.
17. The method of any one of claims 14-16, wherein the method results in one or more of the following: i) improved HSCs/HSPCs migrate towards SDF-1; ii) improved HSCs/HSPCs homing to bone marrow in the subject iii) improved HSCs/HSPCs engraftment in the bone marrow, optionally measured by higher donor myeloid chimerism in the bone marrow and/or in the peripheral blood; and/or iv) improved neutrophil and/or platelet recovery following administration of the modified cell.
18. The method of any one of claims 14-17, 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.
19. The method of claim 18, wherein the cancer is a solid tissue cancer or a blood cancer.
20. The method of claim 19, wherein the blood cancer is a leukemia, a lymphoma, or a myelodysplastic syndrome.
21. The method of claim 20, wherein the leukemia is acute myeloid leukemia (AML).
22. The method of claim 18, wherein the immunodeficiency is severe combined immunodeficiency (SCID).
23. The method of claim 18, wherein the genetic disorder is sickle cell disease or Fanconi anemia.
24. The method of any one of claims 14-23, further comprising administering to the subject a therapeutic agent for treatment of the disease or disorder.
25. A modified mRNA encoding a CXCR4 polypeptide, including variants thereof, wherein the modified mRNA comprises one or more nucleoside substitutions and/or a cap sequence.
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