WO2024036214A2 - 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
WO2024036214A2
WO2024036214A2 PCT/US2023/071937 US2023071937W WO2024036214A2 WO 2024036214 A2 WO2024036214 A2 WO 2024036214A2 US 2023071937 W US2023071937 W US 2023071937W WO 2024036214 A2 WO2024036214 A2 WO 2024036214A2
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polypeptide
modified
nucleic acid
exogenous
cell
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PCT/US2023/071937
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French (fr)
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WO2024036214A3 (en
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Wendy PANG
Rajiv TIWARI
Hye-Sook Kwon
Judith SHIZURU
Robert Sikorski
Leopold D’ESPAUX
Song Eun Lee
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Jasper Therapeutics, Inc.
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Publication of WO2024036214A2 publication Critical patent/WO2024036214A2/en
Publication of WO2024036214A3 publication Critical patent/WO2024036214A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/10—Protein-tyrosine kinases (2.7.10)
    • C12Y207/10001—Receptor protein-tyrosine kinase (2.7.10.1)

Definitions

  • the present disclosure relates to modified or engineered hematopoietic stem and progenitor cells, and their use for hematopoietic cell transplantation.
  • 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.
  • Hematopoietic cell transplantation may also be performed in the context of gene therapy, in order to provide to a patient hematopoietic stem cells that express a nucleic acid or protein missing or mutated in the patient’s endogenous hematopoietic cells.
  • 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. 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.
  • 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 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 provides inter alia 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 one or more exogenous or introduced nucleic acids encoding a combination of two or more of: a CD47 polypeptide, a modified CD117 polypeptide with constitutive activity, a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning, and/or a CXCR4 polypeptide, or functional fragments or variants thereof.
  • the modified or engineered cell comprises: (1) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity; (2) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning; (3) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (4) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity and an exogenous or introduced nucleic acid encoding a modified CD 117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning; (5) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with
  • each polypeptide is encoded by a different introduced nucleic acid, so the cells may comprise two or more different introduced nucleic acids.
  • two or more polypeptides may be encoded by a single introduced nucleic acid, so the cells may comprise only one introduced nucleic acid.
  • the introduced nucleic acids are transiently present in the cells and/or transiently express the polypeptide(s) in the cells, e.g., for about one day to about 10 days, or about one day to about one week, e.g., for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.
  • the cell is transduced with one or more vectors disclosed herein or one or more mRNAs, e.g., the modified or engineered cell comprises an exogenous or introduced polynucleotide sequence encoding a CD47 polypeptide, one or more modified CD117 polypeptide, and/or a CXCR4 polypeptide.
  • the cell is a stem cell, e.g., an 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 a CD47 polypeptide, a CD117 polypeptide, and/or a CXCR4 polypeptide, optionally wherein the modified cell expresses the two or more CD47, CD 117, and/or CXCR4 polypeptides transiently.
  • the CD47 polypeptides, one or more CD47 polypeptide, and/or CXCR4 polypeptides are human polypeptide, or a variant or fragment thereof.
  • the CD47 polypeptide is a modified CD47 polypeptide, e.g., with increased or constitutive activity as compared to the corresponding wild type CD47 polypeptide.
  • the CD117 polypeptide is a modified CD117 polypeptide, e.g., with constitutive activity as compared to the corresponding wild type CD117 polypeptide.
  • the modified CD117 polypeptides provide for constitutive signaling and/or CD117-mediated kinase activity when expressed in cells, e.g., HSCs and/or HSPCs. Accordingly, in particular embodiments, when expressed in HSCs and/or HSPCs, the modified CD117 polypeptides allow CD117 signaling when bound by antibodies that block SCF binding to CD117.
  • the modified CD117 polypeptide comprises one or more amino acid modifications as compared to a wild type CD117 polypeptide, e.g., one or more amino acid substitutions, insertions, or deletions with increased or constitutive activity as compared to the corresponding wild type CD117 polypeptide.
  • the modified CD117 polypeptide comprises one or more amino acid substitutions, e.g., at one or more of the following amino acids present in wild type human CD117: N505, V559, D816, V568, V570, Y703, or D816, such as, e.g., a D816V substitution and/or a N505I substitution.
  • the CD117 polypeptide is a modified polypeptide, e.g., which does not bind the anti-c-Kit antibody used for HCT conditioning.
  • the modified CD117 polypeptide provides SCF-mediated signaling and/or CD117-mediated kinase activity when expressed in cells, e.g., HSCs and/or HSPCs. Accordingly, in some embodiments, when expressed in HSCs and/or HSPCs, the modified CD117 polypeptides allow CD117 signaling in the presence of antibodies that block SCF binding to wild type CD117.
  • the modified CD117 polypeptide comprises one or more amino acid modifications as compared to a wild type CD117 polypeptide, e.g., one or more amino acid substitutions, insertions, or deletions, e.g., which inhibit binding to an anti-c-Kit antibody used for HCT conditioning.
  • the modified CD117 polypeptide comprises one or more amino acid substitutions, e.g., at one or more of the following amino acids present in wild type human CD117: E73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, or R271, or at an amino acid residue located within 2, within 3, within 4, within 5, within 6, within 7 within 8 within 8 within 10, within 11 or within 12 amino acids of any ofE73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, or R271, e.g., either N-terminal or C-terminal of any of these residues.
  • the one or more amino acid modifications is located within surface exposed amino acid residues of the extracellular domain of the wild type CD117 polypeptide.
  • 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 tl9 deletion corresponding to deletion of the C-terminal 19 amino acid residues of CXCR4.
  • 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.
  • 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 nuclease
  • zinc finger nuclease gene editing technology e.g., gene editing may have been performed to correct a genetic mutation present in the cell, e.g., in the context of autologous HCT.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the modified cells, e.g., HSCs and/or HSPCs, comprising one or more nucleic acids encoding the CD47 polypeptide, the CD117 polypeptide, and/or 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 CD47, CD117, and/or 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 one or more nucleic acid or vector encoding two or more of a CD47 polypeptide, a CD117 polypeptide (constitutively active), a CD117 polypeptide (not bound by an anti-CD117 antibody) and/or 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.
  • HCT hematopoietic cell transplantation
  • the nucleic acid or vector is introduced into the cell by transfection, transduction, infection, electroporation, or nanopore technology.
  • the nucleic acid e.g., mRNA
  • the nucleic acid is introduced into the cell using lipid nanoparticles (LNPs), liposomes, nanomechanical methods, or other modalities.
  • LNPs lipid nanoparticles
  • the nucleic acid, e.g., mRNA may be present within or bound to an LNP or liposome.
  • 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 one or more nucleic acid or vector encoding two or more of a CD47 polypeptide, a CD117 polypeptide (constitutively active), a CD117 polypeptide (not bound by an anti-CD117 antibody) and/or a CXCR4 polypeptide.
  • modified cells e.g., HSCs and/or HSPCs
  • 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).
  • expression of the combination of two or more of CD47, CD117(s), and/or CXCR4 (e.g., transiently) in HSCs and/or HSPCs improves HCT.
  • CD47, CD117, and/or CXCR4 transient expression in HSCs and/or HSPCs improves HSCs/HSPCs ability to bind signal regulatory protein a (SIRPa) in vitro.
  • SIRPa signal regulatory protein a
  • CD47, variant CD117, and/or CXCR4 transient expression in HSCs and/or HSPCs improves the ability of HSCs/HSPCs that are injected intravenously to home to the bone marrow.
  • CD47, variant CD 117, and/or 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.
  • CD47, variant CD117, and/or 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).
  • 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.
  • Figure 1 is a chart depicting illustrative myeloablative, reduced intensity myeloablative, and non-myeloablative conditioning regimens that may be used according to the disclosure, and is 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.
  • FIG. 2 is a graph showing the OD595 of wild type BaF3 cells (BaF3), or BaF3 cells expressing wild-type CD117 (c-Kit) or the CD117-D816V mutant in the presence of the indicated concentrations of stem cell factor (SCF), and in the presence or absence of the anti- CD117 antibody, JSP191.
  • the mRNA construct expressing wild-type CD117 corresponds to ctl80, which includes the CleanCap cap and includes Nlm-pseudouridine instead of uridine.
  • Figures 3A-3C are graphs showing binding of JSP191 (Figure 3A) or AB85 ( Figure 3B (whole view) and Figure 3C (zoomed in view)) to clones of an alanine scanning library. Mean binding value is plotted as a function of expression (represented by clone reactivity) for each clone.
  • Figure 4 is a table showing the results of alanine scanning of JSP191Fab, AB85Fab and 104D2 Mab. Mean binding reactivity (and range) of each to clones comprising the indicated point mutations in CD117 is shown as % binding to wild type CD 117. Critical residues for Ab binding are shaded.
  • Figures 5A-5B show crystal structures of JSP191 Fab (Figure 5A) and AB85 Fab ( Figure 5B). Critical residues of CD117 (dark spheres) are shown on the crystal structure of the target Abs (Yuzawa et al., 2007).
  • Figure 6 is a table listing the critical residues whose mutation gave the lowest reactivities with the JSP191 or AB85 antibody.
  • FIG. 7 is a schematic of the synthesis of 5 CXCR4 mRNA variants by IVT.
  • Figure 8 shows isolation of human CD34+ HSPCs from mobilized peripheral blood using the 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.
  • Figures 9A-9E show electroporation of human CD34+HSPCs with CXCR4 mRNA.
  • Figure 9A shows mRNAs encoding various CXCR4 sequences containing various chemical modifications were electroporated and characterized.
  • Figure 9B shows 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 NlmPsU/5mC modification; CXCR4 wild type with PsU modification; CXCR4 tl9 mutant with NlmPsU modification; and CXCR4 wild type with NlmPsU modification.
  • Figure 9C shows 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 (ct73 with TriLink UTRs and 120 A poly A tail) electroporated over time.
  • the mRNA used was CXCR4 wild type with 5moU modification.
  • Figure 9D shows viability as measured by flow cytometry staining of live cells of CD34+ HSPCs that were not electroporated (Control), mock electroporated, and CXCR4 mRNA (ct73 with TriLink UTRs and 120 A poly A tail) electroporated over time post-electroporation.
  • FIG. 10A is a graph showing CXCR4 mRNAs containing various chemistries and their corresponding expression levels.
  • XI corresponds to ct73, and the others correspond to ct2 with the chemistry as indicated. All mRNAs encode wild type CXCR4.
  • Figure 10B is a bar graph showing various mRNA chemistries led to differences in transwell migration.
  • mRNA “A” contained Nlm-pseudouri dine U-substitution, and CleanCapAG-3’OMe (TriLink).
  • Figures 11A and 11B show the homing of CXCR4 mRNA electroporated human CD34+ HSPCs into the bone marrow of NSG mice shortly after transplantation.
  • Figure 11A shows the frequency of HSPCs homing to the BM at ⁇ 16 hours post injection of mRNA ct73 with TriLink UTRs and chemistry as indicated.
  • mRNAs encoding WT CXCR4 were synthesized with the indicated nucleoside substitutions. All mRNAs encoded wild type CXCR4.
  • Figure 11B shows mRNAs with the same chemistry (NlmPsU) but the indicated CXCR4 mutant sequences (ct74 and ct75) with CleanCapAG-3OMe cap and NlmPsU sub stituti on of Us) .
  • Figure 12 shows CD47 overexpression in human CD34+ cells electroporated with CD47-mr7 mRNA, relative to control cells and mock mRNA.
  • Figures 13 A and 13B show the relative percentages of human CD45+ cells after transplant with CD34+ cells transfected with CD47-mr7 mRNA.
  • Figure 13 A shows the percent of human CD45+ cells in the bone marrow 1 day after transplant
  • Figure 13B shows the percent of human HSC derived granulocytes in the blood system 1 month after transplant.
  • Figure 14 shows CD47 expression relative to each indicated polyA.
  • Figure 15 shows CD47 expression relative to the mRNA used and the dose of each mRNA used.
  • Figures 16A and 16B show CD47 expression relative to the UTR included in the mRNA sequence ( Figure 16A) and the position of each 5’ and 3’ UTR used ( Figure 16B).
  • Figure 17 shows CD47 expression relative to the CD47 sequence used.
  • Figure 18 shows CD47 expression in human CD34+ cells relative to the length of the mRNA polyA tail.
  • Figure 19 shows the design of the segmented polyA tail AMOS (SEQ ID NOs: 206 and 207), in which the 140 adenine bases are segmented into 2x 70 adenine sequences.
  • Figure 20 shows the design of an example plasmid that may be used for in vitro transcription of a CD47 mRNA. Similar plasmid constructs may be used for expression of other proteins, such as CXCR4 or cKIT.
  • Figure 21 provides illustrative sequences to be used in the example plasmid of Figure 20 (SEQ ID NOs: 208-213).
  • Figure 22 shows expression levels of CD117 (cKIT), CXCR4, and CD47 3 hours after transfection in human CD34+ cells with the indicated modified mRNAs.
  • Figure 23 shows expression levels of CD117 (cKIT), CXCR4, and CD47 20 hours after transfection human CD34+ cells with the modified mRNAs.
  • Figure 24 shows expression levels of CD117 (cKIT), CXCR4, and CD47 48 hours after transfection human CD34+ cells with the modified mRNAs shown in Table 1.
  • Figure 25 shows human CD34+ live cell numbers 3 hours after transfection with the indicated modified mRNAs.
  • Figure 26 shows human CD34+ live cell numbers 20 hours after transfection with the modified mRNAs.
  • Figure 27 shows human CD34+ live cell numbers 48 hours after transfection with the modified mRNAs.
  • Figure 28 shows human CD34+ cell viability 20 hours after transfection with the modified mRNAs.
  • Figures 29A and 29B are graphs showing that expression of CD117 E73A/N505I double mutant in Ba/F3 cells led to enhanced growth and JSP191 resistance.
  • Cells in these experiments were transfected with lentiviral constructs encoding wild type, E73 A, N505I, and E73A/N505I double mutant CD117 proteins.
  • Figure 29A shows growth of Ba/F3 cells with wild type and E73A/N505I mutant CD117 expression in the presence of human stem cell factor (hSCF).
  • Figure 29B shows growth of Ba/F3 cells with wild type and E73A/N505I mutant CD117 expression in the presence of the JSP191 antibody and hSCF.
  • Figure 30 is a graph of CD117 expression in human CD34+ cells 3 hours after transfection with the modified CD117 mRNAs.
  • Figure 31 is a graph of CD90 expression in human CD34+ cells, expressing high and low levels of CD117, 3 hours after transfection with the modified wild type CD117 mRNA.
  • Figures 32A-32C are graphs of CD117 expression in human CD34+ cells over time showing two distinct levels of wild type and E73A CD 117 mRNA expression at 3 hours after transfection, which resolve by 20 hours after transfection.
  • Figure 32A shows cell count versus CD117 expression 3 hours after transfection;
  • Figure 32B shows cell count versus CD117 expression 20 hours after transfection;
  • Figure 32C shows cell count versus CD117 expression 48 hours after transfection.
  • Figure 33 is a graph of the level of CD117 (cKit) expression for null control (Ctrl), mock electroporation (Mock EP), wild type (WT), E73A, N505I and E73A/N505I double mutant CD117 mRNA in human CD34+ cells post transfection.
  • the various constructs are the same as described for Figure 2 but with the different point mutations introduced.
  • Figure 34 is a graph of the level of CD117 (cKit) expression from modified CD117 mRNAs in human CD34+ cells post transfection.
  • Figure 35 shows cell count versus CXCR4 expression 3 hours after transfection with null control (Ctrl), mock electroporation (Mock EP), wild type (WT), and N119S CXCR4 mutant mRNA in human CD34+ cells.
  • Figure 36 is a graph of the level of CXCR4 expression from modified CXCR4 mRNAs in human CD34+ cells post transfection.
  • Figure 37 is a graph of the level of CXCR4 expression from modified CXCR4 mRNAs in human CD34+ cells post transfection.
  • Figures 38A and 38B are boxplots of human CD34+ cell chimerism in mouse bone marrow, 12 weeks after transplant with human CD34+ cells transfected with modified CXCR4 mRNAs. Also shown is the level of CXCR4 present in the chimeric cells.
  • Figure 39 shows dose-expression correlations of following electroporation of cells with the indicated doses of mRNA constructs encoding wild type of the indicated cKit mutants.
  • Figures 40A and 40B show expression levels of CXCR4, CD47, and cKit three hours following electroporation of cells with the indicate mRNA constructs.
  • PhaRNA and TL cKIT- DV correspond to ctl82 (with chemical modification as indicated, undisclosed UTRs); cKIT WT is ct96, cKIT WT col is ct97, and cKIT_E73A_col is ct98.
  • Figures 41A and 41B show cell number at day 3 ( Figure 41A) and day 12 ( Figure 4 IB) following electroporation of cells with the indicated mRNA constructs and following treatment with the indicated amount of JSP191 antibody. For each antibody concentration shown, the three bars from left to right correspond to control (Ctrl), mock electroporation (Mock EP), and E73 A-N505I.
  • Figure 42 is a graph showing increased expression of CXCR4 and/or CD47 3 hours following electroporation of CD34+ cells with the indicated amounts of mRNA constructs expressing CXCR4 and/or CD47.
  • Figure 43 is a graph showing increased expression of CXCR4 and/or CD47 1 day following administration of CD34+ cells modified via mRNA constructs expressing CXCR4 and/or CD47.
  • Figures 44A and 44B show in vitro expression.
  • Figure 44A is a graph showing cKit expression kinetics following electroporation of mRNAs encoding the indicated cKit into human CD34+ cells. At about 20 hours, the lines from top to bottom correspond to: wild type cKit, unidentified cKit mutant, unidentified cKit mutant, cKit N505I, control, and mock electroporation (EP).
  • Figure 44B shows viability and live cell numbers of CD34+ cells one day following electroporation with the indicated mRNA or control.
  • Figure 45 provides graphs showing CD34+ cell proliferation at 3 days and 12 days following electroporation with the indicated amount of the indicated mRNA construct. For each dose, the bars from left to right correspond to control (Ctrl; not electroporated), mock electroporated (Mock EP), and E73 A-N505I.
  • Figure 46 shows CXCR4 expression at 3 hours post-electroporation.
  • the graph shows CXCR4 expression (fold of baseline) following transfection of each of the indicated CXCR4 mRNA constructs (Jasper ctl23 (wild type), Jasper 119S ctl l4, Jasper 119A ctl25, Jasper 119K ctl25, and Trilink 119S ct75). All tails were A90 unless otherwise specified.
  • Figure 47 shows CXCR4 at various timepoints following electroporation of the indicated CXCR4 mRNA constructs. At about 4 hours post-electroporation, the lines from top to bottom correspond to: N119S_Jasper, WT Jasper, N119S_TriLink, N119A_Jasper, N119K_Jasper, Control, and Mock electroporation (EP).
  • Figure 48 shows CXCR4 expression with various stop codons and UTRs.
  • the left graph shows CXCR4 expression resulting from mRNA constructs comprising the indicated stop codons.
  • the right graph shows CXCR4 expression resulting from mRNA constructs comprising the indicated UTRs:
  • FIG. 49 shows expression of CXCR4 mutants.
  • the top graph shows CXCR4 expression following electroporation with the indicated mRNA construct.
  • the lines from top to bottom correspond to N119S_Jasper, WT Jasper, N119S_TriLink, Control (CTRL), and Mock electroporation (EP).
  • the bottom graph shows cell growth following electroporation of cells with the indicated mRNA constructs.
  • the lines from top to bottom correspond to WT Jasper, N119S_Jasper, Control (CTRL), N119S_TriLink, and Mock electroporation (EP).
  • Figure 50 shows XCR half lives.
  • the top graph shows CXCR4 expression following electroporation of the indicated mRNA constructs. At about 18 hours, the lines from top to bottom correspond to WT Jasper, ctruncl9_Jasper, N119K_Jasper, Control (CTRL), and Mock electroporation (EP).
  • the bottom graph shows CXCR4 expression following electroporation of the indicated mRNA constructs. At about 4 hours, the lines from top to bottom correspond to: N119S_Jasper, N119S-ctruncl9_Jasper, N119S_TriLink, N119A_Jasper, Control, and Mock EP.
  • Figure 51 shows transmigration in a transwell assay.
  • the graph shows the number of migrated cells following electroporation of the cells with the indicated CXCRR mRNA constructs or controls. Quadruple results are provided for each of the following constructs from left to right: Control, Mock EP, and CXCR4 N119S (1 ug), and in the absence of SDF1.
  • Figure 52 shows engraftment of CD34+ cells in NSG mice.
  • the graph shows hCD34 cell chimerism 3 months following transplant of cells electroporated with each of the indicated constructs or controls.
  • CXCR4 expression fold change was 1 for control, 0.96 for mock, 6.43 for CXCR4-WT, and 7.5 for CXCR4-119S.
  • Figure 53 is a graph of CD117 expression from different CD117 mRNAs (Table 1) or controls 20 hours after electroporation.
  • Figures 54A and 54B are graphs of cell viability 20 hours post electroporation with the indicated mRNAs (Table 1) or controls.
  • Figure 54A is a graph of live cell numbers;
  • Figure 54B is a graph of percent viability relative to cell death.
  • Figure 55 is a graph of CD17 expression from different CD117 mRNAs (Table 1) or controls 3 hours after electroporation.
  • Figure 56 is a graph of cell count versus CD117 expression in human CD34+ cells expressing mock, control (null), wild type, and E73A CD117 mRNAs (Table 1).
  • Figures 57A-57C are graphs of CD117 expression in human CD34+ cells over time showing initially two distinct levels of wild type and E73A CD117 expression which resolve by 20 hours after electroporation.
  • Figure 57A shows cell count versus CD117 expression 3 hours after electroporation;
  • Figure 57B shows cell count versus CD117 expression 20 hours after electroporation;
  • Figure 57C shows cell count versus CD117 expression 48 hours after electroporation.
  • Figure 58 is a graph of the level of CD117 (cKit) expression for mock electroporation (Mock EP), null control (Ctrl), wild type (WT), and E73A CD117 expressing cells.
  • Figures 59A and 59B are graphs showing that expression of CD117 E73A mutants in Ba/F3 cells leads to JSP191 resistance.
  • Cells in these experiments were transfected with lentiviral constructs encoding wild type and E73 CD117 proteins.
  • Figure 59A shows growth of Ba/F3 cells based on wild type and E73A CD117 expression in the presence of human stem cell factor (hSCF).
  • Figure 59B shows growth of Ba/F3 cells based on wild type and E73 A CD117 expression in the presence of the JSP191 antibody.
  • Figures 60A-60B are graphs showing the effects of CD117 expression on cell growth in the presence of stem cell factor (SCF) and the JSP91 antibody.
  • Figure 60A shows the growth of human CD34+ cells transfected without mRNA.
  • Figure 60B shows the growth of cells transfected with wild type CD117 mRNA (as encoded by SEQ ID NO: 54).
  • Figure 61 shows cKit expression following electroporation with the indicated amounts of mRNAs encoding WT cKIT or cKit E73 A, or controls.
  • 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. 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.
  • 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.
  • blood stem cell e.g., HSC and/or HSPC
  • the disclosure provides modified or engineered cells, e.g., HSCs and HSPCs, comprising a variant CD117 that is both constitutively active and is not bound by an antibody that binds to CD117, e.g., an antibody used for HCT conditioning, such as JSP191.
  • the modified or engineered cells e.g., HSCs and HSPCs, comprise an introduced or exogenous nucleic acid that encodes the variant CD117.
  • the modified or engineered cells, e.g., HSCs and HSPCs express the variant CD117.
  • the disclosure provides modified or engineered e.g., HSCs and HSPCs, comprising two, three, or four or five modifications selected from: 1) expression of an exogenous or introduced CD47, 2) expression of an exogenous or introduced CXCR4, 3) expression of a variant CD117 that is constitutively active, 4) expression of a variant CD117 that is not bound by an antibody that binds to CD117, and 5) expression of a variant CD117 that is both constitutively active and is not bound by an antibody that binds to CD 117.
  • modified or engineered e.g., HSCs and HSPCs, comprising two, three, or four or five modifications selected from: 1) expression of an exogenous or introduced CD47, 2) expression of an exogenous or introduced CXCR4, 3) expression of a variant CD117 that is constitutively active, 4) expression of a variant CD117 that is not bound by an antibody that binds to CD117, and 5) expression of a variant CD117 that is both constitutively active and is not
  • the modified or engineered e.g., HSCs and HSPCs comprise one or more introduced or exogenous nucleic acid that encodes the CD47, the CXCR4, the constitutively active variant CD117, the variant CD117 that is not bound by an antibody that binds to CD117, and/or a variant CD117 that is both constitutively active and is not bound by an antibody that binds to CD117.
  • the CD47, CXCR4, and/or various variant CD117s may be encoded by different nucleic acids, or two or more may be encoded by a single nucleic acid molecule.
  • the modified or engineered e.g., HSCs and HSPCs express the variant CD117, the CD47, CXCR4, and/or various variant CD 117s.
  • the engineered or modified e.g., HSCs and HSPCs comprises: 1) one or more of the variant CD117s; and 2) expression of introduced or exogenous CD47 and/or expression of introduced or exogenous CXCR4.
  • modified CD117 polypeptides are modified so that they are either: (i) constitutively active or (ii) are not bound by an anti-CD117 antibody used for HCT conditioning, or (iii) both.
  • one or two different modified CD117 polypeptides may be expressed in the modified cells, alone or in combination with CD47 and/or CXCR4.
  • the disclosure provides for compositions and methods for the ex vivo introduction of a polynucleotide encoding a CD47 polypeptide, a CD117 polypeptide, and/or a CXCR4 polypeptide, e.g., human CD47, CD117, and/or CXCR4 polypeptides, by RNA-based and/or DNA-based methods, into cells, e.g., 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.
  • a polynucleotide encoding a CD47 polypeptide, a CD117 polypeptide, and/or a CXCR4 polypeptide, e.g., human CD47, CD117, and/or CXCR4 polypeptides, by RNA-based and/or DNA-based methods, into cells, e.g., HSCs and/
  • compositions and methods disclosed herein are used to introduce the CD47 mRNA, modified CD117 mRNA, and/or CXCR4 mRNA into allogeneic normal HSPCs to improve engraftment and replace a patient’s diseased stem cells with a healthy hematopoietic system.
  • the engineered or modified cells may be transplanted into a subject, e.g., to treat a disease or disorder, including those disclosed herein.
  • Transplantation of these modified or engineered (e.g., genetically engineered) cells, e.g., HSPCs may be done after or in combination with a conditioning regimen, including treatment with antibodies (such as anti-CD117 antibodies, e.g., JSP191).
  • modified or engineered HSPCs may be transplanted alone or in combination with other cells.
  • antibody includes reference to an immunoglobulin molecule immunologically reactive with a particular antigen, and includes both polyclonal and monoclonal antibodies.
  • the term also includes genetically engineered forms such as humanized antibodies, chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies.
  • antibody also includes antigen binding forms of antibodies, including fragments with antigen-binding capability (e.g., Fab', F(ab')2, Fab, Fv and rlgG.
  • the term also refers to recombinant single chain Fv fragments (scFv).
  • the term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies.
  • An antibody binds a particular antigen, and is considered “specific for” the antigen when it preferentially recognizes/binds its target antigen in a complex mixture of proteins and/or macromolecules.
  • An antibody is considered to not bind an antigen when it does not bind the antigen or does not preferentially recognize/bind the antigen in a complex mixture of proteins and/or other macromolecules.
  • an antibody that binds a specific antigen is considered to not bind a modified form of the specific antigen if it binds to the antigen with an at least 5-fold or at least 10-fold lower dissociation constant (KD) than the KD of its binding to the unmodified specific antigen, where increased affinity is associated with lower KD.
  • KD dissociation constant
  • 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 such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • 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.
  • 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.
  • polynucleotide includes, but is not limited to, double- and single-stranded molecules, and mixtures thereof.
  • any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form, whether as RNA or DNA, or a mixture thereof.
  • polypeptide refers to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, to include disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.
  • a polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences.
  • sequence identity refers to the percentage identity obtained when sequences are aligned for maximum correspondence over a comparison window (e.g., a specified region of each of the sequences), which may be calculated by any of the algorithms described herein using default parameters, which are expected to generate the same alignment, in most cases, when applied to similar sequences. Identity is calculated, unless specified otherwise, across the full length of the reference sequence.
  • a sequence-of-interest “shares at least x% identity to” a reference sequence if, when the sequence-of-interest is aligned to the reference sequence, at least x% (rounded down) of the residues in the sequence-of-interest are aligned as an exact match to a corresponding residue in the reference sequence. Gaps may be introduced into the sequence- of-interest and/or the reference sequence to maximize correspondence over the comparison window.
  • Sequence similarity can be determined in a number of different manners.
  • sequences can be aligned using the methods and computer programs, including BLAST, available over the worldwide web at ncbi.nlm.nih.gov/BLAST/. Unless indicated to the contrary, sequence identity is determined using the BLAST algorithm (e.g., bl2seq) with default parameters. Sequence alignments may be performed using the NCBI Blast service (BLAST+ version 2.12.0).
  • FASTA 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.
  • 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. 70: 173-187 (1997).
  • the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).
  • the program has default parameters determined by the sequences inputted to be compared.
  • the sequence identity is determined using the default parameters determined by the program. This program is available also from Genetics Computing Group (GCG) package, from Madison, Wis., USA.
  • GCG Genetics Computing Group
  • FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based upon the following parameters: Mismatch Penalty: 1.00; Gap Penalty: 1.00; Gap Size Penalty: 0.33; and Joining Penalty: 30.0.
  • a "vector” as used herein refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which can be used to mediate delivery of the polynucleotide to a cell.
  • Illustrative vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery vehicles.
  • An "expression vector” as used herein encompasses a vector, e.g., plasmid, minicircle, viral vector, liposome, and the like as discussed herein or as known in the art, comprising a polynucleotide which encodes a gene product of interest, and is used for effecting the expression of a gene product in an intended target cell.
  • An expression vector also comprises control elements operatively linked to the encoding region to facilitate expression of the gene product in the target.
  • control elements e.g., promoters, enhancers, UTRs, miRNA targeting sequences, etc.
  • expression cassette a gene or genes to which they are operably linked for expression.
  • a "promoter” as used herein encompasses a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. Promoters and corresponding protein or polypeptide expression may be ubiquitous, meaning strongly active in a wide range of cells, tissues and species, or it may be cell-type specific, tissue-specific, or species specific. Promoters may be “constitutive,” meaning continually active, or “inducible,” meaning the promoter can be activated or deactivated by the presence or absence of biotic or abiotic factors.
  • “Operatively linked” or “operably linked” refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.
  • mutant refers to a mutant of a reference polynucleotide or polypeptide sequence, for example a native polynucleotide or polypeptide sequence, i.e., having less than 100% sequence identity with the reference polynucleotide or polypeptide sequence.
  • a polypeptide variant comprises at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to a reference polypeptide sequence, e.g., a native polypeptide sequence.
  • a polynucleotide variant comprises at least one nucleobase difference (e.g., substitution, insertion, deletion) relative to a reference polynucleotide sequence, e.g., a native polynucleotide 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 fragments of a reference sequence, 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.
  • Fragments generally comprise less than the full length native nucleic acid or polypeptide sequence, e.g., a fragment may comprise or consist of less than 100%, less than 99%, less than 98%, less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% of the native nucleic acid or polypeptide.
  • the disclosure further includes variants of fragments, e.g., variant having a sequence identity of 50% or more, 60% or more, or 70% or more with a fragment of the native polynucleotide or polypeptide, 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 fragment.
  • polypeptide variants and fragments are considered to be “functional” variants or fragments, if they substantially retain a biological activity of the native polypeptide, e.g., the ability to bind a cognate ligand or receptor, or the ability to modulate a biological process, such as, e.g., a signaling pathway, or cellular proliferation, differentiation, or apoptosis.
  • CD47 polypeptide encompasses native or wildtype CD47, CD 117, and CXCR4 polypeptides, as well as functional variants and functional fragments of a native polypeptide
  • CD47 polynucleotide encompasses native or wild-type polynucleotides and nucleic acids, and variants and fragments thereof, that encode a CD47, CD 117, or CXCR4 polypeptide (including functional fragments and variants thereof).
  • Functional variants retain at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% or more, of a biological activity of a reference polypeptide, e.g., kinase activity or binding activity.
  • 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 used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, e.g., reducing the likelihood that the disease or symptom thereof occurs in the subject, and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal and includes: (a) 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.
  • 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.).
  • mammalian sport animals e.g., horses
  • mammalian farm animals e.g., sheep, goats, etc.
  • mammalian pets dogs, cats, etc.
  • rodents e.g., mice, rats, etc.
  • CD47 Leukocyte surface antigen CD47
  • OA3, integrin associated protein (IAP), and MER6 is a protein that in humans is encoded by the CD47 gene.
  • the CD47 protein is a signal regulatory protein a (SIRPa) and SIRPy receptor.
  • SIRPa signal regulatory protein a
  • HSPC hematopoietic stem and progenitor cells
  • the CD47 polypeptide including functional fragments and variants, binds to SIRPa, which is a regulatory membrane glycoprotein expressed mainly in HSPC and neurons.
  • CD47 functional fragments and variants bind to SIRPa with at least 50%, at least 75%, or at least 90% of the specificity and affinity as a corresponding wild type CD47 protein.
  • CD47 protein including functional fragments or variants thereof, may be used according to aspects of the disclosure.
  • the CD47 polypeptide is a human CD47 polypeptide, while in other embodiments, it is another mammalian CD47 polypeptide. Sequences of human and mammalian CD47 polypeptides are known in the art.
  • the CD47 polypeptide sequence comprises or consists of one of the following amino acid sequences: MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYV KWKFKGRDIYTFDGALNKSTVPTDF S S AKIEVSQLLKGD ASLKMDKSD AVSHTGNY TCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGM DEKTIALL VAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIA ILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQ PPRKAVEEPLNAFKESKGMMNDE (SEQ ID NO: 55);
  • ILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQ PPRKAVEEPLNE SEQ ID NO: 56
  • ILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQ PPRNN (SEQ ID NO: 57) or a variant or fragment thereof of any of these sequences, 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.
  • the CD47 polypeptide sequence, or fragment or variant thereof is encoded by a polynucleotide sequence that comprises or consists of one of the following:
  • Illustrative mRNA constructs/sequences of CD47 are provided in Table 2 below. These sequences generally correspond to the plasmid sequence and typically include the T7 polymerase promoter but do not show the poly A sequence. For all constructs/sequences disclosed herein, in the context of RNA, the Ts are Us, and in particular embodiments, the Ts are fully substituted with NlmPsU. Coding sequence is capitalized. Note that throughout the specification, construct sequences are generally specified T7 promoter 3’UTR and do not include poly A tail sequences, except for AMOS. mRNA constructs electroporated into cells may lack the T7 promoter sequence.
  • the T7 promoter sequence comprises the sequence
  • the T7 polymerase starts transcription at the underlined G in the promoter sequence.
  • the polymerase then transcribes using the opposite strand as a template from 5’-> 3'.
  • the first base in the transcript wifi be a G.
  • Illustrative isoform 2 CD47 mRNA constructs/sequences are provided in Table 3 below Illustrative mRNA constructs/sequences are provided in the table below.
  • Ts are Us, and in particular embodiments, the Ts are fully substituted with NlmPsU. Coding sequence is capitalized.
  • construct sequences are generally specified T7 promoter 3’UTR and do not include poly A tail sequences, except for AMOS.
  • mRNA constructs electroporated into cells may lack the T7 promoter sequence.
  • the T7 promoter sequence comprises the sequence TAATACGACTC/kCTATAG (SEQ II) NO: 86).
  • the T7 polymerase stans transcription at the underlined G in the promoter sequence.
  • the polymerase then transcribes using the opposite strand as a template from 5’->3’.
  • the first base in the transcript will be a G.
  • These mRNAs include the 5’ HBA1 UTR, the 3’ HBB1 UTR, and the TAATAA stop codon.
  • the sequence encoding CD47 is capitalized, and the TAATAA stop codon is capitalized.
  • Ts are Us, and in particular embodiments, the Ts are fully substituted with NlmPsU. Coding sequence is capitalized.
  • construct sequences are generally specified T7 promoter 3’UTR and do not include poly A tail sequences, except for AMOS.
  • mRNA constructs electroporated into cells may lack the T7 promoter sequence.
  • the T7 promoter sequence comprises the sequence TAATACXkACTCACTATAGXSEQJD N0;_J6).
  • the T7 polymerase starts transcription at the underlined G in the promoter sequence.
  • the polymerase then transcribes using the opposite strand as a template from 5’->3’.
  • the first base in die transcript will be a G.
  • CD 117 also known as c-kit or stem cell factor receptor (SCFR) has a molecular weight of 145 kDa as a mature protein and is a member of the type III receptor tyrosine kinase (RTK) family that includes platelet-derived growth factor (PDGF) receptors and the macrophage colony-stimulating factor 1 (CSF-1) (c-fms) receptor.
  • RTK type III receptor tyrosine kinase
  • PDGF platelet-derived growth factor
  • CSF-1 macrophage colony-stimulating factor 1
  • CD117 is essential for the development of normal hematopoietic cells and plays an important role in the survival, proliferation, and differentiation of mast cells, melanocytes, and germ cells. It is expressed by hematopoietic cells in the embryonic liver throughout development, and by more committed progenitors, such as myeloid, erythroid, megakaryocytic, natural killer, and dendritic progenitor cells
  • CD117 includes an approximately 519 amino acid extracellular domain comprised of five immunoglobulin-like domains, a transmembrane segment, a juxtamembrane domain, and a protein kinase domain that contains an insert of about 80 amino acid residues. Approximately 184 amino acids of the extracellular domain are surface exposed, which were identified based on x-ray crystallographic studies.
  • CD117 The crystallographic structure of CD117 is provided in, e.g., Mol, et al., Accelerated Publications, Volume 278, ISSUE 34, P31461-31464, August 22, 2003; Ogg et al., RCSB Protein Data Bank, 6XV9, Crystal structure of the kinase domain of human c-KIT in complex with a type-II inhibitor, DOI: 10.2210/pdb6XV9/pdb; McAuley et al., RCSB Protein Data Bank Alkynyl Benzoxazines and Dihydroquinazolines as Cysteine Targeting Covalent Warheads and Their Application in Identification of Selective Irreversible Kinase Inhibitors, DOI: 10.1021/jacs.9bl3391; Schimpl et al., RCSB Protein Data Bank 6GQM, Crystal structure of human c-KIT kinase domain in complex with a small molecule inhibitor, AZD3229, DO
  • CD117 to its ligand (stem cell factor; SCF) induces receptor dimerization, trans autophosphorylation of the kinase domain, recruitment of signaling proteins via phosphotyrosine binding or Src homology 2 (SH2) domains, and subsequent signal transduction.
  • SCF stem cell factor
  • the disclosure provides modified CD117 polypeptides comprising one or more amino acid modifications as compared to a wild type CD117 polypeptide.
  • the one or more amino acid modifications comprise one or more amino acid substitutions, insertions, or deletions.
  • the one or more amino acid modifications are located in the extracellular domain of the CD117 polypeptide.
  • the one or more amino acid modifications are located in one or more surface exposed amino acids of the CD117 polypeptide’s extracellular domain.
  • the modified CD117 polypeptides comprise one or more deletions, e.g., an N- terminal or C-terminal deletion, optionally wherein the deletion does not substantially impair biological activity, e.g., signaling, of the modified CD117 polypeptide.
  • the modified CD117 polypeptides retain or have at least 90%, at least 95%, at least 98%, or at least 99% sequence homology to the wild type CD117 polypeptide.
  • the one or more amino acid modifications do not substantially inhibit or reduce binding of stem cell factor (SCF) to the modified CD117 polypeptide when expressed in cells, as compared to the binding of SCF to the wild type CD117 polypeptide.
  • SCF stem cell factor
  • the amino acid substitution is a conservative amino acid substitution.
  • conservative substitution denotes that one or more amino acids are replaced by another, biologically similar residue.
  • the wild type CD117 polypeptide upon which the variant is based is a human CD117 polypeptide, while in other embodiments, it is another mammalian CD117 polypeptide. Sequences of human and mammalian CD117 polypeptides are known in the art. Due to alternative splicing of the c-kit gene, the human CD117 polypeptide is expressed as various isoforms, and any of these may be used according to the disclosure.
  • GNNK+ and GNNK- isoforms include two GNNK+ and GNNK- isoforms (also denoted c-Kit and c-KitA, respectively), which differ by the presence or absence of four amino acids, GNNK, and which are coexpressed in most tissues, although the GNNK- isoform usually predominates.
  • the wild type CD 117 polypeptide is the GNNK+ or GNNK- isoform and comprises or consists of one of the following amino acid sequences:
  • the modified CD117 polypeptides substantially retain kinase activity as compared to the wild type CD117 polypeptide, e.g., when bound by stem cell factor (SCF).
  • SCF stem cell factor
  • the modified CD117 polypeptide has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the kinase activity of the wild type CD117 polypeptide, e.g., when bound by SCF.
  • the modified CD117 polypeptide has increased kinase activity as compared to the wild type CD117 polypeptide.
  • the modified CD117 polypeptide has at least 150%, at least 200%, at least 300%, at least 500%, at least 750%, or at least 1000% of the kinase activity of the wild type CD117 polypeptide.
  • Kinase activity may be determined using assays known in the art, including the ADP-GloTM Kinase Assay, which is a luminescent kinase assay that measures ADP formed from a kinase reaction; ADP is converted into ATP, which is converted into light by Ultra-GioTM Luciferase (available from Promega Corporation, Madison, WI).
  • the one or more amino acid modifications do not substantially inhibit or reduce binding of stem cell factor (SCF) to the modified CD117 polypeptide when expressed in cells, as compared to the binding of SCF to the wild type CD117 polypeptide.
  • the modified CD117 retains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of binding to SCF as compared to the corresponding wild type CD117.
  • the one or more amino acid modifications do not result in cells expressing only the modified CD117 having substantially inhibited or reduce CD117 signaling or proliferation or viability, optionally in response to SCF binding, as compared to the signaling in cells only expressing the wild type CD117 polypeptide.
  • the modified CD117 retains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% CD117 signaling and/or proliferation and/or viability, optionally in response to SCF binding, as compared to the corresponding wild type CD117.
  • CD117 signaling or proliferation or viability may be determined using methods standard in the art.
  • CD117 signaling or proliferation e.g., in response to SCF
  • a cell line e.g., Ba/F3 cells
  • Cells are cultured in the presence of IL-3, with or without stem cell factor (SCF), and in the presence or absence of an anti-CD117 antibody, e.g., JSP191.
  • SCF stem cell factor
  • an anti-CD117 antibody e.g., JSP191.
  • Control parental Ba/F3 cells do not proliferate in the absence of IL-3.
  • parental Ba/F3 cells do not express CD117 and are not responsive to SCF signaling. Proliferation in response to SCF binding may this be determined for cells overexpressing the modified CD117, e.g., in the presence and absence of SCF and/or the anti-CD117 antibody.
  • the modified CD117 has constitutive signaling or kinase activity, even in absence of SCF binding to the modified CD117 and/or in the presence of an anti-c-Kit antibody that blocks SCF binding to the modified CD117 polypeptide.
  • the modified CD117 has constitutive autophosphorylation activity, e.g., without bound SCF.
  • a variety of such modified CD117 have been identified, e.g., in cancer cells, and any of these may be used according to the compositions and methods disclosed herein.
  • activating or gain-of-function CD117 modifications include, but are not limited to, N505I, V559D, D816V, D816H, V568F, V570F, or Y703F, modifications or mutation of amino acid residues corresponding to 505, 522, 816, 557, 558, 559, 568, 569, 570, 703, 816, or deletion of codon 579 (Asp). See, e.g., Akin and Metcalfe, Journal of Allergy and Clinical Immunology, Vol. 114, Issue 1, p 13- 19, July 1, 2004; Hirotakoji et al., Science 23, Jan 1998, Vol. 279, Issue 5350, pp.
  • the amino acid modification is in the region between the transmembrane and tyrosine kinase domains. Mutations causing constitutive activation of c-Kit have been shown to be causative in some forms of mastocytosis, and several types of mutations have been associated with myeloproliferative disorders (MPDs), acute myelogenous leukemia (AML), sinonasai lymphomas, and gastrointestinal stromal tumors (GIST).
  • MPDs myeloproliferative disorders
  • AML acute myelogenous leukemia
  • GIST gastrointestinal stromal tumors
  • the one or more amino acid modifications do not result in cells expressing only the modified CD117 having substantially inhibited or reduce c-Kit signaling or proliferation, optionally in response to SCF binding, as compared to the signaling in cells only expressing the wild type CD117 polypeptide.
  • the modified CD117 retains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% c-Kit signaling and/or proliferation, optionally in response to SCF binding, as compared to the corresponding wild type CD117.
  • the modified CD117 retains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% c-Kit signaling and/or proliferation, in the absence of SCF binding, as compared to the corresponding wild type CD117.
  • the one or more amino acid modifications do not substantially inhibit or reduce binding of an anti-c-Kit antibody to the modified CD117 polypeptide expressed in cells as compared to the wild type CD117 polypeptide.
  • the anti-c-Kit antibody comprises the six CDRs present in any one of JSP191, AB85, CDX-0159, or FSI-174.
  • the anti-c-Kit antibody in any one of JSP191, AB85, CDX-0159, or FSI-174.
  • the one or more amino acid modifications comprise one or more amino acid substitutions or deletions of an amino acid residue selected from the following in human CD117: N505, 522, 557, 558, V559, V568, 569, V570, 579, 703, and D816.
  • the amino acid residue is substituted by Alanine.
  • the one or more amino acid modifications is a deletion of amino acid 579.
  • the one of more amino acid substitutions comprises a D816V substitution and/or a N505I substitution.
  • the modified CD117 polypeptide having constitutive kinase activity comprises one or more of the following amino acid substitutions: N505I, V559D, D816V, D816H, V568F, V570F, and/or Y703F.
  • the modified CD117 has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 750%, or at least 1000% of the kinase activity of the wild type CD117 polypeptide in the presence of an anti-c-kit antibody and comprises the amino acid substitutions of N505I.
  • the one or more amino acid modifications do not substantially inhibit or reduce binding of an anti-c-Kit antibody to the modified CD117 polypeptide expressed in cells as compared to the wild type CD117 polypeptide.
  • the modified CD117 polypeptide comprises or consists of either of the following sequences:
  • the constitutively active modified CD117 polypeptides are still bound by an anti-CDl 17 antibody used for HCT conditioning, such as, e.g., JSP191.
  • T7 promoter sequence comprises the sequence TAATACGACTCACTATAG (SEQ ID NO: 86).
  • the T7 polymerase starts transcription at the underlined G in the promoter sequence.
  • the polymerase then transcribes using the opposite strand as a template from 5 ’ ->3 ’ .
  • the first base in the transcript will be a G.
  • the modified CD117 polypeptide does not bind an anti-c-kit antibody used for HCT conditioning, e.g., JSP191 and/or AB85.
  • the modified CD117 binds stem cell factor (SCF), even in the presence of the anti-c-Kit antibody.
  • SCF stem cell factor
  • the modified CD117 polypeptide has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the kinase activity of the wild type CD117 polypeptide, in response to SCF signaling and in the presence of an anti-c-kit antibody.
  • the modified CD117 comprises one or more amino acid substitutions, e.g., atE73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, orR271, or at an amino acid residue located within 2, within 3, within 4, within 5, within 6, within 7 within 8 within 8 within 10, within 11 or within 12 amino acids of any of E73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, or R271, e.g., either N-terminal or C-terminal of any of these residues.
  • amino acid substitutions e.g., atE73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, orR271, or at an amino acid residue located within 2, within 3, within 4, within 5, within 6, within 7 within 8 within 8 within 10, within 11 or within 12 amino acids of any of E73, D121, R122, S123
  • the modified CD117 polypeptide that does not bind a CD117 antibody comprises a substitution or a deletion at one or more of the following residues: E73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, or R271.
  • the modified CD117 that does not bind a CD117 antibody comprises an Alanine substitution at one or more of the following residues: E73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, or R271.
  • the modified CD117 comprises a conservative or a non-conservative amino acid substitution of any one of these amino acid residues.
  • the CD117 modification is at any of E73, D121, R122, S123, Y125, K203, or at an amino acid residue located within 2, within 3, within 4, within 5, within 6, within 7 within 8 within 8 within 10, within 11 or within 12 amino acids of any of these amino acids, and the modified or variant CD117 does not bind the JSP191 antibody.
  • the CD117 modification is at any of K203, Y259, S261, W262, Y269, or R271, or at an amino acid residue located within 2, within 3, within 4, within 5, within 6, within 7 within 8 within 8 within 10, within 11 or within 12 amino acids of any of these amino acids, and the modified or variant CD117 does not bind the AB85 antibody.
  • the modified CD117 polypeptide substantially retains kinase activity as compared to the wild type CD117 polypeptide, and also does not bind an anti-c-Kit- antibody, e.g., JSP191 and/or AB85, used for HCT conditioning.
  • the modified CD117 polypeptide has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the kinase activity of the wild type CD117 polypeptide, in response to SCF signaling and in the presence of an anti-c-kit antibody.
  • the modified CD117 polypeptide has at least 150%, at least 200%, at least 300%, at least 500%, at least 750%, or at least 1000% of the kinase activity of the wild type CD117 polypeptide in the presence of an anti-c-kit antibody, and comprises one or more substitutions or deletions in an amino acid residue selected from the following in human CD117: E73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, and R271.
  • any of the modified CD117 polypeptides not bound by an anti-CD117 antibody used for HCT conditioning such as, e.g., JSP191, is not constitutively active.
  • the mRNA comprises an RNA sequence corresponding to the open reading frame DNA sequence of SEQ ID NO: 5:
  • the mRNA comprises an RNA sequence corresponding to the open reading frame DNA sequence of SEQ ID NO:6:
  • the mRNA comprises an RNA sequence corresponding to the open reading frame DNA sequence of SEQ ID NO: 7:
  • Illustrative modified CD117 mRNA constructs and sequences are provided below in Table 6, with the coding sequence capitalized.
  • the Ts are Us, and in particular embodiments, the Ts are fully substituted with NlmPsU. Coding sequence is capitalized.
  • construct sequences are generally specified T7 promoter 3’UTR and do not include poly A tail sequences, except for AMOS.
  • mRNA constructs electroporated into cells may lack the T7 promoter sequence.
  • the T7 promoter sequence comprises the sequence TAATACXUkC’rCACI'A'fAG (SEQ ID NO: 86).
  • the T7 polymerase starts transcription at the underlined G in the promoter sequence.
  • the polymerase then transcribes using the opposite strand as a template from 5 ’ ->3 ’ .
  • the first base in the transcript will be a G.
  • mRNA sequences and constructs for expressing modified CD117 polypeptides comprising double mutations are provided below in Table 7. .
  • Ts are Us, and in particular embodiments, the Ts are fully substituted with NlmPsU. Coding sequence is capitalized.
  • construct sequences are generally specified T7 promoter 3’UTR and do not include poly A tail sequences, except for AMOS.
  • mRNA constructs electroporated into cells may lack the T7 promoter sequence.
  • the T7 promoter sequence comprises the sequence
  • the T7 polymerase starts transcription at the underlined G in the promoter sequence.
  • the polymerase then transcribes using the opposite strand as a template from 5’->3’.
  • the first base in the transcript will be a G.
  • a modified CD117 comprises both of the two types of modification disclosed above: it has constitutive activity; and it is not bound by an anti-CDl 17 antibody used for HCT conditioning. In particular embodiments, it has constitutive signaling or kinase activity, even in absence of SCF binding to the modified CD117 and in the presence of an anti-c-Kit antibody that blocks SCF binding to the modified CD117 polypeptide.
  • the amino acid modifications comprise: (1) one or more amino acid substitutions or deletions of an amino acid residue selected from the following in human CD117: N505, 522, 557, 558, V559, V568, 569, V570, 579, 703, and D816; and (2) one or more amino acid substitutions of an amino acid residue selected from the following in human CD117: E73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, or R271, or at an amino acid residue located within 2, within 3, within 4, within 5, within 6, within 7 within 8 within 8 within 10, within 11 or within 12 amino acids of any of E73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, or R271, e.g., either N-terminal or C-terminal of any of these residues.
  • the modification is at any of E73, D121, R122, S123, Y125, K203, or at an amino acid residue located within 2, within 3, within 4, within 5, within 6, within 7 within 8 within 8 within 10, within 11 or within 12 amino acids of any of these amino acids, and the modified or variant CD117 does not bind the JSP191 antibody.
  • the CD117 modification is at any of K203, Y259, S261, W262, Y269, or R271, or at an amino acid residue located within 2, within 3, within 4, within 5, within 6, within 7 within 8 within 8 within 10, within 11 or within 12 amino acids of any of these amino acids, and the modified or variant CD117 does not bind the AB85 antibody.
  • the modified CD117 polypeptide comprises one or more of the following amino acid substitutions: N505I, V559D, D816V, D816H, V568F, V570F, and/or Y703F.
  • variant CD117 comprises an modification at D816 and/or a N505, and/or a modification at E73.
  • the amino acid substitutions comprise a D816V substitution and/or a N505I substitution.
  • the amino acid substitutions comprise an E73A substitution.
  • the amino acid substitutions comprise an N505I substitution and/or E73A substitution.
  • the amino acid substitutions comprise an D816V substitution and/or E73A substitution.
  • the modified CD117 has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 750%, or at least 1000% of the kinase activity of the wild type CD117 polypeptide in the presence of an anti-c-kit antibody.
  • an illustrative SCF has the following polypeptide sequence:
  • C-X-C chemokine receptor type 4 (CXCR4), also known as fusin or CD 184 (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 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 chemokine stromal cell derived factor-1
  • 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:
  • the CXCR4 polypeptide sequence, or fragment or variant thereof is encoded by a polynucleotide sequence that comprises or consists of one of the following:
  • GCCATCGTCC ACGCCACCAA CAGTCAGAGG CCAAGGAAGC TGTTGGCTGA AAAGGTGGTC
  • 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
  • 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.
  • the WHIM CXCR4 mutant is WHIMtl9, 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 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
  • CXCR4 mRNA constructs are provided below in Table 8. CXCR4 coding sequences are indicated in capitals. .
  • Ts are Us, and in particular embodiments, the Ts are fully substituted with NlmPsU. Coding sequence is capitalized.
  • construct sequences are generally specified T7 promoter 3’UTR and do not include poly A tail sequences, except for AMOS.
  • mRNA constructs electroporated into cells may lack the T7 promoter sequence.
  • the T7 promoter sequence comprises the sequence The T7 polymerase starts transcription at the underlined G in the promoter sequence. The polymerase then transcribes using the opposite strand as a template from 5 ’->3’. The first base in the transcript will be a G.
  • RNA constructs are shown in the Table 9 below.
  • Ts are Us, and in particular embodiments, the Ts are fully substituted with NlmPsU. Coding sequence is capitalized.
  • construct sequences are generally specified T7 promoter 3’UTR and do not include poly A tail sequences, except for AMOS.
  • mRNA constructs electroporated into cells may lack the T7 promoter sequence.
  • the T7 promoter sequence comprises the sequence TAATACGACTCACTATAG (SEQ ID NO: 86).
  • the T7 polymerase starts transcription at the underlined G in the promoter sequence.
  • the polymerase then transcribes using the opposite strand as a template from 5’->3’.
  • the first base in the transcript will be a G.
  • Modified cells may be made by introducing into the cell one or more polynucleotide or nucleic acid encoding CXCR4, CD47, and/or one or more variant CD117s into the cell.
  • a variety of vectors and nucleic acids may be used, as well as a variety of methods for introducing the vector and/or nucleic acid into the cell.
  • the nucleic acid comprises RNA, DNA, or a combination thereof, and in particular embodiments, the nucleic acid comprises single-stranded and/or double-stranded regions, or a mixture thereof.
  • the nucleic acid is a double-stranded DNA, and in certain embodiments, the nucleic acid is a single stranded RNA, e.g., a messenger RNA (mRNA).
  • the nucleic acid comprises a modified mRNA.
  • the polynucleotides described herein, e.g., modified mRNA are codon-optimized, e.g., to enhance expression of the encoded polypeptide in a host cell.
  • the one or more introduced or exogenous nucleic acids are RNAs, e.g., a modified mRNA.
  • the nucleic acid comprises a modified mRNA.
  • the sequence encoding CXCR4, CD47, and/or a modified CD117 protein comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the polypeptide encoding sequences disclosed herein.
  • the mRNA construct comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the encoded sequences disclosed herein or comprises an mRNA construct sequence disclosed herein, optionally without the promoter sequence and optionally with a poly A tail.
  • polynucleotide variants comprise one or more modified nucleotide or nucleoside.
  • ⁇ Modified mRNAs comprising one or more modified nucleoside have been described as having advantages over unmodified mRNAs, including increase stability, higher expression levels and reduced immunogenicity.
  • Non-limiting examples of modifications to mRNAs that may be present in the nucleic acids are described, e.g., in PCT Patent Application Publication Nos.
  • the modified mRNA comprises one or more nucleoside modification.
  • the modified mRNA sequence comprises at least one modification as compared to an unmodified A, G, U or C ribonucleoside.
  • uridine can a similar nucleoside such as pseudouridine (T) or Nl-methyl-pseudouridine (mlT), and cytosine can be replaced by 5-methylcytosine.
  • the at least one modified nucleosides include Nl-methyl-pseudouridine and/or 5-methylcytidine.
  • one or more uridines are replaced by 5- methoxyuridine (5moU).
  • all uridines in the modified mRNA are replaced with a similar nucleoside such as pseudouridine (T) or Nl-methyl-pseudouridine (mlT), and/or all cytosines in the modified mRNA are substituted with a similar nucleoside such as 5-methylcytosine.
  • a similar nucleoside such as pseudouridine (T) or Nl-methyl-pseudouridine (mlT)
  • all cytosines in the modified mRNA are substituted with a similar nucleoside such as 5-methylcytosine.
  • one or more mRNA comprises a 5’ terminal cap sequence followed by a sequence encoding the polypeptide, followed by a 3’ tailing sequence, such as a poly A or a polyA-G sequence.
  • the nucleic acid sequence encoding the CXCR4, CD47, and/or a modified CD117 protein comprises 5’ and/or 3’ cellular or viral untranslated regions (UTRs) relative to the sequence encoding the CXCR4, CD47, and/or a modified CD117 protein.
  • the UTR improves mRNA stability, localization and/or expression.
  • the UTR is tissue specific.
  • the 5’ UTR comprises a UTR sequence from alpha-globin.
  • the nucleic acid comprises a Kozak sequence.
  • the 3 ’UTR comprises a UTR from an alpha-globin and/or a beta-globin gene, i.e., a 5’ UTR from hemoglobin alpha 1 (HBA1) and/or a 3’ UTR from one or more of HBA1 or hemoglobin beta 1 (HBB1) gene.
  • HBA1 hemoglobin alpha 1
  • HBB1 hemoglobin beta 1
  • the nucleic acid sequence encoding the CXCR4, CD47, and/or a modified CD117 protein comprises a 5’ UTR with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% identity to a 5’UTR sequence of HBA1 :
  • the nucleic acid sequence encoding the CXCR4, CD47, and/or a modified CD117 protein comprises a Kozak sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% identity to the following: GCCGCCACC.
  • the nucleic acid sequence encoding the CXCR4, CD47, and/or a modified CD117 protein comprises a 3’UTR nucleic acid sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% identity to a 3’UTR of HBB1 :
  • the nucleic acid sequence encoding the CXCR4, CD47, and/or a modified CD117 protein comprises a 3’UTR nucleic acid sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% to a 3’UTR of HBA1 :
  • the nucleic acid sequence encoding the CXCR4, CD47, and/or a modified CD117 protein comprises an extra stop codon downstream of TAA to avoid run-off translation of an mRNA.
  • the extra stop codon is TGA.
  • the nucleic acid sequence encoding the CXCR4, CD47, and/or a modified CD117 protein comprises a TCTAGA sequence to linearize a plasmid as a template for transcription.
  • the nucleic acid sequence encoding the CXCR4, CD47, and/or a modified CD117 protein encodes a poly-adenine or poly guanine tail.
  • a poly A or polyA-G tail improves mRNA stability and manufacturability.
  • the polyA tail may be from 20 to 180 adenine bases in length. In some embodiments, the polyA tail may be from 35 to 140 bases in length.
  • the polyA tail is segmented with a linker to reduce recombination during plasmid production in prokaryotic cells.
  • the polyA tail may be from 70 to 150 bases in length. In some embodiments the polyA tail is 70 adenine bases in length.
  • the linker is a series of bases other than adenine. In some embodiments, the linker is a series of bases including adenine. In some embodiments, the linker is about 3 to about 10 bases in length. In some embodiments, the linker is about 5 to about 20 bases in length. In some embodiments the linker comprises the sequence TATGCA.
  • the sequence encoding the CXCR4, CD47, and/or a modified CD117 protein comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the modified CD117 encoding sequences disclosed herein.
  • a modified mRNA comprises a 5’ terminal cap sequence followed by a sequence encoding the CXCR4, CD47, and/or a modified CD117 protein, including one or more 5’ or 3’ UTRs, followed by a 3’ tailing sequence, such as a poly A or a poly A-G tail sequence.
  • the mRNA encoding CXCR4, CD47, and/or a modified CD117 protein comprises a wild type 5’ terminal cap sequence
  • the mRNA encoding CXCR4, CD47, and/or a modified CD117 protein 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).
  • the mRNA encoding the CXCR4, CD47, and/or a modified CD117 protein comprises the modified 5’ terminal cap, 3'-O-Me-m7G(5')ppp(5')G (Anti Reverse Cap Analog (ARCA); APExBIO, USA).
  • a vaccinia virus mRNA cap methyltransferase adds 7- methylguanylate cap structures (Cap-0) to the 5’ end of RNA.
  • a vaccinia Cap 2’-O-Methyltransferase adds a methyl group at the 2 -0 position of the first nucleotide adjacent to the cap structure at the 5’ end of the RNA.
  • an mRNA construct encoding the CXCR4, CD47, and/or a modified CD117 protein comprises the following elements, optionally from 5’ to 3’ : a 5’ HBA1 UTR; a CleanCap Reagent AG 3’ OMe 5’ terminal cap sequence (m7(3'OMeG)(5')ppp(5')(2'OMeA)pG); a sequence encoding the CXCR4, CD47, and/or a modified CD117 protein; a TAATAA stop codon; and a 3’ HBB1 UTR.
  • the sequence encoding the CXCR4, CD47, and/or a modified CD117 protein is codon-optimized.
  • the construct further comprises a polyA sequence, e.g., after the 3’ HBB1 UTR, optionally an AMOS polyA.
  • the mRNA construct comprises any of the sequences disclosed herein. In certain embodiments, it comprises a segmented polyA tail AMOS, in which the 140 adenine bases are segmented into 2x 70 adenine sequences.
  • an mRNA construct expresses two or more polypeptides selected from a CXCR4, a CD47, and/or a modified CD117 protein.
  • a CXCR4 protein, a CD47 protein, and modified CD117 proteins include both wild type and mutant forms of the proteins, including any of the variants and mutants disclosed herein.
  • the nucleic acid encoding a CD47, CD117, and/or CXCR4 polypeptide comprises a sequence with at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the mRNA sequences encoded by or produced by any of the constructs disclosed in the tables herein.
  • the mRNA construct encoding a CD47, CD117, and/or CXCR4 polypeptide comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the CD47 encoding sequences disclosed herein or comprises an mRNA construct sequence disclosed herein, optionally without the promoter sequence and optionally with a polyA tail.
  • the 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-di oleoyl -s//-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.
  • a zwitterionic lipid e.g., 1 ,2-di oleoyl -s//-glycero-3- phosphoethanolamine [DOPE]
  • the one or more nucleic acids encoding CD47, CD 117, and/or CXCR4 polypeptides are present in one or more vectors.
  • a vector is capable of delivering a 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 CD47 polypeptide.
  • the expression vector comprises a promoter sequence that facilitates expression of the encoded CD47, CD117, and/or CXCR4 polypeptides 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 sequences encoding a CD47, CD117, and/or 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.
  • the disclosure provides a modified or engineered cell comprising one or more exogenous or introduced nucleic acids that combined encode a combination of two or more of: a CD47 polypeptide, a modified CD117 polypeptide with constitutive activity, a modified CD 117 polypeptide not bound by an anti-CD 117 antibody used for HCT conditioning (e.g., JSP191), a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning (e.g., JSP191), and/or a CXCR4 polypeptide, or functional fragments or variants thereof.
  • a CD47 polypeptide e.g., a modified CD117 polypeptide with constitutive activity
  • a modified CD 117 polypeptide not bound by an anti-CD 117 antibody used for HCT conditioning e.g., JSP191
  • a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning e.g.,
  • the disclosure provides a modified or engineered cell comprising an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning (e.g., JSP191).
  • an anti-CDl 17 antibody used for HCT conditioning e.g., JSP191.
  • the disclosure provides a modified or engineered cell comprising one or more exogenous or introduced nucleic acids encoding a combination of two or more of: a CD47 polypeptide, a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning, and/or a CXCR4 polypeptide, or functional fragments or variants thereof.
  • the modified or engineered cell comprises two or more exogenous or introduced nucleic acids, each independently encoding one or more of: a CD47 polypeptide, a modified CD117 polypeptide with constitutive activity, a modified CD117 polypeptide not bound by an anti- CD117 antibody used for HCT conditioning (e.g., JSP191), a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CD117 antibody used for HCT conditioning (e.g., JSP191), and/or a CXCR4 polypeptide, or functional fragments or variants thereof.
  • a CD47 polypeptide e.g., a modified CD117 polypeptide with constitutive activity
  • a modified CD117 polypeptide not bound by an anti- CD117 antibody used for HCT conditioning e.g., JSP191
  • a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CD117 antibody used for HCT conditioning e.g., JSP191
  • the modified or engineered cell comprises: (1) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity; (2) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning; (3) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (4) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning; (5) an exogenous or introduced nucleic acid encoding a modified CD117 poly
  • an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning (14) an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning; or (15) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide, and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning.
  • each of the exogenous or introduced polynucleotide is present in the cell or provided to the cell as a separate mRNA construct.
  • two or more of the exogenous or introduced polynucleotides may be present in the cell or provided to the cell in a single mRNA construct, e.g., a bicistronic mRNA.
  • a c-Kit mRNA construct encodes a c-Kit having two or more different point mutations.
  • any of the modifications or combinations of modification to an engineered cells, e.g., HSC or HSPC, disclosed herein may be combined with a further modification of the cell, e.g., to express programmed death-ligand 1 (PD-L1) on the cell surface.
  • PD-L1 programmed death-ligand 1
  • a nucleic acid sequence encoding PDL1, or a functional fragment or variant thereof, may be introduced into the cells using the same vectors, RNAs, and methods as used for the modifications disclosed herein.
  • PD-L1 is an immune checkpoint inhibitor that binds to its receptor PD-1 expressed by T cells and other immune cells to regulate immune responses; ultimately preventing exacerbated activation and autoimmunity.
  • PD-L1 protein sequence is provided below: MEQTFLLVLHVVLWPTLAALFTVEVDSLSHVAEFYGDVTMGCRFQPGSWDPNLSVI WQRVQPLPDVEVYRLDNGQENLTSQNFQYRGRARLVSEELTNGWAKLHVSRLRIN DSGVYRCLVEMGGADYKQTTLTVKATYKTIIKSMQRRGGGEVELACESEGYPLATI NWRDKSLRNIKSNDTVVKTPNQLFHVTSKITVKYSEKNNYTCAFVEKGEAPKGPSAR FDIPDE1PVIESKPNTLSIVLGTTLTVAMIIVATIFGYRRQKGRLRTLKL (SEQ ID NO: 189)
  • the modified cell comprising a modified CD117 polypeptide and/or encoding nucleic acid is a host cell, such as, e.g., an HEK293 cell that may be used to produce modified CD117 polypeptides.
  • a host cell such as, e.g., an HEK293 cell that may be used to produce modified CD117 polypeptides.
  • any host cells may be employed, including but not limited to, for example, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast.
  • any of the modifications disclosed herein may be present in cells that are to be transplanted into a subject, e.g., to treat a disease or disorder in the subject.
  • the modifications are made to cells that would benefit from avoiding immune detection by natural killer (NK) cells or T cells, or from avoiding phagocytosis, when administered to a subject.
  • NK natural killer
  • Illustrative cell types include, but are not limited to, stem cells, induced pluripotent stem cells (iPSCs), T cells, cardiac cells, pancreatic islet cells, NK cells, B cells.
  • the mammalian cells are HSCs and/or HSPCs.
  • the disclosure provides modified cells, e.g., HSCs and/or HSPCs, comprising one or more nucleic acids, which in combination encode two or more CD47, CD117, and/or CXCR4 polypeptides as described herein.
  • the nucleic acid(s) encoding CD47, CD117, and/or CXCR4 polypeptides are transiently present in the modified cell, and/or is not present within the genome of the cell.
  • one or more modified cells expresses and/or comprises a CD47, CD117, and/or CXCR4 polypeptide, and in particular embodiments, a CD47, CD117, and/or 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 an mRNA, e.g., a modified mRNA.
  • the modified HSCs and/or HSPCs transiently or constitutively overexpress the CD47, CD117, and/or CXCR4 polypeptides, or a functional fragment or variant thereof, e.g., at a level at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold higher than a corresponding unmodified cell, e.g., HSC and/or HSPC.
  • 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 one or more nucleic acids encoding a CD47, CD117, and/or 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.
  • 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.
  • Modified HSCs and/or HSPCs may be produced using HSCs and/or HSPCs obtained from a mammalian donor.
  • the donor is a subject in need of a hematopoietic stem cell transplant, e.g., a subject diagnosed with a disease or disorder that can be treated with HCT.
  • the modified HSCs and/or HSPCs may be produced using HSCs and/or HSPCs obtained from a healthy donor, e.g., wherein the modified HSCs and/or HSPCs are to be used to treat a different subject with HCT.
  • the modified HSCs and/or HSPCs may be autologous or allogeneic to a subject in need for HCT.
  • the bone marrow Prior to harvesting 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.
  • G-CSF granulocyte colony-stimulating factor
  • Mobilization of stem cells from the bone marrow into peripheral blood by cytokines such as G-CSF or GM-CSF has led to the widespread adoption of peripheral blood progenitor cell collection by apheresis for hematopoietic stem cell transplantation.
  • the dose of G-CSF used for mobilization may be about 10 ug/kg/day. In autologous donors who are heavily pretreated, however, doses of up to about 40 ug/kg/day can be given.
  • Mozobil may be used in conjunction with G-CSF to mobilize hematopoietic stem cells to peripheral blood for collection.
  • 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.
  • the hematopoietic stem cells 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 modified CD117 polypeptide into the cell.
  • the nucleic acid may be introduced by transfection or infection with a viral vector, or by contact with an mRNA.
  • 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 one or more nucleic acid encoding one or more of: CD47, CXCR4, and/or one or more modified CD117 into the cell, including any of the specific combinations disclosed herein.
  • the introduced nucleic acid is present within a viral vector.
  • the nucleic acid is associated with or present in a lipid nanoparticle, liposome, or the like.
  • the nucleic acid remains present in the modified cell only transiently, or the nucleic acid only transiently expresses the polypeptide in the cell.
  • the method is used to prepare modified cells, e.g., HSCs and/or HSPCs, for HCT treatment of a mammalian subject.
  • the nucleic acid or vector may be introduced into the cell by a variety of methods known in the art, such as transfection, transduction, infection, electroporation, or nanopore technology.
  • mRNA e.g., modified mRNA is introduced into the cells using lipid nucleic acid particles (LNPs) or nanoparticles.
  • LNPs lipid nucleic acid particles
  • cells may be modified by introducing a nucleic acid encoding one or more of: CD47, CXCR4, and/or one or more modified CD117 polypeptide into the HSCs and/or HSPCs according to a variety of methods available in the art, e.g., electroporation.
  • the modified cells e.g., stem cells are further modified to provide a replacement nucleic acid or protein to the cells, e.g., where the cells are obtained from a subject having a genetic disorder resulting in reduced or lack of expression of a gene or protein, or the expression of a mutant form of a gene or protein.
  • the cells may be further genetically altered to correct a genetic defect present in the cells, e.g., HSCs and/or HSPCs.
  • the cells may be contacted with or transduced by a gene therapy vector that results in the insertion into the cellular genome of an expression cassette that expresses a corrected form of a mutated gene or protein, e.g., a wild- type or non-mutated gene or protein.
  • the gene therapy may replace the mutated gene or a mutated region thereof, e.g., via homologous recombination.
  • the HSCs and/or HSPCs are modified to correct a mutated gene using gene editing or base editing methods, such as, e.g., a CRISPR-Cas9 system that targets the mutated gene.
  • the HSCs and/or HSPCs are modified to correct a mutated gene using Zinc-finger nucleases (ZFNs), meganucleases, or transcription activator-like effector nucleases (TALENs) that target the mutated gene. Correction of a genetic mutation may be done prior to, at the same time as, or following introduction of the nucleic acid encoding CD47, or a fragment or variant thereof.
  • the disclosure provides a method of modifying cells, and the resulting modified cells, including stem cells such as HSCs and/or HSPCs, comprising modifying one or more endogenous CD117 genes or alleles within the cells, e.g., by homologous recombination or gene editing according to a variety of methods available in the art.
  • a CD 117 gene in HSCs and/or HSPCs is edited by any of a variety of methods known and available in the art, including but not limited to: transcription activatorlike effector nucleases (TALENs), megaTALs, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) systems, zinc finger nucleases, homing endonucleases, or meganucleases.
  • the CD117 gene is edited by a base editing method.
  • a gene-editing system is a system comprising one or more proteins or polynucleotides capable of editing an endogenous target gene or locus in a sequence specific manner.
  • the gene-editing system is a protein-based gene regulating system comprising a protein comprising one or more zinc-finger binding domains and an enzymatic domain.
  • the protein-based gene regulating system comprises a protein comprising a Transcription activator-like effector nuclease (TALEN) domain and an enzymatic domain.
  • TALENs Transcription activator-like effector nuclease
  • the gene editing system comprises a nucleic acid sequence corresponding to a region of the CD117 gene and comprising a modification thereof.
  • Zinc finger-based systems comprise a fusion protein comprising two protein domains: a zinc finger DNA binding domain and an enzymatic domain.
  • a “zinc finger DNA binding domain”, “zinc finger protein”, or “ZFP” is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion.
  • the zinc finger domain by binding to a target DNA sequence, directs the activity of the enzymatic domain to the vicinity of the sequence and, hence, induces modification of the endogenous target gene in the vicinity of the target sequence.
  • a zinc finger domain can be engineered to bind to virtually any desired sequence.
  • one or more zinc finger binding domains can be engineered to bind to one or more target DNA sequences in the target genetic locus.
  • Expression of a fusion protein comprising a zinc finger binding domain and an enzymatic domain in a cell effects modification in the target genetic locus.
  • a zinc finger binding domain comprises one or more zinc fingers. Miller et al. (1985) EMBO J. 4: 16010-1714; Rhodes (1993) Scientific American Febuary:56-65; U.S. Pat. No. 6,453,242. Typically, a single zinc finger domain is about 30 amino acids in length. An individual zinc finger binds to a three-nucleotide (z.e., triplet) sequence (or a four-nucleotide sequence which can overlap, by one nucleotide, with the four- nucleotide binding site of an adjacent zinc finger).
  • z.e., triplet or a four-nucleotide sequence which can overlap, by one nucleotide, with the four- nucleotide binding site of an adjacent zinc finger.
  • the length of a sequence to which a zinc finger binding domain is engineered to bind (e.g., a target sequence) will determine the number of zinc fingers in an engineered zinc finger binding domain.
  • the DNA-binding domains of individual ZFNs comprise between three and six individual zinc finger repeats and can each recognize between 9 and 18 base pairs.
  • Zinc finger binding domains can be engineered to bind to a sequence of choice. See, for example, Beerli et al. (2002) Nature Biotechnol. 20: 135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416.
  • An engineered zinc finger binding domain can have a novel binding specificity, compared to a naturally-occurring zinc finger protein. Engineering methods include, but are not limited to, rational design and various types of selection.
  • a target DNA sequence for binding by a zinc finger domain can be accomplished, for example, according to the methods disclosed in U.S. Pat. No. 6,453,242. It will be clear to those skilled in the art that simple visual inspection of a nucleotide sequence can also be used for selection of a target DNA sequence. Accordingly, any means for target DNA sequence selection can be used in the methods described herein.
  • a target site generally has a length of at least 9 nucleotides and, accordingly, is bound by a zinc finger binding domain comprising at least three zinc fingers.
  • binding of, for example, a 4-finger binding domain to a 12-nucleotide target site, a 5-finger binding domain to a 15-nucleotide target site or a 6-finger binding domain to an 18-nucleotide target site is also possible.
  • binding of larger binding domains e.g., 7-, 8-, 9-finger and more
  • the zinc finger binding domains bind to a target DNA sequence that is at least 90% identical to a target DNA sequence (e.g., epitope-encoding) within a target locus of a target CD117 gene. In some embodiments, the zinc finger binding domains bind to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence within a target locus of a target gene. In some embodiments, the zinc finger binding domains bind to a target DNA sequence that is 100% identical to a target DNA sequence within a target locus of a target gene.
  • the enzymatic domain portion of the zinc finger fusion proteins can be obtained from any endo- or exonuclease.
  • Exemplary endonucleases from which an enzymatic domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388.
  • Additional enzymes which cleave DNA are known (e.g., 51 Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993).
  • One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains.
  • Exemplary restriction endonucleases (restriction enzymes) suitable for use as an enzymatic domain of the ZFPs described herein are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding.
  • fusion proteins comprise the enzymatic domain from at least one Type IIS restriction enzyme and one or more zinc finger binding domains.
  • An exemplary Type IIS restriction enzyme whose cleavage domain is separable from the binding domain, is Fok I. This particular enzyme is active as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575.
  • two fusion proteins each comprising a FokI enzymatic domain, can be used to reconstitute a catalytically active cleavage domain.
  • a single polypeptide molecule containing a zinc finger binding domain and two FokI enzymatic domains can also be used.
  • TALEN-based systems comprise a protein comprising a TAL effector DNA binding domain and an enzymatic domain. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands).
  • the FokI restriction enzyme described above is an exemplary enzymatic domain suitable for use in TALEN-based gene regulating systems.
  • TAL effectors are proteins that are secreted by Xanthomonas bacteria via their type III secretion system when they infect plants.
  • the DNA binding domain contains a repeated, highly conserved, 33-34 amino acid sequence with divergent 12th and 13th amino acids. These two positions, referred to as the Repeat Variable Diresidue (RVD), are highly variable and strongly correlated with specific nucleotide recognition. Therefore, the TAL effector domains can be engineered to bind specific target DNA sequences by selecting a combination of repeat segments containing the appropriate RVDs.
  • RVD Repeat Variable Diresidue
  • the nucleic acid specificity for RVD combinations is as follows: HD targets cytosine, NI targets adenenine, NG targets thymine, and NN targets guanine (though, in some embodiments, NN can also bind adenenine with lower specificity).
  • the TAL effector domains bind to a target DNA sequence that is at least 90% identical to a target DNA sequence (e.g., epitope-enoding) within a target locus of a CD117 gene. In some embodiments, the TAL effector domains bind to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence within a target locus of a target gene. In some embodiments, the TAL effector domains bind to a target DNA sequence that is 100% identical to a target DNA sequence within a target locus of a target gene.
  • TAL-effector repeats Methods and compositions for assembling the TAL-effector repeats are known in the art. See e.g., Cermak et a/, Nucleic Acids Research, 39:12, 2011 , e82. Plasmids for constructions of the TAL- effector repeats are commercially available from Addgene.
  • the gene-editing system is a combination gene-regulating system comprising a site-directed modifying polypeptide and a nucleic acid guide molecule.
  • a site-directed modifying polypeptide refers to a polypeptide that binds to a nucleic acid guide molecule, is targeted to a target nucleic acid sequence, such as, for example, a DNA sequence, by the nucleic acid guide molecule to which it is bound, and modifies the target DNA sequence (e.g., cleavage, mutation, or methylation of target DNA).
  • a site-directed modifying polypeptide comprises two portions, a portion that binds the nucleic acid guide and an activity portion.
  • a site-directed modifying polypeptide comprises an activity portion that exhibits site-directed enzymatic activity (e.g., DNA methylation, DNA cleavage, histone acetylation, histone methylation, etc.), wherein the site of enzymatic activity is determined by the guide nucleic acid.
  • site-directed enzymatic activity e.g., DNA methylation, DNA cleavage, histone acetylation, histone methylation, etc.
  • the nucleic acid guide comprises two portions: a first portion that is complementary to, and capable of binding with, an endogenous target DNA sequence (referred to herein as a “DNA-binding segment”), and a second portion that is capable of interacting with the site-directed modifying polypeptide (referred to herein as a “proteinbinding segment”).
  • DNA-binding segment and protein-binding segment of a nucleic acid guide are comprised within a single polynucleotide molecule.
  • the DNA-binding segment and protein-binding segment of a nucleic acid guide are each comprised within separate polynucleotide molecules, such that the nucleic acid guide comprises two polynucleotide molecules that associate with each other to form the functional guide.
  • the nucleic acid guide mediates the target specificity of the combined protein/nucleic gene regulating systems by specifically hybridizing with a target DNA sequence comprised within the DNA sequence of a target gene.
  • Reference herein to a target gene encompasses the full-length DNA sequence for that particular gene and a full-length DNA sequence for a particular target gene will comprise a plurality of target genetic loci, which refer to portions of a particular target gene sequence (e.g., an exon or an intron). Within each target genetic loci are shorter stretches of DNA sequences referred to herein as “target DNA sequences” or “target sequences” that can be modified by the gene-regulating systems described herein.
  • each target genetic loci comprises a “target modification site,” which refers to the precise location of the modification induced by the gene-regulating system (e.g., the location of an insertion, a deletion, or mutation, the location of a DNA break, or the location of an epigenetic modification).
  • the gene-regulating systems described herein may comprise a single nucleic acid guide, or may comprise a plurality of nucleic acid guides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleic acid guides).
  • CRISPR/Cas systems described below are exemplary embodiments of a combination protein/nucleic acid system.
  • the gene editing systems described herein are CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR Associated) nuclease systems.
  • the site-directed modifying polypeptide is a CRISPR- associated endonuclease (a “Cas” endonuclease) and the nucleic acid guide molecule is a guide RNA (gRNA).
  • a Cas polypeptide refers to a polypeptide that can interact with a gRNA molecule and, in concert with the gRNA molecule, homes or localizes to a target DNA sequence and includes naturally occurring Cas proteins and engineered, altered, or otherwise modified Cas proteins that differ by one or more amino acid residues from a naturally-occurring Cas sequence.
  • the Cas protein is a Cas9 protein.
  • Cas9 is a multi-domain enzyme that uses an HNH nuclease domain to cleave the target strand of DNA and a RuvC- like domain to cleave the non-target strand.
  • mutants of Cas9 can be generated by selective domain inactivation enabling the conversion of WT Cas9 into an enzymatically inactive mutant (e.g., dCas9), which is unable to cleave DNA, or a nickase mutant, which is able to produce single-stranded DNA breaks by cleaving one or the other of the target or non-target strand.
  • a guide RNA typically comprises two segments, a DNA-binding segment and a protein-binding segment.
  • the protein-binding segment of a gRNA is comprised in one RNA molecule and the DNA-binding segment is comprised in another separate RNA molecule.
  • double-molecule gRNAs or “two-molecule gRNA” or “dual gRNAs.”
  • the gRNA is a single RNA molecule and is referred to herein as a "single-guide RNA" or an "sgRNA.”
  • the term "guide RNA” or "gRNA” is inclusive, referring both to two-molecule guide RNAs and sgRNAs.
  • the protein-binding segment of a gRNA typically comprises, in part, two complementary stretches of nucleotides that hybridize to one another to form a double stranded RNA duplex (dsRNA duplex), which facilitates binding to the Cas protein.
  • dsRNA duplex double stranded RNA duplex
  • the DNA-binding segment (or "DNA-binding sequence") of a gRNA comprises a nucleotide sequence that is complementary to and capable of binding to a specific sequence target DNA sequence or RNA sequence.
  • the protein-binding segment of the gRNA interacts with a Cas polypeptide and the interaction of the gRNA molecule and site-directed modifying polypeptide results in Cas binding to the endogenous DNA or RNA and produces one or more modifications within or around the target DNA sequence.
  • the precise location of the target modification site is determined by both (i) base-pairing complementarity between the gRNA and the target DNA or RNA sequence; and (ii) the location of a short motif, referred to as the protospacer adjacent motif (PAM), in the target DNA sequence.
  • PAM protospacer adjacent motif
  • the PAM sequence is required for Cas binding to the target DNA sequence.
  • a variety of PAM sequences are known in the art and are suitable for use with a particular Cas endonuclease (e.g., a Cas9 endonuclease) are known in the art (See e.g., Nat Methods. 2013 Nov; 10(11): 1116-1121 and S ci Rep. 2014; 4: 5405).
  • the PAM sequence is located within 50 base pairs of the target modification site. In some embodiments, the PAM sequence is located within 10 base pairs of the target modification site.
  • the DNA or RNA sequences that can be targeted by this method are limited only by the relative distance of the PAM sequence to the target modification site and the presence of a unique 20 base pair sequence to mediate sequence-specific, gRNA- mediated Cas binding.
  • the target modification site is located at the 5’ terminus of the target locus. In some embodiments, the target modification site is located at the 3’ end of the target locus. In some embodiments, the target modification site is located within an intron or an exon of the target locus.
  • the guide RNA binds to a CD117 polynucleotide sequence and includes a region complementary to a target CD117 sequence. In certain embodiments, the guide RNA targets or binds a region of CD117 polynucleotide sequence that encodes one of the following amino acid residues: N505 or D816.
  • the present disclosure provides a polynucleotide encoding a gRNA.
  • a gRNA-encoding nucleic acid is comprised in an expression vector, e.g., a recombinant expression vector.
  • the present disclosure provides a polynucleotide encoding a site-directed modifying polypeptide.
  • the polynucleotide encoding a site-directed modifying polypeptide is comprised in an expression vector, e.g., a recombinant expression vector.
  • the site-directed modifying polypeptide is a Cas protein, e.g., a Cas9 protein.
  • Cas molecules of a variety of species can be used in the methods and compositions described herein, including Cas molecules derived from S. pyogenes, S. aureus, N. meningitidis, S. thermophiles, etc.
  • the Cas protein is a Cas9 protein or a Cas9 ortholog and is selected from the group consisting of SpCas9, SpCas9-HFl, SpCas9- HF2, SpCas9-HF3, SpCas9-HF4, SaCas9, FnCpf, FnCas9, eSpCas9, and NmeCas9.
  • the Cas9 protein is a naturally-occurring Cas9 protein. Exemplary naturally occurring Cas9 molecules are described in Chylinski et al., RNA Biology 2013 10:5, 727-737.
  • a Cas9 protein comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a Cas9 amino acid sequence described in Chylinski et al., RNA Biology 2013 10:5, 727-737; Hou et al, PNAS Early Edition 2013, 1-6).
  • a Cas polypeptide comprises one or more of the following activities: [0226] a nickase activity, i.e., the ability to cleave a single strand, e.g., the non- complementary strand or the complementary strand, of a nucleic acid molecule;
  • a double stranded nuclease activity i.e., the ability to cleave both strands of a double stranded nucleic acid and create a double stranded break, which in an embodiment is the presence of two nickase activities;
  • a helicase activity i.e., the ability to unwind the helical structure of a double stranded nucleic acid.
  • the Cas9 is a wildtype (WT) Cas9 protein or ortholog.
  • WT Cas9 comprises two catalytically active domains (HNH and RuvC). Binding of WT Cas9 to DNA based on gRNA specificity results in double-stranded DNA breaks that can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR).
  • NHEJ non-homologous end joining
  • HDR homology-directed repair
  • Cas9 is fused to heterologous proteins that recruit DNA-damage signaling proteins, exonucleases, or phosphatases to further increase the likelihood or the rate of repair of the target sequence by one repair mechanism or another.
  • a WT Cas9 is co-expressed with a nucleic acid repair template to facilitate the incorporation of an exogenous nucleic acid sequence by homology-directed repair.
  • different Cas9 proteins may be advantageous to use in the various provided methods in order to capitalize on various enzymatic characteristics of the different Cas9 proteins (e.g., for different PAM sequence preferences; for increased or decreased enzymatic activity; for an increased or decreased level of cellular toxicity; to change the balance between NHEJ, homology-directed repair, single strand breaks, double strand breaks, etc.).
  • the Cas polypeptides are engineered to alter one or more properties of the Cas polypeptide.
  • the Cas polypeptide comprises altered enzymatic properties, e.g., altered nuclease activity, (as compared with a naturally occurring or other reference Cas molecule) or altered helicase activity.
  • the present disclosure provides guide RNAs (gRNAs) that direct a site-directed modifying polypeptide to a specific target DNA sequence.
  • a gRNA comprises a DNA- targeting segment and protein-binding segment.
  • the DNA-targeting segment of a gRNA comprises a nucleotide sequence that is complementary to a sequence in the target DNA sequence.
  • the DNA-targeting segment of a gRNA interacts with a target DNA in a sequence-specific manner via hybridization (i.e., base pairing), and the nucleotide sequence of the DNA-targeting segment determines the location within the target DNA that the gRNA will bind.
  • the DNA-targeting segment of a gRNA can be modified (e.g., by genetic engineering) to hybridize to any desired sequence within a target DNA sequence.
  • the protein-binding segment of a guide RNA interacts with a site-directed modifying polypeptide (e.g., a Cas9 protein) to form a complex.
  • the guide RNA guides the bound polypeptide to a specific nucleotide sequence within target DNA via the above-described DNA-targeting segment.
  • the protein-binding segment of a guide RNA comprises two stretches of nucleotides that are complementary to one another and which form a double stranded RNA duplex.
  • a gRNA comprises two separate RNA molecules.
  • each of the two RNA molecules comprises a stretch of nucleotides that are complementary to one another such that the complementary nucleotides of the two RNA molecules hybridize to form the double-stranded RNA duplex of the protein-binding segment.
  • a gRNA comprises a single RNA molecule (sgRNA).
  • the specificity of a gRNA for a target loci is mediated by the sequence of the DNA- binding segment, which comprises about 20 nucleotides that are complementary to a target DNA sequence within the target locus. In some embodiments, the corresponding target DNA sequence is approximately 20 nucleotides in length. In some embodiments, the DNA-binding segments of the gRNA sequences of the present invention are at least 90% complementary to a target DNA sequence within a target locus. In some embodiments, the DNA-binding segments of the gRNA sequences of the present disclosure are at least 95%, 96%, 97%, 98%, or 99% complementary to a target DNA sequence within a target locus, e.g., CD117. In some embodiments, the DNA-binding segments of the gRNA sequences of the present invention are 100% complementary to a target DNA sequence within a target locus.
  • the DNA-binding segments of the gRNA sequences bind to a target DNA sequence that is at least 90% identical to a target DNA sequence within a target locus of a CD117 gene. In some embodiments, the DNA-binding segments of the gRNA sequences bind to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence within a target locus of a target gene. In some embodiments, the DNA-binding segments of the gRNA sequences bind to a target DNA sequence that is 100% identical to a target DNA sequence within a target locus of a target gene.
  • the DNA-binding segments of the gRNA sequences described herein are designed to minimize off-target binding using algorithms known in the art (e.g. , Cas- OFF finder) to identify target sequences that are unique to a particular target locus or target gene.
  • algorithms known in the art e.g. , Cas- OFF finder
  • the gRNAs described herein can comprise one or more modified nucleosides or nucleotides which introduce stability toward nucleases.
  • these modified gRNAs may elicit a reduced innate immune as compared to a non-modified gRNA.
  • innate immune response includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.
  • the gRNAs described herein are modified at or near the 5' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of their 5' end).
  • the 5' end of a gRNA is modified by the inclusion of a eukaryotic mRNA cap structure or cap analog (e.g., a G(5 ')ppp(5 ')G cap analog, a m7G(5 ')ppp(5 ')G cap analog, or a 3 '-0-Me-m7G(5 ')ppp(5 ')G anti reverse cap analog (ARCA)).
  • a eukaryotic mRNA cap structure or cap analog e.g., a G(5 ')ppp(5 ')G cap analog, a m7G(5 ')ppp(5 ')G cap analog, or a 3 '-0-Me-m7G(5 ')ppp(5 ')G anti reverse cap
  • an in vitro transcribed gRNA is modified by treatment with a phosphatase (e.g. , calf intestinal alkaline phosphatase) to remove the 5' triphosphate group.
  • a gRNA comprises a modification at or near its 3' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of its 3' end).
  • the 3' end of a gRNA is modified by the addition of one or more (e.g., 25-200) adenine (A) residues.
  • modified nucleosides and modified nucleotides can be present in a gRNA, but also may be present in other gene-regulating systems, e.g., mRNA, RNAi, or siRNA- based systems. In some embodiments, modified nucleosides and nucleotides can include one or more of.
  • alteration e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage;
  • modification of the 3' end or 5' end of the oligonucleotide e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety
  • modification of the sugar [0249] modification of the sugar.
  • the modifications listed above can be combined to provide modified nucleosides and nucleotides that can have two, three, four, or more modifications.
  • a modified nucleoside or nucleotide can have a modified sugar and a modified nucleobase.
  • every base of a gRNA is modified.
  • each of the phosphate groups of a gRNA molecule are replaced with phosphorothioate groups.
  • a software tool can be used to optimize the choice of gRNA within a user's target sequence, e.g., to minimize total off-target activity across the genome.
  • Off target activity may be other than cleavage.
  • software tools can identify all potential off-target sequences (preceding either NAG or NGG PAMs) across the genome that contain up to a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base-pairs.
  • the cleavage efficiency at each off-target sequence can be predicted, e.g., using an experimentally-derived weighting scheme.
  • Each possible gRNA can then be ranked according to its total predicted off-target cleavage; the top-ranked gRNAs represent those that are likely to have the greatest on-target and the least off-target cleavage.
  • Other functions e.g., automated reagent design for gRNA vector construction, primer design for the on-target Surveyor assay, and primer design for high- throughput detection and quantification of off-target cleavage via next-generation sequencing, can also be included in the tool.
  • CRISPR-Cas systems and components thereof are described in, US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233 and US8999641 and applications related thereto; and WO2014/018423, WO2014/093595, WO2014/093622, WO2014/093635, WO2014/093655, WO2014/093661, WO2014/093694, WO2014/093701, WO20 14/093709, WO2014/093712, WO2014/093718, WO2014/145599, WO2014/204723, WO20 14/204724, WO2014/204725, WO2014/204726, WO2014/204727, WO2014/204728, WO20 14/204729, WO2015/065964, WO2015/08
  • the gene editing methods comprise or consist of base editing methods.
  • Base editing activity involves chemically altering a base within a polynucleotide, e.g., converting a first base to a second base.
  • the base editing activity is cytidine deaminase activity, e.g., converting target OG to T*A.
  • the base editing activity is adenosine or adenine deaminase activity, e.g., converting A*T to G»C.
  • the base editing activity is cytidine deaminase activity, e.g., converting target OG to T*A and adenosine or adenine deaminase activity, e.g., converting A*T to G»C.
  • the base editing methods comprise single nucleotide base editing, such as nucleotide deamination, i.e., A->G or C“>T.
  • Base editing systems may edit genomic DNA or transcribed RNA.
  • a variety of base editing methods are known and used in the art, including but not limited to those disclosed in the references cited herein.
  • Adenosine and cytidine base editors that may be used include, but are not limited to, base editors described in Antoni ou P. et al., Base and Prime Editing Technologies for Blood Disorders, Front. Genome Ed., 28 January 2021.
  • base editing methods comprise C ⁇ >G conversion as described in Kurt, I. C. et al. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat. Biotechnol. (2020).
  • dual editors facilitate simultaneous C ⁇ >T and A->G conversion as described in Zhao, D. et al. New base editors change C to A in bacteria and C to G in mammalian cells. Nat. Biotechnol. (2020).
  • a base editor system generally refers to a system for editing a nucleobase of a target nucleotide sequence.
  • a base editor (BE) system comprises: (1) a polynucleotide programmable nucleotide binding domain, a deaminase domain (e.g., cytidine deaminase or adenosine deaminase) for deaminating nucleobases in the target nucleotide sequence; and (2) one or more guide polynucleotides (e.g., guide RNA) in conjunction with the polynucleotide programmable nucleotide binding domain.
  • a deaminase domain e.g., cytidine deaminase or adenosine deaminase
  • the base editor (BE) system comprises a nucleobase editor domain selected from an adenosine deaminase or a cytidine deaminase, and a domain having nucleic acid sequence specific binding activity.
  • the base editor system comprises: (1) a base editor (BE) comprising a polynucleotide programmable DNA binding domain and a deaminase domain for deaminating one or more nucleobases in a target nucleotide sequence; and (2) one or more guide RNAs in conjunction with the polynucleotide programmable DNA binding domain.
  • the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain.
  • the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE) or a cytidine base editor (CBE).
  • Cas9 or Cas9 domain refers to an RNA guided nuclease comprising a Cas9 protein, or a fragment or variant thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9).
  • a Cas9 protein or a fragment or variant thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9).
  • a variety of different Cas9 proteins, and fragments and variants thereof, are known and available in the art.
  • a guide polynucleotide is a polynucleotide that is specific for a target sequence (e.g., specifically hybridizes to a target polynucleotide sequence, such as a CD117 gene or mRNA) and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cpfl).
  • the guide polynucleotide is a guide RNA (gRNA).
  • gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule.
  • gRNA is used to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules gRNAs, and gRNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs).
  • gRNAs that exist as single RNA species may comprise two domains: (1) a domain that shares homology to a target nucleic acid, and thus directs binding of a Cas9 complex to the target nucleic acid; and (2) a domain that binds a Cas9 protein.
  • domain (2) is a sequence known as a tracrRNA, which comprises a stem-loop structure.
  • domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al., Science 337:816-821(2012).
  • gRNAs e.g., those including domain 2 are described, e.g., in US20160208288, entitled “Switchable Cas9 Nucleases and Uses Thereof," and US 9,737,604, entitled “Delivery System For Functional Nucleases.”
  • a gRNA comprises two or more of domains (1) and (2), which may be referred to as an extended gRNA.
  • An extended gRNA will bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions.
  • the gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to the target site, providing the sequence specificity of the nuclease:RNA complex.
  • the base editing method e.g., a single nucleotide base editing method, targets a polynucleotide encoding a CD117 polypeptide.
  • An illustrative CD117 polynucleotide sequence follows:
  • the guide RNA binds to a CD117 polynucleotide sequence and includes a region complementary to a target CD117 sequence.
  • the guide RNA targets or binds a region of CD117 polynucleotide sequence that encodes one of the following amino acid residues: N505 or D816.
  • the base editing method comprises use of a modified CRISPR protein, bound to a guide RNA, and a base editing enzyme, such as a deaminase, wherein the modified CRISPR protein does not cause a doublestranded break.
  • the modified CRISPR protein is a nucleobase editor polypeptide or nucleic acid programmable-DNA binding protein (napDNAbp), as disclosed in PCT Application Publication Nos.
  • the method of base editing a polynucleotide encoding a CD117 polypeptide comprises expressing in a cell a nucleobase editor polypeptide, wherein the nucleobase editor polypeptide comprises a napDNAbp and a deaminase, and contacting the cell with a guide RNA capable of targeting the polynucleotide encoding a CD117 polypeptide.
  • base editing may refer to RNA base editing methods, e.g., as described in Porto E. et al. Base editing: advances and therapeutic opportunities, Nature Reviews Drug Discovery volume 19, pages 839-859 (2020).
  • any of the gene editing including base editing methods disclosed herein or known in the art may be used to modify one or more amino acids within an epitope of wild type human CD117 bound by an anti-CDl 17 antibody, optionally wherein the epitope comprises one or more of the following amino acids present in the wild type human CD117: N505 or D816, including but not limited to any of these recited amino acid residues.
  • the method introduces a A ⁇ >G or C ⁇ >T mutation into one or both alleles of the CD117 gene, which results in the gene encoding a different amino acid by the codon that was mutated.
  • the disclosure provides a modified cell, e.g., HSPC or HSC, that comprises one or more components of a gene editing, e.g., base editing, system disclosed herein.
  • the one or more component comprises a nucleic acid that binds to a CD117 gene or encoded mRNA, e.g., at a site to be modified to result in the encoding and/or expression of a modified CD117 disclosed herein, such as, e.g., a guide RNA.
  • the guide RNA binds to a CD 117 polynucleotide sequence and includes a region complementary to a target CD117 sequence.
  • the guide RNA targets or binds a region of CD117 polynucleotide sequence that encodes one of the following amino acid residues: N505 or D816.
  • the one or more component comprises a base editing enzyme, e.g., any of those disclosed herein or in references cited herein.
  • a modified cell expressing a modified CD117 polypeptide is not substantially inhibited, eliminated, or killed by monoclonal antibodies (mAbs) that bind endogenous or wild-type cell -surface CD117 and inhibit proliferation of or kill a cell expressing only the wild-type CD117 and not a modified CD117 polypeptide disclosed herein.
  • proliferation of the modified cell expressing the modified CD117 polypeptide is inhibited by less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%, as compared to proliferation of the same cell type that is not modified, e.g., only expresses wild-type CD117.
  • a composition comprising modified HSCs and/or HSPCs is administered to a patient.
  • modified 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.
  • the present disclosure also includes pharmaceutical compositions comprising two or more of CD47, modified CD117, and/or CXCR4 polypeptides, one or more polynucleotides or vectors, together comprising one or more sequence encoding two or more of a CD47, CD117, and/or CXCR4 polypeptide (e.g., a modified mRNA), or a modified cell, e.g., HSC and/or HSPC, comprising one or more polynucleotide or vector, together encoding two or more of a CD47, CD 117, and/or CXCR4 polypeptide or fragment or variant thereof and/or expressing two or more of a CD47, CD117, and/or CXCR4 polypeptide, or fragment or variant thereof, in combination with one or more pharmaceutically acceptable diluent, carrier, or excipient.
  • pharmaceutically acceptable diluent, carrier, or excipient comprising two or more of CD47, modified CD117, and/or CXCR4 poly
  • the present invention discloses a pharmaceutical composition
  • a pharmaceutical composition comprising a modified cell comprising two or more exogenously introduced CD47, CD 117, and/or CXCR4 polypeptide (or one or more exogenous or introduced nucleic acid sequences encoding two or more of a CD47, CD117, and/or 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., an HSC and/or HSPC.
  • the pharmaceutical composition further comprises one or more additional active agents.
  • the present disclosure also includes pharmaceutical compositions comprising a variant CD117 comprising amino acid modifications resulting in constitutive activity and not being bound by an antibody to CD117, e.g., JSP191, a polynucleotide or vector a variant CD 117 comprising amino acid modifications resulting in constitutive activity and not being bound by an antibody to CD117, e.g., JSP191, or a modified cell, e.g., HSC and/or HSPC, comprising a polynucleotide or vector a variant CD117 comprising amino acid modifications resulting in constitutive activity and not being bound by an antibody to CD117, e.g., JSP191, in combination with one or more pharmaceutically acceptable diluent, carrier, or excipient.
  • a variant CD117 comprising amino acid modifications resulting in constitutive activity and not being bound by an antibody to CD117, e.g., JSP191, a polynucleotide or vector a variant CD117 comprising amino acid modifications resulting in constitutive
  • the present invention discloses a pharmaceutical composition
  • a pharmaceutical composition comprising a modified cell comprising an exogenously introduced variant CD117 comprising amino acid modifications resulting in constitutive activity and not being bound by an antibody to CD117, e.g., JSP191 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., an 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, disclosed herein, e.g., comprising one or more exogenous or introduced CD47, CD117, and/or CXCR4 polypeptide described herein and/or exogenous or introduced nucleic acids encoding one or more CD47, CD117, and/or CXCR4 polypeptide.
  • modified cells e.g., HSCs and/or HSPCs, disclosed herein, e.g., comprising one or more exogenous or introduced CD47, CD117, and/or CXCR4 polypeptide described herein and/or exogenous or introduced nucleic acids encoding one or more CD47, CD117, and/or CXCR4 polypeptide.
  • the modified or engineered cells comprise one or more exogenous or introduced nucleic acids, which together encode a combination of two or more of: a CD47 polypeptide, a modified CD1 17 polypeptide with constitutive activity, a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning, and/or a CXCR4 polypeptide, or functional fragments or variants thereof.
  • the modified or engineered cell comprises: (1) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity; (2) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning; (3) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (4) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning; (5) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive
  • an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning (14) an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning; or (15) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide, and an exogenous or introduced nucleic acid encoding a
  • 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.
  • HLA matching traditionally, the loci critical for matching are HLA-A, HLA-B, and HLA-DR.
  • HLA-C and HLA-DQ are also now considered when determining the appropriateness of a donor.
  • a completely matched sibling donor is generally considered the ideal donor.
  • a complete match or a single mismatch is considered acceptable for most transplantation, although in certain circumstances, a greater mismatch is tolerated.
  • 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.
  • GVHD graft versus host disease
  • the HCT methods disclosed use modified HSCs and/or HSPCs comprising an exogenous or introduced combination of CD47, CD117, and/or CXCR4 polypeptide or nucleic acids encoding CD47, CD117, and/or CXCR4 polypeptides.
  • the combination of one or more of CD47, CD117, and/or CXCR4 (or variants thereof) may improve engraftment.
  • the modified or engineered cell expressing CD47 improves HSCs/HSPCs ability to migrate towards SDF-1 in vitro and in vivo, and thereby home to the bone marrow.
  • the modified or engineered cell expressing CXCR4 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.
  • a modified or engineered cell expressing the modified CD117 polypeptide is not substantially inhibited, eliminated, depleted, or killed by monoclonal antibodies (mAbs) that bind endogenous or wiki-type cell-surface CD117 and inhibit proliferation of or kill a cell expressing only the wild-type CD117 and not a modified CD117 polypeptide disclosed herein.
  • proliferation of the modified cell expressing the modified CD117 polypeptide is inhibited, eliminated, depleted, or killed by less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%, as compared to proliferation of the same cell type that is not modified, e.g., only expresses wild-type CD117.
  • 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.
  • transient expression of the variant CD117 comprising amino acid modifications resulting in constitutive activity and not being bound by an antibody to CD117, e.g., JSP191, or the combination of CD47, one or more variant CD 117, and/or CXCR4, further improves HSCs/HSPCs engraftment in the bone marrow, measured by higher donor myeloid chimerism in the bone marrow and in the peripheral blood.
  • CD47, CD117, and/or CXCR4 transient expression in HSCs and/or HSPCs may improve 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 one or more polynucleotide sequences encoding either 1) a modified CD 117 polypeptide with constitutive activity that is not bound by an anti-CD 117 antibody used for HCT conditioning, or 2) two or more of: a CD47 polypeptide, a modified CD117 polypeptide with constitutive activity, a modified CD117 polypeptide not bound by an anti- CD117 antibody used for HCT conditioning, and/or a CXCR4 polypeptide into the HSC and/or HSPCs, optionally wherein the CD47, CD117, and/or CXCR4 polypeptides comprises a sequence disclosed herein, or a functional variant or fragment thereof; and ii
  • the introduced polynucleotide sequence(s) 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 CD47 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 CD47 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:
  • VOD Veno-occlusive disease
  • the disclosure provides a method for HCT comprising: providing to a subject in need thereof a population of cells comprising a plurality of modified 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 one or more introduced polynucleotide sequences encoding either 1) a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CD117 antibody used for HCT conditioning, or 2) two or more of: a CD47 polypeptide, a modified CD117 polypeptide with constitutive activity, a modified CD117 polypeptide not bound by an anti-CD117 antibody used for HCT conditioning, and/or a CXCR4 polypeptide into the HSC and/or HSPCs, optionally wherein the CD47, CD117, and/or CXCR4 polypeptides comprises a sequence disclosed herein, or
  • 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 CD47, CD117, and/or 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(s) encoding the CD47, CD117, and/or CXCR4 polypeptides is an mRNA, and it was introduced into the cells by electroporation.
  • 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 one or more polynucleotide sequence encoding the CD47, CD 117, and/or CXCR4 polypeptides 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 one or more polynucleotide sequences encoding the CD47, CD117, and/or CXCR4 polypeptide is one or more mRNA, which is introduced into the cells by electroporation.
  • 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 1, and any of these may be used according to the methods disclosed herein, although the conditioning regimen is not limited to those disclosed in Figure 1.
  • the modified cells are administered to a subject in combination with a non-myeloablative conditioning regimen.
  • the conditioning regimen comprises one or more of: chemotherapy (optionally a nucleoside analog and/or an alkylating agent), monoclonal antibody therapy, and radiation, optionally radiation to the entire body.
  • chemotherapy optionally a nucleoside analog and/or an alkylating agent
  • monoclonal antibody therapy optionally radiation to the entire body.
  • radiation optionally radiation to the entire body.
  • two or more conditioning agents are used, they are administered at the same or different times, or two or more may be administered at the same time, and the other(s) at different times.
  • the various conditioning agents are administered to the subject or present within the subject during an overlapping time period prior to the subject being administered the modified HSPCs/HSCs.
  • the conditioning regimen is milder than would be used if the subject was being administered cells, e.g., HSPCs or HSCs, that did not comprise a modified CD47, CD117, and/or 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 modified cells being administered to the subject according to the methods disclosed herein comprise a modified CD117 polypeptide that is not bound by the ablative anti-c-Kit antibody used for conditioning
  • the disclosed methods do not require a wash-out period, or require only a reduced wash-out period as compared to when unmodified cells are transplanted.
  • the wash-out period is less than five days, less than four days, less than 3 days, less than two days, or less than one day.
  • the method comprises administering the anti-c-Kit antibody and the pharmaceutical composition or modified cells, e.g., modified HSCs and/or HSPCs, during an overlapping period of time or at about the same time.
  • the method comprises also, or alternatively, administering the anti-c-Kit antibody to the subject after administration of the pharmaceutical composition or modified cells, e.g., modified HSCs and/or HSPCs, optionally for a period of time of at least one day, at least two days, at least three days, at least four days, at least five days, or at least one week.
  • modified HSCs and/or HSPCs e.g., modified HSCs and/or HSPCs
  • This may continue to ablate endogenous HSCs and/or HSPCs following administration of the modified HSCs and/or HSPCs, thus allowing greater engraftment.
  • the conditioning regimen comprises providing to the subject an anti-CD117 antibody (also known as an anti-c-kit 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-CDl 17 monoclonal antibody, e.g., JSP191 and TBI and fludarabine, or a combination of an anti-CDl 17 monoclonal antibody, e.g., JSP191 and azacytidine.
  • the conditioning regimen comprises administration of an anti-CDl 17 antibody, wherein the anti-CDl 17 antibody depletes or reduces endogenous HSPCs.
  • the anti-CDl 17 antibody is selected from the group consisting of: SRI, 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 SRI, 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-CDl 17 antibody, particularly monoclonal anti-human CD117 antibodies, e.g., those that block or inhibit binding of SCF to CD117.
  • An anti-CDl 17 antibody may refer to an antibody that binds to CD117, e.g., human CD117, or an antigen-binding fragment thereof.
  • the anti-CDl 17 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. W02019084064, W02020/219748, and W02020/219770.
  • the FSI-174 antibody is disclosed in PCT application Publication No. W02020/112687 and U.S. Patent Application Publication No. 20200165337.
  • the disclosure includes but is not limited to any anti-CDl 17 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-CDl 17 antibody binds to the extracellular region of CD117, i.e., amino acids 26-524.
  • the sequence of this region is shown below: QPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWIT EKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDP EVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVLSE KFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQTKLQ EKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGF INIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENES NIRYVSELH
  • Illustrative anti-CD 117 antibodies include, but are not limited to, SR- 1 , JSP 191 , 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 SRI, 2B8, ACK2, YB5-B8, 57A5, 104D2, etc.
  • 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.
  • the antibody is one disclosed in any of US Pat. Nos. 7,915,391, US 8,436,150, or US 8,791,249.
  • the antibody is one disclosed in US Pat. Application Publ. No 20200165337 or any of PCT Publication Nos. WO 2020/112687, W02020/219748, WO 2020/219770, or WO 2019/084064.
  • the antibody is a humanized form of SRI, 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 IgGl 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.
  • SCF stem cell factor
  • 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 IgGl 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.
  • 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.
  • sequences of the heavy and light chains of JSP191 are:
  • variable heavy domain of JSP191 comprises the following sequence:
  • variable light chain domain of JSP191 comprises the following sequence:
  • DIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMHWYQQKPGQPPKLLIYLASNL ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDP YTFGGGTKVEIK (SEQ ID NO: 23).
  • 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: DIVMTQSPSXKILSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKXK2LIYSASYR YSGVPDRFXK3GSGSGTDFTLTISSLQXK4EDFAXK 5 YXK6CQQYNSYPRTFGGGTKVEIK (SEQ ID NO: 30), wherein XKI 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, XKS 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
  • XHI 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
  • XHS 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
  • XHS is an amino acid with an aliphatic hydroxyl side chain.
  • antibodies e.g., human or humanized antibodies
  • VH CDRs of a VH domain comprising the amino acid sequence QVQLKQSGAELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEWIARIYPG SGNT YYNEKFKGKATLTAEKS SST AYMQLS SLTSED S AVYFC ARGVYYFD YWGQ GTTLTVSS (SEQ ID NO: 32) or QVQLKQSGAELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEWIARIYPG SGNT YYNEKFKGKATLTAEKS SST AYMQLS SLTSED S AVYFC ARGVYYFD YWGQ GTTLTVSA (SEQ ID NO: 33), and
  • VL CDRs of a VL domain comprising the amino acid sequence DIVMTQSQKFMSTSVGDRVSVTCKASQNVRTNVAWYQQKPGQSPKALIYSASYRYS GVPDRFTGSGSGTDFTLTI SNVQSEDLADYFCQQYNSYPRTFGGGTKLEIKR (SEQ ID NO: 34).
  • 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. W02019084064. Epitope analysis of AB85 binding to CD177 is described in PCT Application Publication No. W02020219770, which identified the following two epitopes within CD117:
  • EKAEATNTGKYTCTNKHGLSNSIYVFVRDPA (SEQ ID NO: 35) (amino acids 60-90), and RCPLTDPEVTNYSLKGCQGKP (SEQ ID NO: 36) (amino acids 100-130).
  • variable heavy chain and variable light chains of AB85 are disclosed as SEQ ID NO: 143 and SEQ ID NO: 144 from W02019084064, respectively.
  • the heavy chain variable region (VH) amino acid sequence of AB85 is: EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMAIINPRDS DTRYRPSFOGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYEGYEGAFDI WGQGTLVTVSS (SEQ ID NO: 37).
  • VH CDR amino acid sequences of AB85 are as follows: NYWIG (VH CDR1; SEQ ID NO: 38); IINPRDSDTRYRPSFQG (VH CDR2; SEQ ID NO: 39); and HGRGYEGYEGAFDI (VH CDR3; SEQ ID NO: 40).
  • VL amino acid sequence of AB85 is:
  • VL CDR amino acid sequences of AB85 are as follows: RSSQGIRSDLG (VL CDR1; SEQ ID NO: 42); DASNLET (VL CDR2; SEQ ID NO: 43); and QQANGFPLT (VL CDR3; SEQ ID NO: 44).
  • 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-CDl 17 antibody comprises the three CDRs or variable heavy chain regions present in any of AHI, 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.
  • 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: 190); CDR-H2 with IIYPGDSDTRYSPSFQG (SEQ ID NO: 191); CDR-H3 with HGRGYNGYEGAFDI (SEQ ID NO: 192).
  • CK6 has the following light chain CDR amino acid sequences: CDR-L1 with RASQGISSALA (SEQ ID NO: 193); CDR-L2 with DASSLES (SEQ ID NO: 194); and CDR-L3 with CQQFNSYPLT (SEQ ID NO: 195).
  • 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 light chain DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLES GVPSRFSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT (SEQ ID NO: 197)
  • Ab249 was derived from antibody CK6, as an antagonist anti-CD117 antibody, as disclosed in PCT Application No. W02020092655A1. Ab249 has improved binding characteristics over the parent CK6. Ab249 has the following heavy chain CDRS: TSWIG (VH CDR1; SEQ ID NO: 198) IIYPGDSDTRYSPSFQG (VH CDR2; SEQ ID NO: 199); and HGLGYNGYEGAFDI (VH CDR3; SEQ ID NO: 200).
  • TSWIG VH CDR1; SEQ ID NO: 198
  • IIYPGDSDTRYSPSFQG VH CDR2; SEQ ID NO: 199
  • HGLGYNGYEGAFDI VH CDR3; SEQ ID NO: 200.
  • Ab249 has the following light chain CDRS: RASQGIGSALA (VL CDR1; SEQ ID NO: 201); DASNLET (VL CDR2; SEQ ID NO: 202); and QQLNGYPLT (VL CDR3; SEQ ID NO: 203).
  • Ab249 has the following variable heavy chain sequence (CDRS are underlined): EVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGWVRQMPGKGLEWMGIIYPGDS DTRYSPSFOGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYEGAFDI WGQGTLVTVSS (SEQ ID NO: 204).
  • Ab249 has the following variable light chain sequence (CDRS are underlined): DIQMTQSPSSLSASVGDRVTITCRASOGIGSALAWYQOKPGKAPKLLIYDASNLETGV PSRFSGSGTDFTLTISSLQPEDFATYYCOQLNGYPLTFGQGTRLEIK (SEQ ID NO: 205)
  • 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-CDl 17 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-CDl 17 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-CDl 17 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-CDl 17 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-CDl 17 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-CDl 17 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-CDl 17 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-CDl 17 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-CDl 17 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 SRI, 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 IgGl 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
  • the conditioning regimen comprises an anti-CDl 17 antibody alone.
  • the subject is administered the anti-CDl 17 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). 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 aboutlO 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
  • 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 (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
  • 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 may refer to any anti-cancer drug that targets rapidly dividing cells.
  • Chemotherapy i.e., anti-cancer or anti-neoplastic agents may include, but are not limited to, fludarabine, clorafabine, cytarabine, an anthracycline drug, such as daunorubicin (daunomycin) or idarubicin, cladribine (2-CdA), mitoxantrone, etoposide (VP-16), 6-thioguanine (6-TG), hydroxyurea, 6-mercaptopurine (6-MP), azacytidine, and/or decitabine.
  • the chemotherapy is fludarabine.
  • Chemotherapies may be administered to partially or completely ablate the patient’s bone marrow cells in preparation for donor HSC cell engraftment and/or as part of continuing treatment thereafter.
  • the subject is administered about 10-50 mg/m2/day of chemotherapy, optionally about 30 mg/m2/day, wherein optionally the chemotherapy is fludarabine and/or clofarabine.
  • the subject is administered about 10 to about 50 mg/m2/day of the chemotherapy, optionally 20 mg/m2/day, 25 mg/m2/day, or about 30 mg/m2/day for about one to about six days.
  • the subject is administered about 10-50 mg/m2/day of the chemotherapy, optionally about 30 mg/m2/day of the fludarabine and/or clofarabine about 10 to about 1 days prior to the HCT.
  • the chemotherapy is administered on days -10, -9, -8, -6, -7, - 5 -4, -3, -2, and/or -1 days prior to the HCT. In certain embodiments, the chemotherapy is administered daily during any of these time periods.
  • the disclosure provides methods for conditioning a subject for HCT, the method comprising administering to the subject an 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 aboutlO 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. In other embodiments the TBI is administered the day of the HCT prior to engraftment. In particular embodiments, the TBI is administered once, e.g., on any of the indicated days.
  • the chemotherapy is administered on days -10, -9, -8, -6, -7, - 5 -4, -3, -2, and/or -1 days prior to the HCT. In certain embodiments, the chemotherapy is administered daily during any of these time periods.
  • the 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 (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
  • 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/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.
  • 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.
  • 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/m2/day of the chemotherapy, optionally about 30 mg/m2/day of the fludarabine and/or clofarabine about 10 to about 1 days prior to the HCT.
  • the 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 CD47, CD117, and/or CXCR4 polypeptide and/or nucleic acid encoding a CD47, CD117, and/or 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 1-2 xlO 6 CD34+ cells/kg body weight for autologous and allogeneic transplants.
  • Higher doses can include, for example, at least or about 3xl0 6 , at least or about 4xl0 6 , at least or about 5xl0 6 , at least or about 6xl0 6 , at least or about 7xl0 6 , at least or about 8xl0 6 , at least or about 9xl0 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. 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.
  • 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 exogenous or introduced CD47, CD117, and/or CXCR4 polypeptides and/or one or more of an exogenous or introduced nucleic acid sequence encoding a CD47, CD 117, and/or CXCR4 polypeptide.
  • the nucleic acid sequence is operably linked to a promoter sequence.
  • the HSC or HSPC administered to the subject comprises one or more exogenous or introduced nucleic acid sequences encoding a CD47 and a CD117 polypeptide. In some embodiments, the HSC or HSPC administered to the subject comprises one or more exogenous or introduced nucleic acid sequences encoding a CD47 and a CXCR4 polypeptide. In some embodiments, the HSC or HSPC administered to the subject comprises one or more exogenous or introduced nucleic acid sequences encoding a CD117 and a CXCR4 polypeptide. In some embodiments, the HSC or HSPC administered to the subject comprises one or more exogenous or introduced nucleic acid sequences encoding a CD47, a CD117, and a CXCR4 polypeptide.
  • the exogenous or introduced nucleic acid sequence(s) encoding CD47, CD117, and CXCR4 are in the same HSC and/or HSPC cell to be administered to the subject. In some embodiments, the exogenous or introduced nucleic acid sequence encoding CD47, CD117, and CXCR4 are in separate HSC and/or HSPC cells to be administered to the subject.
  • 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 cells and methods disclosed herein may be used to treat a variety of indications amenable to treatment with stem cell transplantation, including hematological diseases.
  • the cells and methods disclosed herein may be used in the context of any hematopoietic cell transplant to treat any disease or disorder requiring such a transplant. Examples include, gene therapy, cord blood transplant, treatment of leukemias and cancers, and treatment of non-cancer diseases.
  • the modified cells and methods may be used to treat a leukemia or a severe combined immunodeficiency (SCID). They may also be used to treat various bone marrow failure states and diseases, as well as hemoglobinopathies.
  • SCID severe combined immunodeficiency
  • the modified cells and 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 myel
  • 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,
  • the methods disclosed are used to treat a solid tissue cancer or a blood cancer, such as a leukemia, a lymphoma, or a myelodysplastic syndrome.
  • the disease is a blood cancer, optionally a leukemia, a lymphoma, or a myelodysplastic syndrome (MDS).
  • 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).
  • the leukemia is acute myeloid leukemia (AML).
  • 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
  • MDS/MPN myelodysplastic/myeloproliferative neoplasm
  • MDS/MPN myelodysplastic/myeloproliferative neoplasm
  • MDS/MPN myelodysplastic/myeloproliferative neoplasm
  • MDS/MPN myelodysplastic/myeloproliferative neoplasm
  • 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.).
  • 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
  • MDS/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.
  • 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.
  • Myelodysplastic syndromes 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.
  • cytopenia i.e., lower-than-normal peripheral blood cell counts
  • MDS is characterized according to Table 1.
  • MDS with multilineage dysplasia MDS with multilineage dysplasia
  • MDS with ring sideroblasts (MDS-RS): >15% ring sideroblasts in BM or >5% with SF3B1 mutation
  • MDS-RS-SLD MDS with ring sideroblasts and single-lineage dysplasia
  • MDS-RS-MLD MDS with ring sideroblasts and multilineage dysplasia
  • MDS-EB 1 MDS with excess blasts-1
  • MDS-EB2 MDS with excess blasts-2
  • BM bone marrow
  • MDS myelodysplastic syndromes
  • WHO World Health Organization.
  • 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 cell disease or Fanconi anemia.
  • Sickle cell diseases that may be treat include, but are not limited to: HbS disease; drepanocytic anemia; meniscocytosis, and chronic hemolytic anemia.
  • the method further comprises administering to the subject a therapeutic agent for treatment of the disease or disorder being treated by the HCT method.
  • Control parental Ba/F3 cells did not proliferate in the absence of IL-3. Further, parental Ba/F3 cells did not express CD117 and are not responsive to SCF signaling. Therefore, control parental Ba/F3 cells did not proliferate in the presence of increasing concentrations of SCF and there was no effect on viability or proliferation with the addition of JSP191 ( Figure 2).
  • Ba/F3 cell line expressing wild-type human CD117 c-Kit, sequence of ctl80, Table 5
  • Ba/F3 cell line expressing the CD117-D816V mutant was able to proliferate in the absence of SCF and proliferation was not inhibited by the presence of the anti-CDl 17 antibody JSP191 ( Figure 2).
  • HEK-293T cells were transfected with a wild type (WT) construct of the CD117 protein or with vector alone in 384-well format, followed by confirmation of cellular expression via high-throughput flow cytometry.
  • the MAbs tested included JSP191 and AB85; the ligand tested included AF488-conjugated stem cell factor (SCF), and the control MABs tested included YB5.88 (Invitrogen, Cat. No. 14-1179-82) and 104D2 (BioLegend, Cat. No. 313202), all of which bind WT CD117.
  • Shotgun Mutagenesis epitope mapping services were provided by Integral Molecular (Philadelphia, PA) as described in Davidson and Doranz, 2014. Briefly, a mutation library of the target protein was created by high-throughput, site-directed mutagenesis. Each residue was individually mutated to alanine, with alanine codons mutated to serine. The mutant library was arrayed in 384-well microplates and transiently transfected into HEK293T cells. Following transfection, cells were incubated with the indicated antibodies at concentrations predetermined using an independent immunofluorescence titration curve on wild type protein.
  • MAbs were detected using an Alexa Fluor 488-conjugated secondary antibody and mean cellular fluorescence was determined using Intellicyt iQue flow cytometry platform. Mutated residues were identified as being critical to the MAb epitope if they did not support the reactivity of the test MAb but did support the reactivity of the reference MAb. This counterscreen strategy facilitates the exclusion of mutants that are locally misfolded or that have an expression defect.
  • Binding of each test Ab to each mutant clone in the alanine scanning library was determined, in duplicate, by high-throughput flow cytometry. For each point, background fluorescence was subtracted from the raw data, which were then normalized to Ab reactivity with WT target protein. For each mutant clone, the mean binding value was plotted as a function of expression (represented by control reactivity) ( Figures 3A-C). To identify preliminary primary critical clones (lower right quadrant of graphs), a threshold (dashed lines) of >55% WT binding to control Ab and ⁇ 10% WT binding to test Abs was applied for JSP191, and >45% WT binding to control Ab and ⁇ 10% WT binding to test Abs for AB85.
  • 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.
  • 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.
  • IVT was performed to create mRNAs encoding CXCR4 containing various sequence (Table 8) 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 Nl-methyl-pseudouridine, or adding 5’ cap analogs such as CleanCapAG (TriLink). DNA templates were removed by DNase digestion.
  • desired sequences e.g., genes, mutants, non-coding elements such as untranslated regions
  • 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 7). 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).
  • CXCR4 mRNA (Table 8) 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.
  • BM homing varied dramatically by CXCR4 mRNA chemical modification (Figure 11 A).
  • Hyperactive CXCR4 mutant sequences were examined, including the common WHIM mutant tl 9 (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 1 IB).
  • 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 CD 19+ 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 mRNA constructs were optimized by identifying preferred stop codons and UTRs. As shown in Figure 50, the stop codon “TAATAA” was superior to others tested, and the UTR combination of 5’ HBA1 and 3’ HBB1 was superior to others tested.
  • CXCR4 wild type and the N119S mutant were evaluated in vivo using the NSG mice model. As shown in Figure 54, CXCR4-119S mRNA improved engraftment of CD34 cells in NSG mice. Control/CXCR4-Nl 19S % change was 11.8. CXCR4 expression (fold change) as compared to control (1.0) was 0.96 for mock, 6.43 for CXCR4-WT, and 7.50 for CXCR4- 119S.
  • 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-CDl 17 Antibody Synergizes with 5-Azacytidine to Augment Engraftment of Hematopoietic Stem Cells in Mice with Sickle Cell Disease in Transplantation & Cellular Therapy Meetings. 2021.
  • CXCR4 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 shortterm 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).
  • FACS fluorescence activated cell sorting
  • CXCR4 mRNA electroporated, mock electroporated, and non-electroporated CD 117+ 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 (Table 8) 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.
  • TriLink CD47-mr7 mRNA was electroporated into human CD34+ cells.
  • Mr7 is a CD47 encoding mRNA produced by TriLink with TriLink UTRs and 5moU U- substitution (also referred to as ct71, Table 2).
  • Figure 12 shows that electroporation of CD47-mr7 mRNA resulted in CD47 overexpression in human CD34+ cells, as compared to un-electroporated human CD34+ cells (Control) or human CD34+ cells electroporated with no mRNA (Mock). Under these conditions, CD47 expression on the human CD34+ cell surface had a U life of about 1 week.
  • CD47 mRNA expression in hematopoietic cell transplants human CD34+ cells were electroporated with CD47-mr7 mRNA (5 ug mRNA electroporated into IM HSCs) and transplanted intravenously into immunocompromised NOD scid gamma (NSG) mice (0.5 M eHSCs transplanted per mouse).
  • Figure 13 A shows that CD47- mr7 expressing human CD34+ cells transplanted in mice exhibited higher bone marrow homing than did control or mock mRNA expressing human CD34+ cells, 1 day after transplant.
  • Figure 13B shows that CD47 expressing human CD34+ cells demonstrated a 14-fold improvement (p ⁇ 0.02) at 1 month after transplant in the percent of human HSC derived granulocytes in the mouse blood stream, relative to the control and mock mRNA transfected cells. mRNA optimization
  • a segmented polyA tail was developed in which 140 bases were segmented by a linker, of 4-10 varied bases, into two sequences of 70 adenine bases each, to reduce recombination (CD47-A140S).
  • Figure 18 shows the AMOS segmented polyA tail design. mRNA encoding CD47 and comprising the AMOS poly A is shown in ct51 (Table 2).
  • CD47-mr7 mRNA 5 ug of the CD47-mr7 mRNA and varied concentrations of the CD47-A70 mRNA (comprising a polyA tail of 70 adenine bases) were tested (1 ug to 40 ug).
  • the CD47-A70 mRNAs were produced using in vitro transcription with CleanCapAG-30Me 5’ caps and N1 -methylpseudouridine uridine analogs, and CD47 expression was assayed 1 day after electroporation into human CD34+ cells. It was observed that increased dosage of the CD47-A70 mRNA increased CD47 expression in human CD34+ cells (Figure 15).
  • the 5’ and 3’ untranslated regions (UTRs) of the mRNA encoding CD47 were optimized in vitro in human CD34+ cells.
  • CD47-A70 mRNAs encoding CD47 with 5’UTR from HBA1 and 3 ’UTRs from HBA1 and/or HBB1 were tested for CD47 expression.
  • the mRNAs were produced using in vitro transcription with CleanCapAG-3OMe 5’ caps and Nl-methyl-pseudouridine uridine analogs, and CD47 expression was assayed 1 day after electroporation into human CD34+ cells.
  • CD47 expression in vitro in human CD34+ cells was compared between mRNA encoding wild type CD47 and CD47 mutant sequences (Table 2).
  • the E97K mutant is a dead CD47, which does not bind SIRP-a.
  • the K67E mutant is a CD47 variant that exhibits 150% binding of SIRP-a relative to the wild type CD47 protein.
  • mRNAs were produced using in vitro transcription with CleanCapAG-3OMe 5’ caps and Nl-methyl- pseudouridine uridine analogs, and CD47 expression was assayed 1 day after electroporation.
  • the mRNA encoding wild type CD47 exhibited the highest level of expression in human CD34+ cells.
  • Figure 17 shows CD47 expression relative to the CD47 sequence used.
  • Figure 20 shows an example plasmid developed for in vitro mRNA transcription, with sequences presented in Figure 21 (SEQ ID NOs: 208-213) and Table 2.
  • the plasmid based on pUC57-Kan and about 3.5 kb in size, comprises a T7 CleanCapAG promoter, a 5’ UTR from alpha-globin, a consensus Kozak sequence, a CD47 wild type coding sequence, a 3’UTR from beta-globin, an AMOS segmented tail as described above, and an Xbal linearization site.
  • Modified mRNAs encoding wild type and mutant CD47, CD 117 (cKIT), and CXCR4 were developed and tested in CD34+ HSC and Ba/F3 cells with the aim of improving HSC engraftment and chimerism after HCT transplant. Further to this, modified mRNAs encoding an N505I mutant CD117, an E73A/N505I double mutant CD117 and a wild type CXCR4 (Table 7) were tested in HSC and Ba/F3 cells for their ability to confer resistance to the CD117 antibody, JSP191.
  • Figures 24-27 demonstrate human CD34+ live cell numbers and cell viability 3 hours, 20 hours, and 48 hours after transfection with the modified mRNAs provided herein. As shown in Figures 32 and 33, CD117 expression increased ⁇ 15-fold for wild type and E73 mutant 3 hours post transfection. There were also two cell populations with different CD117 expression levels in human CD34+ cells, 3 hours after the cells were electroporated with wild type and E373A mRNAs (Table 7) ( Figure 30). However, the two levels of expression resolved by 20 hours post transfection ( Figures 32A-C).
  • CD 117 mutant and CXCR4 expression provide resistance to JSP191
  • CD117 mRNAs encoding wild type or various mutant CD117s were electroporated into human CD34+ cells, and CD117 expression was analyzed. As shown in Figure 44A, the cells expressed CD117 highly within hours of electroporation and for about one day after electroporation. Wild type CD117 was expressed at higher levels than CD117 N505I, but both showed greater expression than control or mock electroporated cells. Cell viability is shown in Figure 44B.
  • CD34+ cells were electroporated with mRNA constructs expressing CXCR4 or CD47, separately or in combination, and expression levels of CD47 and CXCR4 as compared to baseline were determined.
  • the CD47 mRNA construct was mr37, which corresponds to ctl84 with an A90 polyA tail, NlmPsU replacement of uridine, and CleanCapAG-3OMe cap.
  • the CXCR4 mRNA construct was ctl l4 (Table 8), which also has an A90 polyA tail, NlmPsU replacement of uridine, and CleanCapAG-3OMe cap. As shown in Figure 42, increased amounts of mRNA resulted in increased expression of CXCR4.
  • CXCR4 lug ⁇ 4.3 times
  • CXCR4 2ug ⁇ 8.5 times
  • CXCR4 4ug ⁇ 16 times
  • CXCR4 lug (combination) ⁇ 5 times.
  • the modified CD34+ cells were used to inject was also tested in NBSGW mice, which is a no-irradiation model mice, at 100,000 cells/mice via R/O route of administration.
  • Test groups included: Control (6 mice), CXCR4 lug (6 mice), CXCR4 2ug (6 mice), CXCR4 4ug (6 mice), CD47 4ug (5 mice), and combination of CD47 4 ug and CXCR4 lug (6 mice).
  • hCD34 + cell are expressed with more than 95% across all the samples.
  • CD117 binds stem cell factor (SCF) to regulate HSC survival, self-renewal, and differentiation.
  • SCF stem cell factor
  • human CD34+ and/or Ba/F3 cell lines were transfected with various mRNA constructs encoding wild type CD117 (Table 1, produced from templates comprising SEQ ID NOs: 5 and 6), CD117 E73A from (produced from a template comprising SEQ ID NO: 7), or another CD117 with an amino acid substitution not identified as critical for JSP191 binding, (KG2-DV and KG2-DV-5moU).
  • SEQ ID NOS: 5-7 are the DNA templates and include a T7 promoter, CleanCapAG 3’ OME Kozak sequence, an HBA1 5’ UTR, a TAATGA double stop codon, an HBB1 3’ UTR.
  • SEQ ID NOs: 5-7 do not include the polyadenosine tail of 70 nucleotides that is present in the template and resulting mRNAs.
  • the mRNAs correspond to mRNA expressed from these templates. Controls included no transfection or null transfection where indicated. All mRNAs were generated using Nlm- pseudouridine instead of uridine.
  • cKIT WT col produced from SEQ ID NO: 6
  • cKIT_E73A_col produced from SEQ ID NO: 7
  • PhaRNA and TriLink c-Kit DV correspond to ctl82 (with chemical modifications as indicated);
  • cKit WT col corresponds to ct97, and
  • cKit_E73A_col corresponds to ct98.
  • Figure 54 When measured just 3 hours after electroporation, the mRNAs provided herein demonstrated significant improvement in in CD117 expression levels over the aforementioned mRNAs ( Figure 54). At the same time, the mRNAs provided herein showed similar levels of CD34+ cell viability to the comparison mRNAs (KG2-DV and KG2-DV-5moU) 20 hours after electroporation ( Figures. 55A and 55B).
  • Figure 56 is a graph of cell count versus CD117 expression in human CD34+ cells expressing mock electroporated (mock EP), null (control), wild type CD117, and CD117 E73A mRNAs. Figures.
  • 57A-57C demonstrate that CD34+ cells expressing wild type CD117 and E73A mRNAs exhibited two distinct levels of transient CD117 expression at 3 hours after electroporation, which resolved by 20 hours after electroporation.
  • Figure 58 shows CD117 expression levels over 72 hours following electroporation.
  • E73 A CD117 mutant lentivirus constructs were transfected into Ba/F3 cells.
  • the Ba/F3 cells grew in response to human stem cell factor, and independently of IL3 stimulation ( Figure 59A).
  • the E73A mutant expressing cells were more growth responsive to stem cell factor than were the wild type ( Figure 59A).
  • Expression of E73A led to nearly complete resistance to JSP191 ( Figure 59B), even at higher concentrations.
  • Figures. 60A-60B show the effects of CD117 expression on human CD34+cell growth in the presence of stem cell factor (SCF) and the JSP91 antibody.
  • Figure 60A shows the growth of cells transfected without mRNA.
  • Figure 60B shows the growth of cells transfected with non-codon optimized wild type CD117 mRNA (as encoded by SEQ ID NO: 54).
  • Figure 61 shows cKit dose/expression correlations following electroporation with mRNAs encoding wild type CD117, CD117 E73A (Table 1), or controls. Both wild type CD117 and CD117 E73A showed increased expression as compared to control or mock transfected cells.

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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. 63/396,912, filed August 10, 2022, and U.S. Provisional Patent Application Serial No. 63/485,249, filed February 15, 2023, both of which are incorporated herein by reference in their entireties.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
[0002] The contents of the electronic sequence listing (JATH_010_03WO_SeqList_ST26.xml; Size: 512,568 bytes; and Date of Creation: August 9, 2023) are herein incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0003] The present disclosure relates to modified or engineered 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. Hematopoietic cell transplantation may also be performed in the context of gene therapy, in order to provide to a patient hematopoietic stem cells that express a nucleic acid or protein missing or mutated in the patient’s endogenous hematopoietic cells.
[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 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 modified HSCs and HSPCs and related compositions and methods of use thereof in hematopoietic stem cell transplant.
[0009] In one embodiment, the disclosure provides a modified or engineered cell comprising one or more exogenous or introduced nucleic acids encoding a combination of two or more of: a CD47 polypeptide, a modified CD117 polypeptide with constitutive activity, a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning, and/or a CXCR4 polypeptide, or functional fragments or variants thereof. In certain embodiments, the modified or engineered cell comprises: (1) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity; (2) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning; (3) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (4) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity and an exogenous or introduced nucleic acid encoding a modified CD 117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning; (5) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (6) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CD117 antibody used for HCT conditioning and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (7) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity, and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CD117 antibody used for HCT conditioning; (8) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity, and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (9) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning, and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (10) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning, and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (11) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning, and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (12) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning; (13) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning; (14) an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning; or (15) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide, and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning. In certain embodiments, each polypeptide is encoded by a different introduced nucleic acid, so the cells may comprise two or more different introduced nucleic acids. In certain embodiments, two or more polypeptides may be encoded by a single introduced nucleic acid, so the cells may comprise only one introduced nucleic acid. In certain embodiments, the introduced nucleic acids are transiently present in the cells and/or transiently express the polypeptide(s) in the cells, e.g., for about one day to about 10 days, or about one day to about one week, e.g., for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.
[0010] In some embodiments, the cell is transduced with one or more vectors disclosed herein or one or more mRNAs, e.g., the modified or engineered cell comprises an exogenous or introduced polynucleotide sequence encoding a CD47 polypeptide, one or more modified CD117 polypeptide, and/or a CXCR4 polypeptide. In certain embodiments, the cell is a stem cell, e.g., an 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+.
[0011] 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 a CD47 polypeptide, a CD117 polypeptide, and/or a CXCR4 polypeptide, optionally wherein the modified cell expresses the two or more CD47, CD 117, and/or CXCR4 polypeptides transiently. In certain embodiments, the CD47 polypeptides, one or more CD47 polypeptide, and/or CXCR4 polypeptides are human polypeptide, or a variant or fragment thereof.
[0012] In particular embodiments, the CD47 polypeptide is a modified CD47 polypeptide, e.g., with increased or constitutive activity as compared to the corresponding wild type CD47 polypeptide.
[0013] In particular embodiments, the CD117 polypeptide is a modified CD117 polypeptide, e.g., with constitutive activity as compared to the corresponding wild type CD117 polypeptide. In certain embodiments, the modified CD117 polypeptides provide for constitutive signaling and/or CD117-mediated kinase activity when expressed in cells, e.g., HSCs and/or HSPCs. Accordingly, in particular embodiments, when expressed in HSCs and/or HSPCs, the modified CD117 polypeptides allow CD117 signaling when bound by antibodies that block SCF binding to CD117. In some embodiments, the modified CD117 polypeptide comprises one or more amino acid modifications as compared to a wild type CD117 polypeptide, e.g., one or more amino acid substitutions, insertions, or deletions with increased or constitutive activity as compared to the corresponding wild type CD117 polypeptide. In certain embodiments, the modified CD117 polypeptide comprises one or more amino acid substitutions, e.g., at one or more of the following amino acids present in wild type human CD117: N505, V559, D816, V568, V570, Y703, or D816, such as, e.g., a D816V substitution and/or a N505I substitution. [0014] In particular embodiments, the CD117 polypeptide is a modified polypeptide, e.g., which does not bind the anti-c-Kit antibody used for HCT conditioning. In certain embodiments, the modified CD117 polypeptide provides SCF-mediated signaling and/or CD117-mediated kinase activity when expressed in cells, e.g., HSCs and/or HSPCs. Accordingly, in some embodiments, when expressed in HSCs and/or HSPCs, the modified CD117 polypeptides allow CD117 signaling in the presence of antibodies that block SCF binding to wild type CD117. In some embodiments, the modified CD117 polypeptide comprises one or more amino acid modifications as compared to a wild type CD117 polypeptide, e.g., one or more amino acid substitutions, insertions, or deletions, e.g., which inhibit binding to an anti-c-Kit antibody used for HCT conditioning. In certain embodiments, the modified CD117 polypeptide comprises one or more amino acid substitutions, e.g., at one or more of the following amino acids present in wild type human CD117: E73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, or R271, or at an amino acid residue located within 2, within 3, within 4, within 5, within 6, within 7 within 8 within 8 within 10, within 11 or within 12 amino acids of any ofE73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, or R271, e.g., either N-terminal or C-terminal of any of these residues. In particular embodiments, the one or more amino acid modifications is located within surface exposed amino acid residues of the extracellular domain of the wild type CD117 polypeptide.
[0015] 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 tl9 deletion corresponding to deletion of the C-terminal 19 amino acid residues of CXCR4. [0016] 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.
[0017] In a further related embodiment, the disclosure provides a pharmaceutical composition comprising the modified cells, e.g., HSCs and/or HSPCs, comprising one or more nucleic acids encoding the CD47 polypeptide, the CD117 polypeptide, and/or 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 CD47, CD117, and/or CXCR4 into CD34+ HSPCs selected from mobilized peripheral blood.
[0018] In a related aspect, the disclosure includes a method of modifying a cell, e.g., an HSC or HSPC, comprising introducing one or more nucleic acid or vector encoding two or more of a CD47 polypeptide, a CD117 polypeptide (constitutively active), a CD117 polypeptide (not bound by an anti-CD117 antibody) and/or 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.
[0019] 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 one or more nucleic acid or vector encoding two or more of a CD47 polypeptide, a CD117 polypeptide (constitutively active), a CD117 polypeptide (not bound by an anti-CD117 antibody) and/or a 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).
[0020] In particular embodiments, expression of the combination of two or more of CD47, CD117(s), and/or CXCR4 (e.g., transiently) in HSCs and/or HSPCs improves HCT. In particular embodiments, CD47, CD117, and/or CXCR4 transient expression in HSCs and/or HSPCs improves HSCs/HSPCs ability to bind signal regulatory protein a (SIRPa) in vitro. In particular embodiments, CD47, variant CD117, and/or 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, CD47, variant CD 117, and/or 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, CD47, variant CD117, and/or 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.
[0021] 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
[0022] Figure 1 is a chart depicting illustrative myeloablative, reduced intensity myeloablative, and non-myeloablative conditioning regimens that may be used according to the disclosure, and is 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.
[0023] Figure 2 is a graph showing the OD595 of wild type BaF3 cells (BaF3), or BaF3 cells expressing wild-type CD117 (c-Kit) or the CD117-D816V mutant in the presence of the indicated concentrations of stem cell factor (SCF), and in the presence or absence of the anti- CD117 antibody, JSP191. The mRNA construct expressing wild-type CD117 corresponds to ctl80, which includes the CleanCap cap and includes Nlm-pseudouridine instead of uridine. These results show that the CD117-D816V mutant confers a proliferative advantage to cells, even in the absence of SCF and the presence of the anti-CDl 17 antibody, JSP191.
[0024] Figures 3A-3C are graphs showing binding of JSP191 (Figure 3A) or AB85 (Figure 3B (whole view) and Figure 3C (zoomed in view)) to clones of an alanine scanning library. Mean binding value is plotted as a function of expression (represented by clone reactivity) for each clone.
[0025] Figure 4 is a table showing the results of alanine scanning of JSP191Fab, AB85Fab and 104D2 Mab. Mean binding reactivity (and range) of each to clones comprising the indicated point mutations in CD117 is shown as % binding to wild type CD 117. Critical residues for Ab binding are shaded.
[0026] Figures 5A-5B show crystal structures of JSP191 Fab (Figure 5A) and AB85 Fab (Figure 5B). Critical residues of CD117 (dark spheres) are shown on the crystal structure of the target Abs (Yuzawa et al., 2007).
[0027] Figure 6 is a table listing the critical residues whose mutation gave the lowest reactivities with the JSP191 or AB85 antibody.
[0028] Figure 7 is a schematic of the 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 NlmPsU modification; 2 = CXCR4 tl9 mutant with full NlmPsU modification; 3= CXCR4 119S mutant with full NlmPsU modification; 4 = ShGFP control with full NlmPsU modification; and 5 = GSP control with full NlmPsU modification.
[0029] Figure 8 shows isolation of human CD34+ HSPCs from mobilized peripheral blood using the 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.
[0030] Figures 9A-9E show electroporation of human CD34+HSPCs with CXCR4 mRNA. Figure 9A shows mRNAs encoding various CXCR4 sequences containing various chemical modifications were electroporated and characterized. Figure 9B shows 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 NlmPsU/5mC modification; CXCR4 wild type with PsU modification; CXCR4 tl9 mutant with NlmPsU modification; and CXCR4 wild type with NlmPsU modification. Figure 9C shows 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 (ct73 with TriLink UTRs and 120 A poly A tail) electroporated over time. The mRNA used was CXCR4 wild type with 5moU modification. Figure 9D shows viability as measured by flow cytometry staining of live cells of CD34+ HSPCs that were not electroporated (Control), mock electroporated, and CXCR4 mRNA (ct73 with TriLink UTRs and 120 A poly A tail) electroporated over time post-electroporation. The mRNA used was CXCR4 wild type with 5moU modification. Figure 9E shows representative flow cytometry analyses of CD34+ HSPCs cultured for 4 or 24 hours after no electroporation (Control), mock electroporation, and CXCR4 mRNA (ct73 with TriLink UTRs and 120A polyA tail) electroporation. The mRNA used was CXCR4 wild type with 5moU modification. [0031] Figures 10A and 10B show the effect of mRNA chemistry on CXCR4 expression and in vitro HSPC migration towards SDF-1. Figure 10 A is a graph showing CXCR4 mRNAs containing various chemistries and their corresponding expression levels. The mRNAs correspond to: XI = CleanCapAG, 5moU, TriLink; A = CleanCapAG-3’OMe, NlmPsU (NEB Hi Scribe); B = CleanCapAG, NlmPsU (NEB Hi Scribe); and D = CleanCapAG-3’OMe, NlmPsU (Ambion Mega). XI corresponds to ct73, and the others correspond to ct2 with the chemistry as indicated. All mRNAs encode wild type CXCR4. Figure 10B is a bar graph showing various mRNA chemistries led to differences in transwell migration. mRNA “A” contained Nlm-pseudouri dine U-substitution, and CleanCapAG-3’OMe (TriLink).
[0032] Figures 11A and 11B show the homing of CXCR4 mRNA electroporated human CD34+ HSPCs into the bone marrow of NSG mice shortly after transplantation. Figure 11A shows the frequency of HSPCs homing to the BM at ~16 hours post injection of mRNA ct73 with TriLink UTRs and chemistry as indicated. mRNAs encoding WT CXCR4 were synthesized with the indicated nucleoside substitutions. All mRNAs encoded wild type CXCR4. Figure 11B shows mRNAs with the same chemistry (NlmPsU) but the indicated CXCR4 mutant sequences (ct74 and ct75) with CleanCapAG-3OMe cap and NlmPsU sub stituti on of Us) .
[0033] Figure 12 shows CD47 overexpression in human CD34+ cells electroporated with CD47-mr7 mRNA, relative to control cells and mock mRNA.
[0034] Figures 13 A and 13B show the relative percentages of human CD45+ cells after transplant with CD34+ cells transfected with CD47-mr7 mRNA. Figure 13 A shows the percent of human CD45+ cells in the bone marrow 1 day after transplant, and Figure 13B shows the percent of human HSC derived granulocytes in the blood system 1 month after transplant. [0035] Figure 14 shows CD47 expression relative to each indicated polyA.
[0036] Figure 15 shows CD47 expression relative to the mRNA used and the dose of each mRNA used.
[0037] Figures 16A and 16B show CD47 expression relative to the UTR included in the mRNA sequence (Figure 16A) and the position of each 5’ and 3’ UTR used (Figure 16B). [0038] Figure 17 shows CD47 expression relative to the CD47 sequence used.
[0039] Figure 18 shows CD47 expression in human CD34+ cells relative to the length of the mRNA polyA tail.
[0040] Figure 19 shows the design of the segmented polyA tail AMOS (SEQ ID NOs: 206 and 207), in which the 140 adenine bases are segmented into 2x 70 adenine sequences.
[0041] Figure 20 shows the design of an example plasmid that may be used for in vitro transcription of a CD47 mRNA. Similar plasmid constructs may be used for expression of other proteins, such as CXCR4 or cKIT.
[0042] Figure 21 provides illustrative sequences to be used in the example plasmid of Figure 20 (SEQ ID NOs: 208-213).
[0043] Figure 22 shows expression levels of CD117 (cKIT), CXCR4, and CD47 3 hours after transfection in human CD34+ cells with the indicated modified mRNAs. [0044] Figure 23 shows expression levels of CD117 (cKIT), CXCR4, and CD47 20 hours after transfection human CD34+ cells with the modified mRNAs.
[0045] Figure 24 shows expression levels of CD117 (cKIT), CXCR4, and CD47 48 hours after transfection human CD34+ cells with the modified mRNAs shown in Table 1.
[0046] Figure 25 shows human CD34+ live cell numbers 3 hours after transfection with the indicated modified mRNAs.
[0047] Figure 26 shows human CD34+ live cell numbers 20 hours after transfection with the modified mRNAs.
[0048] Figure 27 shows human CD34+ live cell numbers 48 hours after transfection with the modified mRNAs.
[0049] Figure 28 shows human CD34+ cell viability 20 hours after transfection with the modified mRNAs.
[0050] Figures 29A and 29B are graphs showing that expression of CD117 E73A/N505I double mutant in Ba/F3 cells led to enhanced growth and JSP191 resistance. Cells in these experiments were transfected with lentiviral constructs encoding wild type, E73 A, N505I, and E73A/N505I double mutant CD117 proteins. Figure 29A shows growth of Ba/F3 cells with wild type and E73A/N505I mutant CD117 expression in the presence of human stem cell factor (hSCF). Figure 29B shows growth of Ba/F3 cells with wild type and E73A/N505I mutant CD117 expression in the presence of the JSP191 antibody and hSCF.
[0051] Figure 30 is a graph of CD117 expression in human CD34+ cells 3 hours after transfection with the modified CD117 mRNAs.
[0052] Figure 31 is a graph of CD90 expression in human CD34+ cells, expressing high and low levels of CD117, 3 hours after transfection with the modified wild type CD117 mRNA.
[0053] Figures 32A-32C are graphs of CD117 expression in human CD34+ cells over time showing two distinct levels of wild type and E73A CD 117 mRNA expression at 3 hours after transfection, which resolve by 20 hours after transfection. Figure 32A shows cell count versus CD117 expression 3 hours after transfection; Figure 32B shows cell count versus CD117 expression 20 hours after transfection; and Figure 32C shows cell count versus CD117 expression 48 hours after transfection.
[0054] Figure 33 is a graph of the level of CD117 (cKit) expression for null control (Ctrl), mock electroporation (Mock EP), wild type (WT), E73A, N505I and E73A/N505I double mutant CD117 mRNA in human CD34+ cells post transfection. The various constructs are the same as described for Figure 2 but with the different point mutations introduced. [0055] Figure 34 is a graph of the level of CD117 (cKit) expression from modified CD117 mRNAs in human CD34+ cells post transfection.
[0056] Figure 35 shows cell count versus CXCR4 expression 3 hours after transfection with null control (Ctrl), mock electroporation (Mock EP), wild type (WT), and N119S CXCR4 mutant mRNA in human CD34+ cells.
[0057] Figure 36 is a graph of the level of CXCR4 expression from modified CXCR4 mRNAs in human CD34+ cells post transfection.
[0058] Figure 37 is a graph of the level of CXCR4 expression from modified CXCR4 mRNAs in human CD34+ cells post transfection.
[0059] Figures 38A and 38B are boxplots of human CD34+ cell chimerism in mouse bone marrow, 12 weeks after transplant with human CD34+ cells transfected with modified CXCR4 mRNAs. Also shown is the level of CXCR4 present in the chimeric cells.
[0060] Figure 39 shows dose-expression correlations of following electroporation of cells with the indicated doses of mRNA constructs encoding wild type of the indicated cKit mutants. [0061] Figures 40A and 40B show expression levels of CXCR4, CD47, and cKit three hours following electroporation of cells with the indicate mRNA constructs. PhaRNA and TL cKIT- DV correspond to ctl82 (with chemical modification as indicated, undisclosed UTRs); cKIT WT is ct96, cKIT WT col is ct97, and cKIT_E73A_col is ct98.
[0062] Figures 41A and 41B show cell number at day 3 (Figure 41A) and day 12 (Figure 4 IB) following electroporation of cells with the indicated mRNA constructs and following treatment with the indicated amount of JSP191 antibody. For each antibody concentration shown, the three bars from left to right correspond to control (Ctrl), mock electroporation (Mock EP), and E73 A-N505I.
[0063] Figure 42 is a graph showing increased expression of CXCR4 and/or CD47 3 hours following electroporation of CD34+ cells with the indicated amounts of mRNA constructs expressing CXCR4 and/or CD47.
[0064] Figure 43 is a graph showing increased expression of CXCR4 and/or CD47 1 day following administration of CD34+ cells modified via mRNA constructs expressing CXCR4 and/or CD47.
[0065] Figures 44A and 44B show in vitro expression. Figure 44A is a graph showing cKit expression kinetics following electroporation of mRNAs encoding the indicated cKit into human CD34+ cells. At about 20 hours, the lines from top to bottom correspond to: wild type cKit, unidentified cKit mutant, unidentified cKit mutant, cKit N505I, control, and mock electroporation (EP). Figure 44B shows viability and live cell numbers of CD34+ cells one day following electroporation with the indicated mRNA or control.
[0066] Figure 45 provides graphs showing CD34+ cell proliferation at 3 days and 12 days following electroporation with the indicated amount of the indicated mRNA construct. For each dose, the bars from left to right correspond to control (Ctrl; not electroporated), mock electroporated (Mock EP), and E73 A-N505I.
[0067] Figure 46 shows CXCR4 expression at 3 hours post-electroporation. The graph shows CXCR4 expression (fold of baseline) following transfection of each of the indicated CXCR4 mRNA constructs (Jasper ctl23 (wild type), Jasper 119S ctl l4, Jasper 119A ctl25, Jasper 119K ctl25, and Trilink 119S ct75). All tails were A90 unless otherwise specified.
[0068] Figure 47 shows CXCR4 at various timepoints following electroporation of the indicated CXCR4 mRNA constructs. At about 4 hours post-electroporation, the lines from top to bottom correspond to: N119S_Jasper, WT Jasper, N119S_TriLink, N119A_Jasper, N119K_Jasper, Control, and Mock electroporation (EP).
[0069] Figure 48 shows CXCR4 expression with various stop codons and UTRs. The left graph shows CXCR4 expression resulting from mRNA constructs comprising the indicated stop codons. The right graph shows CXCR4 expression resulting from mRNA constructs comprising the indicated UTRs:
Figure imgf000015_0001
[0070] Figure 49 shows expression of CXCR4 mutants. The top graph shows CXCR4 expression following electroporation with the indicated mRNA construct. At about 4 hours, the lines from top to bottom correspond to N119S_Jasper, WT Jasper, N119S_TriLink, Control (CTRL), and Mock electroporation (EP). The bottom graph shows cell growth following electroporation of cells with the indicated mRNA constructs. At 96 hours, the lines from top to bottom correspond to WT Jasper, N119S_Jasper, Control (CTRL), N119S_TriLink, and Mock electroporation (EP).
[0071] Figure 50 shows XCR half lives. The top graph shows CXCR4 expression following electroporation of the indicated mRNA constructs. At about 18 hours, the lines from top to bottom correspond to WT Jasper, ctruncl9_Jasper, N119K_Jasper, Control (CTRL), and Mock electroporation (EP). The bottom graph shows CXCR4 expression following electroporation of the indicated mRNA constructs. At about 4 hours, the lines from top to bottom correspond to: N119S_Jasper, N119S-ctruncl9_Jasper, N119S_TriLink, N119A_Jasper, Control, and Mock EP.
[0072] Figure 51 shows transmigration in a transwell assay. The graph shows the number of migrated cells following electroporation of the cells with the indicated CXCRR mRNA constructs or controls. Quadruple results are provided for each of the following constructs from left to right: Control, Mock EP, and CXCR4 N119S (1 ug), and in the absence of SDF1.
[0073] Figure 52 shows engraftment of CD34+ cells in NSG mice. The graph shows hCD34 cell chimerism 3 months following transplant of cells electroporated with each of the indicated constructs or controls. CXCR4 expression (fold change) was 1 for control, 0.96 for mock, 6.43 for CXCR4-WT, and 7.5 for CXCR4-119S.
[0074] Figure 53 is a graph of CD117 expression from different CD117 mRNAs (Table 1) or controls 20 hours after electroporation.
[0075] Figures 54A and 54B are graphs of cell viability 20 hours post electroporation with the indicated mRNAs (Table 1) or controls. Figure 54A is a graph of live cell numbers; Figure 54B is a graph of percent viability relative to cell death.
[0076] Figure 55 is a graph of CD17 expression from different CD117 mRNAs (Table 1) or controls 3 hours after electroporation.
[0077] Figure 56 is a graph of cell count versus CD117 expression in human CD34+ cells expressing mock, control (null), wild type, and E73A CD117 mRNAs (Table 1).
[0078] Figures 57A-57C are graphs of CD117 expression in human CD34+ cells over time showing initially two distinct levels of wild type and E73A CD117 expression which resolve by 20 hours after electroporation. Figure 57A shows cell count versus CD117 expression 3 hours after electroporation; Figure 57B shows cell count versus CD117 expression 20 hours after electroporation; and Figure 57C shows cell count versus CD117 expression 48 hours after electroporation.
[0079] Figure 58 is a graph of the level of CD117 (cKit) expression for mock electroporation (Mock EP), null control (Ctrl), wild type (WT), and E73A CD117 expressing cells.
[0080] Figures 59A and 59B are graphs showing that expression of CD117 E73A mutants in Ba/F3 cells leads to JSP191 resistance. Cells in these experiments were transfected with lentiviral constructs encoding wild type and E73 CD117 proteins. Figure 59A shows growth of Ba/F3 cells based on wild type and E73A CD117 expression in the presence of human stem cell factor (hSCF). Figure 59B shows growth of Ba/F3 cells based on wild type and E73 A CD117 expression in the presence of the JSP191 antibody.
[0081] Figures 60A-60B are graphs showing the effects of CD117 expression on cell growth in the presence of stem cell factor (SCF) and the JSP91 antibody. Figure 60A shows the growth of human CD34+ cells transfected without mRNA. Figure 60B shows the growth of cells transfected with wild type CD117 mRNA (as encoded by SEQ ID NO: 54).
[0082] Figure 61 shows cKit expression following electroporation with the indicated amounts of mRNAs encoding WT cKIT or cKit E73 A, or controls.
DETAILED DESCRIPTION OF THE INVENTION
[0083] 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.
[0084] 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.
[0085] 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.
[0086] In certain embodiments, the disclosure provides modified or engineered cells, e.g., HSCs and HSPCs, comprising a variant CD117 that is both constitutively active and is not bound by an antibody that binds to CD117, e.g., an antibody used for HCT conditioning, such as JSP191. In particular embodiments, the modified or engineered cells, e.g., HSCs and HSPCs, comprise an introduced or exogenous nucleic acid that encodes the variant CD117. In particular embodiments, the modified or engineered cells, e.g., HSCs and HSPCs, express the variant CD117. [0087] In certain embodiments, the disclosure provides modified or engineered e.g., HSCs and HSPCs, comprising two, three, or four or five modifications selected from: 1) expression of an exogenous or introduced CD47, 2) expression of an exogenous or introduced CXCR4, 3) expression of a variant CD117 that is constitutively active, 4) expression of a variant CD117 that is not bound by an antibody that binds to CD117, and 5) expression of a variant CD117 that is both constitutively active and is not bound by an antibody that binds to CD 117. In particular embodiments, the modified or engineered e.g., HSCs and HSPCs, comprise one or more introduced or exogenous nucleic acid that encodes the CD47, the CXCR4, the constitutively active variant CD117, the variant CD117 that is not bound by an antibody that binds to CD117, and/or a variant CD117 that is both constitutively active and is not bound by an antibody that binds to CD117. Of the CD47, CXCR4, and/or various variant CD117s may be encoded by different nucleic acids, or two or more may be encoded by a single nucleic acid molecule. In particular embodiments, the modified or engineered e.g., HSCs and HSPCs, express the variant CD117, the CD47, CXCR4, and/or various variant CD 117s.
[0088] In particular embodiments, the engineered or modified e.g., HSCs and HSPCs, comprises: 1) one or more of the variant CD117s; and 2) expression of introduced or exogenous CD47 and/or expression of introduced or exogenous CXCR4. In particular embodiments, modified CD117 polypeptides are modified so that they are either: (i) constitutively active or (ii) are not bound by an anti-CD117 antibody used for HCT conditioning, or (iii) both. Thus, in some embodiments, one or two different modified CD117 polypeptides may be expressed in the modified cells, alone or in combination with CD47 and/or CXCR4.
[0089] In certain embodiments, the disclosure provides for compositions and methods for the ex vivo introduction of a polynucleotide encoding a CD47 polypeptide, a CD117 polypeptide, and/or a CXCR4 polypeptide, e.g., human CD47, CD117, and/or CXCR4 polypeptides, by RNA-based and/or DNA-based methods, into cells, e.g., 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. In particular embodiments, the compositions and methods disclosed herein are used to introduce the CD47 mRNA, modified CD117 mRNA, and/or CXCR4 mRNA into allogeneic normal HSPCs to improve engraftment and replace a patient’s diseased stem cells with a healthy hematopoietic system.
[0090] The engineered or modified cells, e.g., HSCs and HSPCs, may be transplanted into a subject, e.g., to treat a disease or disorder, including those disclosed herein. Transplantation of these modified or engineered (e.g., genetically engineered) cells, e.g., HSPCs, may be done after or in combination with a conditioning regimen, including treatment with antibodies (such as anti-CD117 antibodies, e.g., JSP191). These modified or engineered HSPCs may be transplanted alone or in combination with other cells.
[0091] 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.
[0092] 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.
[0093] 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.
[0094] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
[001] 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 rlgG. The term also refers to recombinant single chain Fv fragments (scFv). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. An antibody binds a particular antigen, and is considered “specific for” the antigen when it preferentially recognizes/binds its target antigen in a complex mixture of proteins and/or macromolecules. An antibody is considered to not bind an antigen when it does not bind the antigen or does not preferentially recognize/bind the antigen in a complex mixture of proteins and/or other macromolecules. In certain instances, an antibody that binds a specific antigen is considered to not bind a modified form of the specific antigen if it binds to the antigen with an at least 5-fold or at least 10-fold lower dissociation constant (KD) than the KD of its binding to the unmodified specific antigen, where increased affinity is associated with lower KD. KD may be measured, e.g., by ELISA. [0095] 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.
[0096] 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. [0097] 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.
[0098] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. As understood in the art, sequence identity refers to the percentage identity obtained when sequences are aligned for maximum correspondence over a comparison window (e.g., a specified region of each of the sequences), which may be calculated by any of the algorithms described herein using default parameters, which are expected to generate the same alignment, in most cases, when applied to similar sequences. Identity is calculated, unless specified otherwise, across the full length of the reference sequence. Thus, a sequence-of-interest “shares at least x% identity to” a reference sequence if, when the sequence-of-interest is aligned to the reference sequence, at least x% (rounded down) of the residues in the sequence-of-interest are aligned as an exact match to a corresponding residue in the reference sequence. Gaps may be introduced into the sequence- of-interest and/or the reference sequence to maximize correspondence over the comparison window.
[0099] Sequence similarity (i.e., identity) can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the worldwide web at ncbi.nlm.nih.gov/BLAST/. Unless indicated to the contrary, sequence identity is determined using the BLAST algorithm (e.g., bl2seq) with default parameters. Sequence alignments may be performed using the NCBI Blast service (BLAST+ version 2.12.0).
[0100] 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).
[0101] Of interest is the BestFit program using the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482-489 (1981) to determine sequence identity. The gap generation penalty will generally range from 1 to 5, usually 2 to 4 and in many embodiments will be 3. The gap extension penalty will generally range from about 0.01 to 0.20 and in many instances will be 0.10. The program has default parameters determined by the sequences inputted to be compared. Preferably, the sequence identity is determined using the default parameters determined by the program. This program is available also from Genetics Computing Group (GCG) package, from Madison, Wis., USA.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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.
[0106] "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.
[0107] 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 polypeptide variant comprises at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to a reference polypeptide sequence, e.g., a native polypeptide sequence. A polynucleotide variant comprises at least one nucleobase difference (e.g., substitution, insertion, deletion) relative to a reference polynucleotide sequence, e.g., a native polynucleotide 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 fragments of a reference sequence, 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. Fragments generally comprise less than the full length native nucleic acid or polypeptide sequence, e.g., a fragment may comprise or consist of less than 100%, less than 99%, less than 98%, less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% of the native nucleic acid or polypeptide. The disclosure further includes variants of fragments, e.g., variant having a sequence identity of 50% or more, 60% or more, or 70% or more with a fragment of the native polynucleotide or polypeptide, 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 fragment. In particular embodiments, polypeptide variants and fragments are considered to be “functional” variants or fragments, if they substantially retain a biological activity of the native polypeptide, e.g., the ability to bind a cognate ligand or receptor, or the ability to modulate a biological process, such as, e.g., a signaling pathway, or cellular proliferation, differentiation, or apoptosis. As used herein, the term “CD47 polypeptide,” “CD117 polypeptide” and “CXCR4 polypeptide” and the like, encompasses native or wildtype CD47, CD 117, and CXCR4 polypeptides, as well as functional variants and functional fragments of a native polypeptide, and the term “CD47 polynucleotide,” “CD117 polynucleotide” and “CXCR4 polynucleotide” and the like, encompasses native or wild-type polynucleotides and nucleic acids, and variants and fragments thereof, that encode a CD47, CD 117, or CXCR4 polypeptide (including functional fragments and variants thereof). Functional variants retain at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% or more, of a biological activity of a reference polypeptide, e.g., kinase activity or binding activity.
[0108] 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.
[0109] 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.
[0110] 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.).
[OHl] 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.
[0112] 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: ALaboratory Manual (2001); Harlow, Lane andHarlow, 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).
CD47 Polypeptides and Polynucleotides
[0113] Leukocyte surface antigen CD47 (CD47), also known antigenic surface determinant protein OA3, integrin associated protein (IAP), and MER6, is a protein that in humans is encoded by the CD47 gene. The CD47 protein is a signal regulatory protein a (SIRPa) and SIRPy receptor. CD47 is expressed by most cells, including hematopoietic stem and progenitor cells (HSPC), and neurons. In certain embodiments, the CD47 polypeptide, including functional fragments and variants, binds to SIRPa, which is a regulatory membrane glycoprotein expressed mainly in HSPC and neurons. The interaction of CD47 with SIRPa inhibits innate immune response signaling and function, e.g., phagocytosis of the cell by macrophages. In certain embodiments, CD47 functional fragments and variants bind to SIRPa with at least 50%, at least 75%, or at least 90% of the specificity and affinity as a corresponding wild type CD47 protein.
[0114] Any CD47 protein, including functional fragments or variants thereof, may be used according to aspects of the disclosure. In certain embodiments, the CD47 polypeptide is a human CD47 polypeptide, while in other embodiments, it is another mammalian CD47 polypeptide. Sequences of human and mammalian CD47 polypeptides are known in the art. In particular embodiments, the CD47 polypeptide sequence comprises or consists of one of the following amino acid sequences: MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYV KWKFKGRDIYTFDGALNKSTVPTDF S S AKIEVSQLLKGD ASLKMDKSD AVSHTGNY TCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGM DEKTIALL VAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIA ILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQ PPRKAVEEPLNAFKESKGMMNDE (SEQ ID NO: 55);
OR
MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYV KWKFKGRDIYTFDGALNKSTVPTDF S S AKIEVSQLLKGD ASLKMDKSD AVSHTGNY TCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGM DEKTIALL
VAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIA
ILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQ PPRKAVEEPLNE (SEQ ID NO: 56);
OR
MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYV KWKFKGRDIYTFDGALNKSTVPTDF S S AKIEVSQLLKGD ASLKMDKSD AVSHTGNY TCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGM DEKTIALL
VAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIA
ILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQ PPRNN (SEQ ID NO: 57) or a variant or fragment thereof of any of these sequences, 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.
[0115] In particular embodiments, the CD47 polypeptide sequence, or fragment or variant thereof, is encoded by a polynucleotide sequence that comprises or consists of one of the following:
1 gcagcctggg cagtgggtcc tgcctgtgac gcgcggcggc ggtcggtcct gcctgtaacg
61 gcggcggcgg ctgctgctcc ggacacctgc ggcggcggcg gcgaccccgc ggcgggcgcg
121 gagatgtggc ccctggtagc ggcgctgttg ctgggctcgg cgtgctgcgg atcagctcag
181 ctactattta ataaaacaaa atctgtagaa ttcacgtttt gtaatgacac tgtcgtcatt
241 ccatgctttg ttactaatat ggaggcacaa aacactactg aagtatacgt aaagtggaaa
301 tttaaaggaa gagatattta cacctttgat ggagctctaa acaagtccac tgtccccact
361 gactttagta gtgcaaaaat tgaagtctca caattactaa aaggagatgc ctctttgaag
421 atggataaga gtgatgctgt ctcacacaca ggaaactaca cttgtgaagt aacagaatta
481 accagagaag gtgaaacgat catcgagcta aaatatcgtg ttgtttcatg gttttctcca
541 aatgaaaata ttcttattgt tattttccca atttttgcta tactcctgtt ctggggacag
601 tttggtatta aaacacttaa atatagatcc ggtggtatgg atgagaaaac aattgcttta
661 cttgttgctg gactagtgat cactgtcatt gtcattgttg gagccattct tttcgtccca
721 ggtgaatatt cattaaagaa tgctactggc cttggtttaa ttgtgacttc tacagggata
781 ttaatattac ttcactacta tgtgtttagt acagcgattg gattaacctc cttcgtcatt
841 gccatattgg ttattcaggt gatagcctat atcctcgctg tggttggact gagtctctgt
901 attgcggcgt gtataccaat gcatggccct cttctgattt caggtttgag tatcttagct
961 ctagcacaat tacttggact agtttatatg aaatttgtgg cttccaatca gaagactata
1021 caacctccta ggaaagctgt agaggaaccc cttaatgcat tcaaagaatc aaaaggaatg
1081 atgaatgatg aataactgaa gtgaagtgat ggactccgat ttggagagta gtaagacgtg
1141 aaaggaatac acttgtgttt aagcaccatg gccttgatga ttcactgttg gggagaagaa 1201 acaagaaaag taactggttg tcacctatga gacccttacg tgattgttag ttaagttttt
1261 attcaaagca gctgtaattt agttaataaa ataattatga tctatgttgt ttgcccaatt
1321 gagatccagt tttttgttgt tatttttaat caattagggg caatagtaga atggacaatt
1381 tccaagaatg atgcctttca ggtcctaggg cctctggcct ctaggtaacc agtttaaatt
1441 ggttcagggt gataactact tagcactgcc ctggtgatta cccagagata tctatgaaaa
1501 ccagtggctt ccatcaaacc tttgccaact caggttcaca gcagctttgg gcagttatgg
1561 cagtatggca ttagctgaga ggtgtctgcc acttctgggt caatggaata ataaattaag
1621 tacaggcagg aatttggttg ggagcatctt gtatgatctc cgtatgatgt gatattgatg
1681 gagatagtgg tcctcattct tgggggttgc cattcccaca ttcccccttc aacaaacagt
1741 gtaacaggtc cttcccagat ttagggtact tttattgatg gatatgtttt ccttttattc
1801 acataacccc ttgaaaccct gtcttgtcct cctgttactt gcttctgctg tacaagatgt
1861 agcacctttt ctcctctttg aacatggtct agtgacacgg tagcaccagt tgcaggaagg
1921 agccagactt gttctcagag cactgtgttc acacttttca gcaaaaatag ctatggttgt
1981 aacatatgta ttcccttcct ctgatttgaa ggcaaaaatc tacagtgttt cttcacttct
2041 tttctgatct ggggcatgaa aaaagcaaga ttgaaatttg aactatgagt ctcctgcatg
2101 gcaacaaaat gtgtgtcacc atcaggccaa caggccagcc cttgaatggg gatttattac
2161 tgttgtatct atgttgcatg ataaacattc atcaccttcc tcctgtagtc ctgcctcgta
2221 ctccccttcc cctatgattg aaaagtaaac aaaacccaca tttcctatcc tggttagaag
2281 aaaattaatg ttctgacagt tgtgatcgcc tggagtactt ttagactttt agcattcgtt
2341 ttttacctgt ttgtggatgt gtgtttgtat gtgcatacgt atgagatagg cacatgcatc
2401 ttctgtatgg acaaaggtgg ggtacctaca ggagagcaaa ggttaatttt gtgcttttag
2461 taaaaacatt taaatacaaa gttctttatt gggtggaatt atatttgatg caaatatttg
2521 atcacttaaa acttttaaaa cttctaggta atttgccacg ctttttgact gctcaccaat
2581 accctgtaaa aatacgtaat tcttcctgtt tgtgtaataa gatattcata tttgtagttg
2641 cattaataat agttatttct tagtccatca gatgttcccg tgtgcctctt ttatgccaaa
2701 ttgattgtca tatttcatgt tgggaccaag tagtttgccc atggcaaacc taaatttatg
2761 acctgctgag gcctctcaga aaactgagca tactagcaag acagctcttc ttgaaaaaaa
2821 aaatatgtat acacaaatat atacgtatat ctatatatac gtatgtatat acacacatgt
2881 atattcttcc ttgattgtgt agctgtccaa aataataaca tatatagagg gagctgtatt
2941 cctttataca aatctgatgg ctcctgcagc actttttcct tctgaaaata tttacatttt
3001 gctaacctag tttgttactt taaaaatcag ttttgatgaa aggagggaaa agcagatgga
3061 cttgaaaaag atccaagctc ctattagaaa aggtatgaaa atctttatag taaaattttt
3121 tataaactaa agttgtacct tttaatatgt agtaaactct catttatttg gggttcgctc
3181 ttggatctca tccatccatt gtgttctctt taatgctgcc tgccttttga ggcattcact
3241 gccctagaca atgccaccag agatagtggg ggaaatgcca gatgaaacca actcttgctc
3301 tcactagttg tcagcttctc tggataagtg accacagaag caggagtcct cctgcttggg
3361 catcattggg ccagttcctt ctctttaaat cagatttgta atggctccca aattccatca
3421 catcacattt aaattgcaga cagtgttttg cacatcatgt atctgttttg tcccataata
3481 tgctttttac tccctgatcc cagtttctgc tgttgactct tccattcagt tttatttatt
3541 gtgtgttctc acagtgacac catttgtcct tttctgcaac aacctttcca gctacttttg
3601 ccaaattcta tttgtcttct ccttcaaaac attctccttt gcagttcctc ttcatctgtg
3661 tagctgctct tttgtctctt aacttaccat tcctatagta ctttatgcat ctctgcttag
3721 ttctattagt tttttggcct tgctcttctc cttgatttta aaattccttc tatagctaga
3781 gcttttcttt ctttcattct ctcttcctgc agtgttttgc atacatcaga agctaggtac
3841 ataagttaaa tgattgagag ttggctgtat ttagatttat cactttttaa tagggtgagc
3901 ttgagagttt tctttctttc tgtttttttt ttttgttttt tttttttttt tttttttttt
3961 tttttttgac taatttcaca tgctctaaaa accttcaaag gtgattattt ttctcctgga
4021 aactccaggt ccattctgtt taaatcccta agaatgtcag aattaaaata acagggctat
4081 cccgtaattg gaaatatttc ttttttcagg atgctatagt caatttagta agtgaccacc
4141 aaattgttat ttgcactaac aaagctcaaa acacgataag tttactcctc catctcagta
4201 ataaaaatta agctgtaatc aaccttctag gtttctcttg tcttaaaatg ggtattcaaa
4261 aatggggatc tgtggtgtat gtatggaaac acatactcct taatttacct gttgttggaa
4321 actggagaaa tgattgtcgg gcaaccgttt attttttatt gtattttatt tggttgaggg
4381 atttttttat aaacagtttt acttgtgtca tattttaaaa ttactaactg ccatcacctg
4441 ctggggtcct ttgttaggtc attttcagtg actaataggg ataatccagg taactttgaa
4501 gagatgagca gtgagtgacc aggcagtttt tctgccttta gctttgacag ttcttaatta
4561 agatcattga agaccagctt tctcataaat ttctcttttt gaaaaaaaga aagcatttgt
4621 actaagctcc tctgtaagac aacatcttaa atcttaaaag tgttgttatc atgactggtg
4681 agagaagaaa acattttgtt tttattaaat ggagcattat ttacaaaaag ccattgttga
4741 gaattagatc ccacatcgta taaatatcta ttaaccattc taaataaaga gaactccagt
4801 gttgctatgt gcaagatcct ctcttggagc ttttttgcat agcaattaaa ggtgtgctat 4861 ttgtcagtag ccattttttt gcagtgattt gaagaccaaa gttgttttac agctgtgtta 4921 ccgttaaagg tttttttttt tatatgtatt aaatcaattt atcactgttt aaagctttga 4981 atatctgcaa tctttgccaa ggtacttttt tatttaaaaa aaaacataac tttgtaaata 5041 ttaccctgta atattatata tacttaataa aacattttaa gctattttgt tgggctattt 5101 ctattgctgc tacagcagac cacaagcaca tttctgaaaa atttaattta ttaatgtatt 5161 tttaagttgc ttatattcta ggtaacaatg taaagaatga tttaaaatat taattatgaa 5221 ttttttgagt ataataccca ataagctttt aattagagca gagttttaat taaaagtttt 5281 aaatcagtcc aa ( SEQ ID NO : 58 )
1 gcagcctggg cagtgggtcc tgcctgtgac gcgcggcggc ggtcggtcct gcctgtaacg
61 gcggcggcgg ctgctgctcc ggacacctgc ggcggcggcg gcgaccccgc ggcgggcgcg
121 gagatgtggc ccctggtagc ggcgctgttg ctgggctcgg cgtgctgcgg atcagctcag
181 ctactattta ataaaacaaa atctgtagaa ttcacgtttt gtaatgacac tgtcgtcatt
241 ccatgctttg ttactaatat ggaggcacaa aacactactg aagtatacgt aaagtggaaa
301 tttaaaggaa gagatattta cacctttgat ggagctctaa acaagtccac tgtccccact
361 gactttagta gtgcaaaaat tgaagtctca caattactaa aaggagatgc ctctttgaag
421 atggataaga gtgatgctgt ctcacacaca ggaaactaca cttgtgaagt aacagaatta
481 accagagaag gtgaaacgat catcgagcta aaatatcgtg ttgtttcatg gttttctcca
541 aatgaaaata ttcttattgt tattttccca atttttgcta tactcctgtt ctggggacag
601 tttggtatta aaacacttaa atatagatcc ggtggtatgg atgagaaaac aattgcttta
661 cttgttgctg gactagtgat cactgtcatt gtcattgttg gagccattct tttcgtccca
721 ggtgaatatt cattaaagaa tgctactggc cttggtttaa ttgtgacttc tacagggata
781 ttaatattac ttcactacta tgtgtttagt acagcgattg gattaacctc cttcgtcatt
841 gccatattgg ttattcaggt gatagcctat atcctcgctg tggttggact gagtctctgt
901 attgcggcgt gtataccaat gcatggccct cttctgattt caggtttgag tatcttagct
961 ctagcacaat tacttggact agtttatatg aaatttgtgg cttccaatca gaagactata
1021 caacctccta ggaaagctgt agaggaaccc cttaatgaat aactgaagtg aagtgatgga
1081 ctccgatttg gagagtagta agacgtgaaa ggaatacact tgtgtttaag caccatggcc
1141 ttgatgattc actgttgggg agaagaaaca agaaaagtaa ctggttgtca cctatgagac
1201 ccttacgtga ttgttagtta agtttttatt caaagcagct gtaatttagt taataaaata
1261 attatgatct atgttgtttg cccaattgag atccagtttt ttgttgttat ttttaatcaa
1321 ttaggggcaa tagtagaatg gacaatttcc aagaatgatg cctttcaggt cctagggcct
1381 ctggcctcta ggtaaccagt ttaaattggt tcagggtgat aactacttag cactgccctg
1441 gtgattaccc agagatatct atgaaaacca gtggcttcca tcaaaccttt gccaactcag
1501 gttcacagca gctttgggca gttatggcag tatggcatta gctgagaggt gtctgccact
1561 tctgggtcaa tggaataata aattaagtac aggcaggaat ttggttggga gcatcttgta
1621 tgatctccgt atgatgtgat attgatggag atagtggtcc tcattcttgg gggttgccat
1681 tcccacattc ccccttcaac aaacagtgta acaggtcctt cccagattta gggtactttt
1741 attgatggat atgttttcct tttattcaca taaccccttg aaaccctgtc ttgtcctcct
1801 gttacttgct tctgctgtac aagatgtagc accttttctc ctctttgaac atggtctagt
1861 gacacggtag caccagttgc aggaaggagc cagacttgtt ctcagagcac tgtgttcaca
1921 cttttcagca aaaatagcta tggttgtaac atatgtattc ccttcctctg atttgaaggc
1981 aaaaatctac agtgtttctt cacttctttt ctgatctggg gcatgaaaaa agcaagattg
2041 aaatttgaac tatgagtctc ctgcatggca acaaaatgtg tgtcaccatc aggccaacag
2101 gccagccctt gaatggggat ttattactgt tgtatctatg ttgcatgata aacattcatc
2161 accttcctcc tgtagtcctg cctcgtactc cccttcccct atgattgaaa agtaaacaaa
2221 acccacattt cctatcctgg ttagaagaaa attaatgttc tgacagttgt gatcgcctgg
2281 agtactttta gacttttagc attcgttttt tacctgtttg tggatgtgtg tttgtatgtg
2341 catacgtatg agataggcac atgcatcttc tgtatggaca aaggtggggt acctacagga
2401 gagcaaaggt taattttgtg cttttagtaa aaacatttaa atacaaagtt ctttattggg
2461 tggaattata tttgatgcaa atatttgatc acttaaaact tttaaaactt ctaggtaatt
2521 tgccacgctt tttgactgct caccaatacc ctgtaaaaat acgtaattct tcctgtttgt
2581 gtaataagat attcatattt gtagttgcat taataatagt tatttcttag tccatcagat
2641 gttcccgtgt gcctctttta tgccaaattg attgtcatat ttcatgttgg gaccaagtag
2701 tttgcccatg gcaaacctaa atttatgacc tgctgaggcc tctcagaaaa ctgagcatac
2761 tagcaagaca gctcttcttg aaaaaaaaaa tatgtataca caaatatata cgtatatcta
2821 tatatacgta tgtatataca cacatgtata ttcttccttg attgtgtagc tgtccaaaat
2881 aataacatat atagagggag ctgtattcct ttatacaaat ctgatggctc ctgcagcact 1141 aaacaagaaa agtaactggt tgtcacctat gagaccctta cgtgattgtt agttaagttt
1201 ttattcaaag cagctgtaat ttagttaata aaataattat gatctatgtt gtttgcccaa
1261 ttgagatcca gttttttgtt gttattttta atcaattagg ggcaatagta gaatggacaa
1321 tttccaagaa tgatgccttt caggtcctag ggcctctggc ctctaggtaa ccagtttaaa
1381 ttggttcagg gtgataacta cttagcactg ccctggtgat tacccagaga tatctatgaa
1441 aaccagtggc ttccatcaaa cctttgccaa ctcaggttca cagcagcttt gggcagttat
1501 ggcagtatgg cattagctga gaggtgtctg ccacttctgg gtcaatggaa taataaatta
1561 agtacaggca ggaatttggt tgggagcatc ttgtatgatc tccgtatgat gtgatattga
1621 tggagatagt ggtcctcatt cttgggggtt gccattccca cattccccct tcaacaaaca
1681 gtgtaacagg tccttcccag atttagggta cttttattga tggatatgtt ttccttttat
1741 tcacataacc ccttgaaacc ctgtcttgtc ctcctgttac ttgcttctgc tgtacaagat
1801 gtagcacctt ttctcctctt tgaacatggt ctagtgacac ggtagcacca gttgcaggaa
1861 ggagccagac ttgttctcag agcactgtgt tcacactttt cagcaaaaat agctatggtt
1921 gtaacatatg tattcccttc ctctgatttg aaggcaaaaa tctacagtgt ttcttcactt
1981 cttttctgat ctggggcatg aaaaaagcaa gattgaaatt tgaactatga gtctcctgca
2041 tggcaacaaa atgtgtgtca ccatcaggcc aacaggccag cccttgaatg gggatttatt
2101 actgttgtat ctatgttgca tgataaacat tcatcacctt cctcctgtag tcctgcctcg
2161 tactcccctt cccctatgat tgaaaagtaa acaaaaccca catttcctat cctggttaga
2221 agaaaattaa tgttctgaca gttgtgatcg cctggagtac ttttagactt ttagcattcg
2281 ttttttacct gtttgtggat gtgtgtttgt atgtgcatac gtatgagata ggcacatgca
2341 tcttctgtat ggacaaaggt ggggtaccta caggagagca aaggttaatt ttgtgctttt
2401 agtaaaaaca tttaaataca aagttcttta ttgggtggaa ttatatttga tgcaaatatt
2461 tgatcactta aaacttttaa aacttctagg taatttgcca cgctttttga ctgctcacca
2521 ataccctgta aaaatacgta attcttcctg tttgtgtaat aagatattca tatttgtagt
2581 tgcattaata atagttattt cttagtccat cagatgttcc cgtgtgcctc ttttatgcca
2641 aattgattgt catatttcat gttgggacca agtagtttgc ccatggcaaa cctaaattta
2701 tgacctgctg aggcctctca gaaaactgag catactagca agacagctct tcttgaaaaa
2761 aaaaatatgt atacacaaat atatacgtat atctatatat acgtatgtat atacacacat
2821 gtatattctt ccttgattgt gtagctgtcc aaaataataa catatataga gggagctgta
2881 ttcctttata caaatctgat ggctcctgca gcactttttc cttctgaaaa tatttacatt
2941 ttgctaacct agtttgttac tttaaaaatc agttttgatg aaaggaggga aaagcagatg
3001 gacttgaaaa agatccaagc tcctattaga aaaggtatga aaatctttat agtaaaattt
3061 tttataaact aaagttgtac cttttaatat gtagtaaact ctcatttatt tggggttcgc
3121 tcttggatct catccatcca ttgtgttctc tttaatgctg cctgcctttt gaggcattca
3181 ctgccctaga caatgccacc agagatagtg ggggaaatgc cagatgaaac caactcttgc
3241 tctcactagt tgtcagcttc tctggataag tgaccacaga agcaggagtc ctcctgcttg
3301 ggcatcattg ggccagttcc ttctctttaa atcagatttg taatggctcc caaattccat
3361 cacatcacat ttaaattgca gacagtgttt tgcacatcat gtatctgttt tgtcccataa
3421 tatgcttttt actccctgat cccagtttct gctgttgact cttccattca gttttattta
3481 ttgtgtgttc tcacagtgac accatttgtc cttttctgca acaacctttc cagctacttt
3541 tgccaaattc tatttgtctt ctccttcaaa acattctcct ttgcagttcc tcttcatctg
3601 tgtagctgct cttttgtctc ttaacttacc attcctatag tactttatgc atctctgctt
3661 agttctatta gttttttggc cttgctcttc tccttgattt taaaattcct tctatagcta
3721 gagcttttct ttctttcatt ctctcttcct gcagtgtttt gcatacatca gaagctaggt
3781 acataagtta aatgattgag agttggctgt atttagattt atcacttttt aatagggtga
3841 gcttgagagt tttctttctt tctgtttttt ttttttgttt tttttttttt tttttttttt
3901 tttttttttg actaatttca catgctctaa aaaccttcaa aggtgattat ttttctcctg
3961 gaaactccag gtccattctg tttaaatccc taagaatgtc agaattaaaa taacagggct
4021 atcccgtaat tggaaatatt tcttttttca ggatgctata gtcaatttag taagtgacca
4081 ccaaattgtt atttgcacta acaaagctca aaacacgata agtttactcc tccatctcag
4141 taataaaaat taagctgtaa tcaaccttct aggtttctct tgtcttaaaa tgggtattca
4201 aaaatgggga tctgtggtgt atgtatggaa acacatactc cttaatttac ctgttgttgg
4261 aaactggaga aatgattgtc gggcaaccgt ttatttttta ttgtatttta tttggttgag
4321 ggattttttt ataaacagtt ttacttgtgt catattttaa aattactaac tgccatcacc
4381 tgctggggtc ctttgttagg tcattttcag tgactaatag ggataatcca ggtaactttg
4441 aagagatgag cagtgagtga ccaggcagtt tttctgcctt tagctttgac agttcttaat
4501 taagatcatt gaagaccagc tttctcataa atttctcttt ttgaaaaaaa gaaagcattt
4561 gtactaagct cctctgtaag acaacatctt aaatcttaaa agtgttgtta tcatgactgg
4621 tgagagaaga aaacattttg tttttattaa atggagcatt atttacaaaa agccattgtt
4681 gagaattaga tcccacatcg tataaatatc tattaaccat tctaaataaa gagaactcca
4741 gtgttgctat gtgcaagatc ctctcttgga gcttttttgc atagcaatta aaggtgtgct 4801 atttgtcagt agccattttt ttgcagtgat ttgaagacca aagttgtttt acagctgtgt 4861 taccgttaaa ggtttttttt tttatatgta ttaaatcaat ttatcactgt ttaaagcttt 4921 gaatatctgc aatctttgcc aaggtacttt tttatttaaa aaaaaacata actttgtaaa 4981 tattaccctg taatattata tatacttaat aaaacatttt aagctatttt gttgggctat 5041 ttctattgct gctacagcag accacaagca catttctgaa aaatttaatt tattaatgta 5101 tttttaagtt gcttatattc taggtaacaa tgtaaagaat gatttaaaat attaattatg 5161 aattttttga gtataatacc caataagctt ttaattagag cagagtttta attaaaagtt 5221 ttaaatcagt ccaa ( SEQ ID NO : 60 ) or a variant or fragment thereof of any of the aforementioned sequences, 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.
[0116] Illustrative mRNA constructs/sequences of CD47 are provided in Table 2 below. These sequences generally correspond to the plasmid sequence and typically include the T7 polymerase promoter but do not show the poly A sequence. For all constructs/sequences disclosed herein, in the context of RNA, the Ts are Us, and in particular embodiments, the Ts are fully substituted with NlmPsU. Coding sequence is capitalized. Note that throughout the specification, construct sequences are generally specified T7 promoter
Figure imgf000031_0001
3’UTR and do not include poly A tail sequences, except for AMOS. mRNA constructs electroporated into cells may lack the T7 promoter sequence. The T7 promoter sequence comprises the sequence The T7 polymerase starts transcription at the
Figure imgf000031_0002
underlined G in the promoter sequence. The polymerase then transcribes using the opposite strand as a template from 5’-> 3'. The first base in the transcript wifi be a G.
Figure imgf000031_0003
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0002
[0117] Illustrative isoform 2 CD47 mRNA constructs/sequences are provided in Table 3 below Illustrative mRNA constructs/sequences are provided in the table below. For all constructs/sequences disclosed herein, in the context of mRNA, the Ts are Us, and in particular embodiments, the Ts are fully substituted with NlmPsU. Coding sequence is capitalized. Note that throughout the specification, construct sequences are generally specified T7 promoter
Figure imgf000041_0001
3’UTR and do not include poly A tail sequences, except for AMOS. mRNA constructs electroporated into cells may lack the T7 promoter sequence. The T7 promoter sequence comprises the sequence TAATACGACTC/kCTATAG (SEQ II) NO: 86). The T7 polymerase stans transcription at the underlined G in the promoter sequence. The polymerase then transcribes using the opposite strand as a template from 5’->3’. The first base in the transcript will be a G. These mRNAs include the 5’ HBA1 UTR, the 3’ HBB1 UTR, and the TAATAA stop codon. The sequence encoding CD47 is capitalized, and the TAATAA stop codon is capitalized.
Figure imgf000041_0003
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
[0118] An illustrative self-amplifying CD47 mRNA construct is shown in Table 4 below.
For all constructs/sequences disclosed herein, in the context of mRNA, the Ts are Us, and in particular embodiments, the Ts are fully substituted with NlmPsU. Coding sequence is capitalized. Note that throughout the specification, construct sequences are generally specified T7 promoter 3’UTR and do not include poly A tail sequences, except for AMOS. mRNA constructs electroporated into cells may lack the T7 promoter sequence. The T7 promoter sequence comprises the sequence TAATACXkACTCACTATAGXSEQJD N0;_J6). The T7 polymerase starts transcription at the underlined G in the promoter sequence. The polymerase then transcribes using the opposite strand as a template from 5’->3’. The first base in die transcript will be a G.
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
CD 117 Variant Polypeptides and Polynucleotides
[0119] CD 117, also known as c-kit or stem cell factor receptor (SCFR), has a molecular weight of 145 kDa as a mature protein and is a member of the type III receptor tyrosine kinase (RTK) family that includes platelet-derived growth factor (PDGF) receptors and the macrophage colony-stimulating factor 1 (CSF-1) (c-fms) receptor. CD117 is essential for the development of normal hematopoietic cells and plays an important role in the survival, proliferation, and differentiation of mast cells, melanocytes, and germ cells. It is expressed by hematopoietic cells in the embryonic liver throughout development, and by more committed progenitors, such as myeloid, erythroid, megakaryocytic, natural killer, and dendritic progenitor cells
[0120] CD117 includes an approximately 519 amino acid extracellular domain comprised of five immunoglobulin-like domains, a transmembrane segment, a juxtamembrane domain, and a protein kinase domain that contains an insert of about 80 amino acid residues. Approximately 184 amino acids of the extracellular domain are surface exposed, which were identified based on x-ray crystallographic studies. The crystallographic structure of CD117 is provided in, e.g., Mol, et al., Accelerated Publications, Volume 278, ISSUE 34, P31461-31464, August 22, 2003; Ogg et al., RCSB Protein Data Bank, 6XV9, Crystal structure of the kinase domain of human c-KIT in complex with a type-II inhibitor, DOI: 10.2210/pdb6XV9/pdb; McAuley et al., RCSB Protein Data Bank Alkynyl Benzoxazines and Dihydroquinazolines as Cysteine Targeting Covalent Warheads and Their Application in Identification of Selective Irreversible Kinase Inhibitors, DOI: 10.1021/jacs.9bl3391; Schimpl et al., RCSB Protein Data Bank 6GQM, Crystal structure of human c-KIT kinase domain in complex with a small molecule inhibitor, AZD3229, DOI: 10.1021/acs.jmedchem.8b00938; and Lin et al., RCSB Protein Data Bank Identification of a Multitargeted Tyrosine Kinase Inhibitor for the Treatment of Gastrointestinal Stromal Tumors and Acute Myeloid Leukemia, DOI: 10.1021/acs.jmedchem.9b01229. Binding of CD117 to its ligand (stem cell factor; SCF) induces receptor dimerization, trans autophosphorylation of the kinase domain, recruitment of signaling proteins via phosphotyrosine binding or Src homology 2 (SH2) domains, and subsequent signal transduction.
[0121] In one aspect, the disclosure provides modified CD117 polypeptides comprising one or more amino acid modifications as compared to a wild type CD117 polypeptide. In particular embodiments, the one or more amino acid modifications comprise one or more amino acid substitutions, insertions, or deletions. In certain embodiments, the one or more amino acid modifications are located in the extracellular domain of the CD117 polypeptide. In certain embodiments, the one or more amino acid modifications are located in one or more surface exposed amino acids of the CD117 polypeptide’s extracellular domain. In particular embodiments, the modified CD117 polypeptides comprise one or more deletions, e.g., an N- terminal or C-terminal deletion, optionally wherein the deletion does not substantially impair biological activity, e.g., signaling, of the modified CD117 polypeptide. In certain embodiments, the modified CD117 polypeptides retain or have at least 90%, at least 95%, at least 98%, or at least 99% sequence homology to the wild type CD117 polypeptide. In particular embodiments, the one or more amino acid modifications do not substantially inhibit or reduce binding of stem cell factor (SCF) to the modified CD117 polypeptide when expressed in cells, as compared to the binding of SCF to the wild type CD117 polypeptide.
[0122] In certain embodiments, the amino acid substitution is a conservative amino acid substitution. The term "conservative substitution" as used herein denotes that one or more amino acids are replaced by another, biologically similar residue.
[0123] In the scheme below, conservative substitutions of amino acids are grouped by the indicated physicochemical properties. I: neutral, hydrophilic, II: acids and amides, III: basic, IV: hydrophobic, V: aromatic, bulky amino acids.
Figure imgf000050_0001
[0124] In the scheme below, conservative substitutions of amino acids are grouped by the indicated physicochemical properties. VI: neutral or hydrophobic, VII: acidic, VIII: basic, IX: polar, X: aromatic.
Figure imgf000051_0001
[0125] In certain embodiments, the wild type CD117 polypeptide upon which the variant is based is a human CD117 polypeptide, while in other embodiments, it is another mammalian CD117 polypeptide. Sequences of human and mammalian CD117 polypeptides are known in the art. Due to alternative splicing of the c-kit gene, the human CD117 polypeptide is expressed as various isoforms, and any of these may be used according to the disclosure. These isoforms include two GNNK+ and GNNK- isoforms (also denoted c-Kit and c-KitA, respectively), which differ by the presence or absence of four amino acids, GNNK, and which are coexpressed in most tissues, although the GNNK- isoform usually predominates. In particular embodiments, the wild type CD 117 polypeptide is the GNNK+ or GNNK- isoform and comprises or consists of one of the following amino acid sequences:
MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKD VS S S VYSTWKRENSQTKLQEKYNSWHHGDFNYERQ ATLTIS S ARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPV DVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKE QIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYID PTQLPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPS AHLTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRK RDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRS VRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNIL LTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYG IFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLK RPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLV HDDV (SEQ ID NO: 1); or MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKD VS S S VYSTWKRENSQTKLQEKYNSWHHGDFNYERQ ATLTIS S ARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPV D VQTLNS SGPPFGKL VVQS SIDS S AFKHNGTVECK AYND VGKTS AYFNF AFKEQIHP HTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQ LPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAH LTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRD SFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRI GSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTH GRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFL WELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRP TFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHD DV (SEQ ID NO:2).
[0126] In certain embodiments, the modified CD117 polypeptides substantially retain kinase activity as compared to the wild type CD117 polypeptide, e.g., when bound by stem cell factor (SCF). In particular embodiments, the modified CD117 polypeptide has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the kinase activity of the wild type CD117 polypeptide, e.g., when bound by SCF. In some embodiments, the modified CD117 polypeptide has increased kinase activity as compared to the wild type CD117 polypeptide. In particular embodiments, the modified CD117 polypeptide has at least 150%, at least 200%, at least 300%, at least 500%, at least 750%, or at least 1000% of the kinase activity of the wild type CD117 polypeptide. Kinase activity may be determined using assays known in the art, including the ADP-Glo™ Kinase Assay, which is a luminescent kinase assay that measures ADP formed from a kinase reaction; ADP is converted into ATP, which is converted into light by Ultra-Gio™ Luciferase (available from Promega Corporation, Madison, WI).
[0127] In particular embodiments, the one or more amino acid modifications do not substantially inhibit or reduce binding of stem cell factor (SCF) to the modified CD117 polypeptide when expressed in cells, as compared to the binding of SCF to the wild type CD117 polypeptide. In particular embodiments, the modified CD117 retains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of binding to SCF as compared to the corresponding wild type CD117.
[0128] In particular embodiments, the one or more amino acid modifications do not result in cells expressing only the modified CD117 having substantially inhibited or reduce CD117 signaling or proliferation or viability, optionally in response to SCF binding, as compared to the signaling in cells only expressing the wild type CD117 polypeptide. In particular embodiments, the modified CD117 retains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% CD117 signaling and/or proliferation and/or viability, optionally in response to SCF binding, as compared to the corresponding wild type CD117.
[0129] CD117 signaling or proliferation or viability may be determined using methods standard in the art. For example, in certain embodiments, CD117 signaling or proliferation (e.g., in response to SCF), of cells comprising a modified CD117 polypeptide is determined using a cell line (e.g., Ba/F3 cells) engineered to express the modified CD117 polypeptide. Cells are cultured in the presence of IL-3, with or without stem cell factor (SCF), and in the presence or absence of an anti-CD117 antibody, e.g., JSP191. Control parental Ba/F3 cells do not proliferate in the absence of IL-3. Further, parental Ba/F3 cells do not express CD117 and are not responsive to SCF signaling. Proliferation in response to SCF binding may this be determined for cells overexpressing the modified CD117, e.g., in the presence and absence of SCF and/or the anti-CD117 antibody.
Constitutively Active CD117
[0130] In particular embodiments, the modified CD117 has constitutive signaling or kinase activity, even in absence of SCF binding to the modified CD117 and/or in the presence of an anti-c-Kit antibody that blocks SCF binding to the modified CD117 polypeptide. In certain embodiments, the modified CD117 has constitutive autophosphorylation activity, e.g., without bound SCF. A variety of such modified CD117 have been identified, e.g., in cancer cells, and any of these may be used according to the compositions and methods disclosed herein. Illustrative examples of activating or gain-of-function CD117 modifications include, but are not limited to, N505I, V559D, D816V, D816H, V568F, V570F, or Y703F, modifications or mutation of amino acid residues corresponding to 505, 522, 816, 557, 558, 559, 568, 569, 570, 703, 816, or deletion of codon 579 (Asp). See, e.g., Akin and Metcalfe, Journal of Allergy and Clinical Immunology, Vol. 114, Issue 1, p 13- 19, July 1, 2004; Hirotakoji et al., Science 23, Jan 1998, Vol. 279, Issue 5350, pp. 577-580; Sanlorenzo et al., J Proteomics 2016 July 20, 144: 140-147, and references cited in any of the aforementioned, all of which are hereby incorporated by reference in their entireties. In particular embodiments, the amino acid modification is in the region between the transmembrane and tyrosine kinase domains. Mutations causing constitutive activation of c-Kit have been shown to be causative in some forms of mastocytosis, and several types of mutations have been associated with myeloproliferative disorders (MPDs), acute myelogenous leukemia (AML), sinonasai lymphomas, and gastrointestinal stromal tumors (GIST). These may be considered activating mutation of two types "regulatory type’ mutations, which affect regulation of the kinase molecule, and ‘enzymatic pocket type’ mutations, which alter the amino acid sequence directly forming the enzymatic site. Either type of mutation may be used according to various embodiments of the disclosure, including any of those disclosed in Longley et al., Leukemia Research, Vol. 25, Issue 7, July 2001, pp. 571-576, and references cited therein, all of which are incorporated herein by reference in its entireties.
[0131] In particular embodiments, the one or more amino acid modifications do not result in cells expressing only the modified CD117 having substantially inhibited or reduce c-Kit signaling or proliferation, optionally in response to SCF binding, as compared to the signaling in cells only expressing the wild type CD117 polypeptide. In particular embodiments, the modified CD117 retains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% c-Kit signaling and/or proliferation, optionally in response to SCF binding, as compared to the corresponding wild type CD117. In particular embodiments, the modified CD117 retains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% c-Kit signaling and/or proliferation, in the absence of SCF binding, as compared to the corresponding wild type CD117.
[0132] In particular embodiments, the one or more amino acid modifications do not substantially inhibit or reduce binding of an anti-c-Kit antibody to the modified CD117 polypeptide expressed in cells as compared to the wild type CD117 polypeptide. In particular embodiments, the anti-c-Kit antibody comprises the six CDRs present in any one of JSP191, AB85, CDX-0159, or FSI-174. In particular embodiments, the anti-c-Kit antibody in any one of JSP191, AB85, CDX-0159, or FSI-174.
[0133] In particular embodiments, the one or more amino acid modifications comprise one or more amino acid substitutions or deletions of an amino acid residue selected from the following in human CD117: N505, 522, 557, 558, V559, V568, 569, V570, 579, 703, and D816. In some embodiments, the amino acid residue is substituted by Alanine. In some embodiments, the one or more amino acid modifications is a deletion of amino acid 579.
[0134] In particular embodiments, the one of more amino acid substitutions comprises a D816V substitution and/or a N505I substitution. In some embodiments, the modified CD117 polypeptide having constitutive kinase activity comprises one or more of the following amino acid substitutions: N505I, V559D, D816V, D816H, V568F, V570F, and/or Y703F. In some embodiments, the modified CD117 has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 750%, or at least 1000% of the kinase activity of the wild type CD117 polypeptide in the presence of an anti-c-kit antibody and comprises the amino acid substitutions of N505I.
[0135] In particular embodiments, the one or more amino acid modifications do not substantially inhibit or reduce binding of an anti-c-Kit antibody to the modified CD117 polypeptide expressed in cells as compared to the wild type CD117 polypeptide.
[0136] In certain embodiments, the modified CD117 polypeptide comprises or consists of either of the following sequences:
MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKD VS S S VYSTWKRENSQTKLQEKYNSWHHGDFNYERQ ATLTIS S ARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPV DVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKE QIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYID PTQLPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPS AHLTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRK RDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRS VRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNIL LTHGRITKICDFGLARDIKNVSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYG IFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLK RPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLV HDDV (SEQ ID NO: 102);
MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKD VS S SVYSTWKRENSQTKLQEKYNSWHHGDFNYERQ ATLTIS S ARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPV D VQTLNS SGPPFGKL VVQS SIDS S AFKHNGTVECK AYND VGKTS AYFNF AFKEQIHP HTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQ LPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAH LTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRD SFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRI GSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTH GRITKICDFGLARDIKNVSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFL WELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRP TFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHD DV (SEQ ID NO: 103); MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKD VS S S VYSTWKRENSQTKLQEKYNSWHHGDFNYERQ ATLTIS S ARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPV DVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFIFAFKGNNKE QIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYID PTQLPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPS AHLTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRK RDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRS VRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNIL LTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYG IFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLK RPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLV HDDV (SEQ ID NO: 104); or
MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLC TDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPA KLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIK SVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFT VTCTIKD VS S SVYSTWKRENSQTKLQEKYNSWHHGDFNYERQ ATLTIS S ARVNDSG VFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPE HQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPV DVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFIFAFKEQIHPH TLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLP YDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLT EREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFI CSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRIGS YIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHG RITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLW ELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRPTF KQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHDD (SEQ ID NO: 105), or a variant or fragment thereof, e.g., having at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.
[0137] In particular embodiments, the constitutively active modified CD117 polypeptides are still bound by an anti-CDl 17 antibody used for HCT conditioning, such as, e.g., JSP191.
[0138] Illustrative CD117 mRNA constructs and sequences are provided in Table 5 below, with the coding sequence capitalized. For all constructs/sequences disclosed herein, in the context of mRNA, the Ts are Us, and in particular embodiments, the Ts are fully substituted with NlmPsU. Coding sequence is capitalized. Note that throughout the specification, construct sequences are generally specified T7 promoter 3’UTR and do not include poly A tail sequences, except for AMOS. mRNA constructs electroporated into cells may lack the T7 promoter sequence. The T7 promoter sequence comprises the sequence TAATACGACTCACTATAG (SEQ ID NO: 86). The T7 polymerase starts transcription at the underlined G in the promoter sequence. The polymerase then transcribes using the opposite strand as a template from 5 ’ ->3 ’ . The first base in the transcript will be a G.
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
CD117 Not Bound by anti-CDl 17 Antibodies [0139] In other embodiments, the modified CD117 polypeptide does not bind an anti-c-kit antibody used for HCT conditioning, e.g., JSP191 and/or AB85. In some embodiments, the modified CD117 binds stem cell factor (SCF), even in the presence of the anti-c-Kit antibody. In particular embodiments, the modified CD117 polypeptide has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the kinase activity of the wild type CD117 polypeptide, in response to SCF signaling and in the presence of an anti-c-kit antibody.
[0140] In certain embodiments, the modified CD117 comprises one or more amino acid substitutions, e.g., atE73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, orR271, or at an amino acid residue located within 2, within 3, within 4, within 5, within 6, within 7 within 8 within 8 within 10, within 11 or within 12 amino acids of any of E73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, or R271, e.g., either N-terminal or C-terminal of any of these residues. In some embodiments, the modified CD117 polypeptide that does not bind a CD117 antibody comprises a substitution or a deletion at one or more of the following residues: E73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, or R271. In some embodiments, the modified CD117 that does not bind a CD117 antibody comprises an Alanine substitution at one or more of the following residues: E73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, or R271. In some embodiments, the modified CD117 comprises a conservative or a non-conservative amino acid substitution of any one of these amino acid residues. In some embodiments, the CD117 modification is at any of E73, D121, R122, S123, Y125, K203, or at an amino acid residue located within 2, within 3, within 4, within 5, within 6, within 7 within 8 within 8 within 10, within 11 or within 12 amino acids of any of these amino acids, and the modified or variant CD117 does not bind the JSP191 antibody. In some embodiments, the CD117 modification is at any of K203, Y259, S261, W262, Y269, or R271, or at an amino acid residue located within 2, within 3, within 4, within 5, within 6, within 7 within 8 within 8 within 10, within 11 or within 12 amino acids of any of these amino acids, and the modified or variant CD117 does not bind the AB85 antibody.
[0141] In further embodiments, the modified CD117 polypeptide substantially retains kinase activity as compared to the wild type CD117 polypeptide, and also does not bind an anti-c-Kit- antibody, e.g., JSP191 and/or AB85, used for HCT conditioning. In particular embodiments, the modified CD117 polypeptide has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the kinase activity of the wild type CD117 polypeptide, in response to SCF signaling and in the presence of an anti-c-kit antibody. In some embodiments, the modified CD117 polypeptide has at least 150%, at least 200%, at least 300%, at least 500%, at least 750%, or at least 1000% of the kinase activity of the wild type CD117 polypeptide in the presence of an anti-c-kit antibody, and comprises one or more substitutions or deletions in an amino acid residue selected from the following in human CD117: E73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, and R271.
[0142] In particular embodiments, any of the modified CD117 polypeptides not bound by an anti-CD117 antibody used for HCT conditioning, such as, e.g., JSP191, is not constitutively active.
[0143] In particular embodiments, the mRNA comprises an RNA sequence corresponding to the open reading frame DNA sequence of SEQ ID NO: 5:
ATGAGAGGCGCTCGCGGCGCCTGGGATTTTCTCTGCGTTCTGCTCCTACTGCTTCG CGTCCAGACAGGCTCTTCTCAACCATCTGTGAGTCCAGGGGAACCGTCTCCACCA TCCATCCATCCAGGAAAATCAGACTTAATAGTCCGCGTGGGCGACGAGATTAGG CTGTTATGCACTGATCCGGGCTTTGTCAAATGGACTTTTGAGATCCTGGATGAAA CGAATGAGAATAAGCAGAATGAATGGATCACGGAAAAGGCAGAAGCCACCAAC ACCGGCAAATACACGTGCACCAACAAACACGGCTTAAGCAATTCCATTTATGTGT TTGTTAGAGATCCTGCCAAGCTTTTCCTTGTTGACCGCTCCTTGTATGGGAAAGA AGACAACGACACGCTGGTCCGCTGTCCTCTCACAGACCCAGAAGTGACCAATTAT TCCCTCAAGGGGTGCCAGGGGAAGCCTCTTCCCAAGGACTTGAGGTTTATTCCTG ACCCCAAGGCGGGCATCATGATCAAAAGTGTGAAACGCGCCTACCATCGGCTCT GTCTGCATTGTTCTGTGGACCAGGAGGGCAAGTCAGTGCTGTCGGAAAAATTCAT CCTGAAAGTGAGGCCAGCCTTCAAAGCTGTGCCTGTTGTGTCTGTGTCCAAAGCA AGCTATCTTCTTAGGGAAGGGGAAGAATTCACAGTGACGTGCACAATAAAAGAT GTGTCTAGTTCTGTGTACTCAACGTGGAAAAGAGAAAACAGTCAGACTAAACTA CAGGAGAAATATAATAGCTGGCATCACGGTGACTTCAATTATGAACGTCAGGCA ACGTTGACTATCAGTTCAGCGAGAGTTAATGATTCTGGAGTGTTCATGTGTTATG CCAATAATACTTTTGGATCAGCAAATGTCACAACAACCTTGGAAGTAGTAGATAA AGGATTCATTAATATCTTCCCCATGATAAACACTACAGTATTTGTAAACGATGGA GAAAATGTAGATTTGATTGTTGAATATGAAGCATTCCCCAAACCTGAACACCAGC
AGTGGATCTATATGAACAGAACCTTCACTGATAAATGGGAAGATTATCCCAAGTC TGAGAATGAAAGTAATATCAGATACGTAAGTGAACTTCATCTAACGAGATTAAA AGGCACCGAAGGAGGCACTTACACATTCCTAGTGTCCAATTCTGACGTCAATGCT GCCATAGCATTTAATGTTTATGTGAATACAAAACCAGAAATCCTGACTTACGACA GGCTCGTGAATGGCATGCTCCAATGTGTGGCAGCAGGATTCCCAGAGCCCACAA TAGATTGGTATTTTTGTCCAGGAACTGAGCAGAGATGCTCTGCTTCTGTACTGCC AGTGGATGTGCAGACACTAAACTCATCTGGGCCACCGTTTGGAAAGCTAGTGGTT CAGAGTTCTATAGATTCTAGTGCATTCAAGCACAATGGCACGGTTGAATGTAAGG CTTACAACGATGTGGGCAAGACTTCTGCCTATTTTAACTTTGCATTTAAAGGTAA CAACAAAGAGCAAATCCATCCCCACACCCTGTTCACTCCTTTGCTGATTGGTTTC GTAATCGTAGCTGGCATGATGTGCATTATTGTGATGATTCTGACCTACAAATATT TACAGAAACCCATGTATGAAGTACAGTGGAAGGTTGTTGAGGAGATAAATGGAA ACAATTATGTTTACATAGACCCAACACAACTTCCTTATGATCACAAATGGGAGTT TCCCAGAAACAGGCTGAGTTTTGGGAAAACCCTGGGTGCTGGAGCTTTCGGGAA GGTTGTTGAGGCAACTGCTTATGGCTTAATTAAGTCAGATGCGGCCATGACTGTC GCTGTAAAGATGCTCAAGCCGAGTGCCCATTTGACAGAACGGGAAGCCCTCATG TCTGAACTCAAAGTCCTGAGTTACCTTGGTAATCACATGAATATTGTGAATCTAC TTGGAGCCTGCACCATTGGAGGGCCCACCCTGGTCATTACAGAATATTGTTGCTA TGGTGATCTTTTGAATTTTTTGAGAAGAAAACGTGATTCATTTATTTGTTCAAAGC AGGAAGATCATGCAGAAGCTGCACTTTATAAGAATCTTCTGCATTCAAAGGAGTC
TTCCTGCAGCGATAGTACTAATGAGTACATGGACATGAAACCTGGAGTTTCTTAT
GTTGTCCCAACCAAGGCCGACAAAAGGAGATCTGTGAGAATAGGCTCATACATA
GAAAGAGATGTGACTCCCGCCATCATGGAGGATGACGAGTTGGCCCTAGACTTA
GAAGACTTGCTGAGCTTTTCTTACCAGGTGGCAAAGGGCATGGCTTTCCTCGCCT
CCAAGAATTGTATTCACAGAGACTTGGCAGCCAGAAATATCCTCCTTACTCATGG
TCGGATCACAAAGATTTGTGATTTTGGTCTAGCCAGAGACATCAAGAATGATTCT
AATTATGTGGTTAAAGGAAACGCTCGACTACCTGTGAAGTGGATGGCACCTGAA
AGCATTTTCAACTGTGTATACACGTTTGAAAGTGACGTCTGGTCCTATGGGATTTT
TCTTTGGGAGCTGTTCTCTTTAGGAAGCAGCCCCTATCCTGGAATGCCGGTCGAT
TCTAAGTTCTACAAGATGATCAAGGAAGGCTTCCGGATGCTCAGCCCTGAACACG
CACCTGCTGAAATGTATGACATAATGAAGACTTGCTGGGATGCAGATCCCCTAAA
AAGACCAACATTCAAGCAAATTGTTCAGCTAATTGAGAAGCAGATTTCAGAGAG
CACCAATCATATTTACTCCAACTTAGCAAACTGCAGCCCCAACCGACAGAAGCCC
GTGGTAGACCATTCTGTGCGGATCAATTCTGTCGGCAGCACCGCTTCCTCCTCCC AGCCTCTGCTTGTGCACGACGATGTCT.
[0144] In particular embodiments, the mRNA comprises an RNA sequence corresponding to the open reading frame DNA sequence of SEQ ID NO:6:
ATGAGAGGCGCCCGGGGCGCATGGGACTTCTTATGTGTATTATTGCTCCTCCTGAGGGTTCAGA
CCGGGAGTAGCCAACCTTCCGTGTCCCCTGGTGAACCATCGCCACCATCGATCCACCCTGGCAA
ATCCGACCTGATCGTGAGGGTCGGCGATGAAATCCGGCTCCTATGCACGGACCCTGGTTTTGTG
AAATGGACCTTTGAGATCCTCGATGAAACCAATGAGAATAAACAGAACGAGTGGATTACAGAAAA
GGCTGAGGCCACAAATACAGGTAAATACACATGCACCAATAAGCACGGCCTCTCGAACTCTATCT
ACGTTTTTGTGCGAGATCCTGCAAAACTTTTCCTCGTCGATCGCTCCTTATACGGAAAGGAGGAC
AATGATACACTGGTCAGATGCCCTTTAACAGACCCCGAGGTTACAAACTATTCACTCAAGGGTTG
TCAGGGTAAACCTCTGCCAAAAGACCTGCGCTTTATCCCTGATCCTAAAGCCGGCATCATGATAA
AATCTGTAAAGCGGGCCTACCACCGTCTCTGCTTGCACTGTAGCGTTGATCAAGAAGGCAAGTC
AGTTTTAAGCGAGAAATTCATCCTGAAGGTGCGCCCAGCCTTCAAGGCTGTGCCTGTAGTGTCA
GTGTCTAAGGCCTCATACCTGCTCCGGGAGGGGGAGGAATTTACTGTGACCTGCACGATAAAAG
ACGTTTCCTCTTCTGTGTACTCTACTTGGAAGAGGGAAAATAGCCAGACCAAGCTGCAGGAGAA
GTACAACAGCTGGCATCATGGTGACTTCAACTACGAGAGACAGGCCACTCTGACTATTTCATCTG
CACGCGTGAATGACTCCGGTGTGTTTATGTGCTACGCAAATAACACCTTCGGCAGTGCCAATGT
GACAACTACCCTGGAAGTCGTTGACAAGGGCTTCATTAACATCTTTCCAATGATCAATACAACCG
TCTTTGTTAACGATGGCGAAAACGTGGACCTGATCGTTGAATATGAAGCATTCCCCAAACCAGAG
CACCAGCAGTGGATCTACATGAATCGCACTTTCACCGACAAGTGGGAAGACTACCCCAAATCCG
AGAACGAGTCTAACATCCGCTATGTGTCGGAACTCCACCTGACTAGACTTAAGGGTACGGAAGG
AGGCACCTACACCTTTTTGGTGAGCAATAGCGACGTGAACGCGGCAATTGCTTTTAACGTATACG
TGAATACGAAACCCGAAATATTGACATATGACCGTCTGGTGAACGGAATGCTTCAGTGTGTGGCC
GCAGGCTTTCCTGAACCAACCATCGACTGGTACTTTTGCCCTGGTACCGAGCAGCGGTGCTCCG
CGAGCGTGCTGCCTGTGGACGTCCAGACGCTAAATTCTAGTGGGCCACCTTTTGGAAAACTGGT
GGTTCAGTCGTCAATTGATTCTTCTGCATTTAAGCATAATGGGACAGTGGAGTGTAAAGCTTACA
ACGATGTGGGGAAGACAAGCGCCTATTTCAACTTTGCCTTTAAGGGGAACAATAAAGAGCAGATT
CATCCACACACCTTGTTCACTCCTTTATTGATCGGGTTTGTGATCGTGGCGGGAATGATGTGTAT
TATCGTTATGATTTTGACTTATAAGTACCTGCAGAAGCCTATGTATGAAGTGCAGTGGAAAGTGGT
GGAAGAGATCAACGGGAACAATTACGTTTATATCGACCCCACCCAGTTGCCATATGACCACAAAT
GGGAATTTCCCAGGAATCGCTTGAGCTTCGGGAAGACACTCGGTGCCGGAGCCTTCGGAAAGG
TAGTAGAAGCAACGGCTTACGGGCTAATCAAGTCAGATGCCGCTATGACTGTTGCGGTGAAAAT
GTTGAAGCCATCGGCTCATCTGACAGAGCGGGAGGCTCTGATGAGCGAACTCAAGGTTCTCAGT
TACCTCGGCAATCACATGAACATTGTAAATCTCCTTGGGGCCTGTACGATCGGCGGTCCCACCC
TCGTCATAACAGAATACTGCTGCTATGGCGATCTGCTGAACTTCCTCCGGCGCAAGAGGGATTC
CTTTATATGTAGCAAACAAGAAGACCACGCGGAGGCCGCTCTATACAAAAACCTGTTACACAGTA
AAGAGTCTTCATGCAGCGACAGTACGAATGAATACATGGACATGAAACCTGGGGTAAGTTATGTT
GTGCCTACTAAAGCCGACAAGCGGCGCAGCGTCAGGATCGGATCCTATATTGAGAGGGACGTG
ACACCTGCTATTATGGAAGATGACGAGTTAGCATTGGACCTCGAGGACCTTCTATCCTTTTCATAT CAGGTGGCCAAGGGCATGGCCTTCCTGGCGTCTAAAAACTGTATTCACCGCGATTTGGCCGCGA
GAAACATCCTGCTCACACATGGAAGAATCACCAAAATTTGCGACTTTGGCCTGGCCAGAGATATC
AAGAACGACTCAAACTATGTGGTGAAGGGAAATGCACGTCTGCCCGTGAAGTGGATGGCACCAG
AGTCAATCTTTAATTGTGTGTATACGTTCGAAAGTGATGTCTGGTCATACGGAATCTTCCTGTGGG
AGTTGTTCTCCCTGGGGTCTTCCCCCTACCCAGGGATGCCTGTGGACTCTAAATTCTACAAGATG
ATCAAGGAGGGCTTCCGCATGTTATCACCAGAGCACGCACCCGCTGAGATGTACGATATTATGA
AAACTTGCTGGGACGCTGATCCCCTAAAGCGGCCAACTTTCAAACAGATTGTTCAGCTTATTGAA
AAGCAGATCAGTGAATCTACAAACCATATCTATAGTAATCTGGCCAATTGCTCACCTAACCGACA GAAGCCCGTGGTTGATCACTCCGTTAGGATCAACTCTGTGGGCAGCACTGCAAGCTCCAGTCAG CCCCTGCTTGTCC ACGATGATGTC .
[0145] In particular embodiments, the mRNA comprises an RNA sequence corresponding to the open reading frame DNA sequence of SEQ ID NO: 7:
ATGAGAGGCGCCCGGGGCGCATGGGACTTCTTATGTGTATTATTGCTCCTCCTGAGGGTTCAGA
CCGGGAGTAGCCAACCTTCCGTGTCCCCTGGTGAACCATCGCCACCATCGATCCACCCTGGCAA
ATCCGACCTGATCGTGAGGGTCGGCGATGAAATCCGGCTCCTATGCACGGACCCTGGTTTTGTG
AAATGGACCTTTGAGATCCTCGATGccACCAATGAGAATAAACAGAACGAGTGGATTACAGAAAA
GGCTGAGGCCACAAATACAGGTAAATACACATGCACCAATAAGCACGGCCTCTCGAACTCTATCT
ACGTTTTTGTGCGAGATCCTGCAAAACTTTTCCTCGTCGATCGCTCCTTATACGGAAAGGAGGAC
AATGATACACTGGTCAGATGCCCTTTAACAGACCCCGAGGTTACAAACTATTCACTCAAGGGTTG
TCAGGGTAAACCTCTGCCAAAAGACCTGCGCTTTATCCCTGATCCTAAAGCCGGCATCATGATAA
AATCTGTAAAGCGGGCCTACCACCGTCTCTGCTTGCACTGTAGCGTTGATCAAGAAGGCAAGTC
AGTTTTAAGCGAGAAATTCATCCTGAAGGTGCGCCCAGCCTTCAAGGCTGTGCCTGTAGTGTCA
GTGTCTAAGGCCTCATACCTGCTCCGGGAGGGGGAGGAATTTACTGTGACCTGCACGATAAAAG
ACGTTTCCTCTTCTGTGTACTCTACTTGGAAGAGGGAAAATAGCCAGACCAAGCTGCAGGAGAA
GTACAACAGCTGGCATCATGGTGACTTCAACTACGAGAGACAGGCCACTCTGACTATTTCATCTG
CACGCGTGAATGACTCCGGTGTGTTTATGTGCTACGCAAATAACACCTTCGGCAGTGCCAATGT
GACAACTACCCTGGAAGTCGTTGACAAGGGCTTCATTAACATCTTTCCAATGATCAATACAACCG
TCTTTGTTAACGATGGCGAAAACGTGGACCTGATCGTTGAATATGAAGCATTCCCCAAACCAGAG
CACCAGCAGTGGATCTACATGAATCGCACTTTCACCGACAAGTGGGAAGACTACCCCAAATCCG
AGAACGAGTCTAACATCCGCTATGTGTCGGAACTCCACCTGACTAGACTTAAGGGTACGGAAGG
AGGCACCTACACCTTTTTGGTGAGCAATAGCGACGTGAACGCGGCAATTGCTTTTAACGTATACG
TGAATACGAAACCCGAAATATTGACATATGACCGTCTGGTGAACGGAATGCTTCAGTGTGTGGCC
GCAGGCTTTCCTGAACCAACCATCGACTGGTACTTTTGCCCTGGTACCGAGCAGCGGTGCTCCG
CGAGCGTGCTGCCTGTGGACGTCCAGACGCTAAATTCTAGTGGGCCACCTTTTGGAAAACTGGT
GGTTCAGTCGTCAATTGATTCTTCTGCATTTAAGCATAATGGGACAGTGGAGTGTAAAGCTTACA
ACGATGTGGGGAAGACAAGCGCCTATTTCAACTTTGCCTTTAAGGGGAACAATAAAGAGCAGATT
CATCCACACACCTTGTTCACTCCTTTATTGATCGGGTTTGTGATCGTGGCGGGAATGATGTGTAT
TATCGTTATGATTTTGACTTATAAGTACCTGCAGAAGCCTATGTATGAAGTGCAGTGGAAAGTGGT
GGAAGAGATCAACGGGAACAATTACGTTTATATCGACCCCACCCAGTTGCCATATGACCACAAAT
GGGAATTTCCCAGGAATCGCTTGAGCTTCGGGAAGACACTCGGTGCCGGAGCCTTCGGAAAGG
TAGTAGAAGCAACGGCTTACGGGCTAATCAAGTCAGATGCCGCTATGACTGTTGCGGTGAAAAT
GTTGAAGCCATCGGCTCATCTGACAGAGCGGGAGGCTCTGATGAGCGAACTCAAGGTTCTCAGT
TACCTCGGCAATCACATGAACATTGTAAATCTCCTTGGGGCCTGTACGATCGGCGGTCCCACCC
TCGTCATAACAGAATACTGCTGCTATGGCGATCTGCTGAACTTCCTCCGGCGCAAGAGGGATTC
CTTTATATGTAGCAAACAAGAAGACCACGCGGAGGCCGCTCTATACAAAAACCTGTTACACAGTA
AAGAGTCTTCATGCAGCGACAGTACGAATGAATACATGGACATGAAACCTGGGGTAAGTTATGTT
GTGCCTACTAAAGCCGACAAGCGGCGCAGCGTCAGGATCGGATCCTATATTGAGAGGGACGTG
ACACCTGCTATTATGGAAGATGACGAGTTAGCATTGGACCTCGAGGACCTTCTATCCTTTTCATAT
CAGGTGGCCAAGGGCATGGCCTTCCTGGCGTCTAAAAACTGTATTCACCGCGATTTGGCCGCGA
GAAACATCCTGCTCACACATGGAAGAATCACCAAAATTTGCGACTTTGGCCTGGCCAGAGATATC
AAGAACGACTCAAACTATGTGGTGAAGGGAAATGCACGTCTGCCCGTGAAGTGGATGGCACCAG
AGTCAATCTTTAATTGTGTGTATACGTTCGAAAGTGATGTCTGGTCATACGGAATCTTCCTGTGGG
AGTTGTTCTCCCTGGGGTCTTCCCCCTACCCAGGGATGCCTGTGGACTCTAAATTCTACAAGATG
ATCAAGGAGGGCTTCCGCATGTTATCACCAGAGCACGCACCCGCTGAGATGTACGATATTATGA
AAACTTGCTGGGACGCTGATCCCCTAAAGCGGCCAACTTTCAAACAGATTGTTCAGCTTATTGAA
AAGCAGATCAGTGAATCTACAAACCATATCTATAGTAATCTGGCCAATTGCTCACCTAACCGACA GAAGCCCGTGGTTGATCACTCCGTTAGGATCAACTCTGTGGGCAGCACTGCAAGCTCCAGTCAG CCCCTGCTTGTCCACGATGATGTC . [0146] Illustrative modified CD117 mRNA constructs and sequences are provided below in Table 6, with the coding sequence capitalized. For all constructs/sequences disclosed herein, in the context of mRNA, the Ts are Us, and in particular embodiments, the Ts are fully substituted with NlmPsU. Coding sequence is capitalized. Note that throughout the specification, construct sequences are generally specified T7 promoter
Figure imgf000072_0001
3’UTR and do not include poly A tail sequences, except for AMOS. mRNA constructs electroporated into cells may lack the T7 promoter sequence. The T7 promoter sequence comprises the sequence TAATACXUkC’rCACI'A'fAG (SEQ ID NO: 86). The T7 polymerase starts transcription at the underlined G in the promoter sequence. The polymerase then transcribes using the opposite strand as a template from 5 ’ ->3 ’ . The first base in the transcript will be a G.
Figure imgf000072_0002
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
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Figure imgf000080_0001
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Figure imgf000085_0001
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Figure imgf000100_0001
Figure imgf000101_0001
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Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0003
[0147] Illustrative mRNA sequences and constructs for expressing modified CD117 polypeptides comprising double mutations, e.gg., E73 and N505 mutations, are provided below in Table 7. . For all constructs/sequences disclosed herein, in the context of mRNA, the Ts are Us, and in particular embodiments, the Ts are fully substituted with NlmPsU. Coding sequence is capitalized. Note that throughout the specification, construct sequences are generally specified T7 promoter
Figure imgf000121_0001
3’UTR and do not include poly A tail sequences, except for AMOS. mRNA constructs electroporated into cells may lack the T7 promoter sequence. The T7 promoter sequence comprises the sequence
Figure imgf000121_0002
The T7 polymerase starts transcription at the underlined G in the promoter sequence. The polymerase then transcribes using the opposite strand as a template from 5’->3’. The first base in the transcript will be a G.
Figure imgf000121_0004
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Constitutively Active CD117 Not Bound by anti-CDl 17 Antibodies
[0148] In certain embodiments, a modified CD117 comprises both of the two types of modification disclosed above: it has constitutive activity; and it is not bound by an anti-CDl 17 antibody used for HCT conditioning. In particular embodiments, it has constitutive signaling or kinase activity, even in absence of SCF binding to the modified CD117 and in the presence of an anti-c-Kit antibody that blocks SCF binding to the modified CD117 polypeptide.
[0149] In particular embodiments, the amino acid modifications comprise: (1) one or more amino acid substitutions or deletions of an amino acid residue selected from the following in human CD117: N505, 522, 557, 558, V559, V568, 569, V570, 579, 703, and D816; and (2) one or more amino acid substitutions of an amino acid residue selected from the following in human CD117: E73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, or R271, or at an amino acid residue located within 2, within 3, within 4, within 5, within 6, within 7 within 8 within 8 within 10, within 11 or within 12 amino acids of any of E73, D121, R122, S123, Y125, K203, Y259, S261, W262, Y269, or R271, e.g., either N-terminal or C-terminal of any of these residues. In some embodiments, the modification is at any of E73, D121, R122, S123, Y125, K203, or at an amino acid residue located within 2, within 3, within 4, within 5, within 6, within 7 within 8 within 8 within 10, within 11 or within 12 amino acids of any of these amino acids, and the modified or variant CD117 does not bind the JSP191 antibody. In some embodiments, the CD117 modification is at any of K203, Y259, S261, W262, Y269, or R271, or at an amino acid residue located within 2, within 3, within 4, within 5, within 6, within 7 within 8 within 8 within 10, within 11 or within 12 amino acids of any of these amino acids, and the modified or variant CD117 does not bind the AB85 antibody.
[0150] In some embodiments, the modified CD117 polypeptide comprises one or more of the following amino acid substitutions: N505I, V559D, D816V, D816H, V568F, V570F, and/or Y703F. In particular embodiments, variant CD117 comprises an modification at D816 and/or a N505, and/or a modification at E73. In particular embodiments, the amino acid substitutions comprise a D816V substitution and/or a N505I substitution. In particular embodiments, the amino acid substitutions comprise an E73A substitution. In some embodiments, the amino acid substitutions comprise an N505I substitution and/or E73A substitution. In some embodiments, the amino acid substitutions comprise an D816V substitution and/or E73A substitution. In some embodiments, the modified CD117 has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 750%, or at least 1000% of the kinase activity of the wild type CD117 polypeptide in the presence of an anti-c-kit antibody. [0151] In particular embodiments, an illustrative SCF has the following polypeptide sequence:
NP 000890.1 kit ligand isoform b precursor [Homo sapiens]
MKKTQTWILTCIYLQLLLFNPLVKTEGICRNRVTNNVKDVTKLVANLPKDYMITLKY VPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECV KENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFWASETSDCWSSTLSPEKDSR VSVTKPFMLPPVAASSLRNDSSSSNRKAKNPPGDSSLHWAAMALPALFSLIIGFAFGA LYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV (SEQ ID NO: 14).
CXCR4 Polypeptides and Polynucleotides
[0152] C-X-C chemokine receptor type 4 (CXCR4), also known as fusin or CD 184 (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.
[0153] 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 YTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIF ANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQ KRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFH CCLNPI LYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS (SEQ ID
NO: 158); or
MSIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGN
GLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKA
VHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIP DFIFAN
VSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKR
KALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCC
LNPIL YAFLGAKFKT S AQHALT S VSRGS SLKIL SKGKRGGHS S VSTESES S SFHS S (SEQ ID NO: 159) 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.
[0154] 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
Figure imgf000127_0001
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 :
160 ) ; 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 : 161 ) 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. [0155] 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 WHIMtl9, 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.
[0156] 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).
[0157] Illustrative CXCR4 mRNA constructs are provided below in Table 8. CXCR4 coding sequences are indicated in capitals. . For all constructs/sequences disclosed herein, in the context of mRNA, the Ts are Us, and in particular embodiments, the Ts are fully substituted with NlmPsU. Coding sequence is capitalized. Note that throughout the specification, construct sequences are generally specified T7 promoter
Figure imgf000129_0001
3’UTR and do not include poly A tail sequences, except for AMOS. mRNA constructs electroporated into cells may lack the T7 promoter sequence. The T7 promoter sequence comprises the sequence The T7 polymerase starts transcription at the
Figure imgf000129_0002
underlined G in the promoter sequence. The polymerase then transcribes using the opposite strand as a template from 5 ’->3’. The first base in the transcript will be a G.
Figure imgf000129_0003
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
[0158] Examples of illustrative self-amplifying RNA constructs are shown in the Table 9 below. For all constructs/sequences disclosed herein, in the context of mRNA, the Ts are Us, and in particular embodiments, the Ts are fully substituted with NlmPsU. Coding sequence is capitalized. Note that throughout the specification, construct sequences are generally specified T7 promoter 3’UTR and do not include poly A tail sequences, except for AMOS. mRNA constructs electroporated into cells may lack the T7 promoter sequence. The T7 promoter sequence comprises the sequence TAATACGACTCACTATAG (SEQ ID NO: 86). The T7 polymerase starts transcription at the underlined G in the promoter sequence. The polymerase then transcribes using the opposite strand as a template from 5’->3’. The first base in the transcript will be a G.
Figure imgf000142_0002
Figure imgf000143_0001
Figure imgf000144_0001
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
Figure imgf000148_0001
Polynucleotides and Vectors
[0159] Modified cells may be made by introducing into the cell one or more polynucleotide or nucleic acid encoding CXCR4, CD47, and/or one or more variant CD117s into the cell. A variety of vectors and nucleic acids may be used, as well as a variety of methods for introducing the vector and/or nucleic acid into the cell. In particular embodiments, the nucleic acid comprises RNA, DNA, or a combination thereof, and in particular embodiments, the nucleic acid comprises single-stranded and/or double-stranded regions, or a mixture thereof. In certain embodiments, the nucleic acid is a double-stranded DNA, and in certain embodiments, the nucleic acid is a single stranded RNA, e.g., a messenger RNA (mRNA). In certain embodiments, the nucleic acid comprises a modified mRNA. In particular embodiments, the polynucleotides described herein, e.g., modified mRNA, are codon-optimized, e.g., to enhance expression of the encoded polypeptide in a host cell. In certain embodiments, the one or more introduced or exogenous nucleic acids are RNAs, e.g., a modified mRNA. 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. [0160] In certain embodiments, the sequence encoding CXCR4, CD47, and/or a modified CD117 protein comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the polypeptide encoding sequences disclosed herein. In certain embodiments, the mRNA construct comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the encoded sequences disclosed herein or comprises an mRNA construct sequence disclosed herein, optionally without the promoter sequence and optionally with a poly A tail.
[0161] In particular embodiments, polynucleotide variants comprise one or more modified nucleotide or nucleoside. ^Modified mRNAs comprising one or more modified nucleoside have been described as having advantages over unmodified mRNAs, including increase stability, higher expression levels and reduced immunogenicity. Non-limiting examples of modifications to mRNAs that may be present in the nucleic acids are described, e.g., in PCT Patent Application Publication Nos. WO2011/130624, WO2012/138453, WO2013052523, WO2013151666, WO2013/071047, WO2013/078199, W02012045075, W02014081507, 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).
[0162] In particular embodiments, the modified mRNA comprises one or more nucleoside modification. In particular embodiments, the modified mRNA sequence comprises at least one modification as compared to an unmodified A, G, U or C ribonucleoside. For example, uridine can a similar nucleoside such as pseudouridine (T) or Nl-methyl-pseudouridine (mlT), and cytosine can be replaced by 5-methylcytosine. In particular embodiments, the at least one modified nucleosides include Nl-methyl-pseudouridine and/or 5-methylcytidine. In certain embodiments, one or more uridines are replaced by 5- methoxyuridine (5moU). In certain embodiments, all uridines in the modified mRNA are replaced with a similar nucleoside such as pseudouridine (T) or Nl-methyl-pseudouridine (mlT), and/or all cytosines in the modified mRNA are substituted with a similar nucleoside such as 5-methylcytosine.
[0163] In particular embodiments, one or more mRNA comprises a 5’ terminal cap sequence followed by a sequence encoding the polypeptide, followed by a 3’ tailing sequence, such as a poly A or a polyA-G sequence.
[0164] In particular embodiments, the nucleic acid sequence encoding the CXCR4, CD47, and/or a modified CD117 protein comprises 5’ and/or 3’ cellular or viral untranslated regions (UTRs) relative to the sequence encoding the CXCR4, CD47, and/or a modified CD117 protein. In some embodiments, the UTR improves mRNA stability, localization and/or expression. In some embodiments the UTR is tissue specific. In some embodiments, the 5’ UTR comprises a UTR sequence from alpha-globin. In some embodiments the nucleic acid comprises a Kozak sequence. In some embodiments the 3 ’UTR comprises a UTR from an alpha-globin and/or a beta-globin gene, i.e., a 5’ UTR from hemoglobin alpha 1 (HBA1) and/or a 3’ UTR from one or more of HBA1 or hemoglobin beta 1 (HBB1) gene.
[0165] In some embodiments, the nucleic acid sequence encoding the CXCR4, CD47, and/or a modified CD117 protein comprises a 5’ UTR with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% identity to a 5’UTR sequence of HBA1 :
ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCACC (SEQ ID NO: 15).
[0166] In some embodiments, the nucleic acid sequence encoding the CXCR4, CD47, and/or a modified CD117 protein comprises a Kozak sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% identity to the following: GCCGCCACC.
[0167] In some embodiments, the nucleic acid sequence encoding the CXCR4, CD47, and/or a modified CD117 protein comprises a 3’UTR nucleic acid sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% identity to a 3’UTR of HBB1 :
GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAA CTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATA AAAAACATTTATTTTCATTGC (SEQ ID NO: 16).
[0168] In some embodiments, the nucleic acid sequence encoding the CXCR4, CD47, and/or a modified CD117 protein comprises a 3’UTR nucleic acid sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% to a 3’UTR of HBA1 :
GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCT CCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCA (SEQ ID NO: 17).
[0169] In some embodiments, the nucleic acid sequence encoding the CXCR4, CD47, and/or a modified CD117 protein comprises an extra stop codon downstream of TAA to avoid run-off translation of an mRNA. In some embodiments, the extra stop codon is TGA. In some embodiments the nucleic acid sequence encoding the CXCR4, CD47, and/or a modified CD117 protein comprises a TCTAGA sequence to linearize a plasmid as a template for transcription.
[0170] In some embodiments, the nucleic acid sequence encoding the CXCR4, CD47, and/or a modified CD117 protein encodes a poly-adenine or poly guanine tail. A poly A or polyA-G tail improves mRNA stability and manufacturability. In some embodiments, the polyA tail may be from 20 to 180 adenine bases in length. In some embodiments, the polyA tail may be from 35 to 140 bases in length. In some embodiments, the polyA tail is segmented with a linker to reduce recombination during plasmid production in prokaryotic cells. In some embodiments, the polyA tail may be from 70 to 150 bases in length. In some embodiments the polyA tail is 70 adenine bases in length. In some embodiments, the linker is a series of bases other than adenine. In some embodiments, the linker is a series of bases including adenine. In some embodiments, the linker is about 3 to about 10 bases in length. In some embodiments, the linker is about 5 to about 20 bases in length. In some embodiments the linker comprises the sequence TATGCA.
[0171] In certain embodiments, the sequence encoding the CXCR4, CD47, and/or a modified CD117 protein comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the modified CD117 encoding sequences disclosed herein.
[0172] In particular embodiments, a modified mRNA comprises a 5’ terminal cap sequence followed by a sequence encoding the CXCR4, CD47, and/or a modified CD117 protein, including one or more 5’ or 3’ UTRs, followed by a 3’ tailing sequence, such as a poly A or a poly A-G tail sequence.
[0173] In particular embodiments, the mRNA encoding CXCR4, CD47, and/or a modified CD117 protein comprises a wild type 5’ terminal cap sequence, and in certain embodiments, the mRNA encoding CXCR4, CD47, and/or a modified CD117 protein comprises a modified 5’ terminal cap, not limited to but including, e.g., m7G(5')ppp(5')(2'OMeA)pG (CleanCap® Reagent AG for co-transcriptional capping of mRNA; TriLink Biotechnologies, USA) or m7(3'OMeG)(5')ppp(5')(2'OMeA)pG (CleanCap Reagent AG (3' OMe) for co-transcriptional capping of mRNA; TriLink Biotechnologies, USA). In certain embodiments, the mRNA encoding the CXCR4, CD47, and/or a modified CD117 protein comprises the modified 5’ terminal cap, 3'-O-Me-m7G(5')ppp(5')G (Anti Reverse Cap Analog (ARCA); APExBIO, USA). In some embodiments, a vaccinia virus mRNA cap methyltransferase adds 7- methylguanylate cap structures (Cap-0) to the 5’ end of RNA. In some embodiments, a vaccinia Cap 2’-O-Methyltransferase adds a methyl group at the 2 -0 position of the first nucleotide adjacent to the cap structure at the 5’ end of the RNA.
[0174] In some embodiments, an mRNA construct encoding the CXCR4, CD47, and/or a modified CD117 protein comprises the following elements, optionally from 5’ to 3’ : a 5’ HBA1 UTR; a CleanCap Reagent AG 3’ OMe 5’ terminal cap sequence (m7(3'OMeG)(5')ppp(5')(2'OMeA)pG); a sequence encoding the CXCR4, CD47, and/or a modified CD117 protein; a TAATAA stop codon; and a 3’ HBB1 UTR. [0175] In particular embodiments, the sequence encoding the CXCR4, CD47, and/or a modified CD117 protein is codon-optimized. In particular embodiments, the construct further comprises a polyA sequence, e.g., after the 3’ HBB1 UTR, optionally an AMOS polyA. In particular embodiments, the mRNA construct comprises any of the sequences disclosed herein. In certain embodiments, it comprises a segmented polyA tail AMOS, in which the 140 adenine bases are segmented into 2x 70 adenine sequences.
[0176] In certain embodiments, an mRNA construct expresses two or more polypeptides selected from a CXCR4, a CD47, and/or a modified CD117 protein. As understood herein, a CXCR4 protein, a CD47 protein, and modified CD117 proteins include both wild type and mutant forms of the proteins, including any of the variants and mutants disclosed herein.
[0177] In certain embodiments, the nucleic acid encoding a CD47, CD117, and/or CXCR4 polypeptide comprises a sequence with at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the mRNA sequences encoded by or produced by any of the constructs disclosed in the tables herein. In certain embodiments, the mRNA construct encoding a CD47, CD117, and/or CXCR4 polypeptide comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the CD47 encoding sequences disclosed herein or comprises an mRNA construct sequence disclosed herein, optionally without the promoter sequence and optionally with a polyA tail.
[0178] 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-di oleoyl -s//-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. [0179] In certain embodiments, the one or more nucleic acids encoding CD47, CD 117, and/or CXCR4 polypeptides (which encompasses functional fragments or variants thereof) are present in one or more vectors. In particular embodiments, a vector is capable of delivering a 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 CD47 polypeptide. In particular embodiments, the expression vector comprises a promoter sequence that facilitates expression of the encoded CD47, CD117, and/or CXCR4 polypeptides 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 sequences encoding a CD47, CD117, and/or CXCR4 polypeptide.
[0180] 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
[0181] In particular embodiments, the disclosure provides a modified or engineered cell comprising one or more exogenous or introduced nucleic acids that combined encode a combination of two or more of: a CD47 polypeptide, a modified CD117 polypeptide with constitutive activity, a modified CD 117 polypeptide not bound by an anti-CD 117 antibody used for HCT conditioning (e.g., JSP191), a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning (e.g., JSP191), and/or a CXCR4 polypeptide, or functional fragments or variants thereof.
[0182] In particular embodiments, the disclosure provides a modified or engineered cell comprising an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning (e.g., JSP191).
[0183] In particular embodiments, the disclosure provides a modified or engineered cell comprising one or more exogenous or introduced nucleic acids encoding a combination of two or more of: a CD47 polypeptide, a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning, and/or a CXCR4 polypeptide, or functional fragments or variants thereof. In particular embodiments, the modified or engineered cell comprises two or more exogenous or introduced nucleic acids, each independently encoding one or more of: a CD47 polypeptide, a modified CD117 polypeptide with constitutive activity, a modified CD117 polypeptide not bound by an anti- CD117 antibody used for HCT conditioning (e.g., JSP191), a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CD117 antibody used for HCT conditioning (e.g., JSP191), and/or a CXCR4 polypeptide, or functional fragments or variants thereof.
[0184] In certain embodiments, the modified or engineered cell comprises: (1) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity; (2) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning; (3) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (4) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning; (5) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (6) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (7) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity, and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning; (8) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity, and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (9) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning, and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (10) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti- CD117 antibody used for HCT conditioning, and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (11) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CD117 antibody used for HCT conditioning, and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide;
(12) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning;
(13) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning; (14) an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning; or (15) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide, and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning. In each case, any of the polypeptides may by variants. [0185] In particular embodiments, each of the exogenous or introduced polynucleotide is present in the cell or provided to the cell as a separate mRNA construct. In certain embodiments, two or more of the exogenous or introduced polynucleotides may be present in the cell or provided to the cell in a single mRNA construct, e.g., a bicistronic mRNA. In certain embodiments, a c-Kit mRNA construct encodes a c-Kit having two or more different point mutations.
[0186] In certain embodiments, any of the modifications or combinations of modification to an engineered cells, e.g., HSC or HSPC, disclosed herein may be combined with a further modification of the cell, e.g., to express programmed death-ligand 1 (PD-L1) on the cell surface. A nucleic acid sequence encoding PDL1, or a functional fragment or variant thereof, may be introduced into the cells using the same vectors, RNAs, and methods as used for the modifications disclosed herein. PD-L1 is an immune checkpoint inhibitor that binds to its receptor PD-1 expressed by T cells and other immune cells to regulate immune responses; ultimately preventing exacerbated activation and autoimmunity. An illustrative PD-L1 protein sequence is provided below: MEQTFLLVLHVVLWPTLAALFTVEVDSLSHVAEFYGDVTMGCRFQPGSWDPNLSVI WQRVQPLPDVEVYRLDNGQENLTSQNFQYRGRARLVSEELTNGWAKLHVSRLRIN DSGVYRCLVEMGGADYKQTTLTVKATYKTIIKSMQRRGGGEVELACESEGYPLATI NWRDKSLRNIKSNDTVVKTPNQLFHVTSKITVKYSEKNNYTCAFVEKGEAPKGPSAR FDIPDE1PVIESKPNTLSIVLGTTLTVAMIIVATIFGYRRQKGRLRTLKL (SEQ ID NO: 189)
[0187] The modifications disclosed herein may be made to any type of cell, e.g., any mammalian cell.
[0188] In certain embodiments, the modified cell comprising a modified CD117 polypeptide and/or encoding nucleic acid is a host cell, such as, e.g., an HEK293 cell that may be used to produce modified CD117 polypeptides. In preparing the subject compositions, any host cells may be employed, including but not limited to, for example, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast.
[0189] 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.
[0190] In a related aspect, the disclosure provides modified cells, e.g., HSCs and/or HSPCs, comprising one or more nucleic acids, which in combination encode two or more CD47, CD117, and/or CXCR4 polypeptides as described herein. In certain embodiments, the nucleic acid(s) encoding CD47, CD117, and/or CXCR4 polypeptides are transiently present in the modified cell, and/or is not present within the genome of the cell. In particular embodiments, one or more modified cells expresses and/or comprises a CD47, CD117, and/or CXCR4 polypeptide, and in particular embodiments, a CD47, CD117, and/or 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 an mRNA, e.g., a modified mRNA. In particular embodiments, the modified HSCs and/or HSPCs transiently or constitutively overexpress the CD47, CD117, and/or CXCR4 polypeptides, or a functional fragment or variant thereof, e.g., at a level at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold higher than a corresponding unmodified cell, e.g., HSC and/or HSPC. [0191] 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.
[0192] 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 one or more nucleic acids encoding a CD47, CD117, and/or 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.
[0193] 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.
[0194] 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.
[0195] 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.
[0196] 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.
[0197] In certain embodiments, the hematopoietic stem cells 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 modified CD117 polypeptide into the cell. For example, the nucleic acid may be introduced by transfection or infection with a viral vector, or by contact with an mRNA.
[0198] 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.
[0199] In certain embodiments, the disclosure provides a method of modifying cells, including HSCs and/or HSPCs, comprising introducing one or more nucleic acid encoding one or more of: CD47, CXCR4, and/or one or more modified CD117 into the cell, including any of the specific combinations disclosed herein. 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 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 one or more of: CD47, CXCR4, and/or one or more modified CD117 polypeptide into the HSCs and/or HSPCs according to a variety of methods available in the art, e.g., electroporation. [0200] In certain embodiments, the modified cells, e.g., stem cells are further modified to provide a replacement nucleic acid or protein to the cells, e.g., where the cells are obtained from a subject having a genetic disorder resulting in reduced or lack of expression of a gene or protein, or the expression of a mutant form of a gene or protein. Thus, the cells, e.g., HSCs and/HSPCs, may be further genetically altered to correct a genetic defect present in the cells, e.g., HSCs and/or HSPCs. In certain embodiments, the cells may be contacted with or transduced by a gene therapy vector that results in the insertion into the cellular genome of an expression cassette that expresses a corrected form of a mutated gene or protein, e.g., a wild- type or non-mutated gene or protein. In certain embodiments, the gene therapy may replace the mutated gene or a mutated region thereof, e.g., via homologous recombination. In certain embodiments, the HSCs and/or HSPCs are modified to correct a mutated gene using gene editing or base editing methods, such as, e.g., a CRISPR-Cas9 system that targets the mutated gene. In certain embodiments, the HSCs and/or HSPCs are modified to correct a mutated gene using Zinc-finger nucleases (ZFNs), meganucleases, or transcription activator-like effector nucleases (TALENs) that target the mutated gene. Correction of a genetic mutation may be done prior to, at the same time as, or following introduction of the nucleic acid encoding CD47, or a fragment or variant thereof.
[0201] In certain embodiments, the disclosure provides a method of modifying cells, and the resulting modified cells, including stem cells such as HSCs and/or HSPCs, comprising modifying one or more endogenous CD117 genes or alleles within the cells, e.g., by homologous recombination or gene editing according to a variety of methods available in the art. In certain embodiments, a CD 117 gene in HSCs and/or HSPCs is edited by any of a variety of methods known and available in the art, including but not limited to: transcription activatorlike effector nucleases (TALENs), megaTALs, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) systems, zinc finger nucleases, homing endonucleases, or meganucleases. In certain embodiments, the CD117 gene is edited by a base editing method. As used herein, a gene-editing system is a system comprising one or more proteins or polynucleotides capable of editing an endogenous target gene or locus in a sequence specific manner. In some embodiments, the gene-editing system is a protein-based gene regulating system comprising a protein comprising one or more zinc-finger binding domains and an enzymatic domain. In some embodiments, the protein-based gene regulating system comprises a protein comprising a Transcription activator-like effector nuclease (TALEN) domain and an enzymatic domain. Such embodiments are referred to herein as “TALENs.” In particular embodiments, the gene editing system comprises a nucleic acid sequence corresponding to a region of the CD117 gene and comprising a modification thereof. [0202] Zinc finger-based systems comprise a fusion protein comprising two protein domains: a zinc finger DNA binding domain and an enzymatic domain. A “zinc finger DNA binding domain”, “zinc finger protein”, or “ZFP” is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The zinc finger domain, by binding to a target DNA sequence, directs the activity of the enzymatic domain to the vicinity of the sequence and, hence, induces modification of the endogenous target gene in the vicinity of the target sequence. A zinc finger domain can be engineered to bind to virtually any desired sequence. Accordingly, after identifying a target genetic locus containing a target DNA sequence at which cleavage or recombination is desired (e.g., a target locus or epitope identified herein), one or more zinc finger binding domains can be engineered to bind to one or more target DNA sequences in the target genetic locus. Expression of a fusion protein comprising a zinc finger binding domain and an enzymatic domain in a cell, effects modification in the target genetic locus.
[0203] In some embodiments, a zinc finger binding domain comprises one or more zinc fingers. Miller et al. (1985) EMBO J. 4: 16010-1714; Rhodes (1993) Scientific American Febuary:56-65; U.S. Pat. No. 6,453,242. Typically, a single zinc finger domain is about 30 amino acids in length. An individual zinc finger binds to a three-nucleotide (z.e., triplet) sequence (or a four-nucleotide sequence which can overlap, by one nucleotide, with the four- nucleotide binding site of an adjacent zinc finger). Therefore the length of a sequence to which a zinc finger binding domain is engineered to bind (e.g., a target sequence) will determine the number of zinc fingers in an engineered zinc finger binding domain. In some embodiments, the DNA-binding domains of individual ZFNs comprise between three and six individual zinc finger repeats and can each recognize between 9 and 18 base pairs.
[0204] Zinc finger binding domains can be engineered to bind to a sequence of choice. See, for example, Beerli et al. (2002) Nature Biotechnol. 20: 135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416. An engineered zinc finger binding domain can have a novel binding specificity, compared to a naturally-occurring zinc finger protein. Engineering methods include, but are not limited to, rational design and various types of selection.
[0205] Selection of a target DNA sequence for binding by a zinc finger domain can be accomplished, for example, according to the methods disclosed in U.S. Pat. No. 6,453,242. It will be clear to those skilled in the art that simple visual inspection of a nucleotide sequence can also be used for selection of a target DNA sequence. Accordingly, any means for target DNA sequence selection can be used in the methods described herein. A target site generally has a length of at least 9 nucleotides and, accordingly, is bound by a zinc finger binding domain comprising at least three zinc fingers. However, binding of, for example, a 4-finger binding domain to a 12-nucleotide target site, a 5-finger binding domain to a 15-nucleotide target site or a 6-finger binding domain to an 18-nucleotide target site, is also possible. As will be apparent, binding of larger binding domains (e.g., 7-, 8-, 9-finger and more) to longer target sites is also possible.
[0206] In some embodiments, the zinc finger binding domains bind to a target DNA sequence that is at least 90% identical to a target DNA sequence (e.g., epitope-encoding) within a target locus of a target CD117 gene. In some embodiments, the zinc finger binding domains bind to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence within a target locus of a target gene. In some embodiments, the zinc finger binding domains bind to a target DNA sequence that is 100% identical to a target DNA sequence within a target locus of a target gene.
[0207] The enzymatic domain portion of the zinc finger fusion proteins can be obtained from any endo- or exonuclease. Exemplary endonucleases from which an enzymatic domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes which cleave DNA are known (e.g., 51 Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains. Exemplary restriction endonucleases (restriction enzymes) suitable for use as an enzymatic domain of the ZFPs described herein are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et a!. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et a!. (1994a) Proc. Natl. Acad. Sci. USA 91 :883-887; Kim et al. (1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise the enzymatic domain from at least one Type IIS restriction enzyme and one or more zinc finger binding domains.
[0208] An exemplary Type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is Fok I. This particular enzyme is active as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575. Thus, for targeted double-stranded DNA cleavage using zinc finger-Fok I fusions, two fusion proteins, each comprising a FokI enzymatic domain, can be used to reconstitute a catalytically active cleavage domain. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two FokI enzymatic domains can also be used. Exemplary ZFPs comprising FokI enzymatic domains are described in US Patent No. 9,782,437. [0209] TALEN-based systems comprise a protein comprising a TAL effector DNA binding domain and an enzymatic domain. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). The FokI restriction enzyme described above is an exemplary enzymatic domain suitable for use in TALEN-based gene regulating systems.
[0210] TAL effectors are proteins that are secreted by Xanthomonas bacteria via their type III secretion system when they infect plants. The DNA binding domain contains a repeated, highly conserved, 33-34 amino acid sequence with divergent 12th and 13th amino acids. These two positions, referred to as the Repeat Variable Diresidue (RVD), are highly variable and strongly correlated with specific nucleotide recognition. Therefore, the TAL effector domains can be engineered to bind specific target DNA sequences by selecting a combination of repeat segments containing the appropriate RVDs. The nucleic acid specificity for RVD combinations is as follows: HD targets cytosine, NI targets adenenine, NG targets thymine, and NN targets guanine (though, in some embodiments, NN can also bind adenenine with lower specificity).
[0211] In some embodiments, the TAL effector domains bind to a target DNA sequence that is at least 90% identical to a target DNA sequence (e.g., epitope-enoding) within a target locus of a CD117 gene. In some embodiments, the TAL effector domains bind to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence within a target locus of a target gene. In some embodiments, the TAL effector domains bind to a target DNA sequence that is 100% identical to a target DNA sequence within a target locus of a target gene.
Methods and compositions for assembling the TAL-effector repeats are known in the art. See e.g., Cermak et a/, Nucleic Acids Research, 39:12, 2011 , e82. Plasmids for constructions of the TAL- effector repeats are commercially available from Addgene.
[0212] In some embodiments, the gene-editing system is a combination gene-regulating system comprising a site-directed modifying polypeptide and a nucleic acid guide molecule. Herein, a "site-directed modifying polypeptide" refers to a polypeptide that binds to a nucleic acid guide molecule, is targeted to a target nucleic acid sequence, such as, for example, a DNA sequence, by the nucleic acid guide molecule to which it is bound, and modifies the target DNA sequence (e.g., cleavage, mutation, or methylation of target DNA). A site-directed modifying polypeptide comprises two portions, a portion that binds the nucleic acid guide and an activity portion. In some embodiments, a site-directed modifying polypeptide comprises an activity portion that exhibits site-directed enzymatic activity (e.g., DNA methylation, DNA cleavage, histone acetylation, histone methylation, etc.), wherein the site of enzymatic activity is determined by the guide nucleic acid.
[0213] In particular embodiments, the nucleic acid guide comprises two portions: a first portion that is complementary to, and capable of binding with, an endogenous target DNA sequence (referred to herein as a “DNA-binding segment”), and a second portion that is capable of interacting with the site-directed modifying polypeptide (referred to herein as a “proteinbinding segment”). In some embodiments, the DNA-binding segment and protein-binding segment of a nucleic acid guide are comprised within a single polynucleotide molecule. In some embodiments, the DNA-binding segment and protein-binding segment of a nucleic acid guide are each comprised within separate polynucleotide molecules, such that the nucleic acid guide comprises two polynucleotide molecules that associate with each other to form the functional guide.
[0214] The nucleic acid guide mediates the target specificity of the combined protein/nucleic gene regulating systems by specifically hybridizing with a target DNA sequence comprised within the DNA sequence of a target gene. Reference herein to a target gene encompasses the full-length DNA sequence for that particular gene and a full-length DNA sequence for a particular target gene will comprise a plurality of target genetic loci, which refer to portions of a particular target gene sequence (e.g., an exon or an intron). Within each target genetic loci are shorter stretches of DNA sequences referred to herein as “target DNA sequences” or “target sequences” that can be modified by the gene-regulating systems described herein. Further, each target genetic loci comprises a “target modification site,” which refers to the precise location of the modification induced by the gene-regulating system (e.g., the location of an insertion, a deletion, or mutation, the location of a DNA break, or the location of an epigenetic modification). The gene-regulating systems described herein may comprise a single nucleic acid guide, or may comprise a plurality of nucleic acid guides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleic acid guides).
[0215] The CRISPR/Cas systems described below are exemplary embodiments of a combination protein/nucleic acid system.
[0216] In some embodiments, the gene editing systems described herein are CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR Associated) nuclease systems. In such embodiments, the site-directed modifying polypeptide is a CRISPR- associated endonuclease (a “Cas" endonuclease) and the nucleic acid guide molecule is a guide RNA (gRNA). [0217] A Cas polypeptide refers to a polypeptide that can interact with a gRNA molecule and, in concert with the gRNA molecule, homes or localizes to a target DNA sequence and includes naturally occurring Cas proteins and engineered, altered, or otherwise modified Cas proteins that differ by one or more amino acid residues from a naturally-occurring Cas sequence.
[0218] In some embodiments, the Cas protein is a Cas9 protein. Cas9 is a multi-domain enzyme that uses an HNH nuclease domain to cleave the target strand of DNA and a RuvC- like domain to cleave the non-target strand. In some embodiments, mutants of Cas9 can be generated by selective domain inactivation enabling the conversion of WT Cas9 into an enzymatically inactive mutant (e.g., dCas9), which is unable to cleave DNA, or a nickase mutant, which is able to produce single-stranded DNA breaks by cleaving one or the other of the target or non-target strand.
[0219] A guide RNA (gRNA) typically comprises two segments, a DNA-binding segment and a protein-binding segment. In some embodiments, the protein-binding segment of a gRNA is comprised in one RNA molecule and the DNA-binding segment is comprised in another separate RNA molecule. Such embodiments are referred to herein as "double-molecule gRNAs" or "two-molecule gRNA" or “dual gRNAs.” In some embodiments, the gRNA is a single RNA molecule and is referred to herein as a "single-guide RNA" or an "sgRNA." The term "guide RNA" or "gRNA" is inclusive, referring both to two-molecule guide RNAs and sgRNAs.
[0220] The protein-binding segment of a gRNA typically comprises, in part, two complementary stretches of nucleotides that hybridize to one another to form a double stranded RNA duplex (dsRNA duplex), which facilitates binding to the Cas protein.
[0221] The DNA-binding segment (or "DNA-binding sequence") of a gRNA comprises a nucleotide sequence that is complementary to and capable of binding to a specific sequence target DNA sequence or RNA sequence. The protein-binding segment of the gRNA interacts with a Cas polypeptide and the interaction of the gRNA molecule and site-directed modifying polypeptide results in Cas binding to the endogenous DNA or RNA and produces one or more modifications within or around the target DNA sequence. The precise location of the target modification site is determined by both (i) base-pairing complementarity between the gRNA and the target DNA or RNA sequence; and (ii) the location of a short motif, referred to as the protospacer adjacent motif (PAM), in the target DNA sequence. The PAM sequence is required for Cas binding to the target DNA sequence. A variety of PAM sequences are known in the art and are suitable for use with a particular Cas endonuclease (e.g., a Cas9 endonuclease) are known in the art (See e.g., Nat Methods. 2013 Nov; 10(11): 1116-1121 and S ci Rep. 2014; 4: 5405). In some embodiments, the PAM sequence is located within 50 base pairs of the target modification site. In some embodiments, the PAM sequence is located within 10 base pairs of the target modification site. The DNA or RNA sequences that can be targeted by this method are limited only by the relative distance of the PAM sequence to the target modification site and the presence of a unique 20 base pair sequence to mediate sequence-specific, gRNA- mediated Cas binding. In some embodiments, the target modification site is located at the 5’ terminus of the target locus. In some embodiments, the target modification site is located at the 3’ end of the target locus. In some embodiments, the target modification site is located within an intron or an exon of the target locus.
[0222] In particular embodiments, the guide RNA binds to a CD117 polynucleotide sequence and includes a region complementary to a target CD117 sequence. In certain embodiments, the guide RNA targets or binds a region of CD117 polynucleotide sequence that encodes one of the following amino acid residues: N505 or D816. In some embodiments, the present disclosure provides a polynucleotide encoding a gRNA. In some embodiments, a gRNA-encoding nucleic acid is comprised in an expression vector, e.g., a recombinant expression vector. In some embodiments, the present disclosure provides a polynucleotide encoding a site-directed modifying polypeptide. In some embodiments, the polynucleotide encoding a site-directed modifying polypeptide is comprised in an expression vector, e.g., a recombinant expression vector.
[0223] In some embodiments, the site-directed modifying polypeptide is a Cas protein, e.g., a Cas9 protein. Cas molecules of a variety of species can be used in the methods and compositions described herein, including Cas molecules derived from S. pyogenes, S. aureus, N. meningitidis, S. thermophiles, etc. In some embodiments, the Cas protein is a Cas9 protein or a Cas9 ortholog and is selected from the group consisting of SpCas9, SpCas9-HFl, SpCas9- HF2, SpCas9-HF3, SpCas9-HF4, SaCas9, FnCpf, FnCas9, eSpCas9, and NmeCas9. In some embodiments, the Cas9 protein is a naturally-occurring Cas9 protein. Exemplary naturally occurring Cas9 molecules are described in Chylinski et al., RNA Biology 2013 10:5, 727-737. [0224] In some embodiments, a Cas9 protein comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a Cas9 amino acid sequence described in Chylinski et al., RNA Biology 2013 10:5, 727-737; Hou et al, PNAS Early Edition 2013, 1-6).
[0225] In some embodiments, a Cas polypeptide comprises one or more of the following activities: [0226] a nickase activity, i.e., the ability to cleave a single strand, e.g., the non- complementary strand or the complementary strand, of a nucleic acid molecule;
[0227] a double stranded nuclease activity, i.e., the ability to cleave both strands of a double stranded nucleic acid and create a double stranded break, which in an embodiment is the presence of two nickase activities;
[0228] an endonuclease activity;
[0229] an exonuclease activity; and/or
[0230] a helicase activity, i.e., the ability to unwind the helical structure of a double stranded nucleic acid.
[0231] In some embodiments, the Cas9 is a wildtype (WT) Cas9 protein or ortholog. WT Cas9 comprises two catalytically active domains (HNH and RuvC). Binding of WT Cas9 to DNA based on gRNA specificity results in double-stranded DNA breaks that can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR). In some embodiments, Cas9 is fused to heterologous proteins that recruit DNA-damage signaling proteins, exonucleases, or phosphatases to further increase the likelihood or the rate of repair of the target sequence by one repair mechanism or another. In some embodiments, a WT Cas9 is co-expressed with a nucleic acid repair template to facilitate the incorporation of an exogenous nucleic acid sequence by homology-directed repair.
[0232] In some embodiments, different Cas9 proteins (i.e., Cas9 proteins from various species) may be advantageous to use in the various provided methods in order to capitalize on various enzymatic characteristics of the different Cas9 proteins (e.g., for different PAM sequence preferences; for increased or decreased enzymatic activity; for an increased or decreased level of cellular toxicity; to change the balance between NHEJ, homology-directed repair, single strand breaks, double strand breaks, etc.).
[0233] In some embodiments, the Cas polypeptides are engineered to alter one or more properties of the Cas polypeptide. For example, in some embodiments, the Cas polypeptide comprises altered enzymatic properties, e.g., altered nuclease activity, (as compared with a naturally occurring or other reference Cas molecule) or altered helicase activity.
[0234] The present disclosure provides guide RNAs (gRNAs) that direct a site-directed modifying polypeptide to a specific target DNA sequence. A gRNA comprises a DNA- targeting segment and protein-binding segment. The DNA-targeting segment of a gRNA comprises a nucleotide sequence that is complementary to a sequence in the target DNA sequence. As such, the DNA-targeting segment of a gRNA interacts with a target DNA in a sequence-specific manner via hybridization (i.e., base pairing), and the nucleotide sequence of the DNA-targeting segment determines the location within the target DNA that the gRNA will bind. The DNA-targeting segment of a gRNA can be modified (e.g., by genetic engineering) to hybridize to any desired sequence within a target DNA sequence.
[0235] The protein-binding segment of a guide RNA interacts with a site-directed modifying polypeptide (e.g., a Cas9 protein) to form a complex. The guide RNA guides the bound polypeptide to a specific nucleotide sequence within target DNA via the above-described DNA-targeting segment. The protein-binding segment of a guide RNA comprises two stretches of nucleotides that are complementary to one another and which form a double stranded RNA duplex.
[0236] In some embodiments, a gRNA comprises two separate RNA molecules. In such embodiments, each of the two RNA molecules comprises a stretch of nucleotides that are complementary to one another such that the complementary nucleotides of the two RNA molecules hybridize to form the double-stranded RNA duplex of the protein-binding segment. In some embodiments, a gRNA comprises a single RNA molecule (sgRNA).
[0237] The specificity of a gRNA for a target loci is mediated by the sequence of the DNA- binding segment, which comprises about 20 nucleotides that are complementary to a target DNA sequence within the target locus. In some embodiments, the corresponding target DNA sequence is approximately 20 nucleotides in length. In some embodiments, the DNA-binding segments of the gRNA sequences of the present invention are at least 90% complementary to a target DNA sequence within a target locus. In some embodiments, the DNA-binding segments of the gRNA sequences of the present disclosure are at least 95%, 96%, 97%, 98%, or 99% complementary to a target DNA sequence within a target locus, e.g., CD117. In some embodiments, the DNA-binding segments of the gRNA sequences of the present invention are 100% complementary to a target DNA sequence within a target locus.
[0238] In some embodiments, the DNA-binding segments of the gRNA sequences bind to a target DNA sequence that is at least 90% identical to a target DNA sequence within a target locus of a CD117 gene. In some embodiments, the DNA-binding segments of the gRNA sequences bind to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence within a target locus of a target gene. In some embodiments, the DNA-binding segments of the gRNA sequences bind to a target DNA sequence that is 100% identical to a target DNA sequence within a target locus of a target gene.
[0239] In some embodiments, the DNA-binding segments of the gRNA sequences described herein are designed to minimize off-target binding using algorithms known in the art (e.g. , Cas- OFF finder) to identify target sequences that are unique to a particular target locus or target gene.
[0240] In some embodiments, the gRNAs described herein can comprise one or more modified nucleosides or nucleotides which introduce stability toward nucleases. In such embodiments, these modified gRNAs may elicit a reduced innate immune as compared to a non-modified gRNA. The term "innate immune response" includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.
[0241] In some embodiments, the gRNAs described herein are modified at or near the 5' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of their 5' end). In some embodiments, the 5' end of a gRNA is modified by the inclusion of a eukaryotic mRNA cap structure or cap analog (e.g., a G(5 ')ppp(5 ')G cap analog, a m7G(5 ')ppp(5 ')G cap analog, or a 3 '-0-Me-m7G(5 ')ppp(5 ')G anti reverse cap analog (ARCA)). In some embodiments, an in vitro transcribed gRNA is modified by treatment with a phosphatase (e.g. , calf intestinal alkaline phosphatase) to remove the 5' triphosphate group. In some embodiments, a gRNA comprises a modification at or near its 3' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of its 3' end). For example, in some embodiments, the 3' end of a gRNA is modified by the addition of one or more (e.g., 25-200) adenine (A) residues.
[0242] In some embodiments, modified nucleosides and modified nucleotides can be present in a gRNA, but also may be present in other gene-regulating systems, e.g., mRNA, RNAi, or siRNA- based systems. In some embodiments, modified nucleosides and nucleotides can include one or more of.
[0243] alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage;
[0244] alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar;
[0245] wholesale replacement of the phosphate moiety with "dephospho" linkers;
[0246] modification or replacement of a naturally occurring nucleobase;
[0247] replacement or modification of the ribose-phosphate backbone;
[0248] modification of the 3' end or 5' end of the oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety; and
[0249] modification of the sugar. [0250] In some embodiments, the modifications listed above can be combined to provide modified nucleosides and nucleotides that can have two, three, four, or more modifications. For example, in some embodiments, a modified nucleoside or nucleotide can have a modified sugar and a modified nucleobase. In some embodiments, every base of a gRNA is modified. In some embodiments, each of the phosphate groups of a gRNA molecule are replaced with phosphorothioate groups.
[0251] In some embodiments, a software tool can be used to optimize the choice of gRNA within a user's target sequence, e.g., to minimize total off-target activity across the genome. Off target activity may be other than cleavage. For example, for each possible gRNA choice using S. pyogenes Cas9, software tools can identify all potential off-target sequences (preceding either NAG or NGG PAMs) across the genome that contain up to a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base-pairs. The cleavage efficiency at each off-target sequence can be predicted, e.g., using an experimentally-derived weighting scheme. Each possible gRNA can then be ranked according to its total predicted off-target cleavage; the top-ranked gRNAs represent those that are likely to have the greatest on-target and the least off-target cleavage. Other functions, e.g., automated reagent design for gRNA vector construction, primer design for the on-target Surveyor assay, and primer design for high- throughput detection and quantification of off-target cleavage via next-generation sequencing, can also be included in the tool.
[0252] Additional information regarding CRISPR-Cas systems and components thereof are described in, US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233 and US8999641 and applications related thereto; and WO2014/018423, WO2014/093595, WO2014/093622, WO2014/093635, WO2014/093655, WO2014/093661, WO2014/093694, WO2014/093701, WO20 14/093709, WO2014/093712, WO2014/093718, WO2014/145599, WO2014/204723, WO20 14/204724, WO2014/204725, WO2014/204726, WO2014/204727, WO2014/204728, WO20 14/204729, WO2015/065964, WO2015/089351, WO2015/089354, WO2015/089364, WO20 15/089419, WO2015/089427, WO2015/089462, WO2015/089465, WO2015/089473 and WO2015/089486, W02016205711, WO2017/106657, WO2017/127807 and applications related thereto.
[0253] In certain embodiments, the gene editing methods comprise or consist of base editing methods. Various base editing methods are well known in the art and may be adapted to edit any target nucleic acid sequence within a cell. Base editing activity involves chemically altering a base within a polynucleotide, e.g., converting a first base to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, e.g., converting target OG to T*A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, e.g., converting A*T to G»C. In another embodiment, the base editing activity is cytidine deaminase activity, e.g., converting target OG to T*A and adenosine or adenine deaminase activity, e.g., converting A*T to G»C. In some embodiments, the base editing methods comprise single nucleotide base editing, such as nucleotide deamination, i.e., A->G or C“>T. Base editing systems may edit genomic DNA or transcribed RNA.
[0254] A variety of base editing methods are known and used in the art, including but not limited to those disclosed in the references cited herein. Adenosine and cytidine base editors that may be used include, but are not limited to, base editors described in Antoni ou P. et al., Base and Prime Editing Technologies for Blood Disorders, Front. Genome Ed., 28 January 2021. In some embodiments, base editing methods comprise C~>G conversion as described in Kurt, I. C. et al. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat. Biotechnol. (2020). In some embodiments, dual editors facilitate simultaneous C~>T and A->G conversion as described in Zhao, D. et al. New base editors change C to A in bacteria and C to G in mammalian cells. Nat. Biotechnol. (2020).
[0255] A base editor system generally refers to a system for editing a nucleobase of a target nucleotide sequence. In certain embodiments, a base editor (BE) system comprises: (1) a polynucleotide programmable nucleotide binding domain, a deaminase domain (e.g., cytidine deaminase or adenosine deaminase) for deaminating nucleobases in the target nucleotide sequence; and (2) one or more guide polynucleotides (e.g., guide RNA) in conjunction with the polynucleotide programmable nucleotide binding domain. In various embodiments, the base editor (BE) system comprises a nucleobase editor domain selected from an adenosine deaminase or a cytidine deaminase, and a domain having nucleic acid sequence specific binding activity. In some embodiments, the base editor system comprises: (1) a base editor (BE) comprising a polynucleotide programmable DNA binding domain and a deaminase domain for deaminating one or more nucleobases in a target nucleotide sequence; and (2) one or more guide RNAs in conjunction with the polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE) or a cytidine base editor (CBE). [0256] Cas9 or Cas9 domain refers to an RNA guided nuclease comprising a Cas9 protein, or a fragment or variant thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). A variety of different Cas9 proteins, and fragments and variants thereof, are known and available in the art. [0257] A guide polynucleotide is a polynucleotide that is specific for a target sequence (e.g., specifically hybridizes to a target polynucleotide sequence, such as a CD117 gene or mRNA) and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cpfl). In certain embodiments, the guide polynucleotide is a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. gRNA is used to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules gRNAs, and gRNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs). gRNAs that exist as single RNA species may comprise two domains: (1) a domain that shares homology to a target nucleic acid, and thus directs binding of a Cas9 complex to the target nucleic acid; and (2) a domain that binds a Cas9 protein. In some embodiments, domain (2) is a sequence known as a tracrRNA, which comprises a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al., Science 337:816-821(2012). Other examples of gRNAs (e.g., those including domain 2) are described, e.g., in US20160208288, entitled "Switchable Cas9 Nucleases and Uses Thereof," and US 9,737,604, entitled "Delivery System For Functional Nucleases.” In some embodiments, a gRNA comprises two or more of domains (1) and (2), which may be referred to as an extended gRNA. An extended gRNA will bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions. The gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to the target site, providing the sequence specificity of the nuclease:RNA complex.
[0258] In some embodiments, the base editing method, e.g., a single nucleotide base editing method, targets a polynucleotide encoding a CD117 polypeptide. An illustrative CD117 polynucleotide sequence follows:
>NM_000222.2 Homo sapiens KIT proto-oncogene, receptor tyrosine kinase (KIT), transcript variant 1 mRNA:
TCTGGGGGCTCGGCTTTGCCGCGCTCGCTGCACTTGGGCGAGAGCTGGAACG TGGACCAGAGCTCGGATCCCATCGCAGCTACCGCGATGAGAGGCGCTCGCG GCGCCTGGGATTTTCTCTGCGTTCTGCTCCTACTGCTTCGCGTCCAGACAGGC TCTTCTCAACCATCTGTGAGTCCAGGGGAACCGTCTCCACCATCCATCCATCC AGGAAAATCAGACTTAATAGTCCGCGTGGGCGACGAGATTAGGCTGTTATGC
ACTGATCCGGGCTTTGTCAAATGGACTTTTGAGATCCTGGATGAAACGAATG
AGAATAAGCAGAATGAATGGATCACGGAAAAGGCAGAAGCCACCAACACCG
GCAAATACACGTGCACCAACAAACACGGCTTAAGCAATTCCATTTATGTGTT
TGTTAGAGATCCTGCCAAGCTTTTCCTTGTTGACCGCTCCTTGTATGGGAAAG
AAGACAACGACACGCTGGTCCGCTGTCCTCTCACAGACCCAGAAGTGACCAA
TTATTCCCTCAAGGGGTGCCAGGGGAAGCCTCTTCCCAAGGACTTGAGGTTT
ATTCCTGACCCCAAGGCGGGCATCATGATCAAAAGTGTGAAACGCGCCTACC
ATCGGCTCTGTCTGCATTGTTCTGTGGACCAGGAGGGCAAGTCAGTGCTGTC
GGAAAAATTCATCCTGAAAGTGAGGCCAGCCTTCAAAGCTGTGCCTGTTGTG
TCTGTGTCCAAAGCAAGCTATCTTCTTAGGGAAGGGGAAGAATTCACAGTGA
CGTGCACAATAAAAGATGTGTCTAGTTCTGTGTACTCAACGTGGAAAAGAGA
AAACAGTCAGACTAAACTACAGGAGAAATATAATAGCTGGCATCACGGTGA
CTTCAATTATGAACGTCAGGCAACGTTGACTATCAGTTCAGCGAGAGTTAAT
GATTCTGGAGTGTTCATGTGTTATGCCAATAATACTTTTGGATCAGCAAATGT
CACAACAACCTTGGAAGTAGTAGATAAAGGATTCATTAATATCTTCCCCATG
ATAAACACTACAGTATTTGTAAACGATGGAGAAAATGTAGATTTGATTGTTG
AATATGAAGCATTCCCCAAACCTGAACACCAGCAGTGGATCTATATGAACAG
AACCTTCACTGATAAATGGGAAGATTATCCCAAGTCTGAGAATGAAAGTAAT
ATCAGATACGTAAGTGAACTTCATCTAACGAGATTAAAAGGCACCGAAGGA
GGCACTTACACATTCCTAGTGTCCAATTCTGACGTCAATGCTGCCATAGCATT
TAATGTTTATGTGAATACAAAACCAGAAATCCTGACTTACGACAGGCTCGTG
AATGGCATGCTCCAATGTGTGGCAGCAGGATTCCCAGAGCCCACAAT
AGATTGGTATTTTTGTCCAGGAACTGAGCAGAGATGCTCTGCTTCTGTACTGC
CAGTGGATGTGCAGACACTAAACTCATCTGGGCCACCGTTTGGAAAGCTAGT
GGTTCAGAGTTCTATAGATTCTAGTGCATTCAAGCACAATGGCACGGTTGAA
TGTAAGGCTTACAACGATGTGGGCAAGACTTCTGCCTATTTTAACTTTGCATT
TAAAGGTAACAACAAAGAGCAAATCCATCCCCACACCCTGTTCACTCCTTTG
CTGATTGGTTTCGTAATCGTAGCTGGCATGATGTGCATTATTGTGATGATTCT
GACCTACAAATATTTACAGAAACCCATGTATGAAGTACAGTGGAAGGTTGTT
GAGGAGATAAATGGAAACAATTATGTTTACATAGACCCAACACAACTTCCTT
ATGATCACAAATGGGAGTTTCCCAGAAACAGGCTGAGTTTTGGGAAAACCCT
GGGTGCTGGAGCTTTCGGGAAGGTTGTTGAGGCAACTGCTTATGGCTTAATT
AAGTCAGATGCGGCCATGACTGTCGCTGTAAAGATGCTCAAGCCGAGTGCCC
ATTTGACAGAACGGGAAGCCCTCATGTCTGAACTCAAAGTCCTGAGTTACCT
TGGTAATCACATGAATATTGTGAATCTACTTGGAGCCTGCACCATTGGAGGG
CCCACCCTGGTCATTACAGAATATTGTTGCTATGGTGATCTTTTGAATTTTTT
GAGAAGAAAACGTGATTCATTTATTTGTTCAAAGCAGGAAGATCATGCAGAA
GCTGCACTTTATAAGAATCTTCTGCATTCAAAGGAGTCTTCCTGCAGCGATAG
TACTAATGAGTACATGGACATGAAACCTGGAGTTTCTTATGTTGTCCCAACC
AAGGCCGACAAAAGGAGATCTGTGAGAATAGGCTCATACATAGAAAGAGAT
GTGACTCCCGCCATCATGGAGGATGACGAGTTGGCCCTAGACTTAGAAGACT
TGCTGAGCTTTTCTTACCAGGTGGCAAAGGGCATGGCTTTCCTCGCCTCCAAG
AATTGTATTCACAGAGACTTGGCAGCCAGAAATATCCTCCTTACTCATGGTC
GGATCACAAAGATTTGTGATTTTGGTCTAGCCAGAGACATCAAGAATGATTC
TAATTATGTGGTTAAAGGAAACGCTCGACTACCTGTGAAGTGGATGGCACCT
GAAAGCATTTTCAACTGTGTATACACGTTTGAAAGTGACGTCTGGTCCTATG
GGATTTTTCTTTGGGAGCTGTTCTCTTTAGGAAGCAGCCCCTATCCTGGAATG
CCGGTCGATTCTAAGTTCTACAAGATGATCAAGGAAGGCTTCCGGATGCTCA
GCCCTGAACACGCACCTGCTGAAATGTATGACATAATGAAGACTTGCTGGGA TGCAGATCCCCTAAAAAGACCAACATTCAAGCAAATTGTTCAGCTAATTGAG AAGCAGATTTCAGAGAGCACCAATCATATTTACTCCAACTTAGCAAACTGCA GCCCCAACCGACAGAAGCCCGTGGTAGACCATTCTGTGCGGATCAATTCTGT CGGCAGCACCGCTTCCTCCTCCCAGCCTCTGCTTGTGCACGACGATGTCTGAG CAGAATCAGTGTTTGGGTCACCCCTCCAGGAATGATCTCTTCTTTTGGCTTCC ATGATGGTTATTTTCTTTTCTTTCAACTTGCATCCAACTCCAGGATAGTGGGC ACCCCACTGCAATCCTGTCTTTCTGAGCACACTTTAGTGGCCGATGATTTTTG TCATCAGCCACCATCCTATTGCAAAGGTTCCAACTGTATATATTCCCAATAGC AACGTAGCTTCTACCATGAACAGAAAACATTCTGATTTGGAAAAAGAGAGG GAGGTATGGACTGGGGGCCAGAGTCCTTTCCAAGGCTTCTCCAATTCTGCCC AAAAATATGGTTGATAGTTTACCTGAATAAATGGTAGTAATCACAGTTGGCC TTCAGAACCATCCATAGTAGTATGATGATACAAGATTAGAAGCTGAAAACCT AAGTCCTTTATGTGGAAAACAGAACATCATTAGAACAAAGGACAGAGTATG AACACCTGGGCTTAAGAAATCTAGTATTTCATGCTGGGAATGAGACATAGGC CATGAAAAAAATGATCCCCAAGTGTGAACAAAAGATGCTCTTCTGTGGACCA CTGCATGAGCTTTTATACTACCGACCTGGTTTTTAAATAGAGTTTGCTATTAG AGCATTGAATTGGAGAGAAGGCCTCCCTAGCCAGCACTTGTATATACGCATC TATAAATTGTCCGTGTTCATACATTTGAGGGGAAAACACCATAAGGTTTCGTT TCTGTATACAACCCTGGCATTATGTCCACTGTGTATAGAAGTAGATTAAGAG CCATATAAGTTTGAAGGAAACAGTTAATACCATTTTTTAAGGAAACAATATA ACCACAAAGCACAGTTTGAACAAAATCTCCTCTTTTAGCTGATGAACTTATTC TGTAGATTCTGTGGAACAAGCCTATCAGCTTCAGAATGGCATTGTACTCAAT GGATTTGATGCTGTTTGACAAAGTTACTGATTCACTGCATGGCTCCCACAGG AGTGGGAAAACACTGCCATCTTAGTTTGGATTCTTATGTAGCAGGAAATAAA GTATAGGTTTAGCCTCCTTCGCAGGCATGTCCTGGACACCGGGCCAGTATCT ATATATGTGTATGTACGTTTGTATGTGTGTAGACAAATATTTGGAGGGGTATT TTTGCCCTGAGTCCAAGAGGGTCCTTTAGTACCTGAAAAGTAACTTGGCTTTC ATTATTAGTACTGCTCTTGTTTCTTTTCACATAGCTGTCTAGAGTAGCTTACCA GAAGCTTCCATAGTGGTGCAGAGGAAGTGGAAGGCATCAGTCCCTATGTATT TGCAGTTCACCTGCACTTAAGGCACTCTGTTATTTAGACTCATCTTACTGTAC CTGTTCCTTAGACCTTCCATAATGCTACTGTCTCACTGAAACATTTAAATTTT ACCCTTTAGACTGTAGCCTGGATATTATTCTTGTAGTTTACCTCTTTAAAAAC AAAACAAAACAAAACAAAAAACTCCCCTTCCTCACTGCCCAATATAAAAGG CAAATGTGTACATGGCAGAGTTTGTGTGTTGTCTTGAAAGATTCAGGTATGTT GCCTTTATGGTTTCCCCCTTCTACATTTCTTAGACTACATTTAGAGAACTGTG GCCGTTATCTGGAAGTAACCATTTGCACTGGAGTTCTATGCTCTCGCACCTTT CCAAAGTTAACAGATTTTGGGGTTGTGTTGTCACCCAAGAGATTGTTGTTTGC CATACTTTGTCTGAAAAATTCCTTTGTGTTTCTATTGACTTCAATGATAGTAA GAAAAGTGGTTGTTAGTTATAGATGTCTAGGTACTTCAGGGGCACTTCATTG AGAGTTTTGTCTTGGATATTCTTGAAAGTTTATATTTTTATAATTTTTTCTTAC ATCAGATGTTTCTTTGCAGTGGCTTAATGTTTGAAATTATTTTGTGGCTTTTTT TGTAAATATTGAAATGTAGCAATAATGTCTTTTGAATATTCCCAAGCCCATGA GTCCTTGAAAATATTTTTTATATATACAGTAACTTTATGTGTAAATACATAAG CGGCGTAAGTTTAAAGGATGTTGGTGTTCCACGTGTTTTATTCCTGTATGTTG TCCAATTGTTGACAGTTCTGAAGAATTCTAATAAAATGTACATATATAAATC AAAAAAAAAAAAAAAA (SEQ ID NO: 18).
[0259] In particular embodiments, the guide RNA binds to a CD117 polynucleotide sequence and includes a region complementary to a target CD117 sequence. In certain embodiments, the guide RNA targets or binds a region of CD117 polynucleotide sequence that encodes one of the following amino acid residues: N505 or D816.
[0260] One example of base editing methods, systems, and components thereof that may be used according to the methods and compositions disclosed herein is described in PCT Application Publication No. WO2021041945. In some embodiments, the base editing method comprises use of a modified CRISPR protein, bound to a guide RNA, and a base editing enzyme, such as a deaminase, wherein the modified CRISPR protein does not cause a doublestranded break. In some embodiments, the modified CRISPR protein is a nucleobase editor polypeptide or nucleic acid programmable-DNA binding protein (napDNAbp), as disclosed in PCT Application Publication Nos. WO2021041945 or WO2021163587In some embodiments, the method of base editing a polynucleotide encoding a CD117 polypeptide comprises expressing in a cell a nucleobase editor polypeptide, wherein the nucleobase editor polypeptide comprises a napDNAbp and a deaminase, and contacting the cell with a guide RNA capable of targeting the polynucleotide encoding a CD117 polypeptide.
[0261] In other embodiments, base editing may refer to RNA base editing methods, e.g., as described in Porto E. et al. Base editing: advances and therapeutic opportunities, Nature Reviews Drug Discovery volume 19, pages 839-859 (2020).
[0262] In particular embodiments, any of the gene editing, including base editing methods disclosed herein or known in the art may be used to modify one or more amino acids within an epitope of wild type human CD117 bound by an anti-CDl 17 antibody, optionally wherein the epitope comprises one or more of the following amino acids present in the wild type human CD117: N505 or D816, including but not limited to any of these recited amino acid residues. In certain embodiments, the method introduces a A~>G or C~>T mutation into one or both alleles of the CD117 gene, which results in the gene encoding a different amino acid by the codon that was mutated.
[0263] In certain embodiments, the disclosure provides a modified cell, e.g., HSPC or HSC, that comprises one or more components of a gene editing, e.g., base editing, system disclosed herein. In particular embodiments, the one or more component comprises a nucleic acid that binds to a CD117 gene or encoded mRNA, e.g., at a site to be modified to result in the encoding and/or expression of a modified CD117 disclosed herein, such as, e.g., a guide RNA. In particular embodiments, the guide RNA binds to a CD 117 polynucleotide sequence and includes a region complementary to a target CD117 sequence. In certain embodiments, the guide RNA targets or binds a region of CD117 polynucleotide sequence that encodes one of the following amino acid residues: N505 or D816. In particular embodiments, the one or more component comprises a base editing enzyme, e.g., any of those disclosed herein or in references cited herein.
[0264] In particular embodiments, a modified cell expressing a modified CD117 polypeptide is not substantially inhibited, eliminated, or killed by monoclonal antibodies (mAbs) that bind endogenous or wild-type cell -surface CD117 and inhibit proliferation of or kill a cell expressing only the wild-type CD117 and not a modified CD117 polypeptide disclosed herein. In certain embodiments, proliferation of the modified cell expressing the modified CD117 polypeptide is inhibited by less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%, as compared to proliferation of the same cell type that is not modified, e.g., only expresses wild-type CD117.
[0265] For engraftment purposes, a composition comprising modified HSCs and/or HSPCs is administered to a patient. Such methods are well known in the art. The modified 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.
[0266] The present disclosure also includes pharmaceutical compositions comprising two or more of CD47, modified CD117, and/or CXCR4 polypeptides, one or more polynucleotides or vectors, together comprising one or more sequence encoding two or more of a CD47, CD117, and/or CXCR4 polypeptide (e.g., a modified mRNA), or a modified cell, e.g., HSC and/or HSPC, comprising one or more polynucleotide or vector, together encoding two or more of a CD47, CD 117, and/or CXCR4 polypeptide or fragment or variant thereof and/or expressing two or more of a CD47, CD117, and/or CXCR4 polypeptide, or fragment or variant thereof, in combination with one or more pharmaceutically acceptable diluent, carrier, or excipient.
[0267] The present invention discloses a pharmaceutical composition comprising a modified cell comprising two or more exogenously introduced CD47, CD 117, and/or CXCR4 polypeptide (or one or more exogenous or introduced nucleic acid sequences encoding two or more of a CD47, CD117, and/or 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., an HSC and/or HSPC. In certain embodiments, the pharmaceutical composition further comprises one or more additional active agents.
[0268] The present disclosure also includes pharmaceutical compositions comprising a variant CD117 comprising amino acid modifications resulting in constitutive activity and not being bound by an antibody to CD117, e.g., JSP191, a polynucleotide or vector a variant CD 117 comprising amino acid modifications resulting in constitutive activity and not being bound by an antibody to CD117, e.g., JSP191, or a modified cell, e.g., HSC and/or HSPC, comprising a polynucleotide or vector a variant CD117 comprising amino acid modifications resulting in constitutive activity and not being bound by an antibody to CD117, e.g., JSP191, in combination with one or more pharmaceutically acceptable diluent, carrier, or excipient.
[0269] The present invention discloses a pharmaceutical composition comprising a modified cell comprising an exogenously introduced variant CD117 comprising amino acid modifications resulting in constitutive activity and not being bound by an antibody to CD117, e.g., JSP191 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., an HSC and/or HSPC. In certain embodiments, the pharmaceutical composition further comprises one or more additional active agents.
[0270] 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
[0271] 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, disclosed herein, e.g., comprising one or more exogenous or introduced CD47, CD117, and/or CXCR4 polypeptide described herein and/or exogenous or introduced nucleic acids encoding one or more CD47, CD117, and/or CXCR4 polypeptide. In particular embodiments, the modified or engineered cells comprise one or more exogenous or introduced nucleic acids, which together encode a combination of two or more of: a CD47 polypeptide, a modified CD1 17 polypeptide with constitutive activity, a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning, and/or a CXCR4 polypeptide, or functional fragments or variants thereof. In certain embodiments the modified or engineered cell comprises: (1) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity; (2) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning; (3) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (4) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning; (5) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (6) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CD117 antibody used for HCT conditioning and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (7) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity, and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning; (8) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity, and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (9) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning, and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (10) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti- CDl 17 antibody used for HCT conditioning, and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (11) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning, and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide;
(12) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning;
(13) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning; (14) an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning; or (15) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide, and an exogenous or introduced nucleic acid encoding a
Yll modified CD117 polypeptide with constitutive activity that is not bound by an anti-CD117 antibody used for HCT conditioning.
[0272] 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.
[0273] 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.
[0274] The HCT methods disclosed use modified HSCs and/or HSPCs comprising an exogenous or introduced combination of CD47, CD117, and/or CXCR4 polypeptide or nucleic acids encoding CD47, CD117, and/or CXCR4 polypeptides. The combination of one or more of CD47, CD117, and/or CXCR4 (or variants thereof) may improve engraftment.
[0275] In particular embodiments, the modified or engineered cell expressing CD47 improves HSCs/HSPCs ability to migrate towards SDF-1 in vitro and in vivo, and thereby home to the bone marrow.
[0276] In particular embodiments, the modified or engineered cell expressing CXCR4 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.
[0277] In particular embodiments, a modified or engineered cell expressing the modified CD117 polypeptide is not substantially inhibited, eliminated, depleted, or killed by monoclonal antibodies (mAbs) that bind endogenous or wiki-type cell-surface CD117 and inhibit proliferation of or kill a cell expressing only the wild-type CD117 and not a modified CD117 polypeptide disclosed herein. In certain embodiments, proliferation of the modified cell expressing the modified CD117 polypeptide is inhibited, eliminated, depleted, or killed by less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%, as compared to proliferation of the same cell type that is not modified, e.g., only expresses wild-type CD117. [0278] 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.
[0279] In some embodiments, transient expression of the variant CD117 comprising amino acid modifications resulting in constitutive activity and not being bound by an antibody to CD117, e.g., JSP191, or the combination of CD47, one or more variant CD 117, and/or CXCR4, further improves HSCs/HSPCs engraftment in the bone marrow, measured by higher donor myeloid chimerism in the bone marrow and in the peripheral blood. CD47, CD117, and/or CXCR4 transient expression in HSCs and/or HSPCs may improve 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.
[0280] 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 one or more polynucleotide sequences encoding either 1) a modified CD 117 polypeptide with constitutive activity that is not bound by an anti-CD 117 antibody used for HCT conditioning, or 2) two or more of: a CD47 polypeptide, a modified CD117 polypeptide with constitutive activity, a modified CD117 polypeptide not bound by an anti- CD117 antibody used for HCT conditioning, and/or a CXCR4 polypeptide into the HSC and/or HSPCs, optionally wherein the CD47, CD117, and/or CXCR4 polypeptides comprises a sequence disclosed herein, or a functional variant or fragment thereof; and iii) optionally, modifying the HSCs and/or HSPCs, e.g., by introducing a gene therapy vector, or by gene editing or base editing, e.g., to correct a gene mutation in the subject; and iv) providing to a recipient subject in need of HCT the modified HSCs and/or HSPCs resulting from step iii or step iv.
[0281] In particular embodiments, the introduced polynucleotide sequence(s) 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 CD47 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 CD47 polypeptide is an mRNA, and it is introduced into the cells by electroporation.
[0282] In particular embodiments of methods of treatment disclosed herein, the method results in one or more of the following clinical outcomes:
(i) Engraftment: >95% (three consecutive days with absolute neutrophil count >500/pL by 28 days post-transplantation);
(ii) Use of non-myeloablative conditioning;
(iii) Oral mucositis: <10% at 30 days;
(iv) Veno-occlusive disease (VOD): <2% at 100 days;
(v) Grade 3-4 acute graft-versus-host disease: <5% at 100 days; or
(vi) Chronic graft-versus-host disease: <5% at 2 years.
[0283] 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 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 one or more introduced polynucleotide sequences encoding either 1) a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CD117 antibody used for HCT conditioning, or 2) two or more of: a CD47 polypeptide, a modified CD117 polypeptide with constitutive activity, a modified CD117 polypeptide not bound by an anti-CD117 antibody used for HCT conditioning, and/or a CXCR4 polypeptide into the HSC and/or HSPCs, optionally wherein the CD47, CD117, and/or CXCR4 polypeptides comprises a sequence disclosed herein, or a functional variant or fragment thereof, wherein the modified HSCs and/or HSPCs express the encoded CD47, CD117, and/or CXCR4 polypeptides; and iii) optionally, were further modified by comprising a gene therapy vector, or by gene or base editing, e.g., to correct a gene mutation in the subject.
[0284] 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 CD47, CD117, and/or 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(s) encoding the CD47, CD117, and/or CXCR4 polypeptides is an mRNA, and it was introduced into the cells by electroporation.
[0285] 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 one or more polynucleotide sequence encoding the CD47, CD 117, and/or CXCR4 polypeptides 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 one or more polynucleotide sequences encoding the CD47, CD117, and/or CXCR4 polypeptide is one or more mRNA, which is introduced into the cells by electroporation.
[0286] 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.
[0287] A variety of conditioning regimens are known and available in the art. These include myeloablative, reduced intensity, and non-myeloablative conditioning regimens. Illustrative conditioning regimens are described in Figure 1, and any of these may be used according to the methods disclosed herein, although the conditioning regimen is not limited to those disclosed in Figure 1. In particular embodiments, the modified cells are administered to a subject in combination with a non-myeloablative conditioning regimen.
[0288] 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.
[0289] In particular embodiments, since the subject is being administered modified cells, e.g., HSCs, comprising a modified CD47, CD117, and/or CXCR4 polypeptide as 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 a modified CD47, CD117, and/or 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%.
[0290] 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. However, when the modified cells being administered to the subject according to the methods disclosed herein comprise a modified CD117 polypeptide that is not bound by the ablative anti-c-Kit antibody used for conditioning, the disclosed methods do not require a wash-out period, or require only a reduced wash-out period as compared to when unmodified cells are transplanted. In certain embodiments, the wash-out period is less than five days, less than four days, less than 3 days, less than two days, or less than one day. In certain embodiments, the method comprises administering the anti-c-Kit antibody and the pharmaceutical composition or modified cells, e.g., modified HSCs and/or HSPCs, during an overlapping period of time or at about the same time. In particular embodiments, the method comprises also, or alternatively, administering the anti-c-Kit antibody to the subject after administration of the pharmaceutical composition or modified cells, e.g., modified HSCs and/or HSPCs, optionally for a period of time of at least one day, at least two days, at least three days, at least four days, at least five days, or at least one week. This may continue to ablate endogenous HSCs and/or HSPCs following administration of the modified HSCs and/or HSPCs, thus allowing greater engraftment.
[0291] In certain embodiments, the conditioning regimen comprises providing to the subject an anti-CD117 antibody (also known as an anti-c-kit 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-CDl 17 monoclonal antibody, e.g., JSP191 and TBI and fludarabine, or a combination of an anti-CDl 17 monoclonal antibody, e.g., JSP191 and azacytidine.
Anti-CDl 17 Antibodies
[0292] In certain embodiments, the conditioning regimen comprises administration of an anti-CDl 17 antibody, wherein the anti-CDl 17 antibody depletes or reduces endogenous HSPCs. In particular embodiments, the anti-CDl 17 antibody is selected from the group consisting of: SRI, 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 SRI, JSP191, described in U.S. Pat. Nos. 8,436,150 and 7,915,391.
[0293] Compositions and methods disclosed herein may be applicable to any anti-CDl 17 antibody, particularly monoclonal anti-human CD117 antibodies, e.g., those that block or inhibit binding of SCF to CD117. An anti-CDl 17 antibody may refer to an antibody that binds to CD117, e.g., human CD117, or an antigen-binding fragment thereof.
[0294] 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, SRI, 2B8, ACK2, YB5-B8, 57A5, 104D2, etc. In certain embodiments, the anti-CDl 17 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. W02019084064, W02020/219748, and W02020/219770. The FSI-174 antibody is disclosed in PCT application Publication No. W02020/112687 and U.S. Patent Application Publication No. 20200165337. The disclosure includes but is not limited to any anti-CDl 17 antibodies and/or CDR sets disclosed in any of the patent application disclosed herein, which are all incorporated by reference in their entireties.
[0295] In certain embodiments, the anti-CDl 17 antibody binds to the extracellular region of CD117, i.e., amino acids 26-524. The sequence of this region is shown below: QPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWIT EKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDP EVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVLSE KFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQTKLQ EKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGF INIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENES NIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGML QCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFK HNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTP (SEQ ID NO: 19).
[0296] Illustrative anti-CD 117 antibodies include, but are not limited to, SR- 1 , JSP 191 , 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 SRI, 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, W02020/219748, WO 2020/219770, or WO 2019/084064.
[0297] In particular embodiments, the antibody is a humanized form of SRI, 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 IgGl 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 IgGl 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. [0298] 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:
MDWTWRVFCLLAVAPGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMH WVRQAPGQGLEWMGVIYSGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDT AVYYCARERDTRFGNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYQSTYRVVSVLTVLHQDWLNGI< EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK (SEQ ID NO: 20) and
Light Chain:
MVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMH WYQQKPGQPPKLLIYLASNLESGVPDRF SGSGSGTDFTLTIS SLQAED VAVYYCQQN NEDPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC (SEQ ID NO: 21)
[0299] In certain embodiments, the variable heavy domain of JSP191 comprises the following sequence:
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSG NGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQG TLVTVSS (SEQ ID NO: 22)
[0300] In certain embodiments, the variable light chain domain of JSP191 comprises the following sequence:
DIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMHWYQQKPGQPPKLLIYLASNL ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDP YTFGGGTKVEIK (SEQ ID NO: 23).
[0301] The CDRs present in JSP191 are as follows: VH CDR1 = YNMH (SEQ ID NO: 24); VH CDR2 = IYSGNGDTSYNQKFKG (SEQ ID NO: 25); VH CDR3 = ERDTRFGN (SEQ ID NO: 26); VL CDR1 = RASES VDIYGNSFMH (SEQ ID NO: 27); VL CDR2 = LASNLES (SEQ ID NO: 28); and VL CDR3 = QQNNEDPYT (SEQ ID NO: 29). [0302] 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: DIVMTQSPSXKILSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKXK2LIYSASYR YSGVPDRFXK3GSGSGTDFTLTISSLQXK4EDFAXK5YXK6CQQYNSYPRTFGGGTKVEIK (SEQ ID NO: 30), wherein XKI 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, XKS 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:
QVQLVQSGAEXHIKKPGASVKXH2SCKASGYTFTDYYINAVVXH3QAPGKGLEWIARI YPGSGNTYYNEKFKGRXH4TXH5TAXH6KSTSTAYMXH7LSSLRSEDXHSAVYFCARGV YYFDYWGQGTTVTVSS (SEQ ID NO: 31), wherein XHI 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, XHS 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 XHS is an amino acid with an aliphatic hydroxyl side chain. In specific aspects, described herein are antibodies (e.g., human or humanized antibodies), including antigen-binding fragments thereof, comprising: (i) VH CDRs of a VH domain comprising the amino acid sequence QVQLKQSGAELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEWIARIYPG SGNT YYNEKFKGKATLTAEKS SST AYMQLS SLTSED S AVYFC ARGVYYFD YWGQ GTTLTVSS (SEQ ID NO: 32) or QVQLKQSGAELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEWIARIYPG SGNT YYNEKFKGKATLTAEKS SST AYMQLS SLTSED S AVYFC ARGVYYFD YWGQ GTTLTVSA (SEQ ID NO: 33), and
[0303] (ii) VL CDRs of a VL domain comprising the amino acid sequence DIVMTQSQKFMSTSVGDRVSVTCKASQNVRTNVAWYQQKPGQSPKALIYSASYRYS GVPDRFTGSGSGTDFTLTI SNVQSEDLADYFCQQYNSYPRTFGGGTKLEIKR (SEQ ID NO: 34).
[0304] 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. W02019084064. Epitope analysis of AB85 binding to CD177 is described in PCT Application Publication No. W02020219770, which identified the following two epitopes within CD117:
EKAEATNTGKYTCTNKHGLSNSIYVFVRDPA (SEQ ID NO: 35) (amino acids 60-90), and RCPLTDPEVTNYSLKGCQGKP (SEQ ID NO: 36) (amino acids 100-130).
[0305] 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 W02019084064, respectively.
[0306] The heavy chain variable region (VH) amino acid sequence of AB85 is: EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMAIINPRDS DTRYRPSFOGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYEGYEGAFDI WGQGTLVTVSS (SEQ ID NO: 37).
[0307] The VH CDR amino acid sequences of AB85 are as follows: NYWIG (VH CDR1; SEQ ID NO: 38); IINPRDSDTRYRPSFQG (VH CDR2; SEQ ID NO: 39); and HGRGYEGYEGAFDI (VH CDR3; SEQ ID NO: 40).
[0308] The light chain variable region (VL) amino acid sequence of AB85 is:
DIQMTQSPSSLSASVGDRVTITCRSSOGIRSDLGWYQQKPGKAPKLLIYDASNLETGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCOQANGFPLTFGGGTKVEIK (SEQ ID NO: 41).
[0309] The VL CDR amino acid sequences of AB85 are as follows: RSSQGIRSDLG (VL CDR1; SEQ ID NO: 42); DASNLET (VL CDR2; SEQ ID NO: 43); and QQANGFPLT (VL CDR3; SEQ ID NO: 44).
[0310] 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-CDl 17 antibody comprises the three CDRs or variable heavy chain regions present in any of AHI, 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.
[0311] In certain embodiments, the CDRs present in FSI-174 and related antibodies are as follows: VH CDR1 = SYNMH (SEQ ID NO: 45); VH CDR2 = VIYSGNGDTSY(A/N)QKF(K/Q)G (SEQ ID NO: 46); VH CDR3 = ERDTRFGN (SEQ ID NO: 26); VL CDR1 = RAS(D/E)SVDIYG(N/Q)SFMH (SEQ ID NO: 47); VL CDR2 = LASNLES (SEQ ID NO: 28); and VL CDR3 = QQNNEDPYT (SEQ ID NO: 29). 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 Hl, H2 and H3 as defined by Kabat: Hl = SYNMH (SEQ ID NO: 45); H2 = VIYSGNGDTSYAQKFKG (SEQ ID NO: 48); H3 = ERDTRFGN (SEQ ID NO: 26); and the CDRs present in the light variable region are CDRs LI, L2 and L3 as defined by Kabat: LI = RASESVDIYGQSFMH (SEQ ID NO: 49); L2 = LASNLES (SEQ ID NO: 28); and L3 = QQNNEDPYT (SEQ ID NO: 29), respectively except that 1, 2, or 3 CDR residue substitutions is/are present selected from N to A at heavy chain position 60, K to Q at heavy chain position 64 and N to Q at light chain position 30, positions being numbered according to Kabat. In certain embodiments, the antibody comprises any of the heavy chain variable region sequences (AH2, AH3, AH4) and/or light chain variable chain region sequences provided below (AL2), or the CDRs therein shown underlined:
AH2: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYMNHWVRQAPGQGLEWMGVIYSG NGDTSYAQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQG TLVTVSS (SEQ ID NO: 50)
AH3: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYMNHWVRQAPGQGLEWMGVIYSG NGDTSYNQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQG TLVTVSS (SEQ ID NO: 51)
AH4 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYMNHWVRQAPGQGLEWMGVIYSG NGDTSYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQG TLVTVSS (SEQ ID NO: 52)
AL2:
DIVMTQSPLSLPVTPGEPASISCRASESVDIYGOSFMHWYQOKPGQPPKLLIYLASNLE SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCOQNNEDPYTFGGGTKVEIK (SEQ ID NO: 53). [0312] 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: 190); CDR-H2 with IIYPGDSDTRYSPSFQG (SEQ ID NO: 191); CDR-H3 with HGRGYNGYEGAFDI (SEQ ID NO: 192). CK6 has the following light chain CDR amino acid sequences: CDR-L1 with RASQGISSALA (SEQ ID NO: 193); CDR-L2 with DASSLES (SEQ ID NO: 194); and CDR-L3 with CQQFNSYPLT (SEQ ID NO: 195).
[0313] 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:
[0314] Example variable heavy chain:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISENGS DTYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGGAVSYFDV WGQGTLVTVSS (SEQ ID NO: 196) [0315] Example variable light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT (SEQ ID NO: 197)
[0316] Ab249 was derived from antibody CK6, as an antagonist anti-CD117 antibody, as disclosed in PCT Application No. W02020092655A1. Ab249 has improved binding characteristics over the parent CK6. Ab249 has the following heavy chain CDRS: TSWIG (VH CDR1; SEQ ID NO: 198) IIYPGDSDTRYSPSFQG (VH CDR2; SEQ ID NO: 199); and HGLGYNGYEGAFDI (VH CDR3; SEQ ID NO: 200). Ab249 has the following light chain CDRS: RASQGIGSALA (VL CDR1; SEQ ID NO: 201); DASNLET (VL CDR2; SEQ ID NO: 202); and QQLNGYPLT (VL CDR3; SEQ ID NO: 203).
[0317] Ab249 has the following variable heavy chain sequence (CDRS are underlined): EVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGWVRQMPGKGLEWMGIIYPGDS DTRYSPSFOGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYEGAFDI WGQGTLVTVSS (SEQ ID NO: 204).
[0318] Ab249 has the following variable light chain sequence (CDRS are underlined): DIQMTQSPSSLSASVGDRVTITCRASOGIGSALAWYQOKPGKAPKLLIYDASNLETGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCOQLNGYPLTFGQGTRLEIK (SEQ ID NO: 205)
[0319] 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-CDl 17 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-CDl 17 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-CDl 17 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-CDl 17 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.
[0320] In certain embodiments, the anti-CDl 17 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-CDl 17 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-CDl 17 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-CDl 17 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-CDl 17 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.
[0321] 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 SRI, 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.
[0322] JSP191 is an aglycosylated IgGl 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-CDl 17 antibody alone. In particular embodiments, the subject is administered the anti-CDl 17 antibody prior to administration of the modified HSCs and/or HSPCs, e.g., as a single dose.
[0323] 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.
[0324] 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 aboutlO days from the day the subject’s anti-c- Kit antibody blood concentration of about 2000 ng/ml or less.
[0325] 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)
[0326] 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.
[0327] 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.
[0328] 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
[0329] 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.
[0330] 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.
[0331] 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 [0332] 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.
[0333] 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 aboutlO days from the day the subject’s anti-c-Kit antibody blood concentration of about 2000 ng/ml or less. [0334] 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.
[0335] 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.
[0336] 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.
[0337] 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.
[0338] 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.
[0339] 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.
[0340] 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.
[0341] 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.
[0342] 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.
[0343] 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.
[0344] 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.
[0345] 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. [0346] 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.
[0347] The dose of stem cells, e.g., modified HSCs and/or HSPCs comprising an exogenous CD47, CD117, and/or CXCR4 polypeptide and/or nucleic acid encoding a CD47, CD117, and/or 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 1-2 xlO6 CD34+ cells/kg body weight for autologous and allogeneic transplants. Higher doses can include, for example, at least or about 3xl06, at least or about 4xl06, at least or about 5xl06, at least or about 6xl06, at least or about 7xl06, at least or about 8xl06, at least or about 9xl06, 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.
[0348] 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.
[0349] 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 exogenous or introduced CD47, CD117, and/or CXCR4 polypeptides and/or one or more of an exogenous or introduced nucleic acid sequence encoding a CD47, CD 117, and/or CXCR4 polypeptide. In particular embodiments, the nucleic acid sequence is operably linked to a promoter sequence.
[0350] In some embodiments, the HSC or HSPC administered to the subject comprises one or more exogenous or introduced nucleic acid sequences encoding a CD47 and a CD117 polypeptide. In some embodiments, the HSC or HSPC administered to the subject comprises one or more exogenous or introduced nucleic acid sequences encoding a CD47 and a CXCR4 polypeptide. In some embodiments, the HSC or HSPC administered to the subject comprises one or more exogenous or introduced nucleic acid sequences encoding a CD117 and a CXCR4 polypeptide. In some embodiments, the HSC or HSPC administered to the subject comprises one or more exogenous or introduced nucleic acid sequences encoding a CD47, a CD117, and a CXCR4 polypeptide.
[0351] In some embodiments, the exogenous or introduced nucleic acid sequence(s) encoding CD47, CD117, and CXCR4 are in the same HSC and/or HSPC cell to be administered to the subject. In some embodiments, the exogenous or introduced nucleic acid sequence encoding CD47, CD117, and CXCR4 are in separate HSC and/or HSPC cells to be administered to the subject.
[0352] 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.
[0353] The cells and methods disclosed herein may be used to treat a variety of indications amenable to treatment with stem cell transplantation, including hematological diseases. In particular embodiments, the cells and methods disclosed herein may be used in the context of any hematopoietic cell transplant to treat any disease or disorder requiring such a transplant. Examples include, gene therapy, cord blood transplant, treatment of leukemias and cancers, and treatment of non-cancer diseases. In particular embodiments, the modified cells and methods may be used to treat a leukemia or a severe combined immunodeficiency (SCID). They may also be used to treat various bone marrow failure states and diseases, as well as hemoglobinopathies. In particular embodiments, the modified cells and 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. 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.
[0354] 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.
[0355] 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).
[0356] 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).
[0357] 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.
[0358] 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.
[0359] 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).
[0360] 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.
[0361] 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. In certain embodiments MDS is characterized according to Table 1.
Table 1 : WHO Classification of MDS • MDS with single-lineage dysplasia (MDS-SLD)
• MDS with multilineage dysplasia (MDS-MLD)
• MDS with ring sideroblasts (MDS-RS): >15% ring sideroblasts in BM or >5% with SF3B1 mutation
• MDS with ring sideroblasts and single-lineage dysplasia (MDS-RS-SLD)
• MDS with ring sideroblasts and multilineage dysplasia (MDS-RS-MLD)
• MDS with excess blasts-1 (MDS-EB 1): blasts in blood 2%-4%, blast in BM 5%-9%
• MDS with excess blasts-2 (MDS-EB2): blasts in blood 5%-l 9%, blasts in BM 10%- 19%
• MDS with isolated 5q-
• MDS, unclassifiable (MDS-U) _
BM: bone marrow; MDS: myelodysplastic syndromes; WHO: World Health Organization.
[0362] In particular embodiments, the methods disclosed are used to treat an immunodeficiency. In particular embodiments, the immunodeficiency is severe combined immunodeficiency (SCID).
[0363] In particular embodiments, the methods disclosed are used to treat a genetic disorder. In particular embodiments, the genetic disorder is sickle cell disease or Fanconi anemia. Sickle cell diseases that may be treat include, but are not limited to: HbS disease; drepanocytic anemia; meniscocytosis, and chronic hemolytic anemia.
[0364] In certain embodiments of any of the HCT methods disclosed, the method further comprises administering to the subject a therapeutic agent for treatment of the disease or disorder being treated by the HCT method.
EXAMPLES
Example 1
PROLIFERATION OF HEMATOPOIETIC CELLS EXPRESSING A CD117 VARIANT IS NOT INHIBITED BY AN ANTI-CD 117 ANTIBODY
[0365] To demonstrate that expression of a mutated CD117 confers a proliferative advantage for hematopoietic cells expressing mutant CD117 vs. wild-type CD117, Ba/F3 cells expressing wild-type human CD117 (c-Kit) and mutant human CD117-D816V (sequences in Table 5) were cultured in the absence of IL-3, in varying concentrations of stem cell factor (SCF), and in the presence or absence of anti-CDl 17 antibody JSP191.
[0366] Control parental Ba/F3 cells did not proliferate in the absence of IL-3. Further, parental Ba/F3 cells did not express CD117 and are not responsive to SCF signaling. Therefore, control parental Ba/F3 cells did not proliferate in the presence of increasing concentrations of SCF and there was no effect on viability or proliferation with the addition of JSP191 (Figure 2). [0367] Ba/F3 cell line expressing wild-type human CD117 (c-Kit, sequence of ctl80, Table 5) showed dose-responsive proliferation to SCF, which was inhibited in the presence of anti- CD117 antibody J SP191 (Figure 2).
[0368] Ba/F3 cell line expressing the CD117-D816V mutant was able to proliferate in the absence of SCF and proliferation was not inhibited by the presence of the anti-CDl 17 antibody JSP191 (Figure 2).
Example 2
IDENTIFICATION OF CD117 EPITOPES
[0369] Epitopes on CD 117 bound by various anti-c-Kit antibodies were identified by alanine scanning mutagenesis of the wild type human CD117 protein.
[0370] HEK-293T cells were transfected with a wild type (WT) construct of the CD117 protein or with vector alone in 384-well format, followed by confirmation of cellular expression via high-throughput flow cytometry. The MAbs tested included JSP191 and AB85; the ligand tested included AF488-conjugated stem cell factor (SCF), and the control MABs tested included YB5.88 (Invitrogen, Cat. No. 14-1179-82) and 104D2 (BioLegend, Cat. No. 313202), all of which bind WT CD117.
[0371] Shotgun Mutagenesis epitope mapping services were provided by Integral Molecular (Philadelphia, PA) as described in Davidson and Doranz, 2014. Briefly, a mutation library of the target protein was created by high-throughput, site-directed mutagenesis. Each residue was individually mutated to alanine, with alanine codons mutated to serine. The mutant library was arrayed in 384-well microplates and transiently transfected into HEK293T cells. Following transfection, cells were incubated with the indicated antibodies at concentrations predetermined using an independent immunofluorescence titration curve on wild type protein. MAbs were detected using an Alexa Fluor 488-conjugated secondary antibody and mean cellular fluorescence was determined using Intellicyt iQue flow cytometry platform. Mutated residues were identified as being critical to the MAb epitope if they did not support the reactivity of the test MAb but did support the reactivity of the reference MAb. This counterscreen strategy facilitates the exclusion of mutants that are locally misfolded or that have an expression defect.
[0372] Serial dilution of each monoclonal antibody (Mab) or ligand were tested for immunoreactivity against cells expressing target protein (WT) or vector alone to determine optimal screening conditions for each Mab or ligand based on raw signal values and signal-to- background calculations. AlexaFluor 488®-labeled goat anti-human IgG antibody was used as secondary detection antibody for JSP191 and AB85, and Al exaFluor 488®-labeled goat antimouse IgG antibody was used as secondary detection antibody for YB5.B8 and 104D2.
[0373] Library screens of very-high-affinity MAbs sometimes fail to yield critical residues for antibody binding. Conversion of a high-affinity MAb to a Fab usually weakens binding sufficiently to allow identification of critical residues for binding. Serial dilution of Fabs of MAbs were also tested for immunoreactivity against cells expressing target protein (WT) or vector alone to determine optimal screening conditions for each Fab based on raw signal values and signal-to-background calculations. The Fabs tested included two JSP191 Fab (JSP191Fab and JSP191Fab2) and an AB85 Fab. AlexaFluor 488®-labeled goat anti-human IgG F(ab’)2 antibody was used as secondary detection antibody for JSP191 and AB85 Fabs.
[0374] Binding of each test Ab to each mutant clone in the alanine scanning library was determined, in duplicate, by high-throughput flow cytometry. For each point, background fluorescence was subtracted from the raw data, which were then normalized to Ab reactivity with WT target protein. For each mutant clone, the mean binding value was plotted as a function of expression (represented by control reactivity) (Figures 3A-C). To identify preliminary primary critical clones (lower right quadrant of graphs), a threshold (dashed lines) of >55% WT binding to control Ab and <10% WT binding to test Abs was applied for JSP191, and >45% WT binding to control Ab and <10% WT binding to test Abs for AB85.
[0375] Mean binding reactivities (and ranges) are listed for all identified critical residues in Figure 2. Critical residues for Ab binding (shaded) were residues whose mutations were negative for binding to test Abs, but positive for binding to control antibody. Although mutant clone R248A showed a loss of binding for AB85, we do not consider it critical, as R248 appears to be an internal residue that is distant from the other epitope residues (see Figures 5A-B).
[0376] Critical residues whose mutation gave the lowest reactivities with specific antibodies are highlighted in bold and underlined in Figure 6. Validated critical residues represent amino acids whose side chains make the highest energetic contributions to the antibody-epitope interaction (Bogan and Thom, 1998; Lo Conte et al., 1999); therefore, the highlighted residues are likely the major energetic contributors to binding.
Example 3
TRANSIENT MRNA EXPRESSION OF CXCR4 IN HSPCS IMPROVES ENGRAFTMENT
[0377] 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)
[0378] IVT was performed to create mRNAs encoding CXCR4 containing various sequence (Table 8) 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 Nl-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 7). 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
[0379] 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 8), 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
[0380] CXCR4 mRNA (Table 8) 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 9B). CleanCapAG, Nl-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 9C). Viability of CD34+ HSPCS remained approximately 90% for -7 days after electroporation, in both CXCR4 mRNA-electroporated and control CD34+ HSPCs (Figure 5D). 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 9E).
[0381] 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 [0382] 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 ofNOD/SCID and N0D/SCID/B2m(null) mice. Blood, 2001. 97(10): p. 3283-91], Electroporation of CXCR4 mRNAs (Table 8) containing different chemistry modifications dramatically affected expression level (Figure 10A), and significantly increased the number of migrated cells vs. cells (Figure 10B).
Improving in vivo short-term engraftment of CXCR4 mRNA-electroporated human CD34+ HSPCs into the bone marrow of NSG mice [0383] 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 11 A). Hyperactive CXCR4 mutant sequences were examined, including the common WHIM mutant tl 9 (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 1 IB).
Long-term engraftment of CXCR4 mRNA-electroporated human CD34+ HSPCs into the bone marrow of NSG mice
[0384] 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 CD 19+ 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.
[0385] Additional experiments were performed, essentially as described above, using various mRNA constructs. Cells were transfected with 2.5 ug of mRNA per 0.1 ml electroporation reaction. As shown in Figure 48, the N119S CXCR4 variant was highly expressed in cells, whereas the N119A and N119K CXCR4 variants were expressed at a lower level 3 hours post electroporation of cells with the mRNA constructs. Figure 49 shows that for most constructs, mRNA expression was substantially reduced by about 16 hours postelectroporation.
[0386] CXCR4 mRNA constructs were optimized by identifying preferred stop codons and UTRs. As shown in Figure 50, the stop codon “TAATAA” was superior to others tested, and the UTR combination of 5’ HBA1 and 3’ HBB1 was superior to others tested.
[0387] The half-lives of various CXCR4 mutants following electroporation of mRNA constructs were determined over 48 hours following electroporation. As shown in Figure 51, the hyperactive mutants N119S andNl 19A (but not ctruncl9 like ec22-125) showed short halflives as compared to wild-type (and inactive mutantNl 19Kn not shown). Cell growth over 120 hours was somewhat consistent for all constructs tested. Figure 52 provides additional data on the half-lives associated with the various constructs tested.
[0388] Constructs were also tested in the transwell assay. As shown in Figure 53, N119S mRNA showed increased migration rate and number of migrated cells.
[0389] CXCR4 wild type and the N119S mutant were evaluated in vivo using the NSG mice model. As shown in Figure 54, CXCR4-119S mRNA improved engraftment of CD34 cells in NSG mice. Control/CXCR4-Nl 19S % change was 11.8. CXCR4 expression (fold change) as compared to control (1.0) was 0.96 for mock, 6.43 for CXCR4-WT, and 7.50 for CXCR4- 119S.
Example 4
AMELIORATION OF DISEASE IN A MOUSE MODEL OF SICKLE CELL DISEASE (SCD) [0390] 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.
[0391] 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-CDl 17 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.
[0392] Initially, electroporated allogeneic mouse CD 117+ HSPCs are evaluated for shortterm 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 CD 117+ 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 (Table 8) 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.
[0393] 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.
Example 5
ENGRAFTMENT OF CD47 MRNA-ENGINEERED HSCS
[0394] To demonstrate the effects of CD47 mRNA transfection and expression in human CD34+ cells, TriLink CD47-mr7 mRNA was electroporated into human CD34+ cells. Mr7 is a CD47 encoding mRNA produced by TriLink with TriLink UTRs and 5moU U- substitution (also referred to as ct71, Table 2). Figure 12 shows that electroporation of CD47-mr7 mRNA resulted in CD47 overexpression in human CD34+ cells, as compared to un-electroporated human CD34+ cells (Control) or human CD34+ cells electroporated with no mRNA (Mock). Under these conditions, CD47 expression on the human CD34+ cell surface had a U life of about 1 week.
[0395] To demonstrate the effects of CD47 mRNA expression in hematopoietic cell transplants, human CD34+ cells were electroporated with CD47-mr7 mRNA (5 ug mRNA electroporated into IM HSCs) and transplanted intravenously into immunocompromised NOD scid gamma (NSG) mice (0.5 M eHSCs transplanted per mouse). Figure 13 A shows that CD47- mr7 expressing human CD34+ cells transplanted in mice exhibited higher bone marrow homing than did control or mock mRNA expressing human CD34+ cells, 1 day after transplant. Notably, Figure 13B shows that CD47 expressing human CD34+ cells demonstrated a 14-fold improvement (p< 0.02) at 1 month after transplant in the percent of human HSC derived granulocytes in the mouse blood stream, relative to the control and mock mRNA transfected cells. mRNA optimization
[0396] Experiments were performed to optimize the function of a CD47 mRNA (Table 2) in vitro using human CD34+ cells.
[0397] In a first set of experiments, polyA tails of different lengths were tested to examine their effects on CD47 expression. mRNA constructs tested were based on ct47 with the indicated various polyA tails. mRNAs were produced using in vitro transcription with CleanCapAG-3OMe 5’ caps and Nl-methyl-pseudouridine uridine analogs (no uridine) and electroporated into human CD34+ cells. CD47 expression was assayed 1 day after electroporation. Figure 14 shows the effect of polyA tail length on CD47 expression: notably, increasing the polyA tail from 35 adenine bases to 140 adenine bases increased CD47 expression. However, tails longer than 80 adenine bases may be difficult to manufacture due to plasmid instability, in which recombination may occur in repetitive regions during E. coli growth of the plasmid. Therefore, a segmented polyA tail was developed in which 140 bases were segmented by a linker, of 4-10 varied bases, into two sequences of 70 adenine bases each, to reduce recombination (CD47-A140S). Figure 18 shows the AMOS segmented polyA tail design. mRNA encoding CD47 and comprising the AMOS poly A is shown in ct51 (Table 2). [0398] To determine the effects of mRNA dose on CD47 expression in human CD34+ cells, 5 ug of the CD47-mr7 mRNA and varied concentrations of the CD47-A70 mRNA (comprising a polyA tail of 70 adenine bases) were tested (1 ug to 40 ug). The CD47-A70 mRNAs were produced using in vitro transcription with CleanCapAG-30Me 5’ caps and N1 -methylpseudouridine uridine analogs, and CD47 expression was assayed 1 day after electroporation into human CD34+ cells. It was observed that increased dosage of the CD47-A70 mRNA increased CD47 expression in human CD34+ cells (Figure 15).
[0399] In another set of experiments, the 5’ and 3’ untranslated regions (UTRs) of the mRNA encoding CD47 were optimized in vitro in human CD34+ cells. CD47-A70 mRNAs encoding CD47 with 5’UTR from HBA1 and 3 ’UTRs from HBA1 and/or HBB1 were tested for CD47 expression. The mRNAs were produced using in vitro transcription with CleanCapAG-3OMe 5’ caps and Nl-methyl-pseudouridine uridine analogs, and CD47 expression was assayed 1 day after electroporation into human CD34+ cells. In these experiments, the mRNA encoding CD47 with a single 3’UTR from HBA1 was found to improve CD47 expression to the greatest extent (Figure 16 A), performing better than other mRNA with single UTRs or a tandem arrangement of HBA1 and HBB1 UTRs at the 3 ’end of the mRNA (diagrammed in Figure 16B).
[0400] In another set of experiments, CD47 expression in vitro in human CD34+ cells was compared between mRNA encoding wild type CD47 and CD47 mutant sequences (Table 2). The E97K mutant is a dead CD47, which does not bind SIRP-a. The K67E mutant is a CD47 variant that exhibits 150% binding of SIRP-a relative to the wild type CD47 protein. mRNAs were produced using in vitro transcription with CleanCapAG-3OMe 5’ caps and Nl-methyl- pseudouridine uridine analogs, and CD47 expression was assayed 1 day after electroporation. In these experiments, the mRNA encoding wild type CD47 exhibited the highest level of expression in human CD34+ cells. Figure 17 shows CD47 expression relative to the CD47 sequence used.
[0401] Figure 20 shows an example plasmid developed for in vitro mRNA transcription, with sequences presented in Figure 21 (SEQ ID NOs: 208-213) and Table 2. The plasmid, based on pUC57-Kan and about 3.5 kb in size, comprises a T7 CleanCapAG promoter, a 5’ UTR from alpha-globin, a consensus Kozak sequence, a CD47 wild type coding sequence, a 3’UTR from beta-globin, an AMOS segmented tail as described above, and an Xbal linearization site.
[0402] The experiments described herein demonstrate higher bone marrow engraftment of human CD34+ cells transfected with mRNA expressing human CD47 when transplanted into mice, and also provide illustrative mRNAs and plasmids that may be used for in vitro transcription of mRNAs encoding human CD47. Example 6
DEVELOPMENT OF MODIFIED MRNAS FOR ENHANCED HSC ENGRAFTMENT AND CD117
ANTIBODY RESISTANCE
[0403] Modified mRNAs encoding wild type and mutant CD47, CD 117 (cKIT), and CXCR4 were developed and tested in CD34+ HSC and Ba/F3 cells with the aim of improving HSC engraftment and chimerism after HCT transplant. Further to this, modified mRNAs encoding an N505I mutant CD117, an E73A/N505I double mutant CD117 and a wild type CXCR4 (Table 7) were tested in HSC and Ba/F3 cells for their ability to confer resistance to the CD117 antibody, JSP191.
[0404] At 3 hours after transfection, high levels of CD 117 (cKIT) were observed to downregulate CD47 expression. Similarly, at 3 hours after transfection, high levels of CXCR4 expression downregulated CD117 expression, demonstrating the cross-talk between the two pathways (Figures 22-24). Higher CD117 was also associated with a reduction in CXCR4 expression, however; CXCR4 was not observed to regulate CD47.
[0405] Figures 24-27 demonstrate human CD34+ live cell numbers and cell viability 3 hours, 20 hours, and 48 hours after transfection with the modified mRNAs provided herein. As shown in Figures 32 and 33, CD117 expression increased ~15-fold for wild type and E73 mutant 3 hours post transfection. There were also two cell populations with different CD117 expression levels in human CD34+ cells, 3 hours after the cells were electroporated with wild type and E373A mRNAs (Table 7) (Figure 30). However, the two levels of expression resolved by 20 hours post transfection (Figures 32A-C).
[0406] It was also observed that CD90 expression was higher in the lower CD117 expressing cells, and lower in the higher CD117 expressing cells (Figure 31).
CD 117 mutant and CXCR4 expression provide resistance to JSP191
[0407] The E73A and N505I CD117 mutants, the E73A/N505I CD117 double mutants, and wild type and N119S mutant CXCR4 were examined with the aim of developing enhanced HSC engraftment and CD117 antibody resistance.
[0408] Expression of the E73 A and N505I CD117 mutants in Ba/F3 cells exhibited enhanced cell growth in response to SCF, with a CD117 E73A/N505I double mutant exhibiting the most pronounced growth (Figure 29A). E73A, N505I, and E73A/N505I CD117 mutants in the presence of 50 ng/ml human SCF also demonstrated resistance to the CD117 antibody, JSP191, as shown in Figure 29B. [0409] Figure 35 shows that wild type CXCR4 and N119S mutant CXCR4 expression increased ~10 fold 3 hours after electroporation. As shown in Figures 36 and 37, expression levels decreased by 24 hours.
CXCR4 human cell chimerism in bone marrow post-HCT
[0410] The relative levels of human CD34+ and human CD45+ cell chimerism in mouse bone marrow, 12 weeks after transplant with human CD34+ HSC transfected with modified wild type and N199S CXCR4 mRNAs is shown in Figures 38A and 38B. Notably the percent chimerism increased by -12% and 25% respectively. Also shown is the level of CXCR4 present in the chimeric cells. The mice were conditioned prior to transplant with the following: 225 cGY TBI. The number of human CD34+ cells transplanted was 500K. The CXCR4 WT mRNA (NlmpSU) and CXCR4 119S mRNA (NlmpSU) were transfected via electroporation in a concentration of 5ug per lOOul per million cells.
Example 7
COMBINATIONS OF EHSC MODIFICATIONS
[0411] The effect of modifying CD47+ cells to express increased levels of CXCR4 and/or CD47 was tested in vitro and in vitro.
[0412] Various CD117 mRNAs encoding wild type or various mutant CD117s were electroporated into human CD34+ cells, and CD117 expression was analyzed. As shown in Figure 44A, the cells expressed CD117 highly within hours of electroporation and for about one day after electroporation. Wild type CD117 was expressed at higher levels than CD117 N505I, but both showed greater expression than control or mock electroporated cells. Cell viability is shown in Figure 44B.
[0413] Overexpression of a cKit containing both an E73A modification and an N505I modification increased hematopoietic cell proliferation in vitro (Tables 5 and 6). As shown in Figure 45, electroporation impairs humanh CD34+ cell proliferation in vitro observed at day 3 following electroporation. However, the E73A-N505I mutant cKit mRNA (Table 7) restored human CD34+ cell proliferation impairment due to electroporation at day 3, and conferred longer term proliferation advantages by day 12 in vitro.
[0414] Freshly isolated CD34+ cells were electroporated with mRNA constructs expressing CXCR4 or CD47, separately or in combination, and expression levels of CD47 and CXCR4 as compared to baseline were determined. The CD47 mRNA construct was mr37, which corresponds to ctl84 with an A90 polyA tail, NlmPsU replacement of uridine, and CleanCapAG-3OMe cap. The CXCR4 mRNA construct was ctl l4 (Table 8), which also has an A90 polyA tail, NlmPsU replacement of uridine, and CleanCapAG-3OMe cap. As shown in Figure 42, increased amounts of mRNA resulted in increased expression of CXCR4. At 3 hrs, CXCR4 shows a dose dependent expression pattern showed expression fold to control as: CXCR4 lug = ~4.3 times; CXCR4 2ug = ~8.5 times; CXCR4 4ug = ~16 times; and CXCR4 lug (combination) = ~5 times. When cells were electroporated with both an mRNA construct encoding CXCR4 and an mRNA contrast encoding CXCR4, both CXCR4 and CD47 showed increased levels of expression as compared to control. CD47 showed about three-fold increase in expression with 4 ug concentration both in individual and in combination. Viability was comparable across all samples at about 80-85% (data not shown). Live cell numbers were also very comparable, although the combination of CXCR4 and CD47 showed a slight decrease in cell number (data not shown). At day 1 following administration, both CXCR4 and CD47 expression levels were increased in the mice as compared to control, as shown in Figure 43. CD47 maintained the about 3X expression with 4ug concentration both in individual and in combination.
[0415] The modified CD34+ cells were used to inject was also tested in NBSGW mice, which is a no-irradiation model mice, at 100,000 cells/mice via R/O route of administration. Test groups included: Control (6 mice), CXCR4 lug (6 mice), CXCR4 2ug (6 mice), CXCR4 4ug (6 mice), CD47 4ug (5 mice), and combination of CD47 4 ug and CXCR4 lug (6 mice). hCD34 + cell are expressed with more than 95% across all the samples.
Example 8
EFFECTS OF CD117 MRNA EXPRESSION
[0416] CD117 (cKit) binds stem cell factor (SCF) to regulate HSC survival, self-renewal, and differentiation. Experiments were performed to assess wild type and modified CD117 mRNAs, expressed from DNA templates provided in below and SEQ ID NOS: 5-7.
Figure imgf000216_0001
[0417] In brief, human CD34+ and/or Ba/F3 cell lines were transfected with various mRNA constructs encoding wild type CD117 (Table 1, produced from templates comprising SEQ ID NOs: 5 and 6), CD117 E73A from (produced from a template comprising SEQ ID NO: 7), or another CD117 with an amino acid substitution not identified as critical for JSP191 binding, (KG2-DV and KG2-DV-5moU). The sequences of SEQ ID NOS: 5-7 are the DNA templates and include a T7 promoter, CleanCapAG 3’ OME Kozak sequence, an HBA1 5’ UTR, a TAATGA double stop codon, an HBB1 3’ UTR. SEQ ID NOs: 5-7 do not include the polyadenosine tail of 70 nucleotides that is present in the template and resulting mRNAs. The mRNAs correspond to mRNA expressed from these templates. Controls included no transfection or null transfection where indicated. All mRNAs were generated using Nlm- pseudouridine instead of uridine. cKIT Expression
[0418] The codon optimized mRNAs provided herein, e.g., cKIT WT col (produced from SEQ ID NO: 6) and cKIT_E73A_col (produced from SEQ ID NO: 7), demonstrated higher expression levels than the PhaRNA (KG2-DV) and TriLINK (KG2-DV-5moU) cKIT mRNAs (Table 1), as determined by CD117 (cKIT) levels measured 20 hours after electroporation (Figure 55) in human CD34+ cells. PhaRNA and TriLink c-Kit DV correspond to ctl82 (with chemical modifications as indicated); cKit WT col corresponds to ct97, and cKit_E73A_col corresponds to ct98.
[0419] When measured just 3 hours after electroporation, the mRNAs provided herein demonstrated significant improvement in in CD117 expression levels over the aforementioned mRNAs (Figure 54). At the same time, the mRNAs provided herein showed similar levels of CD34+ cell viability to the comparison mRNAs (KG2-DV and KG2-DV-5moU) 20 hours after electroporation (Figures. 55A and 55B). Figure 56 is a graph of cell count versus CD117 expression in human CD34+ cells expressing mock electroporated (mock EP), null (control), wild type CD117, and CD117 E73A mRNAs. Figures. 57A-57C demonstrate that CD34+ cells expressing wild type CD117 and E73A mRNAs exhibited two distinct levels of transient CD117 expression at 3 hours after electroporation, which resolved by 20 hours after electroporation. Figure 58 shows CD117 expression levels over 72 hours following electroporation.
Resistance to JSP191
[0420] To determine the effects of the E73A mutant CD117 expression on JSP191 resistance in cells, E73 A CD117 mutant lentivirus constructs were transfected into Ba/F3 cells. The Ba/F3 cells grew in response to human stem cell factor, and independently of IL3 stimulation (Figure 59A). Notably, the E73A mutant expressing cells were more growth responsive to stem cell factor than were the wild type (Figure 59A). Expression of E73A led to nearly complete resistance to JSP191 (Figure 59B), even at higher concentrations.
[0421] Figures. 60A-60B show the effects of CD117 expression on human CD34+cell growth in the presence of stem cell factor (SCF) and the JSP91 antibody. Figure 60A shows the growth of cells transfected without mRNA. Figure 60B shows the growth of cells transfected with non-codon optimized wild type CD117 mRNA (as encoded by SEQ ID NO: 54). Figure 61 shows cKit dose/expression correlations following electroporation with mRNAs encoding wild type CD117, CD117 E73A (Table 1), or controls. Both wild type CD117 and CD117 E73A showed increased expression as compared to control or mock transfected cells.
[0422] The various embodiments described above can be combined to provide further embodiments.
[0423] Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, application and publications to provide yet further embodiments.
[0424] These and other changes can be made to the embodiments in light of the abovedetailed 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
[0425] 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 one, two, or more exogenous or introduced nucleic acid sequence, each encoding: a CD47, a modified CD117, and/or a CXCR4 polypeptide, including functional fragments or variants thereof, optionally wherein the CD47, modified CD 117, and/or CXCR4 polypeptides are transiently expressed in the cell, and optionally wherein the nucleic acid sequence comprises the following elements, optionally from 5’ to 3’ :
(a) a 5’ UTR; a 5’ terminal cap sequence; a sequence encoding the CXCR4, CD47, and/or a modified CD117 protein; a stop codon; and a 3 ’ UTR; or
(b) a 5’ HBA1 UTR; a CleanCap Reagent AG 3’ OMe 5’ terminal cap sequence (m7(3'OMeG)(5')ppp(5')(2'OMeA)pG); a sequence encoding the CXCR4, CD47, and/or a modified CD117 protein; a TAATAA stop codon; and a 3’ HBB1 UTR.
2. The modified cell of claim 1, wherein the cell comprises:
(a) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity;
(b) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD 117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning;
(c) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide;
(d) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning;
(e) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide; (f) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CD117 antibody used for HCT conditioning and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide;
(g) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity, and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning;
(h) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity, and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide;
(i) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a modified CD 117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning, and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide;
(j) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning, and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide;
(k) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity, an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide not bound by an anti-CDl 17 antibody used for HCT conditioning, and an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide;
(l) an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning;
(m) an exogenous or introduced nucleic acid encoding a CD47 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning;
(n) an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning; or (o) an exogenous or introduced nucleic acid encoding a CD47 polypeptide, an exogenous or introduced nucleic acid encoding a CXCR4 polypeptide, and an exogenous or introduced nucleic acid encoding a modified CD117 polypeptide with constitutive activity that is not bound by an anti-CDl 17 antibody used for HCT conditioning.
3. The modified cell of claim 1, wherein the cell is a stem cell or a progenitor cell, optionally 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 two or more of a CD47 polypeptide, a CD117 polypeptide, and/or a CXCR4 polypeptide, optionally wherein the modified cell expresses a CD47, CD117, and/or CXCR4 polypeptide transiently.
9. The modified cell of claim 8, wherein the CD47, CD117, and/or CXCR4 polypeptides are expressed on the cell surface.
10. The modified cell of any one of claims 1-9, wherein the CD47 polypeptide comprises a sequence selected from: SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, or a variant or fragment thereof.
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 a sequence selected from: SEQ ID NO: 58, SEQ ID NO: 59 or SEQ ID NO: 60 or a variant or fragment thereof, 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; Nl- 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 14, 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-CDl 17 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 SIRPa; 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.
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