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WO2023230533A1 - Compositions de cellules souches modifiées et procédés d'utilisation - Google Patents

Compositions de cellules souches modifiées et procédés d'utilisation Download PDF

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Publication number
WO2023230533A1
WO2023230533A1 PCT/US2023/067433 US2023067433W WO2023230533A1 WO 2023230533 A1 WO2023230533 A1 WO 2023230533A1 US 2023067433 W US2023067433 W US 2023067433W WO 2023230533 A1 WO2023230533 A1 WO 2023230533A1
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Prior art keywords
cell
sequence
cells
seq
modified
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Inventor
Wendy PANG
Leopold Daniel D'ESPAUX
Hye-Sook Kwon
Rajiv TIWARI
Song Eun Lee
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Jasper Therapeutics Inc
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Jasper Therapeutics Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells

Definitions

  • the present disclosure relates to modified 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.
  • the present disclosure provides inter alia novel modified HSCs and HSPCs and related compositions and methods of use thereof in hematopoietic stem cell transplant.
  • the disclosure provides a modified or engineered cell comprising an exogenous or introduced nucleic acid encoding a CD47 polypeptide (including variants thereof), optionally wherein the cell is transduced with a vector disclosed herein or a modified mRNA , e.g., the modified cell comprises an exogenous or introduced polynucleotide sequence encoding the CD47 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. In some embodiments, the cell was obtained from a mammalian donor. In certain embodiments, the mammalian donor is a subject in need of a hematopoietic stem cell transplant (autologous donor), wherein in other embodiments, the mammalian donor is not the subject in need of the hematopoietic stem cell transplant (allogeneic donor). In certain embodiments, the cell expresses the CD47 polypeptide, optionally wherein the modified cell expresses the CD47 polypeptide transiently. In certain embodiments, the CD47 polypeptides is a human CD47 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 CD47 polypeptide comprises a K67E mutation.
  • the exogenous or introduced polynucleotide is an mRNA
  • the mRNA is modified, e.g., to comprise one or more of the following modifications: pseudouridine substitution of one or more uridine; 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.
  • 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
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the modified cells, e.g., HSCs and/or HSPCs, comprising the nucleic acid encoding the CD47 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 into CD34+ HSPCs selected from mobilized peripheral blood.
  • HSPCs human allogeneic transiently modified hematopoietic stem and progenitor cells
  • the disclosure includes a method of modifying a cell, e.g., an HSC or HSPC, comprising introducing a nucleic acid or vector encoding a CD47 polypeptide into the cell, optionally wherein the cell is transiently modified, and optionally wherein the method is for preparing modified cells for hematopoietic cell transplantation (HCT) into a mammalian subject.
  • the nucleic acid or vector is introduced into the cell by transfection, transduction, infection, electroporation, or nanopore technology.
  • the nucleic acid e.g., mRNA
  • LNPs lipid nanoparticles
  • the nucleic acid e.g., mRNA
  • the nucleic acid is an mRNA, such as, e.g., chemically modified mRNA, encoding a modified version of CD47.
  • the disclosure includes a method of treating a mammalian subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising modified cells, e.g., HSCs and/or HSPCs, comprising the nucleic acid encoding the CD47 polypeptide.
  • the method further comprises administering to the subject a conditioning regimen to facilitate or increase engraftment of the modified cells, or deplete endogenous, wild-type HSCs or HSPCs, wherein the conditioning regimen is administered prior to or concurrent with the administering of the pharmaceutical composition.
  • the conditioning regimen comprises or consists of an anti-CD117 antibody, optionally JSP191.
  • the conditioning regimen comprises chemotherapy (optionally a nucleoside analog and/or an alkylating agent), monoclonal antibody therapy, or radiation, optionally radiation to the entire body (total body irradiation or TBI).
  • CD47 e.g., transiently
  • HCT HCT
  • CD47 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 transient expression in HSCs and/or HSPCs reduces the risk of phagocytosis of transplanted cells.
  • CD47 transient expression in HSCs and/or HSPCs improves the ability of HSCs/HSPCs that are injected intravenously to home to the bone marrow.
  • CD47 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 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 l 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.
  • Figure 2 shows CD47 overexpression in human CD34+ cells electroporated with CD47- mr7 mRNA (ct71 with Nlm-pseudouri dine instead of uridine; Table 2), relative to control cells and mock mRNA.
  • Figures 3 A and 3B show the relative percentages of human CD45+ cells after transplant with CD34+ cells transfected with CD47-mr7 mRNA (ct71 with Nlm-pseudouri dine instead of uridine).
  • Figure 3 A shows the percent of human CD45+ cells in the bone marrow 1 day after transplant
  • Figure 3B shows the percent of human HSC derived granulocytes in the blood system 1 month after transplant.
  • Figure 4 shows CD47 expression relative to each indicated polyA.
  • the constructs correspond to ct47 with Nlm-pseudouri dine instead of uridine and the indicated poly As).
  • Figure 5 shows CD47 expression relative to the mRNA used and the dose of each mRNA used.
  • the construct corresponds to ct47 with Nlm-pseudouri dine instead of uridine and the indicated polyA.
  • Figures 6A and 6B show CD47 expression relative to the UTR included in the mRNA sequence ( Figure 6A) and the position of each 5’ and 3’ UTR used ( Figure 6B).
  • Figure 7 shows CD47 expression relative to the CD47 sequence used.
  • Figure 8 shows the design of the segmented polyA tail AMOS, in which the 140 adenine bases are segmented into 2x 70 adenine sequences.
  • Figure 9 shows the design of an example plasmid that may be used for in vitro transcription of a CD47 mRNA.
  • Figure 10 shows the name, description, and sequence of sequences in the example plasmid containing a CD47 coding sequence as shown in Figure 9.
  • Figure 11 is a graph showing the colony formation from CD34+ cells, including control cells that were not electroporated, mock cells that were electroporated with no CD47 mRNA, and mr33 (Table 2) cells that were electroporated with the mr33 CD47 mRNA (ct65 with Nlm- pseudouridine instead of uridine).
  • the bottom band is burst-forming erythroid (BFU-E)
  • the large middle bar is granulocyte/macrophage progenitor (GM)
  • GEMM megakaryocyte
  • Figure 12 is a graph showing bone marrow chimerism at three months posttransplantation of the indicated cells.
  • Figure 13 is a graph showing NBSGW mouse blood chimerism two months posttransplantation of the indicated cells.
  • Figure 14 is a graph showing NSG mouse blood chimerism two months post - transplantation of the indicated cells.
  • Figure 15 provides diagrams of experiments conducted using NOD Mice.
  • Figure 16 is a graph showing NOD blood chimerism at 6 weeks post-transplantation.
  • Figure 17 provides the sequence of the mr37 mRNA construct (Table 2), noting that in mRNA, the Ts are Us.
  • Figure 18 is a graph showing CD47 expression (fold of baseline) achieved with each of the indicated mRNA constructs.
  • Figure 19 is a graph showing the effect of CD47 mRNA on the percent chimerism of gene modified CD34+ cells with any of the following conditioning regimens 9 days prior to hematopoietic stem cell transplant: control cells with no JSP191, control cells with 0. 3mg/kg JSP191, mock mRNA with 0.3 mg/kg JSP191, 5 pg CD47 mRNA (mr37) with 0.3 mg/kg JSP191, and 10 pg CD47 mRNA (mr37) with 0.3 mg/kg JSP191.
  • 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 compositions and methods disclosed herein are used to introduce CD47 mRNA into allogeneic normal HSPCs to improve engraftment and replace a patient’s diseased stem cells with a healthy hematopoietic system.
  • the disclosure provides for compositions and methods for the ex vivo introduction of a polynucleotide encoding a CD47 polypeptide, e.g., a human CD47 polypeptide), by RNA-based and/or DNA-based methods, into HSCs and/or HSPCs, including but not limited to CD34+ cells or subsets of CD34+ cells, such that the HSCs and/or HSPCs are able to be successfully transplanted into recipients.
  • Transplantation of these modified or engineered (e.g., genetically engineered) HSPCs may be done after or in combination with a conditioning regimen, including treatment with antibodies (such as anti-CD117 antibodies).
  • These modified or engineered HSPCs may be transplanted alone or in combination with other cells.
  • 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.
  • 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. 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.
  • 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 variant comprises at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to a reference polynucleotide sequence, e.g., a native polynucleotide or polypeptide sequence.
  • a variant may be a polynucleotide having a sequence identity of 50% or more, 60% or more, or 70% or more with a full-length native polynucleotide sequence, e.g., an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the full-length native polynucleotide sequence.
  • a variant may be a polypeptide having a sequence identity of 70% or more with a full-length native polypeptide sequence, e.g., an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the full-length native polypeptide sequence.
  • Variants may also include variant fragments of a reference, e.g., native, sequence sharing a sequence identity of 70% or more with a fragment of the reference, e.g., native, sequence, e.g., an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the native sequence.
  • 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 wild-type CD47 polypeptides, as well as functional variants and functional fragments of a native CD47 polypeptide
  • CD47 polynucleotide encompasses native or wild-type polynucleotides and nucleic acids, and variants and fragments thereof, that encode a CD47 polypeptide (including functional fragments and variants thereof.
  • 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 compositions and method of the present disclosure may be applied to any cell type expressing CD47.
  • the CD47 polypeptide binds to SIRPa, which is a regulatory membrane glycoprotein expressed mainly in HSPC and neurons.
  • SIRPa 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.
  • 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.
  • Any 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. In particular embodiments, the CD47 polypeptide sequence comprises or consists of one of the following amino acid sequences:
  • VAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIA ILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQ PPRKAVEEPLNE SEQ ID NO: 2
  • VAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAI LVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQ PPRNN SEQ ID NO: 3
  • 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:
  • the polypeptide or polynucleotide sequence is humanized.
  • the polynucleotide or nucleic acid sequence comprises RNA, DNA, or a combination thereof, and in particular embodiments, the nucleic acid comprises singlestranded and/or double-stranded regions, or a mixture thereof.
  • the nucleic acid is a double-stranded DNA, and in certain embodiments, the nucleic acid is a single stranded RNA, e.g., a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the nucleic acid sequence encoding CD47 is codon-optimized to improve gene expression and/or increase the translational efficiency of the sequence in an HSC or HSPC. In some embodiments, the nucleic acid sequence encoding CD47 is optimized for degron and/or stabilon sequences to improve degradation or stabilization of the encoded CD47 protein.
  • the nucleic acid sequence encoding a CD47 protein is codon- optimized and 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 following: ATGTGGCCACTCGTTGCCGCCCTTCTCTTGGGTAGCGCATGTTGTGGTAGTGCCC AACTGCTGTTCAACAAGACCAAGAGTGTGGAGTTTACTTTCTGCAACGATACAGT GGTAATCCCCTGTTTCGTGACCAACATGGAAGCCCAGAATACCACAGAGGTGTAT GTCAAATGGAAGTTCAAGGGTAGGGACATCTATACTTTCGACGGCGCCCTCAATA AAAGCACGGTACCGACAGATTTCTCCTCTGCCAAGATAGAGGTGAGCCAGCTCCT GAAGGGCGACGCTTCCCTGAAAATGGACAAATCTGACGCAGTTTCCCATACTGG CAATTATACCTGCGAGGTGACCGAGCTCACTCGCGAGGGAGAGAGCCTCCT GAAGGGCG
  • the nucleic acid sequence encoding a CD47 protein is codon- optimized and 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 following:
  • the sequence encoding a CD47 protein comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the CD47 encoding sequences disclosed herein.
  • the mRNA construct encoding a CD47 protein comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the CD47 encoding sequences disclosed herein or comprises an mRNA construct sequence disclosed herein, optionally without the promoter sequence and optionally with a poly A tail.
  • the nucleic acid sequence encoding CD47 comprises 5’ and/or 3’ cellular or viral untranslated regions (UTRs) relative to the sequence encoding the CD47 polypeptide. In some embodiments, the UTR improves mRNA stability, localization and/or expression.
  • the UTR is tissue specific.
  • the 5’ UTR comprises a UTR sequence from alpha-globin.
  • the nucleic acid comprises a Kozak sequence.
  • the 3 ’UTR comprises a UTR from an alpha-globin and/or a beta-globin gene, i.e., a 5’ UTR from hemoglobin alpha 1 (HBA1) and/or a 3’ UTR from one or more of HBA1 or hemoglobin beta 1 (HBB1) gene.
  • the nucleic acid sequence encoding CD47 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 CD47 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:
  • the nucleic acid sequence encoding CD47 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).
  • the nucleic acid sequence encoding CD47 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 CD47 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 CD47 comprises a TCTAGA sequence to linearize a plasmid as a template for transcription. [0079] In some embodiments, the nucleic acid sequence encoding CD47 encodes a polyadenine 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.
  • the polyA tail may be from 35 to 140 bases in length. In some embodiments, the polyA tail may be from 70 to 150 bases in length. In some embodiments, the polyA tail is segmented with a linker to reduce recombination during plasmid production in prokaryotic cells. In some embodiments the polyA tail is 70 adenine bases in length. In some embodiments, the linker is a series of bases other than adenine. In particular embodiments, the polyA is a segmented polyA as disclosed herein. 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.
  • nucleic acid comprises a modified mRNA.
  • Modified mRNAs comprising one or more modified nucleoside have been described as having advantages over unmodified mRNAs, including increase stability, higher expression levels and reduced immunogenicity.
  • Non-limiting examples of modifications to mRNAs that may be present in the nucleic acids encoding the CD47 polypeptides are described, e.g., in PCT Patent Application Publication Nos.
  • a 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 N1 -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 (m I T), and/or all cytosines in the modified mRNA are substituted with a similar nucleoside such as 5-methylcytosine.
  • T pseudouridine
  • m I T Nl-methyl-pseudouridine
  • a modified mRNA comprises a 5’ terminal cap sequence followed by a sequence encoding the CD47 polypeptide, including one or more 5’ or 3’ UTRs, followed by a 3’ tailing sequence, such as a polyA or a polyA-G tail sequence.
  • the mRNA encoding CD47 (including modified forms or variants thereof) comprises a wild type 5’ terminal cap sequence, and in certain embodiments, the mRNA encoding CD47 (including modified forms or variants thereof) comprises a modified 5’ terminal cap, not limited to but including, e.g., m7G(5')ppp(5')(2'OMeA)pG (CleanCap® Reagent AG for co-transcriptional capping of mRNA; TriLink Biotechnologies, USA) or m7(3'OMeG)(5')ppp(5')(2'OMeA)pG (CleanCap Reagent AG 3' OMe) for co- transcriptional capping of mRNA; TriLink Biotechnologies, USA).
  • m7G(5')ppp(5')(2'OMeA)pG CleanCap® Reagent AG for co-transcriptional capping of mRNA; TriLink Biotechnologies, USA
  • the mRNA encoding CD47 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.
  • a CD47 mRNA construct comprises the following elements, optionally from 5’ to 3’: a 5’ HBA1 UTR; a CleanCap Reagent AG 3’ OMe 5’ terminal cap sequence; a sequence encoding a CD47 polypeptide; a TAATAA stop codon; and a 3’ HBB1 UTR.
  • the sequence encoding CD47 is codon-optimized.
  • the sequence encoding CD47 encodes CD47 isoform 2, e.g., human CD47 isoform 2.
  • 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.
  • RNA self-amplifying RNA
  • saRNA self-amplifying RNA
  • saRNA is similar to mRNA, and saRNA is a linear, single-stranded RNA molecule that is synthesized with a 5' cap, 3' polyA tail and 5' and 3' UTRs.
  • saRNA also encodes four extra proteins in addition to the transgene, e.g., CD47, being expressed. These four extra proteins encode a replicase, which enables amplification of the original strand of RNA upon delivery into the cell, thus yielding a higher amount of protein expression and thus a minimal dose of RNA required.
  • 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 -sn-glycero-3- phosphoethanolamine [DOPE]) that resembles the lipids in the cell membrane; (3) cholesterol to stabilize the lipid bilayer of the LNP; and (4) a polyethylene glycol (PEG)-lipid to lend the nanoparticle a hydrating layer, improve colloidal stability, and reduce protein absorption.
  • DOPE oleoyl
  • PEG polyethylene glycol
  • the nucleic acid encoding the CD47 polypeptide (which encompasses functional fragments or variants thereof) is present in a vector.
  • the plasmid comprises a T7 promoter.
  • the plasmid comprises a T7 CleanCapAGTM promoter.
  • the vector is used for in vitro transcription of an mRNA to be delivered to mammalian HSCs and/or HSPCs or other stem cells.
  • the T7 promoter comprises a nucleic acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following: TAATACGACTCACTATAAGG (SEQ ID NO: 18).
  • the vector is capable of delivering the nucleic acid into mammalian HSCs and/or HSPCs or other stem cells, e.g., into the nucleus of the HSCs, HSPCs or other stem cells.
  • the vector is an episomal vector, e.g., a plasmid.
  • the vector is an expression vector comprising a promoter sequence operatively linked to a nucleic acid sequence encoding the CD47 polypeptide.
  • the expression vector comprises a promoter sequence that facilitates expression of the encoded CD47 polypeptide in HSCs, HSPCs and/or other stem cells.
  • 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 vector encoding CD47 comprises a 5’UTR sequence of HBA1 and a 3’ UTR of HBB1.
  • the vector encoding CD47 comprises a sequence, e.g., a plasmid sequence, with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% to the following:
  • the disclosure provides modified cells, e.g., HSCs and/or HSPCs, comprising a nucleic acid encoding a CD47 polypeptide as described herein.
  • the nucleic acid encoding the CD47 polypeptide is transiently present in the modified cell, and/or is not present within the genome of the cell.
  • the modified cell expresses and/or comprises the CD47 polypeptide, and in particular embodiments, the CD47 polypeptide is present on the cell surface.
  • the modified cell is transduced with or infected with an expression vector, optionally a viral vector.
  • the modified cell is transduced with a modified mRNA.
  • the modified HSCs and/or HSPCs transiently or constitutively overexpress CD47, 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 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 the nucleic acid encoding the CD47 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 pg/kg/day. In autologous donors who are heavily pretreated, however, doses of up to about 40 pg/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.
  • 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. Alternatively, unmanipulated bone marrow or mobilized peripheral blood populations are used. [0099] In certain embodiments, the disclosure provides a method of modifying cells, including HSCs and/or HSPCs, comprising introducing the nucleic acid encoding a CD47 polypeptide into the cell. In particular embodiments, the introduced nucleic acid is present as an mRNA. In particular embodiments, the introduced nucleic acid is present within a viral vector.
  • the nucleic acid is associated with or present in a lipid nanoparticle, liposome, or the like. In certain embodiments, the nucleic acid remains present in the modified cell only transiently, or the nucleic acid only transiently expresses the CD47 polypeptide in the cell. In certain embodiments, the method is used to prepare modified cells, e.g., HSCs and/or HSPCs, for HCT treatment of a mammalian subject. In particular embodiments, the nucleic acid or vector may be introduced into the cell by a variety of methods known in the art, such as transfection, transduction, infection, electroporation, or nanopore technology.
  • mRNA e.g., modified mRNA is introduced into the cells using lipid nucleic acid particles (LNPs) or nanoparticles.
  • LNPs lipid nucleic acid particles
  • cells e.g., HSCs and/or HSPCs, may be modified by introducing a nucleic acid encoding a CD47 polypeptide into the HSCs and/or HSPCs according to a variety of methods available in the art.
  • the modified 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 HSCs and/HSPCs may be further genetically altered to correct a genetic defect present in the HSCs and/or HSPCs.
  • the cells may be contacted with a gene therapy vector that results in the insertion into the cellular genome of an expression cassette that expresses the correct form of a mutated gene or protein.
  • the gene therapy may replace the mutated gene or a mutated region thereof, e.g., via homologous recombination.
  • the HSCs and/or HSPCs are modified to correct a mutated gene using gene editing or base editing methods, such as, e.g., a CRISPR-Cas9 system that targets the mutated gene.
  • the HSCs and/or HSPCs are modified to correct a mutated gene using Zinc- finger nucleases (ZFNs), meganucleases, or transcription activator-like effector nucleases (TALENs) that target the mutated gene.
  • ZFNs Zinc- finger nucleases
  • TALENs transcription activator-like effector nucleases
  • 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.
  • 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.
  • the modified cell comprising a CD47 polypeptide and/or encoding nucleic acid is a host cell, such as, e.g., an HEK293 cell that may be used to produce modified CD47 polypeptides or mRNA encoding a CD47 polypeptide.
  • a host cell such as, e.g., an HEK293 cell that may be used to produce modified CD47 polypeptides or mRNA encoding a CD47 polypeptide.
  • any host cells may be employed, including but not limited to, for example, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast.
  • the disclosure provides a plasmid that may be used to produce an mRNA encoding a CD47 polypeptide, such as, e.g., a plasmid as shown in Figure 9.
  • the plasmid includes a T7 promoter sequence.
  • the plasmid comprises any of the sequences disclosed herein.
  • the present disclosure also includes pharmaceutical compositions comprising one or more CD47 polypeptides, one or more polynucleotides or vectors comprising a sequence encoding a CD47 polypeptide (e.g., a modified mRNA), or a modified cell, e.g., HSC and/or HSPC, comprising a polynucleotide or vector encoding a CD47 polypeptide or fragment or variant thereof and/or expressing a CD47 polypeptide, or fragment or variant thereof, in combination with one or more pharmaceutically acceptable diluent, carrier, or excipient.
  • a CD47 polypeptide e.g., a modified mRNA
  • a modified cell e.g., HSC and/or HSPC
  • the present invention discloses a pharmaceutical composition
  • a pharmaceutical composition comprising a modified cell comprising a CD47 polypeptide (or nucleic acid sequence encoding the CD47 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.
  • polynucleotides, polypeptides, and cells described herein can be combined with pharmaceutically-acceptable carriers, diluents and reagents useful in preparing a formulation that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for mammalian, e.g., human or primate, use.
  • the pharmaceutical composition is a solution or suspension comprising modified cells disclosed herein.
  • carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulations.
  • Solutions or suspensions used for the formulations can include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; detergents such as Tween 20 to prevent aggregation; and compounds for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the pharmaceutical compositions are sterile.
  • suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the carrier can be, e.g., a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the composition is sterile and may be fluid to the extent that easy syringability exists.
  • a pharmaceutical composition include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the internal compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • the disclosure provides methods of treating a mammalian subject in need thereof, comprising administering to the subject modified cells, e.g., HSCs and/or HSPCs, comprising an exogenous or introduced CD47 polypeptide described herein and/or an exogenous or introduced nucleic acid, e.g., an mRNA or plasmid, encoding the CD47 polypeptide.
  • the subject is in need of HCT.
  • the transplant may be autologous, allogeneic, or xenogeneic, including without limitation allogeneic haploidentical stem cells, mismatched allogeneic stem cells, genetically engineered autologous or allogeneic cells, etc.
  • the modified HSCs and/or HSPCs are infused into the subject, e.g., by intravenous infusion, e.g., through a central vein over a period of several minutes to several hours.
  • the HLA type of the donor and recipient may be tested for a match, or haploidentical cells may be used.
  • cells obtained from HLA-haploidentical donors or HLA-identical donors are used.
  • HLA- haploidentical donors can be manipulated by CD34 or CD34/CD90 selection.
  • 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 CD47 polypeptide or nucleic acid encoding the CD47 polypeptide.
  • CD47 expression in HSCs and/or HSPCs improves HSCs/HSPCs ability to migrate towards SDF-1 in vitro and in vivo.
  • CD47 expression in HSCs and/or HSPCs reduces the risk of phagocytosis of transplanted cells.
  • CD47 expression in HSCs and/or HSPCs improves HSCs/HSPCs that are injected intravenously to home to the bone marrow.
  • CD47 transient expression in HSCs and/or HSPCs improves HSCs/HSPCs engraftment in the bone marrow, measured by higher donor myeloid chimerism in the bone marrow and in the peripheral blood.
  • CD47 transient expression in HSCs and/or HSPCs improves neutrophil and platelet recovery following transplantation.
  • the methods of the invention are also believed to provide for improved engraftment of stem cells after transplantation into a recipient.
  • the disclosure provides a method for producing a population of cell comprising a plurality of modified HSCs and/or HSPCs, comprising: i) obtaining HSCs and/or HSPCs from a donor subject, optionally a mammal, e.g., a human; ii) introducing a polynucleotide sequence, e.g., an mRNA or plasmid, encoding a CD47 polypeptide into the HSC and/or HSPCs, optionally wherein the CD47 polypeptide comprises a sequence disclosed herein, or a functional variant or fragment thereof; and iii) optionally, modifying the HSCs and/or HSPCs, e.g., by introducing a gene therapy vector, or by gene editing or base editing, e.g., to correct a gene mutation in the subject; and iv) providing to a recipient subject in need of HCT the modified HSCs and/or HSPCs
  • the introduced polynucleotide sequence is an mRNA that is expressed in the HSCs and/or HSPCs following introduction into the cells.
  • the gene therapy vector or reagents used to perform the gene editing are introduced into cells obtained from a subject to undergo HCT using the modified HSCs and/or HSPCs, i.e., autologous HCT.
  • the polynucleotide sequence encoding the 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 an introduced polynucleotide sequence encoding a CD47 polypeptide into the HSC and/or HSPCs, optionally wherein the CD47 polypeptide comprises a sequence disclosed herein, or a functional variant or fragment thereof, and wherein the modified HSCs and/or HSPCs express the encoded CD47 polypeptide; 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.
  • a donor optionally a mammal, e.g., a human
  • ii) comprise an introduced polynu
  • 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 polypeptide and the gene therapy vector or reagents used for gene editing were introduced into the cells at the same time, or either was introduced before or after the other, optionally within 10 minutes, 20 minutes, 30 minutes, one hour, two hours, four hours, eight hours, 12 hours, 24 hours, or 48 hours of each other.
  • the polynucleotide sequence encoding the CD47 polypeptide 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 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 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 the modified CD47 polypeptide.
  • the conditioning regimen comprises use of an anti-CD117 antibody in combination with chemotherapy (optionally a nucleoside analog and/or an alkylating agent), other monoclonal antibody therapy, and/or radiation
  • the amount of chemotherapy, other monoclonal antibody therapy, and/or radiation is reduced as compared to the amount used when not in combination with an anti-CD117 antibody, such as JSP191.
  • either or both the amount and/or duration of other conditioning therapy may be reduced by at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, or by about 100%.
  • the agents used according to the conditioning regimen are administered to the subject prior to and/or concurrently with the administration of the modified HSPCs/HSCs or pharmaceutical composition to the subject.
  • the period of time required for clearance of the conditioning agent may be empirically determined, or may be based on prior experience of the pharmacokinetics of the agent.
  • the time for clearance was usually the time sufficient for the level of conditioning agent, e.g., anti-c-Kit antibody, to decrease at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold from peak levels, e.g., at least about 100-fold, 1000-fold, 10,000-fold, or more.
  • the wash-out period is between 2 days and three weeks or between 5 days and two weeks, e.g., about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days about 17 days, about 18 days, about 19 days, or about 20 days.
  • the conditioning regimen comprises providing to the subject an anti-CD117 antibody (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-CD117 antibodies and/or CDR sets disclosed in any of the patent application disclosed herein, which are all incorporated by reference in their entireties.
  • the anti-CD117 antibody binds to the extracellular region of CD117, i.e., amino acids 26-524.
  • the sequence of this region is shown below: QPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWIT EKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDP EVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVLSE KFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQTKLQ EKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGF INIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENES NIRYVSELHLT
  • Illustrative anti-CD117 antibodies include, but are not limited to, SR-1, JSP191, 8D7, K45, 104D2, CK6, YB5.B8, AF-2-1, AF11, AF12, AF112, AF-3, AF-1-1, NF, NF-2-1, NF11, NF12, NF112, NF-3, HF11, HF12, and HF112.
  • a number of antibodies contemplated by the disclosure that specifically bind human CD117 are commercially available, including without limitation 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: 24).
  • 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 YSGVPDRFX K3 GSGSGTDFTLTISSLQXK4EDFAX K5 YXK6CQQYNSYPRTFGGGTKVEIK (SEQ ID NO: 31), 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, XK 5 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:
  • 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
  • X H4 is an amino acid with an aliphatic side chain
  • X H5 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: 33) or QVQLKQSGAELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEWIARIYPG SGNT YYNEKFKGKATLTAEKS SST AYMQLS SLTSED S AVYFC ARGVYYFD YWGQ GTTLTVSA (SEQ ID NO: 34), and
  • VL CDRs of a VL domain comprising the amino acid sequence DIVMTQSQKFMSTSVGDRVSVTCKASQNVRTNVAWYQQKPGQSPKALIYSASYRYS GVPDRFTGSGSGTDFTLTI SNVQSEDLADYFCQQYNSYPRTFGGGTKLEIKR (SEQ ID NO: 35).
  • 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 CD117 is described in PCT Application Publication No. W02020219770, which identified the following two epitopes within CD117:
  • EKAEATNTGKYTCTNKHGLSNSIYVFVRDPA (SEQ ID NO: 36) (amino acids 60-90), and RCPLTDPEVTNYSLKGCQGKP (SEQ ID NO: 37) (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 PCT Application Publication No. WO20 19084064, respectively.
  • VH heavy chain variable region amino acid sequence of Ab85 is: EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMAIINPRDS DTRYRPSFOGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYEGYEGAFDI WGQGTLVTVSS (SEQ ID NO: 38).
  • VH CDR amino acid sequences of AB85 are as follows: NYWIG (VH CDR1; SEQ ID NO: 39); IINPRDSDTRYRPSFQG (VH CDR2; SEQ ID NO: 40); and HGRGYEGYEGAFDI (VH CDR3; SEQ ID NO: 41).
  • VL amino acid sequence of AB85 is: DIQMTQSPSSLSASVGDRVTITCRSSOGIRSDLGWYQQKPGKAPKLLIYDASNLETGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANGFPLTFGGGTKVEIK (SEQ ID NO: 42).
  • VL CDR amino acid sequences of AB85 are as follows: RSSQGIRSDLG (VL CDR1; SEQ ID NO: 43); DASNLET (VL CDR2; SEQ ID NO: 44); and QQANGFPLT (VL CDR3; SEQ ID NO: 45).
  • F SI- 174 is an anti-CDl 17 antibody being developed in combination with 5F9 as a nontoxic transplant conditioning regimen, as well as a treatment for targeted hematologic malignancies.
  • the sequences of FSI174 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: 55); CDR-H2 with IIYPGDSDTRYSPSFQG (SEQ ID NO: 56); CDR-H3 with HGRGYNGYEGAFDI (SEQ ID NO: 57).
  • CK6 has the following light chain CDR amino acid sequences: CDR-L1 with RASQGISSALA (SEQ ID NO: 58); CDR-L2 with DASSLES (SEQ ID NO: 59); and CDR-L3 with CQQFNSYPLT (SEQ ID NO: 60).
  • 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: [00143]
  • 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: 63) IIYPGDSDTRYSPSFQG (VH CDR2; SEQ ID NO: 56); and HGLGYNGYEGAFDI (VH CDR3; SEQ ID NO: 64).
  • TSWIG VH CDR1; SEQ ID NO: 63
  • IIYPGDSDTRYSPSFQG VH CDR2; SEQ ID NO: 56
  • HGLGYNGYEGAFDI VH CDR3; SEQ ID NO: 64.
  • Ab249 has the following light chain CDRS: RASQGIGSALA (VL CDR1; SEQ ID NO: 65); DASNLET (VL CDR2; SEQ ID NO: 44); and QQLNGYPLT (VL CDR3; SEQ ID NO: 66).
  • Ab249 has the following variable heavy chain sequence (CDRS are underlined): EVQLVQSGAEVKKPGESLKISCKGSGYRFTTSWIGWVRQMPGKGLEWMGIIYPGDS DTRYSPSFOGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGLGYNGYEGAFDI WGQGTLVTVSS (SEQ ID NO: 67).
  • Ab249 has the following variable light chain sequence (CDRS are underlined): DIQMTQSPSSLSASVGDRVTITCRASOGIGSALAWYQOKPGKAPKLLIYDASNLETGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNGYPLTFGQGTRLEIK (SEQ ID NO: 68).
  • 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 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 about10 days from the day the subject’s anti-c- Kit antibody blood concentration of about 2000 ng/ml or less.
  • the conditioning regimen comprises administration of an anti- CDl 17 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,
  • 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,
  • 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 polypeptide and/or nucleic acid encoding a CD47 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 1-2x106 CD34+ cells/kg body weight for autologous and allogeneic transplants.
  • Higher doses can include, for example, at least or about 3x106, at least or about 4x106, at least or about 5x106, at least or about 6x106, at least or about 7x106, at least or about 8x106, at least or about 9x106, at least or about 107 or more CD34+ cells/kg body weight for autologous and allogeneic transplants.
  • 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 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.
  • cell populations may be selected for expression of CD34 and CD90, which cell populations may be highly purified, e.g., at least about 85% CD34+ CD90+ cells, at least about 90% CD34+ CD90+ cells, at least about 95% CD34+ CD90+ cells and may be up to about 99% CD34+ CD90+ cells or more.
  • the method of treating a subject in need of HCT comprises: i) administering a conditioning regimen to the subject, wherein the conditioning regimen comprises an anti-CD117 monoclonal antibody, e.g., JSP191; and ii) administering modified HSCs and/or HSPCs to the subject, wherein the modified HSCs and/or HSPCs comprise an exogenous or introduced CD47 polypeptide and/or an exogenous or introduced nucleic acid sequence encoding the CD47 polypeptide.
  • the nucleic acid sequence is operably linked to a promoter sequence.
  • the anti-c-Kit antibody and/or chemotherapy may be delivered orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.
  • the anti-c-Kit antibody e.g., JSP191
  • the chemotherapy e.g., fludarabine
  • the TBI is administered in a single dose of radiation.
  • the modified cells and methods disclosed herein may be used to treat a variety of indications amenable to treatment with stem cell transplantation, including hematological diseases.
  • the cells and methods disclosed herein may be used in the context of any hematopoietic cell transplant to treat any disease or disorder requiring such a transplant. Examples include, gene therapy, cord blood transplant, treatment of leukemias and cancers, and treatment of non-cancer diseases.
  • the modified cells and methods may be used to treat a leukemia or a severe combined immunodeficiency (SCID). They may also be used to treat various bone marrow failure states and diseases, as well as hemoglobinopathies.
  • 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 myelom
  • 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 which includes both MDS and AML.
  • MDS Myelodysplastic syndromes
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndromes
  • AML acute myeloid leukemia
  • the disease is characterized by an overproduction of immature blood cells. The resulting lack of mature, healthy blood cells causes anemia and an increased risk for infection and bleeding.
  • 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.
  • Table 1 WHO Classification of MDS
  • MDS-SLD MDS with single-lineage dysplasia
  • 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.
  • TriLink CD47-mr7 mRNA was electroporated into human CD34+ cells.
  • Mr7 shown in Table 2 is a CD47 encoding mRNA produced by TriLink with TriLink UTRs and 5moU U- substitutions.
  • Figure 2 shows that electroporation of CD47-mr7 mRNA resulted in CD47 overexpression in human CD34+ cells, as compared to unelectroporated 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 electroporated cells produced similar types and numbers of colonies as control cell when tested using an in vitro colony formation assay (Figure 11).
  • Figure 11 To demonstrate the effects of CD47 mRNA expression in hematopoietic cell transplants, human CD34+ cells were electroporated with CD47-mr7 mRNA (5 pgg mRNA electroporated into IM HSCs) and transplanted intravenously into immunocompromised NOD scid gamma (NSG) mice pre-conditioned with 225 cGy of irradiation (0.5 M eHSCs transplanted per mouse).
  • NSG immunocompromised NOD scid gamma
  • Figure 3A 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 3B 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.
  • the CD47-mr7 expressing CD34+ cells also showed 2.0 fold higher engraftment versus control cells at three months following transplant, and mock cells also showed elevated CD47 and engraftment (Figure 12).
  • CD47 expression was 1.45-fold higher for mock cells as compared to control, and CD47 expression was 2.03-fold higher for mr7 transfected cells as compared to control cells at three months following transplant.
  • mRNA constructs were prepared with NlmPsU replacement of uridine.
  • CD47 mRNA expression in hematopoietic cell transplants was also tested in NBSGW mice, which is a no-irradiation model.
  • Fresh CD34+ cells were transfected with mock RNA or mr33 CD47 mRNA (shown in Table 2), and 100,000 cells were transplanted per mouse without any conditioning.
  • mr33 transfected cells resulted in about 60% higher engraftment versus control at two months following transplant.
  • CD47 expression was 1.1 fold greater for mock cells as compared to control cells, and 1.5 fold greater for mr33 cells as compared to control at two months following transplant.
  • CD47 mr37 mRNA construct shown in Table 2 and Figure 17.
  • Fresh CD34+ cells were transfected with mock RNA or mr37 RNA and cultured for 16 hours. Cells were then transplanted into NSG mice pre-conditioned with 225 cGy total body irradiation (200,000 cells per mouse). As shown in Figure 14, cells transfected with a higher amount of mr37 mRNA (4.0 pg/ 1 M cells versus 0.75 pg / 1 M cells) showed higher engraftment at two months post-transplantation, demonstrating a dose-dependent improvement in chimerism.
  • CD47 expression from mock cells was 1.3 fold higher than control cells, and CD47 expression from mr37 transfected cells was 1.9 fold higher for 0.75 pgand 3.6 fold higher for 4.0 pg mRNA constructs were prepared with NlmPsU replacement of uridine.
  • mice transplanted with CD47 modified cells showed 4.3 fold higher engraftment versus control at six weeks post-transplant. Mr33 increased CD47 expression about 2-fold at six weeks post-transplant.
  • 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, shown in Table 2 as ct51).
  • Figure 8 shows the AMOS segmented polyA tail design.
  • CD47-mr7 mRNA Assay 1 pgof the CD47-mr7 mRNA (shown in Table 2) and varied concentrations of the CD47-A70 mRNA (comprising a polyA tail of 70 adenine bases) were tested (1 pgto 40 pg).
  • the CD47- A70 mRNAs were produced using in vitro transcription with CleanCapAG-3OMe 5’ caps and Nl-methyl-pseudouri dine 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 5).
  • 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-pseudouri dine uridine analogs, and CD47 expression was assayed 1 day after electroporation into human CD34+ cells.
  • mRNAs were produced using in vitro transcription with CleanCapAG-3OMe 5’ caps and N1 -methylpseudouridine uridine analogs, and CD47 expression was assayed 1 day after electroporation.
  • the mRNA encoding wild type CD47 exhibited a similar level of expression to the K67E mutant in human CD34+ cells.
  • Figures 9 and 10 shows an example plasmid and corresponding sequences developed for in vitro mRNA transcription.
  • 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.
  • CD47 mRNA constructs were further optimized by comparing the mr33 mRNA construct (shown in Table 2) to constructs comprising different stop sequences, CD47 isoforms, and CD47 mutants. As shown in Figure 18, the K67E hyperactive CD47 mutant expressed at similar levels as mr33. Other mutants (dead mutants) did not show expression. The TAATAA stop sequence appeared superior to other stop sequences tested, including the TAATGA stop sequence present in mr33. New isoforms or codon-optimized mRNA sequences did not show superior expression as compared to mr33. However, CD47 isoforms 2 and 3 wild type mRNA sequences appeared to have higher expression than CD47 isoform 1.
  • Table 2 provides illustrative plasmid and mRNA constructs/sequences.
  • 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 the 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: 69) The T7 polymerase
  • Table 3 provides illustrative plasmid and mRNA constructs/sequences.
  • 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 the 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: 69).
  • 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.
  • Table 4 provides illustrative plasmid and mRNA constructs/sequences.
  • 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 the 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: 69).
  • 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.
  • mice with no conditioning were pre-engrafted with 100,000 human CD34+ cells. Subsequently, mice were conditioned with 0.3 mg/kg JSP191 -9 days to HCT, followed by transplant with CD24 gene expressing CD34+ cells from the same human donor, demonstrating an “autologous” donor model. In this model, the measure of chimerism of CD34+ cells was the percentage of cells expressing CD24 at 6 weeks post engraftment.
  • CD47 mRNA (mr37) was introduced at concentrations of 5 pg and 10 pg into the autologous donor CD34+ cells to determine if CD47 mRNA expression improved autologous donor engraftment.
  • Figure 19 shows that both JSP191 pre-conditioning and 5 pg and 10 pg concentrations of CD47 mRNA improved autologous donor engraftment in the mouse model.
  • both CD47 mRNA expression and JSP191 conditioning together are predicted to improve engraftment in autologous gene-modified cell transplants in humans as well.

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Abstract

L'invention concerne des cellules souches modifiées et des procédés d'utilisation pour une greffe de cellules souches.
PCT/US2023/067433 2022-05-25 2023-05-24 Compositions de cellules souches modifiées et procédés d'utilisation Ceased WO2023230533A1 (fr)

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US20190290742A1 (en) * 2012-11-26 2019-09-26 Modernatx, Inc. Terminally modified rna
US20210308183A1 (en) * 2018-07-17 2021-10-07 The Regents Of The University Of California Chimeric antigen receptor t cells derived from immunoengineered pluripotent stem cells
US20220002716A1 (en) * 2020-07-02 2022-01-06 Life Technologies Corporation Trinucleotide cap analogs, preparation and uses thereof

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