SHIZURU JUDITH (US)
SIKORSKI ROBERT (US)
TIWARI RAJIV (US)
KWON HYE-SOOK (US)
WO2020112870A1 | 2020-06-04 | |||
WO2019113437A1 | 2019-06-13 | |||
WO2020112687A2 | 2020-06-04 | |||
WO2021041945A2 | 2021-03-04 |
US20180125839A1 | 2018-05-10 | |||
US20200165337A1 | 2020-05-28 |
Claims 1. A pharmaceutical composition comprising a pharmaceutically acceptable excipient, carrier, or diluent and a modified hematopoietic stem cell (HSC) or a hematopoietic stem and progenitor cell (HSPC), wherein the modified HSC or HSPC comprises a modified CD117 polypeptide, optionally wherein the modified CD117 polypeptide has constitutive c-Kit signaling and/or kinase activity, and optionally wherein the modified cell is capable of proliferation and/or survival when contacted with an anti-c-Kit monoclonal antibody capable of inhibiting proliferation and/or survival of an HSPC expressing only a wild-type CD117. 2. The pharmaceutical composition of claim 1, wherein the c-Kit signaling and/or kinase activity of the modified CD117 is not substantially inhibited by an anti-c-Kit monoclonal antibody. 3. The pharmaceutical composition of claim 2, wherein the anti-c-Kit monoclonal antibody inhibits binding of SCF to CD117. 4. The pharmaceutical composition of claim 2, wherein the anti-c-Kit monoclonal antibody comprises one or more of the six CDRs present in any one of JSP191, AB85, CDX-0159, or FSI- 174. 5. The pharmaceutical composition of claim 2, wherein the anti-c-Kit monoclonal antibody is any one of JSP191, AB85, CDX-0159, or FSI-174. 6. The pharmaceutical composition of claim 5, wherein the anti-c-Kit antibody is JSP191. 7. The pharmaceutical composition of claim 5, wherein the anti-c-Kit antibody is FSI-174. 8. The pharmaceutical composition of any one of claims 1-7, wherein the modified CD117 comprises one or more amino acid modifications as compared to the wild-type CD117 polypeptide. 9. The pharmaceutical composition of claim 8, wherein the one or more amino acid modifications comprise one or more amino acid substitutions, insertions, or deletions. 10. The pharmaceutical composition of claim 8, wherein one or more of the amino acid modifications are present within surface exposed amino acid residues of the extracellular domain, within the membrane spanning domain, or within an intracellular domain of the modified CD117 polypeptide. 11. The pharmaceutical composition of claim 8, wherein the modified CD117 polypeptide comprises substitution or deletion of one or more of the following amino acids present in wild type human CD117: N505 or D816. 12. The pharmaceutical composition of claim 11, wherein the modified CD117 polypeptide comprises a D816V substitution and/or a N505I substitution as compared to wild type human CD117. 13. The pharmaceutical composition of any one of claims 1-12, wherein the modified CD117 polypeptide has at least 90%, at least 95%, at least 98%, or at least 99% sequence homology to wild type CD117 polypeptide. 14. The pharmaceutical composition of any one of claims 8-13, wherein the wild type CD117 polypeptide is a wild type human CD117 polypeptide, optionally having one of the following amino acid sequences: MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDP GFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLV DRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAY HRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVS SSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFG SANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTD KWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILT YDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQS SIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGM MCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPRNRLSFGK TLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNH MNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYKNLLHSK ESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDL LSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGN ARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGF RMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQ KPVVDHSVRINSVGSTASSSQPLLVHDDV (SEQ ID NO:1); or MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDP GFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLV DRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAY HRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVS SSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFG SANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTD KWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILT YDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQS SIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKEQIHPHTLFTPLLIGFVIVAGMMCIIV MILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPRNRLSFGKTLGAG AFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIVN LLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYKNLLHSKESSCS DSTNEYMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSY QVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPV KWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSP EHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVD HSVRINSVGSTASSSQPLLVHDDV (SEQ ID NO:2). 15. The pharmaceutical composition of any one of claims 1-14, wherein the modified cell expresses both the modified CD117 polypeptide and a wild type CD117 polypeptide. 16. The pharmaceutical composition of any one of claims 1-15, wherein the modified cell expresses the modified CD117 polypeptide transiently. 17. The pharmaceutical composition of any one of claims 1-16, wherein the HSC or HSPC is CD34+, optionally wherein the HSPC is CD34+/CD90+, CD34+/CD38-, or CD34+/CD38- /CD90+, or CD34+CD133+. 18. The pharmaceutical composition of any one of claims 1-17, wherein the cell is a human cell. 19. The pharmaceutical composition of any one of claims 1-18, wherein the cell was obtained from a mammalian donor. 20. The pharmaceutical composition of claim 19, wherein the mammalian donor is a subject in need of a hematopoietic cell transplant (HCT). 21. The pharmaceutical composition of claim 19, wherein the mammalian donor is a healthy donor. 22. The pharmaceutical composition of any one of claims 19-21, wherein the cell obtained from the mammalian donor was modified ex vivo. 23. The pharmaceutical composition of any one of claims 1-22, further comprising an anti-CD117 antibody. 24. The pharmaceutical composition of any one of claims 1-23, further comprising one or more anti-CD47, anti-CD40L, anti-CD122, anti-CD4, and/or anti-CD8 antibody. 25. A method of treating a mammalian subject in need thereof, comprising administering to the subject the pharmaceutical composition of any one of claims 1-24. 26. The method of claim 25, wherein the subject is administered a conditioning regimen to facilitate or increase engraftment of the modified cells, wherein the conditioning regimen is administered prior to or concurrent with the administering of the pharmaceutical composition. 27. The method of claim 26, wherein the conditioning regimen is also administered subsequent to the administering of the pharmaceutical composition. 28. The method of claim 26 or claim 27, wherein the conditioning regimen is milder than would be used if the subject was being administered hematopoietic stem cells that did not comprise the modified CD117 polypeptide. 29. The method of claim 25, wherein the subject is not administered a conditioning regimen to facilitate or increase engraftment of the modified cells, prior to or concurrent with the administering of the pharmaceutical composition. 30. The method of any one of claims 25-29, wherein the method results in reduced toxicity, reduced morbidity, or reduced graft-versus-host disease, as compared to a method wherein a subject is administered hematopoietic stem cells that do not comprise the modified CD117 polypeptide in combination with a conditioning regimen. 31. The method of any one of claims 25-30, wherein the conditioning regimen comprises or consists of administration of an anti-CD117 monoclonal antibody, optionally JSP191. 32. The method of any one of claims 25-31, wherein the subject is treated for a disease or disorder selected from the group consisting of: a cancer, a cardiac disorder, a neural disorder, an autoimmune disease, an immunodeficiency, a metabolic disorder, and a genetic disorder. 33. The method of claim 32, wherein the cancer is a solid tissue cancer or a blood cancer. 34. The method of claim 33, wherein the blood cancer is a leukemia, a lymphoma, or a myelodysplastic syndrome. 35. The method of claim 34, wherein the leukemia is acute myeloid leukemia (AML). 36. The method of claim 32, wherein the immunodeficiency is severe combined immunodeficiency (SCID). 37. The method of claim 32, wherein the genetic disorder is sickle cell disease or Fanconi anemia. 38. The method of any one of claims 25-37, further comprising administering to the subject a therapeutic agent for treatment of the disease or disorder. 39. A modified CD117 polypeptide comprising one or more amino acid modifications as compared to a wild type CD117 polypeptide, wherein an anti-c-Kit monoclonal antibody does not substantially inhibit or reduce c-Kit signaling, optionally in response to SCF binding, by the modified CD117 polypeptide expressed in cells as compared to the wild type CD117 polypeptide. 40. The modified CD117 polypeptide of claim 39, wherein the modified CD117 polypeptide substantially retains kinase activity, optionally in response to SCF binding, as compared to the wild type CD117 polypeptide. 41. The modified CD117 polypeptide of claim 39 or claim 40, wherein the modified CD117 polypeptide has constitutive c-Kit signaling and/or kinase activity. 42. The modified CD117 polypeptide of any one of claims 39-41, wherein the modified CD117 polypeptide substantially has increased c-Kit signaling and/or kinase activity, optionally in response to SCF binding, as compared to the wild type CD117 polypeptide. 43. The modified CD117 polypeptide of any one of claims 39-42, wherein the anti-c-Kit antibody comprises one or more of the six CDRs present in any one of JSP191, AB85, CDX-0159, or FSI- 174. 44. The modified CD117 polypeptide of claim 43, wherein the anti-c-Kit antibody is any one of JSP191, AB85, CDX-0159, or FSI-174. 45. The modified CD117 polypeptide of claim 43, wherein the anti-c-Kit antibody comprises one or more, optionally six, CDRs present in JSP191 and/or FSI-174. 46. The modified CD117 polypeptide of claim 45, wherein the anti-c-Kit antibody is JSP191 or FSI-174. 47. The modified CD117 polypeptide of any one of claims 39-46, wherein the one or more amino acid modifications comprise one or more amino acid substitutions, insertions, or deletions. 48. The modified CD117 polypeptide of claim 47, wherein one or more of the amino acid modifications are present within surface exposed amino acid residues of the extracellular domain, within the membrane spanning domain, or within an intracellular domain of the wild type CD117 polypeptide. 49. The modified CD117 polypeptide of claim 47 or claim 48, wherein the one or more amino acid modifications comprise one or more amino acid substitutions or deletions. 50. The modified CD117 polypeptide of claim 49, wherein the one or more amino acid modifications comprise one or more amino acid substitutions. 51. The modified CD117 polypeptide of any one of claims 47-50, wherein the one of more amino acid substitutions or deletions comprises substitution or deletion of one or more of the following amino acids present in wild type human CD117: N505 or D816. 52. The modified CD117 polypeptide of claim 50 or claim 51, wherein the one of more amino acid substitutions comprises a D816V substitution and/or a N505I substitution. 53. The modified CD117 polypeptide of any one of claims 39-52, wherein the modified CD117 polypeptide has at least 90%, at least 95%, at least 98%, or at least 99% sequence homology to the wild type CD117 polypeptide. 54. The modified CD117 polypeptide of any one of claims 39-53, wherein the wild type CD117 polypeptide is a wild type human CD117 polypeptide, optionally having one of the following amino acid sequences: MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDP GFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLV DRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAY HRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVS SSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFG SANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTD KWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILT YDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQS SIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGM MCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPRNRLSFGK TLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNH MNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYKNLLHSK ESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDL LSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGN ARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGF RMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQ KPVVDHSVRINSVGSTASSSQPLLVHDDV (SEQ ID NO:1); or MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDP GFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLV DRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAY HRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVS SSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFG SANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTD KWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILT YDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQS SIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKEQIHPHTLFTPLLIGFVIVAGMMCIIV MILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPRNRLSFGKTLGAG AFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIVN LLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYKNLLHSKESSCS DSTNEYMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSY QVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPV KWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSP EHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVD HSVRINSVGSTASSSQPLLVHDDV (SEQ ID NO:2). 55. A nucleic acid encoding the modified CD117 polypeptide of any one of claims 39-54. 56. The nucleic acid of claim 55, wherein the nucleic acid comprises RNA, DNA, or a combination thereof. 57. The nucleic acid of claim 56, wherein the nucleic acid comprises a modified mRNA. 58. The nucleic acid of any one of claims 55-57, wherein the nucleic acid is associated with one or more lipids, optionally wherein the nucleic acid is present within a lipid nucleic acid particle, a lipid nanoparticle, or a liposome. 59. A vector comprising the nucleic acid of any one of claims 55-58. 60. The vector of claim 59, wherein the vector is an expression vector. 61. The vector of claim 59 or claim 60, wherein the vector is a viral vector, optionally an AAV vector or a lentiviral vector. 62. The vector of any one of claims 59-61, wherein the vector is capable of transducing hematopoietic stem cells. 63. A modified cell comprising the modified CD117 polypeptide of any one of claims 39-54 and/or the nucleic acid of any one of claims 55-58. 64. The modified cell of claim 63, wherein the cell expresses both the modified CD117 polypeptide and a wild type CD117 polypeptide. 65. The modified cell of claim 63 or claim 64 wherein the cell was transduced with the vector of any one of claims 59-62. 66. The modified cell of any one of claims 63-65, wherein the cell is a stem cell or a pluripotent cell. 67. The modified cell of claim 66, wherein the stem cell is a hematopoietic stem cell (HSC) or a hematopoietic stem and progenitor cell (HSPC). 68. The modified cell of any one of claims 63-67, wherein the cell is CD34+, optionally wherein the cell is CD34+/CD90+, CD34+/CD38-, or CD34+/CD38-/CD90+, or CD34+CD133+. 69. The modified cell of any one of claims 63-68, wherein the cell is a human cell. 70. The modified cell of any one of claims 63-69, wherein the cell was obtained from a mammalian donor. 71. The modified cell of claim 70, wherein the mammalian donor is a subject is in need of a hematopoietic cell transplant (HCT). 72. The modified cell of claim 70, wherein the mammalian donor is a healthy donor. 73. The modified cell of any one of claims 70-72, wherein the cell obtained from the mammalian donor was modified ex vivo. 74. The modified cell of any one of claims 63-73, wherein the cell expresses the modified CD117 polypeptide, optionally wherein the modified cell expresses the modified CD117 polypeptide transiently. 75. The modified cell of claim 74, wherein the modified CD117 polypeptide is expressed on the cell surface or in the cell membrane. 76. The modified cell of any one of claims 63-75, wherein the cell is capable of proliferating and/or surviving in the presence of an anti-CD117 antibody. 77. The modified cell of claim 76, wherein the anti-CD117 antibody is capable of inhibiting proliferation and/or survival of a cell expressing only the wild-type CD117. 78. The modified cell of claim 76 or claim 77, wherein the anti-CD117 antibody is selected from the group consisting of: JSP191, CDX-0159, AB85, and FSI-174. 79. A method of modifying a cell, comprising introducing the nucleic acid of any one of claims 55-58 or the vector of any one of claims 59-62 into the cell, optionally wherein the cell is transiently modified, and optionally wherein the method is for preparing modified cells for hematopoietic cell transplantation (HCT) into a mammalian subject. 80. The method of claim 79, wherein the nucleic acid or vector is introduced into the cell by transfection, transduction, infection, electroporation, or nanopore technology. |
[0058] In particular embodiments, the one of more amino acid substitutions comprises a D816V substitution and/or a N505I substitution. [0059] In certain embodiments, the wild type CD117 polypeptide upon which the variant is based is a human CD117 polypeptide, while in other embodiments, it is another mammalian CD117 polypeptide. Sequences of human and mammalian CD117 polypeptides are known in the art. Due to alternative splicing of the c-kit gene, the human CD117 polypeptide is expressed as various isoforms, and any of these may be used according to the disclosure. These isoforms include two GNNK+ and GNNK− isoforms (also denoted c-Kit and c-KitA, respectively), which differ by the presence or absence of four amino acids, 510-GNNK-513 (SEQ ID NO: 25) in the extra-cellular domain adjacent to the trans-membrane domain, and which are coexpressed in most tissues, although the GNNK− isoform usually predominates. Isoforms may also differ in the presence or absence of a Ser residue at position 715 in the inter-kinase domain, and the disclosure also includes isoforms of CD117, including those shown below, in which Ser175 is either present or absent. These isoforms may comprise any of the modifications disclosed herein, including, e.g., an N505I or D816V modification, and variants thereof, e.g., comprising at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. [0060] In particular embodiments, the wild type CD117 polypeptide is the GNNK+ or GNNK- isoform and comprises or consists of one of the following amino acid sequences (the GNNK tetrapeptide (SEQ ID NO: 25), and the N505 and D816 residues are in bold; numbering is based on GNNK- isoform): MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTD P GFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLV DRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAY HRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVS SSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFG SANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTD KWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILT YDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQS SIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGM MCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPRNRLSFGK TLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNH MNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYKNLLHSK ESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDL LSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGN ARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGF RMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQ KPVVDHSVRINSVGSTASSSQPLLVHDDV (SEQ ID NO:1); or MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTD P GFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLV DRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAY HRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVS SSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFG SANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTD KWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILT YDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQS SIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKEQIHPHTLFTPLLIGFVIVAGMMCIIV MILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPRNRLSFGKTLGAG AFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIVN LLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYKNLLHSKESSCS DSTNEYMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSY QVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPV KWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSP EHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVD HSVRINSVGSTASSSQPLLVHDDV (SEQ ID NO:2). [0061] In certain embodiments, the modified CD117 polypeptide comprises or consists of either of the following sequences: MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTD P GFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLV DRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAY HRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVS SSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFG SANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTD KWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILT YDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQS SIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGM MCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPRNRLSFGK TLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNH MNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYKNLLHSK ESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDL LSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNVSNYVVKGN ARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGF RMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQ KPVVDHSVRINSVGSTASSSQPLLVHDDV (SEQ ID NO:3); MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTD P GFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLV DRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAY HRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVS SSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFG SANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTD KWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILT YDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQS SIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKEQIHPHTLFTPLLIGFVIVAGMMCIIV MILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPRNRLSFGKTLGAG AFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIVN LLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYKNLLHSKESSCS DSTNEYMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSY QVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNVSNYVVKGNARLPV KWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSP EHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVD HSVRINSVGSTASSSQPLLVHDDV (SEQ ID NO:4); MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTD P GFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLV DRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAY HRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVS SSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFG SANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTD KWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILT YDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQS SIDSSAFKHNGTVECKAYNDVGKTSAYFIFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMM CIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPRNRLSFGKTL GAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMN IVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYKNLLHSKES S CSDSTNEYMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSF SYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGNARL PVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRML SPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPV VDHSVRINSVGSTASSSQPLLVHDDV (SEQ ID NO:5); or MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTD P GFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLV DRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAY HRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVS SSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFG SANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTD KWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILT YDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQS SIDSSAFKHNGTVECKAYNDVGKTSAYFIFAFKEQIHPHTLFTPLLIGFVIVAGMMCIIV M ILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPRNRLSFGKTLGAGA FGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIVNLL GACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYKNLLHSKESSCSDS TNEYMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQV AKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKW MAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHA PAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSV RINSVGSTASSSQPLLVHDDV (SEQ ID NO:6), or a variant or fragment thereof, e.g., having at least 90%, at least 95%, at least 98%, or at least 99% identity thereto. In particular embodiments, the variant retains the N505I or D816V amino acid substitution present in the modified CD117. In certain embodiments, the CD117 variant comprises a different amino acid modification that confers constitutive activity to the modified CD117. In particular embodiments, a fragment substantially retains CD117 kinase activity, e.g., retains at least 50% CD117 kinase activity as . [0062] In certain embodiments, the modified CD117 polypeptide substantially retains kinase activity as compared to the wild type CD117 polypeptide. In particular embodiments, the modified CD117 polypeptide has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the kinase activity of the wild type CD117 polypeptide when bound by SCF, and in certain embodiments, the modified CD117 has this activity even in the absence of SCF binding. In some embodiments, the modified CD117 polypeptide has increased kinase activity as compared to the wild type CD117 polypeptide. In particular embodiments, the modified CD117 polypeptide has at least 150%, at least 200%, at least 300%, at least 500%, at least 750%, or at least 1000% of the kinase activity of the wild type CD117 polypeptide. In particular embodiments, the modified CD117 has constitutive kinase activity, even in the absence of SCF binding or in the presence of the anti-c-Kit antibody. Kinase activity may be determined using assays known in the art, including the ADP‐Glo™ Kinase Assay, which is a luminescent kinase assay that measures ADP formed from a kinase reaction; ADP is converted into ATP, which is converted into light by Ultra‐Glo™ Luciferase (available from Promega Corporation, Madison, WI). In certain embodiments, the modified CD117 polypeptide constitutively phosphorylates Gab2, Shc, SHP-2 and/or Cbl. [0063] In particular embodiments, the one or more amino acid modifications do not substantially inhibit or reduce binding of stem cell factor (SCF) to the modified CD117 polypeptide when expressed in cells, as compared to the binding of SCF to the wild type CD117 polypeptide. In particular embodiments, the one or more amino acid modifications do not substantially inhibit or reduce binding of stem cell factor (SCF) to the modified CD117 polypeptide expressed in cells as compared to the wild type CD117 polypeptide. [0064] In certain embodiments, the modified CD117 is a modified CD117 having constitutive signaling or kinase activity, e.g., without bound SCF ligand. In certain embodiments, the modified CD117 has constitutive autophosphorylation activity, e.g., without bound SCF. A variety of such modified CD117 have been identified, e.g., in cancer cells, and any of these may be used according to the compositions and methods disclosed herein. Illustrative examples of activating or gain-of- function CD117 modifications include, but are not limited to, N505I, V559D, D816V, D816H, V568F, V570F, or Y703F, modifications or mutation of amino acid residues corresponding to 505, 522, 816, 557, 558, 559, 568, 569, 570, 703, 816, or deletion of codon 579 (Asp). See, e.g., Akin and Metcalfe, Journal of Allergy and Clinical Immunology, Vol. 114, Issue 1, p13-19, July 1, 2004; Hirotakoji et al., Science 23, Jan 1998, Vol.279, Issue 5350, pp.577-580; Sanlorenzo et al., J Proteomics 2016 July 20, 144: 140-147, and references cited in any of the aforementioned, all of which are hereby incorporated by reference in their entireties. In particular embodiments, the amino acid modification is in the region between the transmembrane and tyrosine kinase domains. Mutations causing constitutive activation of c-Kit have been shown to be causative in some forms of mastocytosis, and several types of mutations have been associated with myeloproliferative disorders (MPDs), acute myelogenous leukemia (AML), sinonasal lymphomas, and gastrointestinal stromal tumors (GIST). These may be considered activating mutation of two types — ‘regulatory type’ mutations, which affect regulation of the kinase molecule, and ‘enzymatic pocket type’ mutations, which alter the amino acid sequence directly forming the enzymatic site. Either type of mutation may be used according to various embodiments of the disclosure, including any of those disclosed in Longley et al., Leukemia Research, Vol.25, Issue 7, July 2001, pp.571-576, and references cited therein, all of which are incorporated herein by reference in its entireties. [0065] In particular embodiments, the one or more amino acid modifications do not result in cells expressing only the modified CD117 having substantially inhibited or reduce c-Kit signaling or proliferation, optionally in response to SCF binding, as compared to the signaling in cells only expressing the wild type CD117 polypeptide. In particular embodiments, the modified CD117 retains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% c-Kit signaling and/or proliferation, optionally in response to SCF binding, as compared to the corresponding wild type CD117. In particular embodiments, the modified CD117 retains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% c- Kit signaling and/or proliferation, in the absence of SCF binding, as compared to the corresponding wild type CD117. [0066] In particular embodiments, the one or more amino acid modifications do not substantially inhibit or reduce binding of an anti-c-Kit antibody to the modified CD117 polypeptide expressed in cells as compared to the wild type CD117 polypeptide. [0067] In particular embodiments, the anti-c-Kit antibody comprises the six CDRs present in any one of JSP191, AB85, CDX-0159, or FSI-174. In particular embodiments, the anti-c-Kit antibody in any one of JSP191, AB85, CDX-0159, or FSI-174. [0068] In some embodiments, the anti-c-Kit antibody is JSP191 or comprises the six CDRs present in JSP191. [0069] In some embodiments, the anti-c-Kit antibody is AB85 or comprises the six CDRs present in AB85. [0070] In some embodiments, the anti-c-Kit antibody is CDX-0159 or comprises the six CDRs present in CDX-0159. [0071] In some embodiments, the anti-c-Kit antibody is FSI-174 or comprises the six CDRs present in FSI-174. [0072] The disclosure also provides nucleic acid or polynucleotides encoding a modified CD117 polypeptide disclosed herein. In particular embodiments, the nucleic acid comprises RNA, DNA, or a combination thereof, and in particular embodiments, the nucleic acid comprises single- stranded and/or double-stranded regions, or a mixture thereof. In certain embodiments, the nucleic acid is a double-stranded DNA, and in certain embodiments, the nucleic acid is a single stranded RNA, e.g., a messenger RNA (mRNA). In certain embodiments, the nucleic acid comprises a modified mRNA. In particular embodiments, the polynucleotides described herein, e.g., modified mRNA, are codon-optimized, e.g., to enhance expression of the encoded polypeptide in a host cell. [0073] In particular embodiments, polynucleotide variants comprise one or more modified nucleotide or nucleoside. Modified mRNAs comprising one or more modified nucleoside have been described as having advantages over unmodified mRNAs, including increase stability, higher expression levels and reduced immunogenicity. Non-limiting examples of modifications to mRNAs that may be present in the nucleic acids encoding the modified CD117 polypeptides are described, e.g., in PCT Patent Application Publication Nos. WO2011/130624, WO2012/138453, WO2013052523, WO2013151666, WO2013/071047, WO2013/078199, WO2012045075, WO2014081507, WO2014093924, WO2014164253, US Patent Nos: US 8,278,036 (describing modified mRNAs comprising pseudouridine), US 8,691,966 (describing modified mRNAs comprising pseudouridine and/or N1-methylpseudouridine), US 8,835,108 (describing modified mRNAs comprising 5-methylcytidine, US 8,748,089 (describing modified mRNAs comprising pseudouridine or 1-methylpseudouridine). In particular embodiments, the modified mRNA comprises one or more nucleoside modification. In particular embodiments, the modified mRNA sequence comprises at least one modification as compared to an unmodified A, G, U or C ribonucleoside. For example, uridine can a similar nucleoside such as pseudouridine (Ψ) or N1- methyl-pseudouridine (m1Ψ), and cytosine can be replaced by 5-methylcytosine. In particular embodiments, the at least one modified nucleosides include N1-methyl-pseudouridine and/or 5- methylcytidine. In certain embodiments, all uridines in the modified mRNA are replaced with a similar nucleoside such as pseudouridine (Ψ) or N1-methyl-pseudouridine (m1Ψ), and/or all cytosines in the modified mRNA are substituted with a similar nucleoside such as 5- methylcytosine. In particular embodiments, the modified mRNA comprises a 5’ terminal cap sequence followed by a sequence encoding the modified CD117 polypeptide, followed by a 3’ tailing sequence, such as a polyA or a polyA-G sequence. [0074] In certain embodiments, the nucleic acid, e.g., a modified mRNA, is associated with one or more lipids, e.g., to facilitate delivery across the cell membrane, shield its negative charge, and/or to protect against degradation by nucleases. In certain embodiments, the nucleic acid is associated with or present within a lipid nucleic acid particle, a lipid nanoparticle, or a liposome. In certain embodiments, the lipid nucleic acid particle, a lipid nanoparticle, or a liposome facilitates delivery or uptake of the nucleic acid by a cell. In certain embodiments, mRNA, optionally modified mRNA, is co-formulation into lipid nanoparticles (LNPs). In particular embodiments, mRNA-LNP formulations comprise: (1) an ionizable or cationic lipid or polymeric material bearing tertiary or quaternary amines to encapsulate the polyanionic mRNA; (2) a zwitterionic lipid (e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine [DOPE]) that resembles the lipids in the cell membrane; (3) cholesterol to stabilize the lipid bilayer of the LNP; and (4) a polyethylene glycol (PEG)-lipid to lend the nanoparticle a hydrating layer, improve colloidal stability, and reduce protein absorption. [0075] In certain embodiments, the nucleic acid encoding the modified CD117 polypeptide is present in a vector. In particular embodiments, the vector is capable of delivering the nucleic acid into mammalian HSCs or other stem cells, e.g., into the nucleus of the HSCs or stem cells. In certain embodiments, the vector is an episomal vector, e.g., a plasmid. In particular embodiments, the vector is an expression vector comprising a promoter sequence operatively linked to a nucleic acid sequence encoding the modified CD117 polypeptide. In particular embodiments, the expression vector comprises a promoter sequence that facilitates expression of the encoded modified CD117 polypeptide in HSCs or other stem cells. In particular embodiments, the expression vector comprises 5’ and/or 3’ cellular or viral UTRs or the derivatives thereof upstream and downstream, respectively, of the sequence encoding the modified CD117 polypeptide. [0076] In certain embodiments, the vector is a viral vector, optionally an AAV vector, a cytomegalovirus vector, an adenovirus vector, or a lentiviral vector. In certain embodiments, a viral vector infects an HSC when viral vector and the HSCs are incubated together for at least about 24 hours in a culture medium. Modified Hematopoietic Stem Cells and Pharmaceutical Compositions [0077] In a related aspect, the disclosure provides modified cells, e.g., HSCs and/or HSPCs, comprising a nucleic acid encoding a modified CD117 polypeptide described herein. In certain embodiments, the modified CD117 polypeptide comprises one or more amino acid substitutions, e.g., at one or more of the following amino acids present in wild type human CD117: N505 or D816. In particular embodiments, the modified CD117 polypeptide comprises a D816V substitution and/or a N505I substitution. [0078] In certain embodiments, the nucleic acid encoding the modified CD117 polypeptide is transiently present in the modified cell, and it is not present within the genome of the cell. In particular embodiments, the modified cell expresses and/or comprises the modified CD117 polypeptide, and in particular embodiments, the modified CD117 polypeptide is present on the cell surface, e.g., with the extracellular domain present outside the modified cell. In certain embodiments, the modified cell is transduced with or infected with an expression vector, optionally a viral vector. In particular embodiments, the modified cell expresses and/or comprises both the modified CD117 polypeptide and a wild type, endogenous CD117 polypeptide, and in particular embodiments, both the modified CD117 polypeptide and the wild type, endogenous CD117 polypeptide are present on the cell surface, e.g., with their extracellular domains present outside the modified cell. In certain embodiments, the modified CD117 polypeptide is present on the cell surface, e.g., with its extracellular domain present outside the modified cell. In particular embodiments, the CD 117s are human CD117 or modified forms thereof.
[0079] In certain embodiments, the modified cell comprising a modified CD117 polypeptide and/or encoding nucleic acid is a host cell, such as, e.g., an HEK293 cell that may be used to produce modified CD117 polypeptides. In preparing the subject compositions, any host cells may be employed, including but not limited to, for example, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast.
[0080] In particular embodiments, the modified cell is a stem cell or pluripotent cell, and in certain embodiments, the stem cell is a hematopoietic stem cell (HSC) or an HSPC. In some embodiments, the stem cell is a mammalian cell that has the ability both to self-renew, and to generate differentiated progeny. In certain embodiments, the stem cell is a human cell. The stem cell may have one or more of the following properties: an ability to undergo asynchronous, or symmetric replication, that is where the two daughter cells after division can have different phenotypes; extensive self-renewal capacity; capacity for existence in a mitotically quiescent form; and clonal regeneration of all the tissue in which they exist, for example the ability of hematopoietic stem cells to reconstitute all hematopoietic lineages.
[0081] Hematopoietic stem cells (HSCs) are maintained throughout life (self-renewing). They produce hematopoietic progenitor cells that differentiate into every type of mature blood cell within a well-defined hierarchy. Hematopoietic stem cells 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 hematopoietic progenitors. [0082] The cells may be fresh, frozen, or have been subject to prior culture. They may be fetal, neonate, adult, etc. Hematopoietic stem cells and HSPCs may be obtained from fetal liver, bone marrow, blood, particularly G-CSF or GM-CSF mobilized peripheral blood, or any other conventional source. Cells for engraftment are optionally isolated from other cells, where the manner in which the stem cells are separated from other cells of the hematopoietic or other lineage is not critical to this invention. If desired, a substantially homogeneous population of stem or progenitor cells may be obtained by selective isolation of cells free of markers associated with differentiated cells, while displaying epitopic characteristics associated with the stem cells. [0083] Modified HSCs may be produced using HSCs obtained from a mammalian donor. In particular embodiments, the donor is a subject in need of a hematopoietic stem cell transplant, e.g., a subject diagnosed with a disease or disorder that can be treated with HCT. In other embodiments, the modified HSCs may be produced using HSCs obtained from a healthy donor, e.g., wherein the modified HSCs are to be used to treat a different subject with HCT. Thus, the modified HSCs may be autologous or allogeneic to a subject in need for HCT. [0084] Prior to harvesting stem cells from a donor, the bone marrow can be primed with granulocyte colony-stimulating factor (G-CSF; filgrastim [Neupogen]) to increase the stem cell count. Mobilization of stem cells from the bone marrow into peripheral blood by cytokines such as G-CSF or GM-CSF has led to the widespread adoption of peripheral blood progenitor cell collection by apheresis for hematopoietic stem cell transplantation. The dose of G-CSF used for mobilization may be about 10 ug/kg/day. In autologous donors who are heavily pretreated, however, doses of up to about 40 ug/kg/day can be given. Mozobil may be used in conjunction with G-CSF to mobilize hematopoietic stem cells to peripheral blood for collection. [0085] Among hematopoietic stem cell (HSC) markers, CD34 is well known for its unique expression on HSCs. In certain embodiments, the modified cell is a CD34+ cell. In particular embodiments, the modified cell is a subset of HSCs 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 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. [0086] In certain embodiments, the hematopoietic stem cells and/or HSPCs are obtained from bone marrow, peripheral blood, or umbilical cord blood and subsequently modified by introduction of the nucleic acid encoding the modified CD117 polypeptide into the cell. For example, the nucleic acid may be introduced by transfection or infection with a viral vector, or by contact with an mRNA. [0087] In certain embodiments, the disclosure provides a method of modifying cells, including stem cells such as HSCs and/or HSPCs, comprising introducing the nucleic acid encoding a modified CD117 polypeptide into the cell. In particular embodiments, the introduced nucleic acid is present within a viral vector. In certain embodiments, the nucleic acid is associated with or present in a lipid nanoparticle, liposome, or the like. In certain embodiments, the nucleic acid remains present in the modified cell only transiently, or the nucleic acid only transiently expresses the modified CD117 polypeptide in the cell. In certain embodiments, the method is used to prepare modified cells for HCT treatment of a mammalian subject. In particular embodiments, the nucleic acid or vector may be introduced into the cell by a variety of methods known in the art, such as transfection, transduction, infection, electroporation, or nanopore technology. In particular embodiments, mRNA, e.g., modified mRNA is introduced into the cells using lipid nucleic acid particles (LNPs) or nanoparticles. Thus, cells, e.g., HSCs and/or HSPCS may be modified by introducing a nucleic acid encoding a modified CD117 polypeptide into the HSCs and/or HSPCs according to a variety of methods available in the art. [0088] In particular embodiments, the modified cell expressing the modified CD117 polypeptide is not substantially inhibited, eliminated, depleted, or killed by monoclonal antibodies (mAbs) that bind endogenous or wild-type cell-surface CD117 and inhibit proliferation of or kill a cell expressing only the wild-type CD117 and not a modified CD117 polypeptide disclosed herein. In certain embodiments, proliferation of the modified cell expressing the modified CD117 polypeptide is inhibited, eliminated, depleted, or killed by less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%, as compared to proliferation of the same cell type that is not modified, e.g., only expresses wild-type CD117. [0089] Compositions and methods disclosed herein may be applicable to any anti-c-Kit antibody, particularly monoclonal anti-human c-Kit antibodies. Illustrative anti-c-Kit antibodies include, but are not limited to, SR-1, JSP191, 8D7, K45, 104D2, CK6, YB5.B8, AF-2-1, AF11, AF12, AF112, AF-3, AF-1-1, NF, NF-2-1, NF11, NF12, NF112, NF-3, HF11, HF12, and HF112. A number of antibodies contemplated by the disclosure that specifically bind human CD117 are known in the art and commercially available, including without limitation SR1, 2B8, ACK2, YB5-B8, 57A5, 104D2, etc. In certain embodiments, the anti-CD117 antibody is selected from the group consisting of: JSP191 (Jasper Therapeutics; Redwood City, CA); CDX-0159 (Celldex Therapeutics, Hampton, NJ); MGTA-117 (AB85) (Magenta Therapeutics, Cambridge, MA); CK6 (Magenta Therapeutics, Cambridge, MA); AB249 (Magenta Therapeutics, Cambridge, MA); and FSI-174 (Gilead, Foster City, CA). Antibodies from Magenta Therapeutics contemplated by the disclosure include but are not limited to those that are disclosed in US Patent Application Publication No. 20190153114, PCT Application Publication Nos. WO2019084064, WO2020/219748, and WO2020/219770. The FSI-174 antibody is disclosed in PCT application Publication No. WO2020/112687 and U.S. Patent Application Publication No. 20200165337. The disclosure includes but is not limited to any anti-c-Kit antibodies and/or CDR sets disclosed in any of the patent application disclosed herein, which are all incorporated by reference in their entireties. [0090] In certain embodiments, the anti-c-Kit antibody binds to the extracellular region of CD117, i.e., amino acids 26-524. The sequence of this region is shown below: QPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITE K AEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTN YSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVLSEKFILKVR PAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHH GDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVF VNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLK GTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYF CPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTS AYFNFAFKGNNKEQIHPHTLFTP (SEQ ID NO:7). [0091] In particular embodiments, the antibody is the humanized form of SR1, which is a murine anti-c-Kit antibody disclosed in U.S. Patent Nos. 5,919,911 and 5,489,516. The humanized antibody, referred to as JSP191 (formerly referred to as AMG191), is described in U.S. Pat. Nos. 8,436,150, 8,791,249, and 7,915,391, and U.S. Patent Application Publication No.20110223165. JSP191 is an aglycosylated IgG1 humanized antibody. JSP191 is a humanized monoclonal antibody in clinical development as a conditioning agent to clear hematopoietic stem cells from bone marrow. JSP191 specifically binds to human CD117, a receptor for stem cell factor (SCF), which is expressed on the surface of hematopoietic stem and progenitor cells (HSPCs). JSP191 blocks SCF from binding to CD117 and disrupts critical survival signals, leading to the depletion of hematopoietic stem cells. [0092] The sequences of the heavy chains and light chains of JSP191 are disclosed as SEQ ID NO: 4 in U.S. Patent No.8,436,150 and SEQ ID NO: 2 in U.S. Patent No.8,436,150, respectively. The sequences of the heavy and light chains of JSP191 are: Heavy Chain: MDWTWRVFCLLAVAPGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWV RQAPGQGLEWMGVIYSGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYY CARERDTRFGNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:8) Light Chain: MVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMHW YQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDP YTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C (SEQ ID NO:9) [0093] In certain embodiments, the variable heavy domain of JSP191 comprises the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSGNG DTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQGTLVT VSS (SEQ ID NO:10). [0094] In certain embodiments, the variable light chain domain of JSP191 comprises the following sequence: DIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMHWYQQKPGQPPKLLIYLASNLES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDP YTFGGGTKVEIK (SEQ ID NO:11). [0095] The CDRs present in JSP191 are as follows: VH CDR1 = YNMH (SEQ ID NO: 26); VH CDR2 = IYSGNGDTSYNQKFKG (SEQ ID NO: 27); VH CDR3 = ERDTRFGN (SEQ ID NO: 28); VL CDR1 = RASESVDIYGNSFMH (SEQ ID NO: 29); VL CDR2 = LASNLES (SEQ ID NO: 30); and VL CDR3 = QQNNEDPYT (SEQ ID NO: 31). [0096] 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-c-Kit antibodies are described in U.S. Patent No. 10,781,267, and in particular embodiments, an anti-c-Kit disclosed herein comprises the CDRs of any of the antibodies disclosed therein. In certain embodiments, the anti-c-Kit antibody comprises: (i) a light chain variable region ("VL") comprising the amino acid sequence: DIVMTQSPSX K1 LSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKX K2 LIYSASYRYS GVPDRFXK3GSGSGTDFTLTISSLQXK4EDFAXK5YXK6CQQYNSYPRTFGGGTKVEIK (SEQ ID NO:12), wherein XK1 is an amino acid with an aromatic or aliphatic hydroxyl side chain, XK2 is an amino acid with an aliphatic or aliphatic hydroxyl side chain, X K3 is an amino acid with an aliphatic hydroxyl side chain, XK4 is an amino acid with an aliphatic hydroxyl side chain or is P, XK5 is an amino acid with a charged or acidic side chain, and XK6 is an amino acid with an aromatic side chain; and (ii) a heavy chain variable region ("VH") comprising the amino acid sequence: QVQLVQSGAEX H1 KKPGASVKX H2 SCKASGYTFTDYYINAVVX H3 QAPGKGLEWIARIYP GSGNTYYNEKFKGRXH4TXH5TAXH6KSTSTAYMXH7LSSLRSEDXH8AVYFCARGVYYF D YWGQGTTVTVSS (SEQ ID NO:13), wherein X H1 is an amino acid with an aliphatic side chain, X H2 is an amino acid with an aliphatic side chain, X H3 is an amino acid with a polar or basic side chain, XH4 is an amino acid with an aliphatic side chain, XH5 is an amino acid with an aliphatic side chain, X H6 is an amino acid with an acidic side chain, X H7 is an amino acid with an acidic or amide derivative side chain, and X H8 is an amino acid with an aliphatic hydroxyl side chain. In specific aspects, described herein are antibodies (e.g., human or humanized antibodies), including antigen-binding fragments thereof, comprising: (i) VH CDRs of a VH domain comprising the amino acid sequence: QVQLKQSGAELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEWIARIYPG SGNTYYNEKFKGKATLTAEKSSSTAYMQLSSLTSEDSAVYFCARGVYYFDYWGQ GTTLTVSS (SEQ ID NO:14) or QVQLKQSGAELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEWIARIYPG SGNTYYNEKFKGKATLTAEKSSSTAYMQLSSLTSEDSAVYFCARGVYYFDYWGQ GTTLTVSA (SEQ ID NO:15), and (ii) VL CDRs of a VL domain comprising the amino acid sequence DIVMTQSQKFMSTSVGDRVSVTCKASQNVRTNVAWYQQKPGQSPKALIYSASYRYSGV PDRFTGSGSGTDFTLTI SNVQSEDLADYFCQQYNSYPRTFGGGTKLEIKR (SEQ ID NO:16). [0097] MGTA-117 (AB85) is a CD117-targeted antibody engineered for the transplant setting and conjugated to amanitin, which is being developed for patients undergoing immune reset through either autologous or allogeneic stem cell transplant. MGTA-117 depletes hematopoietic stem and progenitor cells, and this antibody and others contemplated by the disclosure are described in U.S> Application No.20200407440 and/or PCT Application No. WO2019084064. Epitope analysis of AB85 binding to CD177 is described in PCT Application Publication No. WO2020219770, which identified the following two epitopes within CD117: EKAEATNTGKYTCTNKHGLSNSIYVFVRDPA (amino acids 60-90; (SEQ ID NO:17)), and RCPLTDPEVTNYSLKGCQGKP (amino acids 100-130; (SEQ ID NO:18)). [0098] The sequences of the variable heavy chain and variable light chains of AB85 are disclosed as SEQ ID NO: 143 and SEQ ID NO: 144 from PCT Application No. WO2019084064, respectively. [0099] The heavy chain variable region (VH) amino acid sequence of AB85 is: EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMAIINPRDSDT RYRPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHGRGYEGYEGAFDIWGQG TLVTVSS (SEQ ID NO:19). [00100] The VH CDR amino acid sequences of AB85 are as follows: NYWIG (VH CDR1; SEQ ID NO: 32); IINPRDSDTRYRPSFQG (VH CDR2; SEQ ID NO: 33); and HGRGYEGYEGAFDI (VH CDR3; SEQ ID NO: 34). [00101] The light chain variable region (VL) amino acid sequence of AB85 is: DIQMTQSPSSLSASVGDRVTITCRSSQGIRSDLGWYQQKPGKAPKLLIYDASNLETGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQANGFPLTFGGGTKVEIK (SEQ ID NO:20). [00102] The VL CDR amino acid sequences of AB85 are as follows: RSSQGIRSDLG (VL CDR1; SEQ ID NO: 35); DASNLET (VL CDR2; SEQ ID NO: 36); and QQANGFPLT (VL CDR3; SEQ ID NO: 37). [00103] FSI-174 is an anti-cKIT antibody being developed in combination with 5F9 as a non-toxic transplant conditioning regimen, as well as a treatment for targeted hematologic malignancies. The sequences of FSI-174 are disclosed in PCT Application Publication No.2020/112687, U.S. Patent Application Publication No. 20200165337, and U.S. Patent No. 11,041,022. In particular embodiments, an anti-c-Kit antibody comprises the three CDRs or variable heavy chain regions present in any of AH1, AH2, AH3, AH4, or AH5 disclosed therein, and/or the three CDRs or variable heavy chain regions present in any of AL1 or AL2 disclosed therein. [00104] In certain embodiments, the CDRs present in FSI-174 and related antibodies are as follows: VH CDR1 = SYNMH (SEQ ID NO: 38); VH CDR2 = VIYSGNGDTSY(A/N)QKF(K/Q)G (SEQ ID NO: 39); VH CDR3 = ERDTRFGN (SEQ ID NO: 40); VL CDR1 = RAS(D/E)SVDIYG(N/Q)SFMH (SEQ ID NO: 41); VL CDR2 = LASNLES (SEQ ID NO: 42); and VL CDR3 = QQNNEDPYT (SEQ ID NO: 43). A/N and the like indicate that the amino acid position may be either of the two amino acids, in this example, A or N. In certain embodiments, CDRs present in the heavy variable region are CDRs H1, H2 and H3 as defined by Kabat: H1 = SYNMH (SEQ ID NO: 38); H2 = VIYSGNGDTSYAQKFKG (SEQ ID NO: 44); H3 = ERDTRFGN (SEQ ID NO: 39); and the CDRs present in the light variable region are CDRs L1, L2 and L3 as defined by Kabat: L1 = RASESVDIYGQSFMH (SEQ ID NO: 45); L2 = LASNLES (SEQ ID NO: 42); and L3 = QQNNEDPYT (SEQ ID NO: 43), respectively except that 1, 2, or 3 CDR residue substitutions is/are present selected from N to A at heavy chain position 60, K to Q at heavy chain position 64 and N to Q at light chain position 30, positions being numbered according to Kabat. In certain embodiments, the antibody comprises any of the heavy chain variable region sequences (AH2, AH3, AH4) and/or light chain variable chain region sequences provided below (AL2), or the CDRs therein shown underlined: AH2: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYMNHWVRQAPGQGLEWMGVIYSGNG DTSYAQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQGTLVT VSS (SEQ ID NO:21) AH3: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYMNHWVRQAPGQGLEWMGVIYSGNG DTSYNQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQGTLVT VSS (SEQ ID NO:22) AH4: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYMNHWVRQAPGQGLEWMGVIYSGNG DTSYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQGTLVT VSS (SEQ ID NO:23) AL2: DIVMTQSPLSLPVTPGEPASISCRASESVDIYGQSFMHWYQQKPGQPPKLLIYLASNLES G VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQNNEDPYTFGGGTKVEIK (SEQ ID NO:24) [00105] In certain embodiments, the anti-CD117 antibody comprises the full heavy chain and/or full light chain of any of the antibodies disclosed herein, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity to a heavy or light chain disclosed herein, e.g., a JSP191 heavy or light chain. In certain embodiments, the anti-CD117 antibody comprises the variable region of a heavy chain and/or light chain of any of the antibodies disclosed herein, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity to the variable region of a heavy or light chain disclosed herein, e.g., a JSP191 heavy or light chain variable region. In certain embodiments, the anti-CD117 antibody comprises a heavy chain and/or a light chain comprising one or more CDRs of an antibody disclosed herein, e.g., two, three, four, five or six CDRs of an antibody disclosed herein, e.g., a JSP191 antibody. In particular embodiments, the anti-CD117 antibody comprises a heavy chain or variable region thereof comprising one, two, or three heavy chain CDRs disclosed herein, e.g., a JSP191 heavy chain. In particular embodiments, the anti-CD117 antibody comprises a light chain or variable region thereof comprising one, two, or three light chain CDRs disclosed herein, e.g., a JSP191 light chain. [00106] In particular embodiments, the antibody binds to a region of wild-type CD117 or an epitope of wild-type CD117 that is modified in a modified CD117 polypeptides disclosed herein. In particular embodiments, the antibody does not bind a modified CD117 polypeptide disclosed herein, or binds to a modified CD117 polypeptide disclosed herein with reduced affinity, e.g,, less than 50%, less than 25%, or less than 10%. Antibody affinity to a particular polypeptide, such as wild-type CD117 or a modified CD117 may be determined, e.g., by measuring the equilibrium dissociation constant between the antibody and its antigen (KD), which may be determined by routine methods in the art, e.g., by surface plasmon resonance, as described in Hearty, Stephen, Paul Leonard, and Richard O’Kennedy. "Measuring antibody–antigen binding kinetics using surface plasmon resonance." Antibody Engineering: Methods and Protocols, Second Edition (2012): 411-442. [00107] In particular embodiments, the modified cell expressing the modified CD117 polypeptide is capable of proliferating or surviving in the presence of an anti-CD117 antibody, e.g., an anti- CD117 antibody that blocks or inhibits binding of SCF to CD117 on the cell surface. In particular embodiments, proliferation and/or survival of the modified cell expressing the modified CD117 polypeptide, in the presence of an anti-CD117 antibody, e.g., an anti-CD117 antibody that blocks or inhibits binding of SCF to CD117 on the cell surface, is at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, or at least 40% the level of proliferation and/or survival in the absence of the anti-CD118 antibody. In certain embodiments, the anti-CD117 antibody is capable of inhibiting proliferation of or inducing death or apoptosis of a cell expressing only the wild-type CD117 and not a modified CD117 polypeptide disclosed herein. In particular embodiments, the anti-CD117 antibody is selected from the group consisting of: SR1, 2B8, ACK2, YB5-B8, 57A5, 104D2, JSP191, CDX-0159, MGTA-117 (AB85), and FSI-174. In particular embodiments, the antibody is JSP191. Thus, in particular embodiments, the modified CD117 polypeptides disclosed herein, when expressed on a HSC and/or HSPC surface, are capable of substantially binding SCF in the presence of an anti-CD117 antibody that inhibit binding of SCF to endogenous, wild-type CD117 on the cell surface. Similarly, in particular embodiments, the modified CD117 polypeptides disclosed herein, when expressed on an HSC surface, are capable of intracellular signaling when bound by SCF, in the absence of and in the presence of an anti-CD117 antibody that inhibit binding of SCF to endogenous, wild-type CD117 on the cell surface. In particular embodiments, SCF binding and/or SCF-mediating signaling is in not substantially reduced in the presence of the anti- CD117 antibody, e.g., binding and/or signaling of the modified cell expressing the modified CD117 polypeptide is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the level of binding and/or signaling observed in the same cell type that is not modified, e.g., only expresses wild-type CD117. [00108] c-Kit signaling or proliferation or viability may be determined using methods standard in the art. For example, in certain embodiments, c-Kit signaling or proliferation (e.g., in response to SCF), of cells comprising a modified CD117 polypeptide is determined using a cell line (e.g., Ba/F3 cells) engineered to express the modified CD117 polypeptide. Cells are cultured in the presence of IL-3, with or without stem cell factor (SCF), and in the presence or absence of an anti- CD117 antibody, e.g., JSP191. Control parental Ba/F3 cells do not proliferate in the absence of IL-3. Further, parental Ba/F3 cells do not express CD117 and are not responsive to SCF signaling. Proliferation in response to SCF binding may this be determined for cells overexpressing the modified CD117, e.g., in the presence and absence of the anti-CD117 antibody. [00109] The disclosure also provides methods of preparing HSCs and/or HSPCs for HCT, comprising introducing a polynucleotide sequence encoding a modified CD117 described herein into the HSCs and/or HSPCs, In particular embodiments, the polynucleotide sequence encoding the modified CD117 is present within an mRNA or an expression vector, and the modified CD117 is transiently or constitutively expressed after it is introduced into the HSCs and/or HSPCs. The polynucleotide and/or vector may comprise nucleotide modifications, including any of those disclosed herein or known in the art, e.g., to increase expression or stability of the polynucleotide or vector. [00110] For engraftment purposes, a composition comprising HSCs and/or HSPCs, is administered to a patient. Such methods are well known in the art. The stem cells are optionally, although not necessarily, purified. Abundant reports explore various methods for purification of stem cells and subsequent engraftment, including flow cytometry; an isolex system (Klein et al. (2001) Bone Marrow Transplant. 28(11):1023-9; Prince et al. (2002) Cytotherapy 4(2):137-45); immunomagnetic separation (Prince et al. (2002) Cytotherapy 4(2):147-55; Handgretinger et al. (2002) Bone Marrow Transplant. 29(9):731-6; Chou et al. (2005) Breast Cancer. 12(3):178-88); and the like. Each of these references is herein specifically incorporated by reference, particularly with respect to procedures, cell compositions and doses for hematopoietic stem cell transplantation. [00111] The present disclosure also includes pharmaceutical compositions comprising one or more modified CD117 polypeptides, one or more polynucleotides or vectors comprising a sequence encoding a modified CD117 polypeptide (e.g., a modified mRNA), or a modified cell comprising a polynucleotide or vector encoding a modified CD117 polypeptide and/or expressing a modified CD117, in combination with one or more pharmaceutically acceptable diluent, carrier, or excipient. [00112] The present invention discloses a pharmaceutical composition comprising a modified cell comprising a modified CD117 polypeptide (or nucleic acid sequence encoding the modified CD117 polypeptide) described herein and one or more pharmaceutically acceptable diluent, carrier, or excipient. In particular embodiments, the cell is a heterologous cell or an autologous cell obtained from the subject to be treated. In particular embodiments, the cell is a stem cell, e.g., a HSC and/or HSPC. In certain embodiments, the pharmaceutical composition further comprises one or more additional active agents. In certain embodiments, the one or more additional active agent comprises an anti-CD117 antibody. In particular embodiments, the anti-CD117 antibody is selected from the group consisting of: SR1, 2B8, ACK2, YB5-B8, 57A5, 104D2, JSP191, CDX- 0159, MGTA-117 (AB85), and FSI-174. In particular embodiments, the antibody is JSP191. In certain embodiments, the one or more additional active agent comprises one or more anti-CD47, anti-CD40L, anti-CD122, anti-CD4, and/or anti-CD8 antibody. [00113] The polynucleotides, polypeptides, and cells described herein can be combined with pharmaceutically-acceptable carriers, diluents and reagents useful in preparing a formulation that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for mammalian, e.g., human or primate, use. In certain embodiments, the pharmaceutical composition is a solution or suspension comprising modified cells disclosed herein. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulations. Solutions or suspensions used for the formulations can include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; detergents such as Tween 20 to prevent aggregation; and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In particular embodiments, the pharmaceutical compositions are sterile. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In certain embodiments, it is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, e.g., a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. In some cases, the composition is sterile and may be fluid to the extent that easy syringability exists. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In certain embodiments, a pharmaceutical composition include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the internal compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Methods of Use [00114] In further aspects, the disclosure provides methods of treating a mammalian subject in need thereof, comprising administering to the subject modified cells, e.g., HSCs or HSPCs, comprising a modified CD117 polypeptide described herein and/or a nucleic acid encoding the modified CD117 polypeptide. In particular embodiments, the subject is in need of HCT or a hematopoietic stem cell transplant. The transplant may be autologous, allogeneic, or xenogeneic, including without limitation allogeneic haploidentical stem cells, mismatched allogeneic stem cells, genetically engineered autologous or allogeneic cells, etc. In particular embodiments, the modified HSCs or HSPCs are infused into the subject, e.g., by intravenous infusion, e.g., through a central vein over a period of several minutes to several hours. In particular embodiments, the modified HSCs or HSPCs transiently express the modified CD117 polypeptide, which is constitutively active, e.g., has constitutive kinase activity. In certain embodiments, the modified CD117 has constitutive autophosphorylation activity, e.g., without bound SCF. In particular embodiments, the modified HSCs or HSPCs that transiently express the modified CD117 are resistant to ablation by an anti-c-Kit antibody, such as, e.g., JSP191. Accordingly, a subject in need of HCT may be conditioned using an anti-c-Kit monoclonal antibody such as JSP191 prior to, with, or following HCT in order to ablate diseased HSPCs, whereas the transplanted modified HSCs or HSPCs are less susceptible to ablation by any monoclonal antibody in the subject following transplant, since they transiently express the modified CD117, which provides transient constitutive CD117 signaling, even in the presence of the antibody. [00115] Where the donor is allogeneic to the recipient, the HLA type of the donor and recipient may be tested for a match, or haploidentical cells may be used. In certain embodiments, cells obtained from HLA-haploidentical donors or HLA-identical donors are used. HLA-haploidentical donors can be manipulated by CD34 or CD34/CD90 selection. For HLA matching, traditionally, the loci critical for matching are HLA-A, HLA-B, and HLA-DR. HLA-C and HLA-DQ are also now considered when determining the appropriateness of a donor. A completely matched sibling donor is generally considered the ideal donor. For unrelated donors, a complete match or a single mismatch is considered acceptable for most transplantation, although in certain circumstances, a greater mismatch is tolerated. Preferably matching is both serologic and molecular. Where the donor cells are from umbilical cord blood, the degree of tolerable HLA disparity is much greater, and a match of three or four out of the six HLA-A, HLA-B and HLA-DRB1 antigens is typically sufficient for transplantation. Immunocompetent donor T cells may be removed using a variety of methods to reduce or eliminate the possibility that graft versus host disease (GVHD) will develop. [00116] The HCT methods disclosed use modified HSCs comprising a modified CD117 polypeptide or nucleic acid encoding the modified CD117 polypeptide. The methods are believed to result in reduced toxicity, reduced morbidity, or reduced graft-versus-host disease, as compared to HCT wherein a subject is administered HSCs that do not comprise the modified CD117 polypeptide or nucleic acid encoding the modified CD117 polypeptide. The methods of the invention are also believed to provide for improved engraftment of stem cells after transplantation into a recipient. [00117] In particular embodiments of any of the methods of treatment disclosed herein, the subject is administered a conditioning regimen to facilitate or increase engraftment of the modified cells. In certain embodiments, the conditioning regimen depletes endogenous normal or disease HSCs of the subject. Conditioning regimens may be given prior to transplant to reduce the number of blood stem cells in the bone marrow to make space for donor blood stem cells to engraft and cure the patient. Typically, the conditioning regimen is administered prior to and/or concurrent with the administering of the pharmaceutical composition. In certain embodiments, the conditioning regimen comprises administration of an anti-CD117 antibody, wherein the anti-CD117 antibody depletes endogenous HSCs expressing wild-type CD117, but the anti-CD117 antibody does not deplete the administered modified HSCs. In particular embodiments, the anti-CD117 antibody is selected from the group consisting of: SR1, 2B8, ACK2, YB5-B8, 57A5, 104D2, JSP191, CDX- 0159, MGTA-117 (AB85), and FSI-174. In particular embodiments, the antibody is JSP191. In particular embodiments, the conditioning regimen comprises an anti-CD117 antibody alone. In particular embodiments, the subject is administered the anti-CD117 antibody prior to administration of the modified HSCs, e.g., as a single dose. [00118] An effective dose of anti-c-Kit antibody is the dose that depletes endogenous hematopoietic stem cells. The effective dose will depend on the individual and the specific antibody, but it will generally be up to about 100 μg/kg body weight, up to about 250 μg/kg, up to about 500 μg/kg, up to about 750 μg/kg, up to about 1 mg/kg, up to about 1.2 mg/kg, up to about 1.5 mg/kg, up to about 3 mg/kg, up to about 5 mg/kg, up to about 10 mg/kg. In some embodiments, the subject is administered about 0.01 mg/kg to about 2 mg/kg of the anti-c-kit antibody, e.g., JSP191, and optionally the subject is administered about 0.1 mg/kg to about 1 mg/kg of the anti-c-Kit antibody, e.g., JSP191. In some embodiments, anti-c-Kit antibody may be administered to a subject in a dose about 0.01 mg/kg to about 2 mg/kg of the subject’s body weight, or about 0.1 mg/kg to about 1 mg/kg of the subject’s body weight. In some embodiments, the anti-c-Kit signaling antibodies are administered in a dose of about 0.6 mg/kg. [00119] In certain embodiments, the conditioning regimen comprises administration of an anti- CD117 antibody in combination with one or more additional antibodies. In certain embodiments, the one or more additional antibodies comprise one or more of: anti-CD47, anti-CD40L, anti- CD122, anti-CD4, and/or anti-CD8 antibody. [00120] In certain embodiments, the conditioning regimen comprises administration of an anti- CD117 antibody, alone or in combination with a myeloablative (MA) conditioning, reduced intensity conditioning (RIC), or other non-MA (NMA) conditioning regimen. Examples of various conditioning regimens are provided in Fig.2. In certain embodiments, the conditioning regimen is a genotoxic conditioning regimen and/or may comprise one or more of: chemotherapy (optionally a nucleoside analog and/or an alkylating agent), monoclonal antibody therapy, and radiation, optionally radiation to the entire body. In particular embodiments, since the subject is being administered modified cells, e.g., HSCs, comprising a modified CD117 described herein and/or an anti-CD117 antibody, the conditioning regimen is milder than would be used if the subject was being administered cells, e.g., HSCs, that did not comprise the modified CD117 polypeptide. In particular embodiments, wherein the conditioning regimen comprises use of an anti-CD117 antibody in combination with chemotherapy (optionally a nucleoside analog and/or an alkylating agent), other monoclonal antibody therapy, and/or radiation, the amount of chemotherapy, other monoclonal antibody therapy, and/or radiation is reduced as compared to the amount used when not in combination with an anti-CD117 antibody, such as JSP191. For example, either or both the amount and/or duration of other conditioning therapy may be reduced by at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, or by about 100%. [00121] However, in other embodiments, the subject is not administered a myeloablative or genotoxic conditioning regimen prior to or concurrent with the administering of the pharmaceutical composition. For example, the recipient may be immunocompetent, and the transplantation may be performed in the absence of myeloablative conditioning, i.e., in the absence of radiation and/or chemotherapeutic drugs. The recipient may be conditioned with the combined administration a set of agents selected according to the cells and HLA match. [00122] The dose of stem cells, e.g., modified HSCs comprising a modified CD117 polypeptide and/or nucleic acid encoding a modified CD117 polypeptide, administered to a subject may depend on the purity of the infused cell composition, and the source of the cells. In particular embodiments, the dose administered is at least or about 1-2x10 6 CD34+ cells/kg body weight for autologous and allogeneic transplants. Higher doses can include, for example, at least or about 3x10 6 , at least or about 4x10 6 , at least or about 5x10 6 , at least or about 6x10 6 , at least or about 7x10 6 , at least or about 8x10 6 , at least or about 9x10 6 , at least or about 10 7 or more CD34+ cells/kg body weight for autologous and allogeneic transplants. Frequently, the dose is limited by the number of available cells, and the methods disclosed encompass delivering less cells when necessary or limited. Typically, regardless of the source, the dose is calculated by the number of CD34+ cells present. The percent number of CD34+ cells can be low for unfractionated bone marrow or mobilized peripheral blood; in which case the total number of cells administered may be higher. [00123] In certain embodiments, a maximum number of CD3+ cells delivered with the modified HSC composition is not more than about 10 7 CD3+ cells/kg of recipient body weight, not more than about 10 6 CD3+ cells/kg of recipient body weight, not more than about 10 5 CD3+ cells/kg of recipient body weight, or not more than about 10 4 CD3+ cells/kg of recipient body weight. Alternatively, cell populations may be selected for expression of CD34 and CD90, which cell populations may be highly purified, e.g., at least about 85% CD34+ CD90+ cells, at least about 90% CD34+ CD90+ cells, at least about 95% CD34+ CD90+ cells and may be up to about 99% CD34+ CD90+ cells or more.
[00124] In certain embodiments, the disclosure includes a method of treating a mammalian subject in need thereof, comprising administering to the subject modified cells, e.g., HSCs and/or HSPCs, comprising a modified CD117 polypeptide disclosed herein, e.g., a modified CD 117 with constitutive c-Kit signaling or kinase activity. In certain embodiments, the modified CD117 has constitutive autophosphorylation activity, e.g., without bound SCF. In particular embodiments, the modified CD117 polypeptide is transiently expressed in the cells, e.g., for about one day, about two days, about three days, about four days, about five days, or about a week. In particular embodiments, the subject is also administered a conditioning regimen to facilitate or increase engraftment of the modified cells following transplantation, wherein the conditioning regimen is administered prior to or concurrent with the administering of the pharmaceutical composition. In particular embodiments, the conditioning regimen comprises administration of an anti-c-Kit antibody, e.g., any disclosed herein (such as, e.g., JSP191), to the subject. In some embodiments, the anti-c-Kit antibody is administered to the subject prior to administration of the pharmaceutical composition to the subject. In particular embodiments, there is a “washout” period following administration of the anti-c-Kit antibody and before administration of the modified cells (i.e., the HCT). This period of time allows clearance of the anti-c-Kit antibody (or other agent used for conditioning). The period of time required for clearance of the ablative agent may be empirically determined, or may be based on prior experience of the pharmacokinetics of the agent. Historically, the time for clearance was usually the time sufficient for the level of ablative agent, e.g., anti-c- Kit antibody, to decrease at least about 10-fold from peak levels, usually at least about 100-fold, 1000-fold, 10,000-fold, or more. However, since the modified cells being administered to the subject according to the methods disclosed herein comprise a modified CD117 polypeptide that is not bound by the ablative anti-c-Kit antibody used for conditioning, the disclosed methods do not require a wash-out period, or they require only a reduced wash-out period as compared to when unmodified cells are transplanted. In certain embodiments, the wash-out period is less than five days, less than four days, less than 3 days, less than two days, or less than one day. In certain embodiments, the method comprises administering the anti-c-Kit antibody and the pharmaceutical composition or modified cells, e.g., modified HSCs and/or HSPCs, during an overlapping period of time or at about the same time. In particular embodiments, the method comprises administering the anti-c-Kit antibody to the subject after administration of the pharmaceutical composition or modified cells, e.g., modified HSCs and/or HSPCs, optionally for a period of time of at least one day, at least two days, at least three days, at least four days, at least five days, or at least one week. This may continue to ablate endogenous HSCs and/or HSPCs following administration of the modified HSCs and/or HSPCs, thus allowing greater engraftment. [00125] In one embodiment, the method comprises: (i) selectively ablating endogenous hematopoietic stem cells in the subject by administering to the subject an anti-c-Kit antibody, e.g., JSP-191; (ii) optionally, waiting for a period of time following administration of the anti-c-Kit antibody; and (iii) following (ii), administering to the subject the pharmaceutical composition comprising the modified cells, e.g., modified HSCs and/or modified HSPCs, in a dose effective to achieve multilineage peripheral blood chimerism. In particular embodiments, the period of time of step (ii) is less than five days, less than four days, less than 3 days, less than two days, or less than one day, or there is no period of time. In particular embodiments, the modified CD117 has constitutive c-Kit signaling or kinase activity. In particular embodiments, the modified CD117 polypeptide is transiently expressed in the cells, e.g., for about one day, about two days, about three days, about four days, about five days, or about a week. In particular embodiments, the modified CD117 polypeptide has at least 90%, at least 95%, at least 98%, or at least 99% identity to a wild-type human c-Kit polypeptide and comprises an amino acid substitution at a position referred to as N505 or D816, such as, e.g., N505I or D816V. [00126] In certain embodiments, the method of treating a subject in need of HCT comprises: (i) administering a conditioning regimen to the subject, wherein the conditioning regimen comprises an anti-CD117 monoclonal antibody, e.g., JSP191; and (ii) administering modified HSCs to the subject, wherein the modified HSCs comprise a modified CD117 polypeptide, wherein the modified CD117 polypeptide is expressed on the cell surface, and wherein the modified HSCs are not depleted by the conditioning regimen to the same extent as endogenous HSCs that comprise only wild type CD117 polypeptide and/or wherein the modified HSCs have a proliferative advantage as compared to the endogenous HSCs. In particular embodiments, the modified CD117 has constitutive c-Kit signaling or kinase activity. In certain embodiments, the modified CD117 has constitutive autophosphorylation activity, e.g., without bound SCF. In particular embodiments, the modified CD117 polypeptide is transiently expressed in the cells, e.g., for about one day, about two days, about three days, about four days, about five days, or about a week. In particular embodiments, the conditioning regimen comprises a monoclonal anti-c-Kit antibody, e.g., JSP191. In particular embodiments, the modified CD117 polypeptide has at least 90%, at least 95%, at least 98%, or at least 99% identity to a wild-type human c-Kit polypeptide and comprises an amino acid substitution at a position referred to as N505 or D816, such as, e.g., N505I or D816V. The actual location of the “N505” or “D816” modification may differ depending on the particular isotype of c-Kit polypeptide. In particular embodiments, the modified CD117 polypeptide has the sequence shown in any one of SEQ ID NOs:3-6. [00127] In some embodiments, the transplantation is performed in the absence of myeloablative conditioning. In some embodiments the recipient is immunocompetent. The administration of the pre-transplantation conditioning regimen is repeated as necessary to achieve the desired level of ablation. Following transplantation with donor stem cells, the recipient may be a chimera or mixed chimera for the donor cells. [00128] The methods disclosed herein may be used to treat a variety of indications amenable to stem cell transplantation. In particular embodiments, HCT methods disclosed herein are used to treat a disease or disorder selected from the group consisting of: a cancer, a cardiac disorder, a neural disorder, an autoimmune disease, an immunodeficiency, a metabolic disorder, hemoglobinopathies, and a genetic disorder. In particular embodiments, they are used to treat any of the following disorders: multiple myeloma, non-Hodgkin lymphoma, Hodgkin disease, acute myeloid leukemia, neuroblastoma, germ cell tumors, and autoimmune disorders, e.g., systemic lupus erythematosus (SLE), systemic sclerosis, or amyloidosis, for example, by autologous HCT. In particular embodiments, they are used to treat any of the following disorders: acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia; chronic lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndromes, multiple myeloma, non- Hodgkin lymphoma, Hodgkin disease, aplastic anemia, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, thalassemias, thalassemia major, sickle cell anemia, combined immunodeficiency, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, hemophagocytic lymphohistiocytosis (HLH), inborn errors of metabolism (e.g., mucopolysaccharidosis, Gaucher disease, metachromatic leukodystrophies, and adrenoleukodystrophies), epidermolysis bullosa, severe congenital neutropenia, Shwachman- Diamond syndrome, Diamond-Blackfan anemia, leukocyte adhesion deficiency, and the like, for example, by allogeneic HCT. [00129] In particular embodiments, the methods disclosed are used to treat a solid tissue cancer or a blood cancer, such as a leukemia, a lymphoma, or a myelodysplastic syndrome. In particular embodiments, the leukemia is acute myeloid leukemia (AML). [00130] In particular embodiments, the lymphoma is diffuse large B-cell lymphoma. [00131] In particular embodiments, the methods disclosed are used to treat an immunodeficiency. In particular embodiments, the immunodeficiency is severe combined immunodeficiency (SCID). In particular embodiments, the immunodeficiency is immunoglobulin G subclass deficiency, selective immunoglobulin A deficiency, DiGeorge syndrome, hyper-immunoglobulin M (HIGM) syndrome, selective IgM deficiency, Wiskott-Aldrich syndrome, or X-linked agammaglobulinemia (XLA). [00132] In particular embodiments, the methods disclosed are used to treat a genetic disorder. In particular embodiments, the genetic disorder is sickle cell disease or Fanconi anemia. Sickle cell diseases that may be treat include, but are not limited to: HbS disease; drepanocytic anemia; meniscocytosis, and chronic hemolytic anemia. [00133] In certain embodiments of any of the HCT methods disclosed, the method further comprises administering to the subject a therapeutic agent for treatment of the disease or disorder being treated by the HCT method. EXAMPLES Example 1 PROLIFERATION OF HEMATOPOIETIC CELLS EXPRESSING A CD117 VARIANT IS NOT INHIBITED BY AN ANTI-CD117 ANTIBODY [00134] To demonstrate that expression of a mutated CD117 confers a proliferative advantage for hematopoietic cells expressing mutant CD117 vs. wild-type CD117, Ba/F3 cells expressing wild- type human CD117 (c-Kit) and mutant human CD117-D816V were cultured in the absence of IL- 3, in varying concentrations of stem cell factor (SCF), and in the presence or absence of anti- CD117 antibody JSP191. [00135] Control parental Ba/F3 cells did not proliferate in the absence of IL-3. Further, parental Ba/F3 cells did not express CD117 and were not responsive to SCF signaling. Therefore, control parental Ba/F3 cells did not proliferate in the presence of increasing concentrations of SCF, and there was no effect on viability or proliferation with the addition of JSP191.
[00136] Ba/F3 cell line expressing wild-type human CD117 (c-Kit) showed dose-responsive proliferation to SCF, which was inhibited in the presence of anti-CD117 antibody JSP191.
[00137] Ba/F3 cell line expressing the CD117-D816V mutant was able to proliferate in the absence of SCF and proliferation was not inhibited by the presence of the anti-CDl 17 antibody JSP191.
[00138] These studies demonstrate that cells comprising a constitutively active modified CD117 are capable of proliferating in the presence of anti-CDl 17 antibodies that inhibit SCF binding to CD 117, and thus the modified CD117 confers a proliferative advantage to the modified cells as compared to wild type cells, particularly in the presence of the anti-CDl 17 antibody.
[00139] The various embodiments described above can be combined to provide further embodiments.
[00140] Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, patent applications, and publications to provide yet further embodiments.
[00141] These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure
[00142] All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety.