Universal donor stem cells and related methods

Patent metadata

Field	Value
Publication number	US-2021171903-A1
Application number	US-202016908618-A
Country	US
Kind code	A1
Filing date	Jun 22, 2020
Priority date	Feb 15, 2018
Publication date	Jun 10, 2021
Grant date	—

Abstract

Disclosed herein are universal donor stem cells and related methods of their use and production. The universal donor stem cells disclosed herein are useful for overcoming the immune rejection in cell-based transplantation therapies. In certain embodiments, the universal donor stem cells disclosed herein have modulated expression of one or more MHC-I and MHC-II human leukocyte antigens and one or more tolerogenic factors.

First claim

1 .- 56 . (canceled)

57 . A hypoimmunogenic stem cell comprising three exogenous genes encoding PD-L1, HLA-G, and CD47, respectively, wherein the three exogenous genes are inserted into a safe harbor locus of at least one allele of the hypoimmunogenic stem cell.

58 . The hypoimmunogenic stem cell of claim 57 , further comprising reduced expression of HLA-A, HLA-B, HLA-C, and CIITA.

59 . The hypoimmunogenic stem cell of claim 58 , wherein the hypoimmunogenic stem cell does not express HLA-A, HLA-B, HLA-C, and CIITA.

60 . The hypoimmunogenic stem cell of claim 57 , wherein the hypoimmunogenic stem cell retains differentiation potential.

61 . The hypoimmunogenic stem cell of claim 57 , wherein the hypoimmunogenic stem cell elicits a reduced adaptive immune response and a reduced innate immune response in a subject into which the hypoimmunogenic stem cell is transplanted as compared to responses elicited by a wild type stem cell.

62 . The hypoimmunogenic stem cell of claim 57 , wherein the hypoimmunogenic stem cell elicits reduced immune responses from T cells, NK cells, and/or macrophages in a subject into which the hypoimmunogenic stem cell is transplanted as compared to responses elicited by a wild type stem cell.

63 . The hypoimmunogenic stem cell of claim 62 , wherein the T cells are CD8+ T cells.

64 . The hypoimmunogenic stem cell of claim 57 , wherein the safe harbor locus is an AAVS1 locus.

65 . The hypoimmunogenic stem cell of claim 57 , wherein the safe harbor locus is a HPRT locus.

66 . A nucleic acid molecule comprising:
i) an expression cassette comprising:
a) a first nucleic acid sequence encoding PD-L1,
b) a second nucleic acid sequence encoding HLA-G, and
c) a third nucleic acid sequence encoding CD47,
ii) a promoter that induces overexpression of PD-L1, HLA-G, and CD47, and iii) a first arm sequence and a second arm sequence, wherein the first arm sequence is located upstream of the expression cassette and the promoter and the second arm sequence is located downstream of the expression cassette and the promoter, and wherein the arm sequences are homologous to a safe harbor locus of a target stem cell.

67 . The nucleic acid molecule of claim 66 , wherein the safe harbor locus is an AAVS1 locus.

68 . The nucleic acid molecule of claim 66 , wherein the safe harbor locus is a HPRT locus.

69 . The nucleic acid molecule of claim 66 , wherein the promoter is a CAGGS promoter.

70 . A hypoimmunogenic stem cell comprising two exogenous genes encoding PD-L1 and CD47, respectively, wherein the two exogenous genes are inserted into a safe harbor locus of at least one allele of the hypoimmunogenic stem cell.

71 . The hypoimmunogenic stem cell of claim 70 , further comprising reduced expression of HLA-A, HLA-B, HLA-C, and CIITA.

72 . The hypoimmunogenic stem cell of claim 71 , wherein the hypoimmunogenic stem cell comprises no expression of HLA-A, HLA-B, HLA-C, and CIITA.

73 . The hypoimmunogenic stem cell of claim 70 , wherein the hypoimmunogenic stem cell retains differentiation potential.

74 . The hypoimmunogenic stem cell of claim 70 , wherein the hypoimmunogenic stem cell elicits reduced immune responses from T cells and/or macrophages in a subject into which the hypoimmunogenic stem cell is transplanted as compared to immune responses elicited by a wild type stem cell.

75 . The hypoimmunogenic stem cell of claim 74 , wherein the T cells are CD8+ T cells.

76 . The hypoimmunogenic stem cell of claim 70 , wherein the safe harbor locus is an AAVS1 locus.

77 . The hypoimmunogenic stem cell of claim 70 , wherein the safe harbor locus is a HPRT locus.

78 . A nucleic acid molecule comprising:
i) an expression cassette comprising:
a) a first nucleic acid sequence encoding PD-L1, and
b) a second nucleic acid sequence encoding CD47,
ii) a promoter that induces overexpression of PD-L1 and CD47, and iii) a first arm sequence and a second arm sequence, wherein the first arm sequence is located upstream of the expression cassette and the promoter and the second arm sequence is located downstream of the expression cassette and the promoter, and wherein the arm sequences homologous to a safe harbor locus of a target stem cell.

79 . The nucleic acid molecule of claim 78 , wherein the safe harbor locus is an AAVS1 locus.

80 . The nucleic acid molecule of claim 78 , wherein the safe harbor locus is a HPRT locus.

81 . The nucleic acid molecule of claim 78 , wherein the promoter is a CAGGS promoter.

Assignees

• Harvard College

Inventors

• Meissner Torsten B
• Ferreira Leonardo M R
• Strominger Jack L
• Cowan Chad A

Classifications

• C12N5/0606 Primary

  Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696) · CPC title

• C12N2510/00

  Genetically modified cells · CPC title

• C07K14/70532

  B7 molecules, e.g. CD80, CD86 · CPC title

• C07K14/70503

  Immunoglobulin superfamily · CPC title

• C07K14/70539 Primary

  MHC-molecules, e.g. HLA-molecules · CPC title