Universal donor stem cells and related methods
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 do not express one or more MHC-I and MHC-II human leukocyte antigens. Similarly, in certain embodiments, the universal donor stem cells disclosed herein do not express one or more human leukocyte antigens (e.g., HLA-A, HLA-B and/or HLA-C) corresponding to MHC-I and MHC-II human leukocyte antigens, thereby rendering such cells hypoimmunogenic.
1 . A method of generating a genetically modified stem cell comprising:
(i) introducing one or more genetic modifications into a stem cell that reduce cell surface expression of one or more MHC-I human leukocyte antigen molecules relative to an unmodified cell of the same type and/or one or more genetic modifications that reduce cell surface expression of one or more MHC-II human leukocyte antigen molecules relative to an unmodified cell of the same type, and
(ii) introducing a nucleic acid encoding CD47 into the stem cell,
wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell, and
wherein the genetically modified stem cell comprises one or more genetic modifications that reduce cell surface expression of one or more MHC-I human leukocyte antigen molecules relative to an unmodified cell of the same type and/or one or more genetic modifications that reduce cell surface expression of one or more MHC-II human leukocyte antigen molecules relative to an unmodified cell of the same type, and comprise a nucleic acid encoding CD47.
2 . The method of claim 1 , wherein the introduction of the one or more genetic modifications comprises introducing one or more indels in one or more genes encoding an MHC-I human leukocyte antigen molecule, thereby resulting in the reduced cell surface expression of the one or more MHC-I human leukocyte antigen molecules relative to an unmodified cell of the same type.
3 . The method of claim 2 , wherein the introduction of the one or more genetic modifications comprises introducing one or more indels in an HLA-A gene, an HLA-B gene, an HLA-C gene, or a combination thereof in the genome of the genetically modified cell, thereby resulting in the reduced cell surface expression of the one or more MHC-I human leukocyte antigen molecules relative to an unmodified cell of the same type.
4 . The method of claim 2 , wherein the introduction of the one or more genetic modifications comprises introducing one or more indels in a β2M gene in the genome of the genetically modified cell, thereby resulting in the reduced cell surface expression of the one or more MHC-I human leukocyte antigen molecules relative to an unmodified cell of the same type.
5 . The method of claim 4 , wherein the one or more indels comprises a β2M gene knock out.
6 . The method of claim 5 , wherein the genetically modified stem cell is a β2M −/− genetically modified stem cell.
7 . The method of claim 1 , wherein the introduction of the one or more genetic modifications comprises introducing one or more indels in one or more genes encoding a transcriptional regulator of an MHC-I human leukocyte antigen molecule, thereby resulting in the reduced cell surface expression of the one or more MHC-I human leukocyte antigen molecules relative to an unmodified cell of the same type.
8 . The genetically modified cell of claim 7 , wherein the introduction of the one or more genetic modifications comprise introducing one or more indels in a TAPI gene, an NLRC5 gene, an RFX5 gene, an RFXAP gene, an RFXANK gene, an NFY-A gene, an NFY-B gene, an NFY-C gene, an IRF-1 gene, or a combination thereof.
9 . The method of claim 1 , wherein the introduction of the one or more genetic modifications comprises introducing one or more indels in one or more genes encoding an MHC-II human leukocyte antigen molecule, thereby resulting in the reduced cell surface expression of the one or more MHC-II human leukocyte antigen molecules.
10 . The method of claim 1 , wherein the introduction of the one or more genetic modifications comprises introducing one or more indels in one or more genes encoding a transcriptional regulator of an MHC-II human leukocyte antigen molecule, thereby resulting in the reduced cell surface expression of the one or more MHC-II human leukocyte antigen molecules relative to an unmodified cell of the same type.
11 . The method of claim 10 , wherein the introduction of the one or more genetic modifications comprises introducing one or more indels in a class II major histocompatibility complex transactivator (CIITA) gene in the genome of the genetically modified cell, thereby resulting in the reduced cell surface expression of the one or more MHC-II human leukocyte antigen molecules.
12 . The method of claim 11 , wherein the one or more indels comprises a CIITA gene knock out.
13 . The method of claim 12 , wherein the genetically modified stem cell is a CIITA −/− genetically modified stem cell.
14 . The method of claim 1 , wherein the genetically modified stem cell is a β2M −/− CIITA −/− genetically modified stem cell.
15 . The method of claim 1 , wherein the nucleic acid encoding CD47 is inserted into at least one allele of a safe harbor locus of the genetically modified stem cell.
16 . The method of claim 15 , wherein the safe harbor locus comprises an AAVS1 locus.
17 . The method of claim 1 , wherein the introduction of the one or more genetic modifications comprise introducing one or more indels in a TAPI gene, an NLRC5 gene, an RFX5 gene, an RFXAP gene, an RFXANK gene, an NFY-A gene, an NFY-B gene, an NFY-C gene, an IRF-1 gene, or any combination thereof.
18 . The method of claim 1 , further comprising differentiating the genetically modified stem cell into a cardiomyocyte, an endothelial cell, a hepatocyte, a hepatocyte-like cell, a beta cell, a mesenchymal progenitor cell, a neural progenitor cell, a macrophage, or a T cell.
19 . The method of claim 1 , further comprising differentiating wherein the genetically modified stem cell into a T cell.
20 . The method of claim 1 , further comprising differentiating the genetically modified stem cell into a CD8+ T cell.
21 . The method of claim 1 , further comprising differentiating the genetically modified stem cell into a beta cell.