Modified stem cells and uses thereof
Stem cells are modified to express an extracellular component including a tag cassette. The tag cassette can be used to detect, enrich, isolate, activate, track, deplete, or eliminate modified cells. The cells can be administered before or following differentiation into a more committed cell type.
1. A genetically modified stem cell or differentiated cell comprising a nucleic acid encoding a chimeric molecule wherein, when expressed by the genetically modified stem cell or differentiated cell, the chimeric molecule comprises
an extracellular component and an intracellular component connected by a hydrophobic portion, wherein the extracellular component consists essentially of
two or three tag cassettes wherein at least two tag cassettes have the amino acid sequence as set forth in SEQ ID NO.: 1,
two or more linker sequences wherein at least two linker sequences have the amino acid sequence as set forth in SEQ ID NO.: 5, and
one or more spacer regions, wherein at least one spacer region comprises an Fc domain and a human IgG4 heavy chain hinge,
wherein a first tag cassette and a second tag cassette having the amino acid sequence as set forth in SEQ ID NO.: 1 are each adjacent to at least one linker sequence having the amino acid sequence as set forth in SEQ ID NO.: 5,
wherein the chimeric molecule includes from amino-terminus to carboxy-terminus: (i) the first tag cassette having the amino acid sequence as set forth in SEQ ID NO.: 1 adjacent to the at least one linker sequence having the amino acid sequence as set forth in SEQ ID NO.: 5, (ii) the second tag cassette having the amino acid sequence as set forth in SEQ ID NO.: 1 adjacent to a linker sequence having the amino acid sequence as set forth in SEQ ID NO.: 5, (iii) the at least one spacer region, and (iv) the hydrophobic portion.
2. The genetically modified stem cell or differentiated cell of claim 1 , wherein the differentiated cell is a cardiomyocyte, a neuron, an oligodendrocyte, an insulin producing beta cell, or a retinal cell.
3. The genetically modified stem cell or differentiated cell of claim 1 , wherein the first tag cassette having the amino acid sequence as set forth in SEQ ID NO.: 1 and the adjacent at least one linker sequence having the amino acid sequence as set forth in SEQ ID NO.: 5 are within an amino acid sequence as set forth in SEQ ID NO.: 19, SEQ ID NO.: 20, SEQ ID NO.: 21, SEQ ID NO.: 22, SEQ ID NO.: 23, or SEQ ID NO.: 24.
4. The genetically modified stem cell or differentiated cell of claim 1 , wherein the hydrophobic portion comprises a human transmembrane domain.
5. The genetically modified stem cell or differentiated cell of claim 1 , wherein the chimeric molecule consists essentially of from amino-terminus to carboxy-terminus: (i) the first tag cassette having the amino acid sequence as set forth in SEQ ID NO.: 1 adjacent to the at least one linker sequence having the amino acid sequence as set forth in SEQ ID NO.:5, (ii) the second tag cassette having the amino acid sequence as set forth in SEQ ID NO.: 1 adjacent to a linker sequence having the amino acid sequence as set forth in SEQ ID NO.:5, (iii) the at least one spacer region, and (iv) the hydrophobic portion.
6. The genetically modified stem cell or differentiated cell of claim 1 , wherein the hydrophobic portion comprises a CD28 transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, or a CD27 transmembrane domain.
7. The genetically modified stem cell or differentiated cell of claim 1 , wherein the hydrophobic portion comprises a human transmembrane domain.
8. The genetically modified stem cell or differentiated cell of claim 1 , formulated for administration to a subject.
9. The genetically modified stem cell or differentiated cell of claim 2 , wherein the neuron is a motor neuron, a dopaminergic neuron, a glutamatergic neuron, or a GABAergic neuron.
10. The genetically modified stem cell or differentiated cell of claim 1 , wherein the intracellular component comprises an intracellular signaling domain.
11. A genetically modified stem cell or differentiated cell comprising a nucleic acid encoding a chimeric molecule wherein, when expressed by the genetically modified stem cell or differentiated cell, the chimeric molecule comprises
an extracellular component and an intracellular component connected by a hydrophobic portion, wherein the extracellular components consists essentially of
a first tag cassette, a second tag cassette, and a third tag cassette each having the amino acid sequence as set forth in SEQ ID NO.: 1,
three or more linker sequences wherein at least three linker sequences have the amino acid sequence as set forth in SEQ ID NO.: 5, and
two or more spacer regions, wherein at least one spacer region comprises an Fc domain and a human IgG4 heavy chain hinge, and
wherein the first tag cassette, the second tag cassette and the third tag cassette having the amino acid sequence as set forth in SEQ ID NO.: 1 are each adjacent to at least one linker sequence having the amino acid sequence as set forth in SEQ ID NO.: 5,
wherein the chimeric molecule includes from amino-terminus to carboxy-terminus: (i) the first tag cassette having the amino acid sequence as set forth in SEQ ID NO.: 1 adjacent to the at least one linker sequence having the amino acid sequence as set forth in SEQ ID NO.: 5, (ii) the second tag cassette having the amino acid sequence as set forth in SEQ ID NO.: 1 adjacent to a linker sequence having the amino acid sequence as set forth in SEQ ID NO.: 5, (iii) the at least one spacer region, wherein the at least one spacer region is a first spacer region, (iv) the third tag cassette having the amino acid sequence as set forth in SEQ ID NO.: 1 adjacent to a linker sequence having the amino acid sequence as set forth in SEQ ID NO.: 5, (v) a second spacer region comprising an Fc domain and a human IgG4 heavy chain hinge, and (vi) the hydrophobic portion.
12. A genetically modified stem cell or differentiated cell comprising a nucleic acid encoding a chimeric molecule wherein, when expressed by the genetically modified stem cell or differentiated cell, the chimeric molecule comprises
an extracellular component linked to a hydrophobic portion, wherein the extracellular component consists essentially of
one or more tag cassettes wherein at least one tag cassette has the amino acid sequence set as forth in SEQ ID NO.: 1,
one or more linker sequences wherein at least one linker sequence has the amino acid sequence as set forth in SEQ ID NO.: 5, and
one or more spacer regions, wherein at least one spacer region comprises an Fc domain and a human IgG4 heavy chain hinge, and
wherein the at least one tag cassette having the amino acid sequence as set forth in SEQ ID NO.: 1 is adjacent to the at least one linker sequence having the amino acid sequence as set forth in SEQ ID NO.: 5,
wherein the hydrophobic portion links the extracellular component to an intracellular component, and
wherein the chimeric molecule includes from amino-terminus to carboxy-terminus: (i) the at least one tag cassette having the amino acid sequence as set forth in SEQ ID NO.: 1 adjacent to the at least one linker sequence having the amino acid sequence as set forth in SEQ ID NO.: 5, (ii) the at least one spacer region, (iii) the hydrophobic portion; and (iv) the intracellular component.
13. The genetically modified stem cell or differentiated cell of claim 12 , wherein the first tag cassette having the amino acid sequence as set forth in SEQ ID NO.: 1 and the adjacent at least one linker sequence having the amino acid sequence as set forth in SEQ ID NO.: 5 are within an amino acid sequence as set forth in SEQ ID NO.: 19, SEQ ID NO.: 20, SEQ ID NO.: 21, SEQ ID NO.: 22, SEQ ID NO.: 23, or SEQ ID NO.: 24.
14. The genetically modified stem cell or differentiated cell of claim 12 , wherein the differentiated cell is a cardiomyocyte, a neuron, an oligodendrocyte, an insulin producing beta cell, a retinal cell, a myeloid progenitor, or a lymphoid progenitor.
15. The genetically modified stem cell or differentiated cell of claim 14 , wherein the neuron is a motor neuron, a dopaminergic neuron, a glutamatergic neuron, or a GABAergic neuron.
16. The genetically modified stem cell or differentiated cell of claim 12 , wherein the hydrophobic portion comprises a CD28 transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, or a CD27 transmembrane domain.
17. The genetically modified stem cell or differentiated cell of claim 12 , formulated for administration to a subject.