Genomic engineering of pluripotent cells
Provided are methods and compositions for obtaining genome-engineered iPSCs, and derivative cells with stable and functional genome editing at selected sites. Also provided are cell populations or clonal cell lines derived from genome-engineered iPSCs, which comprise targeted integration of one or more exogenous polynucleotides, and/or in/dels in one or more selected endogenous genes.
1. A cell or population thereof, wherein (i) the cell is an induced pluripotent stem cell (iPSC), a clonal iPSC, or an iPSC line cell; and (ii) the cell comprises: (a) one or more exogenous polynucleotides at an endogenous T cell receptor (TCR) alpha locus, wherein at least one of the one or more exogenous polynucleotides encodes a chimeric antigen receptor (CAR), and wherein the one or more exogenous polynucleotides encoding the CAR is expressed under the control of an endogenous promoter of said TCR alpha locus; and (b) introduced or increased expression of CD16, wherein the CD16 is a high affinity non-cleavable CD16 (hnCD16).
2. The cell or population thereof of claim 1 , wherein the one or more exogenous polynucleotides is present in a nucleic acid encoding a constant region in the TCR alpha locus.
3. The cell or population thereof of claim 2 , wherein an endogenous TCR alpha gene is knocked out.
4. The cell or population thereof of claim 3 , wherein the CAR is expressed at about the same level as an endogenous TCR alpha in a reference cell in which the endogenous TCR alpha gene is not knocked out.
5. The cell or population thereof of claim 3 , wherein the CAR is expressed at about the same developmental stage as an endogenous TCR alpha in a reference cell in which the endogenous TCR alpha gene is not knocked out.
6. The cell or population thereof of claim 1 , wherein the CAR comprises a CD19 related CAR.
7. The cell or population thereof of claim 1 , wherein the cell is an iPSC obtained from reprogramming a T cell to a pluripotent state (TiPSC) and genomically editing the TiPSC.
8. The cell or population thereof of claim 7 , wherein the cell is an iPSC obtained from reprogramming a T cell comprising the one or more exogenous polypeptides present in a nucleic acid encoding a constant region in the TCR alpha locus.
9. The cell or population thereof of claim 1 , further comprising deletion or reduced expression in at least one of B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP and any gene in the chromosome 6p21 region; and/or introduced or increased expression in at least one of HLA-E, HLA-G, CD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, Fc receptor, an engager, and a surface triggering receptor for coupling with bi-, multi-specific or universal engagers.
10. The cell or population thereof of claim 1 , further comprising one or more polynucleotides encoding one or more of (i) a CRISPR endonuclease, and (ii) a guide RNA that directs cleavage within a target TCR locus by the CRISPR endonuclease.
11. The cell or population thereof of claim 1 , wherein the one or more exogenous polynucleotides is inserted at the endogenous T cell receptor (TCR) alpha locus using a CRISPR endonuclease.
12. The cell or population thereof of claim 1 , further comprising introduced or increased expression of CD3.
13. A cell or population thereof, wherein (i) the cell is an induced pluripotent stem cell (iPSC), a clonal iPSC, or an iPSC line cell; (ii) the cell comprises a polynucleotide encoding at least one chimeric antigen receptor (CAR) introduced into a T cell receptor (TCR) alpha locus; (iii) an endogenous TCR alpha gene is knocked out; and (iv) expression of the polynucleotide encoding at least one CAR is under control of an endogenous TCR promoter of the TCR alpha locus.
14. The cell or population thereof of claim 13 , wherein the polynucleotide encoding the at least one CAR is present in a nucleic acid encoding a constant region in the TCR alpha locus.
15. The cell or population thereof of claim 14 , wherein the at least one CAR comprises a CD19 related CAR.
16. The cell or population thereof of claim 13 , further comprising deletion or reduced expression in at least one of B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP and any gene in the chromosome 6p21 region; and/or introduced or increased expression in at least one of HLA-E, HLA-G, CD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, Fc receptor, an engager, and a surface triggering receptor for coupling with bi-, multi-specific or universal engagers.
17. The cell or population thereof of claim 13 , further comprising introduced or increased expression of CD16, wherein the CD16 is a high affinity non-cleavable CD16 (hnCD16).
18. The cell or population thereof of claim 13 , further comprising introduced or increased expression of CD3.
19. The cell or population thereof of claim 13 , further comprising deletion or reduced expression of at least one of B2M and CIITA, and optionally introduced or increased expression in HLA-G.
20. The cell or population thereof of claim 13 , wherein the CAR is expressed at about the same level as an endogenous TCR alpha in a reference cell in which the endogenous TCR alpha gene is not knocked out.
21. The cell or population thereof of claim 13 , wherein the CAR is expressed at about the same developmental stage as an endogenous TCR alpha in a reference cell in which the endogenous TCR alpha gene is not knocked out.
22. The cell or population thereof of claim 13 , wherein the cell is an iPSC obtained from reprogramming a T cell to a pluripotent state (TiPSC) and genomically editing the TiPSC.
23. The cell or population thereof of claim 22 , wherein the cell is an iPSC obtained from reprogramming a T cell comprising the polynucleotide present in a nucleic acid encoding a constant region in the TCR alpha locus.
24. A cell or population thereof, wherein (i) the cell is an induced pluripotent stem cell (iPSC), a clonal iPSC, or an iPSC line cell; and (ii) the cell comprises: (a) one or more exogenous polynucleotides present in an endogenous T cell receptor (TCR) alpha locus, wherein the at least one or more exogenous polynucleotides encodes a chimeric antigen receptor (CAR), and the exogenous polynucleotide encoding the CAR is expressed under the control of an endogenous promoter of said TCR alpha locus; and (b) deletion or reduced expression in at least one of B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP and any gene in the chromosome 6p21 region; and/or introduced or increased expression in at least one of HLA-E, HLA-G, CD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2a R, Fc receptor, an engager, and a surface triggering receptor for coupling with bi-, multi-specific or universal engagers.