Genomic engineering of pluripotent cells

1. A method of obtaining a human cell or population thereof,

wherein (i) the human cell is an induced pluripotent stem cell (iPSC); (ii) the iPSC comprises a polynucleotide encoding at least one chimeric antigen receptor (CAR) introduced into a constant region of a T cell receptor (TCR) locus; (iii) an endogenous TCR gene of the iPSC is knocked out, and (iv) the iPSC comprises introduced or increased expression of CD3; and,

wherein the method comprises steps of (I) or (II):

(I):

(i) reprogramming a T cell to an iPSC; and

(ii) genomically editing the iPSC to knock out the TCR, knock in a polynucleotide encoding at least one CAR at the constant region of the TCR locus, and introducing into the genome of the iPSC a polynucleotide encoding CD3;

or, (II):

(i) genomically editing a T cell to knock out the TCR, knock in a polynucleotide encoding at least one CAR at the constant region of the TCR locus, and introducing into the genome of the T cell a polynucleotide encoding CD3, thereby obtaining a genomically edited T cell; and

(ii) reprogramming the genomically edited T cell of step (II)(i) to an iPSC;

thereby obtaining said human cell or population thereof.

2. The method of claim 1 , wherein knocking out the TCR and knocking in the polynucleotide encoding at least one CAR are simultaneous.

3. The method of claim 1 , wherein knocking out the TCR and knocking in the polynucleotide encoding at least one CAR are sequential.

4. The method of claim 1 , wherein the genomically editing further comprises knocking in an additional polynucleotide encoding a protein of interest.

5. The method of claim 1 , wherein expression of the at least one CAR is under control of an endogenous TCR promoter.

6. The method of claim 2 , wherein knocking out the TCR uses a CRISPR endonuclease.

7. The method of claim 2 , wherein knocking in the polynucleotide encoding the at least one CAR uses a CRISPR endonuclease.

8. The method of claim 1 , wherein the at least one CAR comprises a CD19 CAR.

9. The method of claim 1 , wherein the genomic editing of (I)(ii) or (II)(i) further comprises deletion of or reducing 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, 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.

10. The method of claim 9 , wherein the genomic editing of (I)(ii) or (II)(i) further comprises introducing or increasing expression of CD16, wherein the CD16 is a high affinity non-cleavable CD16 (hnCD16).

11. The method of claim 9 , wherein the genomic editing of (I)(ii) or (II)(i) further comprises deletion or reduced expression of at least one of B2M and CIITA, and optionally introduced or increased expression in HLA-G.

12. The method of claim 1 , further comprising cryopreserving said iPSC, wherein the cryopreserved iPSC maintains differentiation potential.

13. The method of claim 1 , further comprising differentiating said iPSC to a derived hematopoietic lineage cell, wherein the derived hematopoietic lineage cell retains the genomic editing of the iPSC.

14. The method of claim 13 , wherein the derived hematopoietic lineage cell comprises a mesodermal cell, a hemogenic endothelium cell, a CD34 cell, a hematopoietic stem and progenitor cell, a hematopoietic multipotent progenitor cell, a T cell progenitor, a NK cell progenitor, a T cell, an NKT cell, an NK cell, or a B cell.

15. The method of claim 13 , wherein the derived hematopoietic lineage cell has a longer telomere length than its primary cell counterpart.

16. The method of claim 1 , wherein the T cell is donor-, disease-, or treatment response-specific.

17. A method of manufacturing therapeutic cells comprising obtaining a human cell or population thereof according to claim 1 , and combining the human cell or population thereof with a pharmaceutically acceptable carrier.

18. The method of claim 4 , wherein the protein of interest is a CD16 or a variant thereof.

19. The method of claim 1 , wherein the method comprises the steps of (i) reprogramming the T cell to the iPSC; and (ii) genomically editing the iPSC to knock out the TCR, knock in the polynucleotide encoding at least one CAR at the constant region of the TCR locus, and introducing into the genome of the iPSC the polynucleotide encoding CD3.

20. The method of claim 19 , further comprising differentiating the iPSC into a cell of a desired cell type.

21. The method of claim 1 , wherein the method comprises the steps of (i) genomically editing the T cell to knock out the TCR, knock in the polynucleotide encoding at least one CAR at the constant region of the TCR locus, and introducing into the genome of the T cell the polynucleotide encoding CD3, thereby obtaining the genomically edited T cell; and (ii) reprograming the genomically edited T cell of step (II)(i) to the iPSC.

22. The method of claim 21 , further comprising differentiating the iPSC into a cell of a desired cell type.

23. A method of manufacturing therapeutic cells comprising obtaining a human cell or population thereof according to claim 13 , and combining the human cell or population thereof with a pharmaceutically acceptable carrier.

24. The method of claim 1 , wherein the CD3 is a surface-expressed CD3.

25. The method of claim 1 , wherein the genomic editing of (I)(ii) or (II)(i) further comprises knocking out B2M.

26. The method of claim 25 , wherein the genomic editing of (I)(ii) or (II)(i) further comprises introducing a polynucleotide encoding HLA-E or HLA-G.

27. The method of claim 1 , wherein the iPSC is capable of differentiating into a T cell.

28. The method of claim 13 , wherein the derived hematopoietic lineage cell is a T cell.