Genomic engineering of pluripotent cells

Abstract

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 method of obtaining a human cell or population thereof, wherein (i) the human cell is an TCR-null induced pluripotent stem cell (iPSC); (ii) the TCR-null 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 TCR-null iPSC is capable of differentiating into a T cell; 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 and knock in a polynucleotide encoding at least one CAR at the constant region of the TCR locus; 
   or, (II):
 (i) genomically editing a T cell to knock out the TCR and knock in a polynucleotide encoding at least one CAR at the constant region of the TCR locus, thereby obtaining a genomically edited T cell; and 
 (ii) reprogramming the genomically edited T cell of step (II)(i) to an iPSC; 
   thereby obtaining the TCR-null iPSC 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, 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. 
     
     
         10 . The method of  claim 9 , wherein the genomic editing of (I)(ii) or (II)(i) further comprises introducing or increasing expression of CD16. 
     
     
         11 . The method of  claim 10 , wherein the CD16 is a high affinity non-cleavable CD16 (hnCD16). 
     
     
         12 . 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. 
     
     
         13 . The method of  claim 1 , further comprising cryopreserving the TCR-null iPSC, wherein the cryopreserved TCR-null iPSC maintains differentiation potential. 
     
     
         14 . The method of  claim 1 , further comprising differentiating the TCR-null iPSC to a derived hematopoietic lineage cell, wherein the derived hematopoietic lineage cell retains the genomic editing of the TCR-null iPSC. 
     
     
         15 . The method of  claim 14 , 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. 
     
     
         16 . The method of  claim 14 , wherein the derived hematopoietic lineage cell has a longer telomere length than its primary cell counterpart. 
     
     
         17 . The method of  claim 1 , wherein the T cell is donor-, disease-, or treatment response-specific. 
     
     
         18 . 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. 
     
     
         19 . A process of using therapeutic human cells made by the method of  claim 18  for adoptive cell therapy, comprising administering to a subject in need of the therapy one or multiple doses of said human cell or population thereof at a therapeutically sufficient amount. 
     
     
         20 . The process of  claim 19 , wherein the subject has an autoimmune disorder, a hematological malignancy, a solid tumor, or an infection associated with HIV, RSV, EBY, CMV, adenovirus, or BK polyomavirus. 
     
     
         21 . The process of  claim 19 , wherein the adoptive cell therapy is autologous. 
     
     
         22 . The process of  claim 19 , wherein the adoptive cell therapy is allogeneic. 
     
     
         23 . A method of administering a cell, the method comprising administering to a subject the cell of the desired cell type obtained according to  claim 14 . 
     
     
         24 . The method of  claim 23 , further comprising administering to the subject an engager. 
     
     
         25 . The method of  claim 24 , wherein the engager is bi-specific or multi-specific. 
     
     
         26 . A method of manufacturing therapeutic cells comprising obtaining a derived hematopoietic lineage cell or a population thereof according to  claim 14 , and combining the derived hematopoietic lineage cell or a population thereof with a pharmaceutically acceptable carrier. 
     
     
         27 . The method of  claim 1 , wherein the genomic editing of (I)(ii) or (II)(i) further comprises introducing or increasing expression of CD3.