Gene-edited natural killer cells

Abstract

The present invention relates to, inter alia, an engineered cell (e.g., iPSC, IPS-derived NK, or NK cell) comprising a disrupted B2M gene and an inserted polynucleotide encoding one or more of SERPINB9, a fusion of IL15 and IL15Rα, and/or HLA-E. The engineered cell can further comprise a disrupted CIITA gene and an inserted polynucleotide encoding a CAR, wherein the CAR can be an anti-BCMA CAR or an anti-CD30 CAR. The engineered cell may further comprise a disrupted ADAM17 gene, a disrupted FAS gene, a disrupted CISH gene, and/or a disrupted REGNASE-1 gene. Methods for producing the engineered cells are also provided, and therapeutic uses of the engineered cells are also described. Guide RNA sequences targeting described target sequences are also described.

1 - 80 . (canceled) 
     
     
         81 . A method for treating a subject in need thereof, comprising administering an engineered cell to the subject, wherein the engineered cell comprises:
 (a) a disrupted beta-2-microglobulin (B2M) gene; and   (b) an insertion of a first polynucleotide and a second polynucleotide in the disrupted B2M gene, the first polynucleotide encoding a SERPINB9 protein and the second polynucleotide encoding a fusion protein of interleukin 15 (IL15) and interleukin 15 receptor subunit alpha (IL15Rα); and 
 wherein the engineered cell expresses the SERPINB9 protein and the fusion protein of IL15 and IL15Rα, and the engineered cell has a disrupted expression of B2M. 
     
     
         82 . The method of  claim 81 , wherein the engineered cell comprises a disrupted Class II major histocompatibility complex transactivator (CIITA) gene and has a disrupted expression of CIITA. 
     
     
         83 . The method of  claim 82 , wherein the engineered cell comprises an insertion of a third polynucleotide encoding a chimeric antigen receptor (CAR) and expresses the CAR. 
     
     
         84 . The method of  claim 83 , wherein the third polynucleotide encoding the CAR is linked to a fourth polynucleotide encoding a human leukocyte antigen E (HLA-E) trimer, and the cell expresses the HLA-E trimer. 
     
     
         85 . The method of  claim 84 , wherein the third polynucleotide and the fourth polynucleotide are inserted in the disrupted CIITA gene. 
     
     
         86 . The method of  claim 85 , wherein the engineered cell comprises a disrupted cytokine-inducible SH2-containing protein (CISH) gene, a disrupted Fas cell surface death receptor (FAS) gene, or both; and wherein the cell has a disrupted expression of CISH, a disrupted expression of FAS, or both. 
     
     
         87 . The method of  claim 81 , the engineered cell is a lineage-restricted progenitor cell or fully differentiated somatic cell. 
     
     
         88 . The method of  claim 87 , comprising obtaining or having obtained the lineage-restricted progenitor cell or fully differentiated somatic cell from an edited stem cell, wherein the edited stem cell comprises:
 (a) a disrupted beta-2-microglobulin (B2M) gene; and   (b) an insertion of a first polynucleotide and a second polynucleotide in the disrupted B2M gene, the first polynucleotide encoding a SERPINB9 protein and the second polynucleotide encoding a fusion protein of interleukin 15 (IL15) and interleukin 15 receptor subunit alpha (IL15Rα).   
     
     
         89 . The method of  claim 88 , wherein the stem cell is an induced pluripotent stem cell (iPSC), a hematopoietic stem cell, an embryonic stem cell, or an adult stem cell. 
     
     
         90 . The method of  claim 87 , wherein the lineage-restricted progenitor cell is an hematopoietic progenitor cell, an mesodermal cell, a definitive hemogenic endothelium cell, a definitive hematopoietic stem or progenitor cell, a CD34+ cell, an multipotent progenitor (MPP) cell, a common lymphoid progenitor cell, a T cell progenitor, a NK cell progenitor, a pancreatic endoderm progenitor cell, a pancreatic endocrine progenitor cell, an mesenchymal progenitor cell, an muscle progenitor cell, a blast cell, or a neural progenitor cell; and wherein the fully differentiated somatic cell is an hematopoietic cell, a pancreatic beta cell, an epithelial cell, an endodermal cell, an macrophage, an hepatocyte, an adipocyte, a kidney cell, a blood cell, a cardiomyocyte, or an immune system cell. 
     
     
         91 . The method of  claim 81 , wherein the engineered cell is a natural killer cell. 
     
     
         92 . The method of  claim 81 , wherein the subject has a cancer. 
     
     
         93 . The method of  claim 92 , wherein the cancer is multiple myeloma, Hodgkin’s lymphoma, lung cancer, leukemia, B-cell acute lymphoblastic leukemia (B-ALL), B-cell non-Hodgkin’s lymphoma (B-NL), chronic lymphocytic leukemia (C-CLL), acute myeloid leukemia (AML), T cell lymphoma, T cell leukemia, clear cell renal cell carcinoma (ccRCC), thyroid cancer, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, melanoma, ovarian cancer, colon cancer, glioblastoma, cervical cancer, or a combination thereof. 
     
     
         94 . The method of  claim 81 , wherein the subject is human. 
     
     
         95 . The method of  claim 81 , wherein the engineered cell is expanded in culture prior to administration to the subject. 
     
     
         96 . The method of  claim 81 , wherein the subject is administered the engineered cell at a dose in the range of about 1 × 10 7  to 1 × 10 9  engineered cells. 
     
     
         97 . The method of  claim 96 , wherein the administration is via injection or infusion. 
     
     
         98 . The method of  claim 86 , the administration is intravenous, intrathecal, intraperitoneal, intraspinal, intracerebrospinal, or intrasternal. 
     
     
         99 . The method of  claim 86 , wherein the subject has no chronic immune suppression. 
     
     
         100 . The method of  claim 86 , wherein the subject has received a lymphodepleting regimen.