# US 12,600,944 B2 · Multiplex genome editing of immune cells to enhance functionality and resistance to suppressive environment > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,600,944 B2 - Document ID: US12600944B2 - Status: Granted - Kind: B2 - Application number: 17/309,408 - Filed: 2019-11-27 - Published: 2022-02-03 - Granted: 2026-04-14 - Earliest claimed: 2018-11-28 - Assignees: Board of Regents, The University of Texas System - Applicants: Board of Regents, The University of Texas System - Inventors: Rafet Basar; Elizabeth Shpall; Katy Rezvani - Web page: https://patentgrep.com/patent/US12600944B2 ## CPC - C12N 5/0636: {T lymphocytes} - A61K 40/15: Natural-killer [NK] cells; Natural-killer T [NKT] cells - A61K 40/31: Chimeric antigen receptors [CAR] - A61K 40/42: Cancer antigens - A61P 35/00: Antineoplastic agents - C07K 14/7051: {T-cell receptor (TcR)-CD3 complex} - C12N 5/0646: {Natural killers cells [NK], NKT cells} - C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} - C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein} - C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} - C12N 15/85: for animal cells - C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR] - C12N 2510/00: Genetically modified cells - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract Provided herein are methods for producing immune cells with disruption of multiple genes. Further provided are methods for inserting a chimeric antigen receptor at a gene locus of an immune cell. ## Claims ### Claim 1 An engineered natural killer (NK) cell with knockout of at least two gene products, wherein the at least two gene products are selected from the group consisting of NK Cell Receptor A (NKG2A) and Cytokine Inducible SH2-Containing Protein (CISH), wherein knockout comprises the elimination or reduction of level of expression of the at least two gene products, compared to the level of expression of the at least two gene products in the absence of the knockout. ### Claim 2 (depends on 1) The engineered NK cell of claim 1, wherein the engineered NK cell is produced according to an in vitro method for the disruption of the at least two genes. ### Claim 3 (depends on 1) The engineered NK cell of claim 1, wherein the engineered NK cell is isolated from cord blood. ### Claim 4 (depends on 1) The engineered NK cell of claim 1, wherein the NK cell is engineered to express a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain that targets one or more tumor associated antigens. ### Claim 5 (depends on 4) The engineered NK cell of claim 4, wherein the CAR is inserted at a gene selected from the group consisting of NKG2A and CISH. ### Claim 6 (depends on 5) The engineered NK cell of claim 5, wherein the CAR is under the control of the endogenous promoter of said gene. ### Claim 7 (depends on 5) The engineered NK cell of claim 5, wherein the CAR was inserted at the gene locus by CRISPR-mediated gene editing. ### Claim 8 (depends on 1) The engineered NK cell of claim 1, wherein the engineered NK cell is engineered to express a heterologous cytokine selected from the group consisting of IL-7, IL-2, IL-15, IL-12, IL-18, IL-21, and a combination thereof. ### Claim 9 (depends on 1) The engineered NK cell of claim 1, wherein the engineered NK cell further comprises a polynucleotide encoding a CAR. ### Claim 10 (depends on 1) The engineered NK cell of claim 1, wherein the engineered NK cell further comprises a polynucleotide encoding interleukin 15 (IL-15). ### Claim 11 (depends on 1) The engineered NK cell of claim 1, wherein the at least two genes are NKG2A and CISH and the engineered NK cell further comprises one or more polynucleotide encoding a CAR and IL-15. ### Claim 12 (depends on 1) The engineered NK cell of claim 1, wherein the knockout comprises one or more insertions, one or more frameshift mutations, one or more missense mutations, one or more deletions, or knock-in of the at least two genes or part of the at least two genes encoding the gene products. ### Claim 13 An engineered NK cell with knockout of at least two gene products, wherein the at least two gene products are Transforming Growth Factor Beta Receptor 2 (TGFBR2) and Nuclear Receptor Subfamily 3 Group C Member 1 (NR3C1) wherein knockout comprises the elimination or reduction of level of expression of the at least two gene products, compared to the level of expression of the at least two gene products in the absence of the knockout, wherein the engineered NK cell is engineered to express a CAR, wherein the CAR comprises one or more antigen-binding domain that targets one or more tumor associated antigens. ### Claim 14 (depends on 13) The engineered NK cell of claim 13, wherein the one or more tumor associated antigen is selected from the group consisting of Tumor-associated calcium signal transducer 2 (TACSTD2), Epidermal Growth Factor Receptor (EGFR), and EGFR variant III (EGFRvIII).