Method for providing immune cells with enhanced function

Abstract

The present disclosure relates to methods for producing immune cells with enhanced function. More specifically, disclosed herein is a method for enhancing the function of an immune cell comprising modifying the immune cell to inhibit the function of at least one gene selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2. Also disclosed herein is a method comprising modifying a stem cell or progenitor cell capable of differentiating into an immune cell to inhibit the function of at least one gene selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1 and TGFBR2. Also disclosed herein are immune cells or stem cells prepared by the methods of the invention and the use of immune cells in therapeutic therapy.

Description

[0001] This application is a divisional application of Chinese Invention Patent Application No. 202080080655.8, filed on November 18, 2020, having a priority date of November 20, 2019, and entitled “Methods for Providing Immune Cells with Enhanced Functionality”.

[0002] Cross Reference to Related Applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 938,022, filed November 20, 2019, the contents of which are incorporated in their entirety. TECHNICAL FIELD

[0004] The present disclosure relates to methods for producing immune cells with enhanced functionality. More specifically, disclosed herein is a method for enhancing immune cell functionality comprising modifying an immune cell to suppress the function of at least one gene selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2. Also disclosed herein is a method comprising modifying a stem cell or progenitor cell capable of differentiating into an immune cell to suppress the function of at least one gene selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2. Also disclosed herein are immune cells or stem cells prepared by the methods of the invention, as well as the use of immune cells in therapeutic treatments.

[0005] SEQUENCE LIST INCORPORATION BY REFERENCE

[0006] The sequence listing in ASCII text file, named 37830WO_ND201903_SequenceListing.txt, size 10 KB, created on November 3, 2020, is incorporated by reference herein. BACKGROUND

[0007] Chimeric antigen receptor-expressing T cells (CAR-T cells) have shown to be very effective at killing tumor cells in diseases such as acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL). Approved products targeting the B-cell antigen CD19 are produced by introducing a CAR gene construct into patient-derived (“autologous”) T cells (Kershaw et al., Gene-engineered T cells for cancer therapy, Nat Rev Cancer, 2013, 13(8):525-41). Additional autologous products are in development that target other hematologic malignancies (e.g., multiple myeloma) to other blood cell markers such as B-cell maturation antigen (BCMA) (Sadelain et al., Therapeutic T cell engineering, Nature, 2017, 545(7655):423-431).

[0008] While clinical results with CAR-T cells in blood-based cancers have been impressive, similar results have not emerged in treating solid tumors. There are multiple reasons for the relative lack of efficacy in solid tumors, including restricted access to the tumor site, immunosuppressive properties of the tumor microenvironment, and lack of solid tumor-specific target antigens. In addition, lack of persistence and “exhaustion” of administered CAR-T cells is a limitation that has been observed (Newick et al., CAR T Cell Therapy for Solid Tumors, Annu Rev Med, 2017, 68: 139-152).

[0009] Inhibitory receptors such as CTLA-4, PD-1 or LAG-3 can attenuate CAR-T cell activation and accelerate T cell exhaustion. After PD-1 is disrupted by genome editing, the anti-tumor activity of T cells is expected to improve (Liu et al., CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells, Cell Res, 2017, 27(1): 154-157). However, ablation of PD-1 on T cells can also increase susceptibility to exhaustion, reduce lifespan and fail to improve anti-tumor effects (Odorizzi et al., Genetic absence of PD-1 promotes accumulation of terminally differentiated exhausted CD8+ T cells, J Exp Med, 2015, 212(7): 1125-37). For these reasons, it is necessary to evaluate on a case-by-case basis whether gene editing in T cells enhances anti-tumor activity.

[0010] CRISPR / Cas9 is an essential component of the bacterial immune system that allows bacteria to remember and destroy bacteriophages. In mammalian cells, CRISPR / Cas9 can be used for gene editing, as other gene editing technologies, such as TALENs and ZFNs. The CRISPR system contains two main components, Cas9 nuclease and guide RNA. Specifically, a designed guide RNA forms a complex with Cas9 nuclease, directing the Cas9-gRNA ribonucleoprotein (RNP) complex to a user-defined cleavage site in the human genome. RNP cleavage results in a double-stranded DNA break in the genome, which is repaired by an error-prone process called non-homologous end joining (NHEJ). In the NHEJ pathway, nucleotide deletions or insertions (“indels”) result in gene disruption or knockout (Addgene, CRISPR 101: A Desktop Resource (2nd Edition), 2017). The on-target efficiency and off-target effects of guide RNA determine the specificity and safety of CRISPR / Cas9 gene targeting applications. Therefore, specially designed guide RNA plays a crucial role in the success of gene disruption.

[0011] Recent studies used CRISPR to perform genome-wide loss-of-function screens for immune modulators and found that ablation of negative regulators such as TCE2, SOCS1, RASA, and CBLB significantly increased T cell cytotoxicity in vitro (Shifrut et al., Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function. Cell, 2018, 175(7): 1958-1971, e15). However, short-term in vitro cytotoxicity provides limited guidance on the impact of gene suppression or deletion on in vivo function or longevity. The potential impact of gene suppression on the function (including their activity or longevity) of immune cells, including T cells, NK cells, NKT cells, and the like, needs to be more broadly assessed in vitro and in vivo.

[0012] While enhanced immune cells are a potential weapon against cancer, there are challenges in numerically generating, expanding, and characterizing immune cell products. Immune cells can be generated from pluripotent stem cells (PSCs). Thus, pluripotent stem cell technology is a very promising technology because pluripotent stem cells, in theory, provide an unlimited, renewable source of cells. The ability to efficiently and indefinitely supply immune cells (which have enhanced capabilities, including a broad target recognition system (TCR / CAR / cytotoxic receptor) that is capable of responding to a variety of pathogens and cancers) directly from stem cells (e.g., induced pluripotent stem cells (iPSCs)) represents a significant business opportunity. Thus, it is also important to understand the impact of suppression of particular genes of interest on the ability of iPSCs to survive, self-renew, proliferate, and differentiate into immune cells. SUMMARY

[0013] It has been demonstrated herein that suppression of several genes enhances the persistence and anti-tumor activity of cytotoxic cells in vivo.

[0014] In one aspect, provided herein is a method for enhancing immune cell function. The method comprises modifying the immune cell to suppress function of at least one gene selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2.

[0015] In another aspect, provided herein is a method for modifying a stem cell capable of differentiating into an immune cell. The method comprises modifying the stem cell to suppress function of at least one gene selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2. In some embodiments, the modified stem cell is further differentiated into an immune cell, wherein function of the at least one gene is suppressed in the immune cell.

[0016] In some embodiments, the inhibition of gene function is achieved by reducing the level or function of an mRNA, optionally by a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), or an antisense nucleic acid.

[0017] In some embodiments, the inhibition of gene function is achieved by reducing the level or activity of a protein encoded by a gene, optionally by using an antibody or a small molecule.

[0018] In some embodiments, the inhibition of gene function is achieved by a gene editing system. In some embodiments, the gene editing system is selected from the group consisting of CRISPR / Cas, TALEN, and ZFN. In some embodiments, the gene editing system is a CRISPR / Cas system comprising a guide RNA-nuclease complex. In some embodiments, the guide RNA targets a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 16. In some embodiments, the CRISPR / Cas system utilizes a guide RNA-dependent nuclease selected from the group consisting of Cpf1, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslOO, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csx1, Csxl5, Csf1, Csf2, Csf3, and CSF4.

[0019] In some embodiments, the immune cell is selected from the group consisting of a T cell (including a cell, e.g., a NKT cell) or an NK cell.

[0020] In some embodiments, the modified cell, e.g., a modified immune cell or a modified stem cell, produced by the methods disclosed herein further comprises a nucleic acid encoding a chimeric antigen receptor (CAR).

[0021] In some embodiments, the modified immune cell produced by the methods disclosed herein recognizes one or more target antigens. In some embodiments, the target antigen is selected from the group consisting of TAG-72, CD19, CD20, CD24, CD30, CD47, folate receptor alpha (FRa), and BCMA.

[0022] In another aspect, provided herein are immune cells produced by the methods disclosed herein.

[0023] In another aspect, provided herein are modified stem cells produced by the methods disclosed herein.

[0024] In one aspect, provided herein are modified immune cells, wherein the function of at least one gene is inhibited in the modified immune cells relative to unmodified immune cells, wherein the at least one (i.e., one or more) gene is selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2. In some embodiments, the RC3H1 gene is inhibited in the modified immune cells. In some embodiments, the RC3H2 gene is inhibited in the modified immune cells. In some embodiments, the A2AR gene is inhibited in the modified immune cells. In some embodiments, the FAS gene is inhibited in the modified immune cells. In some embodiments, the TGFBR1 gene is inhibited in the modified immune cells. In some embodiments, the TGFBR2 gene is inhibited in the modified immune cells. In some embodiments, multiple genes selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2 are inhibited.

[0025] In some embodiments, the inhibition of gene function in the modified immune cells is caused by a decrease in the level or function of mRNA transcribed from the gene, or by the level or activity of a protein encoded by the gene.

[0026] In some embodiments, the inhibition of gene function is caused by a modification of the nucleic acid sequence of the gene.

[0027] In some embodiments, the modified immune cells are selected from the group consisting of T cells (including cells such as NKT cells) or NK cells.

[0028] In some embodiments, the modified immune cells express a chimeric antigen receptor (CAR).

[0029] In some embodiments, the modified immune cells recognize one or more target antigens. In some embodiments, the target antigens are selected from the group consisting of TAG-72, CD19, CD20, CD24, CD30, CD47, folate receptor alpha (FRa), and BCMA.

[0030] In another aspect, provided herein are modified stem cells that are capable of differentiating into immune cells, comprising a modification of a nucleic acid sequence of at least one gene, wherein the modification inhibits the function of the at least one gene, and wherein the at least one gene is selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2.

[0031] In some embodiments, the RC3H1 gene is inhibited in the modified stem cell. In some embodiments, the RC3H2 gene is inhibited in the modified stem cell. In some embodiments, the A2AR gene is inhibited in the modified stem cell. In some embodiments, the FAS gene is inhibited in the modified stem cell. In some embodiments, the TGFBR1 gene is inhibited in the modified stem cell. In some embodiments, the TGFBR2 gene is inhibited in the modified stem cell. In some embodiments, a plurality of genes selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2 are inhibited.

[0032] In some embodiments, the modified stem cell is an induced pluripotent stem cell.

[0033] In some embodiments, the modified stem cell comprises a nucleic acid encoding a chimeric antigen receptor (CAR).

[0034] In another aspect, provided herein are compositions for enhancing immune cell function, comprising a guide RNA-nuclease complex capable of editing a target gene sequence, wherein the guide RNA targets a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 16.

[0035] In some embodiments, the nuclease comprises at least one protein selected from the group consisting of Cpf1, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslOO, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csx1, Csxl5, Csf1, Csf2, Csf3, and Csf4.

[0036] In another aspect, a method for treating a disorder in a subject is provided, comprising administering to the subject a modified immune cell disclosed herein. In some embodiments, the disorder is cancer, an infection, an autoimmune disease, organ fibrosis, or endometriosis.

[0037] The application further relates to the following embodiments:

[0038] 1. A method for enhancing immune cell function, comprising:

[0039] modifying the immune cell to suppress function of at least one gene selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2.

[0040] 2. A method for modifying a stem cell capable of differentiating into an immune cell, comprising:

[0041] modifying the stem cell to suppress function of at least one gene selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2.

[0042] 3. The method of embodiment 2, further comprising differentiating the modified stem cell into an immune cell, wherein the function of the at least one gene is suppressed in the immune cell.

[0043] 4. The method of any one of embodiments 1-3, wherein the suppression of gene function is achieved by a gene editing system.

[0044] 5. The method of embodiment 4, wherein the gene editing system is selected from the group consisting of CRISPR / Cas, TALEN, and ZFN.

[0045] 6. The method of embodiment 4, wherein the gene editing system is a CRISPR / Cas system comprising a guide RNA-nuclease complex.

[0046] 7. The method of embodiment 6, wherein the guide RNA targets a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 16.

[0047] 8. The method of embodiment 6, wherein the CRISPR / Cas system utilizes a guide RNA- dependent nuclease selected from the group consisting of Cpf1, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslOO, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csx1, Csxl5, Csf1, Csf2, Csf3, and Csf4.

[0048] 9. The method of any of the preceding embodiments, wherein the immune cell is selected from a T cell, an NK cell, an NKT cell, or a macrophage.

[0049] 10. The method of embodiment 1 or 2, wherein the inhibition of gene function is achieved by reducing the level or function of an mRNA, optionally by a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), or an antisense nucleic acid.

[0050] 11. The method of embodiment 1 or 2, wherein the inhibition of gene function is achieved by reducing the level or activity of a protein encoded by the gene, optionally by using an antibody or a small molecule.

[0051] 12. The method of any of the preceding embodiments, wherein the modified cell produced by the method further comprises a nucleic acid encoding a chimeric antigen receptor (CAR).

[0052] 13. The method of any of the preceding embodiments, wherein the modified immune cell produced by the method recognizes one or more target antigens.

[0053] 14. The method of embodiment 13, wherein the target antigen is selected from the group consisting of TAG-72, CD19, CD20, CD24, CD30, CD47, folate receptor alpha (FRa), and BCMA.

[0054] 15. The method of any of embodiments 1-14, wherein the at least one gene is RC3H1.

[0055] 16. The method of any of embodiments 1-14, wherein the at least one gene is RC3H2.

[0056] 17. The method according to any one of embodiments 1-14, wherein the at least one gene is A2AR.

[0057] 18. The method according to any one of embodiments 1-14, wherein the at least one gene is FAS.

[0058] 19. The method according to any one of embodiments 1-14, wherein the at least one gene is TGFBR1.

[0059] 20. The method according to any one of embodiments 1-14, wherein the at least one gene is TGFBR2.

[0060] 21. An immune cell produced by the method according to any one of embodiments 1 or 3-20, or differentiated from a modified stem cell produced by the method according to any one of embodiments 2 or 4-20.

[0061] 22. A modified immune cell, wherein the function of at least one gene is inhibited in the modified immune cell, and wherein the at least one gene is selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1 and TGFBR2.

[0062] 23. The modified immune cell according to embodiment 22, wherein the inhibition of gene function is caused by a reduction in the level or function of mRNA transcribed from the gene, or in the level or activity of a protein encoded by the gene.

[0063] 24. The modified immune cell according to embodiment 22, wherein the inhibition of gene function is caused by a modification of the nucleic acid sequence of the gene.

[0064] 25. The modified immune cell according to any one of embodiments 22-24, wherein the modified immune cell is selected from the group consisting of a T cell, an NK cell, an NKT cell or a macrophage.

[0065] 26. The modified immune cell according to any one of embodiments 22-25, wherein the modified immune cell expresses a chimeric antigen receptor (CAR).

[0066] 27. The modified immune cell according to any one of embodiments 22-26, wherein the modified immune cell recognizes one or more target antigens.

[0067] 28. The modified immune cell of embodiment 27, wherein the target antigen is selected from the group consisting of TAG-72, CD19, CD20, CD24, CD30, CD47, folate receptor alpha (FRa), and BCMA.

[0068] 29. The modified immune cell of any one of embodiments 22-28, wherein the at least one gene is RC3H1.

[0069] 30. The modified immune cell of any one of embodiments 22-28, wherein the at least one gene is RC3H2.

[0070] 31. The modified immune cell of any one of embodiments 22-28, wherein the at least one gene is A2AR.

[0071] 32. The modified immune cell of any one of embodiments 22-28, wherein the at least one gene is FAS.

[0072] 33. The modified immune cell of any one of embodiments 22-28, wherein the at least one gene is TGFBR1.

[0073] 34. The modified immune cell of any one of embodiments 22-28, wherein the at least one gene is TGFBR2.

[0074] 35. A modified stem cell capable of differentiating into an immune cell, comprising a modification of a nucleic acid sequence of at least one gene, wherein the modification inhibits the function of the at least one gene, and wherein the at least one gene is selected from the group consisting of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2.

[0075] 36. The modified stem cell of embodiment 35, which is an induced pluripotent stem cell.

[0076] 37. The modified stem cell of embodiment 36, wherein the induced pluripotent stem cell is generated from a donor cell homozygous for three HLA genotypes.

[0077] 38. The modified stem cell of any one of embodiments 35-37, further comprising a nucleic acid encoding a chimeric antigen receptor (CAR).

[0078] 39. The modified stem cell of any one of embodiments 35-38, wherein the at least one gene is RC3H1.

[0079] 40. The modified stem cell of any one of embodiments 35-38, wherein the at least one gene is RC3H2.

[0080] 41. The modified stem cell of any one of embodiments 35-38, wherein the at least one gene is A2AR.

[0081] 42. The modified stem cell of any one of embodiments 35-38, wherein the at least one gene is FAS.

[0082] 43. The modified stem cell of any one of embodiments 35-38, wherein the at least one gene is TGFBR1.

[0083] 44. The modified stem cell of any one of embodiments 35-38, wherein the at least one gene is TGFBR2.

[0084] 45. A composition for enhancing immune cell function, comprising: a guide RNA-nuclease complex capable of editing a target gene sequence,

[0085] wherein the guide RNA targets a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 16.

[0086] 46. The composition of embodiment 45, wherein the nuclease comprises at least one protein selected from the group consisting of Cpf1, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslOO, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4.

[0087] 47. A method of treating a disorder in a subject, comprising administering to the subject the modified immune cell of any one of embodiments 21-34.

[0088] 48. The method of embodiment 47, wherein the disorder is cancer, an infection, an autoimmune disease, organ fibrosis, or endometriosis. BRIEF DESCRIPTION OF DRAWINGS

[0089] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0090] Figures 1A-1B Exemplary strategies to evaluate the anti-tumor activity of modified immune cells. (A) Schematic of the strategy performed to evaluate CAR-T cells, which includes CRISPR knockout of immune regulatory genes, showing a representative timeline of lentiviral CAR transduction, gene targeting, and functional analysis in primary T cells used in the examples. (B) Representative timeline to generate modified NK-92 cells, where CRISPR knockout of immune regulatory genes is followed by lentiviral CAR transduction and functional analysis in NK-92 cells.

[0091] Figures 2A-2B Lentiviral transduction of human primary T cells to generate TAG-72 CAR-T cells. (A) Schematic of the TAG-72 specific CAR construct used in this study. (B) Transduction efficiency of the CAR in human primary T cells. Expression was checked 10 days after transduction with lentiviral vectors. The value embedded in each dot plot represents the frequency of CAR+ events as a percentage of live single cells. (Representative data from T cells of one donor is shown).

[0092] Figure 3Growth curves of TAG-72 CAR-T cells following CRISPR / Cas9 RNP transfection (representative data from T cells from one donor are shown). NT: untransduced T cells; TAG-72 CAR: T cells transduced with TAG-72 CAR; TAG-72 CAR / PD-1 KO T: T cells transduced with TAG-72 CAR and CRISPR / Cas9 RNP targeting PD-1; TAG-72 CAR / A2AR KO T: T cells transduced with TAG-72 CAR and CRISPR / Cas9 RNP targeting A2AR; TAG-72 CAR / FAS KO T: T cells transduced with TAG-72 CAR and CRISPR / Cas9 RNP targeting FAS; TAG-72 CAR / RC3H1 KO T: T cells transduced with TAG-72 CAR and CRISPR / Cas9 RNP targeting RC3H1; TAG-72 CAR / RC3H2 KO T: T cells transduced with TAG-72 CAR and CRISPR / Cas9 RNP targeting RC3H2; TAG-72 CAR / TGFBR1 KO T: T cells transduced with TAG-72 CAR and CRISPR / Cas9 RNP introducing a dominant negative mutation into TGFBR1; TAG-72 CAR / TGFBR2 KO T: T cells transduced with TAG-72 CAR and CRISPR / Cas9 RNP introducing a dominant negative mutation into TGFBR2.

[0093] Figure 4A- Transfection of RNP formed by 4D guide RNA introduced indels into the open reading frame of specific genes in CAR-T cells. The frequency of indels was assessed by CRISPR Editing (ICE) assay. (A) Sanger sequencing trace from CAR-T cells transfected with RC3H2 gRNA ("edited sample") shows a heterogeneous mix of bases downstream of the cleavage site compared to untransfected CAR-T cells ("control sample") (SEQ ID NO: 17 shows 281 to 346 bp from edited sample; SEQ ID NO: 18 shows 281 to 346 bp from control sample). The black underlined region of the control sample represents the guide sequence, and the horizontally red dotted underlined region is the relevant PAM (Protospacer Adjacent Motif) site. The vertical black dotted line on both traces represents the cleavage site. (B) Relative percentage of contribution of each edited sequence in the genomic DNA from CAR-T cells transfected with RC3H2 RNP (normalized). From top to bottom, the sequences are shown in SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, and 26, respectively. (C) Distribution of indel sizes in the whole edited population of CAR-T cells transfected with RC3H2 RNP. The out-of-frame indel percentage is the proportion of indels representing frameshifts or lengths over 21 bp. The R 2 value calculated by Pearson correlation coefficient represents the confidence of the indel percentage. (D) Summary of the results of the ICE assay for CAR-T cells transfected with RNP. The RNP complexes were formed by representative guide RNAs used in this study (PD-1, SEQ ID NO: 1; RC3H1, SEQ ID NO: 2; RC3H2, SEQ ID NO: 4; A2AR, SEQ ID NO: 7; FAS, SEQ ID NO: 9; TGFBR1, SEQ ID NO: 11; TGFBR2, SEQ ID NO: 14); representative data from T cells of one donor are shown.

[0094] FIGS. 5A-5H Gene knockout TAG-72 CAR-T cells mediate potent cell killing of TAG-72hiexpressing target cells (OVCAR-3 cell line) (FIGS. 5A, 5C, 5E, and 5G), but not of TAG-72-neg / low cancer target cells (MES-OV cell line) (FIGS. 5B, 5D, 5F, and 5H). Target cells were allowed to attach to the plate overnight before the addition of CAR-T cells at a 1:1 effector to target ratio. Untransduced T cells (NT) were included as a control in the killing assay. Cell impedance was monitored over 20 hours (mean ± SD, expressed as normalized cell index (NCI)). Target cell proliferation under normal growth conditions (“Target cells only”) was also monitored throughout the process. (Representative data from T cells of one donor are shown in technical triplicates). CAR-T (FIGS. 5A-5H): TAG-72 CAR-T cells; PD-1 (FIGS. 5A-5B): PD-1 knockout TAG-72 CAR-T cells; RC3H1 (FIGS. 5C and 5D): RC3H1 knockout TAG-72 CAR-T cells; RC3H2 (FIGS. 5C and 5D): RC3H2 knockout TAG-72 CAR-T cells; A2AR (FIGS. 5E and 5F): A2AR knockout TAG-72 CAR-T cells; FAS (FIGS. 5E and 5F): FAS knockout TAG-72 CAR-T cells; TGFBR1 (FIGS. 5G and 5H): TGFBR1 dominant negative TAG-72 CAR-T cells; TGFBR2 (FIGS. 5G and 5H): TGFBR2 dominant negative TAG-72 CAR-T cells.

[0095] Figure 6 Tumor growth curve of OVCAR-3 ovarian tumors in NOD scid gamma (NSG) mouse xenograft model. Four NSG mice per group were subcutaneously administered 1x10 7 OVCAR-3 tumor cells (TAG-72 positive). When the tumors grew to approximately 150-200 mm 3 in diameter, two doses of 5x10 6 T cells were adoptively transferred by intravenous injection every 5 days. Values and error bars represent mean tumor size (mm 3 ± SEM). NT: untransduced T cells; TAG-72 CAR-T: T cells transduced with TAG-72 CAR; TAG-72 CAR / PD-1 KO T: PD-1 gene knockout TAG-72 CAR-T cells; mean ± SEM; representative data from T cells of one donor are shown.

[0096] Figure 7Anti-tumor activity of RC3H1 and / or RC3H2 gene knockout CAR-T cells in OVCAR-3 ovarian tumor NSG mouse xenograft model. Four NSG mice per group were subcutaneously administered with 1 x 106 7 OVCAR-3 tumor cells (TAG-72 positive). When tumors grew to approximately 150-200 mm 3 in diameter, two doses of 5 x 106 6 T cells were adoptively transferred by intravenous injection every 5 days. Values and error bars represent mean tumor size (mm 3 ± SEM). NT: untransduced T cells; TAG-72 CAR-T: T cells transduced with TAG-72 CAR; TAG-72 CAR / RC3H1 KO T: RC3H1 gene knockout TAG-72 CAR-T cells; TAG-72 CAR / RC3H2 KO T: RC3H2 gene knockout TAG-72 CAR-T cells; TAG-72 CAR / RC3H1,2 KO T: RC3H1 and RC3H2 double gene knockout TAG-72 CAR-T cells. ** p<0.01, mixed effect analysis with Greisser-Greenhouse correction and Dunnett’s multiple comparison one-way ANOVA comparing all group means to TAG-72 CAR-T control group. Representative data from T cells of one donor are shown.

[0097] Figure 8 Anti-tumor activity of A2AR and FAS gene knockout CAR-T cells in OVCAR-3 ovarian tumor NSG mouse xenograft model. Four NSG mice per group were subcutaneously administered with 1 x 106 7 OVCAR-3 tumor cells (TAG-72 positive). When tumors grew to approximately 150-200 mm 3 in diameter, two doses of 5 x 106 6 T cells were adoptively transferred by intravenous injection every 5 days. Values and error bars represent mean tumor size (mm 3 ± SEM). NT: untransduced T cells; TAG-72 CAR-T: T cells transduced with TAG-72 CAR; TAG-72 CAR / A2AR KO T: A2AR gene knockout TAG-72 CAR-T cells; TAG-72 CAR / FAS KO T: FAS gene knockout TAG-72 CAR-T cells. * p<0.05, ** p<0.01, # p<0.001, two-way ANOVA followed by Dunnett’s multiple comparison test comparing all group means to CAR-T control group. Representative data from T cells of one donor are shown.

[0098] Figure 9 Anti-tumor activity of TGFBR1 and TGFBR2 dominant negative gene mutated CAR-T cells in OVCAR-3 ovarian tumor NSG mouse xenograft model. Four NSG mice per group were subcutaneously administered with 1x10 7 OVCAR-3 tumor cells (TAG-72 positive). When tumors grew to approximately 150-200 mm 3 in diameter, two doses of 5x10 6 T cells were adoptively transferred by intravenous injection every 5 days. Values and error bars represent mean tumor size (mm 3 ± SEM). NT: non-transduced T cells, TAG-72 CAR-T: T cells transduced with TAG-72 CAR, TAG-72 CAR / TGFBR1 KO T: TGFBR1 dominant negative gene knockout TAG-72 CAR-T cells, TAG-72 CAR / TGFBR2 KO T: TGFBR2 dominant negative gene knockout TAG-72 CAR-T cells. * p<0.05, ** p<0.01, *** p<0.001, two-way ANOVA followed by Dunnett’s multiple comparison test comparing all group means to the CAR-T control group. Representative data from T cells of one donor are shown.

[0099] Figures 10A-10B Anti-tumor activity of CD19 CAR-T cells with RC3H1 and / or RC3H2 gene knockout in Raji lymphoma tumor NSG mouse xenograft model. Four NSG mice per group were subcutaneously administered with Raji tumor cells (CD19 positive). Three days after tumor inoculation, mice were treated with a single dose of 5x10 6 CAR-T cells by intravenous injection. (A) Tumor size was monitored for 23 days. Values and error bars represent mean tumor size (mm 3± SEM). Multiple t-tests with Holm-Sidak correction were performed to compare RC3H1 and / or RC3H2 gene knockout CD19 CAR-T cell groups with untransfected CD19 CAR-T cells. (*p<0.005; **p<0.001) (B) Kaplan-Meier survival curves were analyzed using the log-rank (Mantel-Cox) test. NT: untransduced T cells; CD19 CAR: CD19 CAR-T cells; CD19 CAR / RC3H1 KO: RC3H1 gene knockout CD19 CAR-T cells; CD19 CAR / RC3H2 KO: RC3H2 gene knockout CD19 CAR-T cells; CD19 CAR / RC3H1,2 KO: RC3H1 and RC3H2 double gene knockout CD19 CAR-T cells. Representative data from T cells of one donor are shown.

[0100] Figure 11 Expression of activation markers on CD19 CAR-T cells with or without RC3H1 and / or RC3H2 gene KO after continuous activation exposure. Graphs show the expression of activation markers CD25 and CD69 on CAR+ cells after 7 days of antigen exposure. CD19 CAR-T cells were generated from a single healthy donor. Results represent mean ± SD of technical replicates.

[0101] Figure 12 CRISPR knockout analysis of RC3H1 and RC3H2 genes in single and double knockout T cells. RNP formed with RC3H1 and RC3H2 guide RNAs were transfected into human activated T cells to generate RC3H1 or RC3H2 single KO T cells (RC3H1 KO T cells or RC3H2 KO T cells), or RC3H1 and RC3H2 double KO T cells (RC3H1,2 KO T cells). Knockout efficiency was analyzed using ICE analysis. Off-target indel percentage is the proportion of indels representing frameshifts or length over 21 bp.

[0102] Figure 13 Effect of RC3H1 and / or RC3H2 KO on T cells (CD8+, CD4+) function without CAR. T cells ± RC3H1 and / or RC3H2 KO were kept in T cell expansion media in the presence of Dynabeads Human T-Activator CD3 / CD28 beads (Thermofisher, MA, USA) (DB) at a 1 : 1 bead-to-cell ratio for at least 92 h. xCELLigence RT-CES™ SP Dynamic Cell Assay System (ACEA Biosciences, CA, USA) was used to monitor T cell proliferation and activation. TM T cells ± RC3H1 and / or RC3H2 KO were kept in T cell expansion media in the presence of Dynabeads Human T-Activator CD3 / CD28 beads (Thermofisher, MA, USA) (DB) at a 1 : 1 bead-to-cell ratio for at least 92 h. xCELLigence RT-CES™ SP Dynamic Cell Assay System (ACEA Biosciences, CA, USA) was used to monitor T cell proliferation and activation. ®The beads were removed magnetically prior to the assay using effector cells. Effector cells were added to target cancer cells (OVCAR-3 in this example) at a 1:1 effector to target ratio (E:T). The NCIs were monitored for over 20 h. Target cell elimination was observed in all conditions (decrease in NCI was observed). Importantly, cells with RC3H1 and / or RC3H2 gene deletion were able to eliminate target cells more efficiently in vitro after sustained CD3 / CD28-mediated activation. Results represent the average of biological and intra-assay triplicates + SEM.

[0103] Figure 14 CRISPR knockout analysis of RC3H1 and RC3H2 genes in single and double knockout NK-92 cells. RNP formed with RC3H1 and RC3H2 guide RNAs were transfected into NK-92 cells to generate RC3H1 or RC3H2 single KO NK-92 cells (RC3H1 KO NK-92 cells or RC3H2 KO NK-92), or RC3H1 and RC3H2 double KO NK-92 cells (RC3H1,2 KO NK-92). Knockout efficiency was analyzed using ICE analysis. The percentage of off-target indels is the proportion of indels that are frameshift or longer than 21 bp. The R 2 value calculated by Pearson correlation coefficient indicates the confidence of the percentage of indels.

[0104] Figures 15A-15C. Effect of RC3H1 and / or RC3H2 KO on NK-92 cell (with and without TAG-72 CAR) function. The real-time cell monitoring system xCELLigence ®NK cell line NK-92 ± RC3H1 KO (green) or RC3H2 KO (purple) or RC3H1,2 KO (orange) ± TAG-72 CAR ability to eliminate cancer cells in vitro. (A) RC3H1 and / or RC3H2 genes were deleted in the NK-92 cell line using CRISPR / Cas9. The resulting RC3H1 and / or RC3H2 KO NK-92 effector cells were added to target cancer cells (MES-OV (left panel) or OVCAR-3 (right panel) at a 1 : 1 E:T ratio. NCI was monitored over 40 h. Target cell elimination was observed in all conditions (decrease in NCI was observed compared to target cells alone (blue)). Results represent the mean ± SEM of technical triplicates. (B) NK-92 cells were further genetically manipulated to introduce a TAG-72 CAR. Lentiviral transduction was performed after transfection. Transduction efficiency was assessed by flow cytometry after about 72 h of culture, with the value embedded in each dot plot representing the proportion of CAR+ cells as a frequency of live single cells. The resulting TAG-72 CAR / RC3H1 and / or RC3H2 KO NK-92 cells were isolated using fluorescence-activated cell sorting and their in vitro function was assessed as previously described. (C) NCI was monitored over 40 h. Results represent the mean ± SEM, n = 1 -3.

[0105] Figure 16 Generation of CRISPR gene knockout induced pluripotent stem cells (iPSCs) as a cell source for adoptive cell therapy. Workflow to derive gene knockout immune cells from iPSCs. iPSCs are transfected to knockout a gene of interest. These cells are then sequenced to characterize and validate the knockout, and then differentiated into CD34+ cells and immune cells.

[0106] Figures 17A-17B RC3H1 and RC3H2 double KO in iPSCs (RC3H1,2 KO iPSCs) does not affect pluripotency. Characterized by (A) morphology (scale bar = 200 pm) with presence of undifferentiated cells, and (B) flow cytometry analysis of iPSC markers TRA-1-60, TRA-1-81, and SSEA-4. Dead cells, debris, and doublets were excluded, so the histograms show all live cells in culture from untransfected iPSC or RC3H1,2 KO iPSC samples. Over 99% of all live cells expressed all iPSC markers.

[0107] Figures 18A-18CTransfection of RC3H1 and RC3H2 guide RNA formed RNPs introduced indels into the open reading frame of specific genes in iPSCs. Sanger sequencing traces from iPSCs co-transfected with RC3H1 and RC3H2 gRNAs ("edited samples") show a heterogeneous mix of bases downstream of the RC3H1 gene (A) and RC3H2 gene (B) cleavage site compared to untransfected iPSCs ("control sample") (in A, SEQ ID NO: 27 shows 184 to 249 bp from edited sample, SEQ ID NO: 28 shows 183 to 248 bp from control sample; in B, SEQ ID NO: 29 shows 270 to 336 bp from edited sample, SEQ ID NO: 30 shows 272 to 337 bp from control sample). The black underlined region of the control sample represents the guide sequence, and the horizontally red dotted underlined region is the relevant PAM site. The vertical black dotted line on both traces represents the cleavage site. (C) CRISPR knockout analysis of iPSCs co-transfected with RC3H1 and RC3H2 gRNAs. The efficiency of knockout of RC3H1 and RC3H2 genes was assessed using ICE analysis. The percent off-frame indels is the proportion of indels that represent frameshifts or are longer than 21 bp. The R 2 value represents the confidence of the indel percentage calculated by Pearson correlation coefficient.

[0108] Figure 19 RC3H1 and RC3H2 double KO in iPSCs does not prevent differentiation into iCD34+ cells. Unstained cells and cells stained with antibodies against CD34+ were analyzed by flow cytometry. Dead cells, debris, and doublets were excluded, so the histograms show all live CD34+ cells in culture from untransfected iPSC or RC3H1,2 KO iPSC samples. Deletion of both RC3H1 and RCH32 genes does not prevent iPSCs from developing into iCD34 cell subpopulation.

[0109] Figure 20iPSCs containing RC3H1 and RC3H2 double KO were able to differentiate into CD56+ cells expressing NK cytotoxic receptors for NKG2D and NKp46. Dead cells, debris, and doublets were excluded, so the CD56+ histograms show all live cells in the cultures generated. NKp46 and NKG2D plots exclude CD56+ cells. Unstained controls and isotype controls were presented to show positive staining for each antibody for each respective receptor. Co-expression of NK functional receptors (NKp46 or NKG2D) with CD56 indicates that CD56+ cells derived from RC3H1,2 KO iPSCs have the potential to perform NK-mediated cytotoxic functions.

[0110] Figures 21A-21B A2AR KO in iPSCs did not affect pluripotency. It was characterized by (A) morphology (scale bar = 200 pm) with presence of undifferentiated cells, and (B) flow cytometry analysis of iPSC markers TRA-1-60, TRA-1-81, and SSEA-4. Dead cells, debris, and doublets were excluded, so the histograms show all live cells in the cultures from untransfected iPSC or A2AR KO iPSC samples. Over 95% of all live cells expressed all iPSC markers.

[0111] Figures 22A-22C Transfection of A2AR guide RNA-formed RNPs introduced indels into the open reading frame of the A2AR gene in iPSCs. The frequency of indels was assessed by ICE analysis. (A) Sanger sequencing traces from A2AR KO iPSCs (“edited samples”) show a heterogeneous mix of bases downstream of the cleavage site compared to untransfected iPSCs (“control samples”). SEQ ID NO: 31 shows 134 to 199 bp of edited samples; SEQ ID NO: 32 shows 137 to 202 bp from control samples. The black underlined region of the control sample represents the guide sequence, and the horizontally red dashed underlined region is the relevant PAM site. The vertical black dashed line on both traces represents the cleavage site. (B) Relative percentage of contribution of each edited sequence in the genomic DNA from A2AR KO iPSCs (normalized). Sequences from top to bottom are shown in SEQ ID NO: 33, 34, 35, 36, 37, and 38, respectively. (C) Distribution of indel sizes in the entire edited population of RNP-transfected iPSCs. Out-of-frame indel percentage is the proportion of indels representing frameshifts or lengths over 21 bp. R 2 Values represent confidence of indel percentage.

[0112] Figure 23 Inclusion of A2AR KO in iPSCs does not prevent their differentiation into iCD34+ cells. Cells stained with antibodies against CD34 were analyzed by flow cytometry. Unstained cells and cells stained with isotype control were included as controls. Dead cells, debris, and doublets were excluded, so the histograms show all live cells in cultures generated from untransfected iPSC or A2AR KO iPSC samples. Inclusion of the KO does not prevent development of the iCD34+ cell subtype.

[0113] Figure 24 A2AR KO iPSCs are able to differentiate into iNK cells. Unstained cells and cells stained with antibodies against NK cell markers were analyzed by flow cytometry. Dead cells, debris, and doublets were excluded, so the CD56+ histograms show all live cells in cultures generated from untransfected iPSC or A2AR KO iPSC samples. Unstained samples were presented to show clear positive staining of each antibody for each respective receptor. Appropriate isotype controls were also run and were negative. Expression of NK functional receptors (NKp46, NKp30, NKp44, and NKG2D) demonstrate that CD56+ cells derived from A2AR KO iPSCs are iNK cells and are potentially capable of cytotoxic function.

[0114] Figures 25A-25B A2AR KO iPSCs are able to differentiate into functional iNK cells with enhanced killing activity in vitro. iNK cells were derived from untransfected iPSCs and A2AR KO iPSCs. The resulting iNK cells were evaluated for function in vitro using a real-time cell monitoring system (xCELLigence ® ) with OVCAR-3 cells used as targets. A 1 :2 effector to target ratio was used. (A) Changes in NCI were recorded every 15 min during the co-culture period of at least 10 h, where a decrease in NCI indicates target cell death. (B) Results of (A) are shown as the percentage of cytotoxicity of iNK cells against target cells at 5 h (left panel) and 10 h (right panel) of co-culture. Cells were derived from a single iNK differentiation. Each data point represents a technical replicate. DETAILED DESCRIPTION

[0115] Throughout this specification, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0116] In the specification and claims, the terms "a" and "an" should not be construed to mean "at least one" or "one or more" unless expressly stated otherwise. Unless otherwise clear from the context, all percentages will be understood as meaning percent by weight.

[0117] As used herein, a "nucleic acid construct" generally refers to a nucleic acid molecule that is artificially or recombinantly constructed or prepared, and can also be referred to interchangeably as a nucleic acid vector. For example, a nucleic acid construct can be made to include a nucleotide sequence of interest that is desired to be transcribed in a cell, and in some cases, to produce an RNA molecule having a desired function (e.g., an antisense RNA, an siRNA, an miRNA, or a gRNA), and in other cases, to produce an mRNA that is translated into a protein of interest (e.g., a Cas protein). The nucleotide sequence of interest in a nucleic acid construct can be operably linked to a 5' regulatory region (e.g., a promoter such as a heterologous promoter) and / or a 3' regulatory region (e.g., a 3' untranslated region (UTR) such as a heterologous 3' UTR). A nucleic acid construct can be in a circular (e.g., a plasmid) or linear form, can be an integrating nucleic acid (i.e., capable of integrating into the chromosome of a host cell, e.g., a viral vector such as a lentiviral vector), or can remain episomal (e.g., a plasmid).

[0118] General Description

[0119] Disclosed herein are methods for providing immune cells with enhanced functionality by inhibiting the function of one or more selected genes. For example, it has been demonstrated herein that ablation of one or more selected genes using CRISPR / Cas9 gene editing technology enhances the persistence and anti-tumor activity of cytotoxic lymphocytes in vivo. Accordingly, methods are provided by inhibiting the function of one or more selected genes in an immune cell or a stem cell capable of differentiating into an immune cell. Also disclosed herein are immune cells or stem cells prepared by the methods of the invention, as well as the use of immune cells in therapeutic treatments.

[0120] Immune Cells

[0121] As used herein, "immune cells" shall be understood to include cells of the mammalian immune system (e.g., lymphocytes (T cells, B cells, NK cells, and NKT cells), neutrophils, and monocytes (including macrophages and dendritic cells)) as well as cell lines derived from cells of the mammalian immune system. Immune cells can be isolated from a mammalian subject, collected from a cell line culture of immune cells derived from a mammalian subject, or produced by differentiation from a stem cell.

[0122] The present disclosure aims to provide immune cells with enhanced function. By "enhanced function" is meant that the immune cells provided as a result of the modifications or manipulations disclosed herein exhibit enhanced activity (e.g., cytotoxicity), proliferation, survival, persistence, and / or infiltration as compared to control immune cells (i.e., immune cells without the modifications or manipulations). Cytotoxicity of an immune cell refers to the ability of the immune cell to kill target cells, typically through receptor-based mechanisms.

[0123] In some embodiments, the immune cell is a cytotoxic immune cell, e.g., a cytotoxic lymphocyte.

[0124] In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is an NKT cell. In some embodiments, the immune cell is an NK cell.

[0125] Reference to "T cells" is understood to refer to any cell comprising a T cell receptor. In this regard, the T cell receptor can comprise any one or more of an alpha, beta, gamma, or delta chain. As will be appreciated by those skilled in the art, NKT cells also express T cell receptors, and thus target antigen specific NKT cells can also be generated according to the present application. The present application is not intended to be limited to any particular T cell subset, although in one embodiment, the subject T cells express an alpha / beta TCR dimer. In some embodiments, the T cells are CD4+ helper T cells, CD8+ killer T cells, or NKT cells. Without limiting the present application to any one theory or mode of action, CD8+ T cells are also known as cytotoxic cells. As a major part of the adaptive immune system, CD8+ T cells scan the intracellular environment to primarily target and destroy infected cells. Small peptide fragments derived from intracellular contents are processed and transported to the cell surface, where they are presented in the context of MHC class I molecules. However, in addition to responding to viral infections, CD8+ T cells also provide additional immune surveillance by monitoring and clearing damaged or abnormal cells, including cancer. CD8+ T cells recognizing MHC I presented peptides typically result in the release of cytotoxic granules or lymphokines, or activation of the apoptotic pathway through FAS / FASL interactions to destroy the subject cell. On the other hand, CD4+ T cells typically recognize peptides presented by antigen presenting cells in the context of MHC class II, resulting in the release of cytokines aimed at modulating B cell and / or CD8+ T cell immune responses. CD4+ T cells with cytotoxic activity are also observed in various immune responses. Furthermore, CD4+ CAR-T cells show comparable cytotoxicity to CD8+ CAR-T cells in vitro, and even for longer term anti-tumor activity, outperform CD8+ CAR-T cells in vivo (see, e.g., Wang et al., JCI Insight. 2018; 3(10):e99048; Yang et al., Sci Transl Med. 2017 Nov 22;9(417), eaagl209).

[0126] Natural killer T cells (also known as NKT or T / NK cells) are a specialized T cell population that express semi-invariant T cell receptors (TCRa-β) and surface antigens often associated with natural killer cells. The TCR on NKT cells is unique in that it typically recognizes glycolipid antigens presented by the MHC I-like molecule CDld. Most NKT cells express an invariant TCR a chain and one of a few TCR β chains. The TCR present on type I NKT cells typically recognizes the antigen a-galactosylceramide (a-GalCer). Within this group, distinguishable subsets have been identified, including CD4 + CD8 - cells, CD4- CD8 + cells and CD4 - CD8 - cells. Type II NKT cells (or non-invariant NKT cells) express a more diverse TCR a chain and do not recognize a-GalCer antigen. NKT cells produce cytokines with a variety of often opposing effects (e.g., promoting inflammation or inducing immunosuppression, including tolerance). Thus, they can contribute to antibacterial and antiviral immune responses, promote immune surveillance associated with tumors, and suppress or promote the development of autoimmune diseases. Like natural killer cells, NKT cells can also induce perforin, FAS, and TNF-related cytotoxicity. Thus, reference to T cells should be understood to include reference to NKT cells.

[0127] Natural killer (NK) cells are a type of cytotoxic lymphocyte that form part of the innate immune system. NK cells provide a rapid response to cells infected with viruses, acting about 3 days after infection, and also react to tumor formation. Typically, immune cells such as T cells detect the presence of major histocompatibility complex (MHC) on the surface of infected or transformed cells, triggering cytokine release and leading to target cell lysis or apoptosis. NK cells, however, have the ability to recognize stressed cells in the absence of antibodies or MHC, allowing for a faster immune response. This action is particularly important because harmful cells that lack MHC I markers cannot be detected and destroyed by other immune cells such as T cells. In contrast to NKT cells, NK cells do not express TCRs or CD3, but they often express the surface markers CD16 (FcyRIII) and CD56.

[0128] In some embodiments, immune cells to be modified or manipulated according to the methods of the application can be isolated from a mammalian subject, including, for example, blood (whole blood, serum, or plasma), bone marrow, thymus, lymph nodes.

[0129] In some embodiments, immune cells to be modified or manipulated according to the methods of the application can be collected from a cell line culture of immune cells derived from a mammalian subject (e.g., a T cell line).

[0130] In some embodiments, immune cells to be modified or manipulated according to the methods of the application can be differentiated from stem cells or other progenitor cells, e.g., from stem cell cultures and differentiated cells. Methods for differentiating stem cells into immune cells, in particular into T cells or NK cells, are known in the art (Li et al., Human iPSC-Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti-tumor Activity, Cell Stem Cell, 2018, 23(2): 181-192 e5; Themeli et al., Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy, Nat Biotechnol, 2013, 31(10): 928-33; Maeda et al., Regeneration of CD8alphabeta T Cells from T-cell-Derived iPSC Imparts Potent Tumor Antigen-Specific Cytotoxicity, Cancer Res, 2016, 76(23): 6839-6850).

[0131] stem cell

[0132] As used herein, “source cell” refers to a cell that will be transformed into a “derived cell” by reprogramming or differentiation. Examples of source cells suitable for use in the methods disclosed herein include stem cells. Examples of “derived cells” include immune cells, e.g., T cells, NKT cells, and NK cells.

[0133] The term “stem cell” is understood to mean a cell that is capable of self-renewal and exhibits the potential to develop in multiple lineage directions given its particular phenotype, and thereby forms new or regenerates tissues or cell populations of an organism. Stem cells utilized according to the present application are pluripotent and multipotent and are capable of differentiating along two or more lineages, and include, but are not limited to, embryonic stem cells (ESCs), adult stem cells, umbilical cord stem cells, hematopoietic stem cells (HSCs), progenitor cells, precursor cells, multipotent cells, multipotent cells, or dedifferentiated somatic cells (e.g., induced pluripotent stem cells). “Pluripotent” means that the subject stem cell can differentiate to form cells of any of the three germ layers, namely, ectoderm, endoderm, and mesoderm.

[0134] In some embodiments, the source cell also expresses at least one homozygous major HLA genotype. In some embodiments, the source cell expresses at least one homozygous HLA genotype that is a major transplantation antigen and is preferably expressed by a significant proportion of the population, e.g., at least 5%, at least 10%, at least 15%, at least 17%, at least 20% or more of the population. When the homozygous HLA genotype corresponds to a dominant MHC I or MHC II HLA type (in terms of tissue rejection), use of such a cell in the context of a therapeutic regimen results in a significantly reduced problem of tissue rejection in a broader population receiving the cells of the application. In other embodiments, the source cell can be homozygous with respect to more than one HLA antigen, e.g., two, three or more HLA antigens. The HLA antigens of interest can be selected from, e.g., HLA Al, B8, C7, DR17, DQ2, or HLA A2, B44, C5, DR4, DQ8, or HLA A3, B7, C7, DR15, DQ6.

[0135] In some embodiments, the source cell is homozygous with respect to an inhibitory gene.

[0136] In some embodiments, the source cell has been genetically modified in one or more genes identified herein, such that the function of the modified gene is inhibited in the derivative cells differentiated from the genetically modified source cell.

[0137] In some embodiments, the source cell has also been genetically modified to comprise a nucleic acid encoding a CAR (i.e., a chimeric antigen receptor). The nucleic acid encoding the CAR can be introduced into the source cell by methods known in the art.

[0138] In some embodiments, the source cell is a stem cell. In some embodiments, the source cell is an induced pluripotent stem cell (iPSC).

[0139] In some embodiments, progenitor cells capable of differentiating into immune cells are used for modification; e.g., cells cultured from pluripotent stem cells (e.g., iPSCs) that have undergone some differentiation toward immune cells in culture, but have not yet fully differentiated into immune cells.

[0140] iPSC

[0141] iPSCs are typically generated directly from somatic cells. iPSCs can in principle be induced from any nucleated cell, including, for example, mononuclear cells from blood and skin cells. In some embodiments, iPSCs can be generated from fully differentiated T cells; or from precursor T cells (e.g., thymocytes) that have already begun or even completed rearrangement of their TCR and exhibit specificity for a target antigen. In another embodiment, iPSCs are transfected with one or more nucleic acid molecules encoding a TCR (e.g., a rearranged TCR gene) directed against a target antigenic determinant (e.g., a tumor antigenic determinant). In one embodiment, iPSCs are derived from cells that express a rearranged TCR, preferably a rearranged aP TCR. In another embodiment, the cells express a rearranged gammadelta TCR. Examples of cells suitable for generating iPSCs of the application include, but are not limited to, CD4+ T cells, CD8+ T cells, NKT cells, thymocytes, or other forms of precursor T cells.

[0142] In another embodiment, iPSCs are derived from another type of immune cell, e.g., NK cells.

[0143] Methods for generating iPSCs from mature or differentiated cells (e.g., T cells or precursor T cells) are known to those of skill in the art (Themeli, Kloss et al. 2013, Li, Hermanson et al. 2018).

[0144] In some embodiments, the source cell is an induced pluripotent stem cell (iPSC).

[0145] In some embodiments, the source cell is generated from umbilical cord blood PBMCs (peripheral blood mononuclear cells).

[0146] In some embodiments, the subject source cell is a cell that is more differentiated toward an immune cell compared to a pluripotent stem cell.

[0147] Derivative immune cells generated by the methods disclosed herein include hematopoietic lineage cells that are capable of differentiating into immune cells and specific types of immune cells. Examples of derivative immune cells are HEs, pre-HSCs, HSCs, multipotent progenitor cells, common lymphoid progenitor cells, early thymic progenitor cells, pre-T cell progenitor cells, pre-NK progenitor cells, T progenitor cells, NK progenitor cells, macrophages, and other immune cells (e.g., T cells, NK-T cells, and NK cells).

[0148] The present disclosure relates to providing immune cells with enhanced function or derived immune cells produced by differentiation. By "enhanced function" is meant that the immune cells provided as a result of the modifications or manipulations disclosed herein exhibit enhanced activity (e.g., cytotoxicity), proliferation, survival, persistence, and / or infiltration as compared to control immune cells (i.e., immune cells without the modifications or manipulations). Cytotoxicity of an immune cell refers to the ability of the immune cell to kill target cells, typically through receptor-based mechanisms.

[0149] Genes to be inhibited

[0150] According to the present disclosure, inhibition of the function of one or more genes identified herein can enhance the function of an immune cell.

[0151] As used herein, "inhibition of the function of a gene" means that the level and / or activity of the protein encoded by the gene is ultimately reduced or eliminated. Thus, the function of a gene can be inhibited as a result of a manipulation or modification of the genomic DNA sequence of the gene (e.g., resulting in a disruption of the gene), as a result of inhibiting the mRNA (e.g., reducing the level or function of the mRNA, such as by inhibiting transcription or translation), or as a result of inhibiting the protein (e.g., by reducing the level or activity of the protein). In some embodiments, the degree of inhibition is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, when the level and / or activity of the protein encoded by the gene in the modified cell is compared to the level and / or activity of the protein in the unmodified cell.

[0152] In some embodiments, the gene whose function is to be inhibited is selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2. In some embodiments, the inhibition is directed to a single gene selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2; e.g., a single gene of RC3H1, RC3H2, A2AR, FAS, TGFBR1, or TGFBR2. In some embodiments, the inhibition is directed to a single gene selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2, in combination with inhibition of at least one other gene. In some embodiments, the inhibition is directed to two or more of the genes selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2, e.g., RC3H1 and RC3H2 genes, TGFBR1 and TGFBR2 genes, TGFBR1 and RC3H2 genes; and optionally in combination with inhibition of at least one other gene.

[0153] As described below, members of the genome of RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2 are known in the art to be involved in immune cell function. However, it is not known in the art whether inhibiting the function of these genes, alone or in combination, produces adverse consequences. In particular, it is expected that complete removal of the function of these genes (e.g., by gene editing) can have adverse effects on important cellular functions, resulting in reduced cell viability or replicative capacity. Furthermore, it is expected that removal of the function of these genes in stem cells (e.g., iPSCs) has adverse effects on cellular functions, such as viability, self-renewal, pluripotency, ability to differentiate into specific cell types (e.g., immune cells), and to function as these cell types. Those skilled in the art will recognize that maintaining these key cellular functions is a key feature of the present application.

[0154] RC3H1, RC3H2

[0155] RC3H1 is also known as RC3H1, Roquin-1, Ring Finger and CCCH Domain Containing 1, Ring Finger and CCCH-Type Zinc-Finger Domain Containing Protein 1, Ring Finger and C3H Zinc Finger Protein 1, Ring Finger and CCCH-Type Zinc Finger Domain 1, ROQ1, RNF198, or Ring Finger Protein 198.

[0156] RC3H2 is also known as Roquin-2, Roquin2, Ring Finger and CCCH Domain Containing 2, Ring Finger and CCCH-Type Zinc-Finger Domain Containing Protein 2, Ring Finger and CCCH-Type Zinc Finger Domain 2, MNAB, ROQ2, RNF164, or Ring Finger Protein 164.

[0157] The ROQUIN family of proteins includes ROQUIN1 (encoded by RC3H1) and ROQUIN2 (encoded by RC3H2), which are RNA-binding proteins that play important roles in the innate and adaptive immune system (Athanasopoulos, V., R.R. Ramiscal, and C.G. Vinuesa, ROQUIN signalling pathways in innate and adaptive immunity. Eur J Immunol, 2016, 46(5): p. 1082-90). Mutation of Rc3h1 in mice (sanroque mice) leads to increased ICOS expression in T cells, which causes a lupus-like autoimmune syndrome in mice (Yu, D., et al., Roquin represses autoimmunity by limiting inducible T-cell co-stimulator messenger RNA. Nature, 2007, 450(7167): p. 299-303). Although RC3H1 or RC3H2 knockout alone does not produce autoantibodies and lacks autoimmunity in mice, RC3H1 and RC3H2 double knockout mice show similar immunophenotypes as sanroque mice. To date, no human has been found to carry a disease-causing mutation in RC3H1 or RC3H2 (Athanasopoulos, V., R.R. Ramiscal, and C.G. Vinuesa, ROQUIN signalling pathways in innate and adaptive immunity. Eur J Immunol, 2016, 46(5): p. 1082-90). The role of RC3H1 and RC3H2 genes in human T cells, especially their function in cytotoxic cells, was unknown prior to the present disclosure.

[0158] According to the present disclosure, inhibition of the function of one or both of RC3H1 and RC3H2 genes enhances the function of immune cells.

[0159] A2AR

[0160] A2AR is also known as ADORA2A, adenosine A2a receptor, adenosine receptor A2a, ADORA2, adenosine receptor subtype A2a, or RDC8.

[0161] Extracellular adenosine produced by tumor cells is a key immunosuppressive metabolite that limits activation of cytotoxic lymphocytes through the adenosine 2A receptor (A2AR) and inhibits anti-tumor immune responses.

[0162] According to the present disclosure, inhibition of the function of the A2AR gene (e.g. by gene editing (e.g. by CRISPR / Cas9 mediated based on specially designed guide RNA)) enhances the function of the immune cell.

[0163] FAS

[0164] FAS is also known as Fas cell surface death receptor, APT1, CD95, FAS1, APO-1, FASTM, ALPS1A or TNFRSF6.

[0165] The FAS receptor (also known as CD95 and APO-1) induces apoptosis and terminal differentiation of cytotoxic T cells. Engagement of FAS with its ligand FASL can inhibit the anti-tumor activity of CAR-T cells.

[0166] According to the present disclosure, inhibition of the function of the FAS gene (e.g. by gene editing (e.g. by CRISPR / Cas9 mediated)) enhances the function of the immune cell.

[0167] TGFBR1 and TGFBR2

[0168] TGFBRl is also known as TGFRBRI, TGFB Receptor 1, TGF-beta Receptor 1, AAT5, ALK5, ESSl, LDSl, MSSE, SKR4, TBRI, ALK-5, LDS1A, LDS2A, TBR-1, TGFR-1, ACVRLK4, tβR-I, Transforming Growth Factor Beta Receptor 1 or Transforming Growth Factor Beta Receptor I.

[0169] TGFBR2 is also known as TGFBRII, AAT3, FAA3, LDS2, MFS2, RIIC, LDS1B, LDS2B, TAAD2, TBRII, TBR-ii, TGFR-2, TGF -RII, Transforming Growth Factor Beta Receptor 2 or Transforming Growth Factor Beta Receptor II.

[0170] TGF-β exerts a systemic immunosuppressive effect and inhibits host immune surveillance and is considered one of the main factors of the immunosuppressive microenvironment in tumors.

[0171] According to the present disclosure, inhibition of the function of the TGFBRl and / or TGFBR2 gene (e.g. by gene editing (e.g. by CRISPR / Cas9 mediated based on specially designed guide RNA)) enhances the function of the immune cell.

[0172] According to the present disclosure, inhibition of the function of at least one of the genes selected from RC3H1, RC3H2, A2AR, FAS, TGFBR1, and TGFBR2, in combination with inhibition of at least another gene, enhances the function of an immune cell.

[0173] Inhibition of the function of a gene

[0174] Inhibition of the function of a gene can be achieved by a variety of methods, for example by gene editing (inhibition of translation by, for example, RNA interference or antisense oligonucleotides), or by the use of a compound (for example a small molecule or an antibody that directly antagonizes the protein product).

[0175] Inhibition by gene editing

[0176] In some embodiments, inhibition of the function of a gene is achieved by the use of a gene editing system that modifies the genomic sequence of the gene.

[0177] A gene editing system typically includes a DNA-binding protein or DNA-binding nucleic acid coupled to a nuclease. The DNA-binding protein or DNA-binding nucleic acid specifically binds to or hybridizes to a targeted region of the gene, and the nuclease creates one or more double-strand breaks and / or one or more single-strand breaks in the targeted region of the gene. The targeted region can be in the coding region of the gene, for example in an exon, near the N-terminal portion of the coding region (for example, in the first or second exon). The double-strand or single-strand breaks can be repaired by cellular repair processes, for example by non-homologous end joining (NHEJ) or homology-directed repair (HDR). In some cases, the repair process introduces an insertion, a deletion, a missense mutation, or a frameshift mutation (including, for example, a bi-allelic frameshift mutation), resulting in disruption of the gene and inhibition of the function of the gene.

[0178] Examples of gene editing systems include fusions comprising a DNA-binding protein and a nuclease, for example a zinc finger nuclease (ZFN) or a TAL effector nuclease (TALEN), or an RNA-guided nuclease, for example a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas system.

[0179] ZFPs and TALENs

[0180] In some embodiments, inhibition of the function of a gene is achieved by the use of a gene editing system that includes a DNA-binding protein fused to an endonuclease, for example one or more zinc finger proteins (ZFPs) or transcription activator-like proteins (TALs). Examples include ZFNs, TALEs, and TALENs.

[0181] The DNA binding domains of ZFPs and TALs can be "engineered" to bind to a target DNA sequence of interest. For example, one or more amino acids of the recognition helix region of a naturally occurring zinc finger or TALE protein can be modified to bind directly to a predetermined DNA sequence. Criteria for rational design are described in, e.g., U.S. Patent 6,140,081, U.S. Patent 6,453,242, U.S. Patent 6,534,261, WO 98 / 53058, WO 98 / 53059, WO 98 / 53060, WO 02 / 016536, WO 03 / 016496, and U.S. Patent Publication No. 20110301073 Al.

[0182] In some embodiments, the DNA binding protein comprises a zinc finger protein (ZFP) or one or more zinc finger domains of a ZFP. ZFPs or domains thereof bind to DNA in a sequence-specific manner through one or more "zinc fingers," regions of amino acids within the binding domain whose structure is stabilized by coordination of a zinc ion. The sequence specificity of naturally occurring ZFPs can be altered by making amino acid substitutions at certain positions on the zinc finger recognition helix. Additionally, many engineered gene-specific zinc fingers are commercially available (see, e.g., the CompoZr platform for zinc finger construction, developed by Sangamo Biosciences (Richmond, CA, USA) in collaboration with Sigma-Aldrich (St. Louis, MO, USA)). Thus, in some embodiments, the ZFP is engineered to bind to a target sequence within a gene identified herein as being to be suppressed. Typical target sequences include exons, regions near the N-terminal region of a coding sequence (e.g., the first exon, the second exon), and 5' regulatory regions (promoter or enhancer regions). ZFPs are fused to endonucleases or DNA cleavage domains to form zinc finger nucleases (ZFNs). Examples of DNA cleavage domains include DNA cleavage domains of type IIS restriction enzymes.

[0183] In some embodiments, the ZFN is introduced into a cell (e.g., an immune cell or a stem cell) by transfecting a nucleic acid construct comprising a nucleic acid sequence encoding the ZFN. The ZFN is then expressed from the construct in the cell and causes editing and disruption of the target gene. In some embodiments, the ZFN is introduced into the cell in its protein form.

[0184] In some embodiments, the DNA binding protein comprises a naturally occurring or engineered transcription activator-like protein (TAL) DNA binding domain, for example in a transcription activator-like protein effector (TALE) protein. See, e.g., US20110301073 Al, incorporated herein by reference. TALE DNA binding domains are polypeptides comprising one or more TALE repeat sequences, each repeat sequence being 33-35 amino acids in length and containing 1 or 2 DNA binding residues. It has been determined that the HD (histidine-aspartate) sequence at positions 12 and 13 of the TAL repeat sequence results in binding to cytosine (C), NG (asparagine-glycine) binds to T, NI (asparagine-isoleucine) binds to A, and NN (asparagine-asparagine) binds to G or A. See, e.g., US20110301073 Al. In some embodiments, a TALE can be designed to have an array of TAL repeat sequences specific for a target DNA sequence of interest within a gene identified herein as being to be inhibited. Custom designed TALE arrays are also commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA). In some embodiments, the TAL DNA binding domain is fused to an endonuclease to form a TALE-nuclease (TALEN) that cleaves the nucleotide sequence at a target site within a gene identified herein as being to be inhibited.

[0185] In some embodiments, the TALEN is introduced into a cell by transfecting a nucleic acid construct (e.g., a plasmid, mRNA, or lentivirus vector) comprising a nucleic acid sequence encoding the TALEN. The TALEN is then expressed from the construct in the cell and causes editing and disruption of the target gene. In some embodiments, the TALEN is introduced into the cell in its protein form.

[0186] CRISPR / Cas

[0187] In some embodiments, inhibition of gene function is achieved by utilizing the CRISPR (for "Clustered Regularly Interspaced Short Palindromic Repeats") / Cas (for "CRISPR- Associated Nuclease") system for gene editing. CRISPR / Cas is well known in the art, with reagents and protocols readily available (Mali et al., 2013, Science, 339(6121), 823-826; Hsu et al., 2014, Cell, 157.6: 1262-1278; Jiang et al., 2013, Nature Biotechnology, 31, 233-239; Anzalone et al., Nature (2019) doi:10.1038 / s41586-019-1711-4; Komor et al., Nature 533: 420-424, 2016; Gaudelli et al., Nature 551: 464-471 (2017)). Exemplary CRISPR-Cas gene editing protocols are described in Jennifer Doudna and Prashant Mali, 2016, "CRISPR-Cas: A Laboratory Manual" (CSHL Press, ISBN: 978-1-621821-30-4) and Ran et al. 2013, Nature Protocols, 8 (11): 2281-2308.

[0188] The CRISPR / Cas system generally comprises two components: (1) an RNA-dependent DNA nuclease, also referred to herein as a CRISPR endonuclease or Cas protein, such as Cas9, Cas12, or other alternative nuclease; and (2) a non-coding short “guide RNA” comprising a dual RNA containing crRNA (“CRISPR RNA”) and tracrRNA (“trans-activating crRNA”) or a single-stranded full-length guide RNA, and comprising a targeting sequence that directs the nuclease to a target site in the genome. The guide RNA (gRNA) directs the nuclease to the target site, where the nuclease generates a double-stranded break (DSB) in the DNA at the target site. The resulting DSB is then repaired by one of two general repair pathways: the non-homologous end joining (NHEJ) pathway and the homology-directed repair (HDR) pathway. The NHEJ repair pathway is the most active repair mechanism, capable of rapidly repairing DSBs, but often results in small nucleotide insertions or deletions (Indels) at the DSB site, leading to frameshift mutations to knock out functional genes. The HDR pathway is less efficient, but has high fidelity. The double-stranded break is repaired by HDR using a homologous DNA template when the CRISPR endonuclease is provided with a DNA template that is homologous to the region of the break. The HDR pathway allows large gene insert fragments to be inserted into cells along with the RNP.

[0189] The design or selection of a gRNA sequence comprising a sequence that targets a target site in a gene of interest has been described in the art. The target site can include a regulatory region sequence (e.g., a promoter and an enhancer), or a sequence within a coding region (e.g., an exon, such as an exon near the 5’ end, or an exon that encodes a particular domain or region of a protein). In some embodiments, the target site is selected based on its position immediately 5’ of a PAM sequence, such as typically NGG or NAG.

[0190] The guide sequence is designed to include a targeting sequence that has complementarity to a target sequence (nucleotide sequence at the target site). Perfect complementarity is not necessarily required, only sufficient complementarity to cause specific hybridization between the guide sequence and the target sequence and to facilitate formation of a CRISPR complex at the target site. In some embodiments, the degree of complementarity between the targeting sequence of the gRNA and the target sequence is at least 80%, 85%, 90%, 95%, 98%, 99%, or higher (e.g., 100% or perfect complementarity).

[0191] In some embodiments, the guide sequence is at least 15 nucleotides in length, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 or more nucleotides in length. In some embodiments, the guide sequence is no more than 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, or 20 nucleotides in length. In some embodiments, the targeting sequence portion of the guide sequence is about 20 nucleotides in length. Truncated gRNAs with shorter regions of target complementarity (< 20 nucleotides) have been described to effectively improve target specificity (see, e.g., Fu et al., Nature Biotechnol., 32(3): 279-284, 2014). Thus, in some embodiments, the targeting sequence of the guide RNA is 17, 18, 19, or 20 nucleotides in length. In some embodiments, the targeting sequence of the guide RNA is fully complementary to the nucleotide sequence at the target site. In some embodiments where the targeting sequence of the guide RNA is not fully complementary to the nucleotide sequence at the target site, the portion of the targeting sequence proximal to the PAM sequence in the genome (also referred to as the seed region) is fully complementary to the nucleotide sequence at the target site. In other words, some variation of the nucleotides 5' of the guide sequence (i.e., the non-seed region) is permissible. For example, the guide sequence can be designed to comprise a targeting portion that is at least 17 nucleotides in length, e.g., 17, 18, 19, or 20 nucleotides in length, with a seed region of at least 17 nucleotides that is fully complementary to at least 17 nucleotides in the target sequence.

[0192] Examples of target sequences in particular genes are provided in Table 1. In some embodiments, the guide sequence comprises a targeting sequence of 17-20 nucleotides, wherein at least 17 nucleotides in the seed region (3' portion of the targeting sequence) are fully complementary to at least 17 nucleotides in the target sequence, e.g., to the 17 nucleotides 3' of the target sequence.

[0193] Table 1

[0194]

[0195] Databases of gRNAs for CRISPR genome editing are publicly available, which provide exemplary sgRNA target sequences in constitutive genes exons of the human genome or mouse genome (see, e.g., gRNA databases provided by GenScript and Massachusetts Institute of Technology; see also Sanjana et al. (2014) Nat. Methods, 11:783-4). In some embodiments, the gRNA sequence is or comprises a sequence with minimal off-target binding to non-target genes.

[0196] Examples of Cas proteins or CRISPR endonucleases suitable for use herein include Cpfl (Zetsche et al., Cell (2015) 163(3): 759-771), Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx 12), CaslOO, Csy l, Csy2, Csy3, Cse l, Cse2, Csc l, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr l, Cmr3, Cmr4, Cmr5, Cmr6, Csb l, Csb2, Csb3, Csx 17, Csx 14, Csx 10, Csx 16, CsaX, Csx3, Csx l, Csx 15, Csf l, Csf2, Csf3, or Csf4, or a functional derivative thereof (i.e., a mutated form or derivative of a naturally occurring CRISPR endonuclease, e.g., a fragment thereof, which substantially retains the RNA-dependent endonuclease activity of the naturally occurring form). See, e.g., US20180245091A1 and US20190247517A1. In some embodiments, the Cas protein is Cas9, e.g., from S. pyogenes, S. aureus, or S. pneumoniae. In some embodiments, the Cas protein is a Cas9 protein from S. pyogenes having the amino acid sequence provided in the SwissProt database under accession number Q99ZW2.

[0197] In some embodiments, the inhibition of gene function is achieved by CRISPR-mediated gene editing, which includes introducing into a cell (e.g., an immune cell or a stem cell) a first nucleic acid encoding a Cas nuclease and a second nucleic acid encoding a guide RNA (gRNA) specific for a target sequence in a gene identified herein as being to be inhibited. The two nucleic acids can be contained in one nucleic acid construct (or vector), or provided on different constructs (or vectors), to effect expression of the Cas protein and gRNA in the cell. Expression of the Cas nuclease and gRNA in the cell directs formation of a CRISPR complex at the target sequence, which results in DNA cleavage.

[0198] In some embodiments, the inhibition of gene function is achieved by CRISPR-mediated gene editing, which includes introducing a combination or complex of gRNA and Cas nuclease into the cell. In some embodiments, the Cas protein / gRNA combination or complex can be delivered into the cell by, for example, electroporation, biolistics, calcium phosphate transfection, cell compression or squeezing, liposomes, nanoparticles, microinjection, naked DNA plasmid transfer, protein transduction domain-mediated transduction, or viral-mediated (including integrating viral vectors (e.g., retrovirus and lentivirus) and non-integrating viral vectors (e.g., adenovirus, AAV, HSV, vaccinia)).

[0199] Regardless of the particular gene editing method used, to confirm that the gene sequence has been modified and that the gene function has been inhibited, a variety of assays can be performed, including, for example, by examining DNA or mRNA by means of Southern and Northern blotting, PCR including RT-PCR, or nucleic acid sequencing, or by detecting the presence or activity of a particular protein or peptide by means of, for example, immunologically (ELISA and Western blot).

[0200] In some embodiments, the function of at least one of RC3H1, RC3H2, A2AR, and FAS genes is inhibited by introducing indels into early exons of at least one of these genes by the CRISPR / Cas9 system, which results in a frameshift mutation of at least one of these genes such that a functional protein is not translated from the edited gene. In some embodiments, the function of two or more of RC3H1, RC3H2, A2AR, and FAS genes is inhibited by introducing indels into early exons of two or more of these genes using CRISPR / Cas9, which results in a frameshift mutation of two or more of these genes such that a functional protein is not translated from the edited gene. In some embodiments, two or more of RC3H1, RC3H2, A2AR, and FAS genes comprise RC3H2 in combination with another gene, for example, RC3H2 and RC3H1.

[0201] In some embodiments, the function of at least one of the TGFBRl and TGFBR2 genes is inhibited by introducing indels into the exons and upstream of the codon for the start amino acid residue of the intracellular signaling domain for at least one of these genes using the CRISPR / Cas9 system, resulting in a frameshift mutation that removes the intracellular signaling domain, which is a dominant negative mutation. In some embodiments, the function of both the TGFBRl and TGFBR2 genes is inhibited by introducing indels into the exons and upstream of the codon for the start amino acid residue of the intracellular signaling domain for at least one of these genes using the CRISPR / Cas9 system, resulting in a frameshift mutation that removes the intracellular signaling domain, which is a dominant negative mutation.

[0202] By using a nickase (i.e., a Cas9 nickase) and a high-fidelity enzyme, the CRISPR / Cas system can also be used without a double-strand break or a donor DNA. See, e.g., Anzalone, A, et al., Nature (2019) doi:10.1038 / s41586-019-1711-4; Komor et al., Nature 533: 420-424, 2016; Gaudelli et al., Nature 551:464-471 (2017).

[0203] Inhibition by reducing or eliminating the level or function of mRNA

[0204] In some embodiments, the inhibition of gene function is achieved by reducing or eliminating the level or function of mRNA transcribed from the gene (i.e., inhibition of mRNA). Unlike inhibition by gene editing systems, inhibition of mRNA is temporary.

[0205] In some embodiments, the inhibition of mRNA can be achieved by using, for example, an antisense nucleic acid, a ribozyme, a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a miRNA (microRNA), or a precursor thereof, or a nucleic acid construct that can be transcribed in a cell to produce an antisense RNA, an siRNA, an shRNA, a miRNA, or a precursor thereof.

[0206] Antisense-antisense technology is a well-known method. Antisense RNA is an RNA molecule that is complementary to all or part of an endogenous mRNA and blocks translation from the endogenous mRNA by forming a duplex with the endogenous mRNA. Antisense RNA can be prepared synthetically and introduced into a target cell (e.g., an immune cell), or prepared in a target cell by transcription from an exogenously introduced nucleic acid construct, to effect inhibition of expression of a target gene. Antisense RNA need not be complementary to the full-length mRNA from a target gene. However, the antisense RNA should be of sufficient length to form a duplex with the target mRNA and block translation based on the target mRNA. Typically, the antisense RNA is at least 15 nucleotides in length, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 35, 40, 50, 75, 100, 200, 300, 400, 500 nucleotides or more in length. In some embodiments, the antisense RNA is no more than 500, 400, 300, 200, 100, 75, or 50 nucleotides in length. Antisense molecules can also be DNA, DNA analogs, and RNA analogs.

[0207] Ribozymes-ribozymes (i.e., catalytic RNAs) can be designed to specifically pair with a target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. See, e.g., U.S. Patent No. 6,423,885, U.S. Patent No. 5,254,678, and Perriman et al., PNAS 92(13):6175-6179 (1995). Ribozymes can be prepared synthetically and introduced into a target cell (e.g., an immune cell), or prepared in a target cell by transcription from an exogenously introduced nucleic acid construct.

[0208] RNAi (RNA interference) - Inhibition of gene expression or translation by RNAi is known in the art and can be achieved using RNA molecules (e.g., siRNA ("small interfering RNA"), shRNA ("short hairpin RNA"), and miRNA ("microRNA")). siRNAs and shRNAs are known to be involved in the RNA interference pathway and interfere with the expression of specific genes. siRNAs are small (typically 20-25 nucleotides in length) double-stranded RNAs and can be designed to comprise a sequence that is homologous or complementary to a target mRNA (i.e., mRNA transcribed from a gene of interest) or a portion of a target mRNA. shRNAs are cleaved by the ribonuclease DICER to produce siRNAs. Given the sequence of a target gene, siRNAs or shRNAs can be designed and prepared synthetically and introduced into a cell of interest (e.g., an immune cell), or prepared in a cell of interest (e.g., an immune cell) from an exogenously introduced nucleic acid construct encoding such an RNA. miRNAs are also small RNA molecules (typically about 21-22 nucleotides) processed from long precursors transcribed from non-protein-coding genes, and interrupt translation through imperfect base pairing with target mRNAs. miRNAs or their precursors (pri-miRNAs or pre-miRNAs) can be prepared synthetically and introduced into a cell of interest (e.g., an immune cell), or prepared in a cell of interest (e.g., an immune cell) from an exogenously introduced nucleic acid construct encoding the miRNA or its precursor.

[0209] In some embodiments, inhibition of mRNA can be achieved using a modified form of the CRISPR / Cas system, in which a Cas molecule, which is an enzyme-inactivated nuclease, is used in combination with a gRNA targeting a gene of interest. The target site can be in the 5' regulatory region of the gene (e.g., a promoter or enhancer region). In some embodiments, the Cas molecule is an enzyme-inactivated Cas9 molecule comprising a mutation, e.g., a point mutation, that abolishes or significantly reduces DNA cleavage activity (see, e.g., WO2015 / 161276). In some embodiments, the enzyme-inactivated Cas9 molecule is directly or indirectly fused to a transcriptional repressor protein.

[0210] Inhibition by other means

[0211] The present application includes other methods known in the art for inhibiting the function of a gene, including for reducing the level or activity of a protein encoded by the gene, e.g., by introducing into a cell (e.g., an immune cell) a compound (e.g., a small molecule, an antibody, etc.) that directly inhibits the activity of a protein encoded by the gene.

[0212] CAR

[0213] In some embodiments, the cell (e.g., immune cell or stem cell) that is modified to suppress one or more selected genes is also modified to contain a nucleic acid encoding a chimeric antigen receptor (or "CAR").

[0214] In some embodiments, the nucleic acid encoding the CAR can be introduced into the cell before, at the same time, or after the cell is modified to suppress the function of the selected gene. In embodiments where the suppression is transient (e.g., by antisense RNA or RNAi), it is preferred that the nucleic acid encoding the CAR is introduced into the cell before the cell is modified to effect suppression. In embodiments where the suppression is permanent (e.g., by gene editing), the nucleic acid encoding the CAR can be introduced into the cell before, at the same time, or after the cell is modified to effect suppression. In some embodiments, the nucleic acid encoding the CAR is designed to allow insertion by HDR into the target site of the gene editing following introduction of a DSB, i.e., the gene is disrupted by knock-in or insertion of the nucleic acid encoding the CAR.

[0215] In some embodiments, the CAR gene can be introduced into the cell by a variety of techniques, including lentivirus or retrovirus vectors, transposon systems, CRISPR-Cas9 or TALEN-mediated gene knock-in.

[0216] The term "chimeric antigen receptor" ("CAR", also known as "artificial T cell receptor", "chimeric T cell receptor", and "chimeric immunoreceptor") is understood to mean an engineered receptor that grafts an antigen recognition moiety onto an immune cell. Generally, a CAR is composed of an antigen recognition moiety that has specificity for a target antigen, a transmembrane domain, and an intracellular / cytoplasmic signaling domain of a receptor that is naturally expressed on an immune cell, operably linked to each other. By "operably linked" is meant that the various domains are linked to each other such that, upon binding of the antigen recognition moiety to the target antigen, a signal is induced through the intracellular signaling domain to activate the cell expressing the CAR (e.g., a T cell or NK cell) and to have its effector function activated.

[0217] The antigen recognition moiety of a CAR is the extracellular portion of the receptor that recognizes and binds to an epitope of the target antigen. The antigen recognition moiety is typically, but not limited to, an scFv.

[0218] The intracellular domain of a CAR can include a primary cytoplasmic signaling sequence of a naturally occurring receptor of an immune cell and / or a secondary or costimulatory sequence of a naturally occurring receptor of an immune cell. Examples of primary cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the intracellular signaling domain of a CAR comprises a cytoplasmic signaling sequence of CD3-zeta. In some embodiments, the intracellular signaling domain of a CAR can comprise a cytoplasmic signaling sequence of CD3-zeta in combination with a costimulatory signaling sequence of a costimulatory molecule. Examples of suitable costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, TIM3, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and the like. In some embodiments, the cytoplasmic domain of a CAR is designed to contain a signaling domain of CD3-zeta and a signaling domain of CD28.

[0219] The transmembrane domain of a CAR is typically a hydrophobic alpha helix that spans the membrane and can be derived from any membrane-bound or transmembrane protein. The transmembrane domain can be derived from natural sources or synthetic sources. Where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. For example, the transmembrane region can be derived from the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from an immunoglobulin such as IgG4. Alternatively, the transmembrane domain can be synthetic, in which case it comprises primarily hydrophobic residues, such as leucine and valine.

[0220] The term "target antigen" should be understood to refer to any protein or non-protein molecule expressed by a cell that is sought to be targeted by an immune cell (e.g., a T cell or NK cell) expressing a receptor. A target antigen can be a "self" molecule (a molecule expressed in the patient's body) or a non-self molecule (e.g., from an infected microorganism). Target antigens referred to herein are not limited to molecules that are naturally capable of eliciting a T or B cell immune response; rather, "target antigen" refers to any protein or non-protein molecule sought to be targeted. In some embodiments, a target antigen is expressed on the surface of a cell. It should be understood that a target antigen can be expressed only by a target cell, or it can be expressed by non-target cells. In some embodiments, a target antigen is a non-self molecule or a molecule expressed only by, or at a significantly higher level than normal cells by, the cell sought to be targeted. Non-limiting examples of target antigens include the following: differentiation antigens (e.g., MART-1 / Melan A (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2) and tumor-specific multilineage antigens (e.g., MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5); overexpressed glycoproteins, such as MUC1 and MUC16; overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressors, such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; e.g., BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein-Barr virus antigens EBVA and human papilloma virus (HPV) antigens E6 and E7.Other tumor-associated antigens include folate receptor alpha (FRa), EGFR, CD47, CD24, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl 85 erbB2, pl 80 erbB-3, cMet, nm-23Hl, PSA, CA 19-9, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, NB / 70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG-72, TLP, TPS, PSMA, mesothelin, or BCMA.

[0221] In some embodiments, the target antigen is a tumor-associated antigen, in particular a protein, glycoprotein, or non-protein tumor-associated antigen.

[0222] In some embodiments, the target antigen is selected from CD47, folate receptor alpha (FRa), and BCMA.

[0223] In some embodiments, the target antigen is a tumor-associated antigen, for example the tumor-associated antigen TAG-72.

[0224] In other embodiments, the target antigen is a surface protein, for example CD24, and in another embodiment, a surface protein useful for tumor targeting, for example CD19 or CD20.

[0225] Pharmaceutical compositions and therapeutic uses of modified cells

[0226] In another aspect, provided herein are compositions comprising cells produced by the methods disclosed herein, i.e., modified cells in which the function of one or more selected genes has been inhibited.

[0227] In some embodiments, provided herein are pharmaceutical compositions comprising the cells produced herein and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include solvents, dispersion media, isotonic agents, and the like. Examples of carriers include oils, water, salt solutions, gels, lipids, liposomes, resins, porous matrices, preservatives, and the like, or combinations thereof. In some embodiments, the pharmaceutical compositions are prepared and formulated for administration to a patient, for example, for adoptive cell therapy, typically in unit dosage injectable form (solutions, suspensions, emulsions). In some embodiments, the pharmaceutical compositions can employ timed-release, delayed-release, and sustained-release delivery systems.

[0228] In some embodiments, the pharmaceutical composition comprises the cells in an amount effective to treat or prevent a disease or disorder, for example, a therapeutically effective amount or a prophylactically effective amount. In some embodiments, the pharmaceutical composition comprises the modified cells disclosed herein in an amount of about 1 million to about 100 billion cells, for example, at least 1, 5, 10, 25, 50, 100, 200, 300, 400, or 500 million cells, up to about 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 billion cells.

[0229] In some embodiments, the pharmaceutical composition further comprises another active agent or drug, for example, a chemotherapeutic agent.

[0230] In another aspect, provided herein are methods and uses of the modified cells disclosed herein, for example, methods and uses of treatment in adoptive cell therapy.

[0231] In some embodiments, the methods comprise administering the modified cells disclosed herein or a composition comprising the modified cells disclosed herein to a subject having or at risk of developing a disease or disorder.

[0232] In some embodiments, the disease or disorder is a neoplastic disorder (i.e., cancer), a microbial or parasitic infection (e.g., HIV, STD, HCV, HBV, CMV, COVID-19, or antibiotic-resistant bacteria), an autoimmune disease (e.g., rheumatoid arthritis (RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Graves’ disease, Crohn’s disease, multiple sclerosis, asthma), organ fibrosis (e.g., heart, lung, liver, etc.), or endometriosis.

[0233] In some embodiments, the oncological disorder comprises a central nervous system tumor, retinoblastoma, neuroblastoma, pediatric tumors, head and neck cancer (e.g., squamous cell carcinoma), breast and prostate cancer, lung cancer (small cell and non-small cell lung cancer), kidney cancer (e.g., renal cell adenocarcinoma), esophagogastric cancer, hepatocellular carcinoma, pancreaticobiliary tumors (e.g., adenocarcinoma and islet cell tumors), colorectal cancer, cervical and anal cancer, uterine and other genital tract cancer, urinary tract cancer (e.g., ureter and bladder), germ cell tumors (e.g., testicular germ cell tumors or ovarian germ cell tumors), ovarian cancer (e.g., ovarian epithelial cancer), cancer of unknown primary, human immunodeficiency-related malignancies (e.g., Kaposi's sarcoma), lymphoma, leukemia, malignant melanoma, sarcoma, endocrine tumors (e.g., thyroid), mesothelioma and other pleural or peritoneal tumors, neuroendocrine tumors, and carcinoid tumors.

[0234] In some embodiments, the present methods result in treatment of a disorder, i.e., a reduction or amelioration of the disorder or of one or more symptoms of the disorder, e.g., by inhibiting tumor growth and / or metastasis in the context of treating cancer or by reducing viral load and / or transmission in the context of treating a viral infection. The term "treatment" does not necessarily imply complete recovery. In some embodiments, the present methods result in prevention of a disorder, i.e., preventing, reducing the risk of developing, or delaying the onset of the disorder. Similarly, "prevention" does not necessarily mean that the subject will ultimately not have the disorder.

[0235] In some embodiments, the subject (e.g., patient) to whom a cell or composition is administered is a mammal, typically a primate, e.g., a human.

[0236] In some embodiments, the cell or composition comprising the cell is administered parenterally. As used herein, the term "parenterally" includes intravenous, intramuscular, subcutaneous, and intraperitoneal administration.

[0237] The desired dosage of the modified cell or composition comprising the modified cell can be delivered by a single administration, multiple administrations, or continuous infusion administration of the composition. Therapeutic or prophylactic efficacy can be monitored by periodic assessment of treated subjects.

[0238] In some embodiments, the adoptive cell therapy is performed by autologous transfer. Immune cells (e.g., T cells) are isolated and / or otherwise prepared from a subject who is to receive the cell therapy, or from a sample derived from such subject. In some embodiments, immune cells (e.g., T cells or NK cells) are isolated from a subject, modified (to suppress the function of one or more genes) according to the methods disclosed herein, and then administered to the same subject.

[0239] In some embodiments, adoptive cell therapy is performed by allogeneic transfer, in which the cells are isolated and / or otherwise prepared from a donor subject that is different from the subject (recipient subject) that is to receive the cell therapy. In some embodiments, the donor and recipient subjects express the same HLA class or supertype.

[0240] Examples

[0241] In the following examples, it has been demonstrated, but not limited to, that in order to enhance the function of CAR-T cells, T cells, NK cells and derived cells (e.g. iNK cells) for tumor therapy, CRISPR / Cas9 gene editing technology was employed to eliminate negative immune regulators of these immune cells. In the case of CAR-containing T cells, the cells were first transduced by lentiviral CAR vectors after activation, and then Cas9 nuclease complex with specially designed guide RNA was transfected into CAR-T cells to ablate immune regulatory genes (Figure 1A). In the case of CAR-containing NK-92 cells, the cells were first transfected with Cas9 nuclease complex with specially designed guide RNA to ablate immune regulatory genes, and then transduced using lentiviral CAR vectors (Figure IB). The efficiency of gene editing was checked by quantitative based on genomic DNA sequencing. The cytotoxicity and expansion rate were then monitored during the in vitro expansion of the cells. To evaluate the in vivo persistence, CAR-cells (CAR-T cells in the following examples) were adoptively transferred into mouse xenograft tumor models (NOD / SCID / IL2Rγnull mice) and the tumor growth was monitored over time. Figures 1A-1B .

[0242] Example 1 - Generation of second generation TAG-72 CAR-T cells

[0243] TAG-72 is a well-established tumor marker for adenocarcinoma and also a target for CAR-T cells in certain solid tumors. Second generation TAG-72 CAR-T cells were generated as described in WO2017 / 088012, incorporated herein by reference. The TAG-72 CAR expression cassette contains a kappa leader sequence as signal peptide, an anti-TAG-72 scFv as tumor antigen binding moiety, a hinge and transmembrane region from human CD8, and cytoplasmic activation signaling domains of 4-1BB and CD3 zeta. P2A is a signal sequence directing proteolytic cleavage that releases EGFP as a fluorescent reporter protein for CAR expression (Figure 2A). Thus, after lentiviral transduction, CAR transduction efficiency and expression levels in T cells can be detected using GFP flow cytometry (Figure 2B).

[0244] Isolation and culture of human T cells

[0245] Primary human T cells were isolated from fresh whole blood from healthy human donors or buffy coat (unfit / discard material not suitable for clinical purposes) obtained from the Australian Red Cross Blood Service. Informed consent was provided by all patients and healthy donors. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (GE Healthcare, Illinois, USA) centrifugation using Leucosep™ tubes (Greiner, Kremsmünster, Austria) according to the manufacturer’s instructions. PBMCs were cryopreserved prior to use. For use in transduction and transfection, PBMCs were thawed and T cells were isolated using Dynabeads ® Human T-activator CD3 / CD28 beads (Thermofisher, Massachusetts, USA) were used to isolate and activate T cells. Cells and beads were incubated at a 1 :3 ratio for 1 hour at room temperature with constant gentle mixing. Unbound cells were then removed by placing the cell-bead suspension on a magnet for 1-2 minutes. The supernatant was removed and the cell-bead mixture was incubated in T cell media: TexMACS media (Miltenyi Biotech, Bergisch Gladbach, Germany) with 5% human AB serum (Sigma-Aldrich, Missouri, USA) and 100 U / mL IL-2 at 37°C 5% CO2 for approximately 65 hours. T cells were collected by mixing 20-50x to dissociate the cell-bead complex, immediately placed on a magnet for 1-2 minutes and the cell-containing supernatant was collected. MUSE TM The isolated T cell suspension was counted on a Casy® Cell Counter (Merck-Millipore, Massachusetts, USA) and prepared for transfection.

[0246] Lentiviral transduction

[0247] Activated human CD3+T cells were transduced using lentiviral CAR vectors as described in WO2017 / 088012, incorporated herein by reference. To generate lentiviral CAR-T cells, activated human CD3+ / CD28+T cells were incubated with lentiviral particles in RetroNectin ® (Takara Bio Inc) coated plates for 48 hours.

[0248] Flow cytometry of CAR expression.

[0249] To detect expression of CAR constructs in lentivirally transduced CAR-T cells, MACSQuant® flow cytometry was performed on cells stained with anti-CD3 antibody conjugated to phycoerythrin (PE) and anti-CD4 antibody conjugated to fluorescein isothiocyanate (FITC) (BD Biosciences, California, USA) according to the manufacturer’s instructions. ®Flow cytometry analysis was performed on Analyzer 10 (Miltenyi Biotec, Bergischgradbach, Germany). GFP expression was analyzed. Live and dead cells were distinguished using propidium iodide solution (Miltenyi Biotec) or Viobility 405 / 520 dye.

[0250] Example 2 - Generating gene-edited TAG-72 CAR-T cells using CRISPR

[0251] To generate CRISPR knockout (KO) CAR-T cells, 48 ​​hours after lentiviral TAG-72 CAR transduction on day 5, T cells were transfected with representative guide RNAs (PD1 KO, SEQ ID NO: 1; RC3H1 KO, SEQ ID NO: 2; RC3H2 KO, SEQ ID NO: 4; A2AR KO, SEQ ID NO: 7; FAS KO, ​​SEQ ID NO: 9; TGBFBR1 KO, SEQ ID NO: 11; TGFBR2 KO, SEQ ID NO: 14) (Figures 1A to 3). Despite electroporation-induced cell death, the cells remained healthy using the protocol disclosed herein. Figure 3 This method can recover and expand CAR-T cells transfected with RNP and untransfected CAR-T cells. Four days after RNP transfection, genomic DNA from CAR-T cells was extracted for quantitative analysis of gene editing. Gene editing efficiency was analyzed using ICE (Inference of CRISPR Edits) assay (Hsiau et al., Inference of CRISPR Edits from Sanger Trace Data. bioRxiv, 2018, 10.1101 / 251082(251082)). This paper presents RC3H2 gene editing efficiency analysis as a representative result of ICE assay. Figure 4A (To 4C). RC3H2 gRNA (SEQ ID NO: 4) showed high activity in introducing indels (total indel frequency = 92%) into early exons of the RC3H2 gene. Additionally, it caused high-frequency frameshifts within open reading frames (out-of-frame indel frequency = 91%), thereby disrupting the translation of functional RC3H2 proteins. Figure 4Band 4C). In this study, very high gene editing efficiency (total indel percentage = 89% to 96%) and high gene knockout results (out-of-frame indel frequency = 61% to 91%) were achieved for all CAR-T cells that were CRISPR gene edited (Fig. 4D). Taken together, these results demonstrate that the gRNAs used in the study were validated to have high activity to disrupt expression of the corresponding genes in CAR-T cells without interfering with CAR-T cell in vitro expansion.

[0252] CRISPR gene editing of CAR-T cells

[0253] Two days after lentiviral TAG-72 CAR transduction, T cells were washed with dPBS for Cas9 RNP transfection. crRNA and tracrRNA (Synthego or IDT) were annealed to form full-length guide RNA. Cas9 RNP was prepared by incubating Cas9 protein with full-length gRNA at a 1:2 ratio for 10 to 20 minutes at room temperature before transfection. To transfect Cas9 RNP, T cells were electroporated with Neon Transfection Device (Thermofisher) or 4D-Nucleofector Device (Lonza, Basel, Switzerland).

[0254] Quantitative assessment of genome editing

[0255] The efficacy and mutation profile of CRISPR / Cas9 genome editing efficiency were analyzed by ICE assay (Hsiau et al., Inference of CRISPR Edits from Sanger Trace Data. bioRxiv, 2018, 10.1101 / 251082 (251082)). Genomic DNA was extracted from cells using ISOLATE II Genomic DNA Kit (Bioline) 4 days after electroporation following the manufacturer’s instructions. High-fidelity Taq polymerase (New England Biolabs) was used to generate PCR amplicons spanning the gRNA genomic target site. Purified PCR products were subjected to Sanger sequencing, and sequence chromatograms were analyzed with the ICE software available online

[0256] Example 3 - In vitro function of CRISPR gene edited TAG-72 CAR-T cells

[0257] During the expansion phase of the gene edited TAG-72 CAR-T cells, in vitro xCELLigence ®Real-time assay to evaluate tumor killing ability of cells. Genetically edited TAG-72 CAR-T cells were generated and validated as described in Examples 1 and 2.

[0258] T cell in vitro cytotoxicity assay

[0259] The real-time cell monitoring system (xCELLigence ® ) was used to determine the killing efficiency of CAR-T cells in vitro. 10,000 target cells / 100 µL (e.g., ovarian cancer cell line OVCAR-3) were resuspended in culture medium (e.g., RPMI-1640 basal medium) supplemented with 10% - 20% fetal bovine serum and bovine insulin and deposited into an RTCA plate. The target cells were kept at 37°C, 5% CO2 for 3-20 hours to allow cell attachment. After target cell attachment, TAG-72 CAR-T effector cells were added at various target ratios of 1:5 to 5:1. In some cases, effector cells were isolated by FACS based on GFP expression prior to use. In parallel, untransfected T cells were co-cultured with target cells to demonstrate background functionality of T cells in vitro. All co-cultures were kept under optimal growth conditions for at least 20 hours. Cell impedance was monitored throughout; a decrease in impedance indicates cell detachment and eventual cell death.

[0260] To compare the initial ability of genetically edited lentiviral TAG-72 CAR-T cells to lyse tumor cells, tumor cells with high or low TAG-72 expression were incubated with genetically edited CAR-T cells or other negative control effector T cells (untransfected) and monitored for in vitro cytotoxicity by xCELLigence ® All of these genetically edited TAG-72 CAR-T cells killed TAG-72 high tumor cells (OVCAR-3) as efficiently as TAG-72 CAR-T cells (FIGS. 5A, 5C, 5E, and 5G), while no lysis of TAG-72 low tumor cells (MES-OV) was observed (FIGS. 5B, 5D, 5F, and 5H). These results demonstrate that genetically edited TAG-72 CAR-T cells generated using the CRISPR procedure disclosed herein retain the tumor killing ability and specificity of TAG-72 CAR-T cells.

[0261] Example 4 – In vivo function of CRISPR genetically edited TAG-72 CAR-T cells

[0262] Recent studies have shown that TAG-72 CAR-T cells can reduce ovarian tumor burden in vivo, but cannot consistently prevent tumor recurrence (Murad, J.P. et al., Effective Targeting of TAG72(+) Peritoneal Ovarian Tumors via Regional Delivery of CAR-Engineered T Cells. Front Immunol, 2018, 9: p. 2268). The efficacy of the generated and validated TAG-72 CAR-T cells as described in Examples 1, 2 and 3 was evaluated in an in vivo mouse solid tumor (xenograft) model. For this model, approximately 1 x 10 7 Individuals source TAG-72 positive OVCAR-3 cancer cells were injected subcutaneously into the flanks of 6 to 10 week old mice and human tumor cell lines grew on the flanks of NSG mice. Over 7 to 9 weeks, fully formed 150-200 mm 3 tumors developed at the injection site. Once tumors reached this volume, the groups were randomized for treatment. CAR-T cells with different edited genes were administered intravenously to the mice, a total of 5 x 10 6 T cells / injection in 2 injections. Tumor volume, body weight and clinical scores were monitored after CAR-T cell infusion. Mice with tumor sizes of 800 mm 3 to 1000 mm 3 , significant body weight loss or poor clinical scores were culled according to animal ethics approval. In this ovarian cancer tumor model, second generation TAG-72 CAR-T cell treatment initially reduced tumor size, but tumor recurrence was observed about 30 days after CAR-T cell administration (Murad, J.P. et al., Effective Targeting of TAG72(+) Peritoneal Ovarian Tumors via Regional Delivery of CAR-Engineered T Cells. Front Immunol, 2018, 9: p. 2268). Figure 6 TAG-72 CAR-T cells were generated according to the methods described in Examples 1 and 2 and in vivo efficacy was evaluated in the same model. PD-1 gene knockout TAG-72 CAR-T cells did not improve the anti-tumor activity or persistence of TAG-72 CAR-T cells (Murad, J.P. et al., Effective Targeting of TAG72(+) Peritoneal Ovarian Tumors via Regional Delivery of CAR-Engineered T Cells. Front Immunol, 2018, 9: p. 2268). Figure 6 However, knockout of RC3H1 and / or RC3H2 genes resulted in a significant improvement in the anti-tumor activity and persistence of TAG-72 CAR-T therapy. Furthermore, RC3H1 and RC3H2 double gene knockout TAG-72 CAR-T cells (TAG-72 CAR / RC3H1,2 KO T cells) showed the best anti-tumor activity and persistence in these groups, as evidenced by the complete prevention of tumor recurrence in TAG-72 CAR / RC3H1,2 KO T cell treated mice during the monitoring period (Murad, J.P. et al., Effective Targeting of TAG72(+) Peritoneal Ovarian Tumors via Regional Delivery of CAR-Engineered T Cells. Front Immunol, 2018, 9: p. 2268). Figure 7). A2AR and FAS gene knockout also improved the anti-tumor efficacy and persistence of TAG-72 CAR-T therapy, which delayed tumor recurrence in NSG mouse xenograft models ( Figure 8 ). Dominant negative mutations of TGF receptor 1 and 2 guided by CRISPR also enhanced the persistence of TAG-72 CAR-T cells, as evidenced by more persistent control of tumor volume 60 days after CAR-T treatment ( Figure 9 ).

[0263] Example 5 - Generation of RC3H1 and / or RC3H2 gene edited CD19 CAR-T cells using CRISPR and in vivo function

[0264] CD19 CAR-T cell therapy was the first successful CAR-T treatment approved for B cell malignancies (Porter et al., N Engl J Med, 2011. 365(8): p. 725-33). To verify that the anti-tumor activity of CAR-T cells enhanced by CRISPR gene knockout was not limited to the OVCAR-3 tumor model, TAG-72 antigen, or TAG-72 CAR-T cells, CD19 CAR-T cells with RC3H1 and / or RC3H2 gene knockout were also generated for in vivo functional evaluation. As previously described, a CD19 scFv-4-1BB-CD3 zeta CAR expression cassette was constructed (Porter et al., N Engl J Med, 2011. 365(8): p. 725-33; Milone et al., Mol Ther, 2009. 17(8): p. 1453-64; see also WO2017088012). CD19 scFv-4-1BB-CD3 zeta CAR lentivirus vectors were prepared and transduced into human activated T cells to generate CD19 CAR-T cells as described in Example 1, and then transfected by RNP complexes formed from RC3H1 gRNA (SEQ ID NO: 2) and / or RC3H2 gRNA (SEQ ID NO: 4) as described in Example 2 to generate CRISPR RC3H1 and / or RC3H2 gene knockout CD19 CAR-T cells.

[0265] CD19 CAR-T cell in vivo cytotoxicity assay

[0266] In vivo efficacy of T cells was evaluated in a Burkitt’s lymphoma xenograft model. For this model, 5 x 10 5 CD19-positive Raji lymphoma cells were injected subcutaneously into the flank of 6- to 10-week-old NSG mice. Three days after tumor inoculation, each mouse was injected intravenously with a single dose of 5 x 106 CAR-T cells. Tumor volume, body weight, and clinical scores were monitored after CD19 CAR-T cell infusion. Mice with tumor size of 800 mm 3 to 1000 mm 3 , significant body weight loss, or poor clinical scores were culled according to animal ethics approval. In this lymphoma tumor model, CD19 CAR / RC3H1,2KOT cell treatment significantly delayed tumor growth in mice and improved the median survival of tumor-bearing mice compared to CD19 CAR-T cell treatment (p < 0.0001) (Figure 6). Figures 10A-10B This result indicates that knockout of RC3H1 and RC3H2 genes improves the anti-tumor activity of CD19 CAR-T cells in vivo, similar to what was observed with TAG-72 CAR-T cells.

[0267] Example 6 - Activation markers of CD19 CAR / RC3H1 and / or RC3H2 KO T cells after continuous activation exposure

[0268] As described in Example 5, RC3H1 and / or RC3H2 gene knockout CD19 CAR-T cells were generated.

[0269] Evaluation of CD19 CAR T cells + differences in activation markers after antigen exposure Figure 11 Engineered CD19 overexpressing cell line OVCAR-3 (CD19) was irradiated (30 Gy) and plated at 80,000 cells / mL / well in 24-well tissue culture plates. Aliquots of 1x10 6 CAR-T cells (with and without RC3H1 and / or RC3H2 KO) were added to each well; these CAR-T cells were then transferred daily into uncontacted irradiated OVCAR-3 (CD19) cell monolayers over a period of 7 days. After 7 days of continuous antigen exposure, effector cells were washed once by centrifugation and expression of activation markers CD69 and CD25 was evaluated. These markers are associated with early and late activation, respectively, with expression related to TCR ligation. To detect expression of these activation markers on CAR-T cells, flow cytometry analysis was performed using a MACSQuant® Analyzer 10. CAR expression was indirectly detected by detecting co-expressed GFP. Cell surface staining for CD69 and CD25 was performed using standard protocols, with cells incubated with fluorescently conjugated antibodies for 15 minutes at 4°C, protected from light. Cells were washed twice with FACS buffer before analysis. Propidium iodide solution was used to distinguish between live and dead cells. FlowLogic® software was used for data analysis. ® CAR-T cells (with and without RC3H1 and / or RC3H2 KO) were added to each well; these CAR-T cells were then transferred daily into uncontacted irradiated OVCAR-3 (CD19) cell monolayers over a period of 7 days. After 7 days of continuous antigen exposure, effector cells were washed once by centrifugation and expression of activation markers CD69 and CD25 was evaluated. These markers are associated with early and late activation, respectively, with expression related to TCR ligation. To detect expression of these activation markers on CAR-T cells, flow cytometry analysis was performed using a MACSQuant® Analyzer 10. CAR expression was indirectly detected by detecting co-expressed GFP. Cell surface staining for CD69 and CD25 was performed using standard protocols, with cells incubated with fluorescently conjugated antibodies for 15 minutes at 4°C, protected from light. Cells were washed twice with FACS buffer before analysis. Propidium iodide solution was used to distinguish between live and dead cells. FlowLogic® software was used for data analysis. TMsoftware (Miltenyi Biotec) for data analysis.

[0270] After sustained antigen exposure, CD19 CAR / RC3H1 and / or RC3H2 KO T cells lacking one or both genes showed evidence of higher frequency of CAR+ / CD25+ / CD69+expressing cells. While the increase was not statistically significant, this was consistent across all three KO T cells, suggesting increased activation compared to untransfected CD19 CAR-T cells Figure 11 ).

[0271] Example 7 - Generation of RC3H1 and / or RC3H2 KO T cells and in vitro function using CRISPR

[0272] To demonstrate that the method for generating gene knockout immune cells is not limited to CAR-T cells, equivalent CRISPR gene knockouts were also performed in normal T cells.

[0273] To generate CRISPR T cells, human T cells were isolated and activated using CD3 / CD28 beads as described in Example 1. Three days after activation and in vitro expansion, activated human T cells were transfected by RNP complexes formed by RC3H1 gRNA (SEQ ID NO: 2) and / or RC3H2 gRNA (SEQ ID NO: 4) as described in Example 2.

[0274] CRISPR indel frequency and gene knockout efficiency of transfected T cells were analyzed by ICE assay as described in Example 2. ICE assay results indicated that these guide RNAs also showed high activity to introduce indels (including out-of-frame indels) in activated human T cells Figure 12 ).

[0275] In vitro killing was performed by prolonged T cell activation Figure 13

[0276] To determine whether the effect of KO is limited to CAR-T cells, normal T cells were polyclonally activated by their TCR and CD28 co- stimulatory molecules Figure 13 ​). RC3H1 and / or RC3H2 KO T cells were maintained in the presence of aCD3 / aCD28 beads at a 1 : 1 bead to cell ratio for at least 92 hours. Cell counts were performed approximately every 24 hours, with fresh beads added accordingly. Following continued activation, RC3H1 and / or RC3H2 KO T cells showed improved in vitro function compared to untransfected (NT) T cells over a 20 hour monitoring period. While the differences were not statistically significant, each of the three KO T cells killed target tumor cells more effectively than untransfected T cells, suggesting that prolonged activation of KO T cells can not result in “exhaustion” of killing function.

[0277] Example 8 - Generation of RC3H1 and / or RC3H2 KO NK-92 cells (with and without CAR) using CRISPR

[0278] To demonstrate that the method for generating gene knockout immune cells is not limited to T cells, an equivalent CRISPR gene knockout was performed in NK-92 cells (Figure IB). NK-92 is a natural killer (NK) cell line with high cytotoxicity against cancer targets. NK-92 function can be enhanced through genetic modifications, including CAR expression (Klingemann et al., Front Immunol, 2016. 7: p. 91). The NK-92 cell line was maintained and expanded in RPMI-1640 medium with 200 U / mL IL-2 and fetal bovine serum.

[0279] To generate RC3H1 and RC3H2 gene knockout NK-92 cells (RC3H1 KO NK-92 cells and RC3H2 KO NK-92 cells, respectively), NK-92 cells were transfected with RNP complexes formed from RC3H1 gRNA (SEQ ID NO: 2) and / or RC3H2 gRNA (SEQ ID NO: 4) as described in Example 2. Transfected NK-92 cells were also analyzed for CRISPR indel frequency and gene knockout efficiency by ICE assay as described in Example 2. ICE assay results indicated that these guide RNAs can also introduce indels (including out-of-frame indels) at high frequency in NK-92 cells (Table 2). Figure 14 ).

[0280] To generate TAG-72 CAR / RC3H1 and / or RC3H2 KO NK-92 cells, RC3H1 and / or RC3H2 KO NK-92 cells were transduced with a TAG-72 CAR lentivirus vector as described in Example 1.

[0281] Example 9 - In vitro function of RC3H1 and / or RC3H2 KO NK-92 and TAG-72 CAR / RC3H KO NK-92 cells

[0282] As described in Example 8, the resulting RC3H1 and / or RC3H2 KO NK-92 cells were transduced using lentiviral TAG-72 CAR vector. RC3H1 and / or RC3H2 KO NK-92 ± CAR cells were generated in L-glutamine RPMI-1640 supplemented with 10% FBS and 100 U / mL IL-2 and routinely maintained in culture. After at least 3 days in culture, transduction efficiency was assessed by flow cytometry. Additionally, RC3H1 and / or RC3H2 KO NK-92 ± CAR cells were evaluated in vitro for their ability to eliminate cancer cells.

[0283] In vitro killing efficiency of RC3H1 and / or RC3H2 KO NK-92 cells was determined using a real-time cell monitoring system (xCELLigence ® ) SPRTM. Target cells (10,000 target cells / 100 uL) (e.g., ovarian cancer cell lines MES-OV or OVCAR-3) were resuspended in culture medium (e.g., McCoy's 5a or RPMI-1640 basal medium) supplemented with 10-20% FBS with (OVCAR-3) or without (MES-OV) bovine insulin and distributed into RTCA plates. Target cells were kept at 37°C, 5% CO2 for at least 5 hours to allow cell attachment. After target cell attachment, RC3H1 and / or RC3H2 KO NK-92 effector cells were added at a 1:1 E:T ratio. In parallel, untransfected NK-92 cells were co-cultured with target cells to demonstrate background functionality of NK-92 cells in vitro. All co-cultures were kept under optimal growth conditions for at least 40 hours. Cell impedance was monitored throughout.

[0284] To compare the ability of RC3H1 and / or RC3H2 KO NK-92 cells to lyse tumor cells, tumor cells were incubated with RC3H1 and / or RC3H2 KO NK-92 cells or untransfected NK-92 cells and then lysed by xCELLigence ®In vitro cytotoxicity was monitored. When co-cultured with MES-OV cells, all NK-92 cells (Figure 15A, left panel) showed an inhibitory effect on cell growth. This effect was increased using RC3H2 KO NK-92 cells and RC3H1,2 KO NK-92 cells when compared to non-transfected NK-92 controls, demonstrating an enhancement of in vitro functionality. Additionally, when co-cultured with OVCAR-3 cells, all NK-92 cells (Figure 15A, right panel) showed a cytotoxic effect as evidenced by a decrease in NCI. This effect was increased using RC2H2 KO NK-92 cells and RC2H1 / 2 KO NK-92 cells, respectively, when compared to non-transfected NK-92 cell conditions, demonstrating an enhancement of in vitro functionality.

[0285] Similar assays were performed using TAG-72 CAR NK-92 cells. To confirm transduction of TAG-72 CAR NK-92 cells into RC3H1 and / or RC3H2 KO NK-92 cells, flow analysis was performed (as described in Example 1) with GFP used as a proxy for integration and expression of the CAR (Figure 15B). Values represent CAR (GFP) % expressed as a percentage of live cells, with debris and doublets not included in the parental gate.

[0286] To compare the ability of RC3H1 and / or RC3H2 gene knockout TAG-72 CAR-NK-92 cells to lyse tumor cells, cancer cell lines (in this case OVCAR-3) were co-cultured with TAG-72 CAR NK-92 cells + RC3H1 and / or RC3H2 KO co-cultures, and by xCELLigence ® In vitro cytotoxicity was monitored. When co-cultured with OVCAR-3 cells, all NK-92 cells with TAG-72 CAR (Figure 15C) Figure 15C ) had a cytotoxic effect as evidenced by a plateauing or decrease in NCI. This effect was significantly greater using TAG-72 CAR / RC3H1 KO NK-92 cells, TAG-72 CAR / RC3H2 KO NK-92 cells, and TAG-72 CAR / RC3H1,2 KO NK-92 cells over 40 hours of co-culture, demonstrating an enhancement of in vitro functionality.

[0287] Example 10 - Generation of gene KO iPSCs using CRISPR

[0288] Stem cells (e.g., induced pluripotent stem cells (iPSCs)) can self-renew indefinitely and differentiate into various cell types, including hematopoietic stem cells (HSCs) and immune cells. Immune cells such as T cells and NK cells have previously been generated from iPSCs for cancer therapy (Themeli et al., Nat Biotechnol, 2013. 31(10): p. 928-33; Li et al., Cell Stem Cell, 2018. 23(2): p. 181-192 e5). In a similar approach, CRISPR gene knockout T or NK cells can be derived from iPSCs Figure 16 ). To generate CRISPR RC3H1 and RC3H2 gene double knockouts (RC3H1,2 KO iPSCs) and A2AR gene knockout iPSCs (A2AR KO iPSCs), RNP complexes formed by representative gRNAs (RC3H1, SEQ ID NO: 2; RC3H2, SEQ ID NO: 4; A2AR, EQ ID NO: 7) were transfected into iPSCs using the Lonza 4D Nucleofector system. First, 12-well plates were coated with Laminin-521 (STEMCELL Technologies) in PBS and incubated at 37 °C for 2 hours. iPSCs were pre-incubated with mTeSR Plus medium (STEMCELL Technologies) containing RevitaCell Supplement (Life Technologies) for 2 hours prior to transfection. RNPs were prepared by combining full-length gRNAs with Lonza P3 buffer and Cas-9. The RNP mixture was then incubated at room temperature for 10-20 minutes. After pre-incubation, iPSCs were extracted as single cells using Accutase (Life Technologies), 1 x 10 TM cells per reaction were obtained for electroporation. To generate gene knockout iPSCs, cells and RNP mixtures in Lonza P3 buffer were combined into PCR tubes and then loaded into Lonza 4D Nucleofector for electroporation. Thereafter, mTeSR Plus medium (STEMCELL Technologies) with CloneR TM was added to the reaction and incubated at room temperature for 10 minutes. After incubation, cells were added to mTeSR Plus medium with CloneR ® medium (STEMCELL Technologies) and incubated at 37 °C for 48 hours. After 48 hours, iPSCs were passaged using Accutase (Life Technologies) and plated in mTeSR Plus medium with CloneR 6 medium (STEMCELL Technologies) and incubated at 37 °C for 48 hours. After 48 hours, iPSCs were passaged using Accutase (Life Technologies) and plated in mTeSR Plus medium with CloneR TM medium (STEMCELL Technologies) and incubated at 37 °C for 48 hours. After 48 hours, iPSCs were passaged using Accutase (Life Technologies) and plated in mTeSR Plus medium with CloneR TM medium (STEMCELL Technologies) and incubated at 37 °C for 48 hours. After 48 hours, iPSCs were passaged using Accutase (Life Technologies) and plated in mTeSR Plus medium with CloneR TM medium (STEMCELL Technologies) and incubated at 37 °C for 48 hours. After 48 hours, iPSCs were passaged using Accutase (Life Technologies) and plated in mTeSR Plus medium with CloneR TMLaminin-521 pre-coated plates. mTeSR Plus TM Medium changes were performed daily for 72 hours and cells were passaged after reaching approximately 80% confluency (6-7 days after electroporation). RC3H1,2 KO iPSC and A2AR KO iPSC colonies with pluripotent stem cell-like morphology were maintained in culture after transfection (Figures 17A and 21A).

[0289] Untransfected and transfected iPSCs were cultured in mTeSR Plus TM on Laminin-521 and imaged using EVOS ® Brightfield microscope at 10x. Cells were lifted and collected as single cells using Accutase ® according to the manufacturer's recommendations. TRA-1-60, TRA-1-81 and SSEA-4 are surface receptors expressed on pluripotent stem cells and are a considered common practice to characterize iPSCs (Baghbaderani et al. 2015, Stem Cell Reports). Cells were analyzed by MACSQuant ® Flow Cytometer (Miltenyi Biotec) along with unstained samples and appropriate isotype controls. Dead cells (by PI staining), debris and doublets were excluded; FlowLogic TM Histograms were generated (Figures 17B and 21B) and analyzed using FlowLogic Figures 17A-17B , RC3H1,2 KO and Figures 21A-21B , A2AR KO). iPSCs with or without KO showed almost identical TRA-1-60, TRA-1-81 and SSEA-4 pluripotency markers, all markers were co-expressed at >95% (Figures 17B and 21B). There were no visual differences in iPSC morphology (Figures 17A and 21A) indicating that RC3H1 and RC3H2 double KO or A2AR KO had no negative impact on iPSC maintenance and pluripotency.

[0290] CRISPR indel frequency and gene knockout efficiency of transfected iPSCs were analyzed by ICE assay as described in Example 2. ICE assay results indicated that gRNAs produced indels (including out-of-frame indels) in iPSCs at high frequencies Figures 18A-18C , RC3H1,2 KO and Figures 22A-22C , A2AR KO).

[0291] In summary, our data demonstrate that the genetically edited iPSCs described herein can be differentiated into CD34+ / HE / HSCs, and then into immune cells (e.g., NK, NKT, or T cells) using known methods for subsequent cancer treatment.

[0292] Example 11 - RC3H1 and RC3H2 KO (RC3H1,2 KO) iPSCs differentiation into iNK cells (edited iNK cells).

[0293] iCD34+ cells

[0294] Receptor CD34 is expressed on HEs and HSCs, which are the stem cell source that forms the platform to generate immune cells. Differentiation of iPSCs into CD34+ cells is a prerequisite and a necessary condition to be able to generate iPSC-derived immune cells (Sturgeon et al. Nature Biotechnology, 2014 Vol 32 (6) pp 554-561, Knorr et al. STEM CELLS Translational Medicine vol 2 (4) pp 274-283, Zeng et al., Stem Cell Reports, 2017 Vol 9 (6) pp 1796-1812). Characterizing CD34+ expression as an intermediate cell type between iPSCs and immune cells is considered a common practice and is a key step to demonstrate that including a gene-KO in iPSCs does not disrupt any potential differentiation pathway during initial development.

[0295] STEMdiff TM Hematopoietic Kit (STEMCELL Technologies) was used to differentiate non-transfected and transfected iPSCs (containing RC3H1,2 KO) into iCD34+ cells. Cells were isolated and stained using an antibody targeting CD34 (Miltenyi Biotec) following the manufacturer’s recommendations. Cells were analyzed by MACSQuant ® Flow Cytometer (Miltenyi Biotec) using unstained samples and appropriate isotype controls. Dead cells (by PI staining), debris, and doublets were excluded; FlowLogic TM was used for data analysis.

[0296] iPSCs containing RC3H1,2 KO or not containing RC3H1,2 KO Figure 19) all differentiated into iCD34+ cells. These data demonstrate successful generation of iCD34+ cells from RC3H1,2 KO iPSCs and indicate that the key developmental pathways required for conversion from iPSCs through all intermediate phenotypes to a cell population containing CD34-expressing cells are intact.

[0297] iNK cells

[0298] iPSCs containing RC3H1,2 KO were able to differentiate into iNK immune cells.

[0299] Gene knockout iCD34+ (derived from RC3H1,2 KO iPSCs) were further differentiated into iNK cells driven by a cytokine combination including IL-15, FLT3 and IL-7. iNK cells can be prepared using published methods (e.g. methods described in US Patent 9,260,696 B2 (Kaufman, Knorr), Li et al. (Stem Cell, 23 (2018) 181-197) or using the commercially available culture system StemSpan™ NK Cell Generation Kit (Stem Cell Technologies).

[0300] Differentiated cells were isolated and stained using antibodies targeting CD56 (Miltenyi Biotec), NKp46 (Miltenyi Biotec) and NKG2D (Miltenyi Biotec) following the manufacturer’s recommendations. Differentiated cells were analyzed by MACSQuant® Flow Cytometer (Miltenyi Biotec) using unstained samples and appropriate isotype controls. Dead cells (by PI staining), debris and doublets were excluded, and data analyzed using FlowLogic® TM Flow Cytometer (Miltenyi Biotec) using unstained samples and appropriate isotype controls. Dead cells (by PI staining), debris and doublets were excluded, and data analyzed using FlowLogic® TM for data analysis. Figure 20 Expression of NK functional receptors (NKp46 and NKG2D) supports that iCD56+ cells have NK-specific cytotoxic functions.

[0301] Example 12 - A2AR KO iPSCs differentiated into iNK cells (edited iNK cells).

[0302] iCD34+ cells

[0303] STEMdiff® iNK Cell Differentiation Kit (R&D Systems) was used following the manufacturer’s instructions. Cells were cultured in iNK Cell Differentiation Medium (R&D Systems) for 14 days. TMA blood cell generation kit (STEMCELL Technologies) was used to differentiate both untransfected and transfected iPSCs (containing RC3H1, 2 KO) into iCD34+ cells. Cells were isolated and stained using antibodies targeting CD34 (Miltenyi Biotec) following the manufacturer’s recommendations. Cells were analyzed by MACSQuant ® Flow cytometry (Miltenyi Biotec) using unstained samples and appropriate isotype controls. Dead cells (by PI staining), debris, and doublets were excluded; FlowLogic TM Data analysis was performed.

[0304] iPSCs containing A2AR KO or without containing A2AR KO Figure 23 were differentiated into iCD34+ cells. These data demonstrate the successful generation of iCD34+ cells from A2AR KO iPSCs and indicate that the key developmental pathways required for the conversion from iPSCs through all intermediate phenotypes into a cell population containing CD34-expressing cells remain intact.

[0305] iNK cells

[0306] iPSCs containing A2AR KO were able to differentiate into iNK immune cells.

[0307] Untransfected iCD34 (derived from untransfected iPSCs) and knockout iCD34+ (derived from knockout iPSCs) were further differentiated into iNK cells driven by a cytokine combination including IL-15, FLT3, and IL-7. iNK cells can be prepared using published methods (e.g., methods described in U.S. Patent 9,260,696 B2 (Kaufman, Knorr), Li et al. (Stem Cell, 23 (2018) 181-197)) or using the commercially available culture system StemSpan™ NK Cell Generation Kit (Stem Cell Technologies).

[0308] Expression of NK cell markers was assessed by flow cytometry on differentiated cells. Dead cells, debris, and doublets were excluded, making it Figure 24 The CD56+ histogram presented in Figure 6 shows all live cells in cultures generated from untransfected or transfected iPSC samples. Unstained samples were presented to show clear positive staining of each antibody for each respective receptor. Negative for appropriate isotype controls. Expression of NK functional receptors (NKp46, NKp30, NKp44, and NKG2D) confirmed that iCD56+ cells are iNK cells with cytotoxic functionality.

[0309] Example 13 - Functionality of Edited iNK Cells

[0310] A2AR KO iPSCs were generated (Example 10) and differentiated into edited iNK cells (Example 12). The iNK cells were then collected after 20-40 days and used for subsequent functional assays.

[0311] The ability of iNK cells to kill cancer cells was evaluated in vitro using a real-time cell monitoring system (xCELLigence ® ) SPOTRead software. Target cells (10,000 / 100 uL) (e.g., ovarian cancer cell line OVCAR-3) were resuspended in culture medium (e.g., RPMI-1640 and L-glutamine basal medium) supplemented with 10-20% FBS and bovine insulin and dispensed into RTCA plates. The target cells were kept at 37°C, 5% CO2 for at least 5 hours to allow cell attachment. After target cell attachment, iNK effector cells were added at a 1:1 E:T ratio. In parallel, iPSC-derived iNK cells were co-cultured with target cells to demonstrate background functionality of untransfected iNK cells in vitro. All co-cultures were kept under optimal growth conditions for at least 10 hours. Throughout the process, cell impedance was monitored and presented herein as NCI, where normalization occurs at the time of effector cell addition. The percentage of iNK cell cytotoxicity (% cytotoxicity) was calculated after 5 and 10 hours of co-culture using the following equation: + A2AR KO effector cells (test) versus cytotoxicity percentage (% cytotoxicity) of target cells alone (control):

[0312] ((normalized cell index 对照 – normalized cell index 测试 ) / normalized cell index 对照 ) x 100

[0313] To compare the ability of A2AR KO iPSC-derived iNK cells (A2AR KO iNK cells) to lyse tumor cells, tumor cell lines were incubated with A2AR KO iNK cells or NT iNK cells and cytotoxicity was measured by xCELLigence ®In vitro cytotoxicity was monitored. NT iNK cells showed cytotoxic effects when co-cultured with OVCAR-3 target cells (Figure 25A). This effect was increased using A2AR KO iNK cells compared to untransfected controls, demonstrating an enhancement of in vitro function. Furthermore, cytotoxicity after 5 hours (Figure 25B, left panel) and 10 hours (Figure 25B, right panel) of co-culture in A2AR KO iNK showed higher cytotoxicity. In summary, these data show that A2AR KO can enhance anti-tumor activity not only in T cells but also in iNK cells compared to untransfected control cells.

[0314] Throughout this specification various publications, including patents, patent applications, published patent applications, accession numbers, technical articles, and scholarly articles, are cited. Each of these cited publications is incorporated by reference herein in its entirety and for all purposes.

Claims

1. A modified stem cell capable of differentiating into an immune cell, wherein the function of at least one gene is inhibited in the modified stem cell, and wherein the at least one gene is selected from RC3H1 and RC3H2.

2. The modified stem cell of claim 1, wherein the function of one or more genes selected from A2AR, FAS, TGFBR1, and TGFBR2 is further inhibited.

3. The modified stem cell of claim 1 or 2, wherein the inhibition of the function of a gene is caused by a reduction in the level or function of mRNA transcribed from the gene, or by a reduction in the level or activity of a protein encoded by the gene.

4. The modified stem cell of claim 1 or 2, wherein the inhibition of the function of a gene is caused by a modification in the nucleic acid sequence of the gene.

5. The modified stem cell of claim 1 or 2, which is an induced pluripotent stem cell.

6. The modified stem cell of claim 5, wherein the induced pluripotent stem cell is generated from a donor cell homozygous for three HLA genotypes.

7. The modified stem cell of claim 1 or 2, further comprising a nucleic acid encoding a chimeric antigen receptor (CAR).

8. The modified stem cell of claim 7, wherein the CAR comprises an antigen recognition moiety for a target antigen selected from the group consisting of TAG-72, CD19, CD20, CD24, CD47, tissue factor, folate receptor alpha (FRa), and BCMA.

9. The modified stem cell of claim 1 or 2, wherein the at least one gene is RC3H1.

10. The modified stem cell of claim 1 or 2, wherein the at least one gene is RC3H2.

11. The modified stem cell of claim 1 or 2, wherein the function of both RC3H1 and RC3H2 genes is inhibited in the modified stem cell.

12. A modified immune cell, wherein the function of at least one gene is inhibited in the modified immune cell, and the at least one gene is selected from RC3H1 and RC3H2.

13. The modified immune cell of claim 12, wherein the function of one or more genes selected from A2AR, FAS, TGFBR1, and TGFBR2 is further inhibited.

14. The modified immune cell of claim 12 or 13, wherein the inhibition of the function of a gene is caused by a reduction in the level or function of mRNA transcribed from the gene, or by a reduction in the level or activity of a protein encoded by the gene.

15. The modified immune cell of claim 12 or 13, wherein the inhibition of the function of a gene is caused by a modification in the nucleic acid sequence of the gene.

16. The modified immune cell of claim 12 or 13, wherein the modified immune cell is selected from a T cell, an NK cell, an NKT cell, or a macrophage.

17. The modified immune cell of claim 12 or 13, wherein the modified immune cell expresses a chimeric antigen receptor (CAR).

18. The modified immune cell of claim 12 or 13, wherein the modified immune cell recognizes one or more target antigens.

19. The modified immune cell of claim 18, wherein the target antigen is selected from the group consisting of TAG-72, CD19, CD20, CD24, CD30, CD47, tissue factor, folate receptor alpha (FRa), and BCMA.

20. The modified immune cell of claim 12 or 13, wherein the at least one gene is RC3H1.

21. The modified immune cell of claim 12 or 13, wherein the at least one gene is RC3H2.

22. The modified immune cell of claim 12 or 13, wherein the function of both RC3H1 and RC3H2 genes is inhibited in the modified immune cell.

23. The modified immune cell of claim 12 or 13, wherein the modified immune cell is differentiated from a modified stem cell in which the function of the at least one gene is inhibited.

24. A composition for enhancing immune cell function, comprising: a guide RNA-nuclease complex capable of editing a target gene sequence, wherein the guide RNA targets a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO:

6.

25. The composition of claim 24, wherein the nuclease comprises at least one protein selected from the group consisting of Cpf1, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslOO, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4.

26. A method for enhancing immune cell function, comprising: modifying the immune cell to inhibit the function of at least one gene selected from RC3H1 and RC3H2.

27. A method of modifying a stem cell capable of differentiating into an immune cell, comprising: modifying the stem cell to inhibit the function of at least one gene selected from RC3H1 and RC3H2.

28. The method of claim 27, further comprising modifying the stem cell to inhibit the function of one or more genes selected from A2AR, FAS, TGFBR1, and TGFBR2.

29. The method of claim 27 or 28, further comprising differentiating the modified stem cell into an immune cell, wherein the function of the at least one gene is inhibited in the immune cell.

30. The method of claim 27 or 28, wherein the inhibition of the function of a gene is achieved by a gene editing system.

31. The method of claim 30, wherein the gene editing system is selected from the group consisting of CRISPR / Cas, TALEN, and ZFN.

32. The method of claim 30, wherein the gene editing system is a CRISPR / Cas system comprising a guide RNA-nuclease complex.

33. The method of claim 32, wherein the guide RNA targets a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO:

6.

34. The method of claim 32, wherein the CRISPR / Cas system utilizes a guide RNA-dependent nuclease selected from the group consisting of Cpf 1, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslOO, Csy l, Csy2, Csy3, Cse l, Cse2, Csc l, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr l, Cmr3, Cmr4, Cmr5, Cmr6, Csb l, Csb2, Csb3, Csxl7, Csx 14, CsxlO, Csx 16, CsaX, Csx3, Csx l, Csx 15, Csf l, Csf2, Csf3, and Csf4.

35. The method of claim 26, 27, or 28, wherein the inhibition of the function of a gene is achieved by reducing the level or function of mRNA, optionally by a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), or an antisense nucleic acid.

36. The method of claim 26, 27, or 28, wherein the inhibition of gene function is achieved by reducing the level or activity of a protein encoded by the gene, optionally by using an antibody or a small molecule.

37. The method of claim 26, 27, or 28, wherein the immune cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, or a macrophage.

38. The method of claim 26, 27, or 28, wherein the modified cell produced by the method further comprises a nucleic acid encoding a chimeric antigen receptor (CAR).

39. The method of claim 26, 27, or 28, wherein the modified immune cell produced by the method recognizes one or more target antigens.

40. The method of claim 36, wherein the target antigen is selected from the group consisting of TAG-72, CD19, CD20, CD24, CD30, CD47, tissue factor, folate receptor alpha (FRa), and BCMA.

41. The method of claim 26, 27, or 28, wherein the at least one gene is RC3H1.

42. The method of claim 26, 27, or 28, wherein the at least one gene is RC3H2.

43. The method of claim 26, 27, or 28, wherein the function of both RC3H1 and RC3H2 genes are inhibited.

44. Use of the modified cell of any one of claims 1-23 or the modified cell produced by the method of any one of claims 26-43 in the manufacture of a medicament for treating a disorder in a subject.

45. The use of claim 44, wherein the disorder is cancer, an infection, an autoimmune disease, organ fibrosis, or endometriosis.

Images