Method for engineering of immune cells expressing chimeric antigen receptors at immune checkpoint loci for disease treatment

Abstract

A method for inserting a polynucleotide exogenous transgene sequence at a pre- determined endogenous genetic locus in a hose cell genome includes: (i) a donor DNA template including a polynucleotide insert; a 5 '-homology arm; and a 3 '-homology arm. In some embodiments, the 5' homology arm and the 3' homology arm are complementary to the DNA in a target region; and (ii) a ribonucleoprotein complex (RNP) including (1) a Cas nuclease, and at least one small guide RNAs (sgRNA) that is complementary to at least one selected nucleic acid sequence within the pre-determined genetic locus in the host cell genome.

Description

[0001] METHOD FOR ENGINEERING OF IMMUNE CELLS EXPRESSING CHIMERIC

[0002] ANTIGEN RECEPTORS AT IMMUNE CHECKPOINT LOCI FOR DISEASE

[0003] TREATMENT

[0004] This application claims priority to US Provisional Patent Application No. 63 / 535,412, filed on August 30, 2023, which is incorporated by reference for all purposes as if fully set forth herein.

[0005] FIELD OF THE INVENTION

[0006] The present application relates to genome editing in research and therapeutical application, and more specifically, to engineering of immune cells expressing chimeric antigen receptors at immune checkpoint locus for cancer treatment using homology-directed repair donor templates.

[0007] BACKGROUND OF THE INVENTION

[0008] One of the most exciting developments in the fight against cancer is the advent of cellbased immunotherapy, a promising treatment that kills cancer by using body’s own immune cells. Among these, CAR (chimeric antigen receptor) immune cells therapy (CAR-T or CAR- NK) is a type of cancer immunotherapy treatment that uses immune cells like T cells and NK cells that are genetically altered in a lab to enable them in locating and destroying cancer cells more effectively1. To date, 6 CAR-T cell therapies have been approved by FDA for people with certain blood cancers. And many clinical trials of CAR-NK cell therapies are also actively launched. Efforts to develop in vivo CAR-T cell therapies are also ongoing.

[0009] While many challenges still exist in the CAR-T or CAR-NK cell therapies. One of the key challenges is the genetic engineering process and related safety concerns. Currently, lentiviral and retroviral vector-based transduction is mainly used for the engineering process and has been shown to achieve decent gene transfer efficiency in these immune cells, especially NK (natural killer) cells that are relatively hard to transduce. However, it carries several big concerns, including: 1) inconsistent transduction, 2) limitation of cargo size capacity, 3) large- scale manufacturing challenges for clinical use, and 4) safety risks due to random insertional mutagenesis2’3. Accordingly, a more robust, precise, stable and safer genetic toolkit for cell engineering is urgently needed.

[0010] Recent advances of the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats gene associated protein 9) editing technology have reinvigorated interest in genome editing of human T cells and NK cells4. AAV6 (adeno-associated virus type 6) has been reported in use as a prevalent donor carrier for HDR-mediated cell engineering showing high KI efficiency and increased anti-tumor activity. While AAV6 is also associated with safety issues, manufacturing challenges, and restricted applications with its 4.5 Kb packaging limit5. Non-viral approaches such as using double-stranded linear DNA (dsDNA) or singlestranded linear DNA (ssDNA) as donor templates have been tested in cell genome editing while showing low efficiency and high cytotoxicity6.

[0011] SUMMARY OF THE INVENTION

[0012] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.

[0013] In one embodiment, the present application discloses a method for inserting a polynucleotide sequence at a pre-determined endogenous genetic locus in a hose cell genome. The method includes:

[0014] (i) a donor DNA template including a polynucleotide insert; a 5 ’-homology arm; and a 3 ’-homology arm. In some embodiments, the 5’ homology arm and the 3’ homology arm are complementary to the DNA in a target region.

[0015] (ii) a ribonucleoprotein complex (RNP) comprising (1) a Cas nuclease, and (2) at least one small guide RNAs (sgRNA) that is complementary to at least one selected nucleic acid sequence within the pre-determined genetic locus in the host cell genome.

[0016] In another embodiment, the target genomic loci in which the exogenous polynucleotide insert is inserted in, while the endogenous gene was simultaneously disrupted by nuclease cleavage, is an immune checkpoint locus, for example, (e.g., cytokine-inducible SH2- containing protein (CISH)), PI)!, NKG2A, signal regulatory protein a (SIRPa), T cell immunoglobulin mucin family member 3 (TIMS), lymphocyte-activation gene 3 (LAGS), T cell immunoreceptor with Ig and ITIM domains (TIGIT), TACTILE (CD96), or tumor necrosis factor-alpha-induced protein-8 like-2 (TIPE2).

[0017] In another embodiment, the donor vector is selected from a plasmid DNA , linear double-stranded or single-stranded DNA, circular single-stranded DNA, DNA-nanoparticle complex, AAV viral particle, adenovirus particle or lentivirus particle. In one embodiment, the donor vector further includes at least one polynucleotide sequence encoding a CAR that has anti-tumor activity. In one embodiment, the insertion of the DNA insert produces a chimeric antigen receptor targeting at least one selected from the group consisting of B-cell maturation antigen (BCMA), carcinoembryonic antigen (CEA), CD2, CD4, CD5, CD7, CD 19, CD22, CD30, claudin 18.2 (CLDN 18.2), epithelial cellular adhesion molecule (EpCAM), folate receptor 1 (FOLR1), human epidermal growth factor receptor 2 (HER2), mesothelin (MSLN), mucin 1 (MUC1), natural killer group 2D (NKG2D). Chimeric antigen receptors are also described in, for example, Chu et al.(Contemp Oncol (Pozn). 2018 Mar; 22(1A): 73-80). The entirety of the reference is hereby incorporated herein by reference. In some embodiments, the insertion of the DNA insert produces an engineered TCR that has altered antigen specificity compared with the original T-cells.

[0018] In another embodiment, the exogenous transgene is precisely engineered at the checkpoint CISH locus, and activating receptors, chimeric antigen receptors (CARs) or “armored” CARs for enhanced activity by secreting cytokines, express cytokine receptors to modulate a cytokine milieu of a tumor microenvironment or increase resistance to the tumor microenvironment.

[0019] In another embodiment, the exogenous transgene is knocked in at the checkpoint locus for immune-oncology therapy or other immune related disorders.

[0020] In another embodiment, the immune cell is a primary T cell, an NK cell, or a TIL cell.

[0021] In another embodiment, the immune cell is differentiated from an immune checkpoint engineered iPSCs or hematopoietic stem cell and a progenitor cell.

[0022] In another embodiment, the immature immune cell further differentiates to a mature immune cell.

[0023] In another embodiment, the hose cell is isolated from a healthy human and a human with cancer. In another embodiment, the host cell is isolated from a human carrying an inherited disease.

[0024] In another embodiment, the modified host cell is an autologous immune cell isolated form the subject. In another embodiment, the modified host cell is an allogenic immune cell.

[0025] In another embodiment, the immune cell is natural killer (NK) cell; the DNA donor has a length of about 2,000 bases or longer, and the length of the DNA insert is about 1,000 bases or longer; and an editing efficiency of the engineering is 20% or higher based on a total number of the immune cell.

[0026] In another embodiment, the immune cell is a T cell; the DNA donor has a length of about 2,000 bases or longer, and the length of the DNA insert is about 1,000 bases or longer; and an editing efficiency of the non-viral engineering is 40% or higher based on a total number of the immune cell. In another embodiment, the engineering includes: using at least one site-specific nuclease selected from the group consisting of a Cas nuclease, a zinc-finger nuclease, a meganuclease, a transcription activator-like effector nuclease (TALEN).

[0027] In another embodiment, the engineering includes: using at least one site-specific genetic integration from the group consisting of a Prime Editing system, a modified integrase system, a modified Transposon / Transpose system, a recombinase system or other modified mobile genetic elements.

[0028] In another embodiment, the engineering includes: using a class I Cas protein or a class II Cas protein.

[0029] In another embodiment, the engineering includes: using Cas9, Cas3, CaslO, Casl l, CasX, Cas 12a, MAD7, or a catalytic dead form or nickase form thereof.

[0030] In another embodiment, each of a 5 ’-homology arm and a 3 ’-homology arm independently has a length ranging from about 25 nucleotides to about 5000 nucleotides.

[0031] In another embodiment, the method includes: delivering the donor DNA template to the immune cell by a viral vector (such as AAV), a lipid or non-lipid nanoparticle delivery, an exosome delivery, an electroporation delivery, a gene gun delivery, or an injection, with or without ultrasound guidance.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention.

[0034] In the drawings:

[0035] Figure 1. High biocompatibility and HDR-mediated genome knock-in efficiency of cssDNA in primary human NK cells when compared with dsDNA (N=3). (A) Schematic diagram of the cssDNA donor template mediating CRISPR genomic integration of a GFP reporter gene at the RABI 1 A locus. (B) Cell viability on Day 1 after electroporation with various amount of donor templates. (C) Cell viability on Day 9 after electroporation with various amount of donor templates. (D) Knock-in efficiency (GFP+ %) was measured on Day 9 with various amount of donor templates. (E) In vitro proliferation of the electroporated cells from Dayl to Day9 with various amount of donor templates.

[0036] Figure 2. In-house gRNA mediated genomic deletion screen. (A) A specified gRNA sequence was screened out showing high CRISPR genomic disruption of CISH locus (-90% Indel) in human primary NK cells. (B) Screened gRNA mediated CISH disruption in NK cells (from two different healthy donors) improved both NK cells’ in vitro proliferation and cytotoxicity after co-culturing with K562 target cells at various effector to target (E:T) ratios for 4 hrs.

[0037] Figure 3. CD19.CAR specific knock-in at CISH locus in human primary NK cells mediated by cssDNA HDR donor template. (B) Schematic diagram of the cssDNA donor template mediating CRISPR genomic integration of chimeric antigen receptors (CARs) targeting CD 19 at the immune checkpoint CISH locus. (C) Cell viability on Day 1 after electroporation. (D) Cell viability on Day 7 after electroporation. (E) Knock-in efficiency (CD19.CAR+ %) was measured through Flow cytometry analysis at different time points after electroporation. (F) In vitro cytotoxicity of the electroporated cells (CD 19. CAR expressing cells) after co-culturing with CD 19 positive NALM6 target cells at various effector to target (E:T) ratios for 4 hrs. N=3 in panel B, C and E. N=2 in panel D. (G) Left, Schematic diagram of the cssDNA donor template mediating CRISPR genomic integration of chimeric antigen receptors (CARs) targeting CD19 at RABI 1 A locus. Right, Knock-in efficiency (CD19.CAR+ %) at RABI 1 A locus was measured through Flow cytometry analysis on Day 7 and Day 21 after electroporation.

[0038] Figure 4. CD 19. CAR specifical knock-in at CISH locus in human primary T cells mediated by cssDNA HDR donor template. Electroporated T cells were treated with 1 pM M- 3814 for 24 hours. (A) Representative flow cytometry plots for CD19.CAR expression on Day 7 after electroporation. (B) Percentage of CAR+ cells on Day 7 and Day 10 after electroporation. (C) Cell expansion of mock, CISH knocked-out (CISH RNP) and CD19.CAR knocked-in T cells (RNP + cssDNA). (D) In vitro killing of NALM6 acute lymphoblastic leukemia cell line with engineered T cells in comparison to unmodified T cells from the same blood donor after 24 hours of co-culture at various effector to target (E:T) ratios. (E) The growth curve of target NALM6 cells. NALM6-GFP cells were co-cultured with mock or engineered CAR-T cells at 1.5:1 effector to target ratio. The GFP positive target cell counts were plotted over 72 hours during the co-culture. N=3.

[0039] DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

[0040] Reference will now be made in detail to embodiments of the present invention, example of which is illustrated in the accompanying drawings.

[0041] The present invention employs non-viral gene engineering technologies to design scalable, cost-effective, and reliable processes for CAR immune cell production. The inventors recently developed an advanced technology platform (Genome writing catalyst, GATALYST) using circular single-stranded DNA (cssDNA) as an improved donor vector for homology- directed repair (HDR) mediated genome editing7. Preliminary studies showed that GATALYST enables high knock-in (KI) efficiency and low DNA-mediated cytotoxicity in different cell types, including iPSC cells, primary B cells, primary human T cells and primary human NK cells. Specifically, when DNA donor templates, either the cssDNA or the control dsDNA were co-electroporated with ribonucleoprotein (RNP) complex composing of spCas9 protein and single guide RNA (sgRNA) targeting exon 1 of the RAB11A locus in NK cells, the inventors found that cssDNA has been clearly shown much better biocompatibility (lower cytotoxicity) and higher HDR-mediated genome knock-in efficiency (% of GFP reporter expression) (Figure 1).

[0042] In addition to the challenge of the efficiency and safety of the genetic engineering process, particular attention has been paid to increase the therapeutic efficacy such as prolonged cell persistence and sustainable cytotoxicity against refractory or relapsed cancer, especially the solid tumors that includes about 90% of all cancer diagnoses.

[0043] Cytokine-inducible SH2-containing protein (CISH, encoded by CISH a key member of the suppressor of cytokine signaling (SOCS) protein family, is a novel intracellular immune checkpoint and negatively regulates human immune cells (such as T cells, natural killer cells and tumor infiltrating lymphocytes) effector function8. It carries a central SH2 domain, which can bind to molecules that have been phosphorylated by tyrosine kinases, and a SOCS box sequence motif that functions as an adaptor to recruit E3 ligases and ubiquitin signaling molecules, enabling the proteasomal degradation of the SH2 domain interacted molecules. Cytokines such as interleukin 15 (IL- 15) and interleukin 2 (IL-2) are used to stimulate NK cells, and prolonged stimulation may lead to the exhaustion of NK cells by inducing CISH protein. And as feedback, the induction of CISH expression will in turn inhibit cytokine-induced stimulation of NK cells. It has been reported that CISH can inhibit IL-2 / 15 -driven JAK / STAT signaling activation by binding directly to JAK1 and to reduce cellular metabolic fitness, which is mediated by the mTOR signaling pathway9. Also, the modified CISH'1' NK cells exhibit hypersensitivity to cytokines including IL-2 and / or IL-15 and maintain expansion and antitumor functions10’11. In addition to NK cells, CISH has been reported to negatively regulate the anti-tumor activity of T cells such as CD8 T cells and tumor infiltrating lymphocytes (TILs). For example, CISH has been shown to be induced by TCR stimulation in CD8 T cells and inhibits their immune function against tumors by physically interacting with the TCR intermediate PLC-yl and targeting it for proteasomal degradation12. Besides, CISH promotes PD-1 expression by suppressing the expression ofFBXO3813. In another study, single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL, indicating CISH expression was associated with T cell dysfunction. Accordingly, elimination of CISH in primary human TIL has been reported to increase their function and neoantigen recognition14. Furthermore, in a preclinical in vivo model, CISH knockout also increased tumor vulnerability to checkpoint inhibition.

[0044] Accordingly, CISH gene is a potential therapeutic target that can be further utilized as an effective knock-in locus for CARs expression thus to augment cell (e.g., NK and T cell) - mediated cancer immunotherapy.

[0045] The inventors first conducted a gRNA screening and identified a specific one with very high cleavage activity (> 90% indel) (Figure 2A). And these CISH1SQ NK cells show improved in vitro cell proliferation and cytotoxicity against K562 target cells (Figure 2B).

[0046] Next, the inventors sought to generate CAR immune cells such as CAR-T and CAR- NK cells through the single-step, non-viral precision genome-editing GATALYST platform, characterized with an efficient “two-in-one” CISH knock-out (KO) coupled with knock-in (KI) of CARs targeting the tumor specific antigens (Figure 3 A).

[0047] The inventors designed and synthesized a cssDNA donor template containing the payload - CD19.CAR construct with 5’- and 3’- HA sequences for CISH locus-targeting site (Figure 3B). The cssDNA donor template was co-delivered with ribonucleoprotein (RNP) targeting CISH locus into NK cells. The results have shown that the cssDNA mediated KI at a specific dosage (2 pg / reaction) was biocompatible inducing no obvious cytotoxicity when compared with RNP group (Figure 3C and 3D). And surprisingly, the inventors have found that the CAR expression was continuously increasing (from -11% to -70%) along with the cell expansion and proliferation (Figure 3E). In comparison with mock or CISH KO (CISH RNP) cells, the cssDNA engineered NK cells with CD19.CAR knock-in at CISH locus have shown much higher in vitro cytotoxicity against antigen (herein, CD 19) positive expressing target NALM6 cells, a human B cell precursor leukemia cell line (Figure 3F). The increasing trend of CD19.CAR+ cell percentage during cell expansion, and the enhanced cytotoxicity function have been observed in CD19.CAR-NK cell engineered on CAST / locus were confirmed in primary peripheral NK cells from at least 2 healthy donors. The increasing CAR positive NK cells during culturing was specific to CAR knock-in at CISH locus, as this phenomenon was not observed when CD19.CAR was knocked-in a RABI 1A locus (Figure 3G).

[0048] Next, the inventors engineered human primary T cells with CD 19. CARs at CISH locus using the non-viral cssDNA donor template in PBMC derived CD4+ / CD8+ T cells from 3 healthy donors. CD 19. CAR cssDNA targeting the CISH locus was co-electroporated with Cas9 protein and CISH gRNA (CISH RNP). CAR expression was determined on Day 7 or Day 10 post electroporation. Over -60% of CAR knock-in was achieved with 2 pg cssDNA donor templates per 2 million T cells (Figure 4A). The percentage of CAR positive cells increased slightly from Day 7 to Day 10 (Figure 4B). The cell expansion of CA19.CAR engineered at CISH locus was similar to mock cells and CISH RNP cells over 14 days of ex vivo culture (Figure 4C). On Day 7 post engineering, the cytotoxicity of engineered CAR-T cells was examined. In vitro assays demonstrated efficient CD 19. CAR-T cell killing of CD 19+ NALM6 cells at different effector to target ratios, in contrast to the mock engineered T cells from the same donor (Figure 4D). The target cell killing activity of CISH knock-out T cells (CISH RNP group) was similar to that of the mock cells. However, CD19.CAR knock-in at CISH locus target cells presented much higher potency and durability against target B cell leukemia cells (Figure 4D and 4E).

[0049] Based on the above preliminary results, the GATALYST platform offers a flexible and efficient gene editing capability in modulating immune checkpoint pathways, specifically like CISH signaling that mediate immune cell dysfunction, and meanwhile in arming these immune cells (NK and T cells) to express CARs to specifically target tumor cells achieving a “two-in- one” goal facilitating immune cells with better persistence and anti-tumor activity. This “two- in-one” engineering benefit is expected to be applicable to additional immune checkpoint loci, and to additional immune cell types, such as tumor-infiltrating lymphocyte (TIL), cytokine- induced killer (CIK), regulatory T cell (Treg), natural killer T cell (NKT), etc. Moreover, this method can also be applied to engineer iPSCs, which can be subsequently differentiated into immune cells, such as T cells, NK cells and macrophages in immunotherapy for cancer and other diseases such as autoimmune disorders.

[0050] Materials and Methods

[0051] 1. Cell culture:

[0052] K562, NALM6, and B lymphoblastoid cell line initiated by Epstein-Barr virus (EBV) transformation (EBV-LCL) were all directly purchased from ATCC and maintained in RPMI1640 (ThermoFisher) supplemented with 10% FBS (ThermoFisher), 100 U / mL penicillin (ThermoFisher), 100 pg / mL streptomycin (ThermoFisher).

[0053] Primary NK cells were obtained through direct isolation from Human peripheral blood leukopak. Briefly, the fresh leukopak was purchased from either StemCell Technologies or Miltenyi Biotec. And the NK cells were isolated through either negative selection using the EasySep™ Direct Human NK cell Isolation Kit (StemCell Technologies) or positive selection using the CD56 MicroBead Kit (Miltenyi Biotec). Following isolation, the cells can be directly expanded or frozen in CryoStor CS10 Cell Freezing Medium (StemCell Technologies) with ~107cells / ml per cryogenic tube and stored in a liquid N2 tank to be thawed and expanded later. Regarding the NK cells expansion, EBV-LCL cells were used as the feeder cells to co-culture with NK cells at a 10: 1 feeder:NK ratio in NK MACS medium (Miltenyi Biotec) supplemented with 5% heat inactivated human AB serum (Valley Biomedical), 500 lU / mL rhIL-2 (PeproTech) and 100 ng / mL rhIL-21 (PeproTech). Herein, the rhIL-21 was added only once at the beginning of the co-culture. Also, prior to co-culture with NK cells, EBV-LCL cells were treated with 10 pg / mL of mitomycin C (Thermo scientific) for 3 hours and rinsed couple of times with lx PBS (Gibco).

[0054] Human primary T cells were isolated using a StraightFrom Leukopak CD4 / CD8 MB Kit in MultiMCAS Cell24 Separator Plus. T cells were cultured and expanded in TexMACS Medium (Miltenyi Biotec) supplemented with 200 lU / mL Human IL-2 IS (Miltenyi Biotec). T cells were activated for 3 days with T Cell TransAct (Miltenyi Biotec) before electroporation.

[0055] All cells were incubated at 37°C in a humidified 5% CO2 environment. Cell status was closely monitored, and cell counting was performed using a Via2-Cassette in NucleoCounter® NC-202 (ChemoMetec) at specified time points.

[0056] 2. T cells and NK cells electroporation:

[0057] Generally, NK cells used for the electroporation are after ~10 days expansion when almost all the feeder cells got killed and cleared, while primary T cells were used for electroporation 3 days after activation. 5 x 105expanded NK cells or 2 x 106activated T cells were electroporated with a P3 Primary Cell 4D-Nucleofector™ Kit (Lonza)per 25 uL reaction. The indicated amount of HDR donor template (cssDNA or dsDNA) were mixed with the Cas9-gRNA complex (RNP). And electroporation was performed using the Amaxa™ 96-well Shuttle™ with the 4D Nucleofector (Lonza) using the specified pulse code (DN-100 for NK cells, EO-115 for T cells). Following electroporation, the cells were diluted into culture medium in the presence of 1 pM M-3814, and further incubated for 24 hours. After that, the cells were collected, counted, rinsed with medium, and placed into culture plates or G-Rex for continue culture and related functional tests (e.g., knock-out / knock-in efficiency detection and in vitro cytotoxicity evaluation). 3. CISH gRNA screen:

[0058] The CISH gRNA sequences already used in NK and T cells were first literature- reviewed. And gRNA sequences were further designed using CRISPR Design Tool on the Benchling website (http: / / www.benchling.com). On target efficiencies and off-target scores were predicted in silico by the same tool. All the picked gRNAs (SEQ ID NOs: 2, 3, 4, and 5) were synthesized and ordered from Integrated DNA Technologies (https: / / www.idtdna.com). After that, all the formed Cas9-gRNA complexes (RNPs) were mixed with NK cells respectively and went through the electroporation as described above. And the CISH KO efficiency (indel %) was performed by targeted amplicon next-generation sequencing (NGS). The sequence of RABI lA-gRNA is shown in SEQ ID NO: 1.

[0059] 4. The functional evaluation of CISH O NK cells:

[0060] Based on the CISH gRNA screen, one sgRNA with highest cutting efficiency was identified. And the NK cells (derived from two different healthy donors) were electroporated with Cas9-gRNA complex (RNP) and further cultured under NK MACS complete medium (Miltenyi Biotec). The cell number was counted using a Via2-Cassette in NucleoCounter NC- 202 (ChemoMetec) at specified time points. To test the in vitro cytotoxicity of these CISH KO NK cells, either mock NK cells (set as the control) or CISH K NK cells were cocultured with target cells (K562 cells) at various E / T ratios for 4 hours. The in vitro cytotoxicity of the effector cells against target cells was measured via a CCK-8 assay (Apexbio Technology) according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader. Absorbance was measured at 450 nm using a microplate reader. The cytotoxicity was calculated as follows: Cytotoxicity (%) = 100% - [(ODE+T- ODB) - (ODE - 0DB)] / (0DT- ODB) x 100% (OD, absorbance; E, Effector; T, Target; and B, Blank). The sequence of RABI 1 A-GFP donor is shown in SEQ ID NO: 6. The sequence of RABI 1 A-1928z-lxx donor is shown in SEQ ID NO: 7.

[0061] 5. Generation of CD19.CAR specific knock-in at CISH locus in human NK cells (CZS7fKO / KICAR-NK) and T cells (CffiHKO / KICAR-T). cssDNA HDR donor templates containing the payload - CD19.CAR construct with 5’ - and 3’ - HA sequences for CISH locus-targeting site were firstly designed and synthesized. And they were further mixed with Cas9-gRNA complex (RNP) together and coelectroporated into NK cells as described above. The cell viability at different time points was measured using a Via2-Cassette in NucleoCounter® NC-202 (ChemoMetec). And the CAR knock-in efficiency at various time points was detected on an Attune NxT flow cytometer (ThermoFisher Scientific). Briefly, the NK or T cells under culture were collected at specific time points after electroporation, resuspended in FACS buffer (2% FBS, v / v in PBS) and stained with an Alexa Fluor 647 AffiniPure F(ab')2 Fragment Goat Anti-Mouse IgG, F(ab')2 fragment specific antibody (1 : 100 dilution, Jackson ImmunoResearch). When the antibody staining process (30 min at 4°C) got finished, the SYTOX™ Blue live / dead staining (1 : 1000 dilution, Invitrogen) was added and incubated with cells before running through the Flow cytometry. Data analysis was performed using FlowJo_vl0.8.0_CL software. The in vitro cytotoxicity of the effector cells including mock NK cells, CISH KO (RNP) cells, and C7SHKO / KICAR-NK or CAR-T (RNP + cssDNA) against target NALM6 cells was measured via a CCK-8 assay according to the manufacturer’s instructions as described above. The sequence of C757 / -1928z-lxx donor is shown in SEQ ID No: 8.

[0062] 6. Cytotoxicity of engineered NK and T cells

[0063] For CAR-NK, the in vitro cytotoxicity of the effector cells including mock NK cells, CISH O (RNP) cells, and CISHOlYACAR-NK (RNP + cssDNA) cells was measured via a Cell Counting Kit-8 (Apexbio Technology) after co-culturing with target cells at different effector: target (E / T) ratios for 4 hours according to the manufacturer’s instructions. For CAR-T, the cytotoxicity of T cells transduced with a CD 19. CAR was determined by IncuCyte Cytotoxicity Assay with NALM-6 cells that stably express GFP serving as target cells. On Day 7-9 post electroporation, the CAR-T effector (E) and tumor target (T) cells were co-cultured in triplicates at the indicated E / T ratio using flat bottom poly-L-ornithine (Sigma) coated 96-well plates with 1 * 104target cells in a total volume of 200 pL per well in NALM-6 medium. The number of effector CAR-T cells was calculated based on the percentage of CAR-positive cells. Target cells alone were plated at the same cell density to determine cell proliferation. Plates were incubated at 37°C with 5 % CO2 for up to 3 days. Four images were recorded per well every 2 h and analyzed using IncuCyte cell by cell analysis software module (Sartorius). The killing potency of the CAR-T cells was assessed by comparing the percentage of target cells over time relative to the total number of target cells in the wells with NALM6 cells only.

[0064] It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

[0065] References:

[0066] 1. Pan, K. et al. CAR race to cancer immunotherapy: from CAR T, CAR NK to CAR macrophage therapy. J. Exp. Clin. Cancer Res. 41, 119 (2022).

[0067] 2. Persons, D. A. Lentiviral Vector Gene Therapy: Effective and Safe? Mol. Ther. 18, 861-862 (2010).

[0068] 3. Dufait, I. et al. Retroviral and Lentiviral Vectors for the Induction of Immunological Tolerance. Scientifica 2012, (2012).

[0069] 4. Elmas, E. et al. CRISPR Gene Editing of Human Primary NK and T Cells for Cancer Immunotherapy. Front. Oncol. 12, (2022).

[0070] 5. Colella, P., Ronzitti, G. & Mingozzi, F. Emerging Issues in AAV-Mediated In Vivo Gene Therapy. Mol. Ther. Methods Clin. Dev. 8, 87-104 (2017).

[0071] 6. Ghanta, K. S. et al. 5 '-Modifications improve potency and efficacy of DNA donors for precision genome editing. eLife 10, e72216.

[0072] 7. Xie, K. et al. Circular single-stranded DNA is a superior homology-directed repair donor template for efficient genome engineering. 2022.12.01.518578 Preprint at https: / / doi.org / 10.1101 / 2022.12.01.518578 (2022).

[0073] 8. Sobah, M. L., Liongue, C. & Ward, A. C. SOCS Proteins in Immunity, Inflammatory Diseases, and Immune-Related Cancer. Front. Med. 8, 727987 (2021).

[0074] 9. Delconte, R. B. et al. CIS is a potent checkpoint in NK cell-mediated tumor immunity. Nat. Immunol. 17, 816-824 (2016).

[0075] 10. Bernard, P.-L. et al. Targeting CISH enhances natural cytotoxicity receptor signaling and reduces NK cell exhaustion to improve solid tumor immunity. J. Immunother. Cancer 10, e004244 (2022).

[0076] 11. Zhu, H. et al. Metabolic Reprograming via Deletion of CISH in Human iPSC-Derived NK Cells Promotes In Vivo Persistence and Enhances Anti-tumor Activity. Cell Stem Cell 27, 224-237. e6 (2020).

[0077] 12. Palmer, D. C. et al. CISH actively silences TCR signaling in CD8+ T cells to maintain tumor tolerance. J. Exp. Med. 212, 2095-2113 (2015).

[0078] 13. Lv, J. et al. Disruption of CISH promotes the antitumor activity of human T cells and decreases PD-1 expression levels. Mol. Ther. - Oncolytics 28, 46-58 (2023). 14. Palmer, D. C. et al. Internal checkpoint regulates T cell neoantigen reactivity and susceptibility to PD1 blockade. Med 3, 682-704. e8 (2022).

Claims

WHAT IS CLAIMED IS:

1. A method for inserting a polynucleotide exogenous transgene sequence at a predetermined endogenous genetic locus in a hose cell genome ex vivo or in vivo, comprising:(i) a donor DNA template including a polynucleotide insert; a 5 ’-homology arm; and a 3 ’-homology arm. In some embodiments, the 5’ homology arm and the 3’ homology arm are complementary to the DNA in a target region; and(ii) a ribonucleoprotein complex (RNP) comprising (1) a Cas nuclease, and at least one small guide RNAs (sgRNA) that is complementary to at least one selected nucleic acid sequence within the pre-determined genetic locus in the host cell genome.

2. The method of claim 1, wherein the pre-determined genomic locus in which the exogenous polynucleotide is inserted in, while the endogenous gene was simultaneously disrupted by nuclease cleavage, is an immune checkpoint locus, for example, (e.g., cytokineinducible SH2-containing protein (CISH)), PD1, NKG2A, signal regulatory protein a (SIRPa), T cell immunoglobulin mucin family member 3 TIMS), lymphocyte-activation gene 3 (LAGS), T cell immunoreceptor with Ig and ITIM domains (TIGIT), TACTILE (CD96), or tumor necrosis factor-alpha-induced protein-8 like-2 (TIPE2).

3. The method of claim 1, wherein the exogenous polynucleotide is precisely engineered at the checkpoint GISH locus, and activating receptors, chimeric antigen receptors (CARs) or “armored” CARs for enhanced activity by secreting cytokines, express cytokine receptors to modulate a cytokine milieu of a tumor microenvironment or increase resistance to the tumor microenvironment.

4. The method of claim 1, wherein the exogenous polynucleotide is knocked in at the checkpoint (e.g., CISH) locus for immune-oncology therapy or other immune related disorders.

5. The method of claim 1, wherein the donor vector is selected from a plasmid DNA , linear double-stranded or single-stranded DNA, circular single-stranded DNA, DNA- nanoparticle complex, AAV viral particle, adenovirus particle or lentivirus particle.

6. The method of claim 1, wherein the donor vector further comprises at least one polynucleotide sequence encoding a CAR that has anti-tumor activity.

7. The method of claim 1, wherein the insertion of the exogenous polynucleotide produces a chimeric antigen receptor targeting at least one selected from the group consisting of B-cell maturation antigen (BCMA), carcinoembryonic antigen (CEA), CD2, CD4, CD5, CD7, CD 19, CD22, CD30, claudin 18.2 (CLDN 18.2), epithelial cellular adhesion molecule (EpCAM), folate receptor 1 (FOLR1), human epidermal growth factor receptor 2 (HER2), mesothelin (MSLN), mucin 1 (MUC1), natural killer group 2D (NKG2D).

8. The method of claim 1, wherein the donor vector further comprises at least one polynucleotide sequence encoding an immune checkpoint protein or an anti-immune checkpoint protein.

9. The method of claim 1, wherein the donor vector further comprises at least one polynucleotide sequence encoding a reporter gene.

10. The method of claim 1, wherein the donor vector further comprises a 2A selfcleaving sequence, an internal ribosome entry site (IRES) element, a promoter at the 5’ end of the at least one gene coding sequence, or a 3’ regulatory sequence.

11. The method of claim 1, wherein the nuclease is selected from a protein or an RNA molecule encoding the protein.

12. The method of claim 1, wherein the nuclease, is a class I Cas protein or a class II Cas protein.

13. The method of claim 1, wherein the Cas nuclease is Cas9, Cas3, CaslO, Casl l, CasX, Cas 12a, MAD7, or a catalytic dead form or nickase form thereof.

14. The method of claim 1, wherein the engineering comprises: using an at least one site-specific nuclease selected from the group consisting of a Cas nuclease, a zinc-finger nuclease, a meganuclease, and a transcription activator-like effector nuclease (TALEN).

15. The method of claim 1, wherein the immune cell is a primary T cell, an NK cell, or a tumor-infiltrating lymphocyte (TIL) cell.

16. The method of claim 1, wherein the immune cell is differentiated from an immune checkpoint engineered iPSCs or hematopoietic stem cell and a progenitor cell.

17. The method of claim 16, wherein the immature immune cell further differentiates to a mature immune cell.

18. The method of claim 1, wherein the immune cell is an natural killer (NK) cell; the donor DNA template has a length of about 2,000 bases or longer, and the length of the DNA insert is about 2,000 bases or longer; and an editing efficiency of the engineering is 20% or higher based on a total number of the immune cell.

19. The method of claim 1, wherein the immune cell is a T cell; the donor DNA template has a length of about 2,000 bases or longer, and the length of the DNA insert is about 1,000 bases or longer; and an editing efficiency of the engineering is 40% or higher based on a total number of the immune cell.

20. The method of claim 1, wherein each of a 5’-homology arm and a 3’-homology arm independently has a length ranging from about 25 nucleotides to about 5000 nucleotides.

21. The method of claim 1, further comprising: delivering the donor DNA template to the immune cell by a viral vector, a lipid or non-lipid nanoparticle delivery, an exosome delivery, an electroporation delivery, a gene gun delivery, or an injection.

22. The method of any one of claims 1-21, further comprising contacting the cells with a nonhomologous end joining (NHEJ) inhibitor or a DNA recombinase stimulator.

23. The method of claim 22, wherein the NHEJ inhibitor is a DNA-dependent protein kinase (DNA-PK) inhibitor.

24. The method of claim 23, wherein the DNA-PK comprises M-3814, Alt-R HDR enhancer, Alt-R HDR enhancer V2, generic DNA ligase inhibitor comprises SCR7, generic DNA recombinase stimulator comprises RS-1, or combinations thereof.

25. The method of claim 1, further comprising: engineering of an immune cell expressing chimeric antigen receptors at an immune checkpoint locus, while disrupting the immune checkpoint gene for disease treatment, including cancer and autoimmune disorders by using viral or non-viral homology-directed repair donor templates.

26. A modified host cell produced by the method of claim 1.

27. A method for treating a disease in a subject, the method comprising: administering the host cell of claim 1 or the modified host cell of claim 26 to the subject.

28. The method of claim 27, wherein the host cell is an autologous immune cell isolated from the diseased subject.

29. The method of claim 27, wherein the host cell is an allogeneic immune cells isolated from a healthy subject.

30. The method of claim 27, wherein the disease is a hematological cancer, a solid tumor or autoimmune diseases.

31. The method of claim 1, wherein the disease is treated in vivo.

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