Methods and compositions for generating modified cells

Abstract

The present disclosure relates, in part, to modified cells (e.g, modified pluripotent cells, modified myeloid progenitor cells, modified monocytes, and modified macrophages) comprising certain genetic modifications, which modulate the phenotypes of the modified cells or that of macrophages derived therefrom. The present disclosure further relates to compositions and cell populations comprising the modified cells and methods of making and using the same.

Description

METHODS AND COMPOSITIONS FOR GENERATING MODIFIED CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application No. 63 / 595,276 filed November 1, 2023, and United States Provisional Application No. 63 / 598,500 filed November 13, 2023, the contents of each of which are incorporated by reference in their entireties herein.SEQUENCE LISTING

[0002] This application contains a computer readable Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “14735-054-228_SEQLISTJNG.xml”, was created on October 30, 2024 and is 3,724 bytes in size.1. FIELD

[0003] The present disclosure relates, in part, to modified cells (e.g., modified pluripotent cells, modified myeloid progenitor cells, modified monocytes, and modified macrophages) comprising certain genetic modifications, which modulate the phenotypes of the modified cells or that of macrophages derived therefrom. The present disclosure further relates to compositions and cell populations comprising the modified cells and methods of making and using the same.2. BACKGROUND

[0004] Macrophages are among the most abundant immune cells within tumors and can be categorized into different phenotypes, such as M0, Ml, and M2. Each of the phenotypes is associated with different and diverse functions. For example, M0 macrophages are naive macrophages that are not polarized. Ml macrophages are polarized macrophages, which can be activated by IFN-y, lipopolysaccharide, IL-ip, TNF, and / or GM-CSF and can recognize and destroy tumor cells via phagocytosis and cytotoxicity. In addition, Ml macrophages are pro-inflammatory, and can produce pro-inflammatory cytokines that stimulate anti-tumor immunity. By contrast, M2 macrophages are anti-inflammatory and can be induced by Th2 cytokines such as IL-4, IL- 10, IL- 13, and / or M-CSF. M2 macrophages can favor tumorgrowth and promote TME remodeling by producing growth factors, immunosuppressive factors, pro-angiogenic molecules, and proteases.

[0005] Modulating macrophage phenotypes is an important aspect of macrophage-based cell therapy. The use of macrophages as a therapeutic tool has been limited by the lack of effective approaches for modulating the macrophage phenotypes in vitro, in vivo, and ex vivo.

[0006] There remains a need to modulate the phenotypes of macrophages to provide effective treatments for macrophage-related diseases (e.g., infectious diseases and cancer).3. SUMMARY

[0007] In one aspect, the present disclosure provides a modified cell comprising genetic disruption of at least one gene of: (a) a microRNA 146a (MIR146A) gene; (b) a signal transducer and activator of transcription 6 (STAT6) gene; (c) a G protein-coupled receptor 65 (GPR65) gene; (d) an AKT serine / threonine kinase 1 (AKT1) gene; (e) an AKT serine / threonine kinase 2 (AKT2) gene; (f) an interferon regulatory factor 3 (IRF3) gene; (g) an interferon regulatory factor 4 (IRF4) gene; (h) a phosphatidylinositol-4,5-bisphosphate 3- kinase catalytic subunit gamma (PIK3CG) gene; (i) a TSC complex subunit 1 (TSC1) gene; or (j) a V-set and immunoglobulin domain containing 4 (VSIG4) gene, wherein the modified cell is a modified pluripotent cell, a modified myeloid progenitor cell, a modified monocyte, a modified macrophage, or a modified CD1 lb+CD45+cell. In certain embodiments, the modified cell is homozygous or heterozygous for the genetic disruption of the at least one gene.

[0008] In certain embodiments, the modified cell is a modified pluripotent cell. In certain embodiments, the modified pluripotent cell is a modified induced pluripotent stem cell (iPSC). In certain embodiments, the iPSC has been reprogrammed from a peripheral blood mononuclear cell (PBMC), a CD34+cord blood cell, a macrophage, a monocyte, or a fibroblast.

[0009] In certain embodiments, the modified cell is a modified macrophage, a modified monocyte, or a modified CD1 lb+CD45+cell. In certain embodiments, the modified macrophage is an immature macrophage. In certain embodiments, the immature macrophage has not been subjected to any maturation or polarization process. In certain embodiments, the modified CD1 lb+CD45+cell is a CD1 lb+CD45+CD14- cell or a CD1 lb+CD45+CD14+cell.

[0010] In another aspect, the present disclosure provides a modified cell, wherein: i) the modified cell is a modified macrophage, a modified monocyte, or a modified CD1 lb+CD45+cell, and ii) the modified macrophage, monocyte, or CD1 lb+CD45+cell is derived from an iPSC that is homozygous or heterozygous for genetic disruption of at least one gene: (a) an AKT1 gene; (b) an AKT2 gene; (c) a GPR65 gene; (d) an IRF3 gene; (e) an IRF4 gene; (f) an MIR146A gene; (g) a PIK3CG gene; (h) a STAT6 gene; (i) a TSC1 gene; or (j) a VSIG4 gene.

[0011] In certain embodiments, the modified macrophage is an immature macrophage. In certain embodiments, the immature macrophage has not been subjected to any maturation or polarization process. In certain embodiments, the modified CD1 lb+CD45+cell is a CD1 lb+CD45+CD14- cell or a CD1 lb+CD45+CD14+cell.

[0012] In certain embodiments, the modified cell disclosed herein further comprises genetic disruption of a signal regulatory protein alpha (SIRPA) gene and / or a sialic acid-binding Ig- like lectin 10 (SIGLEC10) gene. In certain embodiments, the modified cell is homozygous or heterozygous for the genetic disruption of the SIRPA gene and / or the SIGLEC10 gene.

[0013] In certain embodiments, the modified cell disclosed herein further comprises genetic disruption of a gene encoding a cytokine receptor. In certain embodiments, the gene encoding a cytokine receptor is an IL10RA gene, an IL10RB gene, a TGFBR1 gene, a TGFBR2 gene, or a TGFBR3 gene.

[0014] In certain embodiments, the modified cell disclosed herein further comprises an exogenous polynucleotide encoding a dominant-negative (dn) variant of a cytokine receptor. In certain embodiments, the dominant-negative variant of the cytokine receptor is a dominant-negative variant of IL-10R (dnIL-lOR) or a dominant-negative variant of TGFpR (dnTGFpR).

[0015] In certain embodiments, the modified cell disclosed herein further comprises an exogenous polynucleotide encoding a proinflammatory cytokine. In certain embodiments, the proinflammatory cytokine is IL-12 or IFNy.

[0016] In certain embodiments, the modified cell disclosed herein further comprises a polynucleotide encoding a chimeric antigen receptor (CAR). In certain embodiments, the CAR comprises an antigen recognition moiety, a hinge domain, a transmembrane domain, and an intracellular signaling domain. In certain embodiments, the CAR promotes Ml macrophage polarization. In certain embodiments, the CAR increases secretion of a pro- inflammatory cytokine (e.g., is a CAR described in PCT / US2023 / 071207). In certain embodiments, the intracellular domain of the CAR is selected from the group consisting of a CD40, a TLR, and an IFNyR intracellular domain. In certain embodiments, the antigen recognition moiety of the CAR binds to a solid tumor antigen.

[0017] In certain embodiments, the modified cell disclosed herein further comprises: (i) genetic disruption of a beta-2-microglobulin (B2M) gene, (ii) genetic disruption of a class II major histocompatibility complex transactivator (CIITA) gene, (iii) genetic disruption of a regulatory factor X (RFX) gene, and / or (iv) an exogenous polynucleotide encoding major histocompatibility complex, class I, E (HLA-E).

[0018] In certain embodiments, the modified cell comprises a genetic disruption of at least the MIR146A gene. In certain embodiments, the modified cell comprises a genetic disruption of at least the STAT6 gene. In certain embodiments, the modified cell comprises a genetic disruption of at least the GPR65 gene. In certain embodiments, the modified cell comprises a genetic disruption of at least the AKT1 gene. In certain embodiments, the modified cell comprises a genetic disruption of at least the AKT2 gene. In certain embodiments, the modified cell comprises a genetic disruption of at least the IRF3 gene. In certain embodiments, the modified cell comprises a genetic disruption of at least the IRF4 gene. In certain embodiments, the modified cell comprises a genetic disruption of at least the PIK3CG gene. In certain embodiments, the modified cell comprises a genetic disruption of at least the TSC1 gene. In certain embodiments, the modified cell comprises a genetic disruption of at least the VSIG4 gene.

[0019] In another aspect, the present disclosure provides a method of generating a modified pluripotent cell, comprising genetically disrupting in a pluripotent cell at least one gene of (a) an MIR146A gene; (b) a STAT6 gene; (c) a GPR65 gene; (d) an AKT1 gene; (e) an AKT2 gene; (f) an IRF3 gene; (g) an IRF4 gene; (h) a PIK3CG gene; (i) a TSC1 gene; or (j) a VSIG4 gene.

[0020] In certain embodiments, the modified pluripotent cell is a modified iPSC.

[0021] In certain embodiments, the modified cell comprises genetic disruption of a SIRPA gene and / or a SIGLEC10 gene. In certain embodiments, the modified cell is homozygous or heterozygous for genetic disruption of the SIRPA gene and / or the SIGLEC10 gene.

[0022] In certain embodiments, the method further comprises genetically disrupting in the pluripotent cell a SIRPA gene and / or a SIGLEC10 gene. In certain embodiments, the genetically disrupting creates homozygous or heterozygous of the genetic disruption of the SIRPA gene and / or the SIGLEC10 gene.

[0023] In certain embodiments, the modified cell comprises genetic disruption of a gene encoding a cytokine receptor. In certain embodiments, the gene encoding a cytokine receptor is an IL10RA gene, an IL10RB gene, a TGFBR1 gene, a TGFBR2 gene, or a TGFBR3 gene.

[0024] In certain embodiments, the modified cell comprises an exogenous polynucleotide encoding a dominant-negative variant of a cytokine receptor. In certain embodiments, the dominant-negative variant of the cytokine receptor is a dominant-negative variant of IL- 1 OR (dnIL-lOR) or a dominant-negative variant of TGFpR (dnTGFpR).

[0025] In certain embodiments, the method further comprises genetically disrupting in the pluripotent cell a gene encoding a cytokine receptor. In certain embodiments, the gene encoding a cytokine receptor is an IL10RA gene, an IL10RB gene, a TGFBR1 gene, a TGFBR2 gene, or a TGFBR3 gene.

[0026] In certain embodiments, the method further comprises introducing in the pluripotent cell an exogenous polynucleotide encoding a dominant-negative variant of a cytokine receptor. In certain embodiments, the dominant-negative variant of the cytokine receptor is a dominant-negative variant of IL-10R (dnIL-lOR) or a dominant-negative variant of TGFpR (dnTGFpR).

[0027] In certain embodiments, the modified cell comprises an exogenous polynucleotide encoding a proinflammatory cytokine. In certain embodiments, the method further comprises introducing into the pluripotent cell an exogenous polynucleotide encoding a proinflammatory cytokine. In certain embodiments, the proinflammatory cytokine is IL-12 or IFNy.

[0028] In certain embodiments, the pluripotent cell comprises a polynucleotide encoding a CAR. In certain embodiments, the method further comprises introducing into the pluripotent cell a polynucleotide encoding a CAR. In certain embodiments, the polynucleotide encoding the CAR is introduced into the pluripotent cell prior to, concurrently with, or after the genetically disrupting.

[0029] In certain embodiments, the CAR comprises an antigen recognition moiety, a hinge domain, a transmembrane domain, and an intracellular signaling domain.

[0030] In certain embodiments, the CAR promotes Ml macrophage polarization. In certain embodiments, the CAR increases secretion of a pro-inflammatory cytokine (e.g., is a CAR described in PCT / US2023 / 071207). In certain embodiments, the intracellular domain of the CAR is selected from the group consisting of a CD40, a TLR, and an IFNyR intracellular domain. In certain embodiments, the antigen recognition moiety of the CAR binds to a solid tumor antigen.

[0031] In certain embodiments, the pluripotent cell further comprises: (i) genetic disruption of a B2M gene, (ii) genetic disruption of a CIITA gene, (iii) genetic disruption of an RFX gene, and / or (iv) an exogenous polynucleotide encoding HLA-E. In certain embodiments,the method further comprises (i) genetic disrupting in a B2M gene, (ii) genetic disrupting in a CIITA gene, (iii) genetic disrupting in an RFX gene, and / or (iv) introducing into the pluripotent cell an exogenous polynucleotide encoding HLA-E.

[0032] In certain embodiments, the modified cell comprises a genetic disruption of at least the MIR146A gene. In certain embodiments, the modified cell comprises a genetic disruption of at least the STAT6 gene. In certain embodiments, the modified cell comprises a genetic disruption of at least the GPR65 gene. In certain embodiments, the modified cell comprises a genetic disruption of at least the AKT1 gene. In certain embodiments, the modified cell comprises a genetic disruption of at least the AKT2 gene. In certain embodiments, the modified cell comprises a genetic disruption of at least the IRF3 gene. In certain embodiments, the modified cell comprises a genetic disruption of at least the IRF4 gene. In certain embodiments, the modified cell comprises a genetic disruption of at least the PIK3CG gene. In certain embodiments, the modified cell comprises a genetic disruption of at least the TSC1 gene. In certain embodiments, the modified cell comprises a genetic disruption of at least the VSIG4 gene.

[0033] In another aspect, the present disclosure provides a method of generating a modified myeloid progenitor cell, a modified monocyte, a modified macrophage, or a modified CD1 lb+CD45+cell, comprising differentiating a modified pluripotent cell produced by a method disclosed herein, under conditions sufficient for differentiating the modified pluripotent cell into the modified myeloid progenitor cell, monocyte, macrophage, or CD1 lb+CD45+cell. In certain embodiments, the method comprises differentiating the modified pluripotent cell under conditions sufficient for differentiating the modified pluripotent cell into the modified macrophage, monocyte, or CD1 lb+CD45+cell. In certain embodiments, the modified macrophage is a modified immature macrophage that has not been subjected to any maturation or polarization process. In certain embodiments, the modified macrophage is an immature macrophage. In certain embodiments, the immature macrophage has not been subjected to any maturation or polarization process. In certain embodiments, the modified CD1 lb+CD45+cell is a CD1 lb+CD45+CD14‘ cell or a CDl lb+CD45+CD14+cell.

[0034] In another aspect, the present disclosure provides a method of generating a modified myeloid progenitor cell, a modified monocyte, a modified macrophage, or a modified CD1 lb+CD45+cell, comprising differentiating a modified pluripotent cell disclosed herein, under conditions sufficient for differentiating the modified pluripotent cell into a modified myeloid progenitor cell, monocyte, macrophage, or CD1 lb+CD45+cell. In certainembodiments, the method comprises differentiating the modified pluripotent cell under conditions sufficient for differentiating the modified pluripotent cell into the modified macrophage, monocyte, or CD1 lb+CD45+cell. In certain embodiments, the modified macrophage is an immature macrophage. In certain embodiments, the immature macrophage has not been subjected to any maturation or polarization process. In certain embodiments, the modified CD1 lb+CD45+cell is a CD1 lb+CD45+CD14’ cell or a CD1 lb+CD45+CD14+cell.

[0035] In another aspect, the present disclosure provides a homogenous population of cells comprising: (a) a modified cell disclosed herein; (b) a modified pluripotent cell made by a method disclosed herein; or (c) a modified myeloid progenitor cell, a modified monocyte, a modified macrophage, or a modified CD1 lb+CD45+cell made by a method disclosed herein. In certain embodiments, the homogenous population comprises the modified macrophage, monocyte, or CD1 lb+CD45+cell. In certain embodiments, the modified macrophage is an immature macrophage. In certain embodiments, the immature macrophage has not been subjected to any maturation or polarization process. In certain embodiments, the cells are isogenic. In certain embodiments, the modified CD1 lb+CD45+cell is a CD1 lb+CD45+CD14‘ cell or a CD1 lb+CD45+CD14+cell.

[0036] In another aspect, the present disclosure provides a population of cells wherein (a) at least about 95% of the cells of the population are a modified cell disclosed herein; (b) at least about 95% of the cells of the population are a modified pluripotent cell made by a method disclosed herein; or (c) at least about 95% of the cells of the population are a modified myeloid progenitor cell, a modified monocyte, a modified macrophage, or a modified CD1 lb+CD45+cell made by a method disclosed herein. In certain embodiments, at least 95% of the cells of the population are the modified macrophage, monocyte, or CD1 lb+CD45+cell. In certain embodiments, the modified macrophage is an immature macrophage. In certain embodiments, the immature macrophage has not been subjected to any maturation or polarization process. In certain embodiments, the modified CD1 lb+CD45+cell is a CD1 lb+CD45+CD14‘ cell or a CD1 lb+CD45+CD14+cell. In certain embodiments, the cells are isogenic.

[0037] In another aspect, the present disclosure provides a composition comprising: (i) a modified cell disclosed herein; (ii) a modified pluripotent cell made by a method disclosed herein; (iii) a modified myeloid progenitor cell, a modified monocyte, a modified macrophage, or a modified CD1 lb+CD45+cell made by a method disclosed herein, (iv) a homogeneous population of cells disclosed herein, or (v) a population of cells disclosed herein.

[0038] In certain embodiments, the composition is a pharmaceutical composition comprising an effective amount of (i) the modified cell, (ii) the modified pluripotent cell, (iii) the modified myeloid progenitor cell, modified monocyte, modified macrophage, or modified CD1 lb+CD45+cell, (iv) the homogeneous population of cells or (v) the population of cells; and a pharmaceutically acceptable carrier.

[0039] In another aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and (i) a modified cell disclosed herein; (ii) a modified pluripotent cell made by a method disclosed herein; (iii) a modified myeloid progenitor cell, a modified monocyte, a modified macrophage, or a modified CD1 lb+CD45+cell made by a method disclosed herein, (iv) a homogeneous population of cells disclosed herein, or (v) a population of cells disclosed herein.

[0040] In another aspect, the present disclosure provides a use of (i) a modified cell disclosed herein; (ii) a modified pluripotent cell made by a method disclosed herein; (iii) a modified myeloid progenitor cell, a modified monocyte, a modified macrophage, or a modified CD1 lb+CD45+cell made by a method disclosed herein, (iv) a homogeneous population of cells disclosed herein, (v) a population of cells disclosed herein, or (vi) a pharmaceutical composition disclosed herein, in the manufacture of a medicament for the treatment of cancer in a subject in need thereof.

[0041] In another aspect, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of (i) a modified cell disclosed herein; (ii) a modified pluripotent cell made by a method disclosed herein; (iii) a modified myeloid progenitor cell, a modified monocyte, a modified macrophage, or a modified CD1 lb+CD45+cell made by a method disclosed herein, (iv) a homogeneous population of cells disclosed herein, (v) a population of cells disclosed herein, or (vi) a pharmaceutical composition disclosed herein.4. BRIEF DESCRIPTION OF THE FIGURES

[0042] FIG. 1 is a schematic showing of the path for maturation and polarization of iPSC- derived macrophages (iMACs).

[0043] FIGS. 2A-2C depicts the transcriptome profiles of the iMACs having a single gene knockout (iMAC-KOs). A curated set of genes highly regulated in Ml or M2 cells was included for the transcriptome profiling. FIG. 2A is a schematic showing of the RNA-seq design. FIG. 2B depicts the clustering of M0, Ml, and M2 iMAC-KOs (including AKT1,AKT2, GPR65, IRF3, IRF4, MIR146A, PIK3CG, STAT6, and VSIG4 iMAC-KOs) and wildtype iMAC (iMAC-WT) (TC-1133) based the transcriptome profiles. FIG. 2C depicts the Ml and M2 scores of the iMAC-KOs. Representative iMAC-KOs (e.g., STAT6, AKT2, and GPR65 iMAC-KOs) and iMAC-WT are indicated in FIG. 2C.

[0044] FIGS. 3A-3C depict the protein marker expression profiles of the genetically modified iMACs (iMAC-KOs). FIG. 3A is a schematic showing of the cytometry design for measuring the protein marker expression profiles in iMAC-KOs. FIG. 3B depicts the clustering of MO, Ml, and M2 iMAC-KOs (including AKT2, IRF4, MIR146A, SIPRA, STAT6, and VSIG4 iMAC-KOs) and iMAC-WT (TC-1133) based the protein marker expression profiles. FIG. 3C depicts the protein expression of specific MO, Ml, and M2 markers in iMAC-KOs.

[0045] FIG. 4 depicts the qPCR evaluation of the expression of MIR146A in iMACs.

[0046] FIG. 5 depicts the transcriptome profiles in Ml and M2 of iMAC-WT and MIR146A iMAC -KO. A curated set of genes highly regulated in Ml or M2 cells was evaluated. As indicated in the Z-score scale bar on top of the figure, “B” indicates the genes had negative Z- scores, whereas “R” indicates the genes had positive Z-scores.

[0047] FIG. 6 depicts the clustering of M0, Ml, and M2 of iMAC-WT and MIR146A iMAC -KO based on UMAP (uniform manifold approximation and projection) analysis of high-dimensional datasets.

[0048] FIG. 7 depicts the protein expression of M2 markers, including CD36, CD206, CD163 and CD169, in iMAC-WT and MIR146A iMAC -KO. The bars for each marker group, from left to right, represent iMAC-WT and MIR146A iMAC -KO respectively.

[0049] FIG. 8 depicts the cytokine secretion profiles in iMAC-WT and MIR146A iMAC- KO, shown as the fold change of the cytokine levels of MIR146A iMAC -KO as compared to iMAC-WT.

[0050] FIGS. 9A-9C depict the metabolic profiling of iMAC-WT and MIR146A iMAC -KO. FIG. 9A depicts the measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in M0 of iMAC-WT and MIR146A iMAC -KO. FIG. 9B depicts basal ECAR and maximal ECAR measured in M0, Ml, and M2 of iMAC-WT and MIR146A iMAC -KO. FIG. 9C depicts the OCR / ECAR profiles in M0, Ml, and M2 of iMAC-WT and MIR146A iMAC-KO.

[0051] FIGS. 10A-10C depict the transcriptome profiles of Ml and M2 of iMAC-WT and STAT6 iMAC-KO. FIG. 10A depicts transcript expression of marker genes in Ml and M2 of iMAC-WT and STAT6 iMAC-KO. As indicated in the Z-score scale bar on top of thefigure, “B” indicates the genes had negative Z-scores, whereas “R” indicates the genes had positive Z-scores. FIGS. 10B and 10C depict the Ml and M2 scores of Ml and M2 of iMAC-WT and STAT6 iMAC-KO.

[0052] FIGS. 11A and 11B depict the protein marker expression in MO, Ml, and M2 of iMAC-WT and STAT6 iMAC-KO. FIG. 11A depicts the clustering of MO, Ml, and M2 of iMAC-WT and STAT6 iMAC-KO based on protein marker expression profiles. FIG. 11B depicts the protein expression of CD206, CD169, and CD80 in iMAC-WT and STAT6 iMAC-KO.

[0053] FIGS. 12A and 12B depict the protein expression of chemokine receptors CX3CR1 (FIG. 12A) and CXCR4 (FIG. 12B) in unpolarized day 17 differentiated iMAC-WT and STAT6 iMAC-KO as measured by flow cytometry. Unstained iMAC-WT cells were used as the negative control. X-axis represents the fluorescence intensity for the specific protein tested, and Y-axis represents the percentage of total fluorescence in the tested sample. In each figure, the peaks from left to right are from the unstained iMAC-WT, the stained iMAC- WT, and the stained STAT6 iMAC-KO respectively.

[0054] FIGS. 13A-13C depict the transcriptome profiles of Ml and M2 of iMAC-WT and GPR65 iMAC-KO. FIG. 13A depicts the transcript expression of marker genes in Ml and M2 of iMAC-WT and GPR65 iMAC-KO. As indicated in the Z-score scale bar on top of the figure, “B” indicates the genes had negative Z-scores, whereas “R” indicates the genes had positive Z-scores. FIGS. 13B and 13C depict the Ml and M2 scores of Ml and M2 of iMAC-WT and GPR65 iMAC-KO.

[0055] FIGS. 14A-14F depict the expression of various protein markers for Ml and M2 states in several iMAC-KO lines measured by mass cytometry (CYTOF). M2 markers (including CD36 (FIG. 14A), CD206 (FIG. 14B), CD163 (FIG. 14C), CD304 (FIG. 14D), and CD 169 (FIG. 14E)), and Ml marker (including CXCR3 (FIG. 14F)) were measured and compared in M2 polarized iMAC-KOs, including VSIG, STAT6, SIPRA, MIR146A, IRF4, and AKT2 iMAC-KOs.

[0056] FIGS. 15A-15F depict the effects of knocking in a dominant-negative variant of TGFpR (dnTGFpR) in iMACs. FIG. 15A depicts the expression of dnTGFpR in dnTGFpR knock-in iMACs (iMAC-DNR), isotope control, and iMAC-WT. FIGS. 15B-15C depict the expression of phospho-SMAD2 (pSMAD2) in isotope control, iMAC-DNR, and iMAC-WT, in the absence of TGFP (FIG. 15B) or induced with 20 ng / ml TGFP (FIG. 15C). FIGS. 15D-15F depict the expression of CCR4 (FIG. 15D), CXCR4 (FIG. 15E), and CD86 (FIG. 15F) in iMAC-WT and iMAC-DNR with or without TGFP induction. “DNR” in FIGS. 15A-15F indicates iMAC-DNR; “WT” in FIGS. 15A-15C indicates iMAC-DNR; “iMAC” in FIGS. 15D-15F indicates iMAC-WT. X-axis represents the fluorescence intensity for the specific protein tested, and Y-axis represents the percentage of total fluorescence in the tested sample.5. DETAILED DESCRIPTION

[0057] The present disclosure relates, in part, to modified cells e.g., modified pluripotent cells, modified myeloid progenitor cells, modified monocytes, or modified macrophages) comprising certain genetic modifications, which modulate the macrophage phenotypes of the modified cells or macrophages derived therefrom. In certain embodiments, the modified cells or macrophages derived therefrom have suppressed M2 macrophage polarization and / or promoted Ml macrophage polarization, relative to unmodified cells. In certain embodiments, the modified cell provided herein comprises genetic disruption of at least one gene of:(a) a microRNA 146a (MIR146A) gene;(b) a signal transducer and activator of transcription 6 (STAT6) gene;(c) a G protein-coupled receptor 65 (GPR65) gene;(d) an AKT serine / threonine kinase 1 (AKT1) gene;(e) an AKT serine / threonine kinase 2 (AKT2) gene;(f) an interferon regulatory factor 3 (IRF3) gene;(g) an interferon regulatory factor 4 (IRF4) gene;(h) a phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (PIK3CG) gene;(i) a TSC complex subunit 1 (TSC1) gene; or(j) a V-set and immunoglobulin domain containing 4 (VSIG4) gene.

[0058] In certain embodiments, the modified cell provided herein further comprises genetic disruption of a signal regulatory protein alpha (SIRPA) gene and / or a sialic acid-binding Ig- like lectin 10 (SIGLEC10) gene.

[0059] The present disclosure is based, in part, on the discovery that knocking out certain genes in induced pluripotent stem cells (iPSCs) suppressed M2 macrophage polarization and promoted Ml macrophage polarization. In particular, macrophages derived from such genetically modified iPSC knockout cells (iPSC-KOs), once being exposed to polarization stimuli (e.g., IFNy and LPS as Ml polarization stimuli and IL4 and IL 13 as M2 polarization stimuli), had increased expression of Ml markers and decreased expression of M2 markers,at both transcript and protein levels, relative to macrophages derived from counterpart nonknockout iPSCs. In addition, the polarized knockout macrophages had increased secretion of Ml cytokines and reduced secretion of M2 cytokines, relative to macrophages derived from counterpart non-knockout iPSCs. Moreover, the polarized knockout macrophages resisted M2-induced reduction in metabolic activities.

[0060] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification. All patents, published patent applications and publications cited herein are incorporated by reference as if set forth fully herein. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

[0061] It should be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0062] As used herein, the terms “about” and “approximately” mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0063] As used herein, the term “wildtype” when used in comparison to the modified cells of the present disclosure is intended to mean a cell that does not comprise genetic disruption of the gene(s) of interest (e.g., a MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene) present in the modified cell, but is otherwise genetically identical to the referenced modified cell. It should be appreciated that a wildtype cell may include other modifications aside from genetic disruption of the gene(s) of interest, if those modifications are also present in the modified cell.

[0064] For purposes of clarity of disclosure and not by way of limitation, the detailed description is divided into the following subsections:5.1. Modified Cells;5.2. Methods of Generating Modified Cells;5.3. Chimeric Antigen Receptors;5.4. Polynucleotides;5.5. Vectors;5.6. Polypeptides;5.7. Compositions;5.8. Methods of Treatment;5.9. Differentiation of Modified Cells and5.10. Assays.5.1 Modified Cells

[0065] In one aspect, the present disclosure provides modified cells comprising certain genetic modifications, which modulate macrophage phenotypes of the modified cells or macrophages derived therefrom. In certain embodiments, the modified cells or macrophages derived therefrom have suppressed M2 macrophage polarization. In certain embodiments, the modified cells or macrophages derived therefrom have promoted Ml macrophage polarization. In certain embodiments, the modified cells or macrophages derived therefrom have suppressed M2 macrophage polarization and promoted Ml macrophage polarization.

[0066] In certain embodiments, the modified cell is a modified pluripotent cell (e.g., induced pluripotent stem cell), a modified myeloid progenitor cell, a modified monocyte, a modified macrophage (e.g., a modified immature macrophage). In certain embodiments, the modified cell is a modified CD1 lb+CD45+cell (e.g., a CD1 lb+CD45+CD14‘ cell or a CDl lb+CD45+CD14+cell).

[0067] In certain embodiments, the modified cell provided herein comprises genetic disruption of at least one gene of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene.

[0068] In certain embodiments, the modified cell provided herein further comprises genetic disruption of a SIRPA gene and / or a SIGLEC10 gene.

[0069] In certain embodiments, presented herein is a cell that comprises a genomic edit of one or more cytokine receptors (e.g., IL-10 receptor (IL-10R) or TGFP receptor (TGFpR), e.g., a TGFpRII) that disrupt the signaling of the corresponding cytokines. In certain embodiments, the genomic edit results in a loss of function of a cytokine receptor (e.g., IL- 10R or TGFpR, e.g., a TGFpRII) or a dominant-negative variant of a cytokine receptor (e.g., dominant-negative IL- 10 receptor (dnIL-lOR) or dominant-negative TGFP receptor (dnTGFpR), e.g, a dnTGFpRII). In certain embodiments, such genomic edit, which results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor, suppresses M2 macrophage polarization. In certain embodiments, the cell does not comprise a genomic edit of a MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene,an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene. In particular embodiments, the cell is a pluripotent cell, e.g., an induced pluripotent stem cell, such as a human induced pluripotent stem cell; a myeloid progenitor cell, e.g, a myeloid progenitor cell derived from an induced pluripotent stem cell, such as a human induced pluripotent stem cell; a monocyte, e.g., a monocyte derived from an induced pluripotent stem cell, such as a human induced pluripotent stem cell; a macrophage, for example an immature macrophage, e.g., a macrophage, for example an immature macrophage derived from an induced pluripotent stem cell, such as a human induced pluripotent stem cell. In certain embodiments, presented herein is a cell that is a CD1 lb+CD45+cell, for example, a CD1 lb+CD45+CD14‘ cell or a CD1 lb+CD45+CD14+cell, e.g., a CD1 lb+CD45+cell, for example, a CD1 lb+CD45+CD14‘ cell or a CD1 lb+CD45+CD14+cell, derived from an induced pluripotent stem cell, such as a human induced pluripotent stem cell.

[0070] In certain embodiments, the modified cell provided herein further comprises a genomic edit of certain cytokine receptors (e.g., IL-10 receptor (IL-10R) or TGFP receptor (TGFpR), e.g., a TGFpRII) that disrupts the signaling of the corresponding cytokines. In certain embodiments, the modified cell provided herein comprises a genomic edit of the cytokine receptor and at least one of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene. In certain embodiments, the genomic edit results in a loss of function of a cytokine receptor (e.g., IL-10R or TGFpR, e.g., a TGFpRII) or a dominant-negative variant of a cytokine receptor (e.g., dominant-negative IL- 10 receptor (dnIL-lOR) or dominantnegative TGFP receptor (dnTGFpR), e.g., a dnTGFpRII). In certain embodiments, such genomic edit, which results in a loss of function of a cytokine receptor, or a dominantnegative variant of a cytokine receptor, suppresses M2 macrophage polarization.

[0071] In certain embodiments, a modified cell provided herein further comprises one or more exogenous polynucleotides, including one or more exogenous polynucleotides that encode one or more polypeptides. For ease of description herein, such one or more exogenous polynucleotides may be referred to herein as an “exogenous polynucleotide” however, it should be understood that this term may encompass more than one polynucleotide. For example, in instances wherein an exogenous polynucleotide encodes more than one polypeptide, unless otherwise noted such an “exogenous polynucleotide” encompasses a single polynucleotide that encodes the polypeptides, as well as one or more polynucleotides that in the aggregate encode the polypeptides.

[0072] In certain embodiments, the modified cell provided herein further comprises an exogenous polynucleotide encoding a proinflammatory cytokine (e.g., IL-12 and / or IFNy). In certain embodiments, expression of the proinflammatory cytokine in the modified cell promotes Ml macrophage polarization.

[0073] In certain embodiments, the modified cell provided herein further comprises a polynucleotide encoding a chimeric antigen receptor (CAR). In certain embodiments, the CAR promotes Ml macrophage polarization. In certain embodiments, the CAR increases secretion of a pro-inflammatory cytokine (e.g., is a CAR described in PCT / US2023 / 071207). In certain embodiments, the intracellular domain of the CAR is selected from the group consisting of a CD40, a TLR, and an IFNyR intracellular domain.

[0074] In certain embodiments, the modified cell provided herein is a hypoimmunogenic cell that is able to evade or partially evade an allogeneic host versus graft immune response. For example, in certain embodiments, the modified cell provided herein further comprises: (i) genetic disruption of a beta-2-microglobulin (B2M) gene, (ii) genetic disruption of a class II major histocompatibility complex transactivator (CIITA) gene, (iii) genetic disruption of a regulatory factor X (RFX) gene, and / or (iv) an exogenous polynucleotide encoding major histocompatibility complex, class I, E (HLA-E). In certain embodiments, genetic disruption the B2M gene, the RFX gene, the CIITA gene, and / or expression of HLA-E results in hypoimmunogenicity in the modified cell.

[0075] In certain embodiments, the modified cell is a human cell. In certain embodiments, the modified cell is a mammalian cell. In certain embodiments, the modified cell is a primate cell. In certain embodiments, the modified cell is a non-human primate cell. In certain embodiments, the modified cell is a rodent, e.g., mouse, cell.5.1.1 Genetic Modifications

[0076] In certain embodiments, the modified cell provided herein comprises genetic disruption of at least one gene of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene.

[0077] In certain embodiments, the modified cell provided herein comprises genetic disruption of a microRNA 146a (MIR146A) gene. The MIR146A gene (e.g., homo sapiens NCBI Gene ID: 3662, mus musculus NCBI Gene ID: 11652) encodes microRNA 146a (miR- 146a) that is involved in the control of the inflammatory response of cells of the innate immune system. In certain embodiments, genetic disruption of the MIR146A gene eliminates or reduces the expression of miR-146a. In certain embodiments, the MIR146A gene is ahuman MIR146A gene. In certain embodiments, the MIR146A gene is a non-human MIR146A gene (e.g., a mouse MIR146A gene).

[0078] In certain embodiments, the modified cell provided herein comprises genetic disruption of a signal transducer and activator of transcription 6 (STAT6) gene. The STAT6 gene (e.g., homo sapiens NCBI Gene ID: 6778, mus musculus NCBI Gene ID: 20852) encodes a STAT6 protein that is a transcription factor that mediates signaling pathway required for the development of T-helper type 2 (Th2) cells and Th2 immune response. In certain embodiments, genetic disruption of the STAT6 gene eliminates or reduces the expression of a STAT6 protein. In certain embodiments, the STAT6 gene is a human STAT6 gene. In certain embodiments, the STAT6 gene is a non-human STAT6 gene (e.g., a mouse STAT6 gene).

[0079] In certain embodiments, the modified cell provided herein comprises genetic disruption of a G protein-coupled receptor 65 (GPR65) gene. The GPR65 gene (e.g., homo sapiens NCBI Gene ID: 8477, mus musculus NCBI Gene ID: 14744) encodes a GPR65 protein that enables G protein-coupled receptor activity. In certain embodiments, genetic disruption of the GPR65 gene eliminates or reduces the expression of an GPR65 protein. In certain embodiments, the GPR65 gene is a human GPR65 gene. In certain embodiments, the GPR65 gene is a non-human GPR65 gene (e.g., a mouse GPR65 gene).

[0080] In certain embodiments, the modified cell provided herein comprises genetic disruption of an AKT serine / threonine kinase 1 (AKT1) gene. The AKT1 gene (e.g., homo sapiens NCBI Gene ID: 207, mus musculus NCBI Gene ID: 11651) encodes an AKT1 protein that is one of the three members of the human AKT serine-threonine protein kinase family, which are often referred to as protein kinase B alpha, beta, and gamma. In certain embodiments, genetic disruption of the AKT1 gene eliminates or reduces the expression of an AKT1 protein. In certain embodiments, the AKT1 gene is a human AKT1 gene. In certain embodiments, the AKT1 gene is a non-human AKT1 gene (e.g., a mouse AKT1 gene).

[0081] In certain embodiments, the modified cell provided herein comprises genetic disruption of an AKT serine / threonine kinase 2 (AKT2) gene. The AKT2 gene (e.g., homo sapiens NCBI Gene ID: 208, mus musculus NCBI Gene ID: 11652) is a putative oncogene encoding an AKT2 protein belonging to a subfamily of serine / threonine kinases containing SH2-like (Src homology 2-like) domains. In certain embodiments, genetic disruption of the AKT2 gene eliminates or reduces the expression of an AKT2 protein. In certain embodiments, the AKT2 gene is a human AKT2 gene. In certain embodiments, the AKT2 gene is a non-human AKT2 gene (e.g., a mouse AKT2 gene).

[0082] In certain embodiments, the modified cell provided herein comprises genetic disruption of an interferon regulatory factor 3 (IRF3) gene. The IRF3 gene (e.g., homo sapiens NCBI Gene ID: 3661, mus musculus NCBI Gene ID: 11652) encodes IRF3 protein that is a member of the interferon regulatory transcription factor (IRF) family, which upon serine / threonine phosphorylation forms a complex with CREBBP. In certain embodiments, genetic disruption of the IRF3 gene eliminates or reduces the expression of an IRF3 protein. In certain embodiments, the IRF3 gene is a human IRF3 gene. In certain embodiments, the IRF3 gene is a non-human IRF3 gene (e.g., a mouse IRF3 gene).

[0083] In certain embodiments, the modified cell provided herein comprises genetic disruption of an interferon regulatory factor 4 (IRF4) gene. The IRF4 gene (e.g., homo sapiens NCBI Gene ID: 3662, mus musculus NCBI Gene ID: 11652) encodes an IR.F4 protein that is required in established plasma cells to regulate gene transcription and mitochondrial homeostasis. In certain embodiments, genetic disruption of the IRF4 gene eliminates or reduces the expression of an IRF4 protein. In certain embodiments, the IRF4 gene is a human IRF4 gene. In certain embodiments, the IRF4 gene is a non-human IRF4 gene (e.g., a mouse IRF4 gene).

[0084] In certain embodiments, the modified cell provided herein comprises genetic disruption of a phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (PIK3CG) gene. The PIK3CG gene (e.g., homo sapiens NCBI Gene ID: 5294, mus musculus NCBI Gene ID: 30955) encodes a PIK3CG protein that is a class I catalytic subunit of PI3K, which binds a p85 regulatory subunit to form phosphoinositide 3-kinase (PI3K). In certain embodiments, genetic disruption of the PIK3CG gene eliminates or reduces the expression of a PIK3CG protein. In certain embodiments, the PIK3CG gene is a human PIK3CG gene. In certain embodiments, the PIK3CG gene is a non-human PIK3CG gene (e.g., a mouse PIK3CG gene).

[0085] In certain embodiments, the modified cell provided herein comprises genetic disruption of a TSC complex subunit 1 (TSC1) gene. The TSC1 gene (e.g., homo sapiens NCBI Gene ID: 7248, mus musculus NCBI Gene ID: 64930) encodes a TSC1 protein (also known as hamartin) that interacts with tuberin to negatively regulate mammalian target of rapamycin complex 1 (mTORCl) signaling, which is a major regulator of anabolic cell growth. In certain embodiments, genetic disruption of the TSC1 gene eliminates or reduces the expression of a TSC1 protein. In certain embodiments, the TSC1 gene is a human TSC1 gene. In certain embodiments, the TSC1 gene is a non-human TSC1 gene (e.g., a mouse TSC1 gene).

[0086] In certain embodiments, the modified cell provided herein comprises genetic disruption of a V-set and immunoglobulin domain containing 4 (VSIG4) gene. The VSIG4 gene (e.g., homo sapiens NCBI Gene ID: 11326, mus musculus NCBI Gene ID: 278180) encodes a VSIG4 protein that is a receptor for the complement component 3 fragments C3b and iC3b. In certain embodiments, genetic disruption of the VSIG4 gene eliminates or reduces the expression of a VSIG4 protein. In certain embodiments, the VSIG4 gene is a human VSIG4 gene. In certain embodiments, the VSIG4 gene is a non-human VSIG4 gene (e.g., a mouse VSIG4 gene).

[0087] In certain embodiments, the modified cell provided herein comprises genetic disruption of one gene selected from the group consisting of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, and a VSIG4 gene. In certain embodiments, the modified cell comprises genetic disruption of an MIR146A gene. In certain embodiments, the modified cell comprises genetic disruption of a STAT6 gene. In certain embodiments, the modified cell comprises genetic disruption of a GPR65 gene. In certain embodiments, the modified cell comprises genetic disruption of an AKT1 gene. In certain embodiments, the modified cell comprises genetic disruption of an AKT2 gene. In certain embodiments, the modified cell comprises genetic disruption of an IRF3 gene. In certain embodiments, the modified cell comprises genetic disruption of an IRF4 gene. In certain embodiments, the modified cell comprises genetic disruption of a PIK3CG gene. In certain embodiments, the modified cell comprises genetic disruption of a TSC1 gene. In certain embodiments, the modified cell comprises genetic disruption of a VSIG4 gene.

[0088] In certain embodiments, the modified cell provided herein comprises genetic disruption of two or more genes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or all genes) selected from the group consisting of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, and a VSIG4 gene.

[0089] In certain embodiments, the modified cell provided herein comprises genetic disruption of two genes selected from the group consisting of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, and a VSIG4 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an AKT1 gene and an AKT2 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an AKT1 gene and a GPR65 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an AKT1 gene and an IRF3 gene. In certain embodiments,the modified cell provided herein comprises genetic disruption of an AKT1 gene and an IRF4 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an AKT1 gene and an MIR146A gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an AKT1 gene and a PIK3CG gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an AKT1 gene and a STAT6 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an AKT1 gene and a TSC1 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an AKT1 gene and a VSIG4 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an AKT2 gene and a GPR65 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an AKT2 gene and an IRF3 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an AKT2 gene and an IRF4 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an AKT2 gene and an MIR146A gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an AKT2 gene and a PIK3CG gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an AKT2 gene and a STAT6 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an AKT2 gene and a TSC1 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an AKT2 gene and a VSIG4 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an GPR65 gene and an IRF3 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an GPR65 gene and an IRF4 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an GPR65 gene and an MIR146A gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an GPR65 gene and a PIK3CG gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an GPR65 gene and a STAT6 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an GPR65 gene and a TSC1 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an GPR65 gene and a VSIG4 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an IRF3 gene and an IRF4 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an IRF3 gene and an MIR146A gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an IRF3 gene and a PIK3CG gene. In certain embodiments, the modified cell provided hereincomprises genetic disruption of an IRF3 gene and a STAT6 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an IRF3 gene and a TSC1 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an IRF3 gene and a VSIG4 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an IRF4 gene and an MIR146A gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an IRF4 gene and a PIK3CG gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an IRF4 gene and a STAT6 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an IRF4 gene and a TSC1 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an IRF4 gene and a VSIG4 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an MIR146A gene and a PIK3CG gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an MIR146A gene and a STAT6 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an MIR146A gene and a TSC1 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an MIR146A gene and a VSIG4 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an PIK3CG gene and a STAT6 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an PIK3CG gene and a TSC1 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an PIK3CG gene and a VSIG4 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of an TSC1 gene and a VSIG4 gene.

[0090] In certain embodiments, the modified cell provided herein comprises genetic disruption of three, four, five, six, seven, eight, nine genes selected from the group consisting of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, and a VSIG4 gene. In certain embodiments, the modified cell provided herein comprises genetic disruption of all an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, and a VSIG4 gene.

[0091] In certain embodiments, the modified cell provided herein is homozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene. In certain embodiments, the modified cell provided herein is heterozygous for the geneticdisruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene.

[0092] In certain embodiments, the modified cell provided herein further comprises genetic disruption of a SIRPA gene. In certain embodiments, the modified cell provided herein further comprises genetic disruption of a SIGLEC10 gene. In certain embodiments, the modified cell provided herein further comprises genetic disruption of a SIRPA gene and a SIGLEC10 gene.

[0093] In certain embodiments, the modified cell provided herein further comprises genetic disruption of a SIRPA gene (e.g., homo sapiens NCBI Gene ID: 140885, mus musculus NCBI Gene ID: 19261). The SIRPA gene encodes for a SIRPa protein, which an immunoglobulin-like cell surface receptor for CD47. In certain embodiments, genetic disruption of SIRPA prevents or reduces expression of a SIRPa protein capable of interaction with CD47. In certain embodiments, genetic disruption of SIRPA prevents a SIRPa protein from being capable of interaction with CD47. In certain embodiments, the SIRPA gene is a human SIRPA gene. In certain embodiments, the SIRPA gene is a non-human SIRPA gene (e.g, a mouse SIRPA gene).

[0094] In certain embodiments, the modified cell provided herein further comprises genetic disruption of a SIGLEC10 gene (e.g, Homo sapiens NCBI Gene ID: 89790, mus musculus NCBI Gene ID: 243958). The SIGLEC10 gene is a ligand for CD52, vascular adhesion protein 1 (VAP-1), and CD24. The CD24- SIGLEC10 interaction can serve as an antiphagocytic signal (see, e.g., Barkal AA, et al., Nature. 2019 Aug;572(7769):392-396). Thus, in certain embodiments genetic disruption of SIGLEC10 prevents or reduces expression of a SIGLEC10 protein capable of interaction with CD24 on a target cell. In certain embodiments genetic disruption of SIGLEC10 prevents a SIGLEC10 protein from being capable of interaction with CD24 on a target cell. In certain embodiments, the SIGLEC10 gene is a human SIGLEC10 gene. In certain embodiments, the SIGLEC10 gene is a non-human SIGLEC10 gene (e.g., a mouse SIGLEC10 gene).

[0095] In certain embodiments, the modified cell provided herein is homozygous for the genetic disruption of the SIRPA gene and / or SIGLEC10 gene. In certain embodiments, the modified cell provided herein is heterozygous for the genetic disruption of the SIRPA gene and / or SIGLEC10 gene.

[0096] In certain embodiments, the modified cell provided herein is a hypoimmunogenic cell that can evade or partially evade an allogeneic host versus graft immune response. In certainembodiments, the modified cell further comprises (i) genetic disruption of a beta-2- microglobulin (B2M) gene, (ii) genetic disruption of a class II major histocompatibility complex transactivator (CIITA) gene, (iii) genetic disruption of a regulatory factor X (RFX) gene, and / or (iv) an exogenous polynucleotide encoding major histocompatibility complex, class I, E (HLA-E). In certain embodiments, the modified cell provided herein further comprises genetic disruption of a B2M gene. In certain embodiments, the modified cell provided herein further comprises genetic disruption of a CIITA gene. In certain embodiments, the modified cell provided herein further comprises genetic disruption of an RFX gene. In certain embodiments, the modified cell provided herein further comprises genetic disruption of a B2M gene and a CIITA gene. In certain embodiments, the modified cell provided herein further comprises genetic disruption of a B2M gene and an RFX gene. In certain embodiments, the modified cell provided herein further comprises genetic disruption of a CIITA gene and an RFX gene. In certain embodiments, the modified cell provided herein further comprises genetic disruption of a B2M gene, a CIITA gene and an RFX gene. In certain embodiments, the modified cell provided herein further comprises an exogenous polynucleotide encoding HLA-E. In certain embodiments, the modified cell provided herein further comprises genetic disruption of (i) a B2M gene, a CIITA gene and / or an RFX gene, and (ii) an exogenous polynucleotide encoding HLA-E. In certain embodiments, the modified cell provided herein further comprises genetic disruption of a B2M gene and a CIITA gene (e.g., B2M / CIITA double knockout), and an exogenous polynucleotide encoding HLA-E. In certain embodiments, the modified cell provided herein further comprises genetic disruption of a B2M gene, a CIITA gene, and an RFX gene, and an exogenous polynucleotide encoding HLA-E.

[0097] In certain embodiments, the modified cell provided herein further comprises genetic disruption of a B2M gene. The B2M gene (e.g., homo sapiens NCBI Gene ID: 567, mus musculus NCBI Gene ID: 12010) encodes the beta chain component of MHC class I molecules. Expression of B2M protein is necessary for assembly and function of MHC class I molecules on the cell surface. In certain embodiments, genetic disruption of the B2M gene eliminates or reduces the expression of an B2M protein. In certain embodiments, the B2M gene is a human B2M gene. In certain embodiments, the B2M gene is a non-human B2M gene (e.g., a mouse B2M gene).

[0098] In certain embodiments, the modified cell provided herein further comprises genetic disruption of a CIITA gene. The CIITA gene (e.g., homo sapiens NCBI Gene ID: 4261, mus musculus NCBI Gene ID: 12265) encodes a CIITA protein that is essential for transcriptionalactivity of the HLA class II promoter. In certain embodiments, genetic disruption of the CIITA gene eliminates or reduces the expression of a CIITA protein. In certain embodiments, the CIITA gene is a human CIITA gene. In certain embodiments, the CIITA gene is a non-human CIITA gene (e.g., a mouse CIITA gene).

[0099] In certain embodiments, the modified cell provided herein further comprises genetic disruption of an RFX gene. In certain embodiments, the RFX gene is an RFX1 gene, an RFX2 gene, an RFX3 gene, an RFX4 gene, an RFX5 gene, an RFX6 gene, an RFX7 gene, or an RFX8 gene. In certain embodiments, genetic disruption of the RFX gene eliminates or reduces the expression of an RFX protein. In certain embodiments, the RFX gene is a human RFX gene. In certain embodiments, the RFX gene is a non-human RFX gene (e.g., a mouse RFX gene).

[0100] In certain embodiments, the modified cell provided herein further comprises an exogenous polynucleotide encoding a major histocompatibility complex, class I, E (HLA- E). HLA-E belongs to the HLA class I heavy chain paralogues. HLA-E binds a restricted subset of peptides derived from the leader peptides of other class I molecules.

[0101] In certain embodiments, the modified cell provided herein is homozygous for the genetic disruption of the B2M gene, CIITA gene, and / or RFX gene. In certain embodiments, the modified cell provided herein is heterozygous for the genetic disruption of the B2M gene, CIITA gene, and / or RFX gene.

[0102] In certain embodiments, the cell or modified cell provided herein further comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the cell provided herein comprises a genomic edit that results in a loss of function of a cytokine receptor or a dominant-negative variant of a cytokine receptor, and does not comprise a genomic edit of a MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene. In certain embodiments, the modified cell provided herein comprises a genomic edit that results in a loss of function of a cytokine receptor or a dominant-negative variant of a cytokine receptor, and comprises at least one of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene. In certain embodiments, the cytokine receptor is IL-10 receptor (IL-10R). In certain embodiments, the cytokine receptor is a TGFP receptor (TGFpR). In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII, or TGFpRIII. In certain embodiments, the cytokine receptor is a TGFpRII.

[0103] In certain embodiments, the cell or modified cell provided herein further comprises genetic disruption of a gene encoding IL-10R. In certain embodiments, the cell or modified cell provided herein comprises genetic disruption of an IL 1 ORA gene and / or an IL10RB gene. The interleukin 10 receptor subunit alpha (IL10RA) gene (e.g., homo sapiens NCBI Gene ID: 3587, mus musculus NCBI Gene ID: 16154) encodes IL- 10 subunit a (IL- lORa). The interleukin 10 receptor subunit beta (IL10RB) gene (e.g., homo sapiens NCBI Gene ID: 3588, mus musculus NCBI Gene ID: 16155) encodes IL-10 subunit P (IL-10RP). IL-10R is a heterotetramer complex comprising two IL-lORa (also referred to as IL-10R1) molecules (encoded by the IL 1 ORA gene) and two IL-10RP (also referred to as IL-10R2) molecules (encoded by the IL10RB gene). In certain embodiments, genetic disruption of the gene encoding IL-10R eliminates or reduces the expression of an IL-10R protein (e.g., IL- 10Ra or IL-10RP). In certain embodiments, the IL10RA and / or IL10RB gene is a human IL10RA and / or IL10RB gene. In certain embodiments, the IL10RA and / or IL10RB gene is a non-human IL10RA and / or IL10RB gene (e.g., a mouse IL10RA and / or IL10RB gene).

[0104] In certain embodiments, the cell or modified cell provided herein further comprises genetic disruption of a gene encoding a TGFpR (e.g., TGFpRI, TGFpRII, or TGFpRIII). In certain embodiments, the cell or modified cell provided herein further comprises genetic disruption of a gene encoding a TGFpRII. In certain embodiments, the cell or modified cell provided herein further comprises genetic disruption of a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene. In certain embodiments, the modified cell provided herein further comprises genetic disruption of a TGFBR2 gene.

[0105] The transforming growth factor beta receptor 1 (TGFBR1) gene (e.g., homo sapiens NCBI Gene ID: 7046, mus musculus NCBI Gene ID: 21812) encodes TGFpRI. The transforming growth factor beta receptor 2 (TGFBR2) gene (e.g., homo sapiens NCBI Gene ID: 7047, mus musculus NCBI Gene ID: 21813) encodes TGFpRII. The transforming growth factor beta receptor 3 (TGFBR3) gene (e.g., homo sapiens NCBI Gene ID: 7048, mus musculus NCBI Gene ID: 21814) encodes TGFpRIII. In certain embodiments, genetic disruption of the gene encoding the TGFpR eliminates or reduces the expression of a TGFpR protein (e.g., a TGFpRI protein, a TGFpRII protein, or a TGFpRIIIm protein). In certain embodiments, the TGFBR1, TGFBR2, or TGFBR3 gene is a human TGFBR1, TGFBR2, or TGFBR3 gene. In certain embodiments, the TGFBR1, TGFBR2, or TGFBR3 gene is a non- human TGFBR1, TGFBR2, or TGFBR3 gene (e.g., a mouse TGFBR1, TGFBR2, or TGFBR3 gene).

[0106] In certain embodiments, presented herein is a cell provided that comprises an exogenous polynucleotide encoding a dominant-negative cytokine receptor. In certain embodiments, the cell comprises an exogenous polynucleotide encoding a dominant-negative variant of IL- 1 OR (dnIL-lOR), for example a dominant-negative variant of human IL-10R. In certain embodiments, the cell comprises an exogenous polynucleotide encoding a dominantnegative variant of TGFpR (dnTGFpR), for example a dominant-negative variant of human TGFpR. In certain embodiments, the cell comprises an exogenous polynucleotide encoding a dnTGFpRI, a dnTGFpRII, or a dnTGFpRIII, for example a dominant-negative variant of human TGFpRI, TGFpRII, or TGFpRIII. In certain embodiments, the cell comprises an exogenous polynucleotide encoding a dnTGFpRII, for example, a dominant-negative variant of human TGFpRII. An exemplary dnTGFpRII is disclosed in Immunity. 2000 Feb;12(2): 171-81. In certain embodiments, an exogenous polynucleotide encoding a dnTGFpR disclosed herein comprises a nucleotide sequence set forth in SEQ ID NO: 1. In certain embodiments, a dnTGFpR disclosed herein comprises an amino acid sequence set forth in SEQ ID NO: 2.

[0107] In particular embodiments, the cell is a pluripotent cell, e.g., an induced pluripotent stem cell, such as a human induced pluripotent stem cell; a myeloid progenitor cell, e.g, a myeloid progenitor cell derived from an induced pluripotent stem cell, such as a human induced pluripotent stem cell; a monocyte, e.g., a monocyte derived from an induced pluripotent stem cell, such as a human induced pluripotent stem cell; a macrophage, for example an immature macrophage, e.g., a macrophage, for example an immature macrophage, derived from an induced pluripotent stem cell, such as a human induced pluripotent stem cell. In certain embodiments, presented herein is a cell that is a CD1 lb+CD45+cell, for example, a CD1 lb+CD45+CD14’ cell or a CD1 lb+CD45+CD14+cell, e.g., a CD1 lb+CD45+cell, for example, a CD1 lb+CD45+CD14‘ cell or a CDl lb+CD45+CD14+cell, derived from an induced pluripotent stem cell, such as a human induced pluripotent stem cell. In certain embodiments, the cell does not comprise a genomic edit of a MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene.

[0108] In certain embodiments, the modified cell provided herein further comprises an exogenous polynucleotide encoding a dominant-negative cytokine receptor. In certain embodiments, the modified cell provided herein further comprises an exogenous polynucleotide encoding a dominant-negative variant of IL- 1 OR (dnIL-lOR). In certain embodiments, the modified cell provided herein further comprises an exogenouspolynucleotide encoding a dominant-negative variant of TGFpR (dnTGFpR). In certain embodiments, the modified cell provided herein further comprises an exogenous polynucleotide encoding a dnTGFpRI, a dnTGFpRII, or a dnTGFpRIII. In certain embodiments, the modified cell provided herein further comprises an exogenous polynucleotide encoding a dnTGFpRII. An exemplary dnTGFpRII is disclosed in Immunity. 2000 Feb;12(2): 171-81. In certain embodiments, an exogenous polynucleotide encoding a dnTGFpR disclosed herein comprises a nucleotide sequence set forth in SEQ ID NO: 1. In certain embodiments, a dnTGFpR disclosed herein comprises an amino acid sequence set forth in SEQ ID NO: 2.

[0109] In certain embodiments, the modified cell provided herein further comprises an exogenous polynucleotide encoding a proinflammatory cytokine. In certain embodiments, expression of the proinflammatory cytokine in the modified cell promotes Ml macrophage polarization in the modified cells or macrophages derived therefrom. In certain embodiments, the proinflammatory cytokines are selected from the group consisting of IL-1, IL-2, IL-6, IL- 12, IL- 17, IL- 18, IL-23, IFNy, MCP-1, and TNFa. In certain embodiments, the proinflammatory cytokine is IL-12. In certain embodiments, the proinflammatory cytokine is IFNy.

[0110] In certain embodiments, the modified cells provided herein further comprise (a) a chimeric antigen receptor (CAR) (such as a CAR described in Section 5.3), (b) a polynucleotide encoding a CAR (such as a polynucleotide described in Section 5.4) (c) a vector comprising a polynucleotide encoding a CAR (such as a vector described in Section 5.5), or (d) a CAR polypeptide (such as a polypeptide described in Section 5.6). In certain embodiments, the modified cells provided herein comprises (i) genetic disruption of at least one gene of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene; and (ii) a CAR (such as a CAR described in Section 5.3).

[0111] In certain embodiments, the modified cells provided herein are generated according to the methods provided herein, such as the methods of generating a modified cell described in Section 5.2. In certain embodiments, a polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding the proinflammatory cytokine (e.g., IL-12 and / or IFNy), and / or an exogenous polynucleotide encoding the dominant-negative cytokine receptor (e.g., dnIL-lOR, dnTGFpRI, dnTGFpRII, or dnTGFpRIII) is integrated into one or more genes disclosed herein (e.g., an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CGgene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPA gene, a SIGLEC10 gene, a B2M gene, a CIITA gene, an RFX gene, an IL 1 ORA gene, an IL 1 ORB gene, a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene).

[0112] In certain embodiments, a polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding the proinflammatory cytokine (e.g., IL-12 and / or IFNy), and / or an exogenous polynucleotide encoding the dominant-negative cytokine receptor (e.g., dnIL-lOR, dnTGFpRI, dnTGFpRII, or dnTGFpRIII) is integrated into a modified cell using the SLEEK (SeLection by Essentialgene Exon Knock in) technology, which is described in Allen et al., A highly efficient transgene knock-in technology in clinically relevant cell types. Nat Biotechnol (2023) and International Patent Publication WO2022235811, the content of each of which is incorporated by reference in its entirety. In certain embodiments, the polynucleotide encoding the CAR, the exogenous polynucleotide encoding the proinflammatory cytokine, and / or an exogenous polynucleotide encoding the dominant-negative cytokine receptor is integrated in frame with and downstream (3’) of a coding sequence of an essential gene, and wherein at least part of the essential gene comprises an exogenous coding sequence. In certain embodiments, the correct knock-in cells would retain essential gene function while also integrating the polynucleotide encoding the CAR, the exogenous polynucleotide encoding the proinflammatory cytokine, and / or the exogenous polynucleotide encoding the dominant-negative cytokine receptor. Cells with non-productive insertions and deletions would undergo negative selection. In certain embodiments, the essential gene encodes a gene product that is required for survival and / or proliferation of the cell. In certain embodiments, the essential gene is a housekeeping gene. In certain embodiments, the essential gene encodes glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In certain embodiments, the essential gene is an essential gene as disclosed in International Patent Publication WO2022235811.

[0113] In certain embodiments, a polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding the proinflammatory cytokine, and / or an exogenous polynucleotide encoding the dominantnegative cytokine receptor is integrated into a safe harbor locus, e.g., an AAVS1 locus.

[0114] Any methods known in the art for genetic disruption of a gene can be used with the present disclosure. In certain embodiments, genetic disruption of a gene disclosed herein (e.g., an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPAgene, a SIGLEC10 gene, a B2M gene, a CIITA gene, an RFX gene, an IL 1 ORA gene, an IL1ORB gene, a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene) is performed by a genome editing tool, such as TALEN, ZFN, or CRISPR (e.g., by deletion of one or more exons, introduction of a stop codon, introduction of a null mutation or inactivation of the promoter). In certain embodiments, genetic disruption of a gene disclosed herein is performed using gRNA. In certain embodiments, genetic disruption of a gene disclosed herein is performed using gRNA and a Casl2a protein. In certain embodiments, genetic disruption of a gene disclosed herein is performed using gRNA and a MAD7 protein. In certain embodiments, the gRNA comprises a targeting sequence complementary to a target sequence of a gene disclosed herein. In certain embodiments, the target sequence comprises a coding sequence of a gene disclosed herein, for example mRNA sequence. In certain embodiments, the gRNA targets an exon of a gene disclosed herein. In certain embodiments, the target sequence comprises non-coding sequence. Exemplary non-coding sequence includes intronic, promoter, 5’ untranslated region (UTR), 3’ UTR, or enhancer sequence of a gene disclosed herein. In certain embodiments, genetic disruption of a gene disclosed herein is performed by RNAi. In certain embodiments, genetic disruption of a gene disclosed herein is performed by conditional knockout. In certain embodiments, the genetic disruption of a gene disclosed herein is performed in human cells.

[0115] In certain embodiments, the modified cells provided herein are modified mammalian cells. In certain embodiments, the modified cells provided herein are modified human cells. In certain embodiments, the modified human cells are autologous or allogeneic to a subject receiving administration of the modified cells. In certain embodiments, the modified cells are allogeneic, relative to the subject. In certain embodiments, the modified cells are autologous, relative to the subject. In certain embodiments, the modified cells provided herein are modified non-human mammalian cells.5.1.2 Modified Pluripotent Cells

[0116] The present disclosure further provides a modified pluripotent cell comprising genetic disruption of at least one gene (e.g., one, two, three, four, five, six, seven, eight, nine, or all gene(s)) of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene.

[0117] Pluripotent cells can give rise to a multiplicity of cell types, and include, for example, induced pluripotent stem cells (iPSCs). In certain embodiments, a pluripotent stem cell as described herein is a mammalian pluripotent cell, e.g., a mammalian iPSC. In certain embodiments, a pluripotent stem cell as described herein is a human pluripotent cell, e.g., ahuman iPSC. In certain embodiments, a pluripotent cell as described herein is a multipotent cell, e.g., a human multipotent cell. In certain embodiments, a pluripotent cell as described herein is a hematopoietic stem cell (HSC), e.g., a human HSC. In certain embodiments, a pluripotent cell as described herein is an embryonic stem cell (ESC), for example, a human ESC. In certain non-limiting embodiments, a pluripotent cell as described herein is a parthenogenic stem cell, e.g., a human parthenogenetic stem cell, a primordial germ cell-like pluripotent stem cell, e.g., a human primordial germ cell-like pluripotent stem cell, an epiblast stem cell, e.g., a human epiblast stem cell, an F-class pluripotent stem cell, e.g., a human F-class pluripotent stem cell, a somatic stem cell, e.g., a human somatic stem cell, or any other cell, e.g., human cell, capable of lineage specific differentiation. In certain embodiments, a pluripotent cell described herein is a non-human pluripotent cell. In certain embodiments, a pluripotent cell as described herein is a nonhuman primate pluripotent cell. In certain embodiments, a pluripotent cell as described herein is a rodent, e.g., mouse, pluripotent cell.

[0118] In certain embodiments, a pluripotent cell as described herein is not an embryonic stem cell, for example, is not a human embryonic stem cell. In certain embodiments, a pluripotent cell as described herein is not capable of differentiating or developing into all cell types. For example, in certain embodiments, a human pluripotent cell as described herein is not capable of differentiating or developing into all cell types of the human body.

[0119] In certain embodiments, the modified pluripotent cell provided herein is an iPSC. In certain embodiments, the modified pluripotent cell provided herein is a human induced pluripotent stem cell (hiPSC). In certain embodiments, the modified pluripotent cell provided herein has been reprogrammed from a human umbilical cord blood cell (e.g., a human CD34+cord blood cell). In certain embodiments, the modified pluripotent cell provided herein is a modified TC-1133 cell.

[0120] In certain embodiments, the modified pluripotent cell provided herein is a hematopoietic stem cell, e.g., a human hematopoietic stem cell. In certain embodiments, a hematopoietic stem cell is produced from a modified iPSC as described herein.

[0121] In certain embodiments, the iPSC has been reprogrammed from a peripheral blood mononuclear cell (PBMC), a CD34+cord blood cell, an immune cell, a macrophage, a monocyte, or a fibroblast. In certain embodiments, the iPSC has been reprogrammed from a PBMC. In certain embodiments, the iPSC has been reprogrammed from a CD34+cord blood cell. In certain embodiments, the iPSC has been reprogrammed from an immune cell. Incertain embodiments, the iPSC has been reprogrammed from a macrophage. In certain embodiments, the iPSC has been reprogrammed from a monocyte. In certain embodiments, the iPSC has been reprogrammed from a fibroblast.

[0122] In certain embodiments, the presently disclosed modified pluripotent cells can give rise, for example, to modified myeloid progenitor cells, modified monocytes, and / or modified macrophages. In certain embodiments, the presently disclosed modified pluripotent cells can give rise to modified myeloid progenitor cells. In certain embodiments, the presently disclosed modified pluripotent cells can give rise to modified monocytes. In certain embodiments, the presently disclosed modified pluripotent cells can give rise to modified macrophages. In certain embodiments, the presently disclosed modified pluripotent cells can give rise, for example, to modified CD1 lb+CD45+cells. In certain embodiments, the presently disclosed modified pluripotent cells can give rise to modified CD1 lb+CD45+CD14+cells.

[0123] In certain embodiments, the presently disclosed modified pluripotent cells can give rise to a committed progenitor cell. In certain embodiments, the presently disclosed modified pluripotent cells can give rise to modified granulocyte-monocyte progenitor (GMP) cells. In certain embodiments, the presently disclosed modified pluripotent cells can give rise to modified monocyte-dendritic cell progenitors (MDPs). In certain embodiments, the presently disclosed modified pluripotent cells can give rise to modified monocyte progenitor cells. In certain embodiments, any of the modified cells provided herein can be enriched into a homogenous population. For example, a homogeneous population may be produced using and / or may be assessed via markers known in the art (such as those described in Section 5.10.2).

[0124] In certain embodiments, the modified pluripotent cell provided herein is homozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene. In certain embodiments, the modified pluripotent cell provided herein is heterozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene.

[0125] In certain embodiments, the modified pluripotent cell comprises genetic disruption of an MIR146A gene. In certain embodiments, the modified pluripotent cell comprises genetic disruption of a STAT6 gene. In certain embodiments, the modified pluripotent cell comprises genetic disruption of a GPR65 gene. In certain embodiments, themodified pluripotent cell comprises genetic disruption of an AKT1 gene. In certain embodiments, the modified pluripotent cell comprises genetic disruption of an AKT2 gene. In certain embodiments, the modified pluripotent cell comprises genetic disruption of an IRF3 gene. In certain embodiments, the modified pluripotent cell comprises genetic disruption of an IRF4 gene. In certain embodiments, the modified pluripotent cell comprises genetic disruption of a PIK3CG gene. In certain embodiments, the modified pluripotent cell comprises genetic disruption of a TSC1 gene. In certain embodiments, the modified pluripotent cell comprises genetic disruption of a VSIG4 gene.

[0126] In certain embodiments, the modified pluripotent cell comprises genetic disruption of two genes of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, and a VSIG4 gene. In certain embodiments, the modified pluripotent cell comprises genetic disruption of three, four, five, six, seven, eight, nine, or all genes of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, and a VSIG4 gene.

[0127] In certain embodiments, the modified pluripotent cell provided herein further comprises genetic disruption of a SIRPA gene or a SIGLEC10 gene. In certain embodiments, the modified pluripotent cell provided herein further comprises genetic disruption of a SIRPA gene and a SIGLEC10 gene. In certain embodiments, the modified pluripotent cell provided herein is homozygous for the genetic disruption of the SIRPA gene and / or SIGLEC10 gene. In certain embodiments, the modified pluripotent cell provided herein is heterozygous for the genetic disruption of the SIRPA gene and / or SIGLEC10 gene.

[0128] In certain embodiments, presented herein is a pluripotent cell, for example, an induced pluripotent stem cell, such as a human induced pluripotent stem cell, that comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the cytokine receptor is IL-10R. In certain embodiments, the cytokine receptor is a TGFpR. In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII, or TGFpRIII. In certain embodiments, the cytokine receptor is a TGFpRII. In certain embodiments, the pluripotent stem cell does not comprise a genomic edit of a MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene.

[0129] In certain embodiments, the modified pluripotent cell provided herein further comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the modifiedpluripotent cell provided herein comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor, and comprises at least one of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene. In certain embodiments, the cytokine receptor is IL-10R. In certain embodiments, the cytokine receptor is a TGFpR. In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII, or TGFpRIII. In certain embodiments, the cytokine receptor is a TGFpRII.

[0130] In certain embodiments, the modified pluripotent cell provided herein further comprises genetic disruption of a gene encoding IL-10R. In certain embodiments, the modified pluripotent cell provided herein further comprises genetic disruption of an IL 1 ORA gene and / or an IL 1 ORB gene. In certain embodiments, the modified pluripotent cell provided herein further comprises genetic disruption of a gene encoding a TGFpR (e.g., TGFpRI, TGFpRII, or TGFpRIII). In certain embodiments, the modified pluripotent cell provided herein further comprises genetic disruption of a gene encoding a TGFpRII. In certain embodiments, the modified pluripotent cell provided herein further comprises genetic disruption of a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene. In certain embodiments, the modified pluripotent cell provided herein further comprises genetic disruption of a TGFBR2 gene. In certain embodiments, the modified pluripotent cell provided herein is homozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the modified pluripotent cell provided herein is homozygous for the genetic disruption of the IL10RA gene, IL10RB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene. In certain embodiments, the modified pluripotent cell provided herein is heterozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the modified pluripotent cell provided herein is heterozygous for the genetic disruption of the IL 1 ORA gene, IL 1 ORB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene.

[0131] In certain embodiments, presented herein is a pluripotent cell, e.g., an induced pluripotent stem cell, such as a human induced pluripotent stem cell, that comprises an exogenous polynucleotide encoding a dominant-negative cytokine receptor. In certain embodiments, the pluripotent cell comprises an exogenous polynucleotide encoding a dnlL- 10R. In certain embodiments, the pluripotent cell comprises an exogenous polynucleotide encoding a dnTGFpR. In certain embodiments, the pluripotent cell provided herein comprises an exogenous polynucleotide encoding a dnTGFpRI, a dnTGFpRII, or a dnTGFpRIII. In certain embodiments, the pluripotent cell provided herein comprises anexogenous polynucleotide encoding a dnTGFpRII. An exemplary dnTGFpRII is disclosed in Immunity. 2000 Feb;12(2): 171-81. In certain embodiments, an exogenous polynucleotide encoding a dnTGFpR disclosed herein comprises a nucleotide sequence set forth in SEQ ID NO: 1. In certain embodiments, a dnTGFpR disclosed herein comprises an amino acid sequence set forth in SEQ ID NO: 2. In certain embodiments, the pluripotent stem cell does not comprise a genomic edit of a MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene.

[0132] In certain embodiments, the modified pluripotent cell provided herein further comprises an exogenous polynucleotide encoding a dominant-negative cytokine receptor. In certain embodiments, the modified pluripotent cell provided herein further comprises an exogenous polynucleotide encoding a dnIL-lOR. In certain embodiments, the modified pluripotent cell provided herein further comprises an exogenous polynucleotide encoding a dnTGFpR. In certain embodiments, the modified pluripotent cell provided herein further comprises an exogenous polynucleotide encoding a dnTGFpRI, a dnTGFpRII, or a dnTGFpRIII. In certain embodiments, the modified pluripotent cell provided herein further comprises an exogenous polynucleotide encoding a dnTGFpRII. An exemplary dnTGFpRII is disclosed in Immunity. 2000 Feb;12(2): 171-81. In certain embodiments, an exogenous polynucleotide encoding a dnTGFpR disclosed herein comprises a nucleotide sequence set forth in SEQ ID NO: 1. In certain embodiments, a dnTGFpR disclosed herein comprises an amino acid sequence set forth in SEQ ID NO: 2.

[0133] In certain embodiments, the modified pluripotent cell provided herein further comprises an exogenous polynucleotide encoding a proinflammatory cytokine (e.g., IL-12 and / or IFNy). In certain embodiments, the proinflammatory cytokines are selected from the group consisting of IL-1, IL-2, IL-6, IL-12, IL-17, IL-18, IL-23, IFNy, MCP-1, and TNFa. In certain embodiments, the modified pluripotent cell provided herein further comprises an exogenous polynucleotide encoding IL-12. In certain embodiments, the modified pluripotent cell provided herein further comprises an exogenous polynucleotide encoding IFNy. In certain embodiments, the modified pluripotent cell provided herein further comprises one or more exogenous polynucleotides encoding IL-12 and IFNy.

[0134] In certain embodiments, the modified pluripotent cell provided herein further comprises a CAR (such as a CAR described in Section 5.3). In certain embodiments, the modified pluripotent cell provided herein further comprises a polynucleotide encoding a CAR (such as a CAR described in Section 5.3) or components thereof.

[0135] In certain embodiments, the modified pluripotent cells provided herein comprises (i) genetic disruption of at least one gene of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene; and (ii) a CAR (such as a CAR described in Section 5.3). In certain embodiments, the CAR increases secretion of a pro-inflammatory cytokine (e.g., is a CAR described in PCT / US2023 / 071207). In certain embodiments, the intracellular domain of the CAR is selected from the group consisting of a CD40, a TLR, and an IFNyR intracellular domain.

[0136] In certain embodiments, a polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding the proinflammatory cytokine (e.g., IL-12 and / or IFNy), and / or an exogenous polynucleotide encoding the dominant-negative cytokine receptor (e.g., dnIL-lOR, dnTGFpRI, dnTGFpRII, or dnTGFpRIII) is integrated into one or more genes disclosed herein (e.g., an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPA gene, a SIGLEC10 gene, a B2M gene, a CIITA gene, an RFX gene, an IL 1 ORA gene, an IL 1 ORB gene, a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene).

[0137] In certain embodiments, a polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding the proinflammatory cytokine (e.g., IL-12 and / or IFNy), and / or an exogenous polynucleotide encoding the dominant-negative cytokine receptor (e.g., dnIL-lOR, dnTGFpRI, dnTGFpRII, or dnTGFpRIII) is integrated into the modified pluripotent cell using the SLEEK technology. In certain embodiments, the polynucleotide encoding the CAR, the exogenous polynucleotide encoding the proinflammatory cytokine, and / or the exogenous polynucleotide encoding the dominant-negative cytokine receptor is integrated in frame with and downstream (3’) of a coding sequence of an essential gene, and wherein at least part of the essential gene comprises an exogenous coding sequence. In certain embodiments, the correct knock-in cells would retain essential gene function while also integrating the polynucleotide encoding the CAR, the exogenous polynucleotide encoding the proinflammatory cytokine, and / or the exogenous polynucleotide encoding the dominant-negative cytokine receptor. Cells with non-productive insertions and deletions would undergo negative selection. In certain embodiments, the essential gene encodes a gene product that is required for survival and / or proliferation of the cell. In certain embodiments, the essential gene is a housekeeping gene. In certain embodiments, the essential gene encodes GAPDH. In certain embodiments, theessential gene is an essential gene as disclosed in International Patent Publication WO2022235811. In certain embodiments, the CAR, proinflammatory cytokine or dominantnegative cytokine receptor, and the gene product encoded by the essential gene are expressed from the endogenous promoter of the essential gene.

[0138] In certain embodiments, a polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding the proinflammatory cytokine, and / or an exogenous polynucleotide encoding the dominantnegative cytokine receptor is integrated into a safe harbor locus, e.g., an AAVS1 locus.

[0139] In certain embodiments, the modified pluripotent cell further comprises (i) genetic disruption of a B2M gene, (ii) genetic disruption of a CIITA gene, (iii) genetic disruption of an RFX gene, and / or (iv) an exogenous polynucleotide encoding HLA-E. In certain embodiments, the modified pluripotent cell provided herein further comprises genetic disruption of (i) a B2M gene, a CIITA gene and / or an RFX gene, and (ii) an exogenous polynucleotide encoding HLA-E. In certain embodiments, the modified pluripotent cell provided herein further comprises genetic disruption of a B2M gene and a CIITA gene (e.g., B2M / CIITA double knockout), and an exogenous polynucleotide encoding HLA-E. In certain embodiments, the modified pluripotent cell provided herein further comprises genetic disruption of a B2M gene, a CIITA gene, and an RFX gene, and an exogenous polynucleotide encoding HLA-E.

[0140] In certain embodiments, the modified pluripotent cell provided herein is homozygous for the genetic disruption of the B2M gene, CIITA gene, and / or RFX gene. In certain embodiments, the modified pluripotent cell provided herein is heterozygous for the genetic disruption of the B2M gene, CIITA gene, and / or RFX gene.

[0141] In certain embodiments, genetic disruption of a gene disclosed herein (e.g., an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPA gene, a SIGLEC10 gene, a B2M gene, a CIITA gene, an RFX gene, an IL 1 ORA gene, an IL 1 ORB gene, a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene) is performed by a genome editing tool, such as TALEN, ZFN, or CRISPR (e.g., by deletion of one or more exons, introduction of a stop codon, introduction of a null mutation or inactivation of the promoter).5.1.3 Modified Myeloid Progenitor Cells

[0142] The present disclosure further provides a modified myeloid progenitor cell comprising genetic disruption of at least one gene (e.g., one, two, three, four, five, six, seven, eight, nine, or all gene(s)) of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene.

[0143] A myeloid progenitor cell described herein can give rise to a monocyte and / or macrophage, or progenitors thereof. A modified myeloid progenitor cell described herein can give rise to a modified monocyte and / or modified macrophage as described herein, or progenitors thereof. In certain embodiments, the modified myeloid progenitor cell is a human myeloid progenitor cell. In certain embodiments, the modified myeloid progenitor cell is a mammalian myeloid progenitor cell. In certain embodiments, the modified myeloid progenitor cell is a primate myeloid progenitor cell. In certain embodiments, the modified myeloid progenitor cell is a non-human primate myeloid progenitor cell. In certain embodiments, the modified myeloid progenitor cell is a rodent, e.g., mouse, myeloid progenitor cell.

[0144] In certain embodiments, a modified myeloid progenitor cell, as presented herein, expresses a detectable level of a myeloid progenitor cell marker, such as for example CD34, and CD38, and does not express a detectable level of Lin and / or CD45RA (e.g., Lin' CD34+CD38+CD45RA' cells), or any other myeloid progenitor cell marker(s) known to one of skill in the art or described herein (such as in Section 5.10.2).

[0145] In certain embodiments, the modified myeloid progenitor cell is derived from a modified pluripotent cell (such as a modified pluripotent cell described in Section 5.1.2). For example, in certain embodiments, the modified pluripotent cell is generated according to the methods provided herein, such as the methods described in Section 5.2.1, and the modified pluripotent cell is differentiated into a myeloid progenitor cell. Techniques known to one of skill in the art or described herein (such as in Section 5.9) may be used to differentiate a pluripotent cell into a myeloid progenitor cell. For example, a homogeneous population of modified myeloid progenitor cells or a cell population comprising a modified myeloid progenitor cell disclosed herein (e.g., at least about 80%, 85%, 90%, 95% or more of the cells in the cell population are the modified myeloid progenitor cells) may be produced, selected for and / or may be assessed via using markers known in the art (such as those described in Section 5.10.2).

[0146] In certain embodiments, the modified myeloid progenitor cell provided herein is derived from an induced pluripotent stem cell (iPSC). In certain embodiments, the iPSC has been reprogrammed from a peripheral blood mononuclear cell (PBMC), a CD34+cord blood cell, an immune cell, a macrophage, a monocyte, or a fibroblast. In certain embodiments, the iPSC has been reprogrammed from a PBMC. In certain embodiments, theiPSC has been reprogrammed from a CD34+cord blood cell. In certain embodiments, the iPSC has been reprogrammed from an immune cell. In certain embodiments, the iPSC has been reprogrammed from a macrophage. In certain embodiments, the iPSC has been reprogrammed from a monocyte. In certain embodiments, the iPSC has been reprogrammed from a fibroblast.

[0147] The present disclosure further provides a homogeneous population of the modified myeloid progenitor cells disclosed herein e.g., modified myeloid progenitor cells described in Section 5.1.3). In certain embodiments, the homogeneous population of modified myeloid progenitor cells are isogenic.

[0148] The present disclosure further provides a population of cells comprising a modified myeloid progenitor cell disclosed herein (e.g., a modified myeloid progenitor cell described in Section 5.1.3), wherein at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.8%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 90% to about 100%, from about 95% to about 98%, from about 95% to about 99%, or from about 95% to 100% of the cells in the population are the modified myeloid progenitor cells.

[0149] In certain embodiments, the modified myeloid progenitor cell disclosed herein is homozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene. In certain embodiments, the modified myeloid progenitor cell provided herein is heterozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene.

[0150] In certain embodiments, the modified myeloid progenitor cell comprises genetic disruption of an MIR146A gene. In certain embodiments, the modified myeloid progenitor cell comprises genetic disruption of a STAT6 gene. In certain embodiments, the modified myeloid progenitor cell comprises genetic disruption of a GPR65 gene. In certain embodiments, the modified myeloid progenitor cell comprises genetic disruption of an AKT1 gene. In certain embodiments, the modified myeloid progenitor cell comprises genetic disruption of an AKT2 gene. In certain embodiments, the modified myeloid progenitor cell comprises genetic disruption of an IRF3 gene. In certain embodiments, the modified myeloid progenitor cell comprises genetic disruption of an IRF4 gene. In certain embodiments, the modified myeloid progenitor cell comprises genetic disruption of a PIK3CG gene. In certainembodiments, the modified myeloid progenitor cell comprises genetic disruption of a TSC1 gene. In certain embodiments, the modified myeloid progenitor cell comprises genetic disruption of a VSIG4 gene.

[0151] In certain embodiments, the modified myeloid progenitor cell comprises genetic disruption of two genes of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, and a VSIG4 gene. In certain embodiments, the modified myeloid progenitor cell comprises genetic disruption of three, four, five, six, seven, eight, nine, or all genes of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, and a VSIG4 gene.

[0152] In certain embodiments, the modified myeloid progenitor cell provided herein further comprises genetic disruption of a SIRPA gene or a SIGLEC10 gene. In certain embodiments, the modified myeloid progenitor cell provided herein further comprises genetic disruption of a SIRPA gene and a SIGLEC10 gene. In certain embodiments, the modified myeloid progenitor cell provided herein is homozygous for the genetic disruption of the SIRPA gene and / or SIGLEC10 gene. In certain embodiments, the modified myeloid progenitor cell provided herein is heterozygous for the genetic disruption of the SIRPA gene and / or SIGLEC10 gene.

[0153] In certain embodiments, presented herein is a myeloid progenitor cell, e.g., human myeloid progenitor cell, that comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the cytokine receptor is IL-10R. In certain embodiments, the cytokine receptor is a TGFpR. In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII or TGFpRIII. In certain embodiments, the cytokine receptor is a TGFpRII. In certain embodiments, the myeloid progenitor cell comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor, and does not comprise genetic disruption of a MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene.

[0154] In certain embodiments, the myeloid progenitor cell provided herein comprises genetic disruption of a gene encoding IL-10R. In certain embodiments, the myeloid progenitor cell provided herein comprises genetic disruption of an IL10RA gene and / or an IL10RB gene. In certain embodiments, the myeloid progenitor cell provided herein comprises genetic disruption of a gene encoding a TGFpR (e.g., TGFpRI, TGFpRII, orTGFpRIII). In certain embodiments, the myeloid progenitor cell provided herein comprises genetic disruption of a gene encoding TGFpRII. In certain embodiments, the myeloid progenitor cell provided herein comprises genetic disruption of a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene. In certain embodiments, the myeloid progenitor cell provided herein comprises genetic disruption of a TGFBR2 gene. In certain embodiments, the myeloid progenitor cell provided herein is homozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the myeloid progenitor cell provided herein is homozygous for the genetic disruption of the IL 1 ORA gene, IL10RB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene. In certain embodiments, the myeloid progenitor cell provided herein is heterozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, myeloid progenitor cell provided herein is heterozygous for the genetic disruption of the IL 1 ORA gene, IL 1 ORB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene.

[0155] In certain embodiments, the myeloid progenitor cell provided herein comprises an exogenous polynucleotide encoding a dominant-negative cytokine receptor. In certain embodiments, the myeloid progenitor cell provided herein comprises an exogenous polynucleotide encoding a dnIL-lOR. In certain embodiments, the myeloid progenitor cell provided herein comprises an exogenous polynucleotide encoding a dnTGFpR. In certain embodiments, the myeloid progenitor cell provided herein comprises an exogenous polynucleotide encoding a dnTGFpRI, a dnTGFpRII, or a dnTGFpRIII. In certain embodiments, the myeloid progenitor cell provided herein comprises an exogenous polynucleotide encoding a dnTGFpRII. An exemplary dnTGFpRII is disclosed in Immunity. 2000 Feb;12(2): 171-81. In certain embodiments, an exogenous polynucleotide encoding a dnTGFpR disclosed herein comprises a nucleotide sequence set forth in SEQ ID NO: 1. In certain embodiments, a dnTGFpR disclosed herein comprises an amino acid sequence set forth in SEQ ID NO: 2.

[0156] In particular embodiments, the myeloid progenitor cell is derived from an induced pluripotent stem cell, such as a human induced pluripotent stem cell.

[0157] In certain embodiments, the modified myeloid progenitor cell provided herein further comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the modified myeloid progenitor cell provided herein comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor, andcomprises at least one of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene. In certain embodiments, the cytokine receptor is a TGFpR. In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII or TGFpRIII. In certain embodiments, the cytokine receptor is a TGFpRII.

[0158] In certain embodiments, the modified myeloid progenitor cell provided herein further comprises genetic disruption of a gene encoding IL-10R. In certain embodiments, the modified myeloid progenitor cell provided herein further comprises genetic disruption of an IL10RA gene and / or an IL10RB gene. In certain embodiments, the modified myeloid progenitor cell provided herein further comprises genetic disruption of a gene encoding a TGFpR (e.g., TGFpRI, TGFpRII, or TGFpRIII). In certain embodiments, the modified myeloid progenitor cell provided herein further comprises genetic disruption of a gene encoding TGFpRII. In certain embodiments, the modified myeloid progenitor cell provided herein further comprises genetic disruption of a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene. In certain embodiments, the modified myeloid progenitor cell provided herein further comprises genetic disruption of a TGFBR2 gene. In certain embodiments, the modified myeloid progenitor cell provided herein is homozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the modified myeloid progenitor cell provided herein is homozygous for the genetic disruption of the IL10RA gene, IL10RB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene. In certain embodiments, the modified myeloid progenitor cell provided herein is heterozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the modified myeloid progenitor cell provided herein is heterozygous for the genetic disruption of the IL 1 ORA gene, IL 1 ORB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene.

[0159] In certain embodiments, the modified myeloid progenitor cell provided herein further comprises an exogenous polynucleotide encoding a dominant-negative cytokine receptor. In certain embodiments, the modified myeloid progenitor cell provided herein further comprises an exogenous polynucleotide encoding a dnIL-lOR. In certain embodiments, the modified myeloid progenitor cell provided herein further comprises an exogenous polynucleotide encoding a dnTGFpR. In certain embodiments, the modified myeloid progenitor cell provided herein further comprises an exogenous polynucleotide encoding a dnTGFpRI, a dnTGFpRII, or a dnTGFpRIII. In certain embodiments, the modified myeloid progenitor cell provided herein further comprises an exogenouspolynucleotide encoding a dnTGFpRII. An exemplary dnTGFpRII is disclosed in Immunity. 2000 Feb;12(2): 171-81. In certain embodiments, an exogenous polynucleotide encoding a dnTGFpR disclosed herein comprises a nucleotide sequence set forth in SEQ ID NO: 1. In certain embodiments, a dnTGFpR disclosed herein comprises an amino acid sequence set forth in SEQ ID NO: 2.

[0160] In certain embodiments, the modified myeloid progenitor cell provided herein further comprises an exogenous polynucleotide encoding a proinflammatory cytokine (e.g., IL-12 and / or IFNy). In certain embodiments, the proinflammatory cytokines are selected from the group consisting of IL-1, IL-2, IL-6, IL-12, IL-17, IL-18, IL-23, IFNy, MCP-1, and TNFa. In certain embodiments, the modified myeloid progenitor cell provided herein further comprises an exogenous polynucleotide encoding IL-12. In certain embodiments, the modified myeloid progenitor cell provided herein further comprises an exogenous polynucleotide encoding IFNy. In certain embodiments, the modified myeloid progenitor cell provided herein further comprises one or more exogenous polynucleotides encoding IL-12 and IFNy.

[0161] In certain embodiments, the modified myeloid progenitor cell provided herein further comprises a CAR (such as a CAR described in Section 5.3). In certain embodiments, the modified myeloid progenitor cell provided herein further comprises a polynucleotide encoding a CAR (such as a CAR described in Section 5.3) or a polypeptide of a CAR (such as a CAR described in Section 5.3).

[0162] In certain embodiments, the modified myeloid progenitor cells provided herein comprises (i) genetic disruption of at least one gene of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene; and (ii) a CAR (such as a CAR described in Section 5.3). In certain embodiments, the CAR promotes Ml macrophage polarization. In certain embodiments, the CAR increases secretion of a pro-inflammatory cytokine (e.g., is a CAR described in PCT / US2023 / 071207). In certain embodiments, the intracellular domain of the CAR is selected from the group consisting of a CD40, a TLR, and an IFNyR intracellular domain.

[0163] In certain embodiments, a polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding the proinflammatory cytokine (e.g., IL-12 and / or IFNy), and / or an exogenous polynucleotide encoding the dominant-negative cytokine receptor (e.g., dnIL-lOR, dnTGFpRI, dnTGFpRII, or dnTGFpRIII) is integrated into one or more genes disclosed herein (e.g., an AKT1 gene,an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPA gene, a SIGLEC10 gene, a B2M gene, a CIITA gene, an RFX gene, an IL 1 ORA gene, an IL 1 ORB gene, a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene).

[0164] In certain embodiments, a polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding the proinflammatory cytokine (e.g., IL-12 and / or IFNy), and / or an exogenous polynucleotide encoding the dominant-negative cytokine receptor (e.g., dnIL-lOR, dnTGFpRI, dnTGFpRII, or dnTGFpRIII) is integrated into the modified myeloid progenitor cell using the SLEEK technology. In certain embodiments, the polynucleotide encoding the CAR, the exogenous polynucleotide encoding the proinflammatory cytokine, and / or the exogenous polynucleotide encoding the dominant-negative cytokine receptor is integrated in frame with and downstream (3’) of a coding sequence of an essential gene, and wherein at least part of the essential gene comprises an exogenous coding sequence. In certain embodiments, the correct knock-in cells would retain essential gene function while also integrating the polynucleotide encoding the CAR, the exogenous polynucleotide encoding the proinflammatory cytokine, and / or the exogenous polynucleotide encoding the dominant-negative cytokine receptor. Cells with non-productive insertions and deletions would undergo negative selection. In certain embodiments, the essential gene encodes a gene product that is required for survival and / or proliferation of the cell. In certain embodiments, the essential gene is a housekeeping gene. In certain embodiments, the essential gene encodes GAPDH. In certain embodiments, the essential gene is an essential gene as disclosed in International Patent Publication WO2022235811.

[0165] In certain embodiments, a polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding the proinflammatory cytokine, and / or an exogenous polynucleotide encoding the dominantnegative cytokine receptor is integrated into a safe harbor locus, e.g., an AAVS1 locus.

[0166] In certain embodiments, the modified myeloid progenitor cell further comprises (i) genetic disruption of a B2M gene, (ii) genetic disruption of a CIITA gene, (iii) genetic disruption of an RFX gene, and / or (iv) an exogenous polynucleotide encoding HLA- E. In certain embodiments, the modified myeloid progenitor cell provided herein further comprises genetic disruption of (i) a B2M gene, a CIITA gene and / or an RFX gene, and (ii) an exogenous polynucleotide encoding HLA-E. In certain embodiments, the modified myeloid progenitor cell provided herein further comprises genetic disruption of a B2M geneand a CIITA gene (e.g., B2M / CIITA double knockout), and an exogenous polynucleotide encoding HLA-E. In certain embodiments, the modified myeloid progenitor cell provided herein further comprises genetic disruption of a B2M gene, a CIITA gene, and an RFX gene, and an exogenous polynucleotide encoding HLA-E.

[0167] In certain embodiments, the modified myeloid progenitor cell provided herein is homozygous for the genetic disruption of the B2M gene, CIITA gene, and / or RFX gene. In certain embodiments, the modified myeloid progenitor cell provided herein is heterozygous for the genetic disruption of the B2M gene, CIITA gene, and / or RFX gene.

[0168] In certain embodiments, genetic disruption of a gene disclosed herein (e.g., an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPA gene, a SIGLEC10 gene, a B2M gene, a CIITA gene, an RFX gene, an IL 1 ORA gene, an IL 1 ORB gene, a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene) is performed by a genome editing tool, such as TALEN, ZFN, or CRISPR (e.g., by deletion of one or more exons, introduction of a stop codon, introduction of a null mutation or inactivation of the promoter).5.1.4 Modified Monocytes

[0169] The present disclosure further provides a modified monocyte comprising genetic disruption of at least one gene (e.g., one, two, three, four, five, six, seven, eight, nine, or all gene(s)) of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene.

[0170] In certain embodiments, the modified monocyte is a human monocyte. In certain embodiments, the modified monocyte is a mammalian monocyte. In certain embodiments, the modified monocyte is a primate monocyte. In certain embodiments, the modified monocyte is a non-human primate monocyte. In certain embodiments, the modified monocyte is a rodent, e.g., mouse, monocyte.

[0171] In certain embodiments, the modified monocyte disclosed herein, once exposed to one or more polarization stimuli, has suppressed M2 polarization and / or promoted Ml polarization relative to a non-modified monocyte exposed to the polarization stimulus. In certain embodiments, the one or more polarization stimuli comprise one or more Ml polarization stimuli. In certain embodiments, the one or more Ml polarization stimuli comprise IFNy, LPS, and / or IL-lb. In certain embodiments, the one or more Ml polarization stimuli comprise IFNy and LPS. In certain embodiments, the one or more Ml polarization stimuli comprise LPS and IL-lb. In certain embodiments, the one or more polarization stimuli comprise one or more M2 polarization stimuli. In certain embodiments, the one ormore M2 polarization stimuli comprise IL-10, IL-4, IL-13, TGFP, adenosine, and / or IL-6. In certain embodiments, the one or more M2 polarization stimuli comprise IL-4 and IL-13. In certain embodiments, the one or more M2 polarization stimuli comprise IL- 10 and TGFp. In certain embodiments, the one or more M2 polarization stimuli comprise adenosine and IL-6. In certain embodiments, the one or more M2 polarization stimuli comprise IL- 10.

[0172] In certain embodiments, the modified monocyte that is exposed to the one or more polarization stimuli has increased expression levels (e.g., protein or transcript expression levels) of one or more Ml markers as compared to the expression levels of the one or more Ml markers in an unmodified monocyte (e.g., a wildtype monocyte) that is exposed to the same polarization stimuli. Exemplary Ml markers include CD38, CD40, CD54, CD80, CD86, CD283 (TLR2), and CXCR3. Additional exemplary Ml markers are disclosed in Table 1. In certain embodiments, the expression levels of the one or more Ml markers in the modified monocyte that is exposed to the one or more polarization stimuli are increased to no less than about 150%, no less than about 200%, no less than about 250%, no less than about 300%, no less than about 350%, no less than about 400%, no less than about 500%, no less than about 600%, no less than about 700%, no less than about 800%, no less than about 900%, no less than about 1000%, no less than 1500%, no less than 2000%, or no less than 2500% as compared to the expression levels of the one or more Ml markers in an unmodified monocyte that is exposed to the same polarization stimuli. In certain embodiments, the expression levels of the one or more Ml markers in the modified monocyte that is exposed to the one or more polarization stimuli are increased to between about 150% and about 2500%, between about 200% and about 2000%, between about 500% and about 1000%, between about 200% and about 500%, or between about 150% and about 300% as compared to the expression levels of the one or more Ml markers in an unmodified monocyte that is exposed to the same polarization stimuli. In certain embodiments, the expression levels of the one or more Ml markers in the modified monocyte that is exposed to the one or more polarization stimuli are increased to about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, about 1500%, about 2000%, about 2500% or more as compared to the expression levels of the one or more Ml markers in an unmodified monocyte that is exposed to the same polarization stimuli.

[0173] In certain embodiments, the modified monocyte exposed to the one or more polarization stimuli has reduced expression levels (e.g., protein or transcript expression levels) of one or more M2 markers as compared to the expression levels of the one or moreM2 markers in an unmodified monocyte (e.g., a wildtype monocyte) that is exposed to the same polarization stimuli. Exemplary M2 markers include CD206, CD 163, CD 169, CD36, CD304, and MRC1. Additional exemplary M2 markers are disclosed in Table 2. In certain embodiments, the expression levels of the one or more M2 markers in the modified monocyte that is exposed to the one or more polarization stimuli are reduced to no more than about 5%, no more than about 10%, no more than about 15%, no more than about 20%, no more than about 25%, no more than about 30%, no more than about 40%, no more than about 50%, no more than about 60%, no more than about 70%, no more than about 80%, no more than about 90%, or no more than about 95% as compared to the expression levels of the one or more M2 markers in an unmodified monocyte that is exposed to the same polarization stimuli. In certain embodiments, the expression levels of the one or more M2 markers in the modified monocyte that is exposed to the one or more polarization stimuli are reduced to between about 5% and about 95%, between about 10% and about 90%, between about 20% and about 80%, between about 50% and about 90%, between about 60% and about 80%, between about 30% and about 40%, or between about 20% and about 50% as compared to the expression levels of the one or more M2 markers in an unmodified monocyte that is exposed to the same polarization stimuli. In certain embodiments, the expression levels of the one or more M2 markers in the modified monocyte that is exposed to the one or more polarization stimuli are reduced to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% as compared to the expression levels of the one or more M2 markers in an unmodified monocyte that is exposed to the same polarization stimuli.

[0174] In certain embodiments, the expression levels of the one or more Ml markers are not increased and the expression levels of the one or more M2 markers are decreased. In some embodiments, the expression levels of the one or more M2 markers are decreased and expression levels of the one or more Ml markers are unchanged.

[0175] In certain embodiments, the modified monocyte that is exposed to the one or more polarization stimuli has increased secretion levels of one or more Ml cytokines as compared to the secretion levels of the one or more Ml cytokines in an unmodified monocyte e.g., a wildtype monocyte) that is exposed to the same polarization stimuli. Exemplary Ml cytokines include CXCL9, CXCL10, CXCL11, CCL3, CCL4, IL-lb, IL-2, IL-6, IL-8, IL-12, IL-23, and TNFa. In certain embodiments, the secretion levels of the one or more Ml cytokines in the modified monocyte that is exposed to the one or more polarization stimuli are increased to no less than about 150%, no less than about 200%, no less than about 250%,no less than about 300%, no less than about 350%, no less than about 400%, no less than about 500%, no less than about 600%, no less than about 700%, no less than about 800%, no less than about 900%, no less than about 1000%, no less than 1500%, no less than 2000%, or no less than 2500% as compared to the secretion levels of the one or more Ml cytokines in an unmodified monocyte that is exposed to the same polarization stimuli. In certain embodiments, the secretion levels of the one or more Ml cytokines in the modified monocyte that is exposed to the one or more polarization stimuli are increased to between about 150% and about 2500%, between about 200% and about 2000%, between about 500% and about 1000%, between about 200% and about 500%, or between about 150% and about 300% as compared to the secretion levels of the one or more Ml cytokines in an unmodified monocyte that is exposed to the same polarization stimuli. In certain embodiments, the secretion levels of the one or more Ml cytokines in the modified monocyte that is exposed to the one or more polarization stimuli are increased to about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, about 1500%, about 2000%, about 2500% or more as compared to the secretion levels of the one or more Ml cytokines in an unmodified monocyte that is exposed to the same polarization stimuli.

[0176] In certain embodiments, the modified monocyte exposed to the one or more polarization stimuli has reduced secretion levels (e.g., protein or transcript secretion levels) of one or more M2 cytokines as compared to the secretion levels of the one or more M2 cytokines in an unmodified monocyte e.g., a wildtype monocyte) that is exposed to the same polarization stimuli. Exemplary M2 cytokines include IL-10, MCP-1, TGFP, CCL13, CCL17, CCL18 and CCL22. In certain embodiments, the secretion levels of the one or more M2 cytokines in the modified monocyte that is exposed to the one or more polarization stimuli are reduced to no more than about 5%, no more than about 10%, no more than about 15%, no more than about 20%, no more than about 25%, no more than about 30%, no more than about 40%, no more than about 50%, no more than about 60%, no more than about 70%, no more than about 80%, no more than about 90%, or no more than about 95% as compared to the secretion levels of the one or more M2 cytokines in an unmodified monocyte that is exposed to the same polarization stimuli. In certain embodiments, the secretion levels of the one or more M2 cytokines in the modified monocyte that is exposed to the one or more polarization stimuli are reduced to between about 5% and about 95%, between about 10% and about 90%, between about 20% and about 80%, between about 50% and about 90%, between about 60% and about 80%, between about 30% and about 40%, or between about20% and about 50% as compared to the secretion levels of the one or more M2 cytokines in an unmodified monocyte that is exposed to the same polarization stimuli. In certain embodiments, the secretion levels of the one or more M2 cytokines in the modified monocyte that is exposed to the one or more polarization stimuli are reduced to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% as compared to the secretion levels of the one or more M2 cytokines in an unmodified monocyte that is exposed to the same polarization stimuli.

[0177] In certain embodiments, the secretion levels of the one or more Ml cytokines are not increased and the secretion levels of the one or more M2 cytokines are decreased. In some embodiments, the secretion levels of the one or more M2 cytokines are decreased and secretion levels of the one or more Ml cytokines are unchanged.

[0178] As used herein, cytokines include chemokines, which are a family of low molecular weight chemotactic cytokines that regulate leukocyte migration. Additional Ml and M2 cytokines are disclosed in Kadomoto et al., Macrophage Polarity and Disease Control, Int J Mol Sci. 2021 Dec 23;23(1): 144, the content of which is incorporated herein by reference.

[0179] In certain embodiments, the modified monocyte resists M2-induced reduction in metabolic activities relative to a non-modified monocyte exposed to the polarization stimulus. In certain embodiments, the modified monocyte exposed to the M2 polarization stimulus does not have reduced oxygen consumption rate (OCR) or extracellular acidification rate (ECAR) as compared to the modified monocyte exposed to the Ml polarization stimulus.

[0180] In certain embodiments, a modified monocyte exhibits a monocyte phenotype such as a monocyte phenotype as described in Section 5.10.2. For example, in certain embodiments, a modified monocyte disclosed herein is a non-adherent cell. In certain embodiments, a modified monocyte disclosed herein has a monocyte cell morphology, e.g., the size of the cell is smaller than macrophages, but larger than T-cells, B-cells or natural killer (NK) cells. In certain embodiments, a modified monocyte has a cell morphology that is less granular than a macrophage, e.g., a mature macrophage. In certain embodiments, a modified monocyte disclosed herein is a non-adherent cell that has a cell morphology that is less granular than a macrophage, e.g., a mature macrophage and has a cell size that is smaller than macrophages, but larger than T-cells, B-cells or NK cells.

[0181] In certain embodiments, a modified monocyte presented herein expresses a detectable level of CD1 lb+and CD45+(e.g., is a CD1 lb+CD45+cell). In certainembodiments, a modified monocyte disclosed herein expresses a detectable level of CD14, e.g., is CD1 lb+CD45+CD14+. In certain embodiments, a modified monocyte presented herein does not express a detectable level of CD14 (e.g., is CD1 lb+CD45+CD14‘). In certain embodiments, a modified monocyte presented herein expresses a high level of CD 14 and does not express a detectable level of CD 16 (CD14hlghCD 16"). In certain embodiments, a modified monocyte presented herein expresses a high level of CD 14 and a detectable level of CD16 (CD14hlghCD16+). In certain embodiments, a modified monocyte presented herein express a low level of CD 14 and a high detectable level of CD 16 (CD14lowCD16hlgh) expression.

[0182] In certain embodiments, a modified monocyte disclosed herein has the properties of killing target cells (e.g., via CAR or monoclonal antibody (mAb) targeting), phagocytosing particles (e.g., bacteria), and / or migrating towards chemokines. In certain embodiments, a modified monocyte disclosed herein exhibits increased killing of target cells (e.g., via CAR or monoclonal antibody (mAb) targeting), phagocytosing particles (e.g., bacteria), and / or migrating towards chemokines relative to a non-modified monocyte. Any assays known in the art can be used for measuring these properties, for example, the assays disclosed in Section 5.10.

[0183] In certain embodiments, the modified monocyte is derived from a modified pluripotent cell (such as a modified pluripotent cell described in Section 5.1.2). For example, in certain embodiments, the modified pluripotent cell is generated according to the methods provided herein, such as the methods described in Section 5.2.1, and the modified pluripotent cell is differentiated into a monocyte. In certain embodiments, the modified monocyte provided herein is derived from a modified myeloid progenitor cell (such as a modified myeloid progenitor cell described in Section 5.1.3). For example, in certain embodiments, the modified myeloid progenitor cell is generated according to the methods provided herein (such as the methods described in Section 5.2.2), and the modified myeloid progenitor cell is differentiated into a monocyte. Techniques known to one of skill in the art or described herein (such as in Section 5.9) may be used to differentiate a pluripotent cell into a monocyte, and / or a myeloid progenitor cell into a monocyte. For example, a homogeneous population of modified monocytes or a cell population comprising a modified monocyte (e.g., at least about 95% of the cells in the cell population are the modified monocytes) disclosed herein may be produced using and / or may be assessed via markers known in the art (such as those described in Section 5.10.2).

[0184] In certain embodiments, the modified monocyte provided herein is derived from an induced pluripotent stem cell (iPSC). In certain embodiments, the iPSC has been reprogrammed from a peripheral blood mononuclear cell (PBMC), a CD34+cord blood cell, an immune cell, a macrophage, a monocyte, or a fibroblast. In certain embodiments, the iPSC has been reprogrammed from a PBMC. In certain embodiments, the iPSC has been reprogrammed from a CD34+cord blood cell. In certain embodiments, the iPSC has been reprogrammed from an immune cell. In certain embodiments, the iPSC has been reprogrammed from a macrophage. In certain embodiments, the iPSC has been reprogrammed from a monocyte. In certain embodiments, the iPSC has been reprogrammed from a fibroblast. In certain embodiments, the iPSC is a hiPSC. In certain embodiments, the iPSC is a TC-1133 cell.

[0185] The present disclosure further provides a homogeneous population of the modified monocytes disclosed herein (e.g., a modified monocyte described in Section 5.1.4). In certain embodiments, the homogeneous population of modified monocytes are isogenic.

[0186] The present disclosure further provides a population of cells comprising a modified monocyte disclosed herein (e.g., a modified monocyte described in Section 5.1.4), wherein at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.8% , from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 90% to about 100%, from about 95% to about 98%, from about 95% to about 99%, or from about 95% to 100% of the cells in the population are the modified monocytes.

[0187] In certain embodiments, the modified monocyte disclosed herein is homozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene. In certain embodiments, the modified monocyte provided herein is heterozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene.

[0188] In certain embodiments, the modified monocyte comprises genetic disruption of an MIR146A gene. In certain embodiments, the modified monocyte comprises genetic disruption of a STAT6 gene. In certain embodiments, the modified monocyte comprises genetic disruption of a GPR65 gene. In certain embodiments, the modified monocyte comprises genetic disruption of an AKT1 gene. In certain embodiments, the modifiedmonocyte comprises genetic disruption of an AKT2 gene. In certain embodiments, the modified monocyte comprises genetic disruption of an IRF3 gene. In certain embodiments, the modified monocyte comprises genetic disruption of an IRF4 gene. In certain embodiments, the modified monocyte comprises genetic disruption of a PIK3CG gene. In certain embodiments, the modified monocyte comprises genetic disruption of a TSC1 gene. In certain embodiments, the modified monocyte comprises genetic disruption of a VSIG4 gene.

[0189] In certain embodiments, the modified monocyte comprises genetic disruption of two genes of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, and a VSIG4 gene. In certain embodiments, the modified monocyte comprises genetic disruption of three, four, five, six, seven, eight, nine, or all genes of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, and a VSIG4 gene.

[0190] In certain embodiments, the modified monocyte provided herein further comprises genetic disruption of a SIRPA gene or a SIGLEC10 gene. In certain embodiments, the modified monocyte provided herein further comprises genetic disruption of a SIRPA gene and a SIGLEC10 gene. In certain embodiments, the modified monocyte provided herein is homozygous for the genetic disruption of the SIRPA gene and / or SIGLEC10 gene. In certain embodiments, the modified monocyte provided herein is heterozygous for the genetic disruption of the SIRPA gene and / or SIGLEC10 gene.

[0191] In certain embodiments, provided herein is a monocyte, e.g., a human monocyte, that comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the monocyte comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor, and does not comprise genetic disruption of a MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene. In certain embodiments, the cytokine receptor is IL-10R. In certain embodiments, the cytokine receptor is a TGFpR. In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII, or TGFpRIII. In certain embodiments, the cytokine receptor is a TGFpRII.

[0192] In certain embodiments, the monocyte comprises genetic disruption of a gene encoding IL-10R. In certain embodiments, the monocyte comprises genetic disruption of an IL10RA gene and / or an IL10RB gene. In certain embodiments, the monocyte comprisesgenetic disruption of a gene encoding a TGFpR (e.g., TGFpRI, TGFpRII, or TGFpRIII). In certain embodiments, the monocyte comprises genetic disruption of a gene encoding TGFpRII. In certain embodiments, the monocyte comprises genetic disruption of a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene. In certain embodiments, the monocyte comprises genetic disruption of a TGFBR2 gene. In certain embodiments, the monocyte is homozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the monocyte is homozygous for the genetic disruption of the IL10RA gene, IL10RB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene. In certain embodiments, the monocyte is heterozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the monocyte is heterozygous for the genetic disruption of the IL10RA gene, IL10RB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene.

[0193] In certain embodiments, the monocyte comprises an exogenous polynucleotide encoding a dominant-negative cytokine receptor. In certain embodiments, the monocyte comprises an exogenous polynucleotide encoding a dnIL-lOR. In certain embodiments, the monocyte comprises an exogenous polynucleotide encoding a dnTGFpR. In certain embodiments, the monocyte comprises an exogenous polynucleotide encoding a dnTGFpRI, a dnTGFpRII, or a dnTGFpRIII. In certain embodiments, the monocyte provided herein comprises an exogenous polynucleotide encoding a dnTGFpRII. An exemplary dnTGFpRII is disclosed in Immunity. 2000 Feb;12(2): 171-81. In certain embodiments, an exogenous polynucleotide encoding a dnTGFpR disclosed herein comprises a nucleotide sequence set forth in SEQ ID NO: 1. In certain embodiments, a dnTGFpR disclosed herein comprises an amino acid sequence set forth in SEQ ID NO: 2.

[0194] In particular embodiments, the monocyte is derived from an induced pluripotent stem cell, such as a human induced pluripotent stem cell. In particular embodiments, the monocyte is derived from a myeloid cell progenitor, such as a human myeloid progenitor cell.

[0195] In certain embodiments, the modified monocyte provided herein further comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the modified monocyte provided herein comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor, and comprises at least one of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene. Incertain embodiments, the cytokine receptor is IL-10R. In certain embodiments, the cytokine receptor is a TGFpR. In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII, or TGFpRIII. In certain embodiments, the cytokine receptor is a TGFpRII.

[0196] In certain embodiments, the modified monocyte provided herein further comprises genetic disruption of a gene encoding IL-10R. In certain embodiments, the modified monocyte provided herein further comprises genetic disruption of an IL10RA gene and / or an IL10RB gene. In certain embodiments, the modified monocyte provided herein further comprises genetic disruption of a gene encoding a TGFpR (e.g., TGFpRI, TGFpRII, or TGFpRIII). In certain embodiments, the modified monocyte provided herein further comprises genetic disruption of a gene encoding TGFpRII. In certain embodiments, the modified monocyte provided herein further comprises genetic disruption of a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene. In certain embodiments, the modified monocyte provided herein further comprises genetic disruption of a TGFBR2 gene. In certain embodiments, the modified monocyte provided herein is homozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the modified monocyte provided herein is homozygous for the genetic disruption of the IL 1 ORA gene, IL 1 ORB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene. In certain embodiments, the modified monocyte provided herein is heterozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the modified monocyte provided herein is heterozygous for the genetic disruption of the IL 1 ORA gene, IL 1 ORB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene.

[0197] In certain embodiments, the modified monocyte provided herein further comprises an exogenous polynucleotide encoding a dominant-negative cytokine receptor. In certain embodiments, the modified monocyte provided herein further comprises an exogenous polynucleotide encoding a dnIL-lOR. In certain embodiments, the modified monocyte provided herein further comprises an exogenous polynucleotide encoding a dnTGFpR. In certain embodiments, the modified monocyte provided herein further comprises an exogenous polynucleotide encoding a dnTGFpRI, a dnTGFpRII, or a dnTGFpRIII. In certain embodiments, the modified monocyte provided herein further comprises an exogenous polynucleotide encoding a dnTGFpRII. An exemplary dnTGFpRII is disclosed in Immunity. 2000 Feb;12(2): 171-81. In certain embodiments, an exogenous polynucleotide encoding a dnTGFpR disclosed herein comprises a nucleotide sequence setforth in SEQ ID NO: 1. In certain embodiments, a dnTGFpR disclosed herein comprises an amino acid sequence set forth in SEQ ID NO: 2.

[0198] In certain embodiments, the modified monocyte provided herein further comprises an exogenous polynucleotide encoding a proinflammatory cytokine (e.g., IL-12 and / or IFNy). In certain embodiments, the proinflammatory cytokines are selected from the group consisting of IL-1, IL-2, IL-6, IL-12, IL-17, IL-18, IL-23, IFNy, MCP-1, and TNFa. In certain embodiments, the modified monocyte provided herein further comprises an exogenous polynucleotide encoding IL-12. In certain embodiments, the modified monocyte provided herein further comprises an exogenous polynucleotide encoding IFNy. In certain embodiments, the modified monocyte provided herein further comprises one or more exogenous polynucleotides encoding IL- 12 and IFNy.

[0199] In certain embodiments, the modified monocyte provided herein further comprises a CAR (such as a CAR described in Section 5.3). In certain embodiments, the modified monocyte provided herein further comprises a polynucleotide encoding a CAR (such as a CAR described in Section 5.3) or a polypeptide of a CAR (such as a CAR described in Section 5.3).

[0200] In certain embodiments, the modified monocyte provided herein comprises (i) genetic disruption of at least one gene of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene; and (ii) a CAR (such as a CAR described in Section 5.3). In certain embodiments, the modified monocyte provided herein comprises a CAR (such as a CAR described in Section 5.3) and does not comprise genetic disruption of a MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene. In certain embodiments, the CAR promotes Ml macrophage polarization. In certain embodiments, the CAR increases secretion of a pro- inflammatory cytokine (e.g., is a CAR described in PCT / US2023 / 071207). In certain embodiments, the intracellular domain of the CAR is selected from the group consisting of a CD40, a TLR, and an IFNyR intracellular domain.

[0201] In certain embodiments, a polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding the proinflammatory cytokine (e.g., IL-12 and / or IFNy), and / or an exogenous polynucleotide encoding the dominant-negative cytokine receptor (e.g., dnIL-lOR, dnTGFpRI, dnTGFpRII, or dnTGFpRIII) is integrated into one or more genes disclosed herein (e.g., an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CGgene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPA gene, a SIGLEC10 gene, a B2M gene, a CIITA gene, an RFX gene, an IL 1 ORA gene, an IL 1 ORB gene, a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene).

[0202] In certain embodiments, a polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding the proinflammatory cytokine (e.g., IL-12 and / or IFNy), and / or an exogenous polynucleotide encoding the dominant-negative cytokine receptor (e.g., dnIL-lOR, dnTGFpRI, dnTGFpRII, or dnTGFpRIII) is integrated into the modified monocyte using the SLEEK technology. In certain embodiments, the polynucleotide encoding the CAR, the exogenous polynucleotide encoding the proinflammatory cytokine, and / or the exogenous polynucleotide encoding the dominant-negative cytokine receptor is integrated in frame with and downstream (3’) of a coding sequence of an essential gene, and wherein at least part of the essential gene comprises an exogenous coding sequence. In certain embodiments, the correct knock-in cells would retain essential gene function while also integrating the polynucleotide encoding the CAR, the exogenous polynucleotide encoding the proinflammatory cytokine, and / or the exogenous polynucleotide encoding the dominant-negative cytokine receptor. Cells with non-productive insertions and deletions would undergo negative selection. In certain embodiments, the essential gene encodes a gene product that is required for survival and / or proliferation of the cell. In certain embodiments, the essential gene is a housekeeping gene. In certain embodiments, the essential gene encodes GAPDH. In certain embodiments, the essential gene is an essential gene as disclosed in International Patent Publication WO2022235811.

[0203] In certain embodiments, a polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding the proinflammatory cytokine, and / or an exogenous polynucleotide encoding the dominantnegative cytokine receptor is integrated into a safe harbor locus, e.g., an AAVS1 locus.

[0204] In certain embodiments, the modified monocyte further comprises (i) genetic disruption of a B2M gene, (ii) genetic disruption of a CIITA gene, (iii) genetic disruption of an RFX gene, and / or (iv) an exogenous polynucleotide encoding HLA-E. In certain embodiments, the modified monocyte provided herein further comprises genetic disruption of (i) a B2M gene, a CIITA gene and / or an RFX gene, and (ii) an exogenous polynucleotide encoding HLA-E. In certain embodiments, the modified monocyte provided herein further comprises genetic disruption of a B2M gene and a CIITA gene (e.g., B2M / CIITA double knockout), and an exogenous polynucleotide encoding HLA-E. In certain embodiments, themodified monocyte provided herein further comprises genetic disruption of a B2M gene, a CIITA gene, and an RFX gene, and an exogenous polynucleotide encoding HLA-E.

[0205] In certain embodiments, the modified monocyte provided herein is homozygous for the genetic disruption of the B2M gene, CIITA gene, and / or RFX gene. In certain embodiments, the modified monocyte provided herein is heterozygous for the genetic disruption of the B2M gene, CIITA gene, and / or RFX gene.

[0206] In certain embodiments, genetic disruption of a gene disclosed herein (e.g., an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPA gene, a SIGLEC10 gene, a B2M gene, a CIITA gene, an RFX gene, an IL 1 ORA gene, an IL 1 ORB gene, a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene) is performed by a genome editing tool, such as TALEN, ZFN, or CRISPR (e.g., by deletion of one or more exons, introduction of a stop codon, introduction of a null mutation or inactivation of the promoter).5.1.5 Modified Macrophages

[0207] The present disclosure further provides a modified macrophage comprising genetic disruption of at least one gene (e.g., one, two, three, four, five, six, seven, eight, nine, or all gene(s)) of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene.

[0208] In certain embodiments, the modified macrophage is a human macrophage. In certain embodiments, the modified macrophage is a mammalian macrophage. In certain embodiments, the modified macrophage is a primate macrophage. In certain embodiments, the modified macrophage is a non-human primate macrophage. In certain embodiments, the modified macrophage is a rodent, e.g., mouse, macrophage.

[0209] In certain embodiments, the modified immature macrophage is a human immature macrophage. In certain embodiments, the modified immature macrophage is a mammalian immature macrophage. In certain embodiments, the modified immature macrophage is a primate immature macrophage. In certain embodiments, the modified immature macrophage is a non-human primate immature macrophage. In certain embodiments, the modified immature macrophage is a rodent, e.g., mouse, immature macrophage.

[0210] In certain embodiments, the modified macrophage disclosed herein, once exposed to one or more polarization stimuli, has suppressed M2 polarization and / or promoted Ml polarization relative to a non-modified macrophage exposed to the polarization stimulus. In certain embodiments, the one or more polarization stimuli comprise one or more Mlpolarization stimuli. In certain embodiments, the one or more Ml polarization stimuli comprise IFNy, LPS, and / or IL-lb. In certain embodiments, the one or more Ml polarization stimuli comprise IFNy and LPS. In certain embodiments, the one or more Ml polarization stimuli comprise LPS and IL-lb. In certain embodiments, the one or more polarization stimuli comprise one or more M2 polarization stimuli. In certain embodiments, the one or more M2 polarization stimuli comprise IL-10, IL-4, IL-13, TGFP, adenosine, and / or IL-6. In certain embodiments, the one or more M2 polarization stimuli comprise IL-4 and IL-13. In certain embodiments, the one or more M2 polarization stimuli comprise IL- 10 and TGFp. In certain embodiments, the one or more M2 polarization stimuli comprise adenosine and IL-6. In certain embodiments, the one or more M2 polarization stimuli comprise IL- 10.

[0211] In certain embodiments, the modified macrophage that is exposed to the one or more polarization stimuli has increased expression levels (e.g., protein or transcript expression levels) of one or more Ml markers as compared to the expression levels of the one or more Ml markers in an unmodified macrophage (e.g., a wildtype macrophage) that is exposed to the same polarization stimuli. Exemplary Ml markers include CD38, CD40, CD54, CD80, CD86, CD283 (TLR2), and CXCR3. Additional exemplary Ml markers are disclosed in Table 1. In certain embodiments, the expression levels of the one or more Ml markers in the modified macrophage that is exposed to the one or more polarization stimuli are increased to no less than about 150%, no less than about 200%, no less than about 250%, no less than about 300%, no less than about 350%, no less than about 400%, no less than about 500%, no less than about 600%, no less than about 700%, no less than about 800%, no less than about 900%, no less than about 1000%, no less than 1500%, no less than 2000%, or no less than 2500% as compared to the expression levels of the one or more Ml markers in an unmodified macrophage that is exposed to the same polarization stimuli. In certain embodiments, the expression levels of the one or more Ml markers in the modified macrophage that is exposed to the one or more polarization stimuli are increased to between about 150% and about 2500%, between about 200% and about 2000%, between about 500% and about 1000%, between about 200% and about 500%, or between about 150% and about 300% as compared to the expression levels of the one or more Ml markers in an unmodified macrophage that is exposed to the same polarization stimuli. In certain embodiments, the expression levels of the one or more Ml markers in the modified macrophage that is exposed to the one or more polarization stimuli are increased to about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, about 1500%, about 2000%, about 2500% or more ascompared to the expression levels of the one or more Ml markers in an unmodified macrophage that is exposed to the same polarization stimuli.

[0212] In certain embodiments, the modified macrophage exposed to the one or more polarization stimuli has reduced expression levels (e.g., protein or transcript expression levels) of one or more M2 markers as compared to the expression levels of the one or more M2 markers in an unmodified macrophage (e.g., a wildtype macrophage) that is exposed to the same polarization stimuli. Exemplary M2 markers include CD206, CD 163, CD 169, CD36, CD304, and MRC1. Additional exemplary M2 markers are disclosed in Table 2. In certain embodiments, the expression levels of the one or more M2 markers in the modified macrophage that is exposed to the one or more polarization stimuli are reduced to no more than about 5%, no more than about 10%, no more than about 15%, no more than about 20%, no more than about 25%, no more than about 30%, no more than about 40%, no more than about 50%, no more than about 60%, no more than about 70%, no more than about 80%, no more than about 90%, or no more than about 95% as compared to the expression levels of the one or more M2 markers in an unmodified macrophage that is exposed to the same polarization stimuli. In certain embodiments, the expression levels of the one or more M2 markers in the modified macrophage that is exposed to the one or more polarization stimuli are reduced to between about 5% and about 95%, between about 10% and about 90%, between about 20% and about 80%, between about 50% and about 90%, between about 60% and about 80%, between about 30% and about 40%, or between about 20% and about 50% as compared to the expression levels of the one or more M2 markers in an unmodified macrophage that is exposed to the same polarization stimuli. In certain embodiments, the expression levels of the one or more M2 markers in the modified macrophage that is exposed to the one or more polarization stimuli are reduced to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% as compared to the expression levels of the one or more M2 markers in an unmodified macrophage that is exposed to the same polarization stimuli.

[0213] In certain embodiments, the expression levels of the one or more Ml markers are not increased and the expression levels of the one or more M2 markers are decreased. In some embodiments, the expression levels of the one or more M2 markers are decreased and expression levels of the one or more Ml markers are unchanged.

[0214] In certain embodiments, the modified macrophage that is exposed to the one or more polarization stimuli has increased secretion levels of one or more Ml cytokines as compared to the secretion levels of the one or more Ml cytokines in an unmodifiedmacrophage (e.g., a wildtype macrophage) that is exposed to the same polarization stimuli. Exemplary Ml cytokines include CXCL9, CXCL10, CXCL11, CCL3, CCL4, IL-lb, IL-2, IL-6, IL-8, IL-12, IL-23, and TNFa. In certain embodiments, the secretion levels of the one or more Ml cytokines in the modified macrophage that is exposed to the one or more polarization stimuli are increased to no less than about 150%, no less than about 200%, no less than about 250%, no less than about 300%, no less than about 350%, no less than about 400%, no less than about 500%, no less than about 600%, no less than about 700%, no less than about 800%, no less than about 900%, no less than about 1000%, no less than 1500%, no less than 2000%, or no less than 2500% as compared to the secretion levels of the one or more Ml cytokines in an unmodified macrophage that is exposed to the same polarization stimuli. In certain embodiments, the secretion levels of the one or more Ml cytokines in the modified macrophage that is exposed to the one or more polarization stimuli are increased to between about 150% and about 2500%, between about 200% and about 2000%, between about 500% and about 1000%, between about 200% and about 500%, or between about 150% and about 300% as compared to the secretion levels of the one or more Ml cytokines in an unmodified macrophage that is exposed to the same polarization stimuli. In certain embodiments, the secretion levels of the one or more Ml cytokines in the modified macrophage that is exposed to the one or more polarization stimuli are increased to about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, about 1500%, about 2000%, about 2500% or more as compared to the secretion levels of the one or more Ml cytokines in an unmodified macrophage that is exposed to the same polarization stimuli.

[0215] In certain embodiments, the modified macrophage exposed to the one or more polarization stimuli has reduced secretion levels (e.g., protein or transcript secretion levels) of one or more M2 cytokines as compared to the secretion levels of the one or more M2 cytokines in an unmodified macrophage (e.g., a wildtype macrophage) that is exposed to the same polarization stimuli. Exemplary M2 cytokines include IL-10, MCP-1, TGFP, CCL13, CCL17, CCL18 and CCL22. In certain embodiments, the secretion levels of the one or more M2 cytokines in the modified macrophage that is exposed to the one or more polarization stimuli are reduced to no more than about 5%, no more than about 10%, no more than about 15%, no more than about 20%, no more than about 25%, no more than about 30%, no more than about 40%, no more than about 50%, no more than about 60%, no more than about 70%, no more than about 80%, no more than about 90%, or no more than about 95% as compared to the secretion levels of the one or more M2 cytokines in an unmodified macrophage that isexposed to the same polarization stimuli. In certain embodiments, the secretion levels of the one or more M2 cytokines in the modified macrophage that is exposed to the one or more polarization stimuli are reduced to between about 5% and about 95%, between about 10% and about 90%, between about 20% and about 80%, between about 50% and about 90%, between about 60% and about 80%, between about 30% and about 40%, or between about 20% and about 50% as compared to the secretion levels of the one or more M2 cytokines in an unmodified macrophage that is exposed to the same polarization stimuli. In certain embodiments, the secretion levels of the one or more M2 cytokines in the modified macrophage that is exposed to the one or more polarization stimuli are reduced to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% as compared to the secretion levels of the one or more M2 cytokines in an unmodified macrophage that is exposed to the same polarization stimuli.

[0216] In certain embodiments, the secretion levels of the one or more Ml cytokines are not increased and the secretion levels of the one or more M2 cytokines are decreased. In some embodiments, the secretion levels of the one or more M2 cytokines are decreased and secretion levels of the one or more Ml cytokines are unchanged.

[0217] As used herein, cytokines include chemokines, which are a family of low molecular weight chemotactic cytokines that regulate leukocyte migration. Additional Ml and M2 cytokines are disclosed in Kadomoto et al., Macrophage Polarity and Disease Control, Int J Mol Sci. 2021 Dec 23;23(1): 144, the content of which is incorporated herein by reference.

[0218] In certain embodiments, the modified macrophage resists M2 -induced reduction in metabolic activities relative to a non-modified macrophage exposed to the polarization stimulus. In certain embodiments, the modified macrophage exposed to the M2 polarization stimulus does not have reduced oxygen consumption rate (OCR) or extracellular acidification rate (ECAR) as compared to the modified macrophage exposed to the Ml polarization stimulus.

[0219] In certain embodiments, the modified macrophage is derived from a modified pluripotent cell (such as a modified pluripotent cell described in Section 5.1.2). In certain embodiments, the modified pluripotent cell is generated according to the methods provided herein (such as the methods described in Section 5.2.1), and the modified pluripotent cell is differentiated into a macrophage. In certain embodiments, the modified macrophage provided herein is derived from a modified myeloid progenitor cell (such as a modifiedmyeloid progenitor cell described in Section 5.1.3). For example, in certain embodiments, the modified myeloid progenitor cell is generated according to the methods provided herein (such as the methods described in Section 5.2.2), and the modified myeloid progenitor cell is differentiated into a macrophage. In certain embodiments, the modified macrophage provided herein is derived from a modified monocyte (such as a modified monocyte described in Section 5.1.4). For example, in certain embodiments, the modified monocyte is generated according to the methods provided herein (such as the methods described in Section 5.2.3), and the modified monocyte is differentiated into a macrophage. Techniques known to one of skill in the art or described herein (such as in Section 5.9) may be used to differentiate a pluripotent cell into a macrophage, a myeloid progenitor cell into a macrophage and / or differentiate a monocyte into a macrophage. For example, a homogeneous population of modified macrophages or a cell population comprising a modified macrophage disclosed herein (e.g., at least about 95% of the cells in the cell population are the modified macrophages) may be produced using and / or may be assessed via markers known in the art (such as those described in Section 5.10.2).

[0220] In certain embodiments, a modified macrophage exhibits a macrophage phenotype such as a macrophage phenotype as described in Section 5.10.2. For example, in certain embodiments, a modified macrophage disclosed herein, in a cell culture environment, is an-adherent cell. In certain embodiments, a modified macrophage disclosed herein has a macrophage cell morphology, e.g., the size of the cell is larger than monocytes. In certain embodiments, a modified macrophage has a cell morphology that is more granular than a monocyte. In certain embodiments, a modified macrophage disclosed herein is an adherent cell that has a cell morphology that is more granular than a monocyte and has a cell size that is larger than macrophages.

[0221] In certain embodiments, a modified macrophage presented herein expresses a detectable level of CD1 lb+and CD45+(e.g., is a CD1 lb+CD45+cell). In certain embodiments, a modified macrophage disclosed herein expresses a detectable level of CD14, e.g., is CD1 lb+CD45+CD14+. In certain embodiments, a modified macrophage presented herein does not express a detectable level of CD14 (e.g., is CD1 lb+CD45+CD14‘).

[0222] In certain embodiments, a modified macrophage, e.g., immature macrophage, disclosed herein has the properties of killing target cells (e.g., via CAR or monoclonal antibody (mAb) targeting), phagocytosing particles (e.g., bacteria), and / or migrating towards chemokines. In certain embodiments, a modified macrophage, e.g., immature macrophage, disclosed herein exhibits increased killing of target cells (e.g., via CAR or monoclonalantibody (mAb) targeting), phagocytosing particles (e.g., bacteria), and / or migrating towards chemokines relative to a non-modified macrophage, e.g., immature macrophage. Any assays known in the art can be used for measuring these properties, for example, the assays disclosed in Section 5.10.

[0223] In certain embodiments, the modified macrophage provided herein is derived from an induced pluripotent stem cell (iPSC). In certain embodiments, the iPSC has been reprogrammed from a peripheral blood mononuclear cell (PBMC), a CD34+ cord blood cell, an immune cell, a macrophage, a monocyte, or a fibroblast. In certain embodiments, the iPSC has been reprogrammed from a PBMC. In certain embodiments, the iPSC has been reprogrammed from a CD34+ cord blood cell. In certain embodiments, the iPSC has been reprogrammed from an immune cell. In certain embodiments, the iPSC has been reprogrammed from a macrophage. In certain embodiments, the iPSC has been reprogrammed from a monocyte. In certain embodiments, the iPSC has been reprogrammed from a fibroblast. In certain embodiments, the iPSC is a hiPSC. In certain embodiments, the iPSC is a TC-1133 cell.

[0224] In certain embodiments, the modified macrophage provided herein is an immature macrophage. In certain embodiments, the immature macrophage is derived from a pluripotent cell (e.g., an iPSC). In certain embodiments, the immature macrophage has not been subjected to any maturation or polarization process. In certain embodiments, the immature macrophage is less granular and less adherent in cell culture than a mature macrophage. In certain embodiments, the immature macrophage is smaller in cell culture than a mature macrophage. In certain embodiments, the immature macrophage is more granular and bigger than monocytes obtained from blood.

[0225] In certain embodiments, the modified macrophage provided herein is a tissueresident macrophage (e.g., an adipose-associated macrophage, osteoblast, microglia, motile liver macrophage, perivascular macrophage, meningeal macrophage, intestinal macrophage, Kupffer cell, Langerhans cell, alveolar macrophage or red-pulp macrophage).

[0226] The present disclosure further provides a homogeneous population of the modified macrophage disclosed herein (e.g., a modified macrophage described in Section5.1.5). In certain embodiments, the homogeneous population of modified macrophages are isogenic.

[0227] The present disclosure further provides a population of cells comprising a modified macrophage disclosed herein (e.g., a modified macrophage described in Section5.1.5), wherein at least about 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.8%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 90% to about 100%, from about 95% to about 98%, from about 95% to about 99%, or from about 95% to 100% of the cells in the population are the modified macrophages.

[0228] The present disclosure further provides a population of cells comprising a modified immature macrophage disclosed herein (e.g., a modified immature macrophage described in Section 5.1.5), wherein at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.8%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 90% to about 100%, from about 95% to about 98%, from about 95% to about 99%, or from about 95% to 100% of the cells in the population are the modified immature macrophages.

[0229] In certain embodiments, the modified macrophage disclosed herein is homozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene. In certain embodiments, the modified macrophage provided herein is heterozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene.

[0230] In certain embodiments, the modified macrophage comprises genetic disruption of an MIR146A gene. In certain embodiments, the modified macrophage comprises genetic disruption of a GPR65 gene. In certain embodiments, the modified macrophage comprises genetic disruption of a STAT6 gene. In certain embodiments, the modified macrophage comprises genetic disruption of an AKT1 gene. In certain embodiments, the modified macrophage comprises genetic disruption of an AKT2 gene. In certain embodiments, the modified macrophage comprises genetic disruption of an IRF3 gene. In certain embodiments, the modified macrophage comprises genetic disruption of an IRF4 gene. In certain embodiments, the modified macrophage comprises genetic disruption of a PIK3CG gene. In certain embodiments, the modified macrophage comprises genetic disruption of a TSC1 gene. In certain embodiments, the modified macrophage comprises genetic disruption of a VSIG4 gene.

[0231] In certain embodiments, the modified macrophage comprises genetic disruption of two genes of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene,an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, and a VSIG4 gene. In certain embodiments, the modified macrophage comprises genetic disruption of three, four, five, six, seven, eight, nine, or all genes of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, and a VSIG4 gene.

[0232] In certain embodiments, the modified macrophage provided herein further comprises genetic disruption of a SIRPA gene or a SIGLEC10 gene. In certain embodiments, the modified macrophage provided herein further comprises genetic disruption of a SIRPA gene and a SIGLEC10 gene. In certain embodiments, the modified macrophage provided herein is homozygous for the genetic disruption of the SIRPA gene and / or SIGLEC10 gene. In certain embodiments, the modified macrophage provided herein is heterozygous for the genetic disruption of the SIRPA gene and / or SIGLEC10 gene.

[0233] In certain embodiments, presented herein is a macrophage that comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the macrophage comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor, and does not comprise at least one of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene. In certain embodiments, the cytokine receptor is IL-10R. In certain embodiments, the cytokine receptor is a TGFpR. In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII or TGFpRIII. In certain embodiments, the cytokine receptor is a TGFpRII. In certain embodiments, the macrophage is a human macrophage. In certain embodiments, the macrophage is a mammalian macrophage. In certain embodiments, the macrophage is a primate macrophage. In certain embodiments, the macrophage is a nonhuman primate macrophage. In certain embodiments, the macrophage is a rodent, e.g., mouse, macrophage. In certain embodiments, the immature macrophage is a human immature macrophage. In certain embodiments, the immature macrophage is a mammalian immature macrophage. In certain embodiments, the immature macrophage is a primate immature macrophage. In certain embodiments, the immature macrophage is a non-human primate immature macrophage. In certain embodiments, the immature macrophage is a rodent, e.g., mouse, immature macrophage. In particular embodiments, the macrophage, e.g., immature macrophage, is derived from a pluripotent stem cell, for example a human pluripotent stem cell. In particular embodiments, the macrophage, e.g., immature macrophage, is derived from an induced pluripotent stem cell, for example a human inducedpluripotent stem cell. In particular embodiments, the macrophage, e.g., immature macrophage, is derived from a myeloid progenitor cell, for example a human myeloid progenitor cell. In particular embodiments, the macrophage, e.g., immature macrophage, is derived from a monocyte, for example a human monocyte.

[0234] In certain embodiments, the macrophage comprises genetic disruption of a gene encoding IL-10R. In certain embodiments, the macrophage comprises genetic disruption of an IL10RA gene and / or an IL10RB gene. In certain embodiments, the macrophage comprises genetic disruption of a gene encoding a TGFpR (e.g., TGFpRI, TGFpRII, or TGFpRIII). In certain embodiments, the macrophage comprises genetic disruption of a gene encoding TGFpRII. In certain embodiments, the macrophage comprises genetic disruption of a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene. In certain embodiments, the macrophage comprises genetic disruption of a TGFBR2 gene. In certain embodiments, the macrophage is homozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the macrophage is homozygous for the genetic disruption of the IL10RA gene, IL10RB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene. In certain embodiments, the macrophage is heterozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the macrophage is heterozygous for the genetic disruption of the IL 1 ORA gene, IL 1 ORB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene.

[0235] In certain embodiments, the macrophage comprises an exogenous polynucleotide encoding a dominant-negative cytokine receptor. In certain embodiments, the macrophage comprises an exogenous polynucleotide encoding a dnIL-lOR. In certain embodiments, the macrophage comprises an exogenous polynucleotide encoding a dnTGFpR. In certain embodiments, the macrophage comprises an exogenous polynucleotide encoding a dnTGFpRI, a dnTGFpRII, or a dnTGFpRIII. In certain embodiments, the macrophage comprises an exogenous polynucleotide encoding a dnTGFpRII. An exemplary dnTGFpRII is disclosed in Immunity. 2000 Feb;12(2): 171-81. In certain embodiments, an exogenous polynucleotide encoding a dnTGFpR disclosed herein comprises a nucleotide sequence set forth in SEQ ID NO: 1. In certain embodiments, a dnTGFpR disclosed herein comprises an amino acid sequence set forth in SEQ ID NO: 2.

[0236] In certain embodiments, the modified macrophage provided herein further comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the modifiedmacrophage provided herein comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor, and comprises at least one of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene. In certain embodiments, the cytokine receptor is IL-10R. In certain embodiments, the cytokine receptor is a TGFpR. In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII or TGFpRIII. In certain embodiments, the cytokine receptor is a TGFpRII.

[0237] In certain embodiments, the modified macrophage provided herein further comprises genetic disruption of a gene encoding IL-10R. In certain embodiments, the modified macrophage provided herein further comprises genetic disruption of an IL10RA gene and / or an IL 1 ORB gene. In certain embodiments, the modified macrophage provided herein further comprises genetic disruption of a gene encoding a TGFpR (e.g., TGFpRI, TGFpRII, or TGFpRIII). In certain embodiments, the modified macrophage provided herein further comprises genetic disruption of a gene encoding TGFpRII. In certain embodiments, the modified macrophage provided herein further comprises genetic disruption of a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene. In certain embodiments, the modified macrophage provided herein further comprises genetic disruption of a TGFBR2 gene. In certain embodiments, the modified macrophage provided herein is homozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the modified macrophage provided herein is homozygous for the genetic disruption of the IL 1 ORA gene, IL 1 ORB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene. In certain embodiments, the modified macrophage provided herein is heterozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the modified macrophage provided herein is heterozygous for the genetic disruption of the IL 1 ORA gene, IL 1 ORB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene.

[0238] In certain embodiments, the modified macrophage provided herein further comprises an exogenous polynucleotide encoding a dominant-negative cytokine receptor. In certain embodiments, the modified macrophage provided herein further comprises an exogenous polynucleotide encoding a dnIL-lOR. In certain embodiments, the modified macrophage provided herein further comprises an exogenous polynucleotide encoding a dnTGFpR. In certain embodiments, the modified macrophage provided herein further comprises an exogenous polynucleotide encoding a dnTGFpRI, a dnTGFpRII, or a dnTGFpRIII. In certain embodiments, the modified macrophage provided herein furthercomprises an exogenous polynucleotide encoding a dnTGFpRII. An exemplary dnTGFpRII is disclosed in Immunity. 2000 Feb;12(2): 171-81. In certain embodiments, an exogenous polynucleotide encoding a dnTGFpR disclosed herein comprises a nucleotide sequence set forth in SEQ ID NO: 1. In certain embodiments, a dnTGFpR disclosed herein comprises an amino acid sequence set forth in SEQ ID NO: 2.

[0239] In certain embodiments, the modified macrophage provided herein further comprises an exogenous polynucleotide encoding a proinflammatory cytokine (e.g., IL-12 and / or IFNy). In certain embodiments, the proinflammatory cytokines are selected from the group consisting of IL-1, IL-2, IL-6, IL-12, IL-17, IL-18, IL-23, IFNy, MCP-1, and TNFa. In certain embodiments, the modified macrophage provided herein further comprises an exogenous polynucleotide encoding IL-12. In certain embodiments, the modified macrophage provided herein further comprises an exogenous polynucleotide encoding IFNy. In certain embodiments, the modified macrophage provided herein further comprises one or more exogenous polynucleotides encoding IL-12 and IFNy.

[0240] In certain embodiments, the modified macrophage provided herein further comprises a CAR (such as a CAR described in Section 5.3). In certain embodiments, the modified macrophage provided herein further comprises a polynucleotide encoding a CAR (such as a CAR described in Section 5.3) or a polypeptide of a CAR (such as a CAR described in Section 5.3).

[0241] In certain embodiments, the modified macrophage provided herein comprises (i) genetic disruption of at least one gene of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene; and (ii) a CAR (such as a CAR described in Section 5.3). In certain embodiments, the CAR promotes Ml macrophage polarization. In certain embodiments, the CAR increases secretion of a pro-inflammatory cytokine (e.g., is a CAR described in PCT / US2023 / 071207). In certain embodiments, the intracellular domain of the CAR is selected from the group consisting of a CD40, a TLR, and an IFNyR intracellular domain.

[0242] In certain embodiments, a polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding the proinflammatory cytokine (e.g., IL-12 and / or IFNy), and / or an exogenous polynucleotide encoding the dominant-negative cytokine receptor (e.g., dnIL-lOR, dnTGFpRI, dnTGFpRII, or dnTGFpRIII) is integrated into one or more genes disclosed herein (e.g., an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPA gene, a SIGLEC10 gene, a B2Mgene, a CIITA gene, an RFX gene, an IL 1 ORA gene, an IL 1 ORB gene, a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene).

[0243] In certain embodiments, a polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding the proinflammatory cytokine (e.g., IL-12 and / or IFNy), and / or an exogenous polynucleotide encoding the dominant-negative cytokine receptor (e.g., dnIL-lOR, dnTGFpRI, dnTGFpRII, or dnTGFpRIII) is integrated into the modified macrophage using the SLEEK technology. In certain embodiments, the polynucleotide encoding the CAR, the exogenous polynucleotide encoding the proinflammatory cytokine, and / or the exogenous polynucleotide encoding the dominant-negative cytokine receptor is integrated in frame with and downstream (3’) of a coding sequence of an essential gene, and wherein at least part of the essential gene comprises an exogenous coding sequence. In certain embodiments, the correct knock-in cells would retain essential gene function while also integrating the polynucleotide encoding the CAR, the exogenous polynucleotide encoding the proinflammatory cytokine, and / or the exogenous polynucleotide encoding the dominant-negative cytokine receptor. Cells with non-productive insertions and deletions would undergo negative selection. In certain embodiments, the essential gene encodes a gene product that is required for survival and / or proliferation of the cell. In certain embodiments, the essential gene is a housekeeping gene. In certain embodiments, the essential gene encodes GAPDH. In certain embodiments, the essential gene is an essential gene as disclosed in International Patent Publication WO2022235811.

[0244] In certain embodiments, a polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding the proinflammatory cytokine, and / or an exogenous polynucleotide encoding the dominantnegative cytokine receptor is integrated into a safe harbor locus, e.g., an AAVS1 locus.

[0245] In certain embodiments, the modified macrophage further comprises (i) genetic disruption of a B2M gene, (ii) genetic disruption of a CIITA gene, (iii) genetic disruption of an RFX gene, and / or (iv) an exogenous polynucleotide encoding HLA-E. In certain embodiments, the modified macrophage provided herein further comprises genetic disruption of (i) a B2M gene, a CIITA gene and / or an RFX gene, and (ii) an exogenous polynucleotide encoding HLA-E. In certain embodiments, the modified macrophage provided herein further comprises genetic disruption of a B2M gene and a CIITA gene (e.g., B2M / CIITA double knockout), and an exogenous polynucleotide encoding HLA-E. In certain embodiments, the modified macrophage provided herein further comprises geneticdisruption of a B2M gene, a CIITA gene, and an RFX gene, and an exogenous polynucleotide encoding HLA-E.

[0246] In certain embodiments, the modified macrophage provided herein is homozygous for the genetic disruption of the B2M gene, CIITA gene, and / or RFX gene. In certain embodiments, the modified macrophage provided herein is heterozygous for the genetic disruption of the B2M gene, CIITA gene, and / or RFX gene.

[0247] In certain embodiments, genetic disruption of a gene disclosed herein (e.g., an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPA gene, a SIGLEC10 gene, a B2M gene, a CIITA gene, an RFX gene, an IL 1 ORA gene, an IL 1 ORB gene, a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene) is performed by a genome editing tool, such as TALEN, ZFN, or CRISPR (e.g., by deletion of one or more exons, introduction of a stop codon, introduction of a null mutation or inactivation of the promoter).

[0248] In certain embodiments, the modified macrophage is a pro-inflammatory macrophage. In certain embodiments, the modified macrophage is an Ml macrophage. In certain embodiments, the modified macrophage expresses an increased level of one or more Ml markers relative to non-modified macrophages, for example, expresses an increased level of one or more of CD38, CD40, CD54, CD80, CD86, CD283 (TLR2), and CXCR3. In certain embodiments, the modified macrophage expresses an increased level of one or more markers in Table 1.Table 1: Exemplary human Ml macrophage markers

[0249] In certain embodiments, the modified macrophage is not an anti-inflammatory macrophage. In certain embodiments, the modified macrophage is not an M2 macrophage. In certain embodiments, the modified macrophage expresses a decreased level of one or more M2 markers relative to a non-modified macrophage, for example, expresses a decreased level of one or more of CD206, CD 163, CD 169, CD36, CD304, and MRC1. In certain embodiments, the modified macrophage expresses a decreased level of one or more M2 markers in Table 2.Table 2: Exemplary human macrophage M2 markers.

[0250] In certain embodiments, the modified macrophage expresses an increased level of one or more Ml markers relative to non-modified macrophages, for example, expresses an increased level of one or more of CD38, CD40, CD54, CD80, CD86, CD283, CXCR3, and a marker in Table 1, and also expresses a decreased level of one or more M2 markers relative to non-modified macrophages, for example, expressed a decreased level of one or more of CD206, CD163, CD169, CD36, CD304, MRC1, and a marker in Table 2.

[0251] In certain embodiments, the modified macrophage is an M0 macrophage.5.1.6 Modified CDllb+CD45+Cells

[0252] The present disclosure further provides a modified CD1 lb+CD45+cells comprising genetic disruption of at least one gene (e.g., one, two, three, four, five, six, seven, eight, nine, or all gene(s)) of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.

[0253] In certain embodiments, the modified CD1 lb+CD45+cell is a human CD1 lb+CD45+cell. In certain embodiments, the modified CD1 lb+CD45+cell is a mammalian CD1 lb+CD45+cell. In certain embodiments, the modified CD1 lb+CD45+cell is a primate CD1 lb+CD45+cell. In certain embodiments, the modified CD1 lb+CD45+cell is a non-human primate CD1 lb+CD45+cell. In certain embodiments, the modified CD1 lb+CD45+cell is a rodent, e.g., mouse, CD1 lb+CD45+cell.

[0254] In certain embodiments, the modified CD1 lb+CD45+cell is a CD1 lb+CD45+CD14+cell. In certain embodiments, the modified CD1 lb+CD45+CD14+cell is a human CD1 lb+CD45+CD14+cell. In certain embodiments, the modified CD1 lb+CD45+CD14+cell is a mammalian CD1 lb+CD45+CD14+cell. In certain embodiments, the modified CD1 lb+CD45+CD14+cell is a primate CD1 lb+CD45+CD14+cell. In certain embodiments, the modified CD1 lb+CD45+CD14+cell is a non-human primate CD1 lb+CD45+CD14+cell. In certain embodiments, the modified CD1 lb+CD45+CD14+cell is a rodent, e.g., mouse, CDl lb+CD45+CD14+cell.

[0255] In certain embodiments, the modified CDl lb+CD45+cell disclosed herein, once exposed to one or more polarization stimuli, has suppressed M2 polarization and / or promoted Ml polarization relative to a non-modified CD1 lb+CD45+cell exposed to the polarization stimulus. In certain embodiments, the one or more polarization stimuli comprise one or more Ml polarization stimuli. In certain embodiments, the one or more Ml polarization stimuli comprise IFNy, LPS, and / or IL-lb. In certain embodiments, the one or more Ml polarization stimuli comprise IFNY and LPS. In certain embodiments, the one or more Ml polarization stimuli comprise LPS and IL-lb. In certain embodiments, the one or more polarization stimuli comprise one or more M2 polarization stimuli. In certain embodiments, the one or more M2 polarization stimuli comprise IL-10, IL-4, IL-13, TGFP, adenosine, and / or IL-6. In certain embodiments, the one or more M2 polarization stimuli comprise IL-4 and IL-13. In certain embodiments, the one or more M2 polarization stimuli comprise IL-10 and TGFp. In certain embodiments, the one or more M2 polarization stimulicomprise adenosine and IL-6. In certain embodiments, the one or more M2 polarization stimuli comprise IL- 10.

[0256] In certain embodiments, the modified CD1 lb+CD45+cell that is exposed to the one or more polarization stimuli has increased expression levels (e.g., protein or transcript expression levels) of one or more Ml markers as compared to the expression levels of the one or more Ml markers in an unmodified CD1 lb+CD45+cell (e.g., a wildtype CD1 lb+CD45+cell) that is exposed to the same polarization stimuli. Exemplary Ml markers include CD38, CD40, CD54, CD80, CD86, CD283 (TLR2), and CXCR3. Additional exemplary Ml markers are disclosed in Table 1. In certain embodiments, the expression levels of the one or more Ml markers in the modified CD1 lb+CD45+cell that is exposed to the one or more polarization stimuli are increased to no less than about 150%, no less than about 200%, no less than about 250%, no less than about 300%, no less than about 350%, no less than about 400%, no less than about 500%, no less than about 600%, no less than about 700%, no less than about 800%, no less than about 900%, no less than about 1000%, no less than 1500%, no less than 2000%, or no less than 2500% as compared to the expression levels of the one or more Ml markers in an unmodified CD1 lb+CD45+cell that is exposed to the same polarization stimuli. In certain embodiments, the expression levels of the one or more Ml markers in the modified CD1 lb+CD45+cell that is exposed to the one or more polarization stimuli are increased to between about 150% and about 2500%, between about 200% and about 2000%, between about 500% and about 1000%, between about 200% and about 500%, or between about 150% and about 300% as compared to the expression levels of the one or more Ml markers in an unmodified CD1 lb+CD45+cell that is exposed to the same polarization stimuli. In certain embodiments, the expression levels of the one or more Ml markers in the modified CD1 lb+CD45+cell that is exposed to the one or more polarization stimuli are increased to about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, about 1500%, about 2000%, about 2500% or more as compared to the expression levels of the one or more Ml markers in an unmodified CD1 lb+CD45+cell that is exposed to the same polarization stimuli.

[0257] In certain embodiments, the modified CD1 lb+CD45+cell exposed to the one or more polarization stimuli has reduced expression levels (e.g., protein or transcript expression levels) of one or more M2 markers as compared to the expression levels of the one or more M2 markers in an unmodified CD1 lb+CD45+cell (e.g., a wildtype CD1 lb+CD45+cell) that is exposed to the same polarization stimuli. Exemplary M2 markers includeCD206, CD 163, CD 169, CD36, CD304, and MRC1. Additional exemplary M2 markers are disclosed in Table 2. In certain embodiments, the expression levels of the one or more M2 markers in the modified CD1 lb+CD45+cell that is exposed to the one or more polarization stimuli are reduced to no more than about 5%, no more than about 10%, no more than about 15%, no more than about 20%, no more than about 25%, no more than about 30%, no more than about 40%, no more than about 50%, no more than about 60%, no more than about 70%, no more than about 80%, no more than about 90%, or no more than about 95% as compared to the expression levels of the one or more M2 markers in an unmodified CD1 lb+CD45+cell that is exposed to the same polarization stimuli. In certain embodiments, the expression levels of the one or more M2 markers in the modified CD1 lb+CD45+cell that is exposed to the one or more polarization stimuli are reduced to between about 5% and about 95%, between about 10% and about 90%, between about 20% and about 80%, between about 50% and about 90%, between about 60% and about 80%, between about 30% and about 40%, or between about 20% and about 50% as compared to the expression levels of the one or more M2 markers in an unmodified CD1 lb+CD45+cell that is exposed to the same polarization stimuli. In certain embodiments, the expression levels of the one or more M2 markers in the modified CD1 lb+CD45+cell that is exposed to the one or more polarization stimuli are reduced to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% as compared to the expression levels of the one or more M2 markers in an unmodified CD1 lb+CD45+cell that is exposed to the same polarization stimuli.

[0258] In certain embodiments, the expression levels of the one or more Ml markers are not increased and the expression levels of the one or more M2 markers are decreased. In some embodiments, the expression levels of the one or more M2 markers are decreased and expression levels of the one or more Ml markers are unchanged.

[0259] In certain embodiments, the modified CD1 lb+CD45+cell that is exposed to the one or more polarization stimuli has increased secretion levels of one or more Ml cytokines as compared to the secretion levels of the one or more Ml cytokines in an unmodified CD1 lb+CD45+cell (e.g., a wildtype CD1 lb+CD45+cell) that is exposed to the same polarization stimuli. Exemplary Ml cytokines include CXCL9, CXCL10, CXCL11, CCL3, CCL4, IL-lb, IL-2, IL-6, IL-8, IL-12, IL-23, and TNFa. In certain embodiments, the secretion levels of the one or more Ml cytokines in the modified CD1 lb+CD45+cell that is exposed to the one or more polarization stimuli are increased to no less than about 150%, no less than about 200%, no less than about 250%, no less than about 300%, no less than about350%, no less than about 400%, no less than about 500%, no less than about 600%, no less than about 700%, no less than about 800%, no less than about 900%, no less than about 1000%, no less than 1500%, no less than 2000%, or no less than 2500% as compared to the secretion levels of the one or more Ml cytokines in an unmodified CD1 lb+CD45+cell that is exposed to the same polarization stimuli. In certain embodiments, the secretion levels of the one or more Ml cytokines in the modified CD1 lb+CD45+cell that is exposed to the one or more polarization stimuli are increased to between about 150% and about 2500%, between about 200% and about 2000%, between about 500% and about 1000%, between about 200% and about 500%, or between about 150% and about 300% as compared to the secretion levels of the one or more Ml cytokines in an unmodified CD1 lb+CD45+cell that is exposed to the same polarization stimuli. In certain embodiments, the secretion levels of the one or more Ml cytokines in the modified CD1 lb+CD45+cell that is exposed to the one or more polarization stimuli are increased to about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, about 1500%, about 2000%, about 2500% or more as compared to the secretion levels of the one or more Ml cytokines in an unmodified CD1 lb+CD45+cell that is exposed to the same polarization stimuli.

[0260] In certain embodiments, the modified CD1 lb+CD45+cell exposed to the one or more polarization stimuli has reduced secretion levels (e.g., protein or transcript secretion levels) of one or more M2 cytokines as compared to the secretion levels of the one or more M2 cytokines in an unmodified CD1 lb+CD45+cell (e.g., a wildtype CD1 lb+CD45+cell) that is exposed to the same polarization stimuli. Exemplary M2 cytokines include IL-10, MCP-1, TGFP, CCL13, CCL17, CCL18 and CCL22. In certain embodiments, the secretion levels of the one or more M2 cytokines in the modified CD1 lb+CD45+cell that is exposed to the one or more polarization stimuli are reduced to no more than about 5%, no more than about 10%, no more than about 15%, no more than about 20%, no more than about 25%, no more than about 30%, no more than about 40%, no more than about 50%, no more than about 60%, no more than about 70%, no more than about 80%, no more than about 90%, or no more than about 95% as compared to the secretion levels of the one or more M2 cytokines in an unmodified CD1 lb+CD45+cell that is exposed to the same polarization stimuli. In certain embodiments, the secretion levels of the one or more M2 cytokines in the modified CD1 lb+CD45+cell that is exposed to the one or more polarization stimuli are reduced to between about 5% and about 95%, between about 10% and about 90%, between about 20% and about 80%, between about 50% and about 90%, between about 60% and about 80%,between about 30% and about 40%, or between about 20% and about 50% as compared to the secretion levels of the one or more M2 cytokines in an unmodified CD1 lb+CD45+cell that is exposed to the same polarization stimuli. In certain embodiments, the secretion levels of the one or more M2 cytokines in the modified CD1 lb+CD45+cell that is exposed to the one or more polarization stimuli are reduced to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% as compared to the secretion levels of the one or more M2 cytokines in an unmodified CD1 lb+CD45+cell that is exposed to the same polarization stimuli.

[0261] In certain embodiments, the secretion levels of the one or more Ml cytokines are not increased and the secretion levels of the one or more M2 cytokines are decreased. In some embodiments, the secretion levels of the one or more M2 cytokines are decreased and secretion levels of the one or more Ml cytokines are unchanged.

[0262] As used herein, cytokines include chemokines, which are a family of low molecular weight chemotactic cytokines that regulate leukocyte migration. Additional Ml and M2 cytokines are disclosed in Kadomoto et al., CD1 lb+CD45+cell Polarity and Disease Control, Int J Mol Sci. 2021 Dec 23;23(1): 144, the content of which is incorporated herein by reference.

[0263] In certain embodiments, the modified CD1 lb+CD45+cell resists M2-induced reduction in metabolic activities relative to a non-modified CD1 lb+CD45+cell exposed to the polarization stimulus. In certain embodiments, the modified CD1 lb+CD45+cell exposed to the M2 polarization stimulus does not have reduced oxygen consumption rate (OCR) or extracellular acidification rate (ECAR) as compared to the modified CD1 lb+CD45+cell exposed to the Ml polarization stimulus.

[0264] A CD1 lb+CD45+cell can be any cells (e.g., a myeloid lineage cell) that express detectable levels of CD1 lb and CD45, for example a monocyte, a macrophage, an immature macrophage, a tissue-resident macrophage (e.g., an adipose-associated macrophage, osteoblast, microglia, motile liver macrophage, perivascular macrophage, meningeal macrophage, intestinal macrophage, Kupffer cell, Langerhans cell, alveolar macrophage or red-pulp macrophage), a precursor thereof, or a progenitor thereof.

[0265] In certain embodiments, a modified CD1 lb+CD45+cell disclosed herein expresses a detectable level of CD14 (e.g., CD1 lb+CD45+CD14+). In certain embodiments, the modified CD1 lb+CD45+cell disclosed herein does not express a detectable level of CD14 (e.g., CD1 lb+CD45+CD14‘). In certain embodiments, a modified CD1 lb+CD45+CD14‘ cell disclosed herein can mature into cells that express a detectable level of CD14. In certainembodiments, a modified CD1 lb+CD45+CD14+cell presented herein expresses a high level of CD 14 and does not express a detectable level of CD 16 (CD14hlghCD 16"). In certain embodiments, a modified CD1 lb+CD45+CD14+cell presented herein expresses a high level of CD14 and a detectable level of CD16 (CD14hlghCD16+). In certain embodiments, a modified CD1 lb+CD45+CD14+cell presented herein express a low level of CD14 and a high detectable level of CD 16 (CD14lowCD16hlgh) expression.

[0266] In certain embodiments, a modified CD1 lb+CD45+cell (e.g., CD1 lb+CD45+CD14+cell or CD1 lb+CD45+CD14‘ cell) disclosed herein has the properties of killing target cells (e.g, via CAR or monoclonal antibody (mAb) targeting), phagocytosing particles (e.g, bacteria), and / or migrating towards chemokines. In certain embodiments, a modified CD1 lb+CD45+cell (e.g., CD1 lb+CD45+CD14+cell or CD1 lb+CD45+CD14’ cell) disclosed herein exhibits increased killing of target cells (e.g., via CAR or monoclonal antibody (mAb) targeting), phagocytosing particles (e.g., bacteria), and / or migrating towards chemokines relative to a non-modified CD1 lb+CD45+cell (e.g., a non-modified CD1 lb+CD45+CD14+cell or non-modified CD1 lb+CD45+CD14‘ cell). Any assays known in the art can be used for measuring these properties, for example, the assays disclosed in Section 5.10

[0267] In certain embodiments, the modified CD1 lb+CD45+cell is derived from a modified pluripotent cell (such as a modified pluripotent cell described in Section 5.1.2). In certain embodiments, the modified pluripotent cell is generated according to the methods provided herein (such as the methods described in Section 5.2.1), and the modified pluripotent cell is differentiated into a CD1 lb+CD45+cell. Techniques known to one of skill in the art or described herein (such as in Section 5.9) may be used to differentiate a pluripotent cell into a CD1 lb+CD45+cell. For example, a homogeneous population of modified CD1 lb+CD45+cells or a cell population comprising a modified CD1 lb+CD45+cell disclosed herein (e.g., at least about 95% of the cells in the cell population are the CD1 lb+CD45+cells) may be produced, selected from and / or assessed via CD1 lb and CD45 markers.

[0268] In certain embodiments, the modified CD1 lb+CD45+cell provided herein is derived from an induced pluripotent stem cell (iPSC). In certain embodiments, the iPSC has been reprogrammed from a peripheral blood mononuclear cell (PBMC), a CD34+cord blood cell, an immune cell, a macrophage, a monocyte, or a fibroblast. In certain embodiments, the iPSC has been reprogrammed from a PBMC. In certain embodiments, the iPSC has been reprogrammed from a CD34+cord blood cell. In certain embodiments, the iPSC has beenreprogrammed from an immune cell. In certain embodiments, the iPSC has been reprogrammed from a macrophage. In certain embodiments, the iPSC has been reprogrammed from a monocyte. In certain embodiments, the iPSC has been reprogrammed from a fibroblast. In certain embodiments, the iPSC is a hiPSC. In certain embodiments, the iPSC is a TC-1133 cell.

[0269] The present disclosure further provides a homogeneous population of the modified CD1 lb+CD45+cell disclosed herein (e.g., a modified CD1 lb+CD45+cell described in Section 5.1.6). In certain embodiments, the homogeneous population of modified monocytes are isogenic.

[0270] The present disclosure further provides a population of cells comprising a modified CD1 lb+CD45+cell disclosed herein wherein at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.8%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 90% to about 100%, from about 95% to about 98%, from about 95% to about 99%, or from about 95% to 100% of the cells in the population are the modified CD1 lb+CD45+cells.

[0271] In certain embodiments, the modified CD1 lb+CD45+cell disclosed herein is homozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene. In certain embodiments, the modified CD1 lb+CD45+cell provided herein is heterozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene.

[0272] In certain embodiments, the modified CD1 lb+CD45+cell comprises genetic disruption of an MIR146A gene. In certain embodiments, the modified CD1 lb+CD45+cell comprises genetic disruption of a STAT6 gene. In certain embodiments, the modified CD1 lb+CD45+cell comprises genetic disruption of a GPR65 gene. In certain embodiments, the modified CD1 lb+CD45+cell comprises genetic disruption of an AKT1 gene. In certain embodiments, the modified CD1 lb+CD45+cell comprises genetic disruption of an AKT2 gene. In certain embodiments, the modified CD1 lb+CD45+cell comprises genetic disruption of an IRF3 gene. In certain embodiments, the modified CD1 lb+CD45+cell comprises genetic disruption of an IRF4 gene. In certain embodiments, the modified CD1 lb+CD45+cell comprises genetic disruption of a PIK3CG gene. In certain embodiments, the modifiedCD1 lb+CD45+cell comprises genetic disruption of a TSC1 gene. In certain embodiments, the modified CD1 lb+CD45+cell comprises genetic disruption of a VSIG4 gene.

[0273] In certain embodiments, the modified CD1 lb+CD45+cell comprises genetic disruption of two genes of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, and a VSIG4 gene. In certain embodiments, the modified CD1 lb+CD45+cell comprises genetic disruption of three, four, five, six, seven, eight, nine, or all genes of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, and a VSIG4 gene.

[0274] In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises genetic disruption of a SIRPA gene or a SIGLEC10 gene. In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises genetic disruption of a SIRPA gene and a SIGLEC10 gene. In certain embodiments, the modified CD1 lb+CD45+cell provided herein is homozygous for the genetic disruption of the SIRPA gene and / or SIGLEC10 gene. In certain embodiments, the modified CD1 lb+CD45+cell provided herein is heterozygous for the genetic disruption of the SIRPA gene and / or SIGLEC10 gene.

[0275] In certain embodiments, presented herein is a CD1 lb+CD45+cell, e.g., a CD1 lb+CD45+CD14+cell, that comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the CDl lb+CD45+cell, e.g., CD1 lb+CD45+CD14+cell, comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor, and does not comprise genetic disruption of a MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene. In certain embodiments, the cytokine receptor is IL- 10R. In certain embodiments, the cytokine receptor is a TGFpR. In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII, or TGFpRIII. In certain embodiments, the cytokine receptor is a TGFpRII. In particular embodiments, the CD1 lb+CD45+cell, e.g., CD1 lb+CD45+CD14+cell, is a human CD1 lb+CD45+cell, e.g., a humanCD1 lb+CD45+CD14+cell. In particular embodiments, the CD1 lb+CD45+cell is derived from a pluripotent cell, for example, a human pluripotent cell. In particular embodiments, the CD1 lb+CD45+cell is derived from an induced pluripotent stem cell, for example, a human induced pluripotent stem cell. In particular embodiments, the CD1 lb+CD45+cell is derived from a myeloid progenitor cell, for example, a human myeloid progenitor cell.

[0276] In certain embodiments, the CD1 lb+CD45+cell comprises genetic disruption of a gene encoding IL-10R. In certain embodiments, the CD1 lb+CD45+cell comprises genetic disruption of an IL10RA gene and / or an IL10RB gene. In certain embodiments, the CD1 lb+CD45+cell comprises genetic disruption of a gene encoding a TGFpR (e.g., TGFpRI, TGFpRII, or TGFpRIII). In certain embodiments, the CD1 lb+CD45+cell comprises genetic disruption of a gene encoding TGFpRII. In certain embodiments, the CD1 lb+CD45+cell comprises genetic disruption of a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene. In certain embodiments, the CD1 lb+CD45+cell comprises genetic disruption of a TGFBR2 gene. In certain embodiments, the mCDl lb+CD45+cell is homozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the CD1 lb+CD45+cell is homozygous for the genetic disruption of the IL 1 ORA gene, IL 1 ORB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene. In certain embodiments, the CD1 lb+CD45+cell is heterozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the CD1 lb+CD45+cell is heterozygous for the genetic disruption of the IL10RA gene, IL10RB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene.

[0277] In certain embodiments, the CD1 lb+CD45+cell comprises an exogenous polynucleotide encoding a dominant-negative cytokine receptor. In certain embodiments, the CD1 lb+CD45+cell comprises an exogenous polynucleotide encoding a dnIL-lOR. In certain embodiments, the CD1 lb+CD45+cell comprises an exogenous polynucleotide encoding a dnTGFpR. In certain embodiments, the CD1 lb+CD45+cell comprises an exogenous polynucleotide encoding a dnTGFpRI, a dnTGFpRII, or a dnTGFpRIII. In certain embodiments, the CD1 lb+CD45+cell comprises an exogenous polynucleotide encoding a dnTGFpRII. An exemplary dnTGFpRII is disclosed in Immunity. 2000 Feb;12(2): 171-81. In certain embodiments, an exogenous polynucleotide encoding a dnTGFpR disclosed herein comprises a nucleotide sequence set forth in SEQ ID NO: 1. In certain embodiments, a dnTGFpR disclosed herein comprises an amino acid sequence set forth in SEQ ID NO: 2.

[0278] In certain embodiments, the modified CD1 lb+CD45+cell provided herein comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the modified CD1 lb+CD45+cell provided herein comprises a genomic edit that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor, and comprises at least one of an MIR146A gene, a STAT6 gene, a GPR65 gene, an AKT1 gene, an AKT2 gene, an IRF3 gene, an IRF4 gene, a PIK3CG gene, a TSC1 gene, or a VSIG4 gene. Incertain embodiments, the cytokine receptor is IL-10R. In certain embodiments, the cytokine receptor is a TGFpR. In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII, or TGFpRIII. In certain embodiments, the cytokine receptor is a TGFpRII.

[0279] In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises genetic disruption of a gene encoding IL-10R. In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises genetic disruption of an IL10RA gene and / or an IL10RB gene. In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises genetic disruption of a gene encoding a TGFpR (e.g., TGFpRI, TGFpRII, or TGFpRIII). In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises genetic disruption of a gene encoding TGFpRII. In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises genetic disruption of a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene. In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises genetic disruption of a TGFBR2 gene. In certain embodiments, the modified CD1 lb+CD45+cell provided herein is homozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the modified CD1 lb+CD45+cell provided herein is homozygous for the genetic disruption of the IL10RA gene, IL10RB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene. In certain embodiments, the modified CD1 lb+CD45+cell provided herein is heterozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the modified CD1 lb+CD45+cell provided herein is heterozygous for the genetic disruption of the IL10RA gene, IL 1 ORB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene.

[0280] In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises an exogenous polynucleotide encoding a dominant-negative cytokine receptor. In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises an exogenous polynucleotide encoding a dnIL-lOR. In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises an exogenous polynucleotide encoding a dnTGFpR. In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises an exogenous polynucleotide encoding a dnTGFpRI, a dnTGFpRII, or a dnTGFpRIII. In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises an exogenous polynucleotide encoding a dnTGFpRII. An exemplary dnTGFpRII is disclosed in Immunity. 2000 Feb;12(2): 171-81. In certain embodiments, an exogenous polynucleotide encoding a dnTGFpR disclosed herein comprises a nucleotidesequence set forth in SEQ ID NO: 1. In certain embodiments, a dnTGFpR disclosed herein comprises an amino acid sequence set forth in SEQ ID NO: 2.

[0281] In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises an exogenous polynucleotide encoding a proinflammatory cytokine (e.g., IL-12 and / or IFNy). In certain embodiments, the proinflammatory cytokines are selected from the group consisting of IL-1, IL-2, IL-6, IL-12, IL-17, IL-18, IL-23, IFNy, MCP-1, and TNFa. In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises an exogenous polynucleotide encoding IL-12. In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises an exogenous polynucleotide encoding IFNy. In certain embodiments, the modified CD1 lb+CD45+ cell provided herein further comprises one or more exogenous polynucleotides encoding IL-12 and IFNy.

[0282] In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises a CAR (such as a CAR described in Section 5.3). In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises a polynucleotide encoding a CAR (such as a CAR described in Section 5.3) or a component thereof.

[0283] In certain embodiments, the modified CD1 lb+CD45+cell provided herein comprises (i) genetic disruption of at least one gene of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene; and (ii) a CAR (such as a CAR described in Section 5.3). In certain embodiments, the CAR increases secretion of a pro-inflammatory cytokine (e.g., is a CAR described in PCT / US2023 / 071207). In certain embodiments, the intracellular domain of the CAR is selected from the group consisting of a CD40, a TLR, and an IFNyR intracellular domain.

[0284] In certain embodiments, a polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding the proinflammatory cytokine (e.g., IL-12 and / or IFNy), and / or an exogenous polynucleotide encoding the dominant-negative cytokine receptor (e.g., dnIL-lOR, dnTGFpRI, dnTGFpRII, or dnTGFpRIII) is integrated into one or more genes disclosed herein (e.g., an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPA gene, a SIGLEC10 gene, a B2M gene, a CIITA gene, an RFX gene, an IL 1 ORA gene, an IL 1 ORB gene, a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene).

[0285] In certain embodiments, a polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding theproinflammatory cytokine (e.g., IL-12 and / or ZFNy), and / or an exogenous polynucleotide encoding the dominant-negative cytokine receptor (e.g., dnIL-lOR, dnTGFpRI, dnTGFpRII, or dnTGFpRIII) is integrated into the modified CD1 lb+CD45+cell using the SLEEK technology. In certain embodiments, the polynucleotide encoding the CAR, the exogenous polynucleotide encoding the proinflammatory cytokine, and / or the exogenous polynucleotide encoding the dominant-negative cytokine receptor is integrated in frame with and downstream (3’) of a coding sequence of an essential gene, and wherein at least part of the essential gene comprises an exogenous coding sequence. In certain embodiments, the correct knock-in cells would retain essential gene function while also integrating the polynucleotide encoding the CAR, the exogenous polynucleotide encoding the proinflammatory cytokine, and / or the exogenous polynucleotide encoding the dominant-negative cytokine receptor. Cells with non-productive insertions and deletions would undergo negative selection. In certain embodiments, the essential gene encodes a gene product that is required for survival and / or proliferation of the cell. In certain embodiments, the essential gene is a housekeeping gene. In certain embodiments, the essential gene encodes GAPDH. In certain embodiments, the essential gene is an essential gene as disclosed in International Patent Publication WO2022235811.

[0286] In certain embodiments, a polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding the proinflammatory cytokine, and / or an exogenous polynucleotide encoding the dominantnegative cytokine receptor is integrated into a safe harbor locus, e.g., an AAVS1 locus.

[0287] In certain embodiments, the modified CD1 lb+CD45+cell further comprises (i) genetic disruption of a B2M gene, (ii) genetic disruption of a CIITA gene, (iii) genetic disruption of an RFX gene, and / or (iv) an exogenous polynucleotide encoding HLA-E. In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises genetic disruption of (i) a B2M gene, a CIITA gene and / or an RFX gene, and (ii) an exogenous polynucleotide encoding HLA-E. In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises genetic disruption of a B2M gene and a CIITA gene (e.g., B2M / CIITA double knockout), and an exogenous polynucleotide encoding HLA-E. In certain embodiments, the modified CD1 lb+CD45+cell provided herein further comprises genetic disruption of a B2M gene, a CIITA gene, and an RFX gene, and an exogenous polynucleotide encoding HLA-E.

[0288] In certain embodiments, the modified CD1 lb+CD45+cell provided herein is homozygous for the genetic disruption of the B2M gene, CIITA gene, and / or RFX gene. Incertain embodiments, the modified CD1 lb+CD45+cell provided herein is heterozygous for the genetic disruption of the B2M gene, CIITA gene, and / or RFX gene.

[0289] In certain embodiments, genetic disruption of a gene disclosed herein (e.g., an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPA gene, a SIGLEC10 gene, a B2M gene, a CIITA gene, an RFX gene, an IL 1 ORA gene, an IL 1 ORB gene, a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene) is performed by a genome editing tool, such as TALEN, ZFN, or CRISPR (e.g., by deletion of one or more exons, introduction of a stop codon, introduction of a null mutation or inactivation of the promoter).5.2 Methods of Generating Modified Cells

[0290] Various techniques known to one of skill in the art or described herein may be used to disrupt a gene or expression of the corresponding protein encoded by the gene disclosed herein (e.g., an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPA gene, a SIGLEC10 gene, a B2M gene, a CIITA gene, an RFX gene, an IL 1 ORA gene, an IL 1 ORB gene, a TGFBR1 gene, a TGFBR2 gene, or a TGFBR3 gene).

[0291] Exemplary techniques include genome editing tools, such as TALEN (Transcription Activator-Like Effector Nucleases), Zinc-finger nucleases (ZFN), and CRISPR, as well as variations thereof (e.g., base editing, prime editing, etc.); RNA interference (RNAi) (e.g., shRNA, or siRNA), or Cre / LoxP -based conditional knockout. In certain embodiments, genetic disruption deletes expression of the corresponding protein encoded by the gene. In certain embodiments, genetic disruption prevents or reduces expression of the full-length protein encoded by the gene.

[0292] In certain embodiments, the gene editing system comprises a CRISPR system. In certain embodiments, the CRISPR system comprises a Class 2 CRISPR system. Class 2 systems currently represent a single protein that is categorized into three distinct types (types II, V and VI). Any class 2 CRISPR system suitable for gene editing, for example a type II, a type V, or a type VI system, is envisaged as within the scope of the instant disclosure.Exemplary Class 2 type II CRISPR systems include Cas9, Csn2 and Cas4. Exemplary Class 2, type V CRISPR systems include, Cast 2, Cast 2a (e.g., Cpfl, MAD7), Cast 2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f, Casl2g, Casl2h, Casl2i and Cast 2k (C2c5). Exemplary Class 2 Type VI systems include Cast 3, Cast 3a (C2c2) Cast 3b, Casl3c and Casl3d.

[0293] The endonuclease protein (e.g., nucleic acid-directed nuclease) may be derived from any bacterial or archaeal Cas protein. Any suitable CRISPR system is contemplated as within the scope of the instant disclosure. In certain embodiments, the endonuclease protein comprises one or more of Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Casl2, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. In certain embodiments, the endonuclease protein is a Cas9 protein, a Cpfl protein, a C2cl protein, a C2c2 protein, a C2c3 protein, Cas3, Cas3-HD, Cas5, Cas7, Cas8, CaslO, Cas 12, modified versions thereof, or combinations or complexes of these.

[0294] In certain embodiments, the Class 2 CRISPR system comprises a type V system. In certain embodiments, the Type V system comprises a Casl2a CRISPR / Cas protein. Exemplary Cas 12a proteins include MAD7, isolated from Eubacterium rectale. MAD7 uses T-rich protospacer adjacent motifs (PAMs) such as YTTN. Exemplary MAD7 proteins are described in US 20190360001 and WO 2021119563, the contents of which are incorporated by reference herein in their entireties.

[0295] In general, the nuclease can be delivered to the cell as a protein or a nucleic acid encoding the protein, e.g., a DNA molecule or mRNA molecule. The protein or nucleic acid can be combined with other delivery agents, e.g., lipids or polymers in a lipid or polymer nanoparticle and targeting agents such as antibodies or other binding agents with specificity for the cell. The DNA molecule can be a nucleic acid vector, such as a viral genome or circular double-stranded DNA, e.g., a plasmid. Nucleic acid vectors encoding a nuclease can include other coding or non-coding elements. For example, a nuclease can be delivered as part of a viral genome (e.g., in an AAV, adenoviral or lentiviral genome) that includes certain genomic backbone elements (e.g., inverted terminal repeats, in the case of an AAV genome).

[0296] A CRISPR / Cas nuclease can be delivered to the cell as a protein or a nucleic acid encoding the protein, e.g., a DNA molecule or mRNA molecule. The guide molecule can be delivered as an RNA molecule or encoded by a DNA molecule. A CRISPR / Cas nuclease can also be delivered with a guide molecule as a ribonucleoprotein (RNP) and introduced into the cell via nucleofection (electroporation).

[0297] Methods for delivering the genome editing tools and systems can vary depending on the need. In certain embodiments, the components of a selected genome editing method are delivered as DNA constructs in one or more plasmids. In certain embodiments, the components of a selected genome editing method are delivered in a lipid orpolymer nanoparticle. In certain embodiments, the components are delivered via viral vectors (e.g., adeno-associated viral vectors (e.g., AAV1, AAV2, AAV5, AAV6, AAV8, AAV9), retroviral vectors, lentiviral vectors, or adenoviral vectors (e.g., Ad5F35, Ad5)). Common delivery methods include but is not limited to, electroporation, microinjection, gene gun, impalefection, hydrostatic pressure, continuous infusion, sonication, magnetofection, adeno-associated viruses, envelope protein pseudotyping of viral vectors, replication- competent vectors cis and trans-acting elements, herpes simplex virus, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides).

[0298] In certain embodiments, genetic disruption of a gene disclosed herein (e.g., an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPA gene, a SIGLEC10 gene, a B2M gene, a CIITA gene, an RFX gene, an IL 1 ORA gene, an IL 1 ORB gene, a TGFBR1 gene, a TGFBR2 gene, or a TGFBR3 gene) is performed by a genome editing tool, such as TALEN, ZFN, or CRISPR (e.g., by deletion of one or more exons, introduction of a stop codon, introduction of a null mutation or inactivation of the promoter). In certain embodiments, genetic disruption of a gene disclosed herein is performed using gRNA. In certain embodiments, genetic disruption of a gene disclosed herein is performed using gRNA and a Casl2a protein. In certain embodiments, genetic disruption of a gene disclosed herein is performed using gRNA and a MAD7 protein. In certain embodiments, the gRNA comprises a targeting sequence complementary to a target sequence of a gene disclosed herein. In certain embodiments, the target sequence comprises a coding sequence of a gene disclosed herein, for example mRNA sequence. In certain embodiments, the gRNA targets an exon of a gene disclosed herein. In certain embodiments, the target sequence comprises non-coding sequence. Exemplary non-coding sequence includes intronic, promoter, 5’ untranslated region (UTR), 3’ UTR, or enhancer sequence of a gene disclosed herein. In certain embodiments, genetic disruption of a gene disclosed herein is performed by RNAi. In certain embodiments, genetic disruption of a gene disclosed herein is performed by conditional knockout. In certain embodiments, the genetic disruption of a gene disclosed herein is performed in human cells.

[0299] In certain embodiments, the methods provided herein comprise selecting a modified cell that comprises genetic disruption of a gene disclosed herein e.g., an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPA gene, a SIGLEC10gene, a B2M gene, a CIITA gene, an RFX gene, an IL 1 ORA gene, an IL 1 ORB gene, a TGFBR1 gene, a TGFBR2 gene, or a TGFBR3 gene). In certain embodiments, the methods provided herein comprise selecting a modified cell that comprises genetic disruption of a gene disclosed herein (e.g., an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPA gene, a SIGLEC10 gene, a B2M gene, a CIITA gene, an RFX gene, an IL 1 ORA gene, an IL10RB gene, a TGFBR1 gene, a TGFBR2 gene, or a TGFBR3 gene), and an undetectable level of nonspecific genetic modifications.

[0300] Various techniques known to one of skill in the art or described herein may be used to introduce an exogenous polynucleotide encoding a protein (e.g. a CAR, a proinflammatory cytokine, or a dominant-negative cytokine receptor) in a modified cell disclosed herein. In certain embodiments, a polynucleotide encoding a CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding a proinflammatory cytokine (e.g., IL-12 and / or IFNy), and / or an exogenous polynucleotide encoding a dominant-negative cytokine receptor (e.g., dnIL-lOR, dnTGFpRI, dnTGFpRII, or dnTGFpRIII) is integrated into one or more genes disclosed herein (e.g., an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPA gene, a SIGLEC10 gene, a B2M gene, a CIITA gene, an RFX gene, an IL 1 ORA gene, an IL 1 ORB gene, a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene).

[0301] In certain embodiments, a polynucleotide encoding a CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding a proinflammatory cytokine (e.g., IL-12 and / or fFNy), and / or an exogenous polynucleotide encoding a dominant-negative cytokine receptor (c.g, dnIL-lOR, dnTGFpRI, dnTGFpRII, or dnTGFpRIII) is integrated into a modified cell using the SLEEK technology, which is described in Allen et al., A highly efficient transgene knock-in technology in clinically relevant cell types. Nat Biotechnol (2023) and International Patent Publication WO2022235811, the content of each of which is incorporated by reference in its entirety. In certain embodiments, the polynucleotide encoding the CAR, the exogenous polynucleotide encoding the proinflammatory cytokine, and / or an exogenous polynucleotide encoding the dominant-negative cytokine receptor is integrated in frame with and downstream (3’) of a coding sequence of an essential gene, and wherein at least part of the essential gene comprises an exogenous coding sequence. In certain embodiments, the correct knock-in cells would retain essential gene function while also integrating the polynucleotide encoding theCAR, the exogenous polynucleotide encoding the proinflammatory cytokine, and / or the exogenous polynucleotide encoding the dominant-negative cytokine receptor. Cells with non-productive insertions and deletions would undergo negative selection. In certain embodiments, the essential gene encodes a gene product that is required for survival and / or proliferation of the cell. In certain embodiments, the essential gene is a housekeeping gene. In certain embodiments, the essential gene encodes glyceraldehyde 3 -phosphate dehydrogenase (GAPDH). In certain embodiments, the essential gene is an essential gene as disclosed in International Patent Publication WO2022235811.

[0302] In certain embodiments, a polynucleotide encoding a CAR (such as a polynucleotide described in Section 5.4), an exogenous polynucleotide encoding a proinflammatory cytokine, and / or an exogenous polynucleotide encoding a dominantnegative cytokine receptor is integrated into a safe harbor locus, e.g., an AAVS1 locus.5.2.1 Methods of Generating Modified Pluripotent Cells

[0303] The present disclosure further provides a method of generating a modified pluripotent cell, comprising genetically disrupting in a pluripotent cell at least one gene (e.g., one, two, three, four, five, six, seven, eight, nine, or all gene(s)) of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.

[0304] In certain embodiments, the modified pluripotent cell is homozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene. In certain embodiments, the modified pluripotent cell is heterozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene.

[0305] In certain embodiments, the method comprises genetically disrupting in a pluripotent cell an MIR146A gene. In certain embodiments, the method comprises genetically disrupting in a pluripotent cell a STAT6 gene. In certain embodiments, the method comprises genetically disrupting in a pluripotent cell a GPR65 gene. In certain embodiments, the method comprises genetically disrupting in a pluripotent cell an AKT1 gene. In certain embodiments, the method comprises genetically disrupting in a pluripotent cell an AKT2 gene. In certain embodiments, the method comprises genetically disrupting in a pluripotent cell an IRF3 gene. In certain embodiments, the method comprises genetically disrupting in a pluripotent cell an IRF4 gene. In certain embodiments, the method comprises genetically disrupting in a pluripotent cell a PIK3CG gene. In certain embodiments, themethod comprises genetically disrupting in a pluripotent cell a TSC1 gene. In certain embodiments, the method comprises genetically disrupting in a pluripotent cell a VSIG4 gene.

[0306] In certain embodiments, the method further comprises genetically disrupting in the pluripotent cell a SIRPA gene or a SIGLEC10 gene. In certain embodiments, the method further comprises genetically disrupting in the pluripotent cell a SIRPA gene and a SIGLEC10 gene.

[0307] In certain embodiments, the method further comprises obtaining a pluripotent cell comprising genetic disruption of a SIRPA gene or a SIGLEC10 gene. In certain embodiments, the method further comprises obtaining a pluripotent cell comprising genetic disruption of a SIRPA gene and a SIGLEC10 gene.

[0308] In certain embodiments, the modified pluripotent cell provided herein is homozygous for the genetic disruption of the SIRPA gene and / or SIGLEC10 gene. In certain embodiments, the modified pluripotent cell provided herein is heterozygous for the genetic disruption of the SIRPA gene and / or SIGLEC10 gene.

[0309] In certain embodiments, the method comprises genetically disrupting in a pluripotent cell a gene encoding a cytokine receptor. In certain embodiments, the pluripotent cell comprises genetic disruption of at least one of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene. In certain embodiments, the method further comprises genetically disrupting in the pluripotent cell a gene encoding IL-10R. In certain embodiments, the method further comprises genetically disrupting in the pluripotent cell an IL10RA gene and / or an IL10RB gene. In certain embodiments, the method further comprises genetically disrupting in the pluripotent cell a gene encoding a TGFpR (e.g., TGFpRI, TGFpRII, or TGFpRIII). In certain embodiments, the method further comprises genetically disrupting in the pluripotent cell a gene encoding a TGFpRII. In certain embodiments, the method further comprises genetically disrupting in the pluripotent cell a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene. In certain embodiments, the method further comprises genetically disrupting in the pluripotent cell a TGFBR2 gene.

[0310] In certain embodiments, the method further comprises obtaining a pluripotent cell comprising genetic disruption of a gene encoding a cytokine receptor. In certain embodiments, the method further comprises genetic disrupting in the pluripotent cell at least one of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene. In certainembodiments, the method further comprises obtaining a pluripotent cell comprising genetic disruption of a gene encoding IL-10R. In certain embodiments, the method further comprises obtaining a pluripotent cell comprising genetic disruption of an IL10RA gene and / or an IL10RB gene. In certain embodiments, the method further comprises obtaining a pluripotent cell comprising genetic disruption of a gene encoding a TGFpR (e.g., TGFpRI, TGFpRII, or TGFpRIII). In certain embodiments, the method further comprises obtaining a pluripotent cell comprising genetic disruption of a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene. In certain embodiments, the method further comprises obtaining a pluripotent cell comprising genetic disruption of a TGFBR2 gene.

[0311] In certain embodiments, the modified pluripotent cell provided herein is homozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the modified pluripotent cell provided herein is homozygous for the genetic disruption of the IL10RA gene, IL10RB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene. In certain embodiments, the modified pluripotent cell provided herein is heterozygous for the genetic disruption of the gene encoding IL-10R and / or the gene encoding a TGFpR. In certain embodiments, the modified pluripotent cell provided herein is heterozygous for the genetic disruption of the IL10RA gene, IL10RB gene, TGFBR1 gene, TGFBR2 gene, and / or TGFBR3 gene.

[0312] In certain embodiments, the method disclosed herein further comprises introducing an exogenous polynucleotide encoding a dominant-negative cytokine receptor to the pluripotent cell. In certain embodiments, the pluripotent cell comprises genetic disruption of at least one of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene. In certain embodiments, the method disclosed herein further comprises introducing an exogenous polynucleotide encoding a dnIL-lOR to the pluripotent cell. In certain embodiments, the method disclosed herein further comprises introducing to the pluripotent cell an exogenous polynucleotide encoding a dnTGFpR. In certain embodiments, the method disclosed herein further comprises introducing to the pluripotent cell an exogenous polynucleotide encoding a dnTGFpRI, a dnTGFpRII, or a dnTGFpRIII.

[0313] In certain embodiments, the method further comprises obtaining a pluripotent cell comprising an exogenous polynucleotide encoding a dominant-negative cytokine receptor to the pluripotent cell. In certain embodiments, the method further comprises genetic disrupting in the pluripotent cell at least one of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6gene, a TSC1 gene, or a VSIG4 gene. In certain embodiments, the method disclosed herein further comprises introducing an exogenous polynucleotide encoding a dnIL-lOR to the pluripotent cell. In certain embodiments, the method further comprises obtaining a pluripotent cell comprising an exogenous polynucleotide encoding a dnTGFpR. In certain embodiments, the method further comprises obtaining a pluripotent cell comprising an exogenous polynucleotide encoding a dnTGFpRI, a dnTGFpRII, or a dnTGFpRIII. In certain embodiments, the modified pluripotent cell provided herein comprises an exogenous polynucleotide encoding a dnTGFpRII. An exemplary dnTGFpRII is disclosed in Immunity. 2000 Feb;12(2): 171-81. In certain embodiments, an exogenous polynucleotide encoding a dnTGFpR disclosed herein comprises a nucleotide sequence set forth in SEQ ID NO: 1. In certain embodiments, a dnTGFpR disclosed herein comprises an amino acid sequence set forth in SEQ ID NO: 2.

[0314] In certain embodiments, the method disclosed herein further comprises obtaining a pluripotent cell comprising (i) genetic disruption of a B2M gene, (ii) genetic disruption of a CIITA gene, (iii) genetic disruption of an RFX gene, and / or (iv) an exogenous polynucleotide encoding HLA-E.

[0315] In certain embodiments, the method disclosed herein further comprises (i) genetically disrupting in the pluripotent cell a B2M gene, a CIITA gene, and / or an RFX gene, and / or (ii) introducing an exogenous polynucleotide encoding a HLA-E.

[0316] In certain embodiments, the modified pluripotent cell provided herein is homozygous for the genetic disruption of the B2M gene, CIITA gene, and / or RFX gene. In certain embodiments, the modified pluripotent cell provided herein is heterozygous for the genetic disruption of the B2M gene, CIITA gene, and / or RFX gene.

[0317] In certain embodiments, the method disclosed herein further comprises introducing an exogenous polynucleotide encoding a proinflammatory cytokine (e.g., IL- 12 and / or IFNy) into the pluripotent cell. In certain embodiments, the method further comprises obtaining a pluripotent cell comprising an exogenous polynucleotide encoding a proinflammatory cytokine (e.g., IL-12 and / or IFNy). In certain embodiments, the proinflammatory cytokines are selected from the group consisting of IL-1, IL-2, IL-6, IL-12, IL- 17, IL- 18, IL-23, fFNy, MCP-1, and TNFa.

[0318] In certain embodiments, the method disclosed herein further comprises introducing (a) a polynucleotide encoding a CAR (such as a polynucleotide described in Section 5.4), (b) a vector comprising a polynucleotide encoding a CAR (such as a vector described in Section 5.5), or (c) a CAR polypeptide (such as a polypeptide described inSection 5.6) into the pluripotent cell. In certain embodiments, the pluripotent cell comprises genetic disruption of at least one of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene. In certain embodiments, the CAR promotes Ml macrophage polarization. In certain embodiments, the CAR increases secretion of a pro-inflammatory cytokine (e.g., is a CAR described in PCT / US2023 / 071207). In certain embodiments, the intracellular domain of the CAR is selected from the group consisting of a CD40, a TLR, and an IFNyR intracellular domain.

[0319] In certain embodiments, the method disclosed herein further comprises obtaining a pluripotent cell comprising a CAR (such as a CAR described in Section 5.3). In certain embodiments, the pluripotent cell comprises (a) a polynucleotide encoding a CAR (such as a polynucleotide described in Section 5.4), (b) a vector comprising a polynucleotide encoding a CAR (such as a vector described in Section 5.5), or (c) a CAR polypeptide (such as a polypeptide described in Section 5.6). In certain embodiments, the method further comprises genetic disrupting in the pluripotent cell at least one of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene. In certain embodiments, the CAR promotes Ml macrophage polarization. In certain embodiments, the CAR increases secretion of a pro- inflammatory cytokine (e.g., is a CAR described in PCT / US2023 / 071207). In certain embodiments, the intracellular domain of the CAR is selected from the group consisting of a CD40, a TLR, and an IFNyR intracellular domain.

[0320] In certain embodiments, the polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), the exogenous polynucleotide encoding a proinflammatory cytokine (e.g., IL-12 and / or IFNy), and / or the exogenous polynucleotide encoding a dominant-negative cytokine receptor (c.g, dnIL-lOR, dnTGFpRI, dnTGFpRII, or dnTGFpRIII) is integrated into one or more genes disclosed herein (e.g., an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPA gene, a SIGLEC10 gene, a B2M gene, a CIITA gene, an RFX gene, an IL 1 ORA gene, an IL 1 ORB gene, a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene). In certain embodiments, the polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), the exogenous polynucleotide encoding a proinflammatory cytokine, and / or the exogenous polynucleotide encoding a dominant-negative cytokine receptor is integrated in frame with and downstream (3’) of a coding sequence of an essential gene, and wherein at least part of the essential genecomprises an exogenous coding sequence. In certain embodiments, the essential gene encodes a gene product that is required for survival and / or proliferation of the cell. In certain embodiments, the essential gene is a housekeeping gene. In certain embodiments, the essential gene encodes GAPDH. In certain embodiments, the essential gene is an essential gene as disclosed in International Patent Publication WO2022235811. In certain embodiments, the polynucleotide encoding the CAR (such as a polynucleotide described in Section 5.4), the exogenous polynucleotide encoding a proinflammatory cytokine, and / or the exogenous polynucleotide encoding a dominant-negative cytokine receptor is integrated into a safe harbor locus, e.g., an AAVS1 locus.

[0321] In certain embodiments, introducing the CAR, the exogenous polynucleotide encoding a proinflammatory cytokine, and / or the exogenous polynucleotide encoding a dominant-negative cytokine receptor is performed before, concurrently, or after the genetic disruption of one or more genes disclosed herein (e.g., an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, a VSIG4 gene, a SIRPA gene, a SIGLEC10 gene, a B2M gene, a CIITA gene, an RFX gene, an IL10RA gene, an IL10RB gene, a TGFBR1 gene, a TGFBR2 gene, and / or a TGFBR3 gene).

[0322] Pluripotent cells can give rise to a multiplicity of cell types, and include, for example, induced pluripotent stem cells (iPSCs). In certain embodiments, a pluripotent stem cell as described herein is a mammalian pluripotent cell, e.g., a mammalian iPSC. In certain embodiments, a pluripotent stem cell as described herein is a human pluripotent cell, e.g., a human iPSC. In certain embodiments, a pluripotent cell as described herein is a multipotent cell, e.g., a human multipotent cell. In certain embodiments, a pluripotent cell as described herein is a hematopoietic stem cell (HSC), e.g., a human HSC. In certain embodiments, a pluripotent cell as described herein is an embryonic stem cell (ESC), for example, a human embryonic stem cell. In certain non-limiting embodiments, a pluripotent cell as described herein is a parthenogenic stem cell, e.g., a human parthenogenetic stem cell, a primordial germ cell-like pluripotent stem cell, e.g., a human primordial germ cell-like pluripotent stem cell, an epiblast stem cell, e.g., a human epiblast stem cell, an F-class pluripotent stem cell, e.g., a human F-class pluripotent stem cell, a somatic stem cell, e.g., a human somatic stem cell, or any other cell, e.g., human cell, capable of lineage specific differentiation. In certain embodiments, a pluripotent cell described herein is a non-human pluripotent cell. In certain embodiments, a pluripotent cell as described herein is a nonhuman primate pluripotent cell.In certain embodiments, a pluripotent cell as described herein is a rodent, e.g., mouse, pluripotent cell.

[0323] In certain embodiments, a pluripotent cell as described herein is not an embryonic stem cell, for example, is not a human embryonic stem cell. In certain embodiments, a pluripotent cell as described herein is not capable of differentiating or developing into all cell types. For example, in certain embodiments, a human pluripotent cell as described herein is not capable of differentiating or developing into all cell types of the human body.

[0324] In certain embodiments, the pluripotent cell provided herein is an iPSC. In certain embodiments, the pluripotent cell provided herein is a human induced pluripotent stem cell (hiPSC). In certain embodiments, the modified pluripotent cell provided herein has been reprogrammed from a human umbilical cord blood cell (e.g., a human CD34+cord blood cell). In certain embodiments, the pluripotent cell provided herein is a TC-1133 cell.

[0325] In certain embodiments, the pluripotent cell provided herein is a hematopoietic stem cell, e.g., a human hematopoietic stem cell. In certain embodiments, a hematopoietic stem cell is produced from a modified iPSC as described herein.

[0326] In certain embodiments, the iPSC has been reprogrammed from a peripheral blood mononuclear cell (PBMC), a CD34+cord blood cell, an immune cell, a macrophage, a monocyte, or a fibroblast. In certain embodiments, the iPSC has been reprogrammed from a PBMC. In certain embodiments, the iPSC has been reprogrammed from a CD34+cord blood cell. In certain embodiments, the iPSC has been reprogrammed from an immune cell. In certain embodiments, the iPSC has been reprogrammed from a macrophage. In certain embodiments, the iPSC has been reprogrammed from a monocyte. In certain embodiments, the iPSC has been reprogrammed from a fibroblast.5.2.2 Methods of Generating Modified Myeloid Progenitor Cells

[0327] Also provided herein is a method of generating a homogenous population of modified myeloid progenitor cell or a cell population comprising a modified myeloid progenitor cell disclosed herein (such as the cell populations disclosed described in Section 5.1.3), comprising expanding and differentiating a modified pluripotent cell (such as the modified pluripotent cells described in Section 5.1.2) under conditions sufficient for cell differentiation into a population of myeloid progenitor cells (e.g., the differentiation methods disclosed in Section 5.9). Also provided herein is a method of generating a modified myeloid progenitor cell (such as the modified myeloid progenitor cell described in Section 5.1.3), a homogenous population of modified myeloid progenitor cell or a cell population comprisinga modified myeloid progenitor cell disclosed herein (such as the cell populations disclosed described in Section 5.1.3), comprising differentiating a modified pluripotent cell (such as the modified pluripotent cells described in Section 5.1.2) under conditions sufficient for cell differentiation into a myeloid progenitor cells (e.g., the differentiation methods disclosed in Section 5.9). In certain embodiments, the modified pluripotent cell provided herein is an iPSC. Techniques known to one of skill in the art or described herein (such as in Section 5.9) may be used to differentiate a pluripotent cell into a myeloid progenitor cell.

[0328] The present disclosure also provides a method of generating a modified myeloid progenitor cell, comprising genetically disrupting in a myeloid precursor cell at least one gene (e.g., one, two, three, four, five, six, seven, eight, nine, or all gene(s)) of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.

[0329] In certain embodiments, the modified myeloid precursor cell is homozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene. In certain embodiments, the modified myeloid precursor cell is heterozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene.

[0330] The present disclosure also provides a method of generating a myeloid progenitor cell, for example, human myeloid progenitor cell, comprising genome editing that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the cytokine receptor is a TGFP receptor (TGFpR). In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII, or TGFpRIII. In certain embodiments, the modified myeloid progenitor cell does not comprise genetic disruption of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.

[0331] The present disclosure also provides a method of generating a modified myeloid progenitor cell, comprising genome editing that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the cytokine receptor is a TGFP receptor (TGFpR). In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII, or TGFpRIII. In certain embodiments, the cytokine receptor is a TGFpRII. In certain embodiments, the modified myeloid progenitor cell comprises genetic disruption of at least one of an AKT1 gene, an AKT2 gene, a GPR65gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.

[0332] The present disclosure also provides a method of generating a modified myeloid progenitor cell, comprising introducing a polynucleotide encoding a CAR (such as a CAR described in Section 5.3). In certain embodiments, the CAR promotes Ml macrophage polarization. In certain embodiments, the CAR increases secretion of a pro-inflammatory cytokine (e.g., is a CAR described in PCT / US2023 / 071207). In certain embodiments, the intracellular domain of the CAR is selected from the group consisting of a CD40, a TLR, and an IFNyR intracellular domain. In certain embodiments, the modified myeloid progenitor cell comprises genetic disruption of at least one of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.5.2.3 Methods of Generating Modified Monocytes

[0333] Also provided herein is a method of generating a homogenous population of modified monocytes or a cell population comprising a modified monocyte disclosed herein (such as the cell populations disclosed described in Section 5.1.4), comprising expanding and differentiating a modified pluripotent cell (such as the modified pluripotent cells described in Section 5.1.2) under conditions sufficient for cell differentiation into a population of monocytes (e.g., the differentiation methods disclosed in Section 5.9). In another aspect, provided herein is a method of generating a modified monocyte (such as the modified monocytes described in Section 5.1.4), a homogenous population of modified monocytes or a cell population comprising a modified monocyte disclosed herein (such as the cell populations disclosed described in Section 5.1.4), comprising differentiating a modified pluripotent cell (such as the modified pluripotent cells described in Section 5.1.2) under conditions sufficient for cell differentiation into a monocyte. In a further aspect, provided herein is a method of generating a homogenous population of modified monocytes or a cell population comprising a modified monocyte disclosed herein (such as the cell populations disclosed described in Section 5.1.4), comprising expanding and differentiating a modified myeloid progenitor cell (such as the modified myeloid progenitor cell described in Section 5.1.3) under conditions sufficient for cell differentiation into a population of monocytes (e.g., the differentiation methods disclosed in Section 5.9). In yet another aspect, provided herein is a modified monocytes (such as the modified monocytes described in Section 5.1.4), a homogenous population of modified monocytes or a cell population comprising a modified monocyte disclosed herein (such as the cell populations disclosed described in Section 5.1.4),comprising differentiating a modified myeloid progenitor cell (such as the modified myeloid progenitor cell described in Section 5.1.3) under conditions sufficient for cell differentiation into a monocyte (e.g., the differentiation methods disclosed in Section 5.9). In certain embodiments, the modified pluripotent cell provided herein is an iPSC. Techniques known to one of skill in the art or described herein (such as in Section 5.9) may be used to differentiate a pluripotent cell into a monocyte or a myeloid progenitor cell into a monocyte.

[0334] The present disclosure also provides a method of generating a modified monocyte, comprising genetically disrupting in a myeloid precursor cell at least one gene e.g., one, two, three, four, five, six, seven, eight, nine, or all gene(s)) of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.

[0335] In certain embodiments, the modified monocyte is homozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene. In certain embodiments, the modified monocyte is heterozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene.

[0336] The present disclosure also provides a method of generating a monocyte, for example a human monocyte, comprising genome editing that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the cytokine receptor is a TGFP receptor (TGFpR). In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII, or TGFpRIII. In certain embodiments, the cytokine receptor is a TGFpRII. In certain embodiments, the cytokine receptor is a TGFpRII. In certain embodiments, the modified monocyte does not comprise genetic disruption of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.

[0337] The present disclosure also provides a method of generating a modified monocyte, comprising genome editing that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the cytokine receptor is a TGFP receptor (TGFpR). In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII, or TGFpRIII. In certain embodiments, the cytokine receptor is a TGFpRII. In certain embodiments, the cytokine receptor is a TGFpRII. In certain embodiments, the modified monocyte comprises genetic disruption of at least one of anAKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.

[0338] The present disclosure also provides a method of generating a modified monocyte, comprising introducing a polynucleotide encoding a CAR (such as a CAR described in Section 5.3). In certain embodiments, the CAR promotes Ml macrophage polarization. In certain embodiments, the CAR increases secretion of a pro-inflammatory cytokine (e.g., is a CAR described in PCT / US2023 / 071207). In certain embodiments, the intracellular domain of the CAR is selected from the group consisting of a CD40, a TLR, and an IFNyR intracellular domain. In certain embodiments, the modified monocyte comprises genetic disruption of at least one of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.5.2.4 Methods of Generating Modified Macrophages

[0339] Also provided herein is a method of generating a homogenous population of modified macrophages or a cell population comprising a modified macrophage disclosed herein (such as the cell populations disclosed described in Section 5.1.5), comprising expanding and differentiating a modified pluripotent cell (such as the modified pluripotent cells described in Section 5.1.2) under conditions sufficient for cell differentiation into a population of macrophages (e.g., the differentiation methods disclosed in Section 5.9). In another aspect, provided herein is a method of generating a modified macrophage (such as the modified macrophage described in Section 5.1.5), a homogenous population of modified macrophages or a cell population comprising a modified macrophage disclosed herein (such as the cell populations disclosed described in Section 5.1.5), comprising differentiating a modified pluripotent cell (such as the modified pluripotent cells described in Section 5.1.2) under conditions sufficient for cell differentiation into a macrophage (e.g., the differentiation methods disclosed in Section 5.9). In certain embodiments, the modified macrophage provided herein is an immature macrophage. In certain embodiments, the immature macrophage has not been subjected to any maturation or polarization process. In certain embodiments, the modified macrophage provided herein is a tissue-resident macrophage (e.g., an adipose-associated macrophage, osteoblast, microglia, motile liver macrophage, perivascular macrophage, meningeal macrophage, intestinal macrophage, Kupffer cell, Langerhans cell, alveolar macrophage or red-pulp macrophage).

[0340] In a further aspect, provided herein is a method of generating a homogenous population of modified macrophage or a cell population comprising a modified macrophagedisclosed herein (such as the cell populations disclosed described in Section 5.1.5), comprising expanding and differentiating a modified myeloid progenitor cell (such as the modified myeloid progenitor cell described in Section 5.1.3) under conditions sufficient for cell differentiation into a population of macrophages (e.g., the differentiation methods disclosed in Section 5.9). In yet another aspect, provided herein is a method of generating a modified macrophage (such as the modified macrophage described in Section 5.1.5), a homogenous population of modified macrophages or a cell population comprising a modified macrophage disclosed herein (such as the cell populations disclosed described in Section 5.1.5), comprising differentiating a modified myeloid progenitor cell (such as the modified myeloid progenitor cell described in Section 5.1.3) under conditions sufficient for cell differentiation into a macrophage (e.g., the differentiation methods disclosed in Section 5.9). In certain embodiments, the modified macrophage provided herein is an immature macrophage. In certain embodiments, the immature macrophage has not been subjected to any maturation or polarization process.

[0341] In a further aspect, provided herein is a method of generating a homogenous population of modified macrophage or a cell population comprising a modified macrophage disclosed herein (such as the cell populations disclosed described in Section 5.1.5), comprising expanding and differentiating a modified monocyte (such as the modified monocyte described in Section 5.1.4) under conditions sufficient for cell differentiation into a population of macrophages (e.g., the differentiation methods disclosed in Section 5.9). In yet another aspect, provided herein is a method of generating a modified macrophage (such as the modified macrophage described in Section 5.1.5), a homogenous population of modified macrophages or a cell population comprising a modified macrophage disclosed herein (such as the cell populations disclosed described in Section 5.1.5), comprising differentiating a modified monocyte (such as the modified monocyte described in Section 5.1.4) under conditions sufficient for cell differentiation into a macrophage (e.g., the differentiation methods disclosed in Section 5.9). In certain embodiments, the modified macrophage provided herein is an immature macrophage. In certain embodiments, the immature macrophage has not been subjected to any maturation or polarization process.

[0342] In certain embodiments, the modified pluripotent cell provided herein is an iPSC. Techniques known to one of skill in the art or described herein (such as in Section 5.9) may be used to differentiate a pluripotent cell into a macrophage, a myeloid progenitor cell into a macrophage, or a monocyte into a macrophage.

[0343] The present disclosure also provides a method of generating a modified macrophage, comprising genetically disrupting in a macrophage, e.g., an immature macrophage, at least one gene (e.g., one, two, three, four, five, six, seven, eight, nine, or all gene(s)) of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.

[0344] In certain embodiments, the modified macrophage, e.g., immature macrophage, is homozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene. In certain embodiments, the modified macrophage, e.g., immature macrophage, is heterozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene.

[0345] The present disclosure also provides a method of generating a macrophage, e.g., a human macrophage, such as an immature macrophage, for example, a human immature macrophage, comprising genome editing that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the cytokine receptor is a TGFP receptor (TGFpR). In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII, or TGFpRIII. In certain embodiments, the cytokine receptor is a TGFpRII. In certain embodiments, the cytokine receptor is a TGFpRII. In certain embodiments, the macrophage does not comprise genetic disruption of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.

[0346] The present disclosure also provides a method of generating a modified macrophage, e.g., a human macrophage, such as an immature macrophage, for example, a human immature macrophage, comprising genome editing that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the cytokine receptor is a TGFP receptor (TGFpR). In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII, or TGFpRIII. In certain embodiments, the cytokine receptor is a TGFpRII. In certain embodiments, the cytokine receptor is a TGFpRII. In certain embodiments, the modified macrophage comprises genetic disruption of at least one of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene. In certain embodiments, the modified macrophage does not comprise genetic disruption of an AKT1gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.

[0347] The present disclosure also provides a method of generating a modified macrophage, e.g., a human macrophage, such as an immature macrophage, for example, a human immature macrophage, comprising introducing a polynucleotide encoding a CAR (such as a CAR described in Section 5.3). In certain embodiments, the CAR promotes Ml macrophage polarization. In certain embodiments, the CAR increases secretion of a pro- inflammatory cytokine (e.g., is a CAR described in PCT / US2023 / 071207). In certain embodiments, the intracellular domain of the CAR is selected from the group consisting of a CD40, a TLR, and an IFNyR intracellular domain. In certain embodiments, the modified macrophage comprises genetic disruption of at least one of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.5.2.5 Methods of Generating Modified CDllb+CD45+cells

[0348] Also provided herein is a method of generating a homogenous population of modified CD1 lb+CD45+cells or a cell population comprising a modified CD1 lb+CD45+cells disclosed herein (such as the cell populations disclosed described in Section 5.1.6), comprising expanding and differentiating a modified pluripotent cell (such as the modified pluripotent cells described in Section 5.1.2) under conditions sufficient for cell differentiation into a population of CD1 lb+CD45+cells (e.g., the differentiation methods disclosed in Section 5.9). In another aspect, provided herein is a method of generating a modified CD1 lb+CD45+cells (such as the modified CD1 lb+CD45+cells described in Section 5.1.6), a homogenous population of modified CD1 lb+CD45+cells or a cell population comprising a modified CD1 lb+CD45+cell disclosed herein (such as the cell populations disclosed described in Section 5.1.5), comprising differentiating a modified pluripotent cell (such as the modified pluripotent cells described in Section 5.1.2) under conditions sufficient for cell differentiation into a CD1 lb+CD45+cell (e.g., the differentiation methods disclosed in Section 5.9). Techniques known to one of skill in the art or described herein (such as in Section 5.9) may be used to differentiate a pluripotent cell into a CD1 lb+CD45+cell.

[0349] The present disclosure also provides a method of generating a modified CDl lb+CD45+cell, e.g., a modified CDl lb+CD45+CD14+cell, comprising genetically disrupting in a CD1 lb+CD45+cell, e.g., a modified CD1 lb+CD45+CD14+cell, at least one gene (e.g., one, two, three, four, five, six, seven, eight, nine, or all gene(s)) of an AKT1 gene,an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.

[0350] In certain embodiments, the modified CD1 lb+CD45+cell, e.g., a modified CD1 lb+CD45+CD14+cell, is homozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene. In certain embodiments, the modified CD1 lb+CD45+cell, e.g., a modified CD1 lb+CD45+CD14+cell, is heterozygous for the genetic disruption of the at least one gene of the MIR146A gene, STAT6 gene, GPR65 gene, AKT1 gene, AKT2 gene, IRF3 gene, IRF4 gene, PIK3CG gene, TSC1 gene, or VSIG4 gene.

[0351] The present disclosure also provides a method of generating a CD1 lb+CD45+cell, e.g., a human CD1 lb+CD45+cell, for example, a CD1 lb+CD45+CD14+cell, such as a human CD1 lb+CD45+CD14+cell, comprising genome editing that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the cytokine receptor is a TGFP receptor (TGFpR). In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII, or TGFpRIII. In certain embodiments, the cytokine receptor is a TGFpRII. In certain embodiments, the CD1 lb+CD45+cell does not comprise genetic disruption of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.

[0352] The present disclosure also provides a method of generating a modified CD1 lb+CD45+cell, e.g., a human CD1 lb+CD45+cell, comprising genome editing that results in a loss of function of a cytokine receptor, or a dominant-negative variant of a cytokine receptor. In certain embodiments, the cytokine receptor is a TGFP receptor (TGFpR). In certain embodiments, the cytokine receptor is TGFpRI, TGFpRII, or TGFpRIII. In certain embodiments, the cytokine receptor is a TGFpRII. In certain embodiments, the modified CD1 lb+CD45+cell comprises genetic disruption of at least one of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.

[0353] The present disclosure also provides a method of generating a modified CDl lb+CD45+cell, e.g., a human CDl lb+CD45+cell, comprising introducing a polynucleotide encoding a CAR (such as a CAR described in Section 5.3). In certain embodiments, the CAR promotes Ml macrophage polarization. In certain embodiments, the CAR increases secretion of a pro-inflammatory cytokine (e.g., is a CAR described inPCT / US2023 / 071207). In certain embodiments, the intracellular domain of the CAR is selected from the group consisting of a CD40, a TLR, and an IFNyR intracellular domain. In certain embodiments, the modified CD1 lb+CD45+cell comprises genetic disruption of at least one of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.5.3 Chimeric Antigen Receptors

[0354] A modified cell as provided herein may comprise a chimeric antigen receptor (CAR) or a polynucleotide encoding the CAR and capable of being expressed in the modified cell. In certain embodiments, the CAR promotes Ml macrophage polarization. In some embodiments, the CAR increases secretion of a pro-inflammatory cytokine (e.g., is a CAR described in PCT / US2023 / 071207). In certain embodiments, the intracellular domain of the CAR is selected from the group consisting of a CD40, a TLR, and an IFNyR intracellular domain. In certain embodiments, a CAR provided herein comprises an antigen recognition moiety (such as an antigen recognition moiety described in Section 5.3.1), a hinge domain (such as a hinge domain described in Section 5.3.2), a transmembrane domain (such as a transmembrane domain described in Section 5.3.3), and an intracellular domain (such as an intracellular domain described in Section 5.3.4).5.3.1 Antigen Recognition Moiety

[0355] In certain embodiments, a chimeric antigen receptor (CAR) of the present disclosure comprises an antigen recognition moiety that recognizes and binds to a specific binding element on a target of interest. Non-limiting examples of the antigen recognition moiety include a single-chain variable fragment (scFv), nanobodies, ligands to cognate receptors, native receptors against targets, and small peptides. In certain embodiments, the antigen recognition moiety is a single-chain variable fragment (scFv).

[0356] In certain embodiments, the antigen recognition moiety recognizes a tumor antigen. In certain embodiments, the tumor antigen is a tumor-specific antigen. In certain embodiments, the tumor antigen is a tumor-associated antigen. In certain embodiments, tumor antigen is a solid tumor antigen. In certain embodiments, the antigen recognition moiety recognizes a tumor antigen selected from the group consisting of CAIX, CEA, CD8, CD7, CD10, CD19, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, Erb-B2, Erb-B3, Erb-B4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, k-light chain, KDR, LeY, LI cell adhesion molecule, MAGE-A1, mesothelin, MUC16, MUC1, ERBB2, MAGE A3, CT83 (also known as KK-LC-1), p53, MARTI, GP100, proteinase3(PR1), tyrosinase, survivin, TROP-2, hTERT, EphA2, NKG2D ligands, NY-ESO-1, oncofetal antigen (h5T4), PSCA, PSMA, R0R1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGFR, EGF1R, EGFR-VIII, and CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 oncoprotein, HPV E7 oncoprotein, ERBB, CLDN18.2, GUCY2C, and GPC3.5.3.2 Hinge Domain

[0357] In certain embodiments, the CAR comprises a hinge domain. A hinge domain (also referred to as a spacer) is a structure between the antigen recognition moiety and the cell membrane. Non-limiting examples include, for example, a hinge domain derived from an IgG subclass (such as IgGl and IgG4), IgD, CD28, CSF1R, a Fey receptor, and CD8 domains. In certain embodiments, provided herein is a hinge domain lacking FcyR binding activity. In certain embodiments, provided herein is a hinge domain derived from a native T cell molecule (e.g., CD28, or CD8). In certain embodiments, a CAR provided herein comprises a hinge domain derived from CD8. In certain embodiments, a CAR provided herein comprises a hinge domain derived from an IgG subclass. In certain embodiments, a CAR provided herein comprises a hinge domain derived from IgGl. In certain embodiments, a CAR provided herein comprises a hinge domain derived from IgG2. In certain embodiments, a CAR provided herein comprises a hinge domain derived from IgG3. In certain embodiments, a CAR provided herein comprises a hinge domain derived from IgG4. In certain embodiments, a CAR provided herein comprises a hinge domain derived from IgD.5.3.3 Transmembrane Domain

[0358] In certain embodiments, the CAR comprises a transmembrane domain. A transmembrane domain is a structure that facilitates anchoring the CAR in a cell membrane, and generally consists of a hydrophobic a-helix that spans the cell membrane. Non-limiting examples include, for example, a transmembrane domain derived from an IgG subclass (such as IgGl and IgG4), IgD, CD28, CSF1R, a Fey receptor, and CD8 domains. In certain embodiments, the transmembrane domain is a single-span transmembrane e.g., a transmembrane domain derived from CD4, CD8oc, or CD28). In certain embodiments, a CAR provided herein comprises a transmembrane domain derived from CD8. In certain embodiments, a CAR provided herein does not comprise a transmembrane domain derived from CD8. In certain embodiments, a CAR provided herein comprises a transmembrane domain derived from CD28. In certain embodiments, a CAR provided herein comprises a transmembrane domain derived from CSF1R. In certain embodiments, a CAR providedherein does not comprise a transmembrane domain derived from CD28. In certain embodiments, a CAR provided herein comprises a transmembrane domain derived from CD86. In certain embodiments, a CAR provided herein does not comprise a transmembrane domain derived from CD86. In certain embodiments, a CAR provided herein comprises a transmembrane domain derived from TLR4. In certain embodiments, a CAR provided herein does not comprise a transmembrane domain derived from TLR4. In certain embodiments, the transmembrane domain is derived from the same source as at least one of the intracellular domains. For example, the transmembrane domain can be derived from CD86 and at least one intracellular domain can also be derived from CD86. As a further example, the transmembrane can be derived from DECTIN-1 and at least one intracellular domain can also be derived from DECTIN-1.5.3.4 Intracellular Domain

[0359] In certain embodiments, provided herein is a CAR comprising one or more intracellular domains. In certain embodiments, the intracellular signaling domain comprises a lymphoid or a myeloid intracellular signaling domain. In certain embodiments, the intracellular signaling domain comprises a polypeptide of CD20, CD22, CD79a, CD79b, CD3< CD35, CD3s, CD28, ICOS, 4-1BB / CD137, 0X40, or CD27 (e.g., an intracellular domain of CD20, CD22, CD79a, CD79b, CD3< CD35, CD3s, CD28, ICOS, 4-1BB / CD137, 0X40, or CD27, or a functional portion thereof).

[0360] In certain embodiments, the intracellular signaling domain comprises a nonlymphoid intracellular signaling domain. In certain embodiments, the intracellular signaling domain comprises a polypeptide of BAI-1, CD86 / B7-2, Loxlc, TIM1, TIM4, MEGF10, SCARF 1, SCARF2, CD93, DAP 12, SLAMF7, IFNyR.2, 2B4 / CD244, DECTIN-1, DECTIN- 2, CD206, DECTIN-3, CLEC2, CD40, CD80 / B7-1, LOX1, CDl lb, CSF1R, CSF2R, CSF3R, IL1R, TLR (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8), DC-SIGN, CXCL16, LIMPII, MELLEC, DNGR1, FCER1G, VEGFR2, MARCO, DEC-205, RAGE, STAB1, LANGERIN, CD36, MRC1 / CD206, or STABILIN2 (e.g., an intracellular domain of BAI-1, CD86 / B7-2, Loxlc, TIM1, TIM4, MEGF10, SCARF1, SCARF2, CD93, DAP12, SLAMF7, IFNyR.2, 2B4 / CD244, DECTIN-1, DECTIN-2, CD206, DECTIN-3, CLEC2, CD40, CD80 / B7-1, LOX1, CDl lb, CSF1R, CSF2R, CSF3R, IL1R, TLR (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8), DC-SIGN, CXCL16, LIMPII, MELLEC, DNGR1, FCER1G, VEGFR2, MARCO, DEC-205, RAGE, STAB1, LANGERIN, CD36, MRC1 / CD206, or STABILIN2, or a functional portion thereof).

[0361] In certain embodiments, the intracellular signaling domain comprises a CD40 polypeptide, an IFNy polypeptide, or a TLR polypeptide, or a functional portion thereof

[0362] In certain embodiments, the intracellular signaling domain comprises at least two intracellular signaling domains (e.g., a first intracellular signaling domain, such as a nonlymphoid, lymphoid, or myeloid intracellular signaling domain described herein, and a second intracellular signaling domain comprising one or more immune-receptor-tyrosine- based-activation-motifs (ITAMs)). Exemplary ITAM-containing intracellular signaling domains are polypeptides of CD3(^, CD35, CD3s, and IFNyRI (e.g., intracellular domains of CD3(^, CD35, CD3s, and IFNyRI, and functional portions thereof).5.4 Polynucleotides

[0363] In certain embodiments, a modified cell as provided herein comprises a polynucleotide that encodes a CAR of the present disclosure, such as a CAR described in Section 5.3. In certain embodiments, the polynucleotide comprises DNA (e.g., cDNA). In certain embodiments, the polynucleotide comprises RNA (e.g., mRNA).5.4.1 Promoter

[0364] In certain embodiments, provided herein is a polynucleotide that encodes a CAR (such as a polynucleotide described in Section 5.4), wherein the polynucleotide is operatively linked to a promoter.

[0365] In certain embodiments, the polynucleotide encoding the CAR is operatively linked to an endogenous promoter of an essential gene. As disclosed in Section 5.2, the polynucleotide encoding the CAR can be integrated into the genome of a modified cell using the SLEEK technology. In certain embodiments, the polynucleotide encoding the CAR is inserted within an endogenous coding sequence of an essential gene in the cell’s genome. In certain embodiments, the essential gene encodes a gene product that is required for survival and / or proliferation of the cell. In certain embodiments, the knock-in cassette comprises an exogenous coding sequence for the CAR in frame with and downstream (3’) of an exogenous coding sequence or partial coding sequence encoding the gene product of the essential gene, or a functional variant thereof, and wherein the cell expresses the CAR and the gene product encoded by the essential gene that is required for survival and / or proliferation of the cell, or a functional variant thereof. In certain embodiments, the CAR and the gene product encoded by the essential gene are expressed from the endogenous promoter of the essential gene. In certain embodiments, the essential gene is a housekeeping gene. In certain embodiments, the essential gene encodes GAPDH. In certain embodiments, the essential gene is an essential gene as disclosed in International Patent Publication WO2022235811.

[0366] In certain embodiments, the promoter is a constitutively active promoter. Constitutively active promoters are known in the art, and any suitable constitutively active promoter capable of expressing the CAR in a mammalian cell, such as the modified mammalian cells described in Section 5.1 can be used. Non-limiting examples of a constitutively active promoter include, for example, the elongation factor- 1 alpha (EFla) promoter, the cytomegalovirus (CMV) promoter, the cytomegalovirus (CMV) enhancer fused to the chicken beta-actin (CAG) promoter, the T7 promoter, and the phosphoglycerate kinase (PGK) promoter. In certain embodiments, the polynucleotide provided herein is operably linked to a EFla promoter. In certain embodiments, the polynucleotide provided herein is operably linked to a CAG promoter. In certain embodiments, the polynucleotide provided herein is operably linked to a PGK promoter. In certain embodiments, the polynucleotide provided herein is operably linked to a CAG promoter. In certain embodiments, the polynucleotide provided herein is operably linked to a T7 promoter.

[0367] Also provided herein is a polynucleotide that encodes a CAR (such as a polynucleotide described in Section 5.4), wherein the polynucleotide is operatively linked to a tissue specific promoter. For example, the tissue specific promoter can be a promoter that selectively enhances or facilitates transcription of a gene in certain cell types (e.g., a monocyte and / or macrophage), but not in other cell types (e.g., a non-monotype or a nonmacrophage). A representative non-myeloid reference cell may be a HeLa or a 293T cell.

[0368] In certain embodiments, the promoter is a myeloid-specific promoter. As used herein, the term “myeloid specific promoter” is intended to mean a promoter that enhances or facilitates transcription of a gene in a myeloid cell (e.g., a macrophage or monocyte), relative to a non-myeloid cell as measured by, for example, a reporter gene assay (e.g., a luciferase, or chemiluminescent reporter assay) or measure of the downstream protein encoded by the gene. In certain embodiments, the myeloid specific promoter enhances transcription of a reporter gene (e.g., GFP) in a monocyte or macrophage, relative to a constitutively active promoter (e.g., CMV), and the myeloid specific promoter does not enhance transcription of a reporter gene (e.g., GFP) in a non-myeloid reference cell, relative to a constitutively active promoter (e.g., CMV). A representative non-myeloid reference cell may be, for example, a human intestinal epithelial cell (e.g., Caco-2), a cervix epithelioid carcinoma cell (e.g., HeLa), a human embryonic kidney cell 293 (e.g., HEK-293 or 293 T), a T lymphocyte (e.g., Jurkat), or a mouse osteoblast (e.g., Oct-1).

[0369] In certain embodiments, the myeloid specific promoter is a native macrophage or a native monocyte promoter, or fragment thereof. For example, the promoter can include the full-length, or a fraction thereof, of the promoter of a gene that is expressed in monocytes and / or macrophages (e.g., CD36, CD68, CD1 lb, or CSF1R). In certain embodiments, the promoter can include the full-length, or a fraction thereof, of the promoter of a gene that is selectively expressed in monocytes and / or macrophages relative to a non-monotype or a nonmacrophage, such as, for example, a HeLa or 293 T cell.

[0370] In certain embodiments, the myeloid specific promoter is synthetic promoter. Techniques known to one of skill in the art or described herein may be used to generate and screen synthetic promoters. For example, synthetic promoters can be generated by random ligation of myeloid / macrophage cis elements. In certain embodiments, synthetic promoter is selected from the group consisting of synthetic promoter-146 (SP146) (GenBank: DQ107383.1), synthetic promoter-107 (SP107) (GenBank: DQ107382.1), and synthetic promoter-60 (SP60) (GenBank: DQ107381.1). In certain embodiments, the promoter is a SP146 promoter. In certain embodiments, the promoter is a SP107 promoter. In certain embodiments, the promoter is a SP60 promoter.5.5 Vectors

[0371] In certain embodiments, a modified cell as presented herein comprises a vector comprising a polynucleotide encoding a CAR (such as a polynucleotide described in Section 5.4). In certain embodiments, the vector is a viral vector. In certain embodiments, the viral vector is selected from the group consisting of an adenoviral vector, a lentiviral vector, and a retroviral vector. In certain embodiments, the vector is an adenoviral vector. In certain embodiments, the vector is a lentiviral vector. In certain embodiments, the vector is a retroviral vector.

[0372] In certain embodiments, the vector comprises one or more selection markers. Non-limiting examples of a selection marker include, for example, drug resistance genes for G418 (neo), puromycin (pac), hygromycin B (hph), zeocin (zeo), blasticidin S (bsd), and histidinol (hisD), as well selection markers suitable for fluorescence-activated cell sorting (FACS), such as, green fluorescent protein (GFP), yellow fluorescent protein (YFP), mCherry, and cyan fluorescent protein (CFP).

[0373] In certain embodiments, the one or more selection markers are operably linked to a promoter that is different from the promoter that regulates expression of the CAR. In certain embodiments, the one or more selection markers are operably linked to a promoter selected from the group consisting of EFla, CAG and PGK. In certain embodiments, the oneor more selection markers are operably linked to a EFla promoter. In certain embodiments, the one or more selection markers are operably linked to a CAG promoter. In certain embodiments, the one or more selection markers are operably linked to a PGK promoter. In certain embodiments, the vector comprises constitutive expression or for inducible expression. In certain embodiments, the vector comprises constitutive expression. In certain embodiments, the vector comprises inducible expression. The selection of promoters, e.g., strong, weak, tissue-specific, inducible, and developmental-specific, is within the ordinary skill of the artisan.

[0374] In certain embodiments, the vector comprises two or more selection markers that are muliticistronic. In certain embodiments, one selection marker (e.g., a drug resistance gene) is upstream a 2 A peptide (e.g., P2A, T2A, E2A and F2A) and another selection marker (e.g., a selection marker suitable for fluorescence-activated cell sorting (FACS)) is downstream the 2A peptide. In certain embodiments, one selection marker (e.g., a selection marker suitable for fluorescence-activated cell sorting (FACS)) is upstream a 2A peptide (e.g., P2A, T2A, E2A and F2A) and another selection marker (e.g., a drug resistance gene) is downstream the 2A peptide. In certain embodiments, one selection marker (e.g., a drug resistance gene) is upstream an Internal Ribosome Entry Site (IRES) element and another selection marker (e.g., a selection marker suitable for fluorescence-activated cell sorting (FACS)) is downstream the IRES element. In certain embodiments, one selection marker (e.g., a selection marker suitable for fluorescence-activated cell sorting (FACS)) is upstream a IRES element and another selection marker (e.g., a drug resistance gene) is downstream the IRES element.

[0375] In certain embodiments, the vector is designed for transient expression, stable expression, or both. In certain embodiments, the vector is designed for stable expression. In certain embodiments, the vector is designed for transient expression.

[0376] 5.6 Polypeptides

[0377] The present disclosure further provides a CAR (such as a CAR describe in Section 5.3) comprising an antigen recognition moiety polypeptide (such as an antigen recognition moiety described in Section 5.3.1), a hinge domain polypeptide (such as a hinge domain described in Section 5.3.2), a transmembrane domain polypeptide (such as a transmembrane domain described in Section 5.3.3), and an intracellular domain polypeptide (such as an intracellular domain described in Section 5.3.4), wherein the CAR is present on the surface of a modified cell as presented herein.5.7 Compositions

[0378] The present disclosure further provides a composition comprising a modified cell of the present disclosure (such as a modified cell described in Section 5.1). In certain embodiments, provided herein is a composition comprising a modified pluripotent cell of the present disclosure (such as a modified pluripotent cell described in Section 5.1.2). In certain embodiments, provided herein is a composition comprising a modified myeloid progenitor cell of the present disclosure (such as a modified myeloid progenitor cell described in Section 5.1.3). In certain embodiments, provided herein is a composition comprising a modified monocyte of the present disclosure (such as a modified monocyte described in Section 5.1.4). In certain embodiments, provided herein is a composition comprising a modified macrophage of the present disclosure (such as a modified macrophage described in Section 5.1.5). In certain embodiments, the modified macrophage provided herein is an immature macrophage. In certain embodiments, the immature macrophage has not been subjected to any maturation or polarization process. In certain embodiments, provided herein is a composition comprising a modified CD1 lb+CD45+cell of the present disclosure (such as a modified CD1 lb+CD45+cell described in Section 5.1.6). In certain embodiments, the modified CD1 lb+CD45+cell disclosed herein expresses a detectable level of CD14 (e.g., CD1 lb+CD45+CD14+). In certain embodiments, the modified CD1 lb+CD45+cell disclosed herein does not express a detectable level of CD14 (e.g., CD1 lb+CD45+CD14‘). In certain embodiments, the modified CD1 lb+CD45+CD14‘ cell disclosed herein can mature into cells that express a detectable level of CD14. In certain embodiments, the modified CDl lb+CD45+cell (e.g., CD1 lb+CD45+CD14+cell or CD1 lb+CD45+CD14- cell) disclosed herein has the properties of killing target cells (e.g., via CAR or monoclonal antibody (mAb) targeting), phagocytosing particles (e.g., bacteria), and / or migrating towards chemokines.

[0379] The present disclosure further provides a pharmaceutical composition comprising a modified cell of the present disclosure (such as a modified cell described in Section 5.1) and a pharmaceutically acceptable carrier. In certain embodiments, provided herein is a pharmaceutical composition comprising a modified pluripotent cell of the present disclosure (such as a modified pluripotent cell described in Section 5.1.2) and a pharmaceutically acceptable carrier. In certain embodiments, provided herein is a pharmaceutical composition comprising a modified myeloid progenitor cell of the present disclosure (such as a modified myeloid progenitor cell described in Section 5.1.3) and a pharmaceutically acceptable carrier. In certain embodiments, provided herein is a pharmaceutical composition comprising a modified monocyte of the present disclosure (suchas a modified monocyte described in Section 5.1.4) and a pharmaceutically acceptable carrier. In certain embodiments, provided herein is a pharmaceutical composition comprising a modified macrophage of the present disclosure (such as a modified macrophage described in Section 5.1.5) and a pharmaceutically acceptable carrier. In certain embodiments, the modified macrophage provided herein is an immature macrophage. In certain embodiments, the immature macrophage has not been subjected to any maturation or polarization process. In certain embodiments, provided herein is a pharmaceutical composition comprising a modified CD1 lb+CD45+cell of the present disclosure (such as a modified CD1 lb+CD45+cell described in Section 5.1.6) and a pharmaceutically acceptable carrier.

[0380] The present disclosure further provides a pharmaceutical composition comprising an effective amount of a modified cell of the present disclosure (such as a modified cell described in Section 5.1) and a pharmaceutically acceptable carrier. In certain embodiments, provided herein is a pharmaceutical composition comprising an effective amount of a modified pluripotent cell of the present disclosure (such as a modified pluripotent cell described in Section 5.1.2) and a pharmaceutically acceptable carrier. In certain embodiments, provided herein is a pharmaceutical composition comprising an effective amount of a modified myeloid progenitor cell of the present disclosure (such as a modified myeloid progenitor cell described in Section 5.1.3) and a pharmaceutically acceptable carrier. In certain embodiments, provided herein is a pharmaceutical composition comprising an effective amount of a modified monocyte of the present disclosure (such as a modified monocyte described in Section 5.1.4) and a pharmaceutically acceptable carrier. In certain embodiments, provided herein is a pharmaceutical composition comprising an effective amount of a modified macrophage of the present disclosure (such as a modified macrophage described in Section 5.1.5) and a pharmaceutically acceptable carrier. In certain embodiments, the modified macrophage provided herein is an immature macrophage. In certain embodiments, the immature macrophage has not been subjected to any maturation or polarization process. In certain embodiments, provided herein is a pharmaceutical composition comprising an effective amount of a modified CD1 lb+CD45+cell of the present disclosure (such as a modified CDl lb+CD45+cell described in Section 5.1.6) and a pharmaceutically acceptable carrier.

[0381] In certain embodiments, the modified cell (e.g., a modified cell described in Section 5.1) comprised in the presently disclosed compositions and pharmaceutical compositions (e.g., compositions and pharmaceutical compositions disclosed in Section 5.7) comprises at least one gene of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene,an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene. In certain embodiments, the modified cell comprises genetic disruption of an MIR146A gene. In certain embodiments, the modified cell comprises genetic disruption of a STAT6 gene. In certain embodiments, the modified cell comprises genetic disruption of a GPR65 gene. In certain embodiments, the modified cell comprises genetic disruption of an AKT1 gene. In certain embodiments, the modified cell comprises genetic disruption of an AKT2 gene. In certain embodiments, the modified cell comprises genetic disruption of an IRF3 gene. In certain embodiments, the modified cell comprises genetic disruption of an IRF4 gene. In certain embodiments, the modified cell comprises genetic disruption of a PIK3CG gene. In certain embodiments, the modified cell comprises genetic disruption of a TSC1 gene. In certain embodiments, the modified cell comprises genetic disruption of a VSIG4 gene.

[0382] In certain embodiments, the modified cell provided herein comprises genetic disruption of a gene encoding a cytokine receptor (e.g., IL-10R, TGFpRI, TGFpRII, or TGFpRIII). In certain embodiments, the modified cell provided herein comprises an exogenous polynucleotide encoding a dominant-negative variant of a cytokine receptor (e.g., dnIL-lOR, dnTGFpRI, dnTGFpRII, or dnTGFpRIII). In certain embodiments, the modified cell provided herein further comprises genetic disruption of at least one of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene.

[0383] In certain embodiments, the modified cell provided herein comprises a polynucleotide encoding a chimeric antigen receptor (CAR). In certain embodiments, the modified cell provided herein further comprises genetic disruption of at least one of an AKT1 gene, an AKT2 gene, a GPR65 gene, an IRF3 gene, an IRF4 gene, an MIR146A gene, a PIK3CG gene, a STAT6 gene, a TSC1 gene, or a VSIG4 gene. In certain embodiments, the CAR promotes Ml macrophage polarization. In certain embodiments, the CAR increases secretion of a pro-inflammatory cytokine (e.g., is a CAR described in PCT / US2023 / 071207). In certain embodiments, the intracellular domain of the CAR is selected from the group consisting of a CD40, a TLR, and an IFNyR intracellular domain.

[0384] In certain embodiments, the modified cell provided herein further comprises genetic disruption of a SIRPA gene and / or a SIGLEC10 gene. In certain embodiments, the modified cell provided herein further comprises genetic disruption of a gene encoding a cytokine receptor (e.g., IL-10R, TGFpRI, TGFpRII, or TGFpRIII). In certain embodiments, the modified cell provided herein further comprises an exogenous polynucleotide encoding adominant-negative variant of a cytokine receptor (e.g., dnIL-lOR, dnTGFpRI, dnTGFpRII, or dnTGFpRIII). In certain embodiments, the modified cell provided herein further comprises an exogenous polynucleotide encoding a proinflammatory cytokine (e.g., IL-12 and / or IFNy). In certain embodiments, the modified cell provided herein further comprises a polynucleotide encoding a chimeric antigen receptor. In certain embodiments, the modified cell provided herein further comprises (i) genetic disruption of a B2M gene, (ii) genetic disruption of a CIITA gene, (iii) genetic disruption of an RFX gene, and / or (iv) an exogenous polynucleotide encoding HLA-E.

[0385] As used herein, the term “pharmaceutically acceptable” when used in reference to a carrier, is intended to mean that the carrier, diluent, or excipient is not toxic or otherwise undesirable, (i.e., the material may be administered to a...

Claims

WHAT IS CLAIMED IS:

1. A modified cell comprising genetic disruption of at least one gene of(a) a microRNA 146a (MIR146A) gene;(b) a signal transducer and activator of transcription 6 (STAT6) gene;(c) a G protein-coupled receptor 65 (GPR65) gene;(d) an AKT serine / threonine kinase 1 (AKT1) gene;(e) an AKT serine / threonine kinase 2 (AKT2) gene;(f) an interferon regulatory factor 3 (IRF3) gene;(g) an interferon regulatory factor 4 (IRF4) gene;(h) a phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (PIK3CG) gene;(i) a TSC complex subunit 1 (TSC1) gene; or(j) a V-set and immunoglobulin domain containing 4 (VSIG4) gene, wherein the modified cell is a modified pluripotent cell, a modified myeloid progenitor cell, a modified monocyte, a modified macrophage, or a modified CD1 lb+CD45+cell.

2. The modified cell of claim 1, wherein the modified cell is a modified pluripotent cell.

3. The modified cell of claim 2, wherein the modified pluripotent cell is a modified induced pluripotent stem cell (iPSC).

4. The modified cell of claim 3, wherein the iPSC has been reprogrammed from a peripheral blood mononuclear cell (PBMC), a CD34+cord blood cell, a macrophage, a monocyte, or a fibroblast.

5. The modified cell of claim 1, wherein the modified cell is a modified macrophage, a modified monocyte, or a modified CD1 lb+CD45+cell.

6. The modified cell of claim 5, wherein the modified macrophage is an immature macrophage.

7. The modified cell of claim 5, wherein the modified CD1 lb+CD45+cell is a CD1 lb+CD45+CD14’ cell or a CD1 lb+CD45+CD14+cell.

8. The modified cell of any one of claims 1-7, wherein the modified cell is homozygous or heterozygous for the genetic disruption of the at least one gene.

9. A modified cell, wherein: i) the modified cell is a modified macrophage, a modified monocyte, or a modified CD1 lb+CD45+cell, and ii) the modified macrophage, monocyte, or CD1 lb+CD45+cell is derived from an iPSC that is homozygous or heterozygous for genetic disruption of at least one gene of(a) an MIR146A gene;(b) a STAT6 gene;(c) a GPR65 gene;(d) an AKT1 gene;(e) an AKT2 gene;(f) an IRF3 gene;(g) an IRF4 gene;(h) a PIK3 C G gene ;(i) a TSCl gene; or(j) a VSIG4 gene.

10. The modified cell of claim 9, wherein the modified macrophage is an immature macrophage.

11. The modified cell of claim 9, wherein the modified CD1 lb+CD45+cell is a CD1 lb+CD45+CD14’ cell or a CD1 lb+CD45+CD14+cell.

12. The modified cell of any one of claims 1-11, further comprising genetic disruption of a signal regulatory protein alpha (SIRPA) gene and / or a sialic acid-binding Ig-like lectin 10 (SIGLEC10) gene.

13. The modified cell of claim 12, wherein the modified cell is homozygous or heterozygous for the genetic disruption of the SIRPA gene and / or the SIGLEC10 gene.

14. The modified cell of any one of claims 1-13, further comprising genetic disruption of a gene encoding a cytokine receptor, optionally wherein the gene is an IL 1 ORA gene, an IL10RB gene, a TGFBR1 gene, a TGFBR2 gene, or a TGFBR3 gene.

15. The modified cell of any one of claims 1-13, further comprising an exogenous polynucleotide encoding a dominant-negative (dn) variant of a cytokine receptor, optionally wherein the dominant-negative variant of the cytokine receptor is a dominant-negative variant of IL-10R (dnIL-lOR) or a dominant-negative variant of TGFpR (dnTGFpR).

16. The modified cell of any one of claims 1-15, further comprising an exogenous polynucleotide encoding a proinflammatory cytokine.

17. The modified cell of claim 16, wherein the proinflammatory cytokine is IL-12 or IFNy.

18. The modified cell of any one of claims 1-17, further comprising a polynucleotide encoding a chimeric antigen receptor (CAR).

19. The modified cell of claim 18, wherein the CAR comprises an antigen recognition moiety, a hinge domain, a transmembrane domain, and an intracellular signaling domain.

20. The modified cell of claim 18 or 19, wherein the CAR promotes Ml macrophage polarization.

21. The modified cell of claim 20, wherein the intracellular signaling domain is selected from the group consisting of a CD40, a TLR, and an IFNyR intracellular domain.

22. The modified cell of any one of claims 19-21, wherein the antigen recognition moiety of the CAR binds to a solid tumor antigen.

23. The modified cell of any one of claims 1-22, further comprising:(i) genetic disruption of a beta-2-microglobulin (B2M) gene,(ii) genetic disruption of a class II major histocompatibility complex transactivator (CIITA) gene,(iii) genetic disruption of a regulatory factor X (RFX) gene, and / or(iv) an exogenous polynucleotide encoding major histocompatibility complex, class I, E (HLA-E).

24. The modified cell of any one of claims 1-23, wherein the modified cell comprises a genetic disruption of at least the MIR146A gene.

25. The modified cell of any one of claims 1-23, wherein the modified cell comprises a genetic disruption of at least the GPR65 gene.

26. The modified cell of any one of claims 1-23, wherein the modified cell comprises a genetic disruption of at least the STAT6 gene.

27. The modified cell of any one of claims 1-23, wherein the modified cell comprises a genetic disruption of at least the AKT1 gene.

28. The modified cell of any one of claims 1-23, wherein the modified cell comprises a genetic disruption of at least the AKT2 gene.

29. The modified cell of any one of claims 1-23, wherein the modified cell comprises a genetic disruption of at least the IRF3 gene.

30. The modified cell of any one of claims 1-23, wherein the modified cell comprises a genetic disruption of at least the IRF4 gene.

31. The modified cell of any one of claims 1-23, wherein the modified cell comprises a genetic disruption of at least the PIK3CG gene.

32. The modified cell of any one of claims 1-23, wherein the modified cell comprises a genetic disruption of at least the TSC1 gene.

33. The modified cell of any one of claims 1-23, wherein the modified cell comprises a genetic disruption of at least the VSIG4 gene.

34. A method of generating a modified pluripotent cell, comprising genetically disrupting in a pluripotent cell at least one gene of(a) an MIR146A gene;(b) a STAT6 gene;(c) a GPR65 gene;(d) an AKT1 gene;(e) an AKT2 gene;(f) an IRF3 gene;(g) an IRF4 gene;(h) a PIK3 C G gene ;(i) a TSCl gene; or(j) a VSIG4 gene.

35. The method of claim 34, wherein the modified pluripotent cell is a modified iPSC.

36. The method of claim 34 or 35, wherein the modified cell comprises genetic disruption of a SIRPA gene and / or a SIGLEC10 gene.

37. The method of claim 36, wherein the modified cell is homozygous or heterozygous for genetic disruption of the SIRPA gene and / or the SIGLEC10 gene.

38. The method of claim 34 or 35, further comprising genetically disrupting in the pluripotent cell a SIRPA gene and / or a SIGLEC10 gene.

39. The method of claim 38, wherein the genetically disrupting creates homozygous or heterozygous of the genetic disruption of the SIRPA gene and / or the SIGLEC10 gene.

40. The method of any one of claims 34-39, wherein the modified cell comprises genetic disruption of a gene encoding a cytokine receptor, optionally wherein the gene is an IL10RA gene, an IL 1 ORB gene, a TGFBR1 gene, a TGFBR2 gene, or a TGFBR3 gene.

41. The method of any one of claims 34-39, wherein the modified cell comprises an exogenous polynucleotide encoding a dominant-negative variant of a cytokine receptor, optionally wherein the dominant-negative variant of the cytokine receptor is a dominantnegative variant of IL-10R (dnIL-lOR) or a dominant-negative variant of TGFpR (dnTGFpR).

42. The method of any one of claims 34-39, further comprising genetically disrupting in the pluripotent cell a gene encoding a cytokine receptor, optionally wherein the gene is an IL 1 ORA gene, an IL 1 ORB gene, a TGFBR1 gene, a TGFBR2 gene, or a TGFBR3 gene.

43. The method of any one of claims 34-39, further comprising introducing in the pluripotent cell an exogenous polynucleotide encoding a dominant-negative variant of a cytokine receptor, optionally wherein the dominant-negative variant of the cytokine receptor is a dominant-negative variant of IL-10R (dnIL-lOR) or a dominant-negative variant of TGFPR (dnTGFpR).

44. The method of any one of claims 34-43, wherein the modified cell comprises an exogenous polynucleotide encoding a proinflammatory cytokine.

45. The method of any one of claims 34-43, further comprising introducing into the pluripotent cell an exogenous polynucleotide encoding a proinflammatory cytokine.

46. The method of claim 44 or 45, wherein the proinflammatory cytokine is IL-12 or IFNy.

47. The method of any one of claims 34-46, wherein the pluripotent cell comprises a polynucleotide encoding a CAR.

48. The method of any one of claims 34-46, further comprising introducing into the pluripotent cell a polynucleotide encoding a CAR.

49. The method of claim 48, wherein the polynucleotide encoding the CAR is introduced into the pluripotent cell prior to, concurrently with, or after the genetically disrupting.

50. The method of any one of claims 47-49, wherein the CAR comprises an antigen recognition moiety, a hinge domain, a transmembrane domain, and an intracellular signaling domain.

51. The method of any one of claims 47-50, wherein the CAR promotes Ml macrophage polarization.

52. The method of claim 51, wherein the intracellular signaling domain is selected from the group consisting of a CD40, a TLR, and an IFNyR intracellular domain53. The method of any one of claims 47-52, wherein the antigen recognition moiety of the CAR binds to a solid tumor antigen.

54. The method of any one of claims 34-53, wherein the pluripotent cell further comprises:(i) genetic disruption of a B2M gene,(ii) genetic disruption of a CIITA gene,(iii) genetic disruption of an RFX gene, and / or(iv) an exogenous polynucleotide encoding HLA-E.

55. The method of any one of claims 34-53, further comprising:(i) genetic disrupting in a B2M gene,(ii) genetic disrupting in a CIITA gene,(iii) genetic disrupting in an RFX gene, and / or(iv) introducing into the pluripotent cell an exogenous polynucleotide encoding HLA- E.

56. The method of any one of claims 34-55, wherein the method comprises genetically disrupting in the pluripotent cell at least the MIR146A gene.

57. The method of any one of claims 34-55, wherein the method comprises genetically disrupting in the pluripotent cell at least the STAT6 gene.

58. The method of any one of claims 34-55, wherein the method comprises genetically disrupting in the pluripotent cell at least the GPR65 gene.

59. The method of any one of claims 34-55, wherein the method comprises genetically disrupting in the pluripotent cell at least the AKT1 gene.

60. The method of any one of claims 34-55, wherein the method comprises genetically disrupting in the pluripotent cell at least the AKT2 gene.

61. The method of any one of claims 34-55, wherein the method comprises genetically disrupting in the pluripotent cell at least the IRF3 gene.

62. The method of any one of claims 34-55, wherein the method comprises genetically disrupting in the pluripotent cell at least the IRF4 gene.

63. The method of any one of claims 34-55, wherein the method comprises genetically disrupting in the pluripotent cell at least the PIK3CG gene.

64. The method of any one of claims 34-55, wherein the method comprises genetically disrupting in the pluripotent cell at least the TSC1 gene.

65. The method of any one of claims 34-55, wherein the method comprises genetically disrupting in the pluripotent cell at least the VSIG4 gene.

66. A method of generating a modified myeloid progenitor cell, a modified monocyte, a modified macrophage, or a modified CD1 lb+CD45+cell, comprising differentiating a modified pluripotent cell produced by the method of any one of claims 34-65 under conditions sufficient for differentiating the modified pluripotent cell into the modified myeloid progenitor cell, monocyte, macrophage, or CD1 lb+CD45+cell.

67. The method of claim 66, wherein the method comprises differentiating the modified pluripotent cell under conditions sufficient for differentiating the modified pluripotent cell into the modified macrophage, monocyte, or CD1 lb+CD45+cell.

68. The method of claim 67, wherein the modified macrophage is a modified immature macrophage.

69. The method of claim 67, wherein the modified CD1 lb+CD45+cell is a CD1 lb+CD45+CD14’ cell or a CD1 lb+CD45+CD14+cell.

70. A method of generating a modified myeloid progenitor cell, a modified monocyte, a modified macrophage, or a modified CD1 lb+CD45+cell, comprising differentiating the modified pluripotent cell of any one of claims 1-4, under conditions sufficient for differentiating the modified pluripotent cell into a modified myeloid progenitor cell, monocyte, macrophage, or CDl lb+CD45+cell.

71. The method of claim 70, wherein the method comprises differentiating the modified pluripotent cell under conditions sufficient for differentiating the modified pluripotent cell into the modified macrophage, monocyte, or CD1 lb+CD45+cell.

72. The method of claim 71, wherein the modified macrophage is a modified immature macrophage.

73. The method of claim 71, wherein the modified CD1 lb+CD45+cell is a CD1 lb+CD45+CD14’ cell or a CD1 lb+CD45+CD14+cell.

74. A homogenous population of cells comprising:(a) the modified cell of any one of claims 1-33;(b) a modified pluripotent cell made by the method of any one of claims 34-65; or(c) a modified myeloid progenitor cell, a modified monocyte, a modified macrophage, or a modified CD1 lb+CD45+cell made by the method of any one of claims 66-73.

75. The homogeneous population of cells of claim 74, wherein the homogenous population comprises the modified macrophage, monocyte, or CD1 lb+CD45+cell.

76. The homogeneous population of cells of claim 75, wherein the modified macrophage is a modified immature macrophage.

77. The homogeneous population of cells of claim 75, wherein the modified CD1 lb+CD45+cell is a CD1 lb+CD45+CD14’ cell or a CD1 lb+CD45+CD14+cell.

78. The homogeneous population of cells of any one of claims 74-77, wherein the cells are isogenic.

79. A population of cells, wherein(a) at least about 95% of the cells of the population are the modified cell of any one of claims 1-33;(b) at least about 95% of the cells of the population are a modified pluripotent cell made by the method of any one of claims 34-65; or(c) at least about 95% of the cells of the population are a modified myeloid progenitor cell, a modified monocyte, a modified macrophage, or a modified CD1 lb+CD45+cell made by the method of any one of claims 66-73.

80. The population of claim 79, wherein at least 95% of the cells of the population are the modified macrophage, monocyte, or CD1 lb+CD45+cell.

81. The population of claim 80, wherein the modified macrophage is a modified immature macrophage.

82. The population of claim 80, wherein the modified CD1 lb+CD45+cell is a CD1 lb+CD45+CD14- cell or a CD1 lb+CD45+CD14+cell.

83. The population of any one of claims 79-82, wherein the cells are isogenic.

84. A composition comprising(i) the modified cell of any one of claims 1-33,(ii) a modified pluripotent cell made by the method of any one of claims 34-65;(iii) a modified myeloid progenitor cell, a modified monocyte, a modified macrophage, or a modified CD1 lb+CD45+cell made by the method of any one of claims 66-73;(iv) the homogeneous population of cells of any one of claims 74-78, or(v) the population of cells of any one of claims 79-83.

85. The composition of claim 84, which is a pharmaceutical composition comprising an effective amount of (i) the modified cell, (ii) the modified pluripotent cell, (iii) the modified myeloid progenitor cell, modified monocyte, modified macrophage, or modifiedCD1 lb+CD45+cell, (iv) the homogeneous population of cells or (v) the population of cells; and a pharmaceutically acceptable carrier.

86. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and:(i) the modified cell of any one of claims 1-33;(ii) a modified pluripotent cell made by the method of any one of claims 34-65;(iii) a modified myeloid progenitor cell, a modified monocyte, a modified macrophage, or a modified CD1 lb+CD45+cell made by the method of any one of claims 66- 73;(iv) the homogeneous population of cells of any one of claims 74-78, or(v) the population of cells of any one of claims 79-83.

87. Use of(i) the modified cell of any one of claims 1-33;(ii) a modified pluripotent cell made by the method of any one of claims 34-65;(iii) a modified myeloid progenitor cell, a modified monocyte, a modified macrophage, or a modified CD1 lb+CD45+cell made by the method of any one of claims 66- 73;(iv) the homogeneous population of cells of any one of claims 74-78;(v) the population of cells of any one of claims 79-83; or(vi) the pharmaceutical composition of claim 86, in the manufacture of a medicament for the treatment of cancer in a subject in need thereof.

88. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of:(i) the modified cell of any one of claims 1-33;(ii) a modified pluripotent cell made by the method of any one of claims 34-65;(iii) a modified myeloid progenitor cell, a modified monocyte, a modified macrophage, or a modified CD1 lb+CD45+cell made by the method of any one of claims 66- 73;(iv) the homogeneous population of cells of any one of claims 74-78;(v) the population of cells of any one of claims 79-83; or(vi) the pharmaceutical composition of claim 86.

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